Symposium SB09 : Interfacing Bio/Nano Materials with Cancer and the Immune System

One strategy to decrease the cost and increase the use of nucleic acid amplification tests is developing sample collection technologies that reduce or eliminate the need for a cold chain for storage and transport. Blood is usually collected at a satellite collection site (SCS) via venipuncture and shipped to the testing laboratory. The cost of sample preparation steps are borne by the central lab, and the cost per test varies per result delivered to the patient depending on the local health system. The cost of tansport and associated sample preparation is about 80% of the total cost per result for many important tests in low resource settings. Removing the cold chain for blood sample transport would greatly reduce costs of blood testing for the patient, the SCS and the central lab. Without a cold chain requirement, sample collection can be carried out closer to the patient, reducing travel and opportunity costs. If the SCS does not have to maintain special storage on site, samples can be collected, batched and sent on a regular schedule to the central testing facility, thus reducing costs. If the sample collection method also prepares the sample for direct input to a NAAT, significant costs can be offset at the central lab.

We translated our three dimensional microfluidic blood sample preparation technology (SNAP) into a two dimensional flexible blood sample nucleic acid (NA) sample preparation card (SNAPFlex). The final prototype uses commodity grade self-laminating plastic sheet for the top cover, heat sealing thermal plastic (5 mil) for the waste pad cover and base layers, Ahlstrom 320 thick chromatography paper for the waste pad, and a Millipore 0.7um glass fiber filter without binder for the nucleic acid capture membrane. Everything except the capture membrane was cut on Trotec Speedy 100 laser cutter. The capture membrane was cut on Graphtec FCX200-60VC cutter plotter. The design allows for roll-to roll processing with two pick and place steps to add the waste pad and the sample capture filter. These parts can be die cut from separate materials in another roll-to-roll process.

We demonstrate the utility of SNAPflex in extracting HIV RNA from whole virions spiked into whole blood. Our results indicate consistent recovery across four logs of input virion concentrations. An initial comparison with QIAGEN QIAamp viral extraction kit shows that SNAPflex has superior recovery. QIAGEN extraction requires an initial plasma separation prior to viral RNA extraction requiring centrifugation while SNAPflex can process whole blood using passive wicking only, indicating not only performance improvement but also simplified sample processing. In stability experiments, in vitro transcribed RNA for the HIV gag gene was prepared. Samples were processed on the SNAPFlex devices. For Day 0 and no template control conditions, samples were immediately eluted and analyzed by RT-qPCR analysis. For extended time point samples (Days 1 – 14), after drying, the sample capture membranes were transferred to mylar zip-top bags containing silica packets that were sealed using a heat sealer. At the appropriate time points (1, 7, or 14 days post sample processing), the samples were eluted and analyzed by RT-qPCR. In vitro transcribed RNA for the HIV gag gene was used for standard curve analysis with input concentration ranging from 10⁴ cp/mL to 10⁷ cp/mL. These data indicate that RNA extracted by SNAPflex remains stable on the capture membrane for 14 days, demonstrating it's potential for use in a field-based HIV viral load monitoring program without the need for a cold-chain.

The activation of CAR-T cells is through the binding of CAR to a surface marker such as CD19 expressed on the tumor cells and the subsequent signal transduction to its intracellular domain such as CD28/CD3z to elicit effector responses, which is independent of the conventional T-cell receptor (TCR) and peptide major histocompatibility complex (p-MHC) pathway. Therefore, the mechanism of CAR-T activation could differ substantially from that of classic T cells, which still remains inadequately studied but essential to the quality control of CAR-T products and the design of future CAR therapies. In this talk, I will present single-cell level transcriptional and cytokine profiling of anti-CD19 CAR-T cell activation states upon antigen-specific stimulation. We found that the predominant response is a highly mixed T_H1/T_H2 functional state in the same cell and the regulatory T cell (T_reg) activity, although observed in a small fraction of activated cells, emerges from this hybrid T_H1/T_H2 population. Surprisingly, the cytokine response is largely independent of differentiation status. This explains in part the functional proteomics data generated through collaborations with Novartis and Kite Pharma, showing that ‘polyfunctional’ CAR-T cells correlate with antigen-specific activation as well as the objective response of patients in clinical trials. Our work provides new insights to the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

Systems biology is concerned with the complex interactions within and between cells that animate biology and create organisms that are more than the sum of their parts. This is currently exemplified in the field of single cell transcriptomics, where thousands of cells are analyzed with thousands of degrees of freedom, revealing novel cell types and interactions in heterogeneous tissues and diseases like cancer. One caveat however, is that single cell transcriptomics addresses a single class of molecules, poly-A-tailed mRNA, while systems biology has proven that proteins, lipids, metabolites, and more all play important roles. Here we demonstrate new methods to extend the functionality and dimensionality of single cell transcriptomics methods to address the concurrent biology of single cells beyond transcriptomics. This addresses the critical question of how different classes of molecules interact in single cells, and how this multi-modal heterogeneity manifests in biology. Current methods of multi-modal single cell analysis require interconversions of measurements, resulting in inevitable signal losses. Here we show that a microfluidic chip can integrate proteomic and transcriptomic measurements by embedding location information within transcriptomic measurements. The transcriptome is sequenced, while proteins are measured by fluorescent immunoassays. This is done with a location encoding strategy and microfluidic flow patterning, linking single cell transcriptomics captured by Dropseq-style bead based methods to single cell protein measurements made using DNA-Encoded Antibody Libraries. With this method, we measure full transcriptomes as well as proteins localized to the cytosol, mitochondria, and nucleus within the same single cells. We then show how this generalized strategy can be extended to augment single cell transcriptomics with completely orthogonal measurements, such as fluorescent measurements of glucose uptake and imaging analyses, to extend the reach of multi-modal single cell measurements in new directions.

We present a standalone, reusable, and portable microfluidic device for single-cell transcriptomic analysis. The microchip platform is designed for performing single-cell capture and lysis directly toward 3′ mRNA sequencing. The presented approach offers format flexibility with a simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and cost per sample matching that of the state-of-the-art scRNA-seq platforms. Through this approach, we profiled thousands of single cells from mouse whole breast tumor and successfully identified different cell types from tumor microenvironment (e.g tumor cells, tumor-associated macrophages, fibroblasts, B cells, and T cells). In addition, we studied the heterogeneity in tumor hypoxia in human breast cancer cells both in vitro and ex vivo and found a unique cell subset in the ex vivo model.

Single-cell RNA-sequencing (scRNA-seq) is becoming a popular tool in biology research to examine the heterogeneity of complex samples, identify distinct cell subsets, and dissect cell differentiation processes and lineage commitment. While many platforms are now available utilizing various approaches, one of the most commonly used technique involves co-isolating each single cell with a uniquely barcoded mRNA capture bead as the enabling step for preparing barcoded libraries. Droplet-based microfluidic techniques and microwell arrays have been one of the widely used approaches to achieve such cell-bead co-isolation, however, they require major capital, peripheral equipment, and complex fabrication process, which limits their wide-spread adoption. Therefore, a more accessible method should be developed to be practically used for potential applicability in numerous cell biology and clinical applications.

Our platform is composed of three components: (1) PDMS microwell array, in which single cells and mRNA capture beads are co-isolated; (2) parafilm flow channel, which is to introduce cells and reagents using a laminar flow profile; and (3) acrylic clamp, which is to create a closed environment. This format is particularly advantageous due to increased consistency, higher cell loading efficiency (~50%), reduced cell consumption (<500 cells total), and high beads loading efficiency with minimal waste of barcoded beads (>99% bead loading efficiencies). It also should be noted that the easier fabrication and assembling process can facilitate the wide-spread adoption of this platform across different laboratories.

To validate the device’s overall workflow and its technical performance in resolving populations of cells in
complex primary samples, we profiled thousands of cells from a mouse breast tumor. After sequencing and alignment, unsupervised graph-based clustering was implemented, revealing unique subpopulations in the breast tumor microenvironment, including tumor cells, tumor-associated macrophages, fibroblasts, B cells, and T cells.

Finally, the heterogeneity in tumor hypoxia in human breast cancer was studied both in vitro and ex vivo. Single cells from normoxia and hypoxia conditions were processed by this platform separately, and the downstream data analysis revealed heterogeneous cellular responses under hypoxia environment and found a unique cell subset in the ex vivo model.

Mechanical forces play a prevalent role in the immune system, particularly during the formation and duration of an immune synapse. During the synapse, a T-cell must latch onto a pathogen-primed antigen-presenting cell (APC) while enduring sheer flow forces. These mechanical forces, which range from 10 pN to 100 nN, are thought to be crucial to immune cell-to-cell interactions but directly measuring them remains an open challenge. Traditional methods of measuring cellular forces such as traction force microscopy (TFM) and FRET sensors have limitations when measuring the immune synapse. TFM cannot be employed in vivo or between cells, while FRET sensors generally bleach over the multi-hour-long duration of the synapse.

Here, we present a novel mechanical force sensor based on upconverting nanoparticles (UCNPs), which can detect inter-cellular forces throughout the formation and duration of an immune synapse. These nanoparticles absorb near-infrared light and emit visible light, with a ratiometric color response that depends on the applied force. Using solvothermal techniques, we synthesize ~11 nm diameter UCNPs comprised of alkaline-earth rare-earth fluoride host lattices (CaLuF, SrLuF, and BaLuF) doped with 30% Yb³⁺ and 2.9 % Er³⁺. As a control, we also synthesize cubic-phase NaYF₄ host lattice particles doped with 28% Yb³⁺ and 2.8% Er³⁺. X-ray diffraction and transmission electron microscopy confirm that the synthesized nanoparticles are all phase-pure (cubic) and monodisperse. We record upconversion spectra as a function of applied pressure using a diamond anvil cell. When excited at 980nm, the nanoparticles emit predominately at 550 nm (green) and 660 nm (red), with a red-to-green intensity ratio (I_R/I_G), and hence color that depends on pressure. For the four materials, CaLuF, SrLuF, BaLuF, and NaYF₄, we measure a percent change in (I_R/I_G) per pressure response. The pressure responses for the particles are 38 ± 4, 16.3 ± 0.6, 12.8 ± 0.9, and 40 ± 4 %_IR/IG/GPa for CaLuF, SrLuF, BaLuF,and NaYF₄ respectively. The response of all particles measured remains consistent over three pressure cycles, demonstrating their cyclability. Upconversion quantum yield was also studied to determine the brightness of the particles. The upconversion quantum yields for the CaLuF, SrLuF, and BaLuF particles are 0.18%, 0.53%, 0.19% at 80 W/cm² respectively [1]. We find that SrLuF particles are our most sensitive particles with the highest quantum yield of 0.53% at 80 W/cm² and the lowest noise force response at 16.3 ± 0.6 %IR/IG/GPa. We can detect mechanical pressures down to 37 MPa corresponding to 27 nN of force, a value within the range useful for the immune synapse.

Furthermore, we are exploring how to integrate UCNPs into an immune synapse between T-cells and APCs. Through charge stabilization and adding amino-functionalization groups to our UCNPs we are working towards functionalizing UCNPs to attach to the cell membrane of APCs. We will also investigate the cytotoxicity of our particles. With our highly force sensitive UCNPs, we are working towards unraveling the mechanisms of the immune synapse.

[1]S. Fischer et al., Nano Lett. 19, 3879–3885 (2019).

We are developing segmented magnetic nanowires (MNWs) as a new platform for highly specific biolabels. We found that cancer cells internalize MNWs and package them into exosomes, which are then secreted for several days. These MNWs thus enable magnetic isolation of exosomes, which could prove useful in future diagnosis. Our immediate goals are to study how cells internalize MNWs, to determine the timing and reproducibility of exosome secretion, and to improve the magnetic isolation of exosomes. Following this work, we aim to use ferromagnetic resonance (FMR) to identify specific MNW biolabels, similar to radio frequency identification (RFID). In addition, to compare the isolation efficiency we have employed high quality magnetite nanoparticles synthesized by magnetotactic bacteria (magnetosomes).
In this study, we incubated osteosarcoma (OSCA-8) cells with Fe/Au segmented MNWs with and without PEG coating for 48 hours. Internalization of MNWs as a function of concentration (5-40 μg/ml) was followed by fluorescence and transmission electron microscopy (TEM). We obtained quantitative estimates of MNW internalization by magnetic measurements. Our experiments indicated better internalization of the MNWs using PEG as a capping agent. As we increased the concentration of MNWs, both the number of cells with MNWs and the number of MNWs per cell increased. TEM images show that after uptake by cancer cells, MNWs were predominantly located within lysosomes, and they appeared to be fragmented into small segments of similar size as exosomes. These segments were mostly composed of either Fe or Au, suggesting that fragmentation occurred at or near the boundary of segments. In the case of magnetosomes, we also observed that they were internalized inside lysosomes, but their morphology remained intact.
We used two different methods for exosome isolation: non-magnetic isolation (centrifuge and ExoQuick TC) as a control and magnetic isolation. We incubated OSCA-8 cells with different concentrations of MNWs (0 to 35 μg/ml), and exosomes were isolated for up to 11 days. We analyzed the exosome size distribution using a nanoparticle tracking analyzer (NanoSight) and found that exosomes isolated magnetically, both with MNWs and magnetosomes, had similar size distributions as those isolated non-magnetically. A minimum concentration of 25 μg/ml MNWs in 3×10⁵ cells was required for appreciable magnetic isolation. Increasing the concentration of MNWs progressively; however, isolated microvesicles that had wider and more heterogeneous size distributions. TEM images of exosome isolated magnetically show that these exosomes packaged small pieces of MNWs.
Next, we successfully identified two different types of MNW biolabels by FMR. MNWs were fixed in a polymer that was placed onto an RFID chip. Each type of MNW exhibited a unique RF signature as the sample was exposed to an external magnetic field.
Our preliminary data show that MNWs appear to allow fast, inexpensive magnetic exosome isolation and is our future goal is MNW identification of exosomes. The methodology developed in this study should be transferable to develop comparable approaches to isolate and identify exosomes from virtually any type of cell.

There is a fundamental interdependence of adenosine triphosphate (ATP) dynamics and cancer development, given that ATP is required for a range of biosynthetic pathways during cancer cell proliferation, migration, and invasion. As a facile tool to monitor the ATP dynamics, biologically-deliverable nanoprobes have drawn increasing attention as an alternative to protein-based biosensors that require extensive genetic engineering for sensor-encoded cells. Nonetheless, challenges have remained with in vitro stability and toxicity issues of the nanoprobes, false positive signals that derive from cellular autofluorescence, and difficulties in normalisation of the intensiometric signals. Here, we present a programmable DNA-inorganic hybrid composite (termed as DNA flower, DNF) as a conjugation-free nanoprobe, enabling imaging of ATP dynamics in living cells upon the selective receptor-mediated internalization. The aptasensor-encoded DNF with two dye pairs achieved FRET-based ratio-metric imaging, where ATP recognition events trigger conformational changes of the aptamer within the composites and change the two fluorescence emission profiles. To the best of our knowledge, this system represents the first example of intracellularly deliverable probes that allow ratio-metric, spatial and temporal measurements of ATP levels in living cells. Capitalising on the structural stability of the DNF against nuclease degradation [1, 2], the developed probes achieved reliable and sensitive detection of changes in cytosolic ATP levels under exposure to various therapeutic agents over the time period. Given the versatility and robustness of the proposed DNA-based nanoprobes, it paves the way for facile, semi-quantitative tool-kits to monitor abundance of intracellular biomolecules during biological processes in cells.

References
1. E. Kim, L. Zwi-Dantsis, N. Reznikov, C. S. Hansel, S. Agarwal, M. M. Stevens, Adv. Mater. 2017, 29, 1701086.
2. E. Kim, S. Agarwal, N. Kim, F. Sydow Hage, V. Leonardo, A. Gelmi, M. M. Stevens, ACS Nano 2019, 13, 2888.

The efficacy of chemotherapeutics such as temozolomide and its decomposition, active product 4-amino-5-imidazole-carboxamide is often affected by the timing, quantity and frequency of dosages. There is strong interest in facilitating the ability to monitor efficacy in individual patients for specific subtypes of cancer. Real-time, dynamic measures of potency may also supplement or in some cases replace reliance on bio-imaging. Towards this end, in this work we develop new, synthetic molecular recognition sites for temozolomide, and its decomposition product AIC, grafting them onto near infrared fluorescent nanoparticles capable of forming optode or other biosensor interfaces to monitor drug efficacy in real-time. Infrared fluorescent single-walled carbon nanotubes, which have a specific DNA wrapping, are encapsulated with poly(ethylene glycol) diacrylate hydrogels and enable the selective recognition of an anti-cancer drug, temozolomide, on U-87 MG human glioblastoma cells. In both solution phase and hydrogel form, the sensors were responsive to temozolomide and show detection limit of 30 uM. Furthermore, cells exhibited no changes in viability for 7 days when in interfaced with the hydrogel. The sensors were used to track the progression of glioblastoma death following temozolomide administration. By providing real-time information of local chemotherapeutic concentration, our technology has the potential to increase the efficacy of cancer treatments.

Extracellular vesicles (EVs) are nano-sized vesicles that carry complex cargoes of lipids, proteins, and nucleic acids depending on the biogenesis and the cell origin. Tumor-derived EVs have emerged as a promising circulating biomarker for liquid biopsy application. However, finding tumor-specific EVs among a heterogeneous population of EVs remains as a major challenge. Here, we extend our EV isolation disc to enrich EVs in three different size fractions; large EVs (L-EVs; 200−1,000 nm), medium EVs (M-EVs; 100−200 nm), and small EVs (S-EVs; 20−100 nm). This centrifugal microfluidic platform allows not only enriching EVs in three size portions but also fluorescently staining for the protein profiling of individual EVs. Combining single-particle localization techniques in super-resolution microscopy and machine learning-based classification analysis, we visualize and analyze the biomarker contents in individual EVs. As a proof-of-concept, we analyzed the presence of human epidermal growth factor receptor 2 (HER2) or prostate-specific membrane antigen (PSMA) in breast cancer cell- or prostate cancer cell-derived EVs, respectively, using the three different size fractions at the single-EV level. By reducing the complexity of EV heterogeneity in each size fraction, we found that HER2-positive breast cancer cells showed the greatest expression of HER2 in S-EVs, whereas PSMA expression was the highest in L-EVs derived from prostate cancer cells which were further confirmed using plasma-derived EVs obtained from prostate cancer patients. Our study suggests the EV heterogeneity will be broken down in several populations in specific size fraction, cell type, and biomarker using the size fraction disc with single EV analysis. Our study demonstrated that single-EV analysis could successfully identify potent subpopulations of cancer type-specific EVs, which has promising translational implications for cancer theranostics.

Adipose tissue interstitial fibrosis is implicated in obesity-associated breast cancer, but the underlying mechanisms and functional consequences remain unclear. Using a combination of experimental and computational approaches we investigated the hypothesis that obesity-associated changes of collagen microstructure promote myofibroblastic differentiation of adipose-derived stem cells (ASCs) and that the resulting ASC-mediated changes in ECM remodeling activate macrophage bias towards a pro-tumorigenic phenotype. Indeed, simulation data indicated that ECM networks with thicker fibers as present during obesity exhibit increased strain-stiffening behavior relative to networks with smaller fibers. Accordingly, ASCs cultured in microfabricated collagen gels with thicker vs. thinner fibers assumed a more contractile phenotype that corresponded to increased expression of myofibroblast markers and de novo deposition of more strained fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs cultured within scaffolds with thicker fibers exhibited a more pro-angiogenic phenotype that depended on collagen structure-dependent changes in cell contractility. Consequently, obesity-associated changes in collagen network architecture can mechanically prime the microenvironment for myofibroblast differentiation and fibrosis. Next, we tested whether obesity-associated ECM remodeling by myofibroblastic ASCs affects the relative abundance of pro (M1)- and anti (M2)-inflammatory macrophages in human breast adipose tissue. Using clinical samples, computational approaches, and decellularized ECM models we found that breast adipose tissue contains more M2-biased than M1-biased macrophages across all body mass index (BMI) categories. Obesity further increased M2-biased macrophages but did not affect M1-biased macrophage density. Gene Set Enrichment Analysis (GSEA) suggested that breast tissue macrophages from obese women are more similar to tumor-associated macrophages (TAMs) than macrophages from lean women. These changes positively correlated with adipose tissue interstitial fibrosis, and in vitro experiments indicated that obese ECM directly stimulates M2-biased macrophage functions. Importantly, however, mammographic density cannot be used as a clinical indicator of these changes. Collectively, our data suggest that ECM microarchitecture can be an independent driver of obesity-associated fibrosis, but may also promote tumorigenesis by stimulating a macrophage phenotype similar to TAMs. These studies suggest a novel, ECM-driven link between obesity and breast cancer.

Acknowledgements: This work was performed in collaboration with Vivek Shenoy, University of Pennsylvania, Andrew Dannenberg and Olivier Elemento, Weill Cornell Medicine, Neil Iyengar, Memorial Sloan Kettering Cancer Center, and Delphine Gourdon, University of Ottawa, Canada. Funding by the Center on the Physics of Cancer Metabolism through Award Number 1U54CA210184-01 from the National Cancer Institute.

Three-dimensional (3D) synthetic hydrogels have recently emerged as desirable in vitro cell culture platforms capable of representing the extracellular geometry, elasticity, and water content of tissue in a tunable fashion. Hydrogels are typically decorated with 1-3 peptide moieties to direct cell behavior, which vastly underrepresents the proteins found in the extracellular matrix (ECM) of real tissues and provides a critical limitation in their biological functionality. Further, peptides chosen are ubiquitous in ECM and are not derived from specific proteins. We developed an approach to incorporate the complex protein signature of specific tissues into biomaterial design. Our hydrogel mimics the elasticity of marrow and has 20 marrow-specific cell-instructive peptides. Compared to generic PEG hydrogels, our marrow-inspired hydrogel improves stem cell differentiation and proliferation. Additionally, this hydrogel can be easily tuned to probe ECM-mediated cell signaling. Here, we made a collagen-null marrow hydrogel, and show that prostate cancer cell growth is dependent on this peptide in a nutrient depleted, marrow-like environment. We propose this tissue-centric approach as the next generation of 3D hydrogel design for applications in tissue engineering.

Hypoxia, defined as low oxygen tension, is a characteristic feature of solid tumors and a hallmark of aggressive cancers. Metabolic adaptation to hypoxia leads to tumor cell growth and invasion, resistance to apoptosis, and multi-drug resistance. For decades, a number of solid tumor models have been engineered to emulate key aspects of tumor biology such as hypoxia. However, challenges with tumor formation and reproducibility, inadequate biomechanical cues provided to cells, and uncontrolled oxygen depletion among other limitations led to non-physiological tumor cell responses and inaccurate clinical predictions to anticancer drugs. To model solid tumors more accurately, we have recently developed an innovative approach using porous cryogel scaffolds inducing rapid oxygen depletion while enabling cellular rearrangement into spherical-like cell aggregates within a three-dimensional (3D) polymer network. Our main objectives were: (1) to engineer hypoxia-inducing cryogels to induce cellular hypoxia; (2) to provide a biophysical support enabling tumor cell attachment, proliferation, and remodeling, and (3) to evaluate acquired resistance of hypoxic B16-F10 melanoma cells to anti-cancer drugs.
Engineered oxygen-depleting scaffolds are capable of inducing local hypoxia while promoting tumor cell remodeling and aggressiveness, leading to anti-cancer drug resistance. Our preliminary data suggest that the tumor-cell laden hypoxia-inducing cryogels mimic key aspects of the native tumor microenvironment, making these advanced cellularized scaffolds a promising platform for drug screening in 3D and potentially improving anti-cancer drug development and discovery.

Bone tissue engineering is a promising technology for next generation regenerative medicine based therapies. In addition, these tissue engineered scaffolds can be used as testbeds to create humanoid cancer tumors at primary site and at metastatic site. In particular, breast and prostate cancer have the propensity to metastasize to bone at which point the disease is incurable. The design of tin vitro testbeds is aparticularly attractive due to lack of availability of human samples at this stage of metastasis and failure of animal models (since animals die before metastasis to bone). We have designed an amini acid modified nanoclay based polymeric scaffold for bone tissue engineering. This scaffold uses biomimetic mineralization of hydroxyapatite inside nanoclays galleries mimicking remodeling human, characterizedby low Ca/P stoichiometry bone, a niche to which cancer cells migrate. On seeding the tissue engineered bone scaffolds with prostate and breast cancer cell lines led to the creation of tumoroids of cancer. The tumoroids exhibited a late stage metastasis as indicated by the gene expression and protein expression. Tumors were extracted from r scaffolds and we conducted FTIR experiments as well as nanomechanical experiments at cancer metastasis progression from 0 to 20 days. Softening of tumors as well as increase in plastic deformations were captured at metastasis. In addition unique spectral markers of DNA, RNA, proteins and lipids were obstained that indicate progression of metastasis. Unique bone-biomimetic scaffolds provide new opportunities as testbeds to evaluate cancer metastasis.

Cancer is the second leading cause of death in the US and 92% of such casualties are due to metastasis. Thus, discovering mechanisms employed by cells to promote metastasis and developing strategies to stop them, are the main targets of cancer research. In particular, extracellular vesicles (EVs) shed from cancer cells, have been recently implicated as important mediators of cellular communication and are suspected to play an important role in metastasis. EVs are membrane-encapsulated vesicles known to shed from several types of eukaryotic cells. Extensive research has shown the existence of two major EVs subtypes, exosomes and microvesicles (MVs). Exosomes are vesicles generated from multivesicular bodies and secreted to the extracellular space by fusing with the plasma membrane. Exosomes are between 20 -120 nm in diameter. The second type, MVs, has a larger size range between 120 nm - 1μm in diameter. MVs are directly shed from the plasma membrane into the extracellular space, preserving native properties from the mother cell. EVs of both types contain different cargo including mRNA, DNA, proteins, and receptors. MVs and exosomes differ in biogenesis, cargo, and surface makeup. Research findings suggest that they have different intracellular routes and functions in cell communication. Therefore, it is imperative to distinguish between them since their functions and communication pathways may have different effects on cells. Both populations have been highlighted in research due to their capacity to facilitate intercellular communication. In that manner, interactions between EVs and host cells have been shown to lead to modifications in cellular behavior of the recipient cell. In the context of cancer, EVs are known to influence proangiogenic activity and transformation on adipose derived stem cell (ADSC) leading to cancer progression. How these interactions occur in the molecular level is not clear. Cues most certainly come from both membrane components on EVs, as well as their internal cargo. Yet, there is a lack of techniques to study those interactions in vivo. Hence, we developed an in vitro platform that allows the isolation and study of interactions between EVs surface and ADSCs. Using hybrid supported lipid bilayer techniques developed by our group and EVs derived from cancer cells, MDA-MB-231, we originated an EVs derived supported bilayer (ESB) as a model of EVs membrane. ESB is a planar, tunable platform that can serve as a cell culture substrate to study the effects and insights of interactions between EVs surface and ADSCs. To assure preservation of EVs membrane materials in our generated ESBs, TIRF microscopy was employed to detect expression of EVs markers specific to each EV population. Furthermore, ADSCs were cultured on ESBs and cell adhesion and spreading, cell viability, and cell VEGF production were analyzed. Our results showed that ADSCs cultured on ESBs displayed higher cell spreading, stronger focal adhesions, and higher VEGF production than cells on synthetic supported lipid bilayers. Therefore, we observed that EVs surface interactions with ADSCs enhances cell adhesion, spreading, viability, and proangiogenic activity related to high VEGF production. In all cases, the influence of ESB from both EVs populations followed the same pattern with MVs derived ESB having a larger effect on ADSCs behavior compared to exosomes derived ESBs. Consequently, we present our developed ESB, containing native composition of EVs membranes, as a cell culture platform to study interactions between EVs and stromal cells. Our method allows the production of different EVs populations derived ESBs to study individually the interactions of EVs and stromal cells and to decouple the biological outcomes produced by each of the EVs population. It could be utilized as a method to investigate cell to cell interactions, and extracellular particles to cell interactions for different types of cells and for several disease scenarios.

Our group has recently developed Hybrid Donor-Acceptor Polymer Particles (HDAPPs), nanoparticles composed of the fluorescent polymer poly[(9,9-dihexylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBTDBT10) and the heat-generating polymer poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b’]dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) to create a theranostic platform. HDAPPs can be activated via 450 nm laser exposure for fluorescent imaging within the NIR tissue absorption minimum by exploiting an amplified energy transfer between the PFBDTB10 and PCPDTBSe polymer components, emitting at 825 nm. Additionally, HDAPPs can be activated by 800 nm laser exposure for photothermal ablation of colorectal cancer (CRC). To actively target CRC, HDAPPs were functionalized via hyaluronic acid (HA) coating of the NP surface to exploit overexpressed CD44 receptor on CRC. A major hurdle for the clinical translation of NPs is their interaction with the 3D tumor microenvironment, where NP transport and photothermal heat dosing may be isolated to the outermost shell of the tumor that maintains direct exposure to the nanotherapeutic. To study the targeted fluorescent detection and heating potential of HDAPPs in the 3D tumor microenvironment, we investigated the encapsulation of tumor cells in a specialized hydrogel (3D organoid) in vitro. Further, to monitor HDAPPs-mediated ablation in real-time within organoids, we demonstrated the utility of a unique fiber-optic-based (FOB) imaging system capable of laser-induced point excitation and fluorescent image reconstruction in opaque media.
HDAPPs were first synthesized using the nanoprecipitation technique, where an aqueous phase consisting of Pluronic F127 surfactant is interfaced via sonication with an organic phase of the polymer constituents to create HDAPPs nanoemulsions. HDAPPs were then coated with chitosan and further targeted to CD44 receptor via coupling of HA to the surface. Selective binding and ablation of CT26 CRC cells in 2D was demonstrated with HA-targeted NPs under photothermal activation with 800 nm stimulation, where HA-targeted NPs displayed increased binding relative to non-functionalized NPs and, upon photothermal activation with laser, CT26 cells incubated in 2D with HA-targeted NPs demonstrated a respective decrease of viability by 40%. Photothermal ablation of CT26 cells expressing green fluorescent protein (CT26.GFP) and encapsulated in 3D organoids was next assessed. HA-targeted NPs decreased cell viability in organiods by 60%, though ablation in 3D organoids occurred only under increased laser fluences in comparison to 2D culture, demonstrating decreased efficiency of photothermal therapy in 3D-tumor like conditions. A major therapeutic hindrance in therapy is diffusion limitation in tumors, where HDAPPs diffusion was measured by exposing HDAPPs to the outer surface of organoids and monitoring via confocal microscopy for up to 24 hrs. Diffusion studies showed that at least 12 hours exposure to therapeutic concentrations is necessary to achieve ablative potential. Finally, to demonstrate FOB imaging compatibility with the 3D tumor environment, organoids with CT26.GFP cells and non-functionalized HDAPPs were imaged by FOB laser excitation and fluorescent images were successfully reconstructed that map to control images.
The results of this work demonstrate the performance of a unique NP platform capable of detecting and ablating CRC within the tumor microenvironment. Coupling of organoids with the FOB imaging device demonstrates a preclinical system that can simultaneously assess CRC detection and ablation with NP therapeutics, where we aim to both develop an organoid platform for proper preclinical translation of photothermal nanotherapeutics while investigating the properties of our own HDAPPs.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechanics and biochemistry, since these are both dependent on protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and ligand density can be independently controlled. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, with an optimum occurring at intermediate stiffness. Indeed, mesenchymal morphology exhibits a similar biphasic dependence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

Introduction: Cell polarity, morphology, and migration are regulated bidirectionally by nanotopographical cues from the extracellular matrix (ECM). In particular, ECM architecture may become dysregulated during wound healing or tumor progression with highly aligned and crosslinked fibrillar nanostructures. However, these fibrillar architectures are biophysically coupled to ligand density, stiffness, and other ECM properties, making it challenging to elucidate their individual contributions. Here, we elucidate how cells respond to 3D nanotopography using a composite hydrogel consisting of magnetically aligned ECM-coated nanoparticles embedded within a background hydrogel of gelatin methacryloyl. We show that by systematically varying the biophysical parameters of the fibrillar nanostructures or the background matrix, cell protrusions and morphology can be tuned to direct cell polarization and migration.
Materials & Methods: ECM-coated 300nm magnetic nanoparticles were dispersed in gelatin methacryloyl (GelMA) precursor solution and exposed to a magnetic field gradient, inspired by previous work from Tanner et al (Biomaterials, 197, 101-118, 2019). Nanoparticles assembled into chain-like fibrillar structures under optimized field gradients, which mimicked the dimensions of ECM fibers observed in vivo. Next, the GelMA was photocrosslinked using UV light with LAP as the photoinitiator. Various cell lines (e.g. MDA-MB-231 overexpressing GFP in the cytoplasm) were then cultured in this composite hydrogel and imaged using spinning disk confocal microscopy with environmental control. Time-lapse images were then analyzed using Bitplane Imaris and custom MATLAB code.
Results & Discussion: Nanoparticle assembly and GelMA hydrogel polymerization were optimized using a custom magnetic apparatus to uniformly disperse fibrillar nanostructures over millimeter scales in 3 dimensions. We optimized the length and diameter of these fibrils based on magnetic field gradient, and further characterized GelMA as a “blank slate” with amorphous topography, tunable stiffness, pore size, and RGD-binding sites. Finally, we show that by systematically tuning either the fibrillar topography or the background matrix, cell protrusive activity as well as migration velocities can be altered. For instance, highly metastatic MDA-MB-231 cells exhibit directed migration along fibers, where providing no magnetic alignment yields a uniform dispersion of particles and yields slow, random migration with short protrusions. In comparison, a stiff background matrix of highly cross-linked GelMA limits protrusive activity and migration, while softer more porous background matrix allows cells to extend long protrusion along engineered fibers and enables higher migration velocities. These migratory behaviors were perturbed by biochemical inhibition of integrins and the cytoskeleton, which were profiled using computer vision and machine learning.

Conclusion: We demonstrate a composite 3D biomaterial with orthogonally tunable fibrillar architecture, hydrogel chemistry, and stiffness to elucidate bidirectional interactions between cells and the ECM. We find that cell protrusions, morphology, and migration are biased by varying degrees of magnetic nanoparticle chaining, which dominate over the mechanochemical cues from the surrounding GelMA hydrogel. We implement this in a scalable format that is compatible with multiwell plates, which could be extended for high content screening. Thus, we envision that this technology could be utilized to systematically characterize mechanotransduction of ECM cues in the context of targeted drug perturbations.
Acknowledgments: This work was funded by the NCI Innovative Molecular Analysis Technologies Program (R21CA212932) and Brown University through the Karen T. Romer Undergraduate Research and Teaching Award.

Organ-on-chip systems are useful tools to fundamentally study the physiology of organs and tissues, to evaluate the efficacy or toxicity of drug compounds, or to study the effect of tensors on human tissues. However, to design and fabricate and organ-on-chip system is not a trivial-task. Here we present an example of a step-by-step fabrication and validation of a 3D-printed microfluidic system that can be generically used to culture cells, micro-tissues, and organoids under well-controlled environments. The system is conformed by three compartments. The left and right compartments have one inlet and one outlet each, providing means to continuously feed liquid streams to the system. The central compartment is designed to host a cell-laden hydrogel where a microtissue can be confined and culture. A transparent lid and bottom can be adapted to enable visual inspection under a microscope.
We conducted fluorescent and saline tracer experiments to characterize the hydraulic performance of the system. In addition, we cultured MCF7-spheroids embedded in a cell-laden hydrogel construct (placed in the central chamber), to illustrate the use of this system to sustain long term micro-tissue culture experiments. We present experimental results that illustrate the flexibility and robustness of use of this 3D-printed device for tumor-on-chip experiments.
This “open-source” organ-on-chip system is intended to be a general-purpose resource to facilitate/democratize the development of new organ-on-chip applications.

Simultaneous observation and induction of biomolecular changes in the tumor microenvironment are challenging to accomplish over extended periods of time. The effect of therapeutics on the target site is often obfuscated by systemic on- and off-target effects inherent in drug administration. Thus, localized delivery and high-resolution imaging are necessary to assess cellular response to changes in the tissue microenvironment. Here, we report on the design, fabrication and testing of a novel microfluidic imaging window (MFIW) using glass and SU-8 photoresist microfabrication techniques to actively deliver agents (e.g. drugs, antibodies, inhibitors, etc.) to underlying living tissue. Microfluidic channels were fabricated using multi-layer SU-8 patterning techniques on 12 mm glass coverslips. Point spread functions were compared between standard glass windows and MFIWs and optical performance of the MFIWs was validated with 2-photon microscopy (at 880nm) on transgenic MMTV-PyMT Dendra2 fluorescent tumors, which confirmed device applicability for biological studies. Microfluidic delivery and diffusion of fluorescent dye into tissue surrogates was measured via confocal microscopy and then compared to COMSOL simulations to characterize agent delivery depth and concentration profile. These results demonstrate the ability of the device to deliver sub-microliter quantities of fluid, and the ability to control the depth and diameter of the diffusion profile. Ongoing studies are focused on insertion and testing of devices in mice, including validation of drug delivery to the tumor microenvironment, in vivo.

Immunotherapies such as immune checkpoint inhibitors (ICI) show great promise as more efficacious treatments for various cancers compared to current methods such as chemotherapy. However, preclinical evaluation of immunotherapies typically relies on mouse models that have limited relevance to human responses, or on in vitro culture methods that suffer from small sample sizes, limited cell viability, a lack of physiologically relevant stimuli such as flow, and an absence of tumor-immune cell interactions. To address these limitations with current models, we have developed a scalable 3D printed microfluidic devices capable of capturing multicellular tumor spheroids and perfusing them in the presence of circulating tumor infiltrating lymphocytes (TILs). Finite element modeling was first used to optimize flow conditions for capturing spheroids as well as for extended viability. Next, we experimentally characterized how T cells interacted with multicellular tumor spheroids in varying flow conditions, as well as after anti-PD-1 immune checkpoint treatment. Ultimately, these microfluidic devices will enable researchers and clinicians alike to test various immunotherapies in a scalable, rapid, and in vivo relevant manner.

Self-assembling peptides have been used in recent decades for a variety of medical applications, including but not limited to tissue engineering, drug delivery, and biosensing. One peptide of particular interest, the leucine-zipper of the transcription factor GCN4, can exist in multiple oligomeric states based on the hydrophobic interface. Through functionalizing these GCN4 peptides with metal-binding ligands on the hydrophilic faces of the coiled coil, metal ion-mediated self-assembly can be promoted radially,¹ and by altering the C- and N-termini of the peptide, self-assembly can be facilitated linearly.² A design which merges these two modifications to create crosslinked coiled coil biomaterials will be reported.
¹ Nambiar, M.; Wang, L. S.; Rotello, V.; Chmielewski, J. "Reversible Hierarchical Assembly of Trimeric Coiled-Coil Peptides into Banded Nano- and Microstructures" J. Am. Chem. Soc. 2018, 140, 13028-13033.
² Nepal, M.; Sheeldo, M.; Das, C.; Chmielewski, J. "Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly" J. Am. Chem. Soc. 2016, 138, 11051-11057.

MicroRNAs (miRNAs) are small non-coding nucleotides playing a crucial role in posttranscriptional expression and regulation of target genes in nearly all kinds of cells. In this study, we demonstrate a reliable and efficient capture and purification of miRNAs and intracellular proteins using magnetic nanoparticles functionalized with antisense oligonucleotides. For this purpose, a tumor suppressor miRNA (miR-198), deregulated in several human cancer types, was chosen. Magnetite nanoparticles carrying the complementary sequence of miR-198 (miR-198 antisense) on their surface were delivered into cells and subsequently used for the extracellular transport of miRNA and proteins. The successful capture of miR-198 was demonstrated by isolating RNA from magnetic nanoparticles followed by real-time PCR quantification. Our experimental data showed that antisense-coated particles captured 5-fold higher amounts of miR-198 when compared to the control nanoparticles. Moreover, several proteins that could play a significant role in miR-198 biogenesis were found attached to miR-198 conjugated nanoparticles and analyzed by mass spectrometry. Our findings demonstrate that a purpose-driven vectorization of magnetic nanobeads with target-specific recognition ligands is highly efficient in selectively transporting miRNA and disease-relevant proteins out of cells and could become a reliable and useful tool for future diagnostic, therapeutic and analytical applications.

Non-thermal atmospheric pressure plasma (NTAPP) is described as a partially ionized gas containing charged particles and radicals at atmospheric pressure. Recently, some studied have reported the application of NATPP to regenerative medicine for wound healing and activation of immune responses. In this study, we demonstrates that NTAPP activates the proliferation of various mesoderm-derived adult stem cells including adipose tissue-derived stem cells (ASCs), bone marrow-derived stem cells (BM-MSCs) and hematopoietic stem cells (HSCs) to develop NTAPP as an efficient tool for adult stem cell therapy. In our study, NTAPP induced the proliferation of these adult stem cells by 1.5 to 2-fold, compared with unexposed cells. Also, NTAPP exposure increased the expression of stem cell-specific surface markers, CD44 and CD105, by 5-fold in BM-MSCs, compared with that in unexposed cells in a low glucose medium with a low centration of basic fibroblast growth factor (b-FGF), and augmented the expression of well-known pluripotent genes including Oct4, Sox2 and Nanog in ASCs and BM-MSCs compared to that in unexposed control cells, suggesting that NTAPP activated the proliferation of various adult stem cells without affecting the stem cell properties. The whole genome expression profiles of ASCs whose proliferation is highly activated by NTAPP showed that NTAPP upregulated genes for cytokines and growth factors, while it downregulated genes functioning in the intrinsic apoptotic pathway. With genes that showed more than two-fold increase or decrease in NTAPP-exposed ASCs by whole RNA-sequencing, we also confirmed the increased mRNA expression of leukemia inhibitory factor (LIF), heparin-binding epidermal growth factor (HBEGF), interleukin-6 (IL-6), IL-11, fibroblast growth factor 16 (FGF16), and IL-1β by 2 to 12-fold and decreased expression of bcl-2-binding component 3 (BBC3), death-associated protein kinase 1 (DAPK1), caspase 10 (CASP10), tumor protein p53 (TP53), and receptor interacting serine/threonine kinase 3 (RIPK3) by 2 to 4.5-fold by real-time PCR, compared with the unexposed control. The increased expression of various cytokines and growth factors was highly declined comparing with NTAPP-treated ASCs in the presence of a nitric oxide (NO) scavenger, indicating that NO generated from NTAPP is mainly responsible for the enhanced expression of cytokines and growth factors by NTAPP. Collectively, these results strongly demonstrated that NTAPP would be an excellent tool to control the expression of cytokines and growth factors to trigger the activation of adult stem cells in vitro for stem cell therapy.

High-intensity focused ultrasound (HIFU) guided by Magnetic Resonance Imaging (MRI) is used as a therapeutic technique in clinical practice. To improve its efficacy, it is essential to accurately identify, or spotlight, target tissues for treatment. In this talk, we present a novel contrast enhancement method based on MRI-guided HIFU and mesoporous silica nanoparticles (MSNs). HIFU modulation was applied to MSNs modified with a thermo-responsive and biocompatible polymer to generate MRI T₁ relaxivity changes in regions of interest. A modulation enhancement map (MEM) was reconstructed to spotlight the region of interest with almost two orders of magnitude increase in contrast compared to conventional enhancement methods. HIFU provides a 1.5 mm³ focal point that is spatially controllable in three-dimensions. The data acquisition time for the experiments in our study was 100 s, and it is practical for imaging during clinical applications. The small (2 °C) temperature change would cause minimal tissue damage. This non-invasive method can be applied in improving the identification of target tissues, such as delineation of tumor margins, for MRI-guided HIFU therapies.

Traditional sensing techniques such as enzyme-linked immunosorbent assay (ELISA) offers limits of detection (LOD) in the pM-fM range, however, it is labor-intensive, require multiple washing steps, and most importantly utilize expensive antibodies for analytical detection which limits their utility for point of care (POC) applications. To address these issues, colloidal nanoparticle (NP) biosensors can be designed to provide precise analytical detection without requiring antibodies and extensive post-processing steps. Optical NPs offer a wide range of biosensing applications such as ultrasensitive detection of cancer biomarkers in human serum, multiplexed, point of care (POC) detection of analytes, and in vivo sensing of reactive oxygen species. Optical nanoparticles, such as quantum dots (QDs) have exhibited comparable LOD to ELISA in some cases with high signal to noise ratios (SNR>10). However, the heavy-metal content of QDs results in high cytotoxicity prohibiting the wide-scale use of QDs particularly in clinical applications. Luminescent upconversion NPs have been introduced as biocompatible alternatives with comparable LODs (~6 nM). However, they require expensive, high energy infrared lasers and are limited in their ability for multiplexing. We have recently developed a Bi³⁺-doped YVO₄:Eu³⁺ core-shell nanoarchitecture that can utilize the changes in surface dipole moments as a novel mechanism for detection. It was found that the downconversion luminescent signal of the NPs can be modulated with respect to the dipole moment. The developed NP sensor was then functionalized with D-biotin to be used for label-free detection of avidin. Decorating the surface of the NPs with D-biotin generated a positive dipole moment (i.e., electron withdrawing) resulting in a decreased luminescent signal. The surface coverage of the biotin decorated NPs was evaluated using thermogravimetric analysis (TGA) to perform by time and concentration-dependent studies into how avidin binding further altered the luminescent properties of the NPs. Exposure to avidin resulted in a further decrease in the luminescent signal of the NPs suggesting that the interaction of avidin with biotin resulted in a positive dipole moment with a larger magnitude. This decrease in luminescent signal was found to be specific and concentration-dependent with respect to the free avidin concentration in solution. The downconversion NPs exhibited a similar SNR to upconversion NPs (SNR~15) with a LOD comparable to those of the QDs. This novel approach in biosensing can be expanded to the label-free detection of disease biomarkers or pathogens at low limits of detection in bulk fluid which is paramount for POC applications.

Interstitial flow, the slow fluid flux occurring in the interstitial space of extracellular matrix (ECM), is mostly negligible in normal soft tissues, yet it could play crucial roles in regulating cellular behaviors in some pathological tissues or bone tissue. With the speed up to a few microns per second, the interstitial flow not only provides direct mechanical cues to the resident cells through shear stress and normal stress, but also couples with biomolecular diffusion to induce biochemical cues to the cells. Previous in vitro and in silico studies have implied that interstitial flow can regulate individual cell migration through at least two mechanisms: autologous chemotaxis and integrin-mediated focal adhesion activation [1-2]. The effectiveness of the former depends on interstitial fluid velocity; the effectiveness of the latter depends on both interstitial fluid velocity and ECM permeability. However, even in vitro, these two mechanisms cannot be rigorously examined due to the inevitable inverse correlation between permeability and stiffness of hydrogel matrices. Specifically, stiffness is another important mechanical cue that influences cell migration. Therefore, developing a thorough understanding of cellular behaviors regulated by interstitial flow requires an ability to independently control permeability and stiffness of the matrix, interstitial fluid velocity, as well as other relevant properties, such as ligand composition and density in the matrix; this independent control is still an unmet challenge in existing 3D in vitro models.

To address this challenge, we developed a dual-gel dual-porosity metamaterial integrating with a microfluidic platform for studying the effects of interstitial flow on cellular behaviors. This novel in vitro model, consisting of GelMA-PEGDA microgels and collagen, allows for independently controlling stiffness, cell-binding cites, permeability, and interstitial flow, as well as live imaging of migrating dynamics. We will present our preliminary results showing the effectiveness of our dual-gel metamaterial based cell culture model by characterizing the migration of breast cancer cells under different flow conditions and ECM properties.

References:
[1] W. J. Polacheck, J. L. Charest, R. D. Kamm, Interstitial flow influences direction of tumor cell migration through competing mechanisms, PNAS 2011, 108, 11115.
[2] J. D. Shields, M. E. Fleury, C. Yong, A. A. Tomei, G. J. Randolph, M. A. Swartz, Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling, Cancer Cell 2007, 11, 526.

Increasing the precision and reliability in biosensing is critical for accurate clinical disease diagnosis and rapid implementation of relevant treatment modalities. A key factor in high precision detection is the reduction of the coefficient of variation in a biosensing platform. A variety of polymer-based microbeads have been used as the medium to immobilize probes for a wide range of targets. Microbeads have been an attractive platform for diagnosis of diseases by targeting specific biomarkers in body fluids such as serum, saliva and urine, that are complex media with a large background of large variety of non-specific biomolecules, including proteins, peptides, mRNA and other small and large biopolymers. Among the several problems associated with the bead-based systems, the key issues are related to bead-to-bead signal variation because of the size and the surface versus internal bead structural variations. These limitations also include the variability and inefficiency of immobilization approaches as well as the formation and mechanical properties of the beads.
We have developed a mechanical methodology by which to mass produce agarose gel-based nanoporous microbeads with controlled internal and surface structures which facilitates acloser control over the surface functionalization. We demonstrate herein a covalent biofunctionalization of disease-specific probes and show high signal-to-noise ratio biomarker detection. For proof-of-concept demonstration, two key biomarkers were used, CRP and CTNI, that raise substantially after injury for multiple organ failure, respectively. Specifically, using gloxylation method we biofunctionalized the agarose beads with a capturing antibody protein both on the surface and throughout the porous internal structure to systematically analyze the capture of disease-specific antigens with significantly enhanced signal-to-noise ratio. The ease of manufacturing and performance characteristics of agarose microbeads demonstrated herein show a significant promise in the rapid and high precision diagnostic of disease-specific biomarkers paving the way for potential clinical implementations where the detection of the disease-specific antigens rapidly and with high efficiency is of prime importance. Funding was provided by the NIH through the National Institute of Dental and Craniofacial Research (NIH Grant Nos.3 U01 DE017793-02S1 and 5 U01 DE017793-2) and the ARO - contract No. W911NF-16-P-0030.

Ultrasound spectroscopy has evolved as a great potential non-invasive tool in the osteoporosis diagnosis and bone assessment since ultrasonic parameters such as normalized broadband ultrasound attenuation (nBUA) and speed of sound (SOS) have been used to characterize cancellous bone. However, the bone characterization is limited to the study of their relations to bone mineral density (BMD). An understanding of the underlying mechanisms responsible for the ultrasound propagation in cancellous bone is still lacking. There is also no evidence on the feasible frequency range for the assessment of bone. We have used the ultrasonic spectroscopy technique in transmission geometry to measure nBUA and speed of longitudinal ultrasonic waves in twenty-two beef cancellous bones at frequencies ranging from 2.25-7.5 MHz, with the goal of finding suitable frequency range for bone assessment. Bone porosity is another important parameter which greatly affects interaction or scattering of ultrasound in cancellous bone, which in turn affects ultrasound speed and influences the elastic properties of bone. In our study, bone porosity of each bone specimen is determined using x-ray micro CT scan and the relationships between bone porosity and ultrasonic parameters like nBUA and SOS are explored in the above-mentioned range of ultrasonic frequencies. We have utilized a systematic approach, beginning with the investigation of a polyethylene disk, moving on to the investigation of bone specimens.

Key aspects of designing effective biosensors include i) Tethering probes onto the sensing substrate while maintaining their proper orientation to appropriately bind target molecules, ii) Efficient signal transduction across soft bio/nano interfaces of the sensing surface, and iii) Reusability of the sensor chip. Solid-binding peptides (SBPs) present a viable method for non-covalent immobilization of probes onto single-atomic layer substrates—providing unprecedented prospects for sensor design. This approach delivers functionalization while eschewing the challenges posed by covalent modification, such as the introduction of lattice defects that could adversely affect sensing properties, and is therefore especially relevant to sensors based on two-dimensional materials like graphene and MoS₂. In addition, nucleic acid biomarkers, e.g., microRNA, cell-free DNA, etc., hold great potential for disease diagnosis and prognosis. Herein we design and construct heterofunctional chimeric PNA-SBP biomolecules that facilitate hybridization of nucleic acid targets onto the sensor surface. Using surface plasmon resonance spectroscopy (SPR), we demonstrate detection of DNA with these chimeric probes against a complex biological fluid, e.g., fetal bovine serum, in aqueous environment. We interrogate the effect of steric hindrance on optimal probe assembly using fluorescent microscopy and atomic force microscopy (AFM) imaging techniques, which reveal that lesser probe packing density results in greater target capture. We also demonstrate the sequential biomolecular assembly of the modular PNA-SBP system on the sensor surface and assess its performance under variety of solution conditions. These results provide a highly effective methodology for defect-free functionalization of 2D material-based bionanosensors through non-covalent means while enabling probe immobilization onto the sensing substrate. The latest results include design criteria of the chimeric probes, immobilization/biofunctionalization protocols, sensor architectures, and data acquisition and analysis procedures. Technical help by Rebekah Khajehpour and Jessica Ahrens-Tran were appreciated. The research is supported by NSF-DMREF program through the grant DMR-1629071 as part of Materials Genome Initiative (MGI).

In this talk I will discuss new algorithms and analytical technologies associated with single cell analysis, with an emphasis on capturing and analyzing multiple levels of information (i.e. multi-omic analysis) from the same single cells. In one instance, I will discuss how single cell methods can resolve cancer cell adaptive responses to therapy, and reveal that even isogenic cells can exhibit multiple, independent adaptive responses, each of which can be independently drugged. I will also discuss large library approaches designed for sampling and single cell characterization of antigen-specific T cell populations, with the goal of identifying matched antigen-TCRa/b pairs for T cells that are circulating in the blood, but are tumor-antigen (or neoantigen) experienced.

We recently developed a strategy to target peptide vaccines to lymph nodes, by linking peptide antigens to albumin-binding phospholipid-polymers. Small peptides are normally rapidly dispersed in the bloodstream following parenteral injection, but binding of amphiphile-peptides to endogenous albumin, which constitutively traffics from blood to lymph, retargeted these molecules to lymph nodes. However, these lipid-polymer conjugates bind to albumin with a relatively low affinity, and these molecules can also partition into cell membranes. We hypothesized that by attaching a small molecule, peptide, or protein ligand for a chimeric antigen receptor (CAR) to the same polymer-lipid tail (forming an “amph-vax” molecule), CAR ligands could be delivered efficiently to lymph nodes by albumin and subsequently partition into membranes of resident antigen presenting cells (APCs), thereby co-displaying a CAR T cell ligand from the cell surface together with native cytokine/receptor costimulation. In syngeneic mouse models of adoptive cell therapy, we demonstrated that this approach effectively concentrates CAR T ligands on the surfaces of dendritic cells in lymph nodes, leading to profound expansion of amph-vax-boosted CAR T cells in vivo. Amph-vax boosting safely increased the polyfunctionality of CAR T cells in parallel with T cell expansion. In a syngeneic model of melanoma, this converted a CAR T treatment that had no impact on tumor progression to a therapy that strongly delayed tumor outgrowth and enhanced survival. We have reduced to practice three different strategies to generalize this approach to any CAR of interest. This concept provides a strategy to regulate the expansion and function of CAR T cells directly in vivo to enhance adoptive cell therapy of cancer.

Cell-cell interaction is of fundamental importance for immune system to mediate abnormal stimuli and eliminate pathogens. Understanding the mechanism of cytotoxic T cells in eliminating cancer cells in tumor immunotherapy requires a quantitative analysis of the interaction between T cells and cancer cells. Given the heterogeneity of cancer cells, it is imperative to evaluate the killing efficiency at single cancer cell level. This project uses a microwell device as a high-throughput platform to assess co-cultured OT-1 cells (T cells) and OVA-expressing B16 cells (cancer cells) and evaluate the cytotoxic efficiency based on different T cell numbers. Each micro-well defines the interaction area, to the point where the same group of T cells with the specific single cancer cell are tracked continuously in each well. The cell activity during attacking process is quantified by fluorescence intensity, which indicates the cytotoxic efficiency is proportional to the number of T cells which have communication with cancer cells, and reversely proportional to the cancer cell size. This precise control, high-throughput method, longitudinal observation using micro-well platform can give a new idea to evaluate therapeutic dosage, reduce the cost and at the same time with the same efficacy of immunotherapy.

The cytotoxic activity of lymphocytes is regulated by a gentle balance between activating and inhibitory signals. Understanding the molecular mechanism of this balance is of fundamental importance, and is essential for the rational design of the future based immunotherapies. For this purpose, the exact function of each receptor must be investigated individually and in combination with each other. In particular, to understand the mechanism of the spatial integration of activating and inhibitory receptors, individual receptors should be manipulated and controlled. Spatial control of receptor with molecular resolution has been possible by exposing cells to arrays of patterned nanodots functionalized with the ligands for the studied receptors^1–3. Such state-of-the-art arrays, however, could only control receptors of one type, and therefore could not be used to study how different receptors with complementary function integrate their signals. An experimental platform that allows simultaneous spatial control of two or more different receptors within the cell membrane has not been demonstrate up to date. Here, we developed a novel nanochip approach that allows simultaneous spatial control of individual transmembrane receptors of two types. We applied this nanochip approach to reveal how the nanoscale segregation between NKG2D and KIRDL1, which are the activating and inhibitory receptors in Natural Killer (NK) cells, respectively, regulate the cytotoxicity of NK cells. Our nanochips are based on tunable arrays of paired 10 nm nanodots of different metals, which are selectively functionalized with activating and inhibitory ligands for NK cells. The functionalized arrays, in turn, are used as a stimulation platform for NK cells, which encodes the arrangement of the two receptors within the cell membrane, and allows to monitor the cytotoxic response of NK cells the variations in this arrangement. To realize the nanochips, we first fabricated heterogeneous arrays of Ti and Au nanodots by nanoimprint lithography, followed double angle evaporation of two metals, and liftoff. Here, the spacing between the nanodots, which was ranged from zero to a few tens of nm, is precisely controlled by the metal evaporation angle. We then selectively functionalized Ti and Au nanodots with (i) MHC class I polypeptide-related sequence A (MICA) – a ligand for NKG2D, and (ii) monoclonal antibody for KIR2DL1, using biotin-avidin and Nitryltriacetic acid (NTA)-Histidine conjugations, respectively⁴. We stimulated NK cell on the chip surfaces, and assessed their degree of activation through the expression of CD107a – a commonly used degranulation marker. We found that KIR2DL1-regulated inhibition of NKG2D signaling indeed depends on the spacing between the two receptors, and is mostly effective when the two receptors are separated by 30 nm. Our results shed the light on the way by which the innate immune function is spatially regulated by the activating and inhibitory signaling crosstalk. Furthermore, our novel nanochip technology opens a general pathway to complex, multifunctional nanomaterials which can be used as experimental platforms for the nanoscale study of the function and structure of immunological synapse, as well as other interfaces between cells and their environment.
(1) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem 2004, 5 (3) 383–388.
(2) Schvartzman, M.; Palma, M.; Sable, J.; Abramson, J.; Hu, X.; Sheetz, M. P.; Wind, S. J. Nano Lett. 2011, 11 (3).
(3) Cai, H.; Muller, J.; Depoil, D.; Mayya, V.; Sheetz, M. P.; Dustin, M. L.; Wind, S. J. Nat. Nanotechnol. 2018.
(4) Le Saux, G.; Edri, A.; Keydar, Y.; Hadad, U.; Porgador, A.; Schvartzman, M. ACS Appl. Mater. Interfaces 2018, 10 (14),
11486–11494.

Platelets play a vital physiological role in hemostasis, inflammation and tissue regeneration, which are associated with wound healing as well as cancer development and metastasis. Inspired by this intrinsic ability of platelets and the clinical success of immune checkpoint inhibitors, we have demonstrated that conjugating anti-PDL-1 antibodies (aPDL-1) to the surface of platelets can reduce post-surgical tumor recurrence and metastasis. Using mice bearing partially removed primary melanomas (B16-F10) or triple-negative breast carcinomas (4T1), we found that anti-PDL1 was effectively released on platelet activation by platelet-derived microparticles, and that the administration of platelet-bound anti-PDL1 significantly prolonged overall mouse survival after surgery by reducing the risk of cancer regrowth and metastatic spread. Furthermore, we have shown that systemically delivered blood platelets decorated with anti-PD-1 antibodies (aPD-1) and conjugated to hematopoietic stem cells (HSCs) suppressed the growth and recurrence of leukemia in mice. This cellular conjugate also promoted resistance to re-challenge with leukemia cells. In addition, we genetically engineered platelets from megakaryocyte progenitor cells to express the programmed cell death protein 1 (PD-1). The PD-1 platelet and its derived microparticle could accumulate within the tumor surgical wound for eradication of residual tumor cells. Cyclophosphamide was further loaded into PD-1-expressing platelets to deplete regulatory T cells, enhancing inhibition of tumor recurrence after surgery.

Biological systems endowed with engineered biocircuits have the capacity to augment and reprogram living functions. We create biological bits (bbits) using proteases – a family of pleiotropic, promiscuous enzymes – to construct the biological equivalent of Boolean logic gates, comparators and analog-to-digital converters. We use these modules to assemble a cell-free bioprogram that can combine with bacteria-infected blood, quantify infection burden, and then calculate and unlock a selective drug dose. Inspired by quantum computing, we leverage protease promiscuity as the biological analog of superposition to program three probabilistic bbits that solve all implementations of the two-bit oracle problem, Learning Parity with Noise. Treating a network of dysregulated proteases in a living animal as an oracle, we use this algorithm to resolve the probability distribution of complement and coagulation proteases in vivo, allowing diagnosis of pulmonary embolism with high sensitivity and specificity (AUROC = 0.92) in a mouse model of thrombosis. Our results demonstrate that proteases can be programmed in cell-free systems to carry out classical and probabilistic algorithms for programmable medicine.

Due to its unique physiological features like high blood throughput and high density of narrow capillaries, lung is one of the major organs into which the evaded tumor cells from primary tumor sites can spread. In fact, 30-55% of advanced cancer patients have lung metastasis. In spite of being the mainstay of cancer treatment, chemotherapy has shown limited efficacy for the treatment of lung metastasis due to ineffective targeting and poor tumor accumulation. Here we report a highly effective Erythrocyte Leveraged Chemotherapy (ELeCt) platform, consisting of biodegradable drug nanoparticles assembled onto the surface of erythrocytes, to enable chemotherapy for lung metastasis treatment. The ELeCt platform significantly extended the circulation time of the drug nanoparticles and delivered 10-fold higher drug content to the lung compared to the free nanoparticles. In both the early- and late-stage melanoma lung metastasis models, the ELeCt platform enabled substantial inhibition of tumor growth that resulted in significant improvement of survival. Further, the ELeCt platform can be used to deliver numerous approved chemotherapeutic drugs. Altogether, the findings suggest that the ELeCt platform offers a versatile strategy to enable chemotherapy for effective lung metastasis treatment.

Delivery of therapeutic cargo to hematopoietic stem and progenitor cells (HSPCs) is a challenging problem whose solution would transform the treatment of a wide variety of diseases.[1] To target HSPCs, nanoparticles (NPs) must not only enter the bone marrow, but also specifically bind and deliver cargo to HSPCs, which are extremely difficult to transfect. To date, only a single NP formulation has achieved this goal.[2,3] It consisted of nucleic acid-loaded poly(lactic-co-glycolic acid) (PLGA) NPs, and while it represents a novel advance, the level of HSPC gene modification it achieved was <1%.[2] There exists a substantial need for a more potent delivery system that will overcome physiological barriers to efficiently deliver cargo to HSPCs. Here, we report the development of biomimetic membrane-wrapped PLGA NPs that meet this need. We show that PLGA NPs wrapped with megakaryocyte (Mk)-derived membranes and loaded with model cargo have preferential binding to, and can enter, HSPCs in vitro. Additionally, we show that these Mk membrane-wrapped NPs (MkNPs) can deliver functional siRNA cargo to HSPCs to elicit gene regulation.
DiD fluorophore-loaded PLGA NPs were prepared by a single-emulsion solvent evaporation process [4] and siRNA-loaded PLGA NPs were prepared following a double-emulsion solvent evaporation process. [5]. siCD34 and siNeg were used as proof-of-concept cargo to allow for measurable silencing of CD34 in early-stage HSPCs. For siRNA-loaded NPs, the encapsulation efficiency (EE) was measured by comparing the amount of siRNA remaining in the filtrate after purification to the total added. The EE was >99%, indicating the siRNA is contained in the NPs and not lost during fabrication.
To create Mk membrane-wrapped NPs (MkNPs), the PLGA NPs and Mk membranes extracted from Mk cells were co-extruded with an Avanti Mini Extruder. Membrane wrapping was confirmed by measuring NP size and zeta potential before and after wrapping with dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). These measurements showed that the NPs were monodisperse and spherical, with the wrapped NPs having a diameter 10-20 nm larger than bare NPs and a complete membrane coating. The preferential interaction of DiD-loaded MkNPs with HSPCs as compared to non-targeted HUVECs (human umbilical vein endothelial cells) and MSCs (mesenchymal stem cells) was evaluated by flow cytometry and confocal microscopy. HSPCs internalized DiD-loaded MkNPs within 24 hr of incubation at a much higher rate than than the other two cell types, confirming the Mk membrane coating can facilitate HSPC-specific binding.
To evaluate gene silencing, siCD34-MkNPs and siNeg-MkNPs were cultured with early-stage HSPCs, which express CD34. After 24, 48, 72, and 96 hr incubation, CD34 expression was analyzed by exposing the samples to anti-CD34 antibodies and performing flow cytometry. Since HSPCs acquired from different donors have variable CD34 expression, the percent deviation from control was calculated by comparing CD34 expression in siCD34-MkNP and siNeg-MkNP treated cells to that of the untreated donor control cells in each trial. MkNPs containing siCD34 significantly decreased CD34 expression in HSPCs over a 96 hr time period by >15%, while MkNPs loaded with siNeg had no impact on CD34 expression. These data indicate MkNPs have substantial potential as tools for targeted gene regulation of HSPCs.
In conclusion, MkNPs can be successfully loaded with, and deliver, siRNA cargo to HSPCs in vitro, eliciting gene knockdown. This exciting advance paves the way for further optimization of the system to induce greater levels of HSPC gene regulation, as well as for further examination of their targeting specificity in vivo.
1. Riviere I. Blood 2012;119:1107-16 2. McNeer N. Gene Ther. 2013;20:658-69. 3. McNeer N. Mol. Ther. 2011;19:172-80. 4. Hu C. PNAS 2011;108:10980-85. 5. Pantazis P. Methods Mol Biol. 2012;906:311-19.

Antibodies that antagonize cell signaling pathways specific to their targeted receptors are invaluable tools to treat cancer, but their utility is limited by high production costs and treatment dosages. We and other researchers have shown that antibodies conjugated to nanoparticles display increased affinity for their target relative to freely delivered antibodies due to multivalent binding. In this presentation, I will discuss how my group has exploited this capability to create antibody nanocarriers that are drastically more effective than freely delivered antibodies at suppressing oncogenic signaling. Specifically, we have developed nanoparticles coated with Frizzled7 antibodies and Notch-1 antibodies to suppress Wnt and Notch signaling, respectively, in triple-negative breast cancer cells. Wnt and Notch are developmental signaling pathways that are overactive in triple-negative breast cancer and drive its progression and metastasis. We have shown that our Wnt inhibitory and Notch inhibitory nanoparticles can reduce cell viability, invasion, and stem-like behavior in vitroand hinder primary tumor growth and metastasis in vivo. These exciting observations warrant further investigation of antibody nanocarriers to manage triple-negative breast cancer and other malignancies that suffer from lack of available targeted therapies.

As an essential component of immunotherapy, monoclonal antibodies (mAbs) have emerged as a class of powerful therapeutics for treatment of a broad range of diseases. For central nervous system (CNS) diseases, however, the efficacy remains limited due to their inability to enter the CNS. I'll discuss about a platform technology that enables effective delivery of mAbs to the CNS for brain tumor therapy (Advanced Materials, 1805697, Nature Biomedical Engineering, in press). This is achieved by encapsulating the mAbs within nanocapsules that contain choline and acetylcholine analogues; such analogues facilitate the penetration of the nanocapsules through the brain-blood barrier and the delivery of mAbs to tumor sites. This platform technology uncages the therapeutic power of mAbs for various CNS diseases that remain poorly treated.

Therapy based on personalized medicine—the genomic context of a patient’s disease—has become a leading strategy to treat cancer. Small molecule drugs such as kinase inhibitors, which target key effectors of cancer signaling pathways, constitute a major component of this strategy. However, such drugs can affect the same signaling pathways in healthy tissues, which often leads to dose-limiting toxicities. Increasing the therapeutic index of targeted therapies would greatly improve their effectiveness. We are investigating new targets to localize precision drugs to the microenvironment of primary and metastatic tumors. We developed nanoparticle drug carrier platforms to localize targeted therapies in tumor-associated vasculature and away from healthy tissues to obviate dose-limiting toxicities and concomitantly improve therapeutic index. We targeted MEK and PI3K inhibitors, pan-kinase inhibitors, and sonic hedgehog pathway inhibitors to tumor sites in both primary and metastatic models, resulting in superior anti-tumor efficacy and the striking reduction of toxicities. Moreover, measurements of tumor tissue show prolonged inhibition of downstream effectors in the signaling pathways, constituting a significant modulation of drug pharmacokinetics.

Chirality is ubiquitous in nature that is hard-wired into every living biological system. Despite the critical role, the nexus of chirality engineering on biomaterials has not been explored. Here we designed chiral supraparticles (SPs) that react distinctively to cells and proteins depending on their handedness. SPs coordinated with D- chirality showed at least three times more efficient cell membrane penetrations and anti-cancer properties. We carried out quartz crystal microbalance with dissipation (QCM-D) and isothermal titration calorimetry (ITC) measurements to understand the mechanism, which confirmed that D-SPs had more effective adhesion on lipid layers where most of the phospholipids in nature has D-chirality. The stronger affinity of D-SPs over L-SPs to lipids was because interactions between the same optical isomeric structures are thermodynamically more stable than that of opposites. When it comes to in vivo environments that contain a large, heterogeneous population of proteins, D-SPs showed superior stability and longer biological half-lives due to the incompatible chirality with endogenous proteins including proteases. This study shows that incorporating D-chirality into nanosystems enhances cellular uptake and in vivo stability in blood providing support for the importance of chirality in biomaterials. Chirality engineering will provide “smart” platforms for drug delivery systems, tumor detection markers, biosensors, and many other biomaterial devices.

Triple negative breast cancer (TNBC), which represents 15-20% of all breast cancers, is a devastating breast cancer subtype that occurs more frequently in women under 50 years of age, in African American women and in individuals carrying a breast cancer early onset (BRCA1) gene mutation. To date, TNBC holds the highest mortality rate among all breast cancer subtypes, and a central problem for TNBC therapy is the lack of effective therapeutics that can hinder the growth and spread of breast cancer cells. To address this challenge, here we report the development of a novel non-cationic, deformable and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. CRISPR genome editing is a revolutionary biotechnology that may potentially provide a cure for many genetic diseases including TNBC. We have demonstrated that tNLGs can be used to encapsulate CRISPR genome editing plasmids in a highly facile and efficient manner. Then we showed that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (LCN2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. Furthermore, we performed in vivo CRISPR genome editing of orthotopic TNBC tumors by systemically administering tNLGs, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide the first experimental evidence that tNLGs can be used as a safe, precise and effective delivery approach for in vivo CRISPR genome editing in TNBC.

Since the discovery of chemotherapy in the beginning of the 20^thcentury, researchers around the world have been actively developing new and more effective chemotherapeutic agents to better treat cancer. Traditionally, chemotherapeutic agents work by interfering with cell division. However, by virtue of their mechanism of action, healthy normal cells can also be targeted and destroyed. As a result, while chemotherapy is an effective way of managing cancer, the resulting side effects limits its use. One approach currently taken to reduce these side effects is to deliver the chemotherapy drugs directly to the tumor via transarterial chemoembolization, or other similar procedures. While this has been effective in reducing systemic toxicity, more can be done to improve this. Ideally, a device that could sequester any unreacted chemotherapy agents could be installed "downstream" of the tumor prior to them entering systemic circulation. Such drug-capture materials have only just started to be realized due to the difficulty in achieving materials that have the right surface chemistry and geometry for blood flow.

Working together with medical doctors, computational fluid dynamics experts, chemists, and materials scientists, we report the fabrication of DNA coated 3D printed porous materials that can be used to capture doxorubicin, a commonly used DNA-targeting chemotherapy agent. We discuss the concept behind the device, the use of 3D printed materials as an ideal substrate, and the chemistries considered in drug binding. To achieve scalability of these devices, we developed a method of attaching cheaply available genomic DNA to these materials, a departure from commonly used synthetic DNA. The efficacy of these functionalized materials were demonstrated, where we observed a >70% reduction in doxorubicin concentration over a period of 10 minutes, highlighting the viability of this as a method of drug capture.

Glioblastoma multiforme is an aggressive untreatable brain cancer with a 14.6-month median survival time and a 2% 5-year survival rate after diagnosis¹. The current best practice for treatment is surgical resection followed by radiotherapy and systemic chemotherapy. However, many tumours cannot be removed surgically leaving only the nonspecific treatments of radiotherapy and systemic chemotherapy, further reducing survival rates. Currently the primary chemotherapeutic agent used to treat these tumours is the alkylating agent temozolomide (TMZ). The success of this drug stems from its limited ability to cross the blood brain barrier. Attempts to mechanically cross the blood brain barrier with an implanted device to deliver liquid drug cocktails directly to the tumour have seen limited efficacy due to increasing the intracranial pressure, which can produce oedema and additional brain damage. Recently, ionic drug delivery has been a subject of increased focus using both drug eluting electrodes or electronic ion pumps as the delivery vehicle. Because these devices can deliver drug molecules without a liquid carrier (termed ‘dry delivery’) they cause no increase in the volume to the surrounding tissue. Therefore, this mode of delivery is a strong candidate for the targeted delivery of high concentrations of chemotherapeutic agents deep in the brain, as the side effects associated with the liquid delivery are avoided.

Recently, fully polymeric conductive elastomers fabricated using dispersions of a doped conducting polymer poly(3,4-ethylenedioxythiophene):polystyrene-sulfonate (PEDOT:PSS) in polyurethane, have shown significant promise for applications in bioelectronic interfaces². These materials present a unique platform for the voltage controlled dry release of drug molecules as the elastomer enables the encapsulation of small molecule drugs, and the conductive polymer enables on-demand electronically controlled offloading of the drug. Here we demonstrate the ability for this system to actively release a model drug (fluorescein) in clinically relevant concentrations ranging from 1 µM to 1 mM through the application of a negative electrical potential. Similarly, a positive electrical potential can be applied to stop the release on-demand. Using the same method, we show that the common chemotherapeutic agent doxorubicin can also be released in the same concentrations ranges. Additionally, by tuning multiple material parameters such as the weight percent of PEDOT:PSS in the polyurethane as well as material thickness, further control of the drug release profiles can be achieved. Finally, the translatability of this technology is shown through the fabrication and implantation of a doxorubicin loaded device into an agar tissue phantom using traditional stereotactic methods.

Through the coupling of material parameters with applied electrical potential, we demonstrate a high degree of customizability for the device to be tuned toward patient specific needs. Moving forward, experiments are underway to confirm the active anti-cancer effect of the device in vitro through implantation of the doxorubicin loaded device into rat glioblastoma spheroids, actively releasing doxorubicin, and tracking of the toxic effects through spheroid size reduction over a period of weeks. Ultimately, this technology constitutes a significant step forward in biomaterials-based cancer treatments and shows promise for ease of translation into clinical use to improve the outcome of patients with non-operable glioblastoma multiforme.

1. Przystal, Justyna Magdalena, et al. "Efficacy of systemic temozolomide-activated phage-targeted gene therapy in human glioblastoma." EMBO molecular medicine (2019).
2. Cuttaz, Estelle, et al. "Conductive elastomer composites for fully polymeric, flexible bioelectronics." Biomaterials science (2019).

Glioblastoma multiforme (GBM), the most aggressive brain cancer, has an incidence of ~ 3.2 cases/100,000 persons/year, with a recurrence rate > 90%, a 5-year survival rate of 5.1%, and a mean survival of < 15 months. GBM treatment relies mainly on the combination of maximally safe surgical resection followed by radiation therapy (RT) and concomitant chemotherapy with temozolomide (TMZ).
Local chemotherapy for GBM treatments is the most attractive approaches by enhancing drug biodistribution to the tumor and avoiding systemic toxicities. GBM recurrence mostly within 2 cm of the original lesion makes it local therapy very attractive. Also, it overcomes the limitation posed by the blood-brain barrier. Gliadel®, a carmustine (bis-chloroethyl-nitrosourea (BCNU))-impregnated polyanhydride poly-(1,3 bis[p-carboxyphenoxy] propane-co-sebacic acid) 20:80 polymeric (p(CPP):SA) wafer, is currently the only FDA-approved local therapy device. Although Gliadel releases most of drugs within a week, the triple therapy paired with RT and systemic TMZ administration improved the median survival up to 21 months. Obviously, the use of local chemotherapy is very effective for GBM treatments. Therefore, the use of new polymer structures extending a release time period can further improve the survival and prevent the recurrence while minimizing side effects from cytotoxic agents.
We have developed discs formed from multi-layered core-sheath fiber membranes (‘NanoMesh’) to provide controlled and sustained drug release for long-term periods, leading to excellent in vivo performance in an animal model. Drug encapsulated core-sheath fibers have been fabricated using pCPP:SA / BCNU core and PCL sheath. SEM images show uniform fiber formation with average diameter of ~2.3 µm (sheath of ~ 0.46 µm). Three-dimensional discs were formed from coaxially electrospun fiber membranes by a simple fold-and-punch process and consistent long-term release with no initial burst release was observed in vitro for up to 160 days. NanoMesh discs provide fairly uniform diffusion lengths over time, because aqueous media gradually wets the disc from the outside due to the hydrophobic nature of fiber surfaces. Therefore, consistent long-term release of encapsulated drugs with minimized initial burst release is obtained. This benefit is significant when targeted to long-term release with hydrophilic drugs. NanoMesh reported here can provide long-term release kinetics regardless of the nature of the encapsulated drug.
In vivo survival was assessed following implantation of NanoMesh discs either concurrently with or five days after intracranial implantation of 9L gliosarcoma to F344 rats. Concurrent implantation of NanoMesh and 9L gliosarcoma resulted in statistically significant long-term survival (>150 days). Control (drug-free) NanoMesh confirmed the safety of these novel implants. Similarly, Day 5 studies showed significant median survival, and long-term survival rates of 50%. Brain histology shows no evidence of malignant cells even at Day 150. Animals in all control groups died very early (~12-14 days).
In addition, we have evaluated the release kinetics of multi-drug incorporated NanoMesh to mimic the current systemic ‘cocktail’ chemotherapy with multiple anti-cancer drugs. Different anti-cancer drugs can be incorporated into different layers within the NanoMesh to be released either in concurrent or sequential manner.
Local chemotherapy using NanoMesh implants provides promising means for GBM treatment. Drug delivery with coaxial core-sheath structures benefits from high drug loading, controlled long-term release kinetics, and slow polymer degradation. This represents a promising evolution for the current treatment of GBM. Furthermore, controlled ‘cocktail’ local therapy may bring a new paradigm to the GBM therapy.

Vaccines can take one of several forms, but those based on subunit antigens (representative subunits of the pathogen for which immunity is desired) offer the greatest safety profile and scalability, but elicit weaker, less durable immune responses. Failure to elicit a sufficiently strong response likely arises from inappropriate temporal control over antigen/adjuvant presentation and immune cell activation. Either short-term presentation of these signals, or misalignment of their presentation along different timelines, results in poor affinity maturation of antibodies and poor immune memory responses. When considering the iterative selection process occurring during somatic hypermutation and antibody affinity maturation in B lymphocytes, it is intuitive that prolonged lymphocyte activation and antigen exposure would lead to the generation of higher-affinity antibodies. In this work we exploit a novel injectable hydrogel platform providing unique drug delivery capabilities for the long-term co-delivery of antigen and adjuvant in subunit vaccines and investigate impact of this altered delivery behavior on the humoral immune response and the development of ultra-high-affinity antibodies. We demonstrate that prolonged hydrogel-based immunizations greatly enhance the magnitude and duration of the primary antibody response, enhance and prolong germinal responses, and lead to 1000-fold enhancement in antibody affinity maturation when compared to the same vaccine delivered in bolus. These advanced materials technologies, therefore, have the potential to provide vastly superior vaccine performance through the precise and sustained delivery of subunit vaccines that take advantage of natural immune mechanisms for building long-lasting and potent immunity.

DNA cancer vaccine is one of the potential strategies in cancer immunotherapy. In order to provoke robust antigen-specific adaptive immune response against tumor, DNAs coding tumor antigen are desired to be engulfed by host antigen-presenting cells (e.g. dendritic cells) which subsequently become antigen-activated for T cell priming. However, therapeutic efficacy of this approach is still restricted due to insufficient DNA transfection to host dendritic cells (DCs) and lack of immunogenicity. Herein, we introduce an injectable 3-demensional macroporous scaffold constructing of high aspect ratio mesoporous silica microrods (MSRs) and antigen-coded DNA complexes (designated as MSR-DNA vaccine) for augmenting the efficacy of DNA vaccine. Release of chemoattractant and immune adjuvant from MSRs initiate the migration and maturation of huge amount of host DCs into MSRs scaffold where DNA complexes are present in the interparticle space, thus enhance cellular uptake of DNA complexes by recruited DCs. Subcutaneous immunization of MSR-DNA vaccine elicits more robust host DCs activation and maturation in comparison to the bolus DNA vaccine counterpart, consequently triggers antigen-specific CD8⁺ T cell response in draining lymph node. Interestingly, MSR-DNA vaccine induces Th1-biased immune response through significant secretion of TNF-α and IFN-γ by CD4⁺ and CD8⁺ T cells along with high production of anti-OVA IgG2a in blood serum. In prophylactic study, MSR-DNA vaccine significantly prevents melanoma growth by the generation of CD8⁺ effector memory T cells. Impressively, established lung metastasis can be inhibited with a single injection of MSR-DNA vaccine. Our findings suggest that MSRs could be a novel platform for delivering DNA vaccine to DCs for the effective cancer immunotherapy.

Abnormalities in the expression of cell surface proteins or receptors are promising biomarkers of human diseases, notably cancer. However, the detection and quantification of these biomarkers are often challenging. Molecularly imprinted polymers (MIPs) are tailor-made synthetic receptors (antibody mimics), able to specifically recognize target molecules. They are synthesized by copolymerization of functional and cross-linking monomers in the presence of a template molecule, resulting in the formation of binding sites with affinities and specificities comparable to those of antibodies.

In the present project, we demonstrate the targeting of a cancer protein biomarker with MIP nanoparticles. MIPs were synthesized using a solid-phase approach in which a fragment of the protein was selected as epitope and immobilized on glass beads (solid support) via click chemistry. This configuration allows an oriented immobilization of the template upon which thermoresponsive MIP nanoparticles are synthesized. The binding sites of the resulting MIPs all have the same orientation, thus MIPs synthesized by the solid-phase approach can be considered analogous to monoclonal antibodies.

MIPs were found to bind the epitope with high (nanomolar) affinity and selectivity as demonstrated by equilibrium binding assays with the peptide fluorescently labeled. Fluorescent MIPs were used for cell imaging to reveal the recognition of the target protein on cancer cells. When control cells not expressing the protein were used, the staining was dramatically decreased. In addition, very similar staining patterns were obtained at immunostaining with monoclonal antibodies. The application of MIPs as therapeutic agents is being studied.

Sonodynamic therapy utilizes ultrasound (US)-responsive generation of reactive oxygen species (ROS) from sonosensitizer, and it is a powerful strategy for anti-cancer treatment in combination with chemotherapy. Herein, we designed drug-loaded nanoparticle which could release drug by ultrasound-mediated ROS generation. Doxorubicin (DOX)-coordinated titanium dioxide nanoparticle (TNP) are encapsulated with polymerized phenyboronic acid (pPBA) via phenyl boronic ester coupling between pPBA and DOX. Phenylboronic ester bonding is cleavable by ROS generated from TNP under ultrasound stimulus. The size of nanoparticles is 200 nm, and DOX was released by generated ROS through ultrasound irradiation. In addition, this nanoparticle can target tumor by interaction between PBA and sialylated epitopes of tumor surface. Tumor targeting capability, intracellular ROS generation, and combined therapeutic effect against tumor cells were confirmed in vitro. Furthermore, this nanoparticle showed high tumor accumulation of and efficient tumor growth when treated by ntravenous injection, which have potential as a multi-functional agent for sonodynamic chemotherapy. The nanoparticles have outstanding therapeutic effects owing to several advantages: 1) An appropriate size of the NP makes an effective cancer-specific accumulation through the EPR effect in vivo; 2) Negative charge of remaining carboxylic acid group can increase circulation time in vivo by preventing unnecessary interactions with serum proteins; 3) PBA moieties also have a high affinity to tumors; 4) Since DOX is released in response to US stimulation, it is possible to reduce the non-specific release of DOX and improve the delivery efficiency; and 5) The dual therapeutic effect of generated ROS and released anti-cancer drug by US can reveal combinatorial synergistic anti-cancer effect. In this study, we investigated the physicochemical properties of nanoparticles, tumor targeting ability, US-responsive behavior and its anti-cancer effect in vivo as well as in vitro.

Targeted delivery of pro-apoptotic proteins with tumor-selective properties, including oncolytic virus-derived proteins and other human tumor suppressors has been considered an ideal platform to achieve high therapeutic efficacy and negligible side effects in cancer therapy. Despite notable therapeutic efficacy, however, the facile production and combination of efficient targeted delivery system remain a challenge. Here, we present genetically engineered oncolytic protein nanoparticles comprising a tumor-selective protein and a targeting moiety as a direct protein delivery system for the targeted cancer therapy. Apoptin, which is a 13.6 kDa protein from the chicken anaemia virus (CAV), has attracted considerable attention as a promising anti-cancer therapeutic because it triggers tumour-selective cell death, while leaving normal cells unaffected. Interestingly activity of apoptin proceeds independently of tumor suppressor p53, and its biological function is stimulated by various intracellular oncoproteins. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumor-targeting moiety and a fusion partner for production of apoptin nanoparticles, respectively. The repebody was genetically fused to apoptin, and the resulting construct (repeboy-apoptin, Rb-Apo) was shown to self-assemble into supramolecular apoptin nanoparticles (Rb-Apo-NP) with high homogeneity and stability as a soluble form in E. coli. We examined the whole-body distribution and EGFR specific uptake of the Rb-Apo-NPs, particularly after intravenous administration. Near-infrared fluorescence dye (Cy5.5) was conjugated to the Rb-Apo-NPs and MBP-Apo-NPs, and their distribution in vivo and ex vivo was analyzed through optical imaging in xenograft mouse models with EGFR-overexpressing MDA-MB-468 cells. The Rb-Apo-NPs were shown to be preferentially localized around the tumor cells at 24 h after intravenous administration, and the tumour regions could be clearly distinguished from the normal surrounding tissue. On the other hand, relative low intensity was observed in EGFR-negative MCF7 xenografts. The Rb-Apo-NP showed a remarkable tumor regression in xenografts mice through a targeted delivery by the EGFR-specific repebody and tumor-selective apoptosis by apoptin.

Most nanometer-size drug delivery systems fail to reach their target site (e.g. tumors), largely due to the action of the immune system or elimination by liver or kidney. Furthermore, the drug delivery nanosystems that do reach the tumor are inefficient at killing cells in the tumor neoplasm due to the formidable barrier posed by the tumor microenvironment. The tumor microenvironment limits diffusion of the nanocarriers’cargo (i.e. chemotherapeutics) due to high interstitial pressure and the presence of a dense stroma surrounding the cells. This extracellular environment (stroma) has a plethora of cells like fibroblasts, macrophages, T and B-cells embedded in a network of collagen, fibrin and other protein fibers. This network of fibers, known as the tumor extracellular matrix (ECM), can be remodeled by the tumor cells to their advantage, making it a dynamic obstacle for the drug delivery system to overcome. As a result, the overall delivery efficiency of nanoparticles to tumor cells is less than 1% on average.

Colloidal motors are synthetic particles roughly between 10 nm - 100 μm in size that can propel themselves in an aqueous environment by converting energy stored in their surroundings (chemical, electric/magnetic fields, sound, etc.) into propulsive forces. Steering these colloidal motors is challenging and requires clever engineering, but guidance systems based on magnetic fields have been developed and show promise for navigation in biological environments. Colloidal motors have been shown to carry and deliver cargo (including anticancer drugs) to targeted sites (including cancer cells). However, previous studies have demonstrated these feats in vitro, and successful drug towing and delivery in tumor ECM has yet to be demonstrated.

In this study, we experimentally demonstrate the movement of magnetic helical motors driven by rotating magnetic fields in two in vivo ECM mimics. The first ECM model/mimic used is Matrigel, a commercially available basement membrane commonly used for 2D and 3D cultures. The second is low-molecular weight PEG hydrogels modified with different molar ratios of DTT (dithiothreitol) for tunable pore-sizes. The pore size, shear and storage modulus are measured for both ECM models. The speed is dictated by the rotational frequency of the field and the orientation of the motors depends on the orientation of the field. Using glancing angle deposition (GLAD), helices 70 nm in diameter, 400 nm in length, and 120 nm wide can be fabricated. These are small enough to potentially be able to navigate between the openings in ECM. Fluorescent dyes and quantum dots enable external tracking of helices in both ECM models. The trajectory of the helices and their velocity is determined for different magnetic field properties and ECM parameters. Finally, we explore the viability of these colloidal motors to be tracked using photoacoustic imaging, which has the potential to image and track these particles in vivo. This work takes the first steps toward enabling targeted drug delivery by self-propelled nanocarriers a possibility.

Poly (γ-glutamic acid, PGA) is a poly (amino acid) naturally produced by Bacillus subtilis. It is made of glutamate units connected by amide linkages between α-amino and γ-carboxylic acid groups. As an FDA GRAS material, it is widely used in food and cosmetic industries. In the biomedical field, PGA has been widely studied due to its excellent muco-adhesion, biocompatibility, and biodegradability.^1-2
In this study, a novel PGA-based hydrogel as a local anti-cancer drug delivery matrix for treatment of pancreatic cancer. It is important that drug loaded matrix is stably sufficiently retained on the surface of the tumor to be effective. To achieve this key property, an in situ forming adhesive hydrogel was designed by modifying the carboxyl group of PGA to NHS (N-hydroxysuccinimide) activated ester applying carbodiimide chemistry. Then the NHS activated PGA was mixed with a multi-functional nucleophile (i.e. multi-arm PEG) to form the hydrogel. The hydrogel quickly formed a crosslinked 3-D network structure within 2 sec and showed good adhesive property as well as high burst pressure. Subsequently, anticancer drugs (e.g. paclitaxel, or gemcitabine, etc.) were loaded into the hydrogel (PTX: 0.97%(w/v), GEM: 5.18%(w/v)) and the anti-tumor efficacy was tested in vivo in murine orthotopic pancreatic tumor model. Briefly, the tumor growth was inhibited and the metastasis of pancreatic cancer cells was reduced, without bodyweight loss. Current results demonstrated that local delivery of anticancer drug enhanced an antitumor efficacy. We expect that this hydrogel system as a promising platform to enhance the efficacy of cancer drugs by increasing the therapeutic index.

References
1) I-L. Shih et al., Bioresource Technology. 2011, 79, 207-225.
2) I.Y. Yamamato et al., Surgery. 2007, 141, 678-681.

Although primary melanoma would be easily removed by surgical resection, it could be developed to metastatic melanoma spreading tumor cell from primary melanoma to other tissue, which leads to significant decrease of five-year survival rate of patient. In addition, detection of melanoma at an early stage is difficult due to lack of appropriate tumor markers and absence of symptoms, which causes that the majority of patients with melanoma experience metastasis. Therefore, considering therapeutic aspects of metastatic melanoma, simultaneous treatment of primary and metastatic melanoma would be suitable therapeutic strategies. To achieve desired therapeutic effect, delivery carrier encapsulating anticancer drug should be sufficiently accumulated not only in primary tumor but also in metastatic melanoma that was not detected in early stage of diagnosis, which could completely cure melanoma and increase overall survival rate for patient.
Small interfering RNA (siRNA) has attracted a lot of attention as an alternative anti-cancer drug reducing side effects of chemotherapy owing to their anti-cancer potency and selectivity via tumor-specific RNA interference. Despite their therapeutic potential, the application of siRNAs is limited due to their rapid enzymatic degradation, non-specific accumulation in tissue, and poor cellular uptake in vivo. Thus, successful tumor treatment via siRNA delivery requires the development of a systemic delivery carrier that provides efficient siRNA protection, tissue-targeting ability and enhanced intracellular uptake.
In this study, we prepared a targeted small interfering RNA (siRNA) delivery platform using organic-inorganic hybrid materials capable of specific tissue targeting and efficient target gene silencing. dopa-hyaluronic acid conjugate (d-HA) successfully formed stabilized calcium phosphate (CaP) nanoparticle, which effectively protect siRNA from enzymatic degradation, enhance colloidal stability and provide targeting ability for tumor cell overexpressing CD44. Cellular uptake of the nanoparticles was enhanced via HA receptor-mediated endocytosis in B16F10 melanoma cells, which leads to enhanced gene silencing efficiency. In vivo biodistribution and therapeutic effect demonstrated that the systemically injected nanoparticles formulated with VEGF siRNA was simultaneously accumulated in primary melanoma and metastatic lung tumor, resulting in an improved therapeutic effect by reducing VEGF expression. These results suggested that the systemic targeted nanoparticle formulated with therapeutic siRNA can be considered as a prospective candidate for simultaneous targeted therapy for primary and metastatic melanoma.

Using coarse-grained molecular dynamics simulations, we systematically investigate the receptor-mediated endocytosis of elastic nanoparticles (NPs) with different sizes, ranging from 25 to 100 nm, and shapes, including sphere-like, oblate-like, and prolate-like. Simulation results provide clear evidence that the membrane wrapping efficiency of NPs during endocytosis is a result of competition between receptor diffusion kinetics and thermodynamic driving force. The receptor diffusion kinetics refer to the kinetics of receptor recruitment that are affected by the contact edge length between the NP and membrane. The thermodynamic driving force represents the amount of required free energy to drive NPs into a cell. Under the volume constraint of elastic NPs, the soft spherical NPs are found to have similar contact edge lengths to rigid ones and to less efficiently be fully wrapped due to their elastic deformation. Moreover, the difference in wrapping efficiency between soft and rigid spherical NPs increases with their sizes, due to the increment of their elastic energy change. Furthermore, because of its prominent large contact edge length, the oblate ellipsoid is found to be the least sensitive geometry to the variation in NP’s elasticity among the spherical, prolate, and oblate shapes during the membrane wrapping. In addition, simulation results indicate that conflicting experimental observations on the efficiency of cellular uptake of elastic NPs could be caused by their different mechanical properties. Our simulations provide a detailed mechanistic understanding about the influence of NPs’ size, shape, and elasticity on their membrane wrapping efficiency, which serves as a rational guidance for the design of NP-based drug carriers.

When nanoparticle-based imaging agents are used for targeted tumor contrast, only a fraction of the dose is specifically delivered; the remaining material circulates, passively accumulates in the tumor due to the enhanced permeation and retention (EPR) effect, or accumulates in off-target organs. When multiple contrast agents with near identical chemical and physical properties, yet distinct spectral emissions, are used, dual-probe imaging can be used to discern between the background and target signal. This can be achieved by removing the contribution of the untargeted probe from the image leaving only the signal from the targeted probe. NIR-emitting quantum dots (QDs) provide an excellent opportunity to improve targeted imaging of tumors in murine models for the study of cancer biology, as multiple QDs emitting discrete tissue-penetrating wavelengths can be prepared such that their size and surface properties are perfectly matched except for the presence or absence of a tumor-specific targeting ligand.
We have synthesized multiple near infrared (NIR) emitting cadmium-free quantum dots (QDs) as bright and photostable contrast agents for multiplexed imaging in tissues. These indium phosphide-based QDs have tunable emission across most of the visible wavelength regime and the first near infrared (NIR) tissue imaging window. Our Inverted-Type I structure comprising InP shells on a ZnSe core, capped with ZnS for stability, exhibit extended tunability beyond that of a traditional InP Type-I QD. By controlling the thickness of the InP shell, we can tune the emission peak from 530 nm to 868 nm (2.34 – 1.43 eV). The full width half maximum (FWHM) of the emission peak remains constant as monolayers are added, indicating regular and high-quality shell growth. To confer water solubility, long circulation times, and chemical handles for bioconjugation, the QDs were encapsulated in PEGylated phospholipid micelles functionalized with terminal azido groups. The QDs retain their high quantum yield and monodispersity following encapsulation. Using click chemistry, the terminal azido groups are conjugated to tumor targeting ligands, including anti-HER2 or anti-CXCR4 peptides, as well as a clickable BCN-ethylenediamine modified folic acid. Dual probe imaging of tumors using targeted and untargeted fluorophores improves the imaging contrast of the specific cancer biomarker of interest.
For dual probe imaging to be successful, the signal contributions from each of the fluorophores has to be well resolved and the contribution from autofluorescence accounted for. To accomplish this, we evaluated multiple spectral unmixing algorithms to determine which generates the most accurate representation of true fluorophore concentration at tissue depth. This is done by covering well plates, where wells contain varying ratios of QDs, with skin-mimicking PDMS phantoms of varying thicknesses, and then imaging these well-phantoms using an IVIS sytem. This algorithm is then applied to the in vivo targeted tumor imaging experiments.
This method is being used to determine the biomarker status of two distinct biomarkers in tumor xenografts of BT-474 and MCF-7 cells in nude mice. Excitingly, the number of distinct biomarkers in a tumor that can be probed per imaging session is only limited by the full width half maximum of the emitters and the number of distinct emitters in the first optical tissue window.

With a size smaller then a living cell, nanoparticles became one of the most promising therapeutic tools in a variety of biomedical application fields. In particular, the new optical, physical and chemical properties of nanomaterials offer a new pathway of treating cancer without affecting healthy cells. Thereby, the hyperthermia properties of magnetic or optical active nanoparticles can be used to induce specifically natural cell death. In this context, gold nanoparticles came into the main focus of cancer research over the last decades by showing high surface plasmonic resonance leading to optothermal behavior in the near infrared area. Furthermore, gold is chemical inert and non-toxic to the human body.

Nevertheless, an immense challenge for using nanoparticles in cancer therapy is to ensure a highly specific uptake of nanoparticles for cancer cells. Therefore, we present monodispersed and highly selective nanoconjugates for example iron oxde (Fe₃O₄ with a size of 5 nm) and gold nanorods (mSiO₂@Au with a length of 40 nm and a width of 20 nm), which were vectorized by attaching estrogen molecules at their surface by chemo specific reactions. The as-vectorized nanoconjugates were also loaded with therapeutic agent to investigate its drug loading and drug release capacity. The nanoconjugates showed enhanced cellular uptake for MCF breast cancer cell lines, which was quantified by FACS analysis. The functionalized nanoprobes showed promising accumulation at the tumor side as compared to the healthy organs. Our data shows, that as-prepared nanoconjugates holed potential to be used as targeting specific drug delivery nanoprobes.

Hippo signalling pathway controls multiple cellular functions that are central to tumorigenesis. Its importance in cancer cell proliferation and metastasis has been well recognized. Here we created polypyridyl ruthenium comjugated peptide complex for lipid-raft-targeted molecular assembly. Via hydrolyzation by ovarian cancer biomarker, glycosylphatidylinositol-anchored placental alkaline phosphatase (ALPP), molecules assemble into nanostructures adhere to lipid rafts restricting their dynamics and spatial distributions. Through actin cytoskeleton, the regulation of lipid rafts stimulates Hippo signalling pathway deactivating the core oncogene YAP in cancer cells suppressing cancer cell migration and inducing cancer cell apoptosis.

Despite of an outstanding therapeutic effect, direct administration of chemotherapeutics still involves diverse side effects and lowers its efficacy. Although, nanoparticle-based drug delivery system (DDS) has been a promising strategy to resolve the problems, continuous controversies about the efficiency of EPR, active targeting, and stimuli-responsive delivery have been suggested. Therefore, rather than relying on man-made targeting system, we focused on natural and intrinsic tumor targeting system in our body, natural killer (NK) cells. The anti-cancer effect of NK cells are known to be innate, immediate and cancer specific, yet insufficient to induce complete regression of tumor.
Once circulating NK cells approach to tumor tissue along chemokines from tumor microenvironment, a NK cell would encounter a target cell and initiate the formation of immunological synapse (IS). IS is a pivotal part where panels of activating and inhibitory receptors are bounding whose the signals are integrated, thus NK cell decide whether to release its lytic granules. Inspired by the fact the acidic lytic granules are released toward the IS, we hypothesized that the local acidification of the IS cleft would be a stimuli for controlled behavior of drug delivery. To reinforce the cytotoxic killing effect of NK cells as well as to deliver chemotherapeutics in highly regulated manner, IS formation-responsive drug delivery system was developed.
Herein we propose a NK cell decorated by functional nanoparticle with embedded therapeutic cargo. These nanoparticles are composed of acid degradable polymers in micellar formation which contains Doxorubicin. Micelle disassembly will only occur when local pH lowered by release of lytic granules from the NK cell towards cancer cell during immunological attack, therefore minimizing the non-specific release of cytotoxic DOX. Hence, the anti-cancer activity is enhanced through combination of NK-cell-induced tumor-specific homing and potent chemotherapeutic efficacy. The advantages in its therapeutic efficacy and targeting precision will benefit further trials in development and clinical translation of NK cell mediated immunotherapy of cancer.

Magnetic resonance imaging (MRI) has attracted increasing interest because of its nonradiative and noninvasive character in addition to providing anatomic images with striking spatial resolution and depth. Positive contrast or proton MR images are produced by T1-based contrast agents such as gadolinium (Gd³⁺) or manganese oxide (MnO) nanoparticles, which increase the signal from protons by reducing the spin-lattice relaxation time of nearby water leading to the brightening of the voxel. Through decades of research, various approaches have been developed for the synthesis of manganese oxide nanoparticles. Despite growing interest, T1-based MR imaging remains comparatively limited because of ratiometric complications and low signal intensities. Also, in order to impact their relaxation times, T1 contrast agent need to directly interact with the surrounding water protons. In case of nanospheres, only ions at the surface are effective. Decreasing the particle size could provide larger surface area. Also, by converting the particles to the hollow nanostructures, nanoparticle surface percentage could be increased which will in turn provide higher relaxivity. Hollow nanoparticles can provide a high specific area and, excellent penetration and permeation abilities. Hollow nanoparticles have significantly higher volumetric capacity to access water molecules, thereby, enhancing the MR contrast. Furthermore, ligand exchange can lead to added enhancement of contrast by making the surface hydrophilic as compared to the bilayer coated nanoparticles as, hydrophobic inner coating may inhibit water penetration leading to a lower spin effect. Until now, a few hollow MnO-based nanoparticles have been developed, including MnO nanoparticles loaded with iron oxide nanoparticles or drug molecules. Despite this, hollow MnO nanoparticles have not been optimized for low relaxivity and maximum contrast enhancement.
We have developed various solid and hollow manganese oxide (MnO) nanocubes for T1-weighted MR imaging (MnOEn, MnOEx, MnOEnHo and MnOExHo). The as-synthesized MnO nanocubes were rendered water soluble by ligand exchange and ligand encapsulation. Moreover, particles were further treated with phthalate buffer leading to formation of hollow MnO nanocubes. Further characterizations found that hollowed out-ligand exchanged nanostructures (MnOExHo) exhibit maximum enhancement of the longitudinal relaxation by allowing more exposure of Mn ions to water protons. We confirmed the T1 MRI contrast effect of the balb / c nude mice gastric orthotopic xenograft cancer model using MnOExHo nanocube. In this study, we suggested that these hollow ligands exchanged nanocubes (MnOExHo) could be used for biomedical research as effective T1 contrast agents.

DNA has been utilized as a building block for the formation of a specific structure as well as a fascinating biomaterial for delivery system due to high biocompatibility and the favorable interactions with several biomolecules. Moreover, recent studies have revealed that the DNA can transform the conformation dynamically depending on stimuli such as pH, ion, and biomolecules, suggesting the use of DNA as a functional domain as well. In particular, the G-quadruplex (G4), a guanine-rich sequence which forms the stacked G-quartets structure, can interact with hemin [iron (III)-protoporphyrin] with high specificity, and exhibits a remarkable catalytic property that mimics peroxidase. Owing to the high structural stability and easier handling, DNAzyme has been extensively studied for biosensing, DNA/RNA cleavage, and catalytic reactions.
Graphene oxide (GO) have attracted much attention in biomedical applications due to mechanical and chemical stability, abundant oxygen functional groups on large surface area, and photothermal effect. Although GO possesses many functionalities as itself, modification of GO with several polymers and/or protein is inevitable for the use of nanomedicine because of the fact that the modification increases physiological stability, decreases systemic toxicity, and improves gene/drug loading capability. However, such a modification severely prohibits the degradability of GO by catalytic enzymes including peroxidases due to the steric hindrance of modified materials on the surface of GO.
In this present study, we introduce the G4/hemin complex to take advantage of these modified GOs and induce degradation in the body after the therapy. By applying unique features of catalytic DNAzyme and GO, we propose an ingenious self-catalytic GO nanomedicine for cancer therapy operated by multifunctional DNAzyme and controlled by photo-switch. The surface of GO is decorated by DNA double strand of G4 and its complementary sequence with mucin1 (MUC1) aptamer for targeted delivery. An anticancer drug, doxorubicin (DOX), is loaded on the DNA double strand, while hemin is loaded on the surface of GO. After cellular internalization and turning on the photoswitch, loaded DOX is released by the DNA melting triggered by the photothermal effect of GO. The remained single stranded G4 sequence conjugated on the GO surface forms quartet structure and subsequently the hemin on GO surface binds on the quartet, inducing catalytic peroxidase effect. Due to the high local concentration of H₂O₂ in cancer cells, G4-hemin generated efficient amount of strong oxidant, hypochlorous acid (HOCl), inducing the degradation of GO into the small fragments for potential clearance.

Photodynamic therapy (PDT) is an anticancer therapy based on special drugs/ photosensitizers. The three basic elements of photodynamic therapy are the excitation laser, photosensitizer molecule, and oxygen in tissue. After irradiation, the photosensitizer responds to specific light thus generating reactive oxygen species (ROS) killing cancer cells. Being a potential photocatalyst, g-CNQDs are able to create more electron-hole pairs under visible light illumination thereby resulting in the production of large amount of ROS which is helpful to cause severe cellular damage. Graphitic carbon nitride (g-C₃N₄) has been used for various biomedical applications due to its small sheet size, high dispersity, hydrophilicity and non cytotoxicity. g-C₃N₄ also have the ability to generate ROS towards cancer killing under very low-intensity light irradiation. In this study, the g-CNQDs are synthesized by two step thermal polymerization of melamine and EDTA. The QDs are small in size ranging from 2-7nm and demonstrate no visible cytotoxicity against fibroblast cells (L929 cells) and HuVEC cells upto a concentration of 5mg/mL. The optical absorption lies in the UV region at ~250 nm which is due to π-π^* electronic transitions. The CNQDs exhibit broad emission peaks shifting towards red region when excited at different wavelengths. The g-CNQDs show excellent singlet oxygen generation capability that makes them a suitable photosensitizer. In vitro ROS studies reveal that CNQDs were able to generate 370% ROS as compared to control dark. The phototoxicity of g-CNQDs was assessed using MTT assay and Lactate Dehydrogenase (LDH) assay. In presence of blue light, the g-CNQDs were able to kill almost 75% of C6 glioblastoma at a concentration of 1.25 mg/mL as revealed by MTT assay. This data was supported by LDH assay showed release of 582% of LDH with respect to control. The photosensitized cells were stained with Fluorescein Diacetate (FDA) and Propidium Iodide (PI) in order to determine the percentage of live and dead cells. The cells treated with CNQDs in presence of light exhibited almost 99%. Cell death via apoptotic pathway was observed from Annexin V/FITC PI assay confirming 93.6% of necrotic and apoptotic population. In addition, caspase 3/7 detection assay was performed to detect the caspase mediated apoptosis corresponding to 88% of apoptotic population in light sensitized cells. These data imply blue light irradiated CNQDs exhibited considerable photodynamic effect against cancer cells by caspase activation through the modulation of apoptosis.

Chemotherapy is one of the most effective treatments for cancer, but it has always faced some challenges such as multi-drug resistant (MDR) effect, poor aqueous solubility, and cardiotoxicity of the drug. In the past few decades, micellar drug delivery systems were used to encapsulate anticancer drugs for better therapeutic effects, but they have poor drug loading content. To address this problem, we designed a micellar drug delivery system using benzyl-substituted poly(caprolactone) (PBnCL) as the hydrophobic block for better interaction with anticancer drug Doxorubicin (Dox). Another approach is the co-loading of Dox with other hydrophobic molecules. Quercetin (Que) is a polyphenolic plant-derived flavonoid which has cardioprotective and chemosensitizing properties. Thus, the co-loading of Dox and Que not only enhances the loading capacity of Dox in micelles but also decreases the adverse side effects of anticancer drug and overcomes the MDR effect. Our other approach to improve conventional chemotherapies is developing thermoresponsive micelles. Thermosensitive nanocarriers ideally will retain their load at physiological temperature and release the drug within a tumor once the external temperature is applied to induce local hyperthermia. Polymers exhibiting lower critical solution temperature (LCST) or upper critical solution temperature (UCST) are used as polymers for thermoresponsive drug delivery systems. Herein, we report the synthesis of thermoresponsive polycaprolactones (PCLs) having LCST close to physiological temperature with the advantage of biocompatibility and biodegradability.
Ring-opening polymerization of benzyl- and oligoethylene glycol-substituted ε-caprolactone monomers was performed using tin octanoate as a catalyst and benzyl alcohol as an initiator to generate amphiphilic block copolymers. The synthesized PME_nCL-b-PBnCL polymers were characterized by ¹H NMR analysis. To make a comparison between amphiphilic copolymers, a 50:50 ratio of hydrophilic to hydrophobic block were synthesized. The critical micellar concentration (CMC) that evaluates the thermodynamic stability of micelles in the aqueous solution was measured using the fluorescent hydrophobic probe, pyrene. All polymers have CMC values in the order of 10^-5 g L^-1, which indicates high thermodynamic stability of micelles. The particle size distribution of micelles in aqueous solution was measured by dynamic light scattering. All polymers formed micelles with sizes lower than 100 nm, which is the ideal size for passive targeting of tumor cells. LCST measurements will be performed using a temperature-controlled UV-Vis spectrometer. Que and Dox were loaded in micelles through solvent evaporation method, and the drug loading capacity and encapsulation efficiency were measured using UV-Vis spectroscopy. We are expecting that the co-loaded micelles will demonstrate higher drug loading for both drugs in comparison to individual loading of drugs in micelles due to hydrophobic interactions between drugs.

Adjuvants increase the efficacy of vaccines and can reduce the number of needed immunizations to achieve protection. Mainly aluminium salts are the most used adjuvants, however their immune response is not broad enough (only Th2 response, not Th1 response). For example, aluminium adjuvants are not useable against influenza or malaria. Therefore, it is favourable to search alternative adjuvants. Poly(methyl methacrylate) is useable for medicine-related applications, for example hip replacements or bone cements. Therefore, it is attractive to analyse if it is a possible adjuvant. Previous studies showed that PMMA has good adjuvant activity.¹ The previous works did not mention the molecular weight of the polymer. To allow renal clearance, adjuvants should have a molecular weight below 20 kg/mol. To achieve that, one needs to produce reproducible and controlled polymers with a low molecular weight, so oligomers.
Catalytic chain transfer polymerization (CCTP) is a valuable method to synthesize oligomers. However, mainly cobalt catalysts are used. This is disadvantageous for the use as adjuvant. To create a more biocompatible method, iron catalysts were analysed for CCTP. Four-coordinate (diimine)iron catalysts containing aryl substituents proved successful as CCTP catalysts.² Related to that, four-coordinate iron-diimine complexes with diisopropylphenyl (DIPP) and trimethylphenyl (TMP) as substituents were explored. Also we tried, to produce the catalyst in situ, so without additional synthesis of the catalyst. For that dimethyl glyoxime and diphenyl glyoxime were used as ligands. Iron(II)bromide and iron(II)choride were used to generate the iron complexes.
The reactions were performed with conventional heating and microwave heating (MH), both in bulk and in solvent solution at a reaction temperature of 80^oC.
Both methods display chain-transfer behaviour, noting that molecular weight of polymer products was lower than uncontrolled free radical polymerisations. In case of bulk polymerization one has to take care about the reaction conditions to avoid uncontrolled reactions and the Trommsdorff-Norrish effect. With the right conditions, it can lead to lower molecular weight than the solution polymerizations. Microwave heating proved rewarding for the iron-catalysed CCTP.
On the whole, iron catalysed CCTP is demonstrated to be a favourable way to produce MMA oligomers to be used as adjuvants.

1) Kreuter, Jörg; Haenzel, Ingrid (1978): Mode of Action of Immunological Adjuvants: Some Physicochemical Factors Influencing the Effectivity of Polyacrylic Adjuvants. In Infection and Immunity (19 (2)), pp. 667–675.


2) Gibson, Vernon C.; O'Reilly, Rachel K.; Wass, Duncan F.; White, Andrew J. P.; Williams, David J. (2003): Polymerization of Methyl Methacrylate Using Four-Coordinate (α-Diimine)iron Catalysts. Atom Transfer Radical Polymerization vs Catalytic Chain Transfer. In Macromolecules 36 (8), pp. 2591–2593. DOI: 10.1021/ma034046z.

Recently, researches on the relation between anti-oxidant activity and cancer have been much attracted. Astaxanthin (ATX) has been regarded as an strong anti-oxidant that is several hundred times stronger than vitamin C. Photothermal therapy (PTT) is the usage of heat produced by electromagnetic radiation for cancer treatment. PTT is utilized to maximize the synergistic effect of anticancer drug. Near-infrared fluorescent dye, IR780 iodide (IR), was utilized for performing PTT. In regarding to lipophilic property of both ATX and IR, there are limitation for bio-applications. Hyaluronic acid (HA) is widely used hydrophilic polymer to overcome the this limitation. HA based micelles are beneficial for cancer treatments owing to their biocompatibility and tumor targeting ability to CD44-overexpressing tumors.
In this study, we fabricated HA micelles by self-assembly method and co-loaded with IR and ATX (IR-ATX-HA) for selective tumor targeting in CD44 overexpressed mouse head and neck squamous carcinoma cell line (SCC7). The physicochemical characterization of IR-ATX-HA micelles showed that the IR and ATX were successfully co-loaded into HA micelles. Furthermore, IR-ATX-HA micelles were destabilized upon laser irradiation and were able to release ATX to be utilized for cancer therapy. Intracellular uptake analysis of IR-ATX-HA micelles in SCC7 demonstrated CD44-based targeting. Moreover, cytotoxicity studies proved the improved anti-tumor activity aided by both ATX and PTT therapies compared to without NIR laser irradiation. In conclusion, IR-ATX-HA based micelles were effective in targeting SCC7 cancer cell and able to elicit enhanced cancer therapy effect of ATX and PTT.

The therapeutic use of copper oxide nanoparticles (CuO NPs) as effective anti-cancer drug is a promising alternative to conventional treatments. In contrast to insoluble carrier nanoparticles, e.g. iron oxide, copper oxide readily dissolves in cellular nutrient-rich environments, even at physiological pH. Intracellular released Cu²⁺ leads to a redox imbalance and binds to available reaction partners such as amino acids, polypeptides or proteins. At the same time, lattice oxygen will result in the formation of reactive oxygen species causing oxidative stress. Regulatory mechanisms to maintain copper homeostasis involve transport and excretion of copper via Cu-ATPase. If the Cu²⁺ release kinetics are too fast for a regulation, elevated copper levels cause severe damage such as proteasome inhibition leading to apoptosis of cancer and peripheral cells.

Due to differences (i.e. metabolism and pH) between maleficent and peripheral cells determining the release kinetics, we postulated that finely tuned release kinetics of CuO NPs can open a therapeutic window for cancer treatment. To test our hypothesis, we synthesized a library of pure and iron-doped CuO NPs using our in-house flame spray pyrolysis process. The incorporation of iron resulted in a bond-length variation and a strong Jahn-Teller distortion with a decreasing release of copper in biotical environments¹. The Cu²⁺ release kinetics of pure and Fe-doped CuO NPs were investigated on the long-term (250 hours, in agreement with the cell and mice studies) in biological test media containing amino acids present in common growth media, e.g. RPMI or DMEM. In contrast to the fast burst-like release of pure CuO, the iron doping resulted in a two-step dissolution: (1) an initial release on the time scale of hours and (2) a long-term release on the time scale of weeks. Material characterization of the as-prepared particles and the remaining particles after long-term dissolution showed that only copper is released from the particle, while iron remains on the surface. Based on these findings, a release kinetic model was developed to explain the two-step dissolution. The model includes the release of copper until all surface available copper is released, followed by a solid state diffusion limited long-term release.

Three materials (pure, 6% and 10% Fe-doped CuO NPs) with a fast, an intermediate and a slow release kinetic, respectively, where chosen to investigate our hypothesis in-vitro and in-vivo. While the cell viability of tumor and peripheral cells was affected in case of pure CuO, 6% Fe-doped CuO NPs only reduced the cell viability of tumor cells, even for doxorubicin (conventional chemotherapy) resistant cell types. In a combined treatment with an immune activator (IDO1), the 6% Fe-doped CuO NPs were successfully tested in-vivo, resulting in complete tumor remission in a syngeneic subcutaneous mouse model (KLN-205).

1. Naatz, H., et al. Safe-by-Design CuO Nanoparticles via Fe-Doping, Cu–O Bond Length Variation, and Biological Assessment in Cells and Zebrafish Embryos. ACS Nano 11, 501-515 (2017).

The field of nanoparticles for biomedical applications has become one of the most promising and most studied topics in the last years. In terms of therapeutic applications, especially drug delivery vehicles have been intensively studied. In this case, hollow mesoporous silica capsules (HMSC) have aroused tremendous interest due to their unique properties such as high biocompatibility and stability in biological milieu, large surface areas, low densities and high loading capacities due to a protected hollow core.
HMSC were synthesized through a hard template-based method. For that purpose, ellipsoidal hematite particles were synthesized in a solvothermal process, coated with silica in a sol gel process and subsequently the iron oxide core was etched resulting in HMSC. The porosity of as-prepared particles was analyzed using nitrogen adsorption-desorption method revealing a pore size of circa 4 nm and a high surface area of 308.8 m²/g. Cytotoxicity was determined using cell viability test (MTT) towards human kidney cells (HEK293) which clearly demonstrates that no reduction of cell viability was observed even at high concentrations of 100 µg/ml. Uptake studies using confocal microscopy were carried out using human cervical cancer cells (HeLa) which could show the successful internalization over a period of 24 hours. For testing their capability as drug delivery vehicle, a hydrophilic antibiotic (ciprofloxacin) and a hydrophobic anticancer (curcumin) compound were loaded and a pH dependent release under physiological conditions at 37°C was monitored via UV-Vis spectroscopy. Ciprofloxacin-loaded HMSC with a concentration of 10 µg/ml were also tested towards gram negative bacteria (E.coli) revealing a complete growth inhibition over 18 hours. This study demonstrates the suitability of as-prepared hollow silica capsules as drug delivery vehicles for a broad range of drugs.

The need for the development of nanoparticles for the early detection and treatment of cancer provides the motivation for specific targeting of cancer. This paper presents results of an atomic force microscopy and molecular dynamics study of the adhesion between Triple Negative Breast Cancer Cells (TNBC) (MDA-MB-231 and MDA-MB-468) cells and biosynthesized gold nanoparticles (BGNP). The BGNPs are conjugated with Luteinizing Hormone Releasing Hormone (LHRH), which is known to target LHRH receptors that are over–expressed on the surfaces of breast cancer cells. The adhesion forces between the LHRH-conjugated gold nanoparticles and the breast cancer cells are found to be about 4-5 times greater than those between normal breast cells and LHRH-conjugated gold nanoparticles. The increase in the adhesion of LHRH to breast cancer cells is shown to be associated with an increase in the LHRH receptor density, which is revealed via confocal microscopy. The implications of the results are then discussed for the development of ligand-conjugated gold nanoparticles for the detection and treatment of cancer.

Despite significant advances in cancer therapeutics, breast cancer (BC) remains a leading cause of cancer-related death in women worldwide. This could be partly due to the lack of early detection required for improved prognosis, while treatment complications (e.g. high systemic toxicity of anticancer drugs or drug resistance) often lead to treatment failure. In particular, triple-negative breast cancers (TNBCs) are highly aggressive, lack validated therapeutic targets and have high risk of metastasis, representing an urgent unmet clinical need for new treatment options [1]. Here, a novel multifunctional theranostic nanostructure for TNBC is presented, combining for the first time metal-enhanced fluorescence (MEF) imaging with multimodal cancer treatment, through hyperthermia and chemotherapeutic drug delivery.

This integrated theranostic platform comprises a gold (Au) nanobipyramid (AuNBP) core as the MEF amplification component. MEF is an optical process in which the near-field interaction of fluorophores with metallic nanoparticles (NPs) can, under specific conditions, produce large fluorescence enhancements [2]. Efforts are underway to exploit this light amplification to considerably increase detection sensitivity in fluorescence bioimaging, particularly in the biological near-infrared (NIR; wavelengths 650-900 nm) window [3], where bright and biocompatible fluorophores are lacking. In the present work, we focus on AuNBPs, a type of elongated Au nanoparticle (NP) with two sharp tips, equipped with several features that make them attractive candidates for MEF. For instance, their high monodispersity compared to other anisotropic NPs, affords lower inhomogeneous spectral broadening of their localized surface plasmon resonance (LSPR) peaks, while their sharp edges allow significant enhancements of the local electric fields. Here, AuNBPs with tunable sizes are synthesized, allowing LSPRs tunable in the NIR window. We show that NIR fluorophore conjugation to AuNBPs through a mesoporous silica (MS) spacer allows several times fluorescence enhancement in the NIR window. Using time-resolved fluorescence measurements to semi-quantitatively deconvolute excitation enhancement from emission enhancement, as well as electric field modeling, we provide important insights into the mechanism of MEF from AuNBPs.

We then explore the in vitro chemotherapeutic efficacy of doxorubicin, loaded within the pores of the MS layer as a strategy to facilitate local drug delivery to BC cells. In parallel, the AuNBP core is applied for photothermal therapy via NIR laser irradiation, as a synergistic treatment modality. Our results demonstrate that hyperthermia amplifies the potency of doxorubicin against BC cells. Finally, we use folate functionalization of the MS-coated AuNBPs for active targeting of the folate receptor alpha (FRα). FRα is a protein overexpressed in 86% of metastatic TNBC patients, and its expression is associated with poor clinical prognosis, making it a good candidate for TNBC targeting [4]. Using fluorescence imaging, we show that folate functionalization enables selective detection of TNBC cells over cells with low FRα expression. We also show that folate targeting enhances NP accumulation in TNBC cells and potentiates TNBC cell killing.

In summary, the insights gained through this work could guide further development of bright NIR-MEF nanoprobes. Our study also demonstrates the potential clinical utility of folate conjugation to enable selective detection and targeted treatment of aggressive BC subtypes.

References:
1. Hudis C. et al., The Oncologist 2011, 16, 1-11.
2. (a) Pang, J.; Theodorou, I.G. et al., ACS Appl. Mater. Interfaces 2019. (b) Theodorou, I.G. et al., Chem. Mater. 2017, 29, 6916-26. (c) Pang, J.; Theodorou, I.G. et al., J. Mater. Chem. C, 2017, 5, 917-25.
3. (a) Theodorou, I.G. et al., Nanoscale, 2019,11, 2079-88. (b) Theodorou, I.G. et al., Nanoscale, 2018, 10, 15854-64.
4. Cheung, A. et al. Oncotarget 2016, 7, 52553-74.

Nanodiamond (ND) particles are nano-scale materials that have recently been attracting major attention for biological purposes, owing to their high biocompatibility and chemical stability. ND particles, however, have their disadvantage to form micro-scale aggregates, which could lead to the loss of the advantageous properties of nanoparticles. An ND-based MRI contrast agent is an example of the material that holds such a problem as mentioned above. Nano-sized contrast agents were fabricated in our previous studies by the complexation of ND particles and gadolinium chelates (Gd-DTPA). The primary size of the gadolinium-complexed ND (Gd-DTPA-ND) particles was set at around 5 nm to achieve the selective imaging of the lymphatic system. This was because MRI contrast agents with a diameter of 3–10 nm could be selectively ingested through lymphatic vessels and filtered in the kidney. However, ordinary contrast agents were smaller than 1 nm in diameter, and thus the MR imaging of the lymphatic system had not been successfully achieved. The primary Gd-DTPA-ND particles possessed an ideal size for the lymphatic imaging, but these particles could easily aggregate in distilled water. As a result, such aggregates exceeded the size required for the selective lymphatic MR imaging. To address this problem, we attempted to improve the dispersity of the Gd-DTPA-ND particles. In this study, carboxylated nanodiamond (CND) particles were used as a platform for the subsequent condensation of gadolinium-complexes (Gd-DTPA-CND). The carboxyl groups introduced by oxidation induced hydrophilicity and hence negative charge to the ND surface, resulting in the high dispersity of the gadolinium-complexed CND particles. The dispersity of Gd-DTPA-CND particles in distilled water was evaluated by the dynamic light scattering, revealing higher dispersity for the fabricated particles than for the Gd-DTPA-ND particles. The MRI visibility of the Gd-DTPA-CND particles in distilled water was also evaluated by the 1.5T MRI. It was found that high-contrast imaging could be established by the Gd-DTPA-CND particles, showing that the Gd-DTPA-CND particles possessed high MRI visibility in distilled water. Furthermore, the Gd-DTPA-CND particles also presented high dispersity and MRI visibility even in human serum, suggesting that the particles could be used intravitally. Finally, Gd-DTPA-CND particle solution was injected in a tail vein of a rat to investigate the MRI visibility of the Gd-DTPA-CND particles in vivo. As a result, the injection of the Gd-DTPA-CND particles produced a strong contrast in the blood circulatory system. A bladder was eventually imaged, suggesting successful filtration of particles in the kidney and assembly in the bladder. The results revealed that Gd-DTPA-CND particles could realize high contrast imaging in living bodies and the particles could be filtered in the kidney for the final excretion in the bladder. It was, therefore, concluded that Gd-DTPA-CND particles could be promising MRI contrast agents for the imaging of the lymphatic system through subcutaneous injection.

The structure and the role of the lymphatic system in the human body have not yet been fully explored in the medical field.This is mainly due to the microscopic diameter of lymphatic vessels, which makes it highly difficult to inject contrast agents directly into vessels. However, it has recently been reported that a macromolecular contrast agentcan be uptaken selectively by lymphatic vessels through simple subcutaneous injection to perform high-resolution imaging of the lymphatic system. This is because particles larger than 3 nm in diameter can be drained by lymphatic vessels, but aredifficultto penetrate through venular endothelia, resulting in selective lymphatic drainage. Particles smaller than 10 nm in diameter would be uptaken by vessels and filtrated in the kidney for the final excretion. Therefore, fabricating MRI contrast agents with 3–10nm in size could be a major pathway to address this problem. Previously, we fabricated MRI contrast agents of a few nanometers in diameter with the condensation of gadolinium complexes and ND particles (Gd-DTPA-ND). The primary particles of these contrast agents possessed a diameter of approximately 5 nm, which was considered to be an adequate size for contrast agents to be selectively uptaken by the lymphatic vessels before the excretion in the kidney. However, these particles formed tight aggregations in water, resulting in micro-scale aggregates. The aggregates were too large to be well uptaken and excreted, and the agents could not be used for lymphatic imaging. In this work, the dispersity of the Gd-DTPA-ND particles was analyzed, which was improved by employingND particles with abundanthydrophilic carboxyl groups on their surface by a pre-oxidation step (CND). The surface carboxyl groups of the CND particles are expected to introduce negative charge on the particles’ surface, inducing the electrostatic repulsion that enables the gadolinium-complexed particles (Gd-DTPA-CND) to disperse in aqueous solution. The dispersity of the fabricated Gd-DTPA-CND particles in distilled water was evaluated by the dynamic light scattering (DLS). The results revealed a hydrodynamic diameter of around 4–5 nm. The transmission electron micrographs (TEM) showed that no aggregation was formed for Gd-DTPA-CND particles, suggesting the improved dispersity in distilled water. Furthermore, the T-1 weighted images of the Gd-DTPA-CND particles in distilled water and human serum presented strong contrast, confirming the high MRI visibility of the particles. Finally, the MRI visibility and the excretion ability of the Gd-DTPA-CND particles were evaluated by injecting the particle solution in a tail vein of a rat. As a result, the blood circulatory system of the rat was clearly imaged, and the bladder was subsequently imaged owing to the filtering of the particles in the kidney. The new Gd-DTPA-CND particles were, therefore, expected as excellent MRI contrast agents for the achievement of the selective MR imaging of the lymphatic system.

Gold-based nanomaterials have shown outstanding physicochemical properties in biomedical applications due to their inherent low toxicity, and their tunable localized surface plasmon resonance (LSPR) wavelength from the visible to near-infrared (NIR) “biological window” region by simply adjusting the size and shape of the nanoparticles. This has enabled the building of fascinating gold-based nanostructures theranostic nanoplatforms for cellular imaging and photothermal therapy (PTT). Herein, we report the synthesis of raspberry gold nanostructures using a polyelectrolyte coated protein (albumin) nanoparticles templates. Albumin nanoparticles were synthesized by desolvation technique using conventional cross-linker (glutaraldehyde) for stable and uniform albumin nanoparticles and then were coated with positively charge poly-L-lysine to enrich the interface with gold anion, exposed to a reducing agent to form raspberry gold nanoshell. The protein templated nanostructures were characterized by dynamic light scattering, filed-emission scanning electron microscope, transmission electron microscopy and visible-near-infrared spectroscopy. The critical parameters for photothermal efficiency, including the concentration and irradiation time, were evaluated. Moreover, the good biocompatibility and the low cytotoxicity of raspberry nanostructures, together with their superior photothermal ablation effect on A549 cancer cells have also been confirmed. Furthermore, we also report the nanostructures for surface-enhanced Taman scattering (SERS) detection of cancer cells with the bioconjugation of different Raman tags. Our study indicates that the protein-templated gold nanoplatform could be a potential candidate PTT and plasmonic sensing in future.

Cationic lipid nanoparticles (LNPs) are well known as drug delivery system (DDS) of nucleic acids and proteins owing to their easy preparation and high cellular uptake efficiency. Many researches have shown various designs of the lipid component to enhance the cellular internalization in different types of cells and meanwhile reduce the cytotoxicity. However, there still lacks the investigation on whether or how cationic LNPs interact with the immune cells.
The NLRP3 inflammasome senses danger signals such as pathogens to alert the immune system by releasing interleukin (IL)-1β. Some cationic NPs and conventional adjuvant alum are reported to have the potential in stimulating the NLRP3 inflammasome. To study the rational of lipid structure in stimulating immune cells, we have investigated a series of cationic LNPs with lysine or arginine head groups but varied lengths of hydrophobic chains and spacers in between in terms of the NLRP3 inflammsomes activation. We found a preferable lipid backbone, ie, ditetradecyl fatty acids with propyl spacer, in either lysine- or arginine-based liposomes that showed high potency in the NLRP3 inflammasome activation in both human and murine macrophages. In addition, these liposomes also exhibited much enhanced antigen presentation in murine dendritic cells when they loaded the ovalbumin antigen on the surface in comparison with other liposomes. The antigen presentations were mediated by both major histocompatibility complex (MHC)-I and MHC-II molecules without interfering co-stimulatory molecules such as CD40, CD80 and CD86. Therefore, OT-I and OT-II lymphocytes (T cells) were sufficiently activated in vitro with significantly increased IL-2 secretion and cell division.
In summary, we have investigated the structure effect of cationic lipids in activating the innate immune cells and further verified their efficiency as antigen carriers in promoting antigen presentation.

Reference:
1. Tianshu Li, et al. Lysine-containing cationic liposomes activate the NLRP3 inflammasome: Effect of a spacer between the head group and the hydrophobic moieties of the lipids. Nanomedicine.2018;14(2):279-288.
2. Tianshu Li, et al. NLRP3 inflammasome-activating arginine-based liposomes promote antigen presentations in dendritic cells. Int J Nanomedicine.2019;14: 3503-3516.

Nitric oxide (NO) is a signalling molecule produced by endothelial cells that serves important biological functions in immune system, cardiovascular system, and central nervous system.¹ However, radical NO species are short-lived. For this reason, controlled delivery of precise amounts of NO with spatiotemporal resolution is highly challenging. Zinc oxide (ZnO) particles offer unique properties that make them excellent tools in biomedical diagnostic and therapeutic fields. In this study, we discovered enzyme-mimicking activities of ZnO and their ability to catalytically decompose endogenous NO prodrugs to generate NO (nM - µM) at physiological conditions. We report a simple approach to synthesize ZnO particles via a chemical precipitation method using poly(vinylpyrrolidone) as the directing agent to control ZnO morphology.² We showed that NO can be locally synthesized and the amount generated can be up-/down-regulated simply by tuning the concentration of ZnO (enzyme mimics) and NO precursors. The catalytic approach enables NO delivery on demand, which can be initiated when needed by external administration of the precursors or prodrugs. ZnO preserved their catalytic activity for at least 6 months and enabled sustained delivery of NO. The present study offers opportunities to overcome current challenges of NO delivery in tissue engineering and regenerative medicine.

References:
1. Yang T., Zelikin A.N., Chandrawati, R. Progress and promise of nitric oxide-releasing platforms. Advanced Science 2018, 5, 1701043.
2. Yang T., Oliver S., Chen Y., Boyer C., Chandrawati R. Tuning the crystallization and morphology of zinc oxide with polyvinylpyrrolidone: formation mechanisms and antimicrobial activity. Journal of Colloid and Interface Science 2019, 546, 43-52.

Diseases may arise if there is a physiological inability to make enough key protective factors when most needed. Nano-bio-manufacturing affords the opportunity to synthesize specific factors in situ and deliver to cells while employing controls on timing and spatial delivery that cannot be achieved by biological systems. Our main objective here is to develop an on-demand production and release of therapeutic proteins at the site of immunological action. Here, we have developed a nano-bio-manufacturing platform that safely augments the immune response to cancers. As a proof of concept, we chose cytokine interleukin 2 (IL2) as our synthetic target which well known to help activate cytotoxic T cells to fight infections and cancer. Here, the microfluidic platform was used to make a library of monodisperse nanoliposomes of variable sizes that can encapsulate IL2 plasmid DNA and extracts of prokaryotic or eukaryotic cells with a high yield. DMPC and DOPC were used as lipid sources. Incorporation of fluorescent GreenLys or GFP-expressing plasmids were used to monitor protein production in real time. Activated caged ATP (DMNPE-caged ATP) was used to block the expression. UV exposure at 360–480 nm wavelength (OmniCure S2000) for 10 s and 80 mW/cm² at pH 7.4, 37 °C uncaged the ATP, and IL2-GFP was successfully synthesized. We then first fabricate artificial cells that mimic the size, shape, and mechanics of lymphocytes like T cells. Then we contain the synthetic machinery for transcription and translation of functional IL2 protein and elements that allow for precisely controlled activation and release of the cytokine. The biosynthesis of proteins in vivo from artificial cells allows for the production of cytokines with number of features not attainable by conventional biological systems: tunable initiation to eliminate basal expression and systemic toxicity; controlled release to locally focus the site of the cytokines’ activity; and targeting to attach the artificial cells to T cells or cancer sites. Here we optimize in vitro synthesis of IL2 cytokine in order to tune T cell fate. First, we encapsulated cell-free protein biosynthesis systems into liposome nanoparticles to make IL2 cytokine. We have established a microfluidic platform to generate monodisperse nanoparticles of various sizes. By controlling the flow rates, we tuned the size of liposomes over a broad range of size (50 - 400 nm). These nanoliposomes encapsulated the cell-free synthesis extract and desired plasmids with efficiency dependent on the DNA size of the cargo. 340 nm particles were selected as the optimized size of nanoliposomes and were loaded inside alginate-based microgels. Stimuli-responsive production and sustain release of IL2 showed improve activation and performance of CD4 and CD8 T cells both in vitro and in B16 melanoma cancer model in vivo. We have developed the de novo design of synthetic immune cells to eliminate systemic toxicities through the local manufacture and sustained release of target cytokines (e.g. IL-2), and eliminating basal production by initiating the synthesis specifically upon an external stimulus.

Despite the many promising results that adaptive T cell therapy has offered to cancer therapy the major hurdle that exists in its clinical translation is the difficulties associated with delivery of trained lymphocytes to tumor sites, along with lack of presence of a support for cells to allow their proper expansion in the immunosuppressive environment of tumor. To bring adaptive T cell therapy one step closer to clinical translation we seek to improve three aspects of current therapies. The first step is to improve capacity and efficiency of adaptive T cells. To tackle this here we have proposed a 3D biopolymer implant as an artificial niche that provides a support for better expansion of pre-trained T cells. We have developed biopolymer-based scaffolds based on the biocompatible Alginate and to further encourage cell trafficking within these structures we have decorated Alginate with RGD peptides. The prorosity of scaffolds then modified to creates micro-pores within these implants that both allows for maximizing the loading capacity for delivering T cells and facilitates their expansion as well. To pursue proliferation which for that we have improved this artificial niche by embedding mesoporous silica microparticles within this scaffold to present cytokine (here IL-2) signal in order to improve T cell proliferation. Surface of these particles also decorated with anti-CD3/anti-CD28 to provide T cells with activation signals. One of the main reasons for immunosuppressive environment of tumors in the abundance of TGFb signal that pursues formation of regulatory T cells (Tregs) which then results in suppression of T killer cells. To overcome this issue, we have empowered our artificial niche with PLGA nanoparticles that engulf TGFbi and suppresses formation of Tregs and gives the trained CD8 T cells a better chance to engage and fight cancer cells. Overall, these bioimplants can be most useful to treat inoperable tumors or in situation were it is near to impossible to remove the whole tumor.

Cell invasion into the surrounding matrix from non-vascularized primary tumors is the main mechanism by which cancer cells migrate to nearby blood vessels and metastasize to eventually form secondary tumors. This process is mediated by an intricate coupling between intracellular and extracellular forces that depend on the stiffness of the surrounding stroma and the alignment of matrix fibers. A multiscale model is used to elucidate the two-way feedback loop between stress-dependent cell contractility and matrix fiber realignment and strain stiffening, which enables the cells to polarize and enhance their contractility to break free from the tumor and invade into the matrix. Importantly, our model can be used to explain how morphological and structural changes in the tumor microenvironment, such as elevated rigidity and fiber alignment prior to cell invasion, are prognostic of the malignant phenotype. The model also predicts how the alignment of matrix fibers can recruit macrophages, which are among the first responders of the innate immune system following organ injury and are crucial for repair, resolution, and re-establishing homeostasis of damaged tissue. I will discuss how the deformation of the nucleus during migration can lead to changes in the spatial organization of chromosomes and their intermingling which can result in genetic mutations and genomic instability. I will also discuss how targeting extracellular matrix mechanics, by preventing or reversing tissue stiffening or interrupting the cellular response in cancer and fibrosis, is a therapeutic approach with clinical potential.

BIO: Vivek Shenoy is the Eduardo D. Glandt President’s Distinguished Professor in the School of Engineering and Applied Sciences at the University of Pennsylvania. Dr. Shenoy's research focuses on developing theoretical concepts and numerical methods to understand the basic principles that control the behavior of both engineering and biological systems. He has used rigorous analytical methods and multiscale modeling techniques, ranging from atomistic density functional theory to continuum methods, to gain physical insight into a myriad of problems in materials science and biomechanics. Dr. Shenoy's honors include a National Science Foundation CAREER Award (2000), the Richard and Edna Solomon Assistant Professorship (2002-2005) and the Rosenbaum Visiting Fellowship from the Isaac Newton Institute of Mathematical Science, University of Cambridge and the Heilmeier award for excellence in faculty research (2019). He is the principal investigator and director of the NSF-funded Science and Technology Center for Engineering Mechanobiology established in 2016. He also serves the editor of the Biophysical Journal and is a fellow of the American Institute for Medical and Biological Engineering.

Sculpting of structure and function of three-dimensional multicellular tissues depend critically on the spatial and temporal coordination of cellular physical properties. Yet the organizational principles that govern these events, and their disruption in disease, remain poorly understood. Here, I will introduce our recent progress performing biomechanical imaging to quantify cell and extracellular matrix (ECM) mechanics, as well as their mechanical interaction. By integrating confocal microscopy with optical tweezers, we have developed a platform to map in three dimensions the spatial and temporal evolution of positions, motions, and physical characteristics of individual cells throughout a growing mammary cancer organoid model. Compared with cells in the organoid core, cells at the organoid periphery and the invasive front are found to be systematically softer, larger and more dynamic. These mechanical changes are shown to arise from supracellular fluid flow through gap junctions, suppression of which delays transition to an invasive phenotype. Together, these findings highlight the role of spatiotemporal coordination of cellular physical properties in tissue organization and disease progression.

Epithelial tumors exhibit dysregulated cell-cell and cell-matrix adhesions as they invade into the surrounding extracellular matrix. In particular, the epithelial-mesenchymal transition (EMT) is associated with weakened cell-cell adhesions and strengthened cell-matrix adhesions, resulting in multicellular dissemination. Traction force microscopy enables new insights into the cell-generated forces that mediate these behaviors, but has primarily been applied to individual cells in 3D. Here, we elucidate the collective tractions of multicellular clusters in 3D matrix after induction of the EMT master regulator Snail. We find that multicellular clusters exhibit characteristic spatial signatures that can be used for mechanophenotypic profiling. In particular, EMT results in highly localized “hotspots” of strong cell-matrix adhesion, associated with high contractility and front / back polarization. We further show that chemotherapeutics and targeted inhibitors can perturb clusters towards more epithelial or mesenchymal-like mechanophenotype. We envision that this 3D culture assay will enable high content preclinical screening of targeted anticancer compounds as well as to predict the clinical response of human patient samples.

Carcinoma cells tend to migrate in collective strands, ducts, sheets or clusters (Friedl, & Gilmour, Nat. Rev. Mol. Cell. Bio. 2009). To migrate collectively, the epithelial collective has been argued to overcome geometric constraints attributable to cell jamming (Atia et al., Nat. Phys. 2018). If so, then the greater is the degree of cellular jamming, then the more would be the extent to which each individual cell becomes caged by its neighbors, and therefore, the less rapidly it would be able to migrate (Park et al., Nat Mat. 2015). The jamming hypothesis, however, has never been tested in the context of cancer cell invasiveness. Using classical in vitro cultures of six breast cancer models, here we investigate structural signatures of jamming, dynamical signatures of jamming, and the relationship between them. In order of increasing invasiveness, the cell lines examined included MCF10A, MCF10A.vector; MCF10A.14-3-3z; MCF10.Erb2, MCF10AT; and MCF10CA1a. Across all models tested, cell shape and shape variability from cell-to-cell conformed well to structural signatures of cell layer jamming. In all cases but one, migratory dynamics changed roughly in concert with expectations based on structural signatures –as the strength of structural signatures of unjamming increased, the rapidity of migratory dynamics tended to progressively increased. The exception was the case of MCF10CA1a, wherein structure signified a moderately jammed state whereas migratory dynamics were excessively rapid and therefore discordant with structure. Closer examination of migratory dynamics of MCF10CA1a showed anomalously large migratory persistence, but the mechanism of discordance in this case remains unclear. A hallmark of cancer is multiple dimension of heterogeneity. Nevertheless, each of the diverse cases examined here reveals that cell jamming imposes an overriding geometric constraint.

Progression to metastatic breast cancer requires cancer cells to invade from a solid tumor into the surrounding stroma and escape into a lymphatic or blood vessel. To understand the biophysical and biochemical parameters that define the kinetics of invasion and escape, we engineered a three-dimensional model of human breast microtumors embedded within native extracellular matrix. We previously found that interstitial fluid pressure (IFP) determines the invasive response of human breast microtumors: specifically, interstitial hypertension (i.e., elevated IFP) prevents invasion, whereas interstitial hypotension (i.e., lowered IFP) promotes invasion. We have now used this system to examine the effects of matrix density, proteolysis, proliferation, and IFP on the kinetics of tumor cell escape into an empty cavity. Our data suggest that the physical microenvironment of a tumor dictates the rates of two early steps in the metastatic cascade, namely, invasion of the surrounding interstitium and escape into an open space. These physical features dictate whether escape results from a ballistic or diffusive invasion process. Furthermore, acute changes in interstitial pressure can suppress tumor cell escape after invasion has already occurred. Our results point to the possibility of using physical therapies to delay or prevent metastatic progression in breast cancer.

Cancer metastasis, i.e., the spreading of cells from the primary tumor to distant organs, is responsible for more than 80% of all cancer deaths. During cancer cell invasion and metastasis, tumor cells migrate through interstitial spaces and transendothelial openings substantially smaller than the diameter of the cell. Recent research has made it apparent that cells migrating in such confined three-dimensional (3D) environments face substantially physical challenges. In particular, the cell nucleus is the largest and stiffest organelle, making nuclear deformation a rate-limiting factor in the passage of cells through confined 3D environments. We have used micro- and nano-fabrication approaches to generate microfluidic devices that closely mimic the physical constraints of physiological interstitial environments, while providing precise control over the constriction geometry and enabling live-cell imaging at high spatial and temporal resolution. Using these devices, we demonstrated the importance of available pore size and nuclear deformability on the ability of cells to move through 3D environments. We combined these devices with fluorescent reporters for nuclear envelope rupture and DNA damage to assess the functional consequences of the physical forces exerted on the nucleus during confined migration. In addition, we developed a microfluidic micropipette aspiration device to rapidly measure nuclear stiffness in large numbers of cells. We found that highly metastatic breast cancer cells had decreased levels of the nuclear envelope proteins lamin A/C, which determine nuclear deformability, compared to less aggressive tumor cells, and that the increased nuclear deformability promoted migration through tight spaces. Increasing expression of lamin A in breast cancer cells with normally low levels of lamin A/C significantly impaired their invasive properties, while depletion of lamin A/C increased invasive potential through micron-scale microfluidic constrictions and dense collagen matrices. Importantly, analysis of human breast tumor tissue microarrays showed that low levels of lamin A/C correlated with reduced disease-free survival, demonstrating the clinical relevance of our findings. Taken together, these studies indicate that downregulation of lamin A/C could promote both cancer cell invasion and metastasis in breast cancer while highlighting the appeal of engineered materials and microenvironments to study tumor cell mechanobiology. Insights gained from this work could improve prognostic approaches; ultimately, targeting regulator pathways associated with altered lamin expression may offer novel therapeutic avenues to control metastatic disease in breast cancer.

The U.S. National Cancer Institute (NCI) leads, conducts, and supports cancer research across the nation to advance scientific knowledge and help people live longer, healthier lives. Many advancements in cancer research in areas of progression, metastasis, and treatment response have been enabled by the development of innovative technologies, including novel biomaterials, microfluidics, and biomimetic engineered technologies. Several programs at the NCI have helped foster cancer technology development in these areas as well as the overall convergence of approaches and perspectives from the physical sciences and engineering into cancer research. Over the past decade, the NCI has supported the NCI Physical Sciences – Oncology Network (PS-ON), which is comprised of nearly 30 transdisciplinary teams that integrate physical sciences perspectives with cancer research to complement and expand on our current understanding of cancer across many biological length- and time-scales. Thematic areas under investigation in the PS-ON include transcriptional dynamics and genomic architecture, modeling evolutionary dynamics of treatment response, cancer mechanobiology and the physical microenvironment, and multi-scale computational modeling approaches to integrate data across length scales. PS-ON investigators and those in the affiliated Cancer Tissue Engineering Collaborative (TEC) research program are utilizing biomaterials and biofabrication for experimental model systems of cancer that recapitulate the tumor microenvironment and tumor-stromal interactions. There is also sophisticated incorporation of bioreactors and microfluidic culture to mimic perfusion, lymphatics, interstitial pressure, and molecular gradients. Other research initiatives supported by the NCI to promote convergent, cross-disciplinary research that include projects incorporating novel biomaterials are the Cancer Systems Biology Consortium (CSBC) and the Innovative Molecular Analysis Technologies (IMAT). The NCI demonstrates its interest in supporting materials science and engineering in cancer research by investment in these areas through investigator-initiated research and the targeted programs. The continued investigation of the physical dynamics of cancer and incorporation of novel biomaterials and biomimetic engineered technologies will be important, focusing on understanding the complex and dynamic multiscale interactions of the tumor, host, and immune system. Innovative technology development will continue to be critical for unprecedented measurements and discoveries in cancer research.

The nucleus links physically to cytoskeleton, adhesions, and extracellular matrix – all of which are subject to stresses and strains. We have taken various materials-intensive approaches to study nuclear rupture in tumors [1], embryonic organs [2], and various in vitro models, and we find rupture results from high nuclear curvature, leading to cytoplasmic mis-localization of multiple DNA repair factors and transcription factors that impact cell fate and function. Curvature is imposed by external probe [1], by migrating quickly (not slowly) through constricting micropores [3,4], or by cell attachment to either aligned matrix or stiff matrix [1], and theory indicates rupture pores from by a heterogeneous nucleation mechanism [5]. Mis-localization of nuclear factors is greatly enhanced by nucleoskeleton depletion (soft nuclei), requires many hours for nuclear re-entry, and correlates with pan-nucleoplasmic foci of DNA damage and with electrophoretic breaks. Excess DNA damage is rescued in ruptured nuclei by co-overexpression of multiple DNA repair factors as well as by soft matrix or inhibition of either actomyosin tension or oxidative stress – with combination treatments needed to rescue a cell cycle checkpoint [4]. Increased contractility has the opposite effect, and stiff tumors with softened nuclei indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Normal differentiation processes of myogenesis and ostoegenesis are also affected by migration through constricting pores, suggesting general effects on cell fates [6]. Mis-repair of DNA is further suggested by two cancer lines that, after constricted migration, exhibit greater genome variation [1,3]. References: [1] Y Xia, … DE Discher. Nuclear rupture at sites of high curvature compromises retention of DNA repair factors J Cell Biol (2018). [2] S Cho … Discher DE. Mechanosensing by the lamina protects against nuclear rupture, DNA damage, and cell cycle arrest. Dev Cell (2019). [3] J Irianto … DE Discher. DNA damage follows repair factor depletion and portends genome variation in cancer cells after pore migration. Curr Biol (2017). [4] Y Xia … Discher DE. Rescue of DNA damage after constricted migration reveals bimodal mechano-regulation of cell cycle. J Cell Biol (2019). [5] D Deviri … Discher DE, Safran SA. Scaling laws indicate distinct nucleation mechanisms of holes in the nuclear lamina. Nature Physics (2019). [6] LR Smith … Discher DE. Constricted migration modulates stem cell differentiation. Mol Biol of the Cell (2019).

Tumors show increased tissue level forces and a present with a chronically stiffened extracellular matrix (ECM), and transformed cells exhibit a perturbed oncogene-stimulated and ECM-tuned mechanophenotype. We have been studying how these aberrant cell and tissue level forces promote malignant transformation and drive tumor metastasis, and how they modulate tumor recurrence and treatment resistance in breast and pancreatic cancer and glioblastoma. We use two and three dimensional culture models with tuned extracellular matrix stiffness, as well as transgenic and syngeneic mouse models, human PDX models and human biospecimens, in which ECM crosslinking and stiffness and integrin mechanosignaling can be quantified and modified. Our studies have thus far revealed that the ECM in all tumors is progressively remodeled and stiffened by stromal fibroblasts and that this occurs prior to malignant transformation. We determined that ECM remodeling and stiffening is mediated very early during malignancy by stromal fibroblasts that are activated by factors including TGFb that are secreted by infiltrating pro inflammatory macrophages. The stromal-fibroblast stiffened ECM disrupts tissue organization, promotes cell growth and survival and drives cell invasion. A chronically stiffened tissue stroma drives angiogenesis, and activates STAT3 to induce key cytokines and chemokines that promote pro-tumor immunity to foster tumor growth and dissemination and impede tumor treatment. The stiffened ECM also drives an epithelial to mesenchymal transition and primes the metastatic niche to foster metastasis. I will discuss the dynamic and reciprocal interplay between tissue tension and innate and acquired immunity and how this can not only force tumor aggression and metastasis but may also initiate tumor progression.

Hydrogels, highly hydrated cross-linked polymer networks, have emerged as powerful synthetic analogs of extracellular matrices for basic cell studies as well as promising biomaterials for regenerative medicine applications. A critical advantage of these synthetic matrices over natural networks is that the biophysical and biochemical properties of the material can be tuned with high control and precision. For example, bioactive functionalities, such as cell adhesive sequences and growth factors, can be incorporated in precise densities. We have engineered poly(ethylene glycol) [PEG]-maleimide hydrogels that support improved pancreatic islet engraftment, vascularization and function in diabetic models. Two biomaterial strategies will be discussed. We have developed proteolytically degradable synthetic hydrogels, functionalized with vasculogenic factors, engineered to deliver islet grafts to extrahepatic transplant sites via in situ gelation. These hydrogels induce differences in vascularization and innate immune responses among subcutaneous, small bowel mesentery, and epididymal fat pad transplant sites with improved vascularization and reduced inflammation at the epididymal fat pad site. This biomaterial-based strategy improves the survival, engraftment, and function of a single pancreatic donor islet mass graft compared to the current clinical intraportal delivery technique. In a second application, we have developed a localized immunomodulation strategy using hydrogels presenting an apoptotic form of Fas ligand (SA-FasL) that results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosts the immunomodulatory efficacy of SA-FasL-hydrogels, resulting in acceptance and function of allografts over 200 days. Survivors generate normal systemic responses to donor antigens, implying immune privilege of the graft, and have increased T-regulatory cells in the graft. Current studies focus on evaluating this immunomodulatory strategy in a large animal model of type 1 diabetes. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

It has long been recognized that tissue mechanical properties are altered in cancer, and this can serve as the basis for early diagnosis. While the impact of changes in tissue stiffness has been the focus of research to date, the role of tissue viscoelasticity has not been widely explored. We have developed hydrogels which allow for independent control over elastic moduli and stress relaxation/creep, and can mimic the fibrillar architecture of native collagenous matrices. These material systems are being utilized in 3D cell culture models of cancer and immunotherapy, and demonstrate that the gene expression of various cell types present in tumors, including cancer cells, mesenchymal cells, and immune cells is profoundly impacted by the viscoelastic properties of their matrix.

Mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine applications due to their ability to self-renew, capacity for multipotent differentiation and secretion of a diverse array of cytokines and growth factors (the MSC secretome). These cells are frequently utilized in conjunction with biomaterial scaffolds designed to encourage cellular retention and direct the cells’ regenerative properties. However, hydrogel carriers have not yet yielded significant results in the clinic in part due to a lack of understanding of how hydrogel biophysical and biochemical properties impact cellular function. Although many synthetic hydrogels incorporate short peptides (e.g. RGD) to support integrin-mediated cell adhesion, the impact of hydrogel adhesive properties on transplanted cell function remains unknown.

We engineered integrin-specific hydrogels for the delivery of MSCs by tethering either the ubiquitous RGD cell adhesion motif or the type-1 collagen derived GFOGER adhesion motif into synthetic poly(ethylene) glycol (PEG)-based hydrogels. Integrin-specificity was confirmed using blocking antibodies and a custom spinning disk platform in which cells attached to hydrogel disks are exposed to well-defined hydrodynamic shear forces allowing for sensitive measurements of the force required to detach the cell from the substrate. The effects of integrin-specific adhesive peptide presentation on MSC secretome and MSC-macrophage interactions were evaluated in vitro using Luminex multiplex technology. Finally, integrin-specific hydrogel directed MSC bone tissue regeneration was assessed over the course of 8 weeks in a critical size radial bone defect in an NSG mouse model.

Spinning disk analysis shows that cell adhesion to hydrogels presenting the adhesive peptides RGD and GFOGER are specific to αvβ3/β1 and α2 integrins respectively. Luminex data shows that the secretome of MSCs encapsulated in integrin-specific hydrogels cluster distinctly based on peptide and secretion of IL-6, IL-8, and VEGF is increased in GFOGER functionalized gels compared to RGD and non-adhesive controls. Further, in a co-culture assay, we show that macrophage cytokine secretion is differentially modulated by MSCs encapsulated in integrin-specific hydrogels, including an increase in IL-10 secretion by macrophages interacting with GFOGER encapsulated MSCs. Finally, MSCs delivered in GFOGER functionalized hydrogels significantly enhance repair of critical size bone defects in vivo compared to RGD and non-adhesive controls. Taken together, our results demonstrate that integrin specificity can be engineered into synthetic hydrogel systems resulting in modulation of the MSC secretome, differential MSC-macrophage interactions and improved tissue healing.

Acute myeloid leukemia (AML) is a malignancy of hematopoietic origin with limited therapeutic options. The standard-of-care cytoreductive chemotherapy can rapidly induce an apparent remission but relapse occurs in the majority of patients, highlighting the difficulty in eradicating all AML cells. A highly immunosuppressive AML microenvironment in the bone marrow and a paucity of suitable cell surface immunotherapy targets on AML cells precludes the induction of an effective endogenous adaptive immune response, which contributes to disease relapse. AML cells generally have a relatively low mutational load, are weak stimulators of host immune cells and often possess mechanisms that prevent induction of an effector T cell response. However, the anthracyclines used for treating AML induce cell death and promote dendritic cell (DC) cross-priming of tumor-associated antigens for T cell by the release of damage-associated molecular patterns. This effect is broadly associated with stimulating an antitumor T cell immune response and may partially contribute to the efficacy of the treatment. The susceptibility of AML to effector T cells is supported by the finding that AML, like many other types of cancer cells, displays tumor antigens that have the potential to trigger immune responses. Therefore, therapeutic vaccines have the potential of achieving a lasting AML-specific immune response capable of eradicating the residual disease that remains following chemotherapy. The development of cancer vaccines requires T cell activation resulting from effective presentation of one or more tumor antigens in the context of co-stimulation. To promote a robust and durable immune-response against AML, we developed a biomaterial-based vaccine which provided a sustained release of GM-CSF to concentrate dendritic cells (DCs), TLR agonist CpG-ODN and one or more leukemia antigens in the form of a peptide antigen, cell lysates or sourced from in vivo recruited AML cells. The vaccine induced local cell infiltration and activated DCs to evoke a potent anti-AML immune response. Prophylactic vaccination alone prevented the engraftment of AML cells. Moreover, mice were able to overcome a re-challenge, indicating the potential of these vaccines to establish a long-term immunity. Combining induction chemotherapy (iCt) and the biomaterial vaccine maximized efficacy to eradicate established disease, even without exogenous delivery of a defined vaccine antigen. We found that by recruiting DCs and sustaining their activation in an otherwise immunosuppressive AML environment, the biomaterial vaccine can harness a broad range of tumor antigens arising from chemotherapy-induced AML cell death, including those not delivered through the vaccine. Furthermore, iCt in combination with the biomaterial vaccine transiently decreased bone marrow FoxP3⁺CD25⁺ T_regs and enhanced tumor-specific T cells. The CD8⁺ T cell/T_reg ratio was higher in the bone marrow of mice treated with iCt and the antigen-free vaccine, compared to the antigen-free vaccine alone. The combination treatment depleted AML cells and generated durable long-term effector T cell responses, and immunized transplanted mice against AML. The biomaterial vaccine treatment was well-tolerated and promoted AML rejection without the indication of pancytopenia or autoimmunity in the studies. Our findings suggest that induction of a potent anti-AML immune response in such a setting might prevent the life-threatening evolution of this disease. In contrast to neoantigen-based vaccination, a scaffold generating tumor specific immune responses in situ can be an off-the-shelf approach for treating patients post-iCt. The results from this experimental mouse model of AML demonstrate the capacity of a biomaterial-based vaccination approach to induce a potent immune response to deplete AML and prevent relapse.