November 29-December 4, 2015 | Boston
Meeting Chairs: T. John Balk, Ram Devanathan, George G. Malliaras, Larry A. Nagahara, Luisa Torsi
The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. Here, we present a new device for CTC analysis that captures these cells and spatially arranges the cells according to their surface phenotype. Antibody-functionalized magnetic nanoparticles facilitate CTC sorting, and permit deconvolution of phenotypic subpopulations. Working with patient blood samples and animal models of cancer, we obtain profiles that elucidate the heterogeneity of CTCs and dynamically track how these cells change as a function of tumor growth.
While metastic disease causes 90% of all cancer deaths, determining the role of various cancer cells in the metastatic process has been difficult due to the rare nature of these cells and the continuum of phenotypes they possess. To assist in studies based on the use of rare CTCs that may be invovled in metastasis, a microfluidic fabricated via micro-replication from a polymer material was used to affinity select different CTC types. The microfluidic contained a series of 190 nL fluidic channels (50-500) configured in a serpentine geometry. The walls of the channels were decorated with antibodies used to recognize and select various subpopulations of CTCs. The covalent attachment to a polymeric surface could be affected by activating the polymer surface using UV/O3 light, which creates a surface scaffold of carboxylic acids that could subsequently be reacted with antibodies via EDC/NHS chemistry. The microfluidic could process 7.5 mL of whole blood in ~20 min with a recovery of 97% and purity >80%. These performance metrics were highly dependent on the type of polymer used as the substrate material for the microfluidic. Following selection from whole blood, the CTCs could be released from the surface-immobilized antibodies; this was affected by using single-stranded DNA linkers that contained a uracil residue that could be enzymatically cleaved releasing the CTCs. The CTCs were then enumerated by measuring impedance signatures of single cells that traversed through a pair of electrodes. The use of the microfluidic for determining the molecular characteristics of CTC subpopulations involved in an epithelial-to-mesenchymal transition were evaluated using pancreatic cancer as the example disease. In this presentation, we will discuss the microfluidic used for the CTC selection and the role of the selection of polymeric material on the performance of the device.
Specific cell separation, further enumeration and further characterization (e.g. molecule analysis and drug response within patient whole bloods) of tumor cells are necessary in a variety of immunology, neuroscience, stem cell, and cancer research. Here we present on the rapid and direct quantification of specific cell captures using a micro-patterned streptavidin (STR)-functionalized silicon nanowire (SiNW) platform, which was prepared by Ag-assisted wet chemical etching and a photo-lithography process. This platform operates by high-affinity cell capture rendered by the combination of antibody-epithelial cell surface-binding, biotin-STR binding, and the topologically enhanced cell-substrate interaction on a 3-dimensional SiNWs array.
Cell migration is a tightly regulated function critical to the normal development of organs and tissues. Cell migration is abnormally activated in a wide range of human diseases, including cancer metastasis, immunological responses, and wound healing. Most of what we know about eukaryotic cell migration at a mechanistic level has stemmed from well-controlled studies of cell migration on flat dishes. However, the onset of migration in disease and development often forces cells to negotiate, exert pulling forces on, and more through a 3D matrix.Recent work has highlighted how 3D migration is fundamentally different from its 2D counterpart, in particular the biophysical forces driving 3D migration. Migration on 2D dishes, which induces an artificial baso-apical polarization of the cell, is driven by actomyosin contraction of stress fibers between focal adhesions and the formation of a wide lamellipodium terminated by thin filopodial protrusions at the leading edge. The same cells in a more physio-pathologically relevant collagen-rich 3D matrix do not display a lamellipodium or filopodia. Instead, cells display highly dendritic protusions controlled by distinct proteins that do not rely nearly as much on acto-myosin contractility, but rather microtubule assembly/disassembly dynamics. Recent work has also shown how cells in 3D can alternate between a mesenchymal and an amoeboid phenotype depending on the physical properties of the matrix (density, pore size, fiber alignment). Several biophysical assays have recently been developed and validated to monitor and measure cell motility in 3D microenvironments for applications in basic life science, biomedical research, and drug screening and testing.
The presence of Circulating Tumor Cells (CTCs) in bloodstream of patients with epithelial cancers is an important intermediate step in cancer metastasis and can provide valuable insights into disease detection, staging and personalized treatment. As compared to obtaining a tissue biopsy which is invasive and painful, ‘‘liquid biopsy&’&’ for CTCs detection can be easily performed via a routine blood draw. The presence and number of CTCs in peripheral blood has been associated with the severity of the disease and have potential use for early detection, diagnosis, prognosis and treatment monitoring purposes. The isolation of CTCs using microfluidics is attractive as the flow conditions can be accurately manipulated to achieve an efficient separation. Here, we demonstrate several effective separation methods by utilizing the unique differences in size and deformability of cancer cells from that of blood cells. By exploiting the fluid dynamics in specially designed microfluidic channels, CTCs which are generally stiffer and larger can be physically separated from the more deformable blood constituents. Using this label-free approach, we are able to retrieve viable CTCs that are not only suitable for downstream molecular analysis such as genetic or RNA sequencing, but also for expansion and culture. With blood specimens from cancer patients, we not only confirmed successful detection, isolation and retrieval of CTCs, but also identification of actionable mutation which will enable precision medicine and personalized treatment of cancer patients.
Circulating tumor cells (CTCs) are the cancer cells disseminated from a tumor into the blood stream. Presence of these cells in blood is known to be associated with metastasis of the disease. Therefore isolation and detection of CTCs have diagnostic and prognostic importance. However, due to their low abundance in blood only highly sensitive detection methods can be potentially utilized for clinical applications. Despite the many successful examples of clinical trials on importance of detection of rare tumor cells, they are yet to be widely considered as a routine clinical test or for prognosis and screening purposes. Most of the current methods for detection of cancer cells are based on immunostaining and require experienced technician. Here we report an integrated chip design that enables isolation and detection of rare cancer cells, using a simple yet sensitive electrochemical ELISA method. With this design, detection of very low number of isolated cancer cells is achieved in whole blood, which demonstrates the clinically relevant specificity and sensitivity of the device.
It is important to understand various biomolecular events that contribute towards classification of programmed cell death (apoptosis) and how tumors evade apoptotic death. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) are two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. With new drugs being formulated to control abnormal cell proliferation (cancer) caused by mutations, effective drug-induced apoptosis will yield the relationship between cancer genetics and treatment sensitivity. The objective of this study was to build non-invasive techniques (without altering inherent cell properties) to isolate cells that are undergoing varying stages of apoptosis for a high-throughput screening application. We have used dielectrophoresis to determine and isolate various stages of apoptotic cells (early, mid and late) for two NSCLCs adenocarcinoma cell lines (HCC1833 and H1755). Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where exposed to a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over a period of 48 hours upon exposure of varying concentrations of ABT-263 ranging between 50 nM to 500 nM. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance, measured using dielectrophoretic technique. In addition, zeta potential (potential at the electrical double layer of cell-buffer interface) of these fixed isolated cells were measured to obtain direct correlation to biomoelcular events. Western blot was used to examine the changes in the expression levels of Bax, Bcl-2, caspase-3, Hsp-90. As a control, the obtained results were compared against standard apoptotic assays for respective stages; JC-1, a mitochondrial membrane potential dye for marking early stage apoptosis, Annexin-V for the detection of PS as a mid-stage marker and defragmentation of DNA, verified by immunofluorescence staining using fluorescein-deoxyuridnie triphosphate (FITC-dUTP) and flow cytometry. The use of non-invasive dielectrophoresis is to provide a sophisticated method to characterize and isolate cells for potential downstream analysis. Effective separation of carcinoma cells at different apoptotic stages should enable a more rational approach to anticancer drug design and therapy.
Compared to tissue biopsy, the standard method for cancer diagnosis, circulating tumor cells (CTCs) provide easy and non-invasive access to tumor cells and have proven to be valuable biomarkers for disease diagnosis and progression. Furthermore, CTCs represent the heterogeneous nature of tumor and hence potential surrogates of tumor cells for functional and genomic analysis. However, CTCs occur at very low frequency (1-10 cells/ml) in blood and are obscured by millions of blood cells, which makes their isolation difficult and, hence, has impeded their clinical application. Over the last decade, different technologies and devices have been developed to facilitate isolation and enumeration of these rare cells. Most technologies are based on micro/nanostructures coated with antibodies against the cell surface markers to aid cell capture. However, this makes device fabrication costly and time consuming. Most such devices also lack the mechanism for releasing the captured cells thereby making downstream analysis difficult.We developed a planar microfluidic device based on tunable polymer-graphene oxide (GO) nanocomposite film made through simple drop-casting on a patterned and surface modified glass substrate. The platform architecture is composed of functionalized GO as the carrier of anti-EpCAM, a tumor cell recognition unit, and embedded in matrix of a thermo-responsive polymer. The polymer is designed to provide selective dissolution only below 13°C (lower critical solution temperature (LCST)) which allows the device to be used for cell capture at room temperature and cell release at temperature lower than the LCST. The non-fouling PEG monolayer-modified substrate further aids the release of the cells upon film dissolution. Since the captured cells are tethered to graphene sheets and not the polymer chains, cell isolation is easier and loss of cell viability is minimized. To test the performance of these devices, fluorescently labeled human breast cancer cell line MCF-7 cells (1000 cells/mL) were spiked into buffer and flowed at different flow rates (1-10mL/h). An average capture efficiency of >82% was achieved in the range of 1-3mL/h flow rate. Cell release experiments showed that on average >90% of the captured cells were released upon flowing cold PBS. Over 90% of the cells remained viable after release. We also tested clinical samples from ten metastatic breast cancer and three pancreatic cancer patients. CTCs were isolated from eight breast and two pancreatic cancer patient samples in the range of 2-20 CTCs/mL. Additionally, to demonstrate the potential for single cell genomic analysis of released CTCs, Fluorescence in situ hybridization (FISH) was conducted using probes for HER2 and chromosome 17 control probe, revealing HER2 amplification in one breast cancer patient. Effective downstream analysis of rare CTC populations facilitated by easy operation of the fabricated device highlights its potential for clinical application.
RuO2 doped nanoparticles TiO2 supported on disordered mesoporous silica (DMS-1) was synthesized by the sol-gel method, its ex-situ modification with TiO2 nanoparticles. The photoactivity was examined for methylene blue photodegradation in aqueous medium. To assist in interpreting the photocatalytic behavior of 0.76%RuO2-40%TiO2/DMS-1 and 0.79%RuO2-30TiO2/DMS-1, reference systems consisting of x%TiO2/DMS-1 photocatalysts undoped RuO2 and TiO2 pure (Degussa P-25). The load RuO2 <1.0 wt% was prepared by impregnation method and vary amount of TiO2 (30 and 40 wt.%). The photocatalysts were characterized by N2 adsorption-desorption isotherms, X-ray diffraction (XRD), UV-vis diffuse reflectance spectroscopy (UV-vis DRS) and transmission electron microscopy (TEM). The results showed that RuO2 on TiO2 present greater activity photocatalytic due to the presence of RuO2 that promoting efficiently charge separation, causes inhibition of recombination of electron-hole pairs.
Three dimensional (3D) aligned mesoporous α-Fe2O3/CdS heterostructures are prepared for solar-driven, real-time, and selective photoelectrochemical sensing of Cu2+ in the living cells. Fabrication of aligned mesoporous α-Fe2O3 substrates is realized by an interfacial aligned growth and self-assembly process based on the van der drift model and subsequent selective in situ growth of CdS nanocrystals. The as-prepared mesoporous iron oxide/CdS heterostructures achieve significant enhancement (~2-fold) in the photocurrent intensity compared with mesoporous iron oxide. The electrochemical impedance spectroscopy measurement and the density functional theory calculation exhibit that the considerably improved performance of 3D aligned mesoporous α-Fe2O3/CdS heterostructures is attributed to the enhancement of charge transfer and the increase of charge carrier density. This photocurrent increase is ascribed to unique 3D Fe2O3 and CdS with multiple features including their excellent flexibility, high surface area (~ 87 m2/g) and large pore size (~ 20 nm), which give rise to enhancement of the PEC performance by facilitating ion transport and providing more active electrochemical reaction sites. In addition, the introduction of Cu2+ enables the activation of quenching the charge transfer efficiency, thus leading to sensitive photoelectrochemical recording of Cu2+ level in buffer and cellular environments. Furthermore, real-time monitoring (~ 0.5 nM) of Cu2+ released from apoptotic HeLa cell is performed using the as-prepared 3D aligned mesoporous iron oxide/CdS biosensor, suggesting the capability of studying the nanomaterial-cell interfaces and illuminating the role of Cu2+ as trace element.
Recently, nanostructured surfaces are finding applications in rare cell analysis and stem cell engineering via nanoscale cell-surface interactions. Studies have shown that nanoscale cellular structures, such as, focal adhesion and integrin, interact with the nanotopography of the substrate, which in turn affects cell behavior such as cell morphology, adhesion, spreading, motility, gene expression, and differentiation. Here, we report the use of a novel nanotopographic substrate - single-crystalline nanoporous GaN substrate fabricated over a large area via electrochemical reaction and showing precisely tunable pore size for the study of human mesenchymal stem cell function and differentiation. This material is not only an optically active semiconductor but also relatively biocompatible and non-toxic. Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20-100 nm. We found that the ability for human mesenchymal stem cells (hMSCs) to adhere uniformly on these substrates shows a maximum with the substrate pore size of 30 nm. Substantial cell elongation was observed on the films with pore size of 80 nm. Interestingly, the osteogenic differentiation of hMSCs also occurs preferentially on the substrate with 30 nm sized nanopores, which is correlated with the optimized conditions for uniform cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are inter-linked and can be co-regulated by nanotopography.
MicroRNAs (miRNAs) are a group of small RNAs that are important regulators of normal development, immune defense and a range of human diseases including cancer. The level, variety and kinetics of miRNAs in a cell population could contribute to the formation of substantial non-genetic cell heterogeneity and alters the collective response of, i.e., tumor cells, to treatment. Measurement of an array of oncogenic and tumor-suppressive miRNAs from a population of cancer cells at single-cell resolution will enable the possibility to quantify cellular heterogeneity and corresponding miRNA profiles in cancer. Herein we developed a microchip platform for amplification-free, multiplexed detection of miRNAs at the single cell level. This microchip consists of a nanoliter microchamber array to isolate single cells and a high-density DNA oligomer array to perform multiplexed detection of miRNA. We tested two different approaches on this microchip platform. It employed a microfluidic double-T unit to conduct on-chip cell lysis and release of cytoplasmic miRNAs, which are detected directly by the DNA oligomers immobilized in the microchambers via hybridization and ligation. Our preliminary results demonstrated up to 12-plexed measurements of miRNA targets from single human monocytes (THP-1) and identified the high expression of miR-16, miR-150, miR-223 and miR221, and an appreciable level of cellular heterogeneity. This microchip platform has the potential to be utilized to invetigate the heterogeneity of cancer cells and the associated microRNA signatures from clinical specimens.
Cancer is one of the major health problems of modern society with predicted 12.7 million newly diagnosed cases per year (2008).1 Its early detection could significantly improve medical therapy and thus reduce morbidity and mortality rates. Promising breath markers for lung2 and breast cancer3 are aldehydes as their increased levels were detected in affected patients. Portable and simple#8209;in#8209;use aldehyde analyzers could contribute significantly to the implementation and widespread application of breath analysis for medical self-diagnostics and simple body parameter monitoring. Chemo-resistive gas sensors are ideal as they offer sufficient analyte sensitivity, fast response and recovery times for real-time monitoring, simple operation and they have been tested already in a portable design.4 Well-known chemo-resistive materials, however, suffer from insufficient aldehyde selectivity in the complex5 analyte matrix of human breath. To overcome this challenge, arrays of multiple non-specific sensors are a viable option where selectivity is obtained by statistical analysis of the sensor responses.6Here, we present a microsensor array consisting of four differently doped SnO2 films for selective formaldehyde (FA) detection. The microsensor substrates are silicon wafer-based and fabricated by state-of-the-art micro-processing technology. Rapid flame aerosol synthesis is applied to produce the sensing nanoparticles (~10 nm) that are accurately deposited onto the substrates in designated areas (d = 500 mu;m). The resulting nanostructured and highly porous film morphology offers high sensitivity that is quite attractive for breath analysis. These sensors are combined in arrays featuring a compact size and low power consumption (~2 W) at operational conditions. The derived arrays are tested on simulated breath mixtures at 90% rh. Breath-relevant FA concentrations (30 - 200 ppb) are clearly identified that could allow detection of cancer. Such microsensor arrays have high potential for further development towards a portable breath analyzer. The compact size and low power consumption make them attractive for integration into portable electronic devices (e.g. smart phone).(1) Bray, F.; Jemal, A.; Grey, N.; Ferlay, J.; Forman, D. The Lancet Oncology2012, 13, 790-801.(2) Fuchs, P.; Loeseken, C.; Schubert, J.K.; Miekisch, W. Int. J. Cancer2010, 126, 2663-2670.(3) Ebeler, S.E.; Clifford, A.J.; Shibamoto, T. J. Chromatogr. B. Biomed. Appl.1997, 702, 211-215.(4) Righettoni, M.; Tricoli, A.; Gass, S.; Schmid, A.; Amann, A.; Pratsinis, S.E. Anal. Chim. Acta2012, 738, 69-75.(5) De Lacy Costello, B.; Amann, A.; Al-Kateb, H.; Flynn, C.; Filipiak, W.; Khalid, T.; Osborne, D.; Ratcliffe, N.M. J. Breath Res.2014, 8, 014001 (29 pp.)(6) Di Natale, C.; Paolesse, R.; Martinelli, E.; Capuano, R. Anal. Chim. Acta2014, 824, 1-17.
Tumor suppressor genes inhibit cancer development and oppose oncogene function providing a physiological balance for the regulation of cell proliferation. Due to random stochastic events, the levels of tumor suppressor proteins (TSP) may fluctuate to very low levels even when one of the alleles remains functional giving the cells an evolutionary advantage to lock them in the low-TSP-expression states over multiple generations. As a result, the cells constantly experience a condition similar to complete deletion/mutation of both TSG alleles. To test this hypothesis, we need to follow the same individual cancer cells over multiple divisions and simultaneously monitor the fluctuation of tumor suppressor protein expression, which is challenging at the population level. We have developed a novel high-throughput single-cell capture, culture, and perfusion microfluidic system to correlate cell proliferation and the level of a given tumor suppressor protein for thousands of single cells. The capture chambers (length 60 um x width 20 um x depth 20 um) provide sufficient space for cell division in the x-plane while communication channels between each chamber and the inlet/outlet will provide continuous nutrient and gas perfusion. Passive perfusion will allow the transportation of cytokines and other factors promoting an environment mimicking growth on culture flasks preferred by adherent cell types. Gravity driven fluid transport in-between the inlet/outlet of the microfluidic network will be modified using height adjustment of the media reservoir since shear force exerted by fluid flow has shown to influence cell attachment and growth. By combining this cell segregation ability with other powerful analytics such as live cell protein level tracking and in situ hybridization, we will be able to correlate the fitness of individual cells and their progenies, and correlate with the level of TSP in real time. Using this platform, we can answer whether or not the initial neoplastic (uncontrolled growth) transition can indeed occur even when the TSG are not completely deleted generating new insight for cancer prevention and control.
As interests in the application of noble metal nanoparticles, especially gold nanoparticles, in medical theranostics rapidly expand in many cutting-edge areas, there is an increasing need for the development of effective strategies for the fabrication of nanomaterials and the detection of the biomolecules. One important strategy is to establish an effective plasmonic coupling of gold-based nanoparticles for an enhanced detection of proteins or biomarkers associated with cancer. This report describes recent findings of an investigation of gold-based nanoparticles to construct a microfluidic platform for the detection of proteins or DNA in the biomolecular recognition process. In addition to building a well-defined gold-based nanoparticle array on a substrate, one important emphasis is the understanding of molecular and biomolecular interactions at the interfaces of the array, which creates “hot-spots” for surface enhanced Raman scattering of reporters labeled on the nanoparticles through controlled small aggregates via specific protein binding activities. Examples of gold-based nanoprobes conjugated with biomolecules such as proteins and DNA sequences linked to the regulatory region of a cell cycle gene will be discussed.
Epigenetic alterations involving DNA methylation are early and frequently observed events in carcinogenesis. Hypermethylation is reported to be associated with cancers of the prostate, colon, lung, liver, breast head and neck, and further correlated with metastatic potential in many other tumor types. Thus, methylation analysis in DNA can play a critical role in the diagnosis of cancer, especially at an early, pre-cancerous stage. A simple, rapid, and reliable method to detect epigenetic modification of DNA, which uses small samples and eliminates bisulfite treatment and PCR amplification, has potential to impact cancer diagnostics. We drilled single nanopores in free standing 10nm thick SiN membranes supported on Silicon substrate. The nanopore measurements were performed in 1M KCl at pH 7.6 or in 0.2 M NaCl at pH 7.6 containing 10mM Tris and 1mM EDTA. The methylated-DNA/MBP complexes were prepared and incubated for 15 minutes at room temperature (25 ± 2 °C) immediately before nanopore experiment. Nanopore current traces were recorded using Axopatch 200B and Digidata 1440A at 10 kHz built-in low pass Bessel filter and 10 µs sampling rates. Instrumental control and data analysis was performed using Clampex 10.2 and Clampfit 10.2. We demonstrate the detection of methylation in 30, 60, and 90 bp double stranded oligos. Hypermethylated DNA (hyMethDNA) were selectively labeled using methylation specific proteins (MBP), and can be detected without the need for any further processes, such as bisulfite conversion, tagging with fluorescent agent, or sequencing. Discrimination of hyMethDNA fully bound with MBP mixed with unmethylated DNA (unMethDNA) revealed through nanopore ionic signatures of current blockage and duration. Specifically at 300 mV, 90 bp hyMethDNA/MBP was discriminated from 90 bp unMethDNA by a 6.5-fold difference in current blocking and a 23-fold difference in transport duration. This approach could be compatible with small amounts of genomic extracts and direct methylation detection without fluorescence-labeling and bisulfite-conversion. However, nanopore-based methylation assay should improve efficiency for low sample volume obtained from body fluids.
Our research program is broadly directed at assay development for post-translational modifications (PTMs), with a focus on protein phosphorylation by tyrosine kinases. Protein tyrosine kinases play key roles in disease and are particularly important in cancer: mutations in several protein tyrosine kinase genes have been identified as drivers of many tumor types and drugs targeted at inhibiting these enzymes represent ~20% (>$9 billion) of the current oncology market. We use a “decoy” substrate biosensor approach in which an artificial, optimized substrate peptide is designed to report the activity of a specific enzyme in living cells. Delivery is achieved using cell-penetrating peptides, and enzymatic modification is measured using a range of readout strategies—some that require extraction of the cell contents and some that leave the cell intact. Targeting the function of the enzyme in its intracellular environment preserves protein-protein interactions, localization, and scaffolding-dependent activation, and decoy substrates provide a snapshot of enzymatic activity that circumvents the need for pre-knowledge of every endogenous substrate site. We also develop multiplex-compatible readouts, so we can use a suite of biosensors for different enzymes in order to profile pathways. We have established our approach and laid the groundwork of a substrate development workflow to expand our repertoire of biosensors for kinases and other enzymes. Long term, our lab will maintain a pipeline of biosensor and read-out technology development while also taking an active role in studying signaling biology with our tools.
The development of electrical techniques for monitoring of biological phenomena is a field that is fast gathering pace. Advantages of electrical techniques are manifold, including the fact that they are label-free, and have the potential to be very efficient transducers, since the signal measured is already in an electrical readout format. Electronic methods for live-cell sensing can be applied to applications involving extracellular recording of electrical activity from electrically active cells (neurons/myocytes), but also for monitoring of non-electrically active cells and tissue assemblies. Electrical impedance sensing (EIS) has emerged as a dynamic method, with demonstrated potential for use in monitoring barrier function, cellular adhesion, proliferation, micro motion, and wound healing many of which are processes that are fundamentally altered in cancer. Although significant progress has been made in developing systems for electrical monitoring of cells in 2D, the transition to 3D cell models, which has been shown repeatedly to be necessary for representative cell models of cancer, presents many challenges. The advent of organic bioelectronics has the potential to meet these challenges. Organic bioelectronics refers to the coupling of conducting polymer based devices with biological systems, proven repeatedly in the last decade to provide numerous advantages to a wide variety of biomedical applications in terms of sensitivity, specificity and most importantly, bridging of the biotic/abiotic interface. Flexibility in design, patterning and processing means that the materials are amenable for integration into novel device formats, compatible with complex 3D cell culture models. Two key examples will be used to show the literal flexibility of organic electronic materials and devices: 1. The adaptation of an organic electronic device for monitoring of 3D cysts and spheroids, and 2. The integration of an ‘organ on chip&’ cell model with microfluidics and embedded electronics. In both cases the device format is adapted to suit the 3D format of the cell model in question. Continuous electronic monitoring provides a very useful dynamic parameter to assess cell growth and health, while the optical transparency of the polymeric materials allows continuous optical monitoring.
The radioisotope palladium (103Pd; 20kV photons; 17-days half-life), encapsulated in millimetre-size seed implants, is widely used in prostate cancer brachytherapy (internal radiotherapy). A new strategy under development to replace millimetre-size implants, consist in injecting radioactive nanoparticles (NPs) in the affected tissues. In particular, the interaction of low-energy 103Pd photons (20 keV) with gold (Au), produce a large number of photoelectric events, that strongly enhance the dose to tumors (the "radiosensitization effect"). Therefore, injections of 103Pd-containingAu NPs could represent an alternative to millimetre-sized implants, which are associated to discomfort and dose heterogenities. However, the production of 103Pd-containing Au NPs requires the development of NP synthesis procedures that are a) highly efficient (to minimise radioactive waste), b) rapid (to minimise the exposure of manipulators), c) performed in water (to avoid tedious ligand exchange), d) precise (Pd/Au ratios, size distribution and core-shell characteristics), and e) performed with biocompatible molecules. In this study, an original NP synthesis procedure allowing the production of 103Pd@Au NPs, was explored for optimal efficiencies, production rates and volumes, core-shell characteristics, and overall 103Pd radioisotope encapsulation efficiency.1,2 First, ultra-small cores of Pd were synthesized by using an original and direct reduction route in water (NP measured by high-resolution TEM and dynamic light scattering - DLS), using dimercaptosuccinic acid (DMSA) as a surface ligand and ascorbic acid as a reductor. Comparisons were made between non-radioactive and radioactive Pd chloride solutions. The synthesis of Pd cores (sim;10 nm core size) was found to be highly susceptible to pH, with synthesis efficiencies as high as 87% in optimised conditions. Then, Pd NPs were purified from the non-reacted synthesis products, and used as seeds for the growth of gold (Au) coatings, followed by steric stabilisation by polyethylene-glycol (thiol reaction). Thus-generated Pd@Au and 103Pd@Au core-shell nanoparticles (sim; 30 nm) were analysed in high-resolution TEM (particle size distribution, core-shell characteristics by EDX mapping), XPS, FTIR, UV-Visible and DLS. The imaging properties of radioactive nanoparticles were assessed in computed tomography (CT; attenuation by high-Z elements) and single-photon emission computed tomography (SPECT; radioactive emissions from 103Pd; for in vivo tracking of the 103Pd@Au NPs). Overall, this study confirms the conditions allowing optimal production of 103Pd@Au radioactive nanoparticles for more precise, uniform, and less invasive brachytherapy procedures.1. Djoumessi, Laprise-Pelletier, Fortin et al, RSC J. Mater. Chem. B, (2015), 3, 2192-2205.2. Laprise-Pelletier, Fortin et al; IUPESM World Congress (WC) on Medical Physics (MP) and Biomedical Engineering (BME); Toronto June 10, 2015.
Gold (Au) nanostructures as a multifaceted nanoplatform have shown their versatility in the fields of biomedical, bionanoscience and material science applications. Based on their unique optical properties they are well explored for several applications including live cellular- imaging, therapy and optoelectronics. Au nanoparticles of various shapes are capable of displaying Localized Surface Plasmon Resonance (LSPR) in the near- infrared region spanning to a range of 600- 1200 nm . Au based nanoparticles are profoundly used for cancer therapeutic applications as they are less cytotoxic and highly biocompatible in nature. Here, we demonstrate the synthesis of a gold- silver nanocage based theragnostic nanoparticles passivated with extremophilic polysaccharide, functionalized with antibodies for combinatorial therapy of breast cancer. Silver (Ag) nanocubes were made as sacrificial templates for the preparation of Au nanocages by employing Galvanic Replacement (GR) reaction; characterization was performed using JEM-ARM200F Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM). On replacement of Ag by Au, several random holes were appeared on the edges and faces of the nanocubes. Statistical analysis revealed a dominance of 220-240 nm sized Au nanocages with a small population of cuboid shaped particles. 3D tomographic analysis of nanoparticles revealed the overall structure of nanoparticle with encapsulation of the drug and the functionalizing moieties; using computational analysis we have analyzed the plausible locations of nanoparticle adsorption and accumulation in the cells. The formulation would be a pivotal step in the combinatorial therapeutic models for breast cancer treatment.References S. Jain, D. G. Hirst, J. M. O&’Sullivan, Br. J. Radiol.2012, 85, 101. X. Xia, Y. Xia, Front. Phys.2014, 9, 378. J. P. M. Almeida, E. R. Figueroa, R. A. Drezek, Nanomedicine Nanotechnology, Biol. Med.2014, 10, 503.
With the recent advances in nanomedicine, there is an increasing expectation for the development of multifunctional nanostructures combining specificity with diagnostic and therapeutic properties. These so-called theranostic materials represent the state-of-the-art in the development of nanoscale-based materials for fighting cancer. Gold nanorods (AuNRs) have found promising applications in medicine, mainly because of the two surface plasmon resonance (SPR) bands resulting from the coherent motion of the conduction band electrons along the short (visible region) and long axes (near infrared region) of the particle. The absorption in the near infrared region makes them appropriate for in vivo photothermal applications due to the maximum radiation penetration through tissue. However, a major obstacle related to the use of AuNRs is related to their low blood circulation time and, consequently, their low accumulation in the tumor. To overcome these challenges, we developed a nanosystem comprising cell membrane-coated AuNRS, which have been synthetized by colloidal seed-mediated, surfactant-assisted approach, followed by coating with human lung adenocarcinoma epithelial cell (A549) membrane. The nanoconjugates presented higher toxicity to cancer cells compared to healthy fibroblasts. The incorporation of gold nanorods into real membrane monolayers was also studied using Langmuir techniques via kinetics absorption and surface pressure measurements and revealed significant differences on how the AuNRs interact with the cell membranes depending on the size of the gold nanorods, indicating that the lipids present in the covering membrane exerted high influence on the uptake process. These results revealed the potential of cell membrane-coated nanomaterials and open opportunities for the development of more efficient nanosystems for cancer applications.
This paper presents the results of an experimental study of uniquely biosynthesized magnetite nanoparticles (BMNPs) using Magnetospirillum magnetotacticum bacteria. The BMNPs were functionalized by conjugation to luteinizing hormone releasing hormone (LHRH), a molecular recognition unit (MRU) with chemically synthesized nanoparticles (CMNPs) as control. The resulting nanoparticle structure, morphology and characteristics properties were examined using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Raman Spectroscopy, Fourier Transform Infrared (FTIR) Spectroscopy as well as quantitative image analysis. Insights on the adhesive forces of the functionalized magnetite nanoparticles needed to overcome the hydrodynamic and shear forces to target the breast cancer cells were explored. The adhesion forces between BMNPs and human breast cancer cells (MDA-MB-231 cell line) for improved selectivity and specificity were demonstrated using the Atomic Force Microscope (AFM). The BMNPs constituents had adhesion forces to breast cancer cells that were greater than those of CMNPs. The implications of the results are very useful for the development of nano-targets and magnetite nanoparticles for the detection and treatment of breast cancer.
Polymeric nanoparticle (NP) drug delivery systems possess several advantages over conventional small molecule chemotherapeutics. Among these is the ability to provide controlled and sustained drug release, specific drug targeting and delivery, and high loading of insoluble agents such as paclitaxel (Pax). Pax is one of the most widely used chemotherapeutic agents for a variety of solid organ malignancies (lung, ovarian, breast, head and neck cancers, and advanced forms of Kaposi&’s sarcoma). However, despite its widespread use, Pax suffers from poor solubility, rapid systemic clearance, limited tumor exposure, and low target tissue concentrations (~0.4% of the systemically administered dose). Due to its poor aqueous solubility, Pax is often delivered in a Cremophor EL (C/E) adjuvant; and C/E itself is known to cause adverse side-effects and hypersensitivity reactions. We have engineered a novel poly(1,2-glycerol carbonate)-graft-succinic-acid-paclitaxel (PGC-Pax) NP system in which Pax can be incorporated at high loadings (>60 wt%). Additionally, the polymer backbone is readily degradable and biocompatible, with glycerol, succinic acid, and carbon dioxide as the degradation byproducts. We herein demonstrate the synthesis and characterization of PGC-Pax NPs with tunable release kinetics, in vitro cytotoxic activity, and in vivo efficacy in a small animal cancer model. We also show the cellular internalization of rhodamine labeled PGC-Pax (PGC-Pax-Rho) NPs via flow cytometric analysis and confocal microscopy.
Three dimensional culture systems are enabling greater insights into tumor biology and angiogenesis in in vitro studies. Many biological processes are clearly different when cells are cultured in 3D systems rather than on 2D surfaces. The majority of 3D culture materials utilized have been extracellular matrix proteins. These mimic many aspects of in vivo biology, but offer little ability to control mechanics or biochemical interactions. We have been working with synthetic hydrogels based on photocrosslinkable poly(ethylenegylcol) diacrylate derivatives that are modified with proteolytically degradable peptides to enable cell-mediated material degradation and with integrin ligand peptides to allow cell adhesion. The mechanical properties of the hydrogels can be tuned over a physiologically relevant range by adjusting either the molecular weight between crosslinks or the polymer concentration in the hydrogel network. Since polyethylene glycol is quite bioinert, cellular interactions are largely limited to the peptides covalently attached to the polymers, allowing one to determine the types and extents of interactions through material design. Examining responses of lung carcinoma cells, we have observed profound impacts of both hydrogel stiffness and [RGDS] adhesion peptide. Tumor spheroid diameters and cell proliferation were greatest in the softest hydrogel materials and in hydrogels with the lowest concentrations of immobilized RGDS. Additional studies have included co-cultures of lung carcinoma cells with endothelial cells and mural cells to study tumor angiogenesis. The lung carcinoma cells utilized have been found to secrete significant levels of angiogenic factors such as VEGF, bFGF and PDGF. We have observed rapid morphological changes and increased invasiveness of tumor spheroids immediately upon contact with a microvessel, and direct contact appears to be required for these changes to occur.
Large genomic structural variations (SV, > 1 kb), known to be associated with complex traits and diseases, are found more prevalent than we previously thought. In spite of advancement in high-throughput short read next-generation sequencing in the past decade, a fair portion of the human genome architecture remains unresolved or ambiguously characterized as gaps and unknown structural or heterozygous information as the “dark matter” of the genome. Rapid whole genome mapping in nanochannel arrays represents a new standard of single-molecule platform independent of yet complementary to DNA sequencing for accurate genome assembly and structural variation analysis. Extremely long intact DNA molecules of hundreds to thousands of kilobases fluorescently labeled at sequence motifs and linearized in true nanofluidic channels enable direct image interrogation of comprehensive genome architecture at a high resolution. De novo assembly of these single molecules yields unprecedented long contiguous genome maps, advantageous in spanning over highly repetitive regions and complex structures in their native form.We present here results from analysis on human and cancer genome, non-model and large complex genomes. Multiple methods were shown to be employed, by de novo genome mapping process, or via direct alignment of the long raw molecules against digitally digested and “barcoded”reference genome for detecting chromosomal abnormalities such as translocations, arm breakpoints or other lesions. Ultra long molecules used in irys system could be used to bridge unknown long distance SV event, such as chromothripsis that often incorporating fragmented chromosomesarms pulverized during cancer transformation. We detected hundreds of large structural variants per genome and haplotype differences in these genomes, revealing the locations, orientations and copy numbers of these complex structural variants that are biologically and clinically relevant; especially able to precisely map viral component integration sites within host/human genome, believed to be linked to genome instability and oncogenesis. For the first time, population scale comparative whole genome study to identify comprehensive genomic structural variation on a single platform with a large patient cohorts is feasible due to the standardized high quality data, automated efficiency of data generation and low cost enabled by nanochannel technology.Comprehensive genome mapping provides extreme valuable structural information otherwise hard or impossible to decipher with short read sequencing data alone, and paves the road for generating true golden standard medical grade personalized genome information.
As of last count, there are about 100 risk factors for breast cancer. Some of these risk factors are genetic, such as mutations in the BRCA1 and 2 genes. Other risk factors are based on bulk tissue characteristics such as the degree to which the tissue attenuates x-rays ("mammographic density"). Finally, risk and outcomes are also correlated with tissue mechanics and specific micro-anatomical features, such as collagen lines or tracts that extend radially outwards from the tumor-stromal interface. Despite significant progress in discovering individual risk factors, it is not understood if and how these risk factors interact. We have developed a simple biological model system for studying the interplay of genes, mechanics, and geometry during transition to invasive phenotypes. In this model system, we grow mammary acini in 3D culture and we then place these acini on different substrates such as collagen 1 gels. We have found that in certain specific conditions, pairs or groups of Ras-transformed mammary acini can generate collagen lines that then coordinate and accelerate transition to an invasive phenotype. Overall, we find that pairs or groups of mammary acini can interact mechanically over mm-scale distances through the collagen matrix and that these directed mechanical interactions are necessary for rapid transition to an invasive phenotype. Most recently, we have been varying additional biological control parameters such as freely diffusing signaling molecules and we have been quantifying intracellular signaling with genome edited cell lines. In my talk, I&’ll brief you on the broader biological context and also on some of the control and imaging tools we have found to be useful for mechanobiology.
It has become evident that tumor cells are responsive to mechanical forces in vivo, which prove critical for tumor cell proliferation and death. Recent work has shown that tumor cells exposed to fluid shear forces can exhibit increased receptor-mediated signaling. Here, we show that biocompatible, polymeric micro- and nanoparticles conjugated to the tumor cell surface via free amine coupling act as mechanical amplifiers in presence of fluid shear forces to increase mechanotransduction, and can be exploited to increase therapeutic efficacy of the TNF-related apoptosis-inducing ligand (TRAIL) both in vitro and in vivo in mice. NHS crosslinker chemistry was used to conjugate polymeric particles to the tumor cell surface, without affecting cell viability or the ability for TRAIL to interact with tumor cell surface receptors. In the presence of fluid shear forces, polymeric particles mechanically amplified the therapeutic affect of TRAIL, as evidenced by increased receptor-mediated apoptosis and decreased tumor cell viability. Additionally, amplification of TRAIL-mediated apoptosis increased with increased particle size, suggesting that increased force exerted on the cell surface with larger particles amplified the therapeutic response. Annexin-V apoptosis assays showed that conjugation of polymeric particles to the cell surface nearly doubled tumor cell apoptosis in the presence of TRAIL under shear forces, and inhibition assays revealed the response to be caspase-dependent apoptosis. Conjugation of particles to the tumor cell surface in vivo increased therapeutic efficacy in a mouse model of lung metastasis, evidenced by decreased circulating tumor cells (CTCs) in the bloodstream and reduced tumor burden within mouse lungs. Together, these data demonstrate that polymeric particles, both degradable and non-degradable, act as mechanical amplifiers of mechanotransduction and TRAIL-mediated apoptosis in the presence of shear forces. Clinically, this approach shows that modulation of mechanical force applied to the tumor cell surface can increase sensitivity to therapeutic ligands.
Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the aberrant proliferation and differentiation of myeloid lineages. The patients with MPN have significantly elevated levels of pro-inflammatory cytokines in blood, and the JAK inhibitor therapy is associated with reduction of systemic cytokine levels. However, the mechanisms by which bone marrow cells with aberrant cytokine production contribute to or even drive MPN pathogenesis and how JAK inhibition act on the hematopoietic cells to achieve therapeutic benefit remain largely unknown due in part to the complexity of all hematopoietic cell lineages affected by MPN, thus requiring single cell resolution measurement of cytokine functions. Here, we developed a novel microfluidic single-cell cytokine profiling technology to characterize the cytokine profiles of various hematopoietic cell types from MPLW515L-derived MPN mice as well as patients with MPN to elucidate the roles of cytokine production in pathogenesis. We optimized the microchip to enable multiplexed measurement of a large panel of cytokines (up to 42) from over 1,000 human and murine hematopoietic cells at the single-cell resolution. Our data showed bone marrow cells from the myelofibrosis mice exhibit aberrant cytokine programs characterized by not only elevated levels of pro-inflammatory cytokine secretion but also increased diversity/multifunctionality of cytokines co-secreted by the same cells, which is a more profound distinction between disease and healthy control. Measuring sorted cell populations with or without activating mutations and from specific differentiation lineages further revealed that both malignant (with mutations) and non-malignant hematopoietic cells contribute to aberrant cytokine production that drive pathogenesis and that JAK inhibition for both populations is required to achieve clinical efficacy. In this work, our technology served as an enabling tool to study the cellular mechanism in the complex bone marrow compartment driving MPN pathogenesis. It also has the potential to discover and measure new biomarkers for detecting or monitoring MPN development and therapeutic response.
Second window near-infrared (NIR-II) imaging provides fluorescence-based anatomical information deep to skin as NIR-II light (1000 nm to 1700 nm) features deep tissue penetration, reduced tissue scattering, and negligible tissue autofluorescence. NIR-II fluorescence imaging probes; including down-conversion particles, quantum dots, single-walled nanotubes, and organic dyes, are formulated in biocompatible layer-by-layer (LbL) nanoparticles. 150 - 200 nm size range is obtained and hyaluronic acid is used as outer layer for extended blood circulation. Systemic delivery of LbL nanoparticles in Balb/c mice demonstrates that down-conversion particles have least autofluorescence due to its relative longer emission wavelength (>1500 nm). Organic dyes with emission at 1100 nm show most autofluorescence and elevated tissue scattering. Both quantums dots and single-walled nanotubes give moderate autofluorescence and tissue scattering. Pharmacokinetics studies of the LbL probes indicat!e that layer-by-layer formulation extends the blood circulation with half-life from 14 ~ 23 hrs. Tissue histology suggests none of the LbL probes present major toxicity. A further imaging involving orthotopic ovarian tumor is conducted using down-conversion particles due to its relative low autofluorescence. At 96 hrs, tumor is visualized in deep intraparatoneal cavity with high resolution. In this work, we present a first example of head-to-head comparison of all current NIR-II probes using LbL formulation. In vivo imaging performance is evaluated and suggests NIR-II imaging is versatile and robust technique for biomedical applications.
Nanotechnology has been providing novel, paradigm shifting solutions to medical problems and to cancer, in particular. In order to further these research goals, NCI formed a program called Alliance for Nanotechnology in Cancer which was initiated in 2004. The Alliance funds Centers of Cancer Nanotechnology Excellence, the development of nanotechnology platforms, and two training programs: Cancer Nanotechnology Training Centers and Path to Independence Awards. An intramural arm of the Alliance - Nanotechnology Characterization Laboratory provides a characterization support to evaluate clinically promising nanomaterials and establish their physical, pharmacological and toxicological characteristics.In this presentation I will discuss a current status of cancer nanotechnology efforts funded by the program and also describe future opportunities and strategies in this field. Further progress is likely to follow two parallel tracks. First one will be associated with on-going translation to the clinical environment; while the second with the development of new tools and techniques in research arena. It is expected that small molecule drugs in nanoparticle-based formulations currently undergoing clinical trials will be joint by other modes of therapy including siRNAs, kinase inhibitors, and others. Active targeting, when appropriate will be used more frequently. In order to make translational efforts more wide spread, access to reliable GLP characterization and GMP manufacturing facilities will need to become more available.Imaging techniques based on nanoparticles will be designed to operate in multi-functional manner; whether it is ability to probe and monitor tumor microenvironment in addition to imaging tumor mass itself, capability of multi-modality imaging, or use of theranostic functions of diagnosis and subsequent treatment. The use of nanotechnologies in intra-operative imaging to guide real time surgery is also expected to expand. In vitro diagnostic devices have matured to a stage in which the development of additional device modalities with new transduction methods does not seem necessary. These devices will be however, increasingly used to collect data for sophisticated multi-parameter analysis allowing to correlate levels of different biomarkers, optimize reliable panels which are required to determine presence of the disease, and determine response of individual patients to different modes of therapies.
Exosomes show potential for cancer and immune diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires substantial sample purification and labeling. We have developed a number of different technologies to purify and analyze exosomes with a particular interest in clinical translation. For example, I will describe a label-free, high-throughput approach for nano-plasmonic exosome (nPLEX) analysis. This assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. Together with other approaches to measure exosomal mRNA (IMER) and single exosome techniques we are now able to ask important clinical questions. I will present data on ovarian, pancreatic and brain cancer where extensive profiling has been done.
Proper function of RNA is critical to the health and maintenance of a cell, and its misregulation plays a critical role in the development of many disorders. Despite this, the ability to study RNA has been severely limited. Many analytical techniques are only capable of quantifying expression levels of transcripts (e.g., PCR and microarray), and do not offer insight into the dynamics of RNA transport or localization. Recently, the Mirkin group has developed a novel nanoconjugate, termed the “Stickyflare”, capable of reporting on both of these critical components in live cells, with the intent to enable a more complete picture of RNA function than any other analytical techniques to date.The Stickyflare, based on the spherical nucleic acid (SNA) platform, consists of a 13nm gold nanoparticle core densely functionalized with DNA oligonucleotides that are designed to be the same sequence as a unique portion of the target transcript. A longer, fluorophore labeled “flare” oligonucleotide is then hybridized to this sequence via complementary base pairing, such that the fluorophore label is antisense to the target RNA. While bound, the fluorophore is held in close proximity to the gold, which acts as an efficient quencher of fluorescence. However, upon interacting with a fully complementary target, the flare forms a longer, more stable duplex with the target and is removed from the gold surface. This displacement from the gold surface results in a quantifiable turn-on of fluorescence, and the “tagging” of the RNA transcript, which can then be tracked in real-time via fluorescence microscopy. Importantly, the Stickyflares enter live cells without the need for harmful transfection agents, quantify target RNA expression with single cell resolution, and allow for real-time analysis of the transport and localization of endogenous RNA. We believe this nanoconjugate will be valuable for studying the function of endogenous RNA in healthy and diseased cells, as well as offer insight into how a change of environment (i.e., drug treatments, hypoxia, starvation, etc.) affect the dynamics of RNA expression.
Circulating tumor cells (CTCs), shed from a primary tumor into the bloodstream, are putative diagnostic/prognostic biomarkers that contain actionable genetic information for tumor diagnosis and treatment. The rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and elaborate downstream molecular analysis. Genetic data extraction from CTCs currently suffers from a lack of reliable analytical methods capable of handling typically low numbers of CTCs which are harvested from cancer patients, esp. early stage patients who have a better chance of cure. Additionally, advanced stage cancer patients can be treated more effectively if tumor mutational analysis can be performed with peripheral blood samples instead of tumor tissue samples. These unmet needs require developing new diagnostic platforms that can either detect cancer at an early stage or monitor tumor progression based on molecular analysis of CTCs or other blood based biomarkers.We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) increased capture efficiency at high flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (~200 pores/mm2), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). These massive single-cell arrays are able to isolate up to thousands of single lung cancer cells to measure gene expression at a given timepoint and to observe the time course of single cancer cells.Our pilot data of lung cancer patients versus control samples indicate that the nanotechnologies based on the MagSifter and Nanowell devices are very promising for obtaining single CTC molecular profiles which can be used to detect early and late stage lung cancers with excellent diagnostic accuracy. This work is supported in part by National Cancer Institute through the Stanford Center for Cancer Nanotechnology Excellence (U54CA151459) and grant R21CA185804.
Over the past several years we have been developing the single cell barcode chip (SCBC) platform for the quantitative analysis of a panel of functional proteins from statistical numbers of single cells, with applications associated with advanced immune monitoring, the analysis of phosphoprotein signaling pathways within heterogeneous tumors, as well as fundamental biological and biophysical studies. In this talk, I will discuss extending these types of platforms to include assay for panels of proteins and metabolites from the same single cells, or for the analysis of genes and proteins from same single cells. Much of this work is driven by specific biomedical challenges, which will also be discussed.For the analysis of heterogeneous tumors, we have concentrated on measuring metabolites that are associated with energy consumption (glucose, glutathione, glutaamine, etc.), together with panels of phosphoproteins associated with metabolic signaling, or associated with the signaling pathways that are hyperactivated for tumor maintenance and growth.For the analysis of tumor-infilatrating lymphocytes, we have concentrated on micro and nano technologies for sorting tumor-antigen specific tumor infiltrating lymphocytes according to antigen (and neoantigen) specificity, and for sequencing the T cell receptor gene a/b sequences from those individual cells, thus permitting a direct pairing of specific tumor antigens with specific T cell receptors.
We previously described shear sensitive nanoparticle aggregates (NPAs) that are capable of targeted disintegration at sites of vascular stenosis, which provide safer and more effective drug delivery in vascular occlusive diseases (1). Here, we show that this novel mechanically-activated drug delivery technology also can be activated by application of low-energy ultrasound (US) radiation to specifically deliver drugs and increase their concentration at tumor sites. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were loaded with the chemotherapy agent, doxorubicin, during their fabrication, were assembled into NPAs by spray drying. The NPA formulations were analyzed for their breakability (release of individual nanoparticles) in vitro with or without exposure to different levels of low intensity US (1 cm2 probe, 2.2 W/cm2, 3 MHz, 10 to 50% duty cycle) for 5 min (i.e., a time that corresponds to their clearance time in mice; 1). When the internalization dynamics of these US-activated NPAs in 4T1 breast cancer cells were evaluated in vitro, we observed greater internalization of the US-stimulated NPAs compared to unstimulated NPAs, leading to an equivalent internalization as that obtained by using the same concentration of free PLGA nanoparticles. The Dox-NPAs also produced cytotoxicity in the 4T1 cells, confirming that its efficacy was retained during the particle synthesis and the subsequent spray drying process. Finally, tumor growth over time was evaluated utilizing 4T1 breast cancer cells implanted into the mammary fat pad of BALB/c mice. These studies demonstrated a robust response to low dose doxorubicin using US-activated Dox-NPAs without any detectable systemic toxicity. This effect was significantly superior to the same dose of free doxorubicin or doxorubicin nanoparticles. Moreover, a 20-fold higher dose of soluble doxorubicin was required to produce a similar response, and this produced significant toxicity leading to death in 40% of the animals. Ultrasound sensitive-NPAs could therefore represent a novel targeted therapy for treatment of localized primary or metastatic cancers with greatly reduced toxic side effects.1. N. Korin, M. Kanapathipillai, B. D. Matthews, M. Crescente, A. Brill, T. Mammoto, K. Ghosh, S. Jurek, S. A. Bencherif, D. Bhatta, A. U. Coskun, C. L. Feldman, D. D. Wagner, D. E. Ingber, Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels. Science337, 738-742 (2012).
Introduction: Incomplete surgical removal of malignant tissue can result in recurrent disease, potential changes in treatment regimen, and ultimately poor patient prognosis. Technologies that indicate the presence of tumor remaining the patient, intraoperatively, could be of great benefit to decrease recurrent disease. For example, near infrared (NIR) fluorescent dyes like indocyanine green (ICG) can be detected by fluorescence image-guided surgery systems. We have previously reported the development of an image-guided surgery (IGS) system that detects near infrared fluorophores, e.g. ICG  and nanoparticle that entrap of ICG for image-guided surgery . Here, we compare NIR dye that is physically entrapped in a nanoparticle (derived from hyaluronic acid (HLA)) versus chemically conjugated in terms of self-assembly, optical properties, cytotoxicity, cell uptake, relative time-dependent biodistribution, and detection in an intraoperative imaging system.Materials and Methods: Hydrophobic ligand, either amino-propyl-pyrenebutanamide or amino-propyl-5-β-cholanamide was conjugated to HLA (10 or 100 kDa) at either 10 or 30wt%. ICG was loaded by first dialyzing in DMSO:H2O and then H2O, yielding NanoICG, whereas Cy7.5 was directly conjugated to amphiphilic HLA prior to self-assembly, yielding NanoCy7.5. Hydrodynamic diameter (HD) was measured using dynamic light scattering. Optical properties were determined by absorbance and fluorescence spectroscopy. Cytotoxicity was examined by a WST-8 assay. Nanoparticle uptake was determined either by fluorescence microscopy of flow cytometry. Using mice bearing MDA-MB-231 breast tumor xenografts, the impact on time-dependent contrast-enhancement was determined as a function of dye entrapment vs. conjugation, the structure and conjugation ratio of hydrophobic ligand, and molecular weight of HLA.Results and Discussion: Conjugation of each ligand drove NP self-assembly in aqueous solution. Average NP HD ranged from 90-150 nm and depended on the structure and conjugation ratio of the hydrophobic ligand. Exposure of cells to NanoICG, NanoCy7.5, or NP vehicle (no dye present) resulted in no decrease in metabolic activity. Uptake of all nanoparticles was decreased at 4°C compared to 37°C suggesting endocytotic uptake. Nanoparticle uptake in CD44 (receptor for HLA)-expressing cells could be inhibited by excess HLA, but only for nanoparticles composed of 100 kDa HLA. Overall, NanoCy7.5 conjugates were significantly brighter in vivo compared to NanoICG and showed significantly higher contrast-enhancement that increased over time. Using a breast phantom with optical properties consistent to human breast tissue, tumors harvested from mice and embedded in the phantom could be detected at greater than 5 mm in an image-guided surgery system.This research was supported by NIH grants R00 CA153916 and R01 EB019449 to AMM. Mohs AM, et al. Anal Chem 2010, 82, 9058-65. Hill TK, et al. Bioconjug Chem 2015, 26, 1416-24.
Current therapy for malignant brain tumors, such as glioblastoma multiforme (GBM), is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two advances in technology: 1) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery (CED); and 2) re-purposed, FDA-approved compounds, which were identified through library screening to target BCSCs. Using fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI), we demonstrate that brain-penetrating nanoparticles can be delivered intracranially to large volumes in both rat and pig. We identified several FDA-approved agents that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by CED, one of these agents dramatically increased survival in rats bearing BCSC-derived xenografts. This new approach to controlled delivery in the brain should have a significant impact on treatment of GBM and suggests new routes for drug and gene delivery to treat other diseases of the CNS.
Surgery, chemotherapy and radiation have been our central approaches to treating cancer. Indeed, in combination these approaches have made a significant difference to our ability to manage several cancers. Aggressive brain tumors however remain a clinical challenge. Our laboratory is exploring several approaches that may be classified as biomedical devices and/or engineered bacterial nanobots to increase the therapeutic repertoire of clinicians combating invasive gliomas. Specifically, I will discuss the design of a device that can move inoperable deep brain tumors from their inoperable locations to a more operable location. In a different, perhaps orthogonal approach, tumor seeking bacterial nanobots engineered to reach small, metastatic tumor nodules in the brain will also be discussed.
Introduction: Tumor pH has been shown to potentiate the effectiveness of chemotherapy . Clinicians require a means to repeatedly quantify the pH in one or more locations within a tumor to enable patient-specific cancer treatments based on tumor pH. Clinically available probe-based methods for measuring pH require reinsertion of the probe for each measurement which can lead to inaccuracies due to the spatial variability of tumor pH. Imaging-based methods are not currently suitable for repeated quantitative pH characterization.Hydrogel-based solid-state magnetic resonance imaging contrast agents enable quantitative pH measurements. These materials can be injected into tumors through a small bore needle, without the need for surgery, and avoid migration once injected. The contrast agents would enable repeated quantitative and non-invasive measurements over the duration of treatment. The tumor pH is measured using 1H magnetic resonance imaging which is widely available in clinical settings.Materials and Methods: Solid-state contrast agents were synthesized through a solution polymerization reaction. Monomers (hydroxyethyl methacrylate, acrylic acid, and dimethylaminoethyl methacrylate), cross-linker (methylenebisacrylamide or ethylene glycol dimethacrylate) and initiators (ammonium persulfate and tetramethylethylenediamine) were dissolved in deionized water. Cross-linked hydrogels were then soaked in buffer solution to modify the pH of the material for testing. Measurements were made using a Bruker minispec (0.5 T) and Varian magnetic resonance imager (7 T).Results and Discussion: The solid-state contrast agents utilize the chemical exchange process in which protons are rapidly exchanged between the contrast agent and the water molecules. The exchange rate is a pH dependent process that is catalyzed in both acidic and basic conditions. The properties of the solid-state contrast agents are designed to specifically have sensitivity in the physiologically relevant range of pH 6.0 to 7.4.Conclusions: Hydrogels tuned to control the pH dependence of chemical exchange serve as solid-state contrast agents for quantitative measurements of pH. These materials are imaged non-invasively using magnetic resonance imaging. Solid-state pH contrast agents will enable physicians to guide cancer therapy based on real-time feedback of the tumor microenvironment.Acknowledgements: This research is funded by the National Cancer Institute through the Centers of Cancer Nanotechnology Excellence (5U54CA151884-02).References:1: Kuin, A et al., Br J Cancer, 1999, 79 (5-6): 793-801.
Molecular trafficking within cells, tissues, and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ~15 µm. Here we present a new 3D tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 mu;s in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built upon spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for 3D particle tracking and allows for two-photon, multi-color imaging. 3D tracking of epidermal growth factor receptor (EGFR) complexes at a depth of ~100 µm in tumor spheroids is demonstrated.Our 3D tracking microscope is built upon spatiotemporally multiplexed two-photon excitation and uses time-gated analysis via a photon counting histogram to discern the molecular 3D position. Feedback control then steers the excitation to lock-on to the single molecule as it travels at a high speed. The molecular trajectories are reconstructed from the recorded actuator positions from the feedback control loop operating at 1-5 ms. Dynamics down to 50 µs can be inferred from analysis of the photon counting histogram. In our method, the first PMT channel is used for particle tracking while the second and the third PMT channels can be used for traditional two-photon scanning microscopy, colocalization analysis, and energy transfer studies. We have coined this technique TSUNAMI (Tracking Single particles Using Nonlinear And Multiplexed Illumination).By following a fixed bead moved in a prescribed pattern, we have verified the tracking accuracy of TSUNAMI to be 16 nm in XY, 35 nm in Z direction, with a sustained tracking speed limit around 6 µm/s. We have also tracked EGFRs tagged with fluorescent beads in A431 skin cancer spheroids (~100 µm thick) for up to 10 minutes. Three phases of the EGFR internalization process can be clearly differentiated in the acquired single-molecule trajectories. Our system will allow researchers to explore new questions in receptor transport and dynamic processes directly in 3D tissues.Reference:1. E. Perillo et al. "Deep and high-resolution 3D tracking of single particles using nonlinear and multiplexed illumination (TSUNAMI). Nature Communications, 2015, in press.
Tumor microenvironments display aberrant microstructure and materials properties, including increased fibril density, anisotropy and stiffness. These hierarchical nanoarchitectures display characteristic length scales ranging from tens of nanometers to tens of microns. Remarkably, modern nanomaterial fabrication techniques enable controlled topographies with sizes and geometries comparable to those observed in vivo. Here, we demonstrate a facile technique for large area patterning of aligned biomimetic topographies by wet deposition of graphene oxide on prestretched elastomeric substrates. The resulting 2.5D wrinkled features direct the morphology of fibroblasts and cancer cells towards increasingly polarized and invasive phenotypes. These features are unusually sharp and deviate from continuum theories, perhaps due to the extreme mechanical properties of graphene. We further use high content imaging to quantify single cell heterogeneity on these substrates. Overall, these novel cell culture surfaces may provide a more physiologically relevant biomaterial context to elucidate cancer cell biology.
Shortwave infrared light (SWIR: 950-1,400 nm) is promising for in vivo fluorescence imaging due to deep tissue penetration and minimal scattering losses compared with visible and near-infrared (NIR) light; fluorescence contrast agents detectable at SWIR wavelengths would offer excellent signal-to-noise performance and provide superior resolution and detection depth to existing clinically-used fluorophores. With their intrinsic fluorescence in the SWIR regime and lack of photobleaching, single-walled carbon nanotubes (SWNTs) are potentially attractive contrast agents to detect tumors. Here, we developed targeted M13 virus-stabilized SWNTs as fluorophores to visualize deep, disseminated ovarian tumors in animal models. M13 serves as a scaffold to stably disperse SWNTs while expressing peptides against SPARC biomarker overexpressed in particular ovarian cancers for targeted imaging. The nanoprobe demonstrates excellent tumor-to-background and exhibits higher normalized signal-to-noise performance compared with visible and NIR dyes for visualizing tumor nodules. Further, SWNT-based image guidance improved detection and excision of tumors by a gynecological surgeon and led to the identification of sub-millimeter tumors (post-analysis). Initial serum chemistry studies in preclinical models indicate no systemic acute toxicity after administration of the probe, which suggest these materials could be potentially translatable into the clinic with further chronic toxicity studies. We will also discuss considerations for additional SWIR fluorophores with potentially improved optical properties compared to existing SWNT-based fluorophores to improve detection. Collectively, these findings demonstrate the promise of targeted SWIR nanoprobes for noninvasive disease monitoring and guided surgical interventions.
Biomedical imaging is an integral part of clinical decision-making during screening, diagnosis, staging, therapy planning and guidance, treatment and real-time monitoring of patient response. The main clinical challenge, however, is to be able to image biological features with sufficient sensitivity for detection at the cellular level. The most promising technique for high-resolution deep-tissue whole body imaging, using relatively safe molecular probes and excitation sources, at a reasonably low cost, appears to be optical imaging.In recent years, there has been tremendous interest in the exploration of optical imaging in vivo, due to the development of near-infrared fluorescent probes, effective targeting agents, and custom-built imagers. Despite the recent development of various NIR-II fluorescent probes and custom built imagers based on InGaAs detectors, there are three significant challenges to imaging in the NIR-II wavelength domain with InGaAs detectors. The first challenge is the limited selection of fluorescent probes which emit in the NIR-II range favorable for medical imaging. The second challenge is to maximize the S/N ratio for high-sensitivity deep-tissue imaging. The third challenge is diffuse light scattering by heterogeneous turbid biological media, imposing trade-off between depth and resolution.In this work, we designed an optical system in NIR-II window that combines both hyperspectral and hyperdiffuse measuring. HSI resolves the first two challenges of SNR by providing information in frequency domain, and allows novel type of investigation (including pixel-wise spectral analysis and 3-D reconstruction) as well as improves result confidence. HDI resolves the third challenge, by not only excluding the emission scattering in the processed results, also presenting pixel-wise diffuse scattering information for contrast imaging and 3-D reconstruction. The hyperspectral and diffuse imaging system (900-1700 nm) has the capability to distinguish the optical signatures of the primary pump laser, background, various types of tissue autofluorescence, reporter fluorescence, as well as the diffuse scattering effect of the fluorescence signal upon transport through heterogeneous turbid optical media. Further by applying our imaging system to various tissue and whole-animal, we demonstrated that it is capable of (1) detecting 1 mm sized particles up to 9 cm depth through a breast-tissue mimic phantom; (2) identifying spectral and diffuse signature of various tissues; (3) reconstructing 3-D fluorescent images based on newly interpreted information from HSC and HDC. This study opens up exciting new possibilities for clinical translation of NIR-II imaging as a viable platform theranostic technology; for early diagnostics, as a real-time surgical assistance tool, and for monitoring patient response to therapies.
Positron emission tomography (PET) imaging has received special attention owing to its higher sensitivity, temporal resolution, and unlimited tissue penetration. The development of tracers that target specific molecules is therefore essential for PET procedures. However, 64Cu as a PET imaging agent generally has been introduced into biomaterials through macrocyclic chelators, which may lead to the misinterpretation of PET imaging results due to the detachment and transchelation of 64Cu in vivo. In this study, we have developed ultrasmall chelator-free radioactive [64Cu]Cu clusters using bovine serum albumin (BSA) as a scaffold for PET imaging in an orthotopic lung cancer model. We preconjugated the tumor target peptide luteinizing hormone releasing hormone (LHRH) to BSA molecules to prepare [64Cu]CuNC@BSA-LHRH. The prepared [64Cu]Cu clusters showed high radiolabeling stability, ultrasmall size, and rapid deposition and diffusion into tumor, as well as predominantly renal clearance. [64Cu]CuNC@BSA-LHRH showed 4 times higher tumor uptake compared with that of [64Cu]CuNC@BSA by analyzing the64Cu radioactivity of tissues via gamma counting. The PET imaging using [64Cu]Cu clusters as tracers showed more sensitive, accurate, and deep penetration imaging of orthotopic lung cancer in vivo compared with near-infrared fluorescence imaging. The clusters provide biomedical research tools for PET molecular imaging.
Chemotherapy is one of the major cancer therapies. To achieve effective chemotherapy, there are many efforts to develop new anticancer drugs. In spite of many efforts, it is difficult to select correct drugs from numerous candidates by in vitro screening because the chemoresistance decreases in in vitro culture of cancer cells. A various cell culture systems have been proposed to improve chemoresistance of cancer cells in vitro. Utilizing extracellular matrix (ECM) proteins is one of the useful approaches. ECM is composed of many proteins and carbohydrates and the signaling activated by these molecules are integrated to regulate cellular functions including chemoresistance. Therefore, it is expected that cell culture substratum mimicking in vivo ECM in tumor tissue highly increase cancer chemoresistance. In this study, we prepared "staged tumorigenesis-mimicking matrices" mimicking in vivo ECM in tumor tissue at different malignancy to increase chemoresistance in in vitro culture.Staged tumorigenesis-mimicking matrices were prepared by breast tumor and colorectal tumor cell lines possessing different malignancy. Breast tumor cell lines, MDA-MB-231 (metastatic), MCF-7 (non-metastatic), and MCF-10A (benign), and colorectal tumor cell lines, HT-29 (metastatic), SW480 (non-metastatic), and CCD-841-CoN (benign), were cultured on tissue culture polystyrene (TCPS), poly (2-methoxyethyl acrylate) (PMEA), and poly (tetrahydrofurfuryl acrylate) (PTHFA) for 2 weeks to form ECM beneath the cells. After the culture, the cells were specifically removed by decellularization treatment and remnant ECM was used as staged tumorigenesis-mimicking matrices. The cells were then reseeded on the matrices and were cultured for 1 day. After 1 day of the culture, the cells were exposed to 5-fluorouracil (5-FU) for 3 days, and cell growth was evaluated by WST-8 assay.The highest resistance of MDA-MB-231 and HT-29 against 5-FU was exhibited on the matrices derived from corresponding cells, suggesting that chemoresistance increased on only the ECM derived from original tissues. Compared the resistance of MCF-7 and SW480 against 5-FU, the highest resistance was exhibited on the matrices derived from MDA-MB-231 and HT-29, respectively. These results suggested that ECM derived from the cells with higher malignancy strongly increases the chemoresistance. Finally, we compared the chemoresistance on the matrices prepared on different initial substrata. The resistance against 5-FU increased on the matrices prepared on PTHFA as an initial substratum.In conclusion, we prepared staged tumorigenesis-mimicking matrices successfully. And the chemoresistance increased on the matrices derived from original tissues at higher malignancy. Moreover, we demonstrated that PTHFA is a suitable initial substratum to increase the chemoresistance. The results might provide the optimal condition to increase chemoresistance on cell-derived decellularized matrices.
In the United States, non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths. NSCLCs are sensitive to tyrosine kinase inhibitors (TKIs). However, sustained treatment of EGFR mutant tumors eventually leads to acquired resistance. Using an impedance based microchip assay, we measured the real time response to drug treatments. This device consists of an array of interdigitated microelectrodes that measure the electric impedance of adhered cells and enable real time monitoring of cell division and growth dynamics. We use a parental lung carcinoma cell line and two drug resistance derivative lines to study the gain/loss of drug resistance in NSCLC in correlation with the drug response of cell growth, which was measured via an ACEA RTCA system. We observed that drug resistant cells grow slower than the parental cells but do not show significant inhibition of growth upon drug treatment. In 1-2 days, the growth of drug resistant cells is further activated by the presence of TKI and surpasses the levels of untreated cells. Furthermore, by synchronizing cell cycles, we are able to observe the breakdown of growth rates in real time. We further performed the study of signaling in Pi3K-Akt-mTOR and MAPK pathways and identified a constant rebound of MAPK signaling that correlates with the inflection of proliferation behavior. Our results indicate a role for the signaling cascades downstream of tyrosine kinase receptor to dampen the therapeutic benefit of TKI treatment and a persistent response may require a combination treatment targeting on MAPK signaling to suppress the reaction of drug resistant cells.
Physical drug delivery methods provide an avenue to overcome the selectivity of the cell membrane via physical forces that disrupt cell membranes and drive drug molecules into the cytosol. A recently developed laser-mediated method of drug delivery has shown improved efficacy of intracellular delivery. When carbon black nanoparticles in suspension with cancer cells and small molecules are exposed to nanosecond-pulsed laser light, high uptake and cell viability are observed [1-3]. It has been hypothesized that the laser-carbon nanoparticle interaction causes thermal expansion and local vaporization that results in the release of acoustic waves into the surrounding medium. These combined energy transduction mechanisms, phenomena called transient nanoparticle energy transduction (TNET), are responsible for permeabilization of the cell membrane and the observed bioeffects of high viability and high drug uptake.The overall objective of this work is to investigate TNET and the bioeffects associated with physical disruption of cell membranes for drug delivery via laser-carbon nanoparticle interactions. Carbon nanoparticles with different thermal properties, geometries, and positive peak pressures are evaluated in vitro. By comparing the performance of carbon black nanoparticles, single-walled carbon nanotubes, and multi-walled carbon nanotubes, which have different aforementioned properties, we demonstrate that long-range effects of TNET, i.e. cumulative pressure output, is not a primary mode of energy transduction. The implications of size and surface area to volume ratio are also investigated via 13, 25, and 75 nm primary particle sizes of carbon black nanoparticles. Additionally, the short-range effects of heat transfer in TNET are studied by variation of the interparticle-cell spacing in vitro. A fundamental understanding of TNET will eventually allow us to harness such mechanisms to improve the therapeutic efficacy of this system and incorporate it with future in vivo and possible clinical applications for the treatment of cancer .References Chakravarty, P., Qian, W., El-Sayed, M. & Prausnitz, M. R. Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses. Nat. Nanotechnol. (2010). Sengupta, A., Kelly, S. C., Dwivedi, N., Thadhani, N. N. & Prausnitz, M. R. Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles. ACS Nano. (2014). Sengupta, A., Mezencev, R., McDonald, JF., & Prausnitz, M.R. Delivery of siRNA to ovarian cancer cells using laser-activated carbon nanoparticles, Nanomedicine. (in press).
In recent years, studies have found that selenium is capable of selectively targeting cells expressing mutated p53 or lacking p53. When delivered to the body in supplementary form, selenium has been shown to allow for higher tolerable doses of chemotherapeutic medications. Additionally, when coupled covalently to polyethylene glycol (PEG), a nanoparticle anti-aggregate, selenium nanoparticles are capable of inducing apoptosis in drug-resistant cancer cells through the destabilization of the mitochondrial membrane potential. Selenium nanoparticles have therefore been deemed a viable candidate for study of their effect on head and neck squamous cell carcinoma (HNSCC). Red selenium nanoparticles (rSeNPs) were made in the lab via the mixing of sodium selenite and glutathione in an alkali environment and were characterized to be 100 nm in diameter and with a zeta potential of -22.05mV. Viability assays were performed on human dermal fibroblasts (HDF) and head and neck squamous cell carcinoma (SCC-9) cell lines treated with varying concentrations of rSeNP. Results indicated a capacity for red selenium nanoparticles to induce apoptosis in HNSCC cells at minimum concentrations of 5 µg/mL after 3 days, while minimizing damage to surrounding healthy fibroblasts. The in vitro treatment of HNSCC with red selenium nanoparticles display promising effectiveness, while simultaneously minimizing cytotoxicity to dermal fibroblasts. Similar viability assays were performed using PEG as the treatment. Results showed that HDF experienced no cytotoxic effects from PEG, while HNSCC experienced mild cytoxicity. Further studies will be conducted to fully characterize the nanoparticles. The rSeNPs will then be functionalized with PEG to reduce the probability of agglomeration. These new particles will be characterized and their cytoxicity will be studied. Fluorescence microscropy will be used to study uptake and localization of the particles within the cells. Laser scanning cytometry will be used the study the effect of the selenium nanoparticles on intracellular superoxide generation, mitochondrial membrane potential destabilization, and cell stage proportions. Fischer, Joshua L, et al. Molecular Cancer Therapeutics, 2007, 6, 355 - 361. Zheng, Shanyuan, et al. International Journal of Nanomedicine, 2012, 7, 3939 - 3949.
Multifunctional nanomaterials are sought out in novel theranostic (therapy and diagnostic) applications. For example, plasmonic nanomaterials such as Au have the unique property of absorbing light and converting it into heat at high efficiency. This so-called photothermal effect can be used for thermal destruction of undesired cells (e.g. tumors). Furthermore, imaging techniques for early diagnosis, such as magnetic resonance imaging (MRI), require efficient contrast agents. Superparamagnetic iron oxides show desirable magnetic properties and are therefore promising. The combination of materials with different properties leads to advanced multifunctional systems that are attractive in an array of biomedical applications. Here, multifunctional high purity SiO2-coated Au/Fe2O3 nanoaggregates are produced via scalable flame spray pyrolysis. The application of this nanomaterial as MRI contrast agent is investigated.The potential of the flame-made high purity multifunctional system for photothermal tumor killing is analysed in vitro. A local destruction of cancerous cells due to the arising temperature increase during laser irradiation of the nanoparticles is reported. The applications of the multifunctional nanoparticles as photothermal agents is further investigated via MRI thermometry. The NIR-laser irradiation of the material within the bore of a MRI allows the in-situ temperature detection via phase changes in the recorded signal. This presents the simultaneous diagnostic and therapeutic applications of the flame-made SiO2-coated Au/Fe2O3 nanoparticles and proofs their promising multifunctional properties for biomedical applications.
During the last decade, intensive research in the field of nanoparticles enabled the development of nanoprobes with desired physicochemical properties which show promising characteristics in terms of cancer diagnostics and therapy. However, in order to guarantee appropriate treatment adapted to the patient&’s conditions, optimal visualization of the malignant tissue is necessary. Several imaging methods are commonly used for the localization and quantification of tumor cells in vivo and all of them comprise characteristic sensitivities and resolutions. In addition, combinations of multiple imaging methods are expected to lead to improved imaging capabilities and hence result in a faster and better understanding of the malignant side by revealing cellular processes and metabolic mechanisms which finally facilitate a diagnosis.Herein we report the simultaneous employment of magnetic resonance imaging and positron emission tomography by using iron oxide nanoparticles coupled to a radionuclide. Therefore, bimodal nanoparticle-based imaging platforms were produced starting from the preparation of magnetite nanoparticles via a one-pot solvothermal process using catechol derivatives as capping agents followed by a green and efficient click chemistry approach. This was enabled by the attachment of terminal azide functionalities at the particle surface which were then labeled by a click reaction to 18F as positron emitting source.In a second approach, radionuclides were incorporated by using a chelating ligand such as DOTA. In this case, 68Ga can be used for diagnostic purposes, while 177Lu, due to its long half-life, can also be employed for therapy. In general, all of the as-prepared crystalline magnetic nanoparticles show promising properties for bimodal cancer diagnosis and may additionally be used for hyperthermia treatment.
Supramolecular hydrogel based peptides by self-assembly have attracted extensive research interests in recent years, which have shown big potential in three dimensional (3D) cell culture, controllable delivery of therapeutic agents, immune boosting, sensing, and regenerative medicine. Therefore, we designed a series of small molecule to make them applied in the field of drug delivery. At first, we developed a molecular hydrogel formed by Folic acid (with the ability of targeting to cancer cell)-Taxol (the widely used anti-cancer drug) conjugate through adding GSH or DTT to reduce disulfide bond for local delivery and sustained release of Taxol. As a result, the hydrogel injected into tumor exhibited the better therapy effect than the 4 times dosage of clinical Taxol and high biocompatibility. Whereafter, we developed the first supramolecular hydrogelator of curcumin. Here, we founded that the pro-gelator possessed the better capability to inhibit cancer cells and tumor than hydrogel and curcumn (dissolved in an excipient mixture of polyethylene glycol 400, propylene glycol, and polysorbate 80 (40:58:2)) injected into mice through the caudal vein. And then we concluded that the hydrogel might only be applied for the topical treatment of cancers. From the above, useful information for design of nano-materials to deliver anti-cancer drug has been presented through our study.
The process of anti-cancer drug development is expensive and time consuming. A key limitation is pre-clinical testing using animals since such tests are often not predictive of human response (i.e. only 11% of drugs that exist from animal trials become approved for human use). The development of surrogates for human response in preclinical testing could improve the drug development process by identifying the drugs most likely to be successful in human clinical trials, this is particularly true for chemotherapeutic drugs to treat cancer.The distribution and potential response of the human body to drugs can be modeled using PBPK-PD (physiologically-based pharmacokinetic-pharmacodynamic) mathematical models. However, these models often fail because they do not capture all of the interactions of drugs with the body, as many of these interactions are unknown. We construct “Body-on-a-Chip” systems (also known as microphysiological systems) combining the techniques of microfabrication and cell culture to make an experimental system that mimics the human body. Since such biological constructs emulate many of the undiscovered interactions of the drugs and its metabolites with human tissue, a physical model based on the use of tissue constructs can predict human response to drugs. Combining a PBPK-PD model with a physical device allows prediction of human response.In this talk we will describe this methodology and demonstrate its potential usefulness for predicting human response to drugs. We construct these systems combining the techniques of microfabrication and tissue engineering. These microfluidic systems can be operated for up to 28 days. We describe how they can be used to emulate human response to chemotherapeutic drugs such as doxorubicin and Tegafur.
Our lack of ability to replicate the complex milieu of human cancer is a critical barrier to controlled studies of human tumor initiation, progression and metastasis, and the evaluation of therapeutic targets and anti-cancer drugs. In vitro, cancer cells are known to rapidly lose their cancer phenotype, while animal models often lack to recapitulate human tumors (for example, the mouse bone is not permissive to invasion of metastatic human cells). A new generation of bioengineered tumors is now emerging in response to these limitations, with the potential to transform cancer research. By recapitulating the in vivo tumor environment, these models allow us to grow physiologically relevant tumors from the patient&’s own cells and to study tumor initiation, progression and metastasis.We first describe a bioengineered model of human Ewing&’s sarcoma that mimics the native bone tumor niche and results in re-expression of focal adhesion and cancer pathways] genes that was lost in monolayer culture. The bioengineered model also recovered the original hypoxic and glycolytic tumor phenotype, and displayed the angiogenic and vasculogenic mimicry features. Then we discuss the integration of these tumor models into the micro-tissue platforms for personalized drug screening. Finally, we demonstrate the utility of bioengineered models of cancer for studies of tumor exosomes. We found that the architecture, composition and stiffness of the scaffold regulated the exosomes released by the cancer cells, by mechanisms involving tension forces. As a result, the exosomes released from bioengineered tumors were similar to those released into the plasma of Ewing sarcoma patients, and significantly different from exosomes released into the supernatants of cell monolayers. While tissue models are just emerging as a new tool for cancer research and drug discovery, they are already demonstrating potential for recapitulating the native behavior of human tumors.
Little is currently known about the detailed process by which tumor cells escape from the microvasculature to establish a metastatic tumor. This likely involves adhesion to the endothelial wall or becoming lodged in a small capillary, transmigration across the endothelium and basement membrane, migration into the surrounding ECM, and recolonization. Our group has established several microfluidic models that enable high resolution imaging of each stage of extravasation, providing new insights into mechanisms. In one model, an endothelial monolayer is grown on the side of a central gel region, and tumor cells are flowed into the endothelialized channel, adhere, and transmigrate. In a second model, a microvascular network is grown within a central gel region and tumor cell containing medium is flowed through the network. Cells adhere to or become lodged in the network, and subsequently extravasate. The third model consists of a flat monolayer on top of a thin (~100 mu;m) layer of matrix. These systems have been used to examine the role of β1 integrins, demonstrating its critical role in all stages of extravasation. In separate experiments, we examine the migration of a tumor cell as it probes the cell-cell junctional complex, and often exists at vertices where 3 or more cells join together. As the cell transmigrates, the VE-cadherin junctional proteins transiently disperse from the junction, then reassemble once the tumor cell transmigrates. [Support of the National Cancer Institute (5R33CA174550) is gratefully acknowledged.]
Existing anticancer therapies are confounded by tumor invasion and metastasis, as well as the emergence of drug resistance. It has been hypothesized that the epithelial-mesenchymal transition (EMT) can transform adherent cells into a invasive and drug-resistant phenotype. As a consequence, EMT may drive cells at the tumor invasion front to detach and locally disseminate into surrounding tissues. Here, we profile heterogeneous invasion dynamics after EMT in engineered microenvironments. Using single cell tracking in space and time, we classify collective epithelial and individual mesenchymal subpopulations. These emergent dynamics can be understood using a physical analogy with phase transitions during the solidification of binary alloys, suggesting the importance of cell sorting as well as phenotypic interconversion. We perturb these behaviors using migration-inhibiting compounds, revealing that different invasion phenotypes are correlated with differences in drug sensitivity.
3D in vitro tumour models promise more reliable preclinical drug screening results, shorter drug development time, and higher success rates of drug candidates during animal testing and clinical trials. Even so, 2D in vitro cell culture methods persist because current 3D cell culture materials and systems are expensive, lack an even distribution of nano-topological features, and don't have established protocols. Recreating the nanofibrous network of the extracellular matrix, while simultaneously providing a macroscopic structure that can be tailored to suit various cell sizes is crucial for a functional 3D model. To-date, it has been a challenge to produce in large-scale using a single technology, an affordable 3D cell culture scaffold that can provide both microfibrous and nanofibrous features. Here we present an affordable 3D cell culture cellulosic fibre scaffold that can integrate and disperse both microfibres and nanofibres throughout the material. The material is generated by the recently developed and commercialised method of shear-spinning. The polymeric fibre production technique uses precipitation of polymer solutions sheared in an aqueous viscous medium. We show three representative cancer cell lines, HEK293, A549 and MCF-7, are highly viable (viability > 99.6%) when cultured in the scaffold for 40 days. We optimise the nanofibre composition and show a ratio of 10% nanofibres to 90% microfibres is the most desirable for cell-scaffold attachment, cellular growth and infiltration. We also demonstrate a straightforward protocol for the handling of the materials and present differences in proliferation rates of the A549 and MCF-7 cell lines when cultured in 3D versus those in 2D.
Breast cancer progression to secondary metastasis still remains major cause of mortality in women worldwide. Current studies focusing on the mechanisms of metastatic breast tumor progression are widely carried out in 2D cultures and xenograft models by manipulating various signaling pathways and molecular targets involved. For instance, tumor aggressiveness is induced in 2D monolayer cells and 3D multicellular aggregates by exposure to artificial hypoxic environments or over-expression/silencing of signaling molecules or using different non-invasive and invasive cell lines. These methods do not recapitulate naturally occurring events of tumor growth-induced changes in tumor microenvironments such as hypoxia, metabolic gradients, eventually leading to metastasis. Breast tumor biopsy samples and tissue microarrays can be useful in identifying metastatic and aggressive tumor phenotype. However, the unavailability of biologically matched early and advanced stage tumor biopsies limits our understanding of molecular mechanisms related to size-dependent metastatic progression in clinical samples. In this study, we have established three-dimensional (3D) preclinical in vitro model that can capture in vivo tumor progression in real time in the same, non-invasive cell line model. We have generated size-controlled multicellular aggregates (microtumors) of subtype-specific breast cancer cell lines by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwell arrays of defined sizes (150-600 mm). The developed 3D model recapitulated in vivo tumor progression with development of hypoxic gradient and characteristic spatial distribution of necrotic cells in a size-dependent manner. Of note, larger microtumors showed aggressive phenotype and collective cell migration unlike smaller ones. Interestingly, hormone receptor positive microtumors (MCF7, T47D, BT474) showed size-dependent upregulation of mesenchymal markers (Snail, Slug, Vimentin and Fibronectin) with no change in epithelial characteristics (no loss of E-cadherin), at transcriptional and translational level. Conversely, microtumor size did not show significant differences in mesenchymal marker upregulation in triple negative breast cancer (TNBC) cell lines. In conclusion, the developed size-controlled 3D microtumor model successfully recapitulates positive association of tumor size with aggressive phenotype of hormone receptor positive breast microtumors, observed clinically. More importantly, the 3D in vitro model also successfully captures the clinically proven poor correlation of tumor size with metastasis in TNBC. Such clinically relevant 3D model can serve as a controlled system for studying tumor progression and drug screening assays within the short duration of time.
In the United States around 40% of the overall population encounters at least one form of cancer in their lifetime and cancer is the second leading cause of death. Many cancers including prostate cancer often metastasis to bone but the molecular mechanisms as well as the microstructural organization of the metastasis is unknown. Thus often bone like environments are created to study the migration, attachment and proliferation of cancer cells. Cancer research is primarily based on in vivo models and 2D substrates which do not represent human body microenvironment. In this study, we have developed a 3D bone mimicking bone in vitro model to study the interactions between prostate cancer cells and bone microenvironment. We have a developed a novel nanoclay based scaffold system that is fabricated by biomineralization of hydroxyapatite inside nanoclay galleries through use of amino acid modifiers. Such hydroxyapatite nanoclay is used to fabricate composites with polycapralactone. A novel sequential cell culture with human mesenchymal stem cells and prostate cancer cells on nanoclay based scaffolds provide a unique bone mimetic environment. Human mesenchymal stem cells differentiate to the osteoblastic lineage on the polycapralactone insitu hydroxyapatite nanoclay (PCL/HAPClay) scaffolds followed by seeding of cancer cells. The cancer cells develop to form 3D cancer tumoroids with organized tight cellular junctions, indistinguishable cellular boundaries and even surfaces. Such tumoroids are not observed on osteoblast seeded nanoclay scaffolds. The formation of the tumoroids on mesenchymal stem cell seeded scaffolds indicate the role of new bone microenvironment on cancer development. These scaffolds with the sequential seeding can be used as model testbeds to evaluate the metastasis of cancer as well as evaluation of anticancer drugs.
Solid tumors house an assortment of complex and dynamically changing microenvironments in which signaling events between multiple cell types are known to play a critical role in tumor progression, invasion, and metastasis. To deepen our understanding of this biology, it is desirable to accurately model these structures in vitro for basic studies and for drug screening, however, current systems fall short of mimicking the complex organization of cells and matrix in vivo. Here we demonstrate the generation of spatially-organized 3D hydrogels of cells and matrix produced from a simple concentric flow device in a single step. Multiple cell types are pre-seeded in different spatial domains such as concentric regions of vessel-like tubular structures to reproducibly establish heterotypic cellular environments in 3D. Using macrophages and breast adenocarcinoma cells as an example of a paracrine loop that regulates metastasis, we explored the effects of clinical drug treatments and observed a dose-dependent modulation of cellular migration. This versatile and tunable approach for tissue fabrication will enable a means to study a wide range of microenvironments and may provide a clinically-viable solution for personalized assessment of patient response to therapeutics.
Two pathways for activating macrophages (MPhi;) exist. One of these routes is termed the classically activated M1 pathway is achieved through exposure to lipopolysaccharide. M1 MPhi;s are part of the type 1 T helper (Th1) response and are known as pro-inflammatory cells.1 The other pathway is reached through interleukin-4 and is known as the alternatively activated M2 pathway. M2 MPhi; produce pro-angiogenic factors.2 Tumor-associated MPhi; (TAMs) are of the M2 pathway and promote tumor growth through the release of angiogenic molecules. Our goal is to use polymeric drug delivery systems to reprogram TAMs such that the produce pro-inflammatory molecules. These polymers will be eventually used to delivery anti-cancer therapeutics to the tumor.MPhi;s were polarized to the M1 and M2 phenotypes and markers of the Th1 response - tumor necrosis factor-α (TNF-α) - and the Th2 response - interleukin-10 - were monitored upon exposure to polymers modified with various functional groups. The different mechanisms by which MPhi;s internalize the particles were also determined through selective blocking. We compared the ability of the materials to alter MPhi; phenotype to material properties.A library of hydrogels was synthesized and exposed to M1, M2, and non-activated MPhi;. Viability assays showed that these materials had >80% cytocompatibility. The materials were found to be able to induce different shifts in the polarization of the MPhi;s depending on the initial phenotype and the material functional group. For example, some of one material was able to polarize MPhi; to the M1 phenotype by increasing the amount of TNF-α secreted 2.5 fold for non-activated and M2 MPhi;s.Additionally, the phagocytic capacity of MPhi;s was found to depend both on phenotype and the functional group. In M1 MPhi;s, the hydrophilicity positively correlated with phagocytic activity. However, there was no such correlation for the M2 phenotype. We also examined the effect of complement activation, zeta potential, and glass transition temperatures of the polymers on their ability to alter MPhi;sPlasticity of macrophages and their phagocytic activity play an important role in the resolution of non-specific host response. In the foreign body response macrophages may promote biocompatibility of the implanted medical device by suppressing pro-inflammatory cytokines and phagocytosis in macrophages, while shifting towards a wound-healing M2 phenotype. Here, we show how different biomaterials affect both M1 and M2 macrophages resulting in different cytokines and phagocytosis levels. The ability to control this polarization presents applications in anti-cancer therapeutics.
In the drug discovery process, the number of insoluble small molecular drug candidates has increased dramatically in recent years, with almost 70% of new drug candidates showing poor water solubility, which impedes effective treatment. Increasing the dissolution rate of poorly soluble drugs and enhancing their bioavailability is an important challenge to pharmaceutical scientists. A common approach to increase drug solubility involves reduction of particle size and modification of crystal habit. However, for nanopowders (particles < 500 nm), homogenization of drug powder is problematic due to particle aggregation.Here we present a new approach to increasing surface to volume ratio in small molecular pharmaceuticals without aggregation. We use Organic Vapor Jet Printing (OVJP) to achieve high surface area nanostructured films of small molecular medicines. OVJP utilizes a carrier gas (e.g. nitrogen) to transport sublimated organic vapor towards a cooled substrate in the form of a focused gas jet. High quality coatings can be obtained, with >99.99% purity, with surface feature morphology ranging from molecularly flat to high surface area, with feature sizes in the micrometer or even nanometer regimes. Importantly, OVJP works without liquid solvents or vacuum, and allows for controlled degree of crystallization in the organic films.We use OVJP to deliver controlled amounts of caffeine, ibuprofen, paracetamol, and other compounds onto various substrates in film form. We study the dissolution kinetics of the deposited drug film in aqueous solutions, monitoring the process using a fluorescent probe (fluorescein). We study crystal structure and chemical stability of the resulting films as function of deposition conditions. The film structure and dissolution behavior are characterized using microscopy (optical, fluorescent and electron), X-Ray diffraction (XRD), optical spectroscopy, FTIR and HPLC techniques. We demonstrate an improved bioavailability of cancer treatment drugs - tamoxifen base and BAY 11-7082 through in-vitro testing of cancer cell cultures. We also demonstrate the jetting of small molecular organic vapor directly into aqueous solutions, demonstrating how precise drug concentrations can be rapidly reached, without the need for additional solvents and/or powder preparation. The thermodynamics of direct dissolution using this approach are discussed, along with possible applications.References:O. Shalev, S. Biswas, Y. Yang, T. Eddir, O. Anatohu, W. Lu, R. Clarke & M. Shtein, Nat. Comm. 5, 5204 (2014)K.T. Savjani, A.K. Gajjar & J. K. Savjani, ISRN Pharmaceutics 2012, 195727 (2012)S. Hoelder, P.A. Clarke, P. Workman., Molecular Oncology 6, 155 (2012)
Tumor organoids embedded in a biomimetic 3D scaffold are highly promising as a physiologically relevant culture model that bridges the gap between 2D monolayer culture and small animal models. This approach is relevant for high-throughput preclinical drug screening and ultimately to culture patient samples ex vivo. A particular application of interest is the epithelial-mesenchymal transition (EMT), which transforms adherent cells to an invasive and drug resistant phenotype. Here, we report the development of a tunable silk fibroin-collagen I hydrogel culture system and its application to EMT. These hydrogels are highly reproducible and relatively inexpensive, enabling long term organoid culture for extended durations over weeks to months. These controlled 3D microenvironmental conditions facilitate reliable induction of EMT to elucidate single cell heterogeneity and plasticity. We further demonstrate the use of this system to measure differential sensitivity to conventional chemotherapeutics and targeted inhibitors.
A monolayer differs significantly from the multicellular nature and three-dimensional growth demonstrated by a tumor. As a result of both of these disparities, cells in a monolayer are genetically different from their counterparts. In order to examine the effect of three-dimensional culture and tumor macrostructure on drug response, we studied three models composed of MDA-MB 231 cells, a post-metastatic, triple negative breast cancer cell line. The first was a traditional monolayer. The second consisted of cells diffusely seeded within a collagen gel, thus a three-dimensional culture system. The third model comprised of a multicellular spheroid embedded within a collagen gel, thus incorporating both tumor macrostructure and a three-dimensional nature.Based upon this premise, we examined how the models responded to paclitaxel, a first line chemotherapeutic agent clinically used in the treatment of breast cancer. When treated with paclitaxel at a concentration of 10 ng/mL for 72 hours, the viability of the monolayer fell below 20%, with a slightly more robust response from the diffuse model with 30% viability. The spheroid model contained two distinct populations, a core, a dense aggregate of cells, and a periphery, cells that have grown into the surrounding collagen. The viability of the periphery was approximately 30%, which was not statistically different from the diffuse model, thus indicating their similar drug responses. Despite an effect against the periphery, the core demonstrated a robust response, with an overall viability of 90%.Paclitaxel&’s mechanism of action focuses on cell division by preventing microtubule disassembly. Since cells in the core are quiescent due to gradients of metabolites and oxygen, they might remain relatively unaffected by treatment. However, paclitaxel was unable to prevent subsequent division of cells in the the core. After treatment with paclitaxel, the core was removed from the system and embedded into new collagen. It&’s subsequent growth, while retarded, was not prevented. While unable to effectively treat the core, the fluorescent conjugate, Paclitaxel Oregon Green was able to penetrate the core, indicating an issue of efficacy as opposed to availability.This research demonstrated the dual response of the spheroid model to treatment with a chemotherapeutic agent. Loosely aggregated cells, invading the collagen were susceptible to treatment, in contrast to the dense, tightly packed core population
Nanoparticle (NP) size, charge, and surface chemistry are accepted as key determinants of NP/cellular interactions that ultimately dictate biodistribution and tumor localization, however, the relative importance of these diverse NP physicochemical properties in regulating interactions with various biological systems remains incompletely understood, and, while the existing paradigm dictates that decreased size and neutral or negative zeta potential are correlated with increased circulation time, the manner in which these combined physicochemical properties conspire to direct in vivo cellular interactions has not been elucidated through careful systematic studies. Here using a library of stable, monosized mesoporous silica nanoparticles (MSN) and MSN-supported lipid bilayers (aka protocells) with systematically varying sizes and surface chemistries, we have directly imaged/evaluated in vivo stability, non-specific binding and uptake by the innate immune system using the highly vascularized exo ovo chick chorioallantoic membrane model (CAM). We demonstrate that two types of cationic MSN that are identical in primary and hydrodynamic particle size, shape, pore structure, colloidal stability, and charge but differ in the arrangement and exposure of charge have profoundly different interactions with cells and tissues. While both particles are ~50 nm in diameter, PEGylated, and positively charged (zeta; = +40mV), PEG-PEI (MSNs modified with exposed polyamines), but not PEG-NMe3+ (MSNs modified with distributed, obstructed amines) rapidly bind serum proteins, diverse cells types in vitro, and endothelial and white blood cells in vivo. Accordingly PEG-NMe3+ had greatly increased circulation times even when the particle size was increased to 150-nm. Quantitative, longitudinal SPECT imaging of the corresponding 111In-labled MSNs injected in rats showed the CAM results to be predictive of biodistribution. PEG-NMe3+ showed greatly increased circulation times and, after 24 hours, 10x reduced accumulation in the liver and 3x less accumulation in the spleen. (Liver and spleen accumulation were also significantly less than for moderately negatively charged 50-nm MSN). On the other hand PEG-NMe3+ accumulated more rapidly in the bladder and were excreted in the urine as confirmed by TEM (despite exceeding the supposed 10-nm limit on renal clearance). By largely avoiding uptake by the liver and spleen PEG-NMe3+ showed a much greater enhanced permeability and retention (EPR) effect in a prostate cancer xenograft model. Measurements of hydrodynamic size after exposure to serum or recovery from blood (mouse or rat) suggest that the difference in behavior is at least partially attributable to ‘opsonization neutrality&’ of the PEG-NMe3+ MSN, i.e. they largely avoid the formation of a protein corona. Overall we conclude that the generally measured physicochemical parameters, size and zeta potential, are not by themselves reliable predictors of in vivo behavior.
Organ-on-a-chip devices facilitate the study of physiology, disease modelling and drug testing. They have the potential to improve healthcare by cutting costs and providing insight into organ and disease physiology. A large number of organ-on-a-chip devices are fabricated using polydimethylsiloxane (PDMS). A key component of these devices are perforated PDMS membranes that allow cells to be cultured and separated in different /separate compartments while still allowing them to interact. A preferred fabrication method for perforated PDMS membranes is molding. This method, however, is complicated and has low yields, making it impractical for large-volume fabrication, posing a barrier to the wide-spread use of organ-on-a-chip devices.An alternative method to molding is reactive ion etching. In that method a protective mask is applied on top of the material and the substrate is exposed to a chemical mixture that reacts with the base material forming volatile products. Thus the areas that are not protected by the masking material are attacked and removed. Since PDMS is a polymer consisting of a siloxane backbone with methyl side groups, an etching mixture consisting of fluorine and oxygen has been envisioned as an efficient etching agent. Results from such experiments have been reported to successfully etch PDMS. The main disadvantage is the relatively low etch rates. This leads to longer processing times and consequently higher production costs.We have explored two etching processes aiming at improving the efficiency of the etching process in terms of etch rate.The first approach uses the already mentioned fluorine chemistry. In this case we used a combination of CF4 and SF6 etching gases. We got etch rates that were superior to the ones reported in literature. Furthermore we established that the addition of oxygen (supposed to help with the removal of the methyl side groups) seemed to reduce the etch rate and best results were obtained in a gas mixture where no oxygen was present. The etch profile was anisotropic and particulate remnants were observed at the etched areas (concurrent with previously reported observations).The second approach relied on Cl based chemistry. A mixture of Cl and a small amount of CF4 was used. The experiments showed that the etch rate is approx. 1.5 µm/min (approximately 3 fold increase over previous results with F based chemistry). The etching profile is anisotropic and no remnants after etching were observed.
Prostate cancer is the second leading cause of cancer death in men in the US besides lung cancer. Current screening tests performed in clinic are inadequate and often provide false positives or fail to detect tumors early resulting in metastatic progression to distant organs or bones. Nanoparticle based diagnostics and therapeutics have recently emerged as a novel platform for management and mitigation of cancer at all stages. Gold nanostructures, specifically, have multiple characteristics that make them ideal for cancer theranostics including high biocompatibility, ease of bioconjugation, ability to tune their plasmon resonance to absorb tissue penetrating near infrared light, their use as contrast agents, and ability to convert light to heat when excited at the plasmon resonance for photothermal ablation of cancer cells. In this work we demonstrate the use of near-infrared light absorbing multibranched gold nanoantennas (MGNs) to simultaneously deliver diagnostic and therapeutic (theranostic) capabilities in prostate adenocarcinoma (LNCaP) cells. MGNs consist of a core that absorbs light and protrusions that serve as emitters confining light into a localized area generating enhanced local fields via the nanoantenna effect and intense photothermal response. By exploiting these optical properties of MGNs we have utilized them for ultrasensitive surface enhanced Raman scattering (SERS) imaging to visualize LNCaP cells with single-cell resolution in vitro and subsequently performed photothermal therapy to enable cell death using low laser powers of < 5W/cm2. By staining the cells with various dyes we have delineated cell death by apoptosis and necrosis to distinguish the two cell death mechanisms. We have also demonstrated the feasibility of theranostic MGNs in vivo as dual-modal contrast agents for SERS and photothermal optical coherence tomography (PTOCT) combining the high sensitivity of SERS and the high resolution and high tissue penetration depths of PTOCT within a single nanoagent.
Adoptive cell transfer (ACT) with autologous tumor-reactive T cells is a promising strategy in cancer immunotherapy, but treatment of solid tumors is limited by the rapid decline in function of the transplanted T cells. In order to maintain high numbers of viable antigen-specific cytotoxic T cells in tumors, co-administration of supporting immunostimulant agents together with transferred cells is often necessary in clinical practice. However, the high systemic doses of such agents needed to enhance T cell functionality can also result in serious side effects. Here, we developed a carrier-free strategy to deliver cytokines specifically to adoptively transferred T cells for cancer immunotherapy. IL2-Fc or an IL-15 superagonist were chemically crosslinked with a disulfide linker to form protein nanogels (NGs), which were conjugated to the plasma membrane of ACT T-cells. These NGs had exceptionally high loading of cytokines (~70 wt%) and released native protein in physiological conditions in a sustained manner through breakdown of the degradable disulfide linker in response to the activated T cell surface reduction activity. Cytokine-NGs were chemically conjugated onto the plasma membrane of donor T cells, enabling continuous pseudo-autocrine release of cytokine for stimulation of transferred CD8+ T cells. Transferred pmel-1 CD8+ T cells with optimized number of NGs conjugated per cell showed enhanced expansion and long persistence in B16F10 tumor bearing mice. Quantification of transferred Thy1.1+CD8+ T cells in tumors at Day 13 showed that T cells with conjugated cytokine-NGs expanded ~80 fold more than the T cells with systemically administered free cytokine. We demonstrated that the cytokine-NG-T cell conjugation strategy could augment transferred T-cell expansion efficiently and specifically in vivo, and thus improve the therapeutic efficacy. This T cell-NG “back pack” approach provides a readily generalizable strategy to provide autocrine protein drug support to donor cells to enhance the safety and efficacy of ACT.
Chemotherapy, the most common drug delivery method for cancer treatment, leads to severe side effects because a strong toxic drug is inevitably delivered to the healthy organisms of patient. Therefore, there is a high demand to encapsulate anti-cancer drugs into a carrier for localized, targeted and controlled delivery. Drug encapsulation provides a sustained release in controlled manner and protection from degrading by the ambient environment.Electrospun nanofiber membranes are one of most promising drug carrier approaches in cancer treatment because of the localized delivery of drugs to the targeted site. Importantly, multi-axial electrospinning can provide encapsulation functionality enabling both one-step drug incorporation and its sustained release without significant initial burst release. In these compound fibers one can control release kinetics by manipulating properties of outer and intermediate layers. Previously, we have demonstrated a dual delivery system using triaxially electrospun fibers encapsulating two different dye molecules, indicating the potential to be used as a novel drug delivery system(1).Here we report on single and multi-axial electrospun membranes encapsulating the mycophenolic acid (MPA) and present initial results on its effectiveness against cancer cell growth in vitro. MPA is an FDA-approved immunosuppressor and has been used clinically for over 20 years. MPA inhibits IMPDH [inosine monophosphate dehydrogenase], which is a rate- limiting enzyme for de novo synthesis pathway for guanosine triphosphate (GTP), an essential energy molecule for cell proliferation. Our research and that of others indicate MPA&’s high potential(2) for treating multiple cancer cells. However, MPA has a short half-life in blood (~ 6h), which challenges its drug repositioning to anti-cancer drug, because continuous exposure of MPA is critical to suppress cancer cell growth. Towards this end, we used a TFA - DMF mixture to dissolve the host polymer with MPA in solution for electrospinning of fiber membranes. The fiber diameter of MPA-incorporated electrospun fibers is ~ 1.6 ± 0.15 µm. Based on cell culture experiments with and without MPA-incorporated membranes, we have demonstrated that MPA in fibers retains its properties without any degradation during electrospinning and subsequent storage up to several weeks. After 5-day cell culturing, it is observed that MPA incorporated fibers have significantly suppressed cancer cell growth compared to the control membrane without MPA. This effectiveness can be further improved by adding another drug into one of multi-axial layers in fiber to trigger a synergetic effect.(1) Han, D.; Steckl, A. J., Triaxial Electrospun Nanofiber Membranes for Controlled Dual Release of Functional Molecules. ACS Applied Materials & Interfaces 2013, 5 (16), 8241-8245.(2) Majd, N.,; Sasaki, A., et al., A Review of the Potential Utility of Mycophenolate Mofetil as a Cancer Therapeutic. J. Cancer Research, 2014, 423401.
Spherical nucleic acids (SNAs) are a nanotechnology platform that enables a powerful approach to cancer therapeutics, by way of engaging, activating, and targeting the immune system to attack tumors. SNAs consist of a 10-30 nm nanoparticle functionalized with a dense, spherical shell of radially oriented oligonucleotides. SNAs efficiently enter immune system cells without the need for transfection agents or chemical modifications, show enhanced resistance towards degradation by cellular nucleases relative to linear oligonucleotides, and show minimal toxicity and off-target immunogenicity.By taking advantage of these properties, we have developed a new nanoparticle technology that can treat cancer by activation of the immune system against tumor cells. The therapeutics consist of a 30 nm liposomal core functionalized with immune-activating oligonucleotides inserted into the lipid bilayer via hydrophobic functional groups introduced at their termini, and cancer-associated peptides. When this architecture is employed as a cancer vaccine, it shows significant advantages over the “free” molecular components (CpG oligonucleotide and antigens). Compared to the free components, liposomal SNAs exhibit up to 80-fold increase in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen and improve median survival in mice with lymphomas through antigen specific tumor inhibition. These properties originate from the ability of the SNAs to enhance the rates of cellular uptake and potency of TLR stimulation, through the nanoscale architecture and polyvalent binding to TLRs enabled by the unique structure of SNAs. Combined with their high serum stability, at-scale synthesis from readily available and FDA-approved materials and minimal toxicity profile, liposomal SNAs hold great promise for the development of cancer vaccines.
We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (1) emergence of genes with high de novo substitution densities (i.e. hot genes). and (2) genes never substituted (i.e. cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never substituted genes were biased in age towards the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.
We interrogate cancer noninvasively through the development of targeted nanoscale probes. By exploiting both the physics (changes in electronic, magnetic, and optical properties) and the biology (changes in trafficking) of the nanoscale, we demonstrated the ability to sensitively detect solid tumors, and explored the role of the tumor microenvironment in nanoparticle homing to both xenograft and genetically-engineered mouse models of cancer. We currently develop technologies that would extend beyond detection to interrogate enzymatic activity within the tumor microenvironment, such as that which would occur in tumor infiltration of surrounding ECM. Using these novel nanoparticle probes, we detected colorectal tumors that could not be identified by secreted CEA. Based on our knowledge of nanoparticle trafficking in the liver, we also study the liver microenvironment as it underwent fibrosis. We found a single synthetic biomarker could be used to monitor fibrosis in response to both xenobiotic and genetic injury, reflecting a conserved tissue response despite distinct underlying etiologies.
Introduction: The aim of this study was to evaluate the efficiency of internalization and bioactivity of doxorubicin (DOX) encapsulating alginate microspheres by human lung carcinoma cells (A549) as compared to passive delivery of free DOX to provide a better understanding of chemotherapeutic delivery mechanisms. Methods: Methacrylated alginate Alg-MA was synthesized utilizing an anhydrous reaction between alginate and methacrylic anhydride. Aqueous solutions of Alg-MA were mixed with photoinitiators for UV (Irgacure D2959) or visible green light activation (eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone). 2% (w/v) Alg-MA solutions were mixed with 0.1% (w/v) DOX and formed into sub-microspheres using a water/oil emulsion. Covalent crosslinking was performed using either 10 minutes UV or green light exposure alone, or in combination with ionic crosslinking using 0.5 M CaCl2 solution. Dynamic light scattering quantified hydrodynamic diameters of the microspheres (buffered saline, pH 7.4). Alexa Fluor® 647 cadaverine was conjugated onto the surface of sub-microspheres; flow cytometry was used to determine populations of A549 cells containing Alg-MA sub-microspheres after 36 hours of culture. Encapsulation efficiency and DOX release profiles were assessed using photospectrometry. To verify the added therapeutic effects of intracellular DOX delivery, sub-microspheres containing DOX were compared to free DOX in solution in the death of cancer cells over time. DOX bioactivity was evaluated using an MTT-based in vitro cytotoxicity assay after 24, 48, 72, and 96 hours of culture with A549 cells. Sub-microspheres were cultured at 10, 50 and 100 mu;g/mL, and free DOX was dosed at varied concentrations from 0.015-5 mu;g/mL. Results and Discussion: Average diameters for the treatment groups were between 300-450 nm. Flow cytometry confirmed that microspheres were readily internalized by A549 cells; compared to control cell populations, nearly 100% of the cell population cultured with the sub-microsphere groups internalized the sub-microspheres. The UV-crosslinked sub-microspheres were internalized more readily. Alg-MA microspheres, i.e., not containing DOX, were non-cytotoxic (~90% MTT activity for various groups) to A549 cells. DOX-encapsulated microspheres successfully delivered bioactive drug, inducing cell death increasingly over a four-day test period. Sub-microsphere groups with high DOX encapsulation efficiencies were more effective at killing cells compared to free DOX. Conclusions: Covalent and ionically crosslinked Alg-MA microspheres successfully delivered a bioactive chemotherapeutic intracellularly to cancer cells. The use of single or dual-activated crosslinking mechanisms varied the efficacy of DOX-encapsulating Alg-MA microspheres in inducing cell death of A549 cells after 24-96 hours of culture. Future work will investigate the efficacy of internalized DOX-encapsulating microspheres in inducing cell death in DOX-resistant cell types.
More than 70% of ovarian cancers are found at an advanced stage due to a lack of effective screening methods. Definite diagnosis of ovarian cancer requires an invasive surgery, but results generally lag behind. A non-invasive and cost-effective screening method for ovarian cancer, thus applicable to general population, is desirable. Recently, tumor-derived extracellular vesicles (EVs) in bodily fluids have emerged as a new biomarker for cancer diagnostic. EVs shed from cancer cells carry molecular information of their parental tumor, and they are stable and abundant in serum (more than 1e9 EVs/mL). Despite the clinical potential of EVs, their widespread use has been hampered by time-consuming sample preparation and a need for expensive specialized equipment. Here, we describe a cost-effective platform that enables detection and profiling of EVs directly from patient samples. This system is based on immunomagnetic separation and electrochemical detection of tumor-specific EVs. It can detect less than 100,000 tumor-specific EVs in serum, and the total assay can be finished within an hour. Equipped with eight channels, this platform can profile multiple tumor-associated transmembrane proteins (e.g., EpCAM, EGFR, and HER2). Furthermore, the platform has a small form factor, which allows the exosomal analysis to be performed in routine clinical settings. In this talk, the detailed information of our diagnostic system and profiling assay will be presented.
High-intensity focused ultrasound (HIFU) has become a promising treatment modality for a variety of cancers including prostate, colon, kidney, liver, and breast cancer. HIFU functions by applying acoustic energy to a small region in the tumor which can lead to tissue damage. HIFU can be used for cancer treatment by locally ablating inoperable tumors via 1) thermal deposition or 2) mechanical damage of the tissue. Our studies have shown that mechanical tumor damage may be controlled by using gas filled silica nanoshells in combination with HIFU. Biodegradable 500 nm perfluoropentane-filled iron-silica nanoshells were synthesized as a sensitizing agent for HIFU therapies. When nanoshells were combined with low duty cycle HIFU, rapid tissue damage occurred in tumor-bearing mice from mechanical rather than thermal effects. High duty cycle resulted in thermal ablation even in the absence of nanoshells. Additionally, nanoshells may be functionalized to have other properties such as immunostimulatory properties. An immune adjuvant was functionalized onto the surface of nanoshells; the nanoshell-adjuvant conjugate showed enhanced immune stimulation in vitro and in vivo. These nanoshell conjugates can be used in future studies as immune adjuvants in combination with HIFU in order to enhance the antitumor immune response.