Symposium F.SM07—Biomaterials for Studying and Controlling the Immune System

Introduction: Angiogenesis is largely directed by macrophages, immune cells that can change their phenotype in response to external stimuli. Macrophages precisely control the timing and dosage of angiogenic growth factors, and also direct the innate immune response to biomaterials. Therefore, biomaterial-mediated macrophage modulation is an attractive strategy to recover dysfunctional angiogenesis. Macrophage phenotypes encompass a wide spectrum, but in vitro are commonly categorized as M0 before activation, then M1 or M2 post-activation, with several identified subtypes under the M2 umbrella. Although each phenotype’s exact role in angiogenesis is not well understood, there is a growing body of evidence suggesting that early M1 macrophages initiate blood vessel sprouting, and subsequent M2 activity stabilizes these structures. Recent studies also indicate that M1 macrophages that switch to an M2 phenotype may have enhanced angiogenic properties. Furthermore, in pathologies where angiogenesis is inhibited, such as chronic wounds, M1 macrophages are insufficiently activated and fail to switch to an M2 phenotype. Thus, biomaterial strategies that seek to recover dysfunctional angiogenesis should aim to initially promote a robust M1 macrophage phenotype.
Previous work from Vegas et al. established a library of alginate hydrogels modified with chemical functionalities, then ranked it by inflammatory activity, as measured by levels of cathepsin activity in vivo. This study was conducted to test the capabilities of the inflammatory alginate hydrogels to promote an M1 phenotype in primary human macrophages in vitro. Here we seeded M0 macrophages onto alginate hydrogels, then used Nanostring, a high-throughput gene expression assay, to measure expression of M1 markers, M2 markers, and genes associated with angiogenesis or fibrosis.
Methods: 5 types of alginate hydrogels were fabricated by the Vegas lab at Boston University: 3 inflammatory, 1 anti-inflammatory, and 1 non-modified. Primary monocytes were purchased from the Human Immunology Core at the University of Pennsylvania and cultured with 20 ng/mL of macrophage colony stimulating factor (MCSF) to induce differentiation into macrophages. On day 5, 200,000 macrophages were seeded onto each 5 mm section of hydrogel in a 24-well plate. Samples were incubated for 1 hour at 37°C to allow cell attachment. 800 µL of complete media with MCSF was added to each scaffold-containing well. M0 and M1 control samples were refreshed with complete media with MCSF. 100 ng/mL of human recombinant IFNg and LPS were added to the M1 control samples to induce M1 activation. On day 7, media was removed and scaffolds were mechanically degraded and lysed. RNA was extracted and quantified. For samples with at least 20 ng/µL of RNA, 100 ng were hybridized with a 72-gene custom Nanostring Codeset targeting M1, M2, angiogenic, and fibrotic genes. Nanostring sample preparation and reading was conducted according to Nanostring’s standard protocol.
Results: Nanostring gene counts were normalized to the Nanostring in-house controls and fold changes over the M0 controls were calculated. Of the M1 markers measured, two (CD80 and CCL2) were upregulated in macrophages seeded on the inflammatory alginate hydrogels. Interestingly, these genes were also highly upregulated in macrophages seeded on non-modified alginate, and were even upregulated in macrophages seeded on anti-inflammatory alginate, though to a lesser extent.
Conclusion: These findings show that alginate hydrogels may be able to induce an M1-like phenotype in macrophages, via upregulation of CD80 and CCL2. However, proinflammatory modification of these hydrogels did not seem to enhance this upregulation. Instead, the results of this study suggest that alginate may be a naturally inflammatory material. Further investigation of the inflammatory properties of alginate are necessary to explore its potential as an M1 phenotype-promoting biomaterial.

The field of nanoparticles for biomedical applications has become one of the most promising and most studied topics in the last years because it offers potential enhancement in pharmaceuticals, medical imaging and diagnosis, cancer treatment, implantable materials and tissue regeneration. In terms of therapeutic applications, especially drug delivery vehicles have been intensively studied. In this case, hollow particles have aroused tremendous interest due to their unique properties such as large surface areas, low densities and high loading capacities due to a protected hollow core. The transport of therapeutic, which can be either, encapsulated, covalently attached or adsorbed onto hollow particles, has been shown to be highly beneficial for multiple reasons compared to simple drug administration e.g., the cargo is protected against enzymatic degradation and the total drug administration can be reduced, which minimizes the risk of side effects.
HMSC were synthesized through a hard template-based method. First, ellipsoidal hematite particles were synthesized in a solvothermal process and coated with silica in a sol gel process afterwards. The iron oxide core was removed by etching resulting in HMSC. The porosity of as-prepared particles was analyzed using nitrogen adsorption-desorption method revealing a pore size of circa 4 nm and a high surface area of 308.8 m^₂/g. Cytotoxicity was determined using cell viability test (MTT) towards human kidney cells (HEK293) which clearly demonstrates that no reduction of cell viability was observed even at high concentrations of 100 µg/ml. Uptake studies using confocal microscopy were carried out using human cervical cancer cells (HeLa) which could show the successful internalization over a period of 24 hours. For testing their capability as drug delivery vehicle, a hydrophilic antibiotic (ciprofloxacin) and a hydrophobic anticancer (curcumin) compound were loaded and a pH dependent release under physiological conditions at 37°C was monitored via UV-Vis spectroscopy. After the drug release a reduction in shell thickness of HMSC was confirmed via SEM and TEM images. This study demonstrates the suitability of as-prepared hollow silica capsules as drug delivery vehicles for a broad range of drugs, hydrophobic as well as hydrophilic. Furthermore, it would be possible to modify the surface of drug loaded HMSC with proteins, e.g. antibodies as they are often used in immunotherapeutic regimens.

In this study, the phytochelatin synthase gene of Rhizobium tropici was assembled by PCR, inserted into pUC19 vector, and further transformed into E coli DH5α cells. Our preliminary data showed that these engineered E coli DH5α cells synthesized more selenium nanoparticles than DH5α cells transformed with pUC19 vector. The scanning electron microscope (SEM) examination of the nanoparticles revealed that the nanoparticles produced by both groups of cells had a size of 100-200nm with a spherical conformation, compositional analysis using Energy-dispersive X-ray spectroscopy identified that the nanoparticles only contain selenium. The capacity of phytochelatin synthase gene transformed bacteria to produce other metallic nanoparticles, including silver and gold will be tested and the physical properties of the nanoparticles will be further analyzed using the transmission electron microscope (TEM) analysis. The research results will be reported in the MRS meeting.

Sepsis, a condition caused by severe infections, affects more than 30 million people worldwide every year and remains the leading cause of death in hospitals. Moreover, antimicrobial resistance has become an additional challenge in the treatment of sepsis, and thus, alternative therapeutic approaches are urgently needed. Macrophages are one of the most efficient pathogen scavengers. Indeed, in septic patients, impaired macrophages seem to be primarily responsible for insufficient antimicrobial defense. Nevertheless, immunostimulatory agents for reversing deactivated macrophages did not significantly improve the patient survival in clinical trials. This may be attributed to that immunostimulatory agents are not able to completely restore the function of impaired macrophages. Moreover, many bacteria, such as Staphylococcus aureus and Escherichia coli, have evolved immune escape mechanisms for thwarting phagolysosomal killing, resulting in intracellular survival. In addition, the prevalence of antibiotic resistance contributes to recurrent infections. Based on these research findings, we hypothesized that adoptive transfer of macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) may enable the immunocompromised sepsis host to boost innate immunity, prevent bacterial immune evasion, and eliminate multi-drug resistant (MDR) bacteria.

To test our hypothesis, we first designed the AMP-CatB mRNA encoding an antimicrobial peptide IB367 (AMP-IB367), a cathepsin B (CatB), and a CatB-sensitive linker. AMP-IB367 is a broad-spectrum AMP with rapid bactericidal activity. CatB, an endogenous lysosomal protein, is first translated as an inactive precursor in the cytoplasm. After translocation into the lysosome, the precursor is processed into mature CatB. Here, the CatB is incorporated to transport the AMP-IB367 into lysosomes, where the linker is cleaved by CatB and thus the APM-IB367 is released. When phagosomes encapsulating bacteria fuse with lysosomes, the ingested bacteria are exposed to both pre-stored AMP-IB367 and lysosomal antimicrobial components. Although immune evasion strategies may protect MDR bacteria from phagolysosomal killing mechanisms, the AMP-IB367 is able to kill these bacteria. In order to equip macrophages with the AMP-CatB mRNA, we synthesized a series of vitamin-derived lipid nanomaterials. We found that the mRNA delivery efficiency of V_CLNPs was 10-fold better than Lipofectamine 3000 and 50-fold better than electroporation in RAW264.7 macrophages. After optimizing the molar ratio of V_C-Lipid:DOPE:Cholesterol and the mass ratio of V_C-Lipid:mRNA, we identified the optimal V_CLNPs formulation with improved mRNA delivery efficiency over 7-fold than its initial formulation. Because V_CLNPs were also effective to deliver mRNA in bone-marrow-derived macrophages (BMDMs), we chose BMDMs for further antibacterial studies because of the clinical translatability. In vitro bactericidal results showed that MACs eliminated over 80% multi-drug resistant Staphylococcus aureus (MDRSA) or multi-drug resistant Escherichia coli (MDR E.coli) at 12 h. Next, we tested the therapeutic effects of MACs in MDRSA- and MDR E.coli-induced sepsis mice with immunosuppression. Analysis of blood at 24 h showed that MACs were more effective in cleaning bacteria than PBS-BMDMs. Furthermore, survival rate of MACs (83%) was significantly higher than that of PBS-BMDM group (40%).

In summary, we have developed macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs) via V_CLNPs-mediated delivery of mRNA. Our results prove the concept that adoptive transfer of MACs beneficially reduce bacterial burden and improve survival in MDR bacteria-induced sepsis mice with immunosuppression by restoring innate immune defense, preventing bacterial immune evasion, and eradicating infections.

According to the National Nanotechnology Initiative of America, titanium dioxide (TiO₂) is one of the most commonly manufactured nanoparticles. These nanoparticles are widely used in food, pharmaceutical, cosmetic and chemical industries as pigments, catalysts, and photovoltaic sources. The latter property is also exploited in water purification where UV-irradiated TiO₂ is a common anti-bacterial agent. However, there is no consensus on the toxicological profile of TiO₂ nanoparticles (NPs), and multiple concerns have surfaced on the potential harmful effects on human health¹. Experiments conducted on HeLa cells indicated that cells cultured with 0.1 mg/ml rutile and anatase TiO₂ NPs for 24 hrs prior to exposure to Staphylococcus aureus (S. aureus) had 350 and 250% respectively more bacteria per cell, while macrophages exposed to TiO₂ NPs ingested 40% fewer S. aureus, further increasing the risk of infection when present in vivo². The particles were observed to sequester within the cytoplasm of phagocytes simply occupying space which would normally be used for ingesting bacteria. Ingestion of TiO₂ NPs in HeLa cells resulted in an increase in membrane rigidity and extracellular LDH which was attributed to the increase in bacteria².
Another possible explanation may be in the concentration distribution of membrane cholesterol. Specifically, we demonstrate an increase in cholesterol concentration of the outer plasma membrane leaflet accompanied by the decrease in cholesterol inner leaflet concentration. Alteration of the inner/outer leaflet cholesterol ratio may also explain the increase in bacterial infectivity NPs since S. aureus requires cholesterol for proper membrane attachment and virulence. This effect was absent in ABCA1 knockout HeLa cells suggesting an interaction between TiO₂ and the ABCA, a known cholesterol membrane efflux regulatory protein that facilitates cholesterol transport across the cellular membrane. The hypothesis has also been confirmed by the RT-PCR data detecting the RNA expressions of ABCA1 and ABCG1. We found that compared to the control group, cells exposed to TiO₂ for 24 hrs had higher RNA expression levels to 240% and 170% for ABCA1 and ABCG1, respectively. These data indicate that cells exposed to TiO₂ NPs have altered cholesterol distribution within their membranes, which results in increased rigidity and enhanced bacterial infection. Further work is in progress to evaluate this effect in cells obtained from healthy human tissue.

¹Pan, Zhi, et al. "Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells." Small 5.4 (2009): 511-520.
²Xu, Yan, et al. "Exposure to TiO₂ nanoparticles increases Staphylococcus aureus infection of HeLa cells." Journal of nanobiotechnology 14.1 (2016): 34.

Introduction: Protein adsorption to a material is a complex process which causes thrombus formation, infection, and inflammation and can lead to failure of devices¹. Surface properties such as chemistry, topography, and mechanics influence protein adsorption and structural changes. Fibrinogen plays a dominant role in mediating platelet and leukocyte interactions with surfaces and its degree of unfolding can impact the extent of cell activation². Macrophages are essential in the immune response to biomaterials and the effect of protein conformational changes on their activation is not fully understood. This work aims to characterize protein adsorption and conformational changes on planar and patterned polydimethylsiloxane (PDMS) with varying elastic moduli and determine the relationship to macrophage response.
Methods: Microscale patterns were designed with three sizes of the following height : groove width : ridge width ratio: (1) 20:20:10 μm; (2) 20:20:20 μm and; (3) 10:10:10 μm. Photolithography and soft lithography are used with PDMS substrates of varying mixing ratios of Sylgard 184 and 527 to investigate the combined effect of surface topography and elastic modulus on fibrinogen adsorption and degree of unfolding. A micro-BCA assay was used to determine the amounts of fibrinogen adsorption (2.0 mg/mL in PBS) on PDMS with the range of stiffnesses. An antibody binding assay using a primary antibody to the fibrinogen gamma chain was developed to quantify the amount of fibrinogen adsorbed with exposure of the gamma chain. This assay will be extended to determine RGD density and further conformational changes of protein on PDMS. We are in the process of further interrogating the role of protein conformation on immune cell interactions using the RAW 264.7 macrophage cell line.
Results: Micropattern designs of three dimensions were prepared for photolithography and soft lithography. Contact angle measurements of PDMS substrates with varying stiffnesses indicated that ratios had relatively similar surface wettabilities with the 1:5 ratio slightly more and the 1:1 ratio slightly less hydrophobic. According to the BCA assay results, the 1:1 ratio of Syl 184 & 527 PDMS polymers adsorbed the most fibrinogen with an average of 10.50 ± 0.26 µg/cm², while the 1:5 ratio of Syl 184 & 527 adsorbed the least with an average of 8.88 ± 0.48 µg/cm². The antibody binding assay was completed and is being optimized to compare absorbance values based on the amount of fibrinogen determined from the BCA assay. This will provide an indication of changes in orientation and conformation as a result of varying surface topography and modulus.
Discussion and Conclusions: This work focuses on understanding the mechanisms underlying protein adsorption and subsequent cellular regulation abilities of microstructures and elastic modulus. The designed micropatterns provide a way to investigate the combined effect of topography and stiffness on protein adsorption and macrophage adhesion. The BCA assay indicates that varying the surface modulus has an influence on fibrinogen adsorption. The antibody binding assay will investigate whether protein conformational changes play a role in this varied amount. Future work is being completed to determine the establishment of macrophage focal adhesions and cytokine secretion profiles in response to micropatterned topography and elastic modulus. Controlling these surface properties provides a method to monitor macrophage activation and regulation of immune cell response.
References:
1. I Firkowska Boden., et al. Advanced healthcare materials, 7 (1), p.1700995, 2018.
2. P Koegler., et al. Advanced drug delivery reviews 64 (15) pp.1820-1839, 2012.

Multidrug resistance (MDR), accounting for therapeutic failure, remains a major challenge to manage cancer effectively. Currently, there are only a few strategies that are executed to conquer MDR by restoring chemosensitivity. In this respect, we report an immune suppressing nanoparticle decorated with alpha-1 acid glycoprotein (AGP), an anti-inflammatory protein, to serve as an effective chemoresistant reversal agent to modulate MDR and resensitize aggressive breast tumour cells. Hyaluronic acid-chitosan nanoparticles decorated with AGP (AGP-HA NPs) were synthesized by a sequential ionic gelation method consisting of spray drying and AGP-surface adsorption. The morphology, size, surface charge density, and composition of the synthesized NPs were characterized by utilizing SEM, DLS, Zeta potential, and FTIR, respectively. We have investigated the potential efficacy of AGP-HA NPs in reversing the MDR phenotype of MDA-MB-231 in response to anti-cancer drug doxorubicin (DOX). Our findings showed synergistic antitumor effects between HA and AGP protein as evident from the significantly enhanced efficacy of DOX post switching drug resistance phenotype of the cells to chemo-sensitive phenotype. Overall, our results suggest that AGP-HA NPs represent a viable and effective treatment option to strengthen the anticancer effects of chemotherapeutic agents and potentially contribute to improving the survival rate of patients with metastatic breast cancer.

The role of nanomedicine as an enhanced pharmaceutical strategy to deliver drugs has been studied for quite a few decades. The emergence of pH-sensitive nanoscale particles is beneficial due to its ability to release its cargo only at a high pH environment of the colon, which helps for direct targeting of the inflamed tissues in inflammatory bowel disease (IBD). Hence, we have designed the formulation of biodegradable polymer poly (lactic-co-glycolic acid) nanoparticles coated with Eudragit® S100 (EPNPS) for the delivery of novel polyphenolic compounds to reduce the inflammation caused by proinflammatory cytokines. The EPNPs were prepared by a single emulsion solvent evaporation technique and were characterized for shape and surface morphology using a scanning electron microscope (SEM) and transmission electron microscopy (TEM). The in vitro drug release was investigated using phosphate buffer saline (PBS) simulating different gut pH. The in vitro cytotoxicity of the EPNPs also evaluated and found to be highly biocompatible in nature. The inhibition of proinflammatory cytokines production also conducted using lactate dehydrogenase (LDH) assay. The characterization of EPNPs using SEM and TEM revealed spherical shape and smooth surface of the nanoparticles and was found to be 200-300nm in size. The EPNPs showed substantial improvement in the release of the drug, specifically at colonic pH. Results suggested that pH-sensitive EPNP nanoparticles as a promising candidate in the oral delivery to the colonic inflamed tissues.

Combinatory modulation of the physical and biochemical characteristics of nanocarrier delivery systems is an emergent topic in the field of nanomedicine. Here, using the combined effects of incorporation of active targeting moieties, nanoscale morphology and shape, and physical properties for anti-cancer drugs function. We investigated a novel complex made of lipid bicelles nanocarrier for the efficient delivery of a combination of Paclitaxel (PTX). In addition, we hypothesized that Parthenolide (PTL) can be used as a chemosensitizers to restore the proper apoptotic signaling and to overcome chemotherapy resistance in human lung and breast cancer cells. We synthesized and characterized self-assembled bicelles (nanodiscs) and nanovesicles which were made up of short- (DHPC) and long-chain (DPPC) lipids encapsulating PTL and PTX molecules. By using simultaneous self-assembly encapsulation, we have encapsulated Parthenolide and Paclitaxel as two cancer drugs. Dynamic light scattering, small angle X-ray scattering (SAXS), Fluorescence-activated cell sorting (FACS), and fluorescence confocal optical microscopy (FCOM) were used to quantify and visualize the cellular uptake of folate-conjugated NPs, respectively and in vivo subcutaneous rat studies were performed to investigate the samples. Self-assembled lipid discoidal and vesicular nanoparticles with low-polydispersity ~30 nm size range and identical chemical compositions were synthesized, characterized, and correlated with in vitro cancer cellular internalization, in vivo tumor accumulation and cancer treatments. Folate targeted nanodiscs not only have shown a higher tumor uptake and therapeutic efficiency, but also exhibited well-defined size, robust formation, and high-efficiency encapsulation of hydrophobic molecules, which enable the scalable manufacturing of a generalized in vivo multimodal delivery platform. A comprehensive evaluation of different effective factors, such as loading rate, surface chemistry, nanocarriers shapes, and drug combination ratios were evaluated and a comprehensive regiment for an effective treatment is introduced. The negatively charged bicelles with a PTL:PTX:Lipid molar ratio of 2.5:1:500 yielded a discoidal shape with a uniform diameter of 30 nm and a bilayer thickness of 5 nm, indicated the best anti-cancer function against both regular and drug resistive lung and breast cancer cells. SAXS analysis was also performed to provide insight into how the hydrophobic PTL and PTX molecules interact within the bicelles. Further, flow cytometry followed by confocal microscopy analyses substantiates the superior transfection efficiency of bicelles. We further analyzed how the co-encapsulation and molar ratios of drug and nanocarrier may affect the anti-cancer efficiency in vivo and in vitro. Several important discoveries in this report are summarized below. Detailed structural analysis and loading capacity of bicelles with two hydrophobic drug molecules, PTX and PTL, are performed. The structural variation of discs strongly depends on the molecular size and shape of the payload. Generally speaking, bicelle shows great potential as a nanocarrier for hydrophobic drug molecules. Both in vitro and in vivo studies on A549 cell line using drug-loaded bicelles indicate high efficacy (high cellular uptake, low IC₅₀ and MIC₉₀) for cancer treatment. Moreover, we demonstrate strong synergy with PTX and PTL with IC₅₀ and MIC₉₀ are magnitudes lower than those of the free drugs in DMSO or Cremophor El. The significant synergetic effect is observed in the MDR A549 cell line as well. The research outcome aims to provide an efficacious nanocarrier platform, which can potentially be generalized for transporting therapeutics and diagnostics to other types of cancers or diseased cells.
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Our research combines immunology and biomaterials to understand the interactions between synthetic materials and immune tissues, and to design more selective therapeutic vaccines for cancer and autoimmunity. This presentation will highlight our recent efforts toward these goals combining materials science and bioengineering tools, cell culture, animal models, and samples from human patients. In one example I will discuss new degradable polymer depots that could improve the selectivity of therapies for autoimmune diseases such as multiple sclerosis and diabetes by locally reprogramming the function of lymph nodes – tissues that coordinate immune function. A second area will present the lab's efforts to self-assemble immune signals into modular nanostructures. This rational design approach allows activation of programmable combinations and levels of immune pathways triggered. Modular control over these aspects of immune signaling could help improve the efficacy of vaccines for cancer and infectious disease, and enhance the efficiency of vaccine translation.

Introduction: Inflammatory monocytes and neutrophils contribute to deleterious inflammatory disease and tissue damage in a number of pathological conditions, such as trauma, viral infection, autoimmune disease, sepsis, and cancer. Tissue destruction results from the influx of these inflammatory monocytes and neutrophils, which secrete pro-inflammatory cytokines, reactive oxygen species (ROS). Critically, a number of agents such as steroids or cytokine-depleting antibodies are associated with unfavorable side effects, such as sepsis, gastrointestinal bleeding, and thromboembolism, indicating that improved methods are needed. Systemic depletion of neutrophils or monocytes has either not altered or has had a small effect on outcomes. Because monocytes and neutrophils are necessary for wound healing and tissue regeneration after injury, reprogramming the immune response could be a more effective strategy to minimize loss of function and enable repair.
Results: Herein, we describe our efforts at employing NPs to reprogram inflammatory cells intravascularly, which can alter their trafficking and polarization. NPs (500 nm diameter, zeta potential < -30 mV) and intercept the immune cells prior to their extravasation and redirect some to the spleen, with a fraction trafficking to the site of trauma or disease. Importantly, the NPs are attenuating the innate response yet do not alter the adaptive immune response and also support enhanced regeneration and functional recovery at sites of trauma or disease. These particles have been applied to models of viral infection and spinal cord injury to limit injury and tissue degeneration, and in models of metastatic cancer to alter metastasis.
Conclusions: NP infusion can reduce inflammation resulting in increasing tissue regeneration and normal tissue function. NPs-mediated rapid reprogramming of innate immune cells has a sustained impact on microenvironment modulation, which initiates a cascade of events, including induction of gene expression profiles associated with regeneration. Collectively, this strategy of targeting the innate immune cells in the vasculature prior to their extravasation to a injury or disease site may have broad applications to attenuating inflammation-mediated tissue damage.
Acknowledgements: This study was supported by the National Institutes of Health (NIH) R01 EB013198 and R01 AI148076.
Conflicts: LDS serves as a consultant and has a financial interest in Cour Pharmaceutical.

This presentation highlights results from our molecular based approaches to direct the immune system toward tolerance and suppression. We have developed tissue-anchored enzymes – chimeric enzyme-carbohydrate binding fusion proteins in order to direct localized immunometabolism toward suppression. Specifically, indoleamine 2,3 dioxygenase, which depletes tryptophan through the kynurenine pathway, is fused to galectin 3, which binds extracellular glycans and affords prolonged tissue retention. This approach has provided potent, confined metabolic programming promoting tissue homeostasis in multiple inflammation models including osteoarthritis and periodontal disease.

Introduction: Deficient vascularization of engineered tissues is one of the major causes of inadequate tissue integration and function in vivo. A potential solution to this problem is the implantation of pre-vascularized biomaterials, such as those prepared from human adipose microvascular endothelial cells (HAMECS) and adipose-derived mesenchymal stromal cells (MSC) [1-2]. However, these pre-vascularized constructs will encounter an inflammatory environment upon implantation, and it is not known how this will affect the engineered blood vessels. Macrophages, the primary cells of the inflammatory response, are key regulators of tissue vascularization and integration of implanted biomaterials. Even though macrophages can exist as different phenotypes, each with distinct effects on blood vessels, they are also very plastic cells that are able to switch phenotypes depending on microenvironmental stimuli [3]. In the first phase of wound healing, macrophages are primarily pro-inflammatory (M1), whereas the second stage is characterized by a pro-wound resolution phenotype (M2). M2 macrophages can be derived from non-activated (M0) macrophages or they may switch from M1 (resulting in M1-M2). The diversity of these populations and their crosstalk with blood vessels are not known. Therefore, the purpose of this study was to assess the phenotypic subsets of primary human macrophages polarized to pro-inflammatory (M1) and pro-wound resolution (M2 and M1-M2) phenotypes, and how they change when cultured with 3D tissue-engineered human blood vessels in vitro.
Methods: Tissue-engineered blood vessels were formed by co-culturing HAMECs and MSCs within porous gelatin scaffolds (Surgifoam®). M0, M1, M2 and M1-M2 primary macrophages were seeded alone on the scaffolds or in co-culture with the engineered blood vessels as previously described [3]. Then, the scaffolds were digested to generate a single cell suspension and stained with a 10-marker flow cytometry panel: macrophage general marker (CD45), M1 macrophage markers (CCR7, HLA-DR, CD83, CD38 and PD-L1), and M2 macrophage markers (CD206, CD209, CD163 and CXCR4). Data were acquired on BD Fortessa™ flow cytometer. Live, single and CD45 positive cells from all the different conditions were down sampled and merged into a single expression matrix prior to dimensionality reduction analysis in FlowJo®. Cluster analysis was done using manual gating or different plugins such as FlowSom.
Results: Using this approach, we were able to differentiate the effect of the biomaterial itself from the effects of interactions with blood vessels on macrophage phenotype. While macrophages polarized to the different phenotypes were relatively homogenous in 2D culture, after 24 hours in 3D culture, even without tissue-engineered blood vessels, they became a heterogeneous population comprising 3-4 main clusters, each comprising cells that expressed varying levels of each marker. Following crosstalk with tissue-engineered blood vessels, the prior polarization states of the macrophages affected their response. In particular, M1 and M1-M2 macrophages became enriched in clusters characterized by high levels of M1 markers (such as CD38, CD80 and CD38), while M2 and M0 macrophages became enriched in clusters that were predominant in M2 markers (such as CD206, CD163 and CXCR4).
Conclusions: These findings suggest that macrophage phenotype is strongly affected by co-culture with tissue-engineered blood vessels in a way that depends on the prior polarization state of the macrophages.
Acknowledgements: This project was supported by NHLBI HL130037 to KLS. The NanoBioCel group acknowledges SAF2017-82292-R (MINECO/AEI/FEDER, UE). B.Hernaez thanks the Basque Government for the PhD grant. Flow cytometry was conducted in Sidney Kimmel Cancer Center Flow Cytometry Facility (Philadelphia)
References: [1]Freiman et al. Stem Cell Research & Therapy (2016) [2]Ben-Shaul et al. PNAS (2019) [3]Graney, P.L. et al. Science Advances (2020)

Super-hydrophilic zwitterionic polymers are a class of non-fouling materials capable of effectively resisting any nonspecific interactions with biological systems. Beyond conventional zwitterionic polymers that are biologically inert, we have designed a functional zwitterionic polymer by achieving a tradeoff between the non-specific interaction for non-fouling property and the specific interaction for bioactive functionality. Built from phosphoserine, an immune-signaling molecule in nature, this zwitterionic polymer exhibits both non-fouling and immunomodulatory properties. Conjugation of this zwitterionic polymer to uricase, a highly immunogenic enzyme, shows to proactively eradicate all the unwanted immune response, outperforming the inert zwitterionic polymers. On the other hand, this polymer could significantly prolong the half-life of uricase in vivo, overcoming the innate drawback of phosphoserine in inducing accelerated clearance. As the first demonstration of a non-fouling zwitterionic material with build-in immunomodulatory functionality, this work not only provides new insights into the fundamental design of biomaterials, but also contains far-reaching implications for broad biomedical applications including drug delivery, implants and cell therapy.

Antibiotic–resistant bacteria are a major and rising global health threat. Although this threat affects all ages, pediatric patients, and aged individuals are at increased risk of antibiotic–resistant bacterial infections. Of concern are Klebsiella pneumoniae (K. pneumoniae ) that fail to induce in vivo germinal center B cell responses, which facilitate antibody production to fight infection. Immunotherapies using antibodies targeting antibiotic–resistant bacteria are emerging as promising alternatives, however, they cannot be efficiently derived ex vivo, necessitating the need for immune technologies to develop therapeutics. In this talk, I will discuss how organoid polymer functionality and mode of Klebsiella pneumoniae membrane antigen presentation regulates ex vivo germinal center epigenetics in young and aged B cells. In this work, polyethylene glycol (PEG)–based immune organoids are developed to elucidate the effects of polymer endpoint chemistry, integrin ligands, and mode of K. pneumoniae antigen presentation on germinal center B cell phenotype and epigenetics, to better define the lymph node microenvironment factors regulating ex vivo germinal center dynamics. Notably, both PEG vinyl sulfone or acrylate fail to sustain primary immune cells, but functionalization with maleimide (PEG–4MAL) leads to B cell expansion and germinal center–like induction. Single cell RNA sequencing analysis of lymph node stromal and bulk sequencing of germinal center B cells show niche associated heterogeneity of integrin–related genes. Incorporation of niche–mimicking peptides reveals that collagen–1 promotes germinal center–like dynamics and epigenetics. PEG–4MAL organoids elucidate the impact of K. pneumoniae outer membrane–embedded protein antigen versus soluble antigen presentation on germinal centers and preserve the response across young and aged mice. Finally, I will discuss ongoing efforts to engineer immune organoids from primary B cells from non–human primates and humans.

A critical element of the immune system is tissue-level spatial organization. Cells and extracellular matrix elements are all highly organized in the lymph node, spleen, and thymus, as well as in the peripheral organs where immune cells infiltrate. Furthermore, secreted molecules are arranged in local patterns that depend on diffusion and interstitial flow. To begin to probe the functional outcomes of this intricate organizational structure, our laboratory has focused on combining live tissues and engineered biomaterials with microfluidic and micropatterning for fluidic and spatial control. We have developed microsystems to deliver drugs locally to specific regions of spatially structured tissues, to co-culture multiple structured organs under recirculating flow, and to begin to build a micropatterned lymph node. Here we will discuss our recent progress towards modeling complex immune function outside the body, to gain insight into the spatiotemporal dynamics of inflammation and immunity.

Immune monitoring approaches in human patients and animal models primarily rely on peripheral blood analysis, as the standard method for immunophenotyping. Although this strategy mirrors important information of the immune responses of the host, blood draws fail to track major immune cell populations preferentially residing in peripheral tissues, including barrier tissues such as the skin. Moreover, invasive approaches for sampling and analyzing immune cell populations from lymph nodes or skin are apparatus intensive and require special training for use. On the other hand, minimally invasive methods for quantitative monitoring of the immune status of the skin or other barrier tissues could provide valuable information in the context of patients with genetic and acquired immunodeficiency disorders, organ transplants, and vaccination, but such approaches are currently lacking. To overcome these challenges, we developed microneedle (MN) skin patches, coated with an ionically-crosslinked biocompatible polymer, which swells upon skin insertion, absorbing interstitial fluid (ISF) and enabling migration of cells into the patch in a minimally invasive, cost-effective manner. These MN arrays were used to monitor antigen-specific tissue-resident lymphocytes in the OVA-vaccinated and LPS-infected C57Bl/6 mice skin. We demonstrated that by applying the patches to skin for 12 hours, several thousand live cells are recovered without immunizing or altering pre-existing T cell or B cell responses against target antigens. In response to local intradermal LPS injection, without prior vaccination, MN arrays were able to sample more abundant lymphocytes and inflammatory cytokines compared to naïve, untreated mice. Our data strongly suggest that the sampling MN skin patches could be harnessed as a powerful tool to quantitatively elucidate immune signatures of infectious or autoimmune diseases in the future.

Cancer is one of the most frequent causes of death globally, and cancer biomarkers are promising for detecting cancers early.¹ This disease kills more than 8 million people per year, and most probably, we may need to expect this number to increase by more than 50% in the coming decades.¹ Early diagnosed cancers are relatively easier to treat and cure. There are various techniques to screen cancers as early as possible. Liquid biopsy is a promising approach since it is used to diagnose tumor-specific biomarkers non-invasively.² Moreover, it can detect genetic material (circulating tumor nucleic acids, ctNA), which is important in various applications, including cancer screening.^3,2 Detecting ctNA in the blood is challenging, because of the low ctNA concentration and the low frequency of mutations compared to wild-type sequences.² Nanotechnology methods can help to address this challenge. In particular, the Scanning Tunneling Microscopic (STM)-assisted break junctions method (STM-BJ)⁴ has recently allowed the first demonstration of detection and identification of RNA via single-molecule conductance.³ This is an ideal method for liquid biopsy since it is non-invasive and could be helpful to detect cancers without the use of surgery.² Early detection of the genetic material in cancer biomarkers, such as ctDNA and ctRNA², through liquid biopsy using nanotechnology may help save many lives from cancer patients.
In this work, we focus on characterizing ctNAs and investigate an effective method for their ultra-sensitive detection in complex samples. The main hypothesis of the study is that the sequences of ctNAs can be used to detect cancers as the literature supports.⁵ The selected biomarkers are KRAS, BRAF, and Nras, in agreement with data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA).^5,6 We use STMBJ to measure and compare the conductivity of these ctNAs, comparing the conductivity of wild type and mutated biomarker sequences of different lengths. Detecting electrical fingerprints from biomarkers will greatly impact the early detection of cancers, allowing beginning treatments early, potentially saving many lives.

References
1. Campbell PJ, Getz G, Korbel JO, et al. Pan-cancer analysis of whole genomes. Nature. 2020;578(7793):82-93. doi:10.1038/s41586-020-1969-6
2. Das J, Kelley SO. High-Performance Nucleic Acid Sensors for Liquid Biopsy Applications. Angew Chemie - Int Ed. 2019. doi:10.1002/anie.201905005
3. Li Y, Artés JM, Demir B, et al. Detection and identification of genetic material via single-molecule conductance. Nat Nanotechnol. 2018;13(12):1167-1173. doi:10.1038/s41565-018-0285-x
4. Xu B, Tao NJ. Measurement of single-molecule resistance by repeated formation of molecular junctions. Science (80- ). 2003;301(5637):1221-1223. doi:10.1126/science.1087481
5. Rheinbay E, Nielsen MM, Abascal F, et al. Analyses of non-coding somatic drivers in 2,658 cancer whole genomes. Nature. 2020;578(7793):102-111. doi:10.1038/s41586-020-1965-x
6. Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA) in: European Journal of Endocrinology Volume 154 Issue 2 (2006). https://eje.bioscientifica.com/view/journals/eje/154/2/1540341.xml. Accessed June 8, 2020.

In immunotherapy, cytotoxic T cells orchestrate an organized macromolecular assembly of T cell receptors (TCRs) in the immunological synapse (IS) to successfully commit antigen recognition and effector functions. Shortly after the contact of T cells with the APCs or cancer cells, the complex of the ligated TCR and costimulatory molecules will dynamically cluster in the IS. Initially, the TCR-CD3-pMHC complexation would generate biochemical and mechano-cues, augmented by the CD28 co-stimulation through the PI3K signaling pathway. Then the ligated TCRs transfer to the central SMAC to trigger the downstream tyrosine kinase cascades. Eventually, the dynamic interactions between the TCR clusters and the actomyosin cytoskeleton lead to the maturation of the IS and the full activation of T cells. Recent researches have shown that the enhanced flexibility of the immunoreceptors would improve the T cell activation and proliferation through an enhanced mechanosensing of the TCR. Lambert et al. showed that the CD4+/CD8+ T cell populations showed an elevated proliferation and robust cytokine production with a decrease in the rigidity of PDMS stimulating microsphere. Moreover, an artificial T cell-stimulating matrix with two stimulating signals and low mechanical stiffness was reported to better promote the CD3 clustering, T cell proliferation, and cytotoxic functionality of the CD8+ T cells. Though not yet fully proven, the high flexibility and mobility of the stimulating ligands should contribute to the augmented outcomes of the activated T cell. Inspired by this idea, we emulated the dynamic process of TCR ligation and clustering by utilizing the supramolecular host-guest interactions to conjugate the activating ligands onto an APC-mimetic substrate, where the cyclodextrin-modified ligands are likely to be relocated under the retrograde actin flow-mediated force during the TCR clustering. First, we synthesized the adamantane-modified methacrylated hyaluronic acid (MeHA-ADA) macromers and separately conjugated the anti-CD3ε (stimulation signal 1) and anti-CD28 (co-stimulation signal 2) antibodies to the b-cyclodextrins. Then both signal molecules attach to the backbones of MeHA-ADA through the non-covalent reversible interactions between the b-cyclodextrins and adamantanes. Control substrates were fabricated by chemically crosslinking the antibodies onto the macromer through thiol-ene reactions. The dynamic host-guest T cell activating substrate was characterized to have similar mechanical stiffness and to bear the non-significantly different amounts of antibodies and RGD peptides compared with those of the control substrates. Then the freshly isolated murine naïve CD8+ T cells were cultured on all substrates for 7 days before the verification of their effects on T cell activation and proliferation. The RT-qPCR results showed that the expression levels of IFNγ, IL-2, TNFα, and NFATc1 were significantly promoted in the CD8+ T cells cultured on the dynamic host-guest substrate compared to those of the control substrates and free-adding antibodies. Besides, the flow cytometry results showed that the surface markers of activated T cells, CD44 and CD69, were significantly elevated on the T cells cultured on dynamic host-guest substrates. Furthermore, over 68% of the naïve T cells cultured on the dynamic host-guest substrates differentiate into the CD45R+ effector T cells and CD45R+/CD62L+ effector memory T cells, accompanied by robust proliferation and cytokine secretion. These results indicate that the reversible nature of supramolecular interactions offers great prospects for the design of T cell-activating platforms, combining the soft mechanical properties of the lymphoid tissues and complex ligand presentation of the APC membrane to promote the activation of cytotoxic T cells for the future clinical use. Our work provides valuable insights for the biomimetic design of platforms with wide therapeutic applications in immunotherapy.

Nanoparticle-based constructs have shown enhanced performance when delivering immunostimulatory molecules in cancer immunotherapy – an emerging approach to treat cancer. Their endosomal organization, however, has received little attention for targeting intracellular receptors. This talk will describe how the spatial organization of endosome-targeted constructs can influence their ability to induce downstream cell signaling. As a model system, Toll-like receptor 9 activation of macrophage induced by CpG-constructs will be examined. First, we will discuss how endosomal organizations of these constructs can be controlled by the surface curvature. Second, the spatial organizations will be correlated with immunostimulatory activity of the constructs. Finally, we will describe the implications and promises of these results for different targeted-delivery systems.

The immune activity of cytotoxic lymphocytes cells is regulated by many chemical and physical cues, whose integration mechanism is still obscure. As other lymphocytes, Cytotoxic lymphocytes act in a highly heterogeneous and diverse environment, with rapidly and abruptly changing physical properties and chemical compositions. Here, we engineered a new nanoscale platform which allows to control and integrate chemical, mechanical, and spatial stimuli for Natural Killer (NK) cells, and screen the individual and cumulative effect of these stimuli on NK cell morphology and cytotoxic activity. This platform is based on nanowires produced from the bottom-up, which are confined within micron-scale stimulating domains. These domains mimic to a certain extent professional antigen-presenting cells, such as dendritic cells. The nanowires within the domains provide the mechanical stimulation of NK cells, as we recently reported [1]. Whether the enhanced activation is due to the high compliance of the ultra-flexible nanowires to the mechanical forces, or to the nanoscale topography that the nanowires impose on the cell membrane, is still unclear.
To address this question here, we compared NK cells stimulated on domains of nanowires with different diameters. These nanowires comply differently to the mechanical forces applied by NK cells while producing similar topographical environments. In our platform, we also mimicked the spatial discontinuity of antigens into nanosized clusters, by selective immobilization onto the nanowire tips. We seeded primary NK cells on these heterogeneous surfaces and found two distinct subpopulations split by physical location: either on the top of the nanowire islands or between the islands. The two subpopulations vastly differed in morphological features of the cells, including circularity, length, spreading, and orientation. Furthermore, the cells in the two subpopulations were different in their activation levels, which we quantified via expression of lysosomal Lysosomal-associated membrane protein 1 (LAMP-1): only cells belonging to the subpopulation sitting on atop the nanowire islands showed enhanced activation. Finally, we showed that by independently varying nanowire flexibility via their diameter, as well as the presence of stimulating antigens, the degree of activation of NK cells could be fine-tuned.
To further explore the effect of the nanowire flexibility on the activation of cytotoxic lymphocytes, we synthesized nanowires with systematically varied length – from 2 microns to 20 microns, chemically coated them with antigens, and used them as a stimulation platform for primary NK cells and T cells. In both cases, we found that the activation of these cells assessed via the expression of LAMP-1 and the release of interferon-gamma increased with the increase in the nanowire length. These results confirm that the nanoscale mechanical compliance of the interface between NK cells and T cells plays a critical role in their immune stimulation.
Our novel platform demonstrates an unprecedented level of simultaneous control over chemical and physical stimuli, with an unparalleled spatial resolution at the nanometric scale. The observed immune response of NK cells to the fine variations in both physical and chemical stimuli provides an important insight into the mechanism of their cytotoxic activity. Besides, the demonstrated microenvironment for NK cell stimulation, which is engineered with features that independently tune chemical and physical stimuli, paves the way to future technologies for the ex vivo expansion and activation of cytotoxic lymphocytes in immunotherapy.
[1] Le Saux et al, G. Le Saux, N. Bar Hanin*, A. Edri, U. Hadad , A. Porgador, and M. Schvartzman Adv. Mater. 31,1805954 (2019)

The control or eradication of numerous infectious diseases relies on widespread vaccination, which in turn currently depends on cold chain continuity and the availability of trained medical personnel across the globe. Supramolecular vaccines based on short peptides have been previously found to be highly thermally stable, potentially reducing requirements for a continuous cold chain. Here we describe recent efforts to render peptide self-assemblies immunogenic when delivered via mucosal routes, along with additional strategies for designing easily administered and stable tabletized forms that are immunogenic sublingually (under the tongue). Combinations of self-assembly, modification with the mucus-inert polymers polyethylene glycol (PEG) or proline-alanine-serine peptides (PAS), mucosal adjuvants, and sublingual delivery enabled systemic immune responses against short peptide epitopes, which otherwise are poorly immunogenic sublingually. Stabilized self-assemblies were formulated into highly porous, thermally stable, and easily administered tablets which rapidly dissolved under the tongue to deliver peptide self-assemblies and elicit systemic immune responses. In mice, sublingual immunizations with tabletized self-assembling peptides elicited antibody responses against both model epitopes and an epitope from M. tuberculosis. Further, the tablets’ immunogenicity was not diminished after being heated for 1 week at 45 degrees C, in contrast to traditional carrier-based vaccines that were thermally sensitive. Together these strategies are aimed at reducing barriers for global vaccine distribution and administration by reducing dependence on continuous cold chains and highly trained medical personnel.

Adjuvants play critical roles in shaping the immune response to vaccines. Here we describe a novel nanoparticle adjuvant, saponin-MPLA nanoparticles (SMNP), formed by the immunostimulatory compound saponin, the Toll like receptor (TLR)-4 agonist monophosphoryl lipid A (MPLA), cholesterol, and phospholipids. These components self-assemble into striking ~40 nm diam. cage-like structures reminiscent of ISCOMs (Immunostimulatory complexes) previously reported for saponin/phospholipid complexes, but have synergistic immunostimulatory properties from the combined action of saponins and MPLA. SMNP exhibited striking potency in both mouse and non-human primate (NHP) preclinical animal models of vaccination with a variety of subunit vaccines. Protein vaccines adjuvanted with SMNP elicited robust germinal center B cell and Tfh responses, leading to strong serum IgG responses and (in NHPs) HIV tier 2 neutralizing antibody responses. Strikingly, SMNP exhibited distinctive mechanisms of action, altering lymph flow and promoting antigen entry into draining lymph nodes, in a manner dependent on co-assembly of MPLA and saponin together in the same nanoparticle. Altogether, these findings suggest SMNP may be a promising novel adjuvant for subunit vaccines in settings of HIV, COVID-19, and other infectious diseases.

Immunomodulatory therapies such as cancer immunotherapies and prophylactic vaccines often depend on adjuvants to drive potent and directed immune responses. Innate immune receptor agonists have become a popular category of adjuvants due to their specificity and robust effects. A subset of these agonists (e.g. CpG ODN, poly(I:C), and cyclic dinucleotides) are nucleic acid derivatives that activate cytosolic or endosomal receptors in immune cells, including several classes of dendritic cells (DCs). Stimulated DCs can then present antigen and stimulate naïve T cells for generation of the appropriate adaptive immune response. Unfortunately, these agonists are susceptible to degradation by nucleases in vivo, and thus enhanced stabilization and targeting should prolong their function and enhance their effect. We hypothesized that encapsulation of these negatively charged agonists in cationic polymeric nanoparticles would provide an increased agonist half-life and promote targeting to intracellular compartments of immune cells. Here, I discuss the development of methods to encapsulate nucleic acid agonists in biodegradable cationic poly(beta amino-ester) (PBAEs) nanoparticles as well as the specific particle properties (i.e., size, surface charge, polymer chemistry) that lead to a 13-fold increase in innate immune cell activation as measured by type I interferon production. By controlling stability, targeting, and release profiles of this promising class of nucleic acid agonists, we were able to direct the downstream adaptive immune response to a subunit vaccine, significantly enhancing both antibody concentration (1.5-fold) and affinity (14-fold). This nanoparticle platform can be used for the delivery of a range of therapeutic and stimulatory nucleic acids for improved cancer immunotherapies and infectious disease vaccines.

Cancer immunotherapy can also be augmented with innate immune cell-targeting adjuvants to elicit potent immune activation and a strong anti-cancer response. Despite their potential in both vaccines and as part of cancer immunotherapies, many small molecule toll-like receptor (TLR) agonists have been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. In this work, we overcome these shortcomings by generating PEG-PLA nanoparticles presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesized that the pharmacokinetic profile of the nanoparticles would minimize systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor draining lymph nodes. In conjunction with anti-programmed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a nanoparticles slowed tumor growth, extended survival, and decreased systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These nanoparticles constitute a modular platform for controlling pharmacokinetics of small molecule immunostimulatory molecules, resulting in increased potency and decreased toxicity. Together both of these studies demonstrate the value of polymer nanoparticle delivery systems for targeted delivery of different adjuvants (many of which have not made it to the clinic due to toxicity or poor stability) to enhance safety and efficacy for use in either prophylactic vaccines or as part of cancer immunotherapies.

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.

In the current era of the COVID-19 pandemic, there is no greater need than vaccines. Dengue, a mosquito-borne viral disease, is a neglected disease in the tropical world, where an estimated 2.5 billion people live at risk of infection each year¹. To date, no safe and effective vaccine exists for Dengue. Most virus vaccines are live-attenuated vaccines (LAVs) containing a weakened virus that produce an effective immune response. However, LAVs typically suffer from safety limitations and, more importantly, require cold chain storage². In contrast to LAVs, protein-based subunit vaccines are considered safer. However, subunit proteins as vaccine antigens are typically poorly immunogenic and require adjuvants that require both co-delivery and controlled release for maximum efficacy³. In this talk, I will describe our efforts to achieve cold chain independence and vaccine delivery using biomaterials.

Here, we design and formulate microneedle-based intradermal vaccines based on the live virus as well as subunit proteins for protection against the Dengue virus. Microneedles are miniaturized needles (100 μm to 1mm) typically assembled as an array and applied onto the skin. Specifically, I will talk about the design and development of (i) LAV² and (ii) protein subunit³ microneedle-based vaccines using the Dengue virus as a model system. In the first part of my talk, I will discuss the development of polymeric matrix to formulate the LAV vaccine on microneedles for improving the viability at room temperature for up to 4 weeks without loss of efficacy. During the second part, I will talk about developing polymeric multilayer thin films on microneedles encapsulating a subunit vaccine (protein antigen and two adjuvants) for achieving controlled release from days to weeks.

References
1. Bhatt S et al. Nature, 2013, 496, 504-507.
2. Turvey ME et al., Bioeng. Transl. Med., 2019, 4, e10127.
3. DSSM Uppu et al., J. Control. Release., 2020, 317, 130.

Human Immunodeficiency Virus (HIV) transfers from special, non-endolysosomal compartments in myeloid dendritic cells and macrophages to T cells in a process called trans-infection. It has been demonstrated that lipid-wrapped gold nanoparticles that contain the ganglioside, GM3, can recapitulate viral segregation in non-endolysosomal compartments in CD169-expressing dendritic cells and macrophages, and some findings suggest that GM3-presenting nanoparticles can even reconstitute a virological synapse after addition of T cells. However, for a number of reasons, including low payload and their non-degradable nature, gold nanoparticles are not ideal for exploiting the GM3-CD169 mediated accumulation of nanoparticles at the contact between antigen presenting cells and T cells for important applications. In this presentation, we will provide an overview of our recent progress in utilizing lipid-wrapped polymer nanoparticles as HIV-mimicking nanoparticles. We have assembled GM3-presenting, lipid-wrapped polymer nanoparticles with different polymer cores. Due to differences in the core composition, the NP had different glass transition temperatures that resulted in different stiffnesses at physiological temperatures. We mapped the intracellular distribution of the nanoparticles in CD169-expressing macrophages through confocal microscopy. Our findings suggest that in addition to the presentation of the ganglioside GM3 in the membrane around the nanoparticle core, the stiffness of the core plays an important role for successfully mimicking viral behavior. We found that while soft nanoparticles (Young’s modulus of < 100 MPa) exclusively enter an endolysosomal pathway, for nanoparticles with a stiff core (Young’s modulus ≈1.4 GPA) a significant fraction of cells shows a different trafficking pattern that results in a segregation in non-endolysosomal compartments. The staining patterns of these compartments resemble those of virus containing compartments (VCCs) known to be reservoir sites for HIV.

Interventions that engage the power and specificity of the immune system have enormous potential to improve human health, yet the efficacy, safety, and utility of many promising immunomodulatory agents is limited by critical drug delivery barriers. This talk will describe current research in my laboratory focused on engineering nanostructured polymeric materials for intracellular delivery of immunomodulatory proteins, nucleic acids, and small molecules. Specifically, I will discuss our recent efforts in developing environmentally-responsive “smart” materials that open access cytosolic immunoregulatory machinery and how these materials can be leveraged to improve responses to cancer immunotherapy and infectious disease vaccines.

Immunotherapeutic approaches are effective by either harnessing the ability of the host immune system to recognize and eliminate cells that display tumor antigens, providing external stimuli to ramp up the destruction of tumor cells in an antigen-specific manner, or by negating the ability of the tumor cells to inhibit host immune cell activation. The latter option is particularly attractive as it avoids constitutive activation of immune cells, which can result in collateral damage to healthy tissues. This can be accomplished by exploiting molecular signals within the tumor microenvironment (TME), so that the tumor shifts from being “immunologically cold” and imune-evasive to “immunologically hot” and susceptible to immune elimination (e.g. checkpoint inhibitor - Keytruda®; anti-PD-1). Although these new immunotherapeutic approaches are a significant improvement on more conventional therapies, they still fail to rid the patient of tumor burden in most cases. As such, active research is underway to uncover other molecules that engender the ‘cold’ climate of the TME. One molecule that is ubiquitous in the TME and now emerging as a major source of immune suppression is lactate. Lactic acid is an intermediate in glucose metabolism and at physiological pHs is found as the conjugate base, lactate. Lactate is surprisingly both a byproduct and energy source for cancer cells, depending on the availability of oxygen. Within the TME, lactate drives the differentiation of immunosuppressive cell subsets, such as myeloid derived suppressor cells, which have the ability to prevent the maturation of dendritic cells (DCs), thereby halting subsequent anti-tumor adaptive immune responses. Lactate also impairs the differentiation of monocytes into macrophages (MΦ) and DCs and the LPS maturation of MΦs and DCs in the TME. Evidently, lactate immunometabolism is critical to the host’s immune response to cancer. However, we lack a defined model to replicate lactate conditions seen in the TME. In vitro models of solid tumors vary in complexity and range from tumor-derived cell lines to 3D models of the tumor microenvironment. More often than not, these models fail to recapitulate the complex milieu of immune factors and immune cells that are present in the in vivo setting and crucial to evaluating the efficacy of immunotherapies. Moreover, current cancer models fail to isolate the effects of lactate alone, which can conflate interpretation of results and leaves the efficacy of therapeutic strategies targeting lactate unanswered. Moreover, a combinatorial model, where lactate can be incorporated in combination with other molecules/ conditions of interest (e.g. hypoxia) is highly desired. Biomaterials offer a potential strategy to recapitulate the biochemistry of the TME, including lactate bioavailibility. Here, we engineered a TME-mimicking system that is able to replicate one critical component of the TME, endogenous lactate levels, to understand the direct effect of localized lactate concentrations on immune cells. This system utilizes peptide hydrogels loaded with GM-CSF and degradable lactate-based polymeric microparticles. The hydrogel environment encapsulates the microparticles in a hydrated peptide mesh which allows for its hydrolysis into monomeric lactate and the subsequent accumulation of lactate in the hydrogel. Additionally, GM-CSF, a suppressive secretory chemokine produced by many tumors, serves to recruit innate immune cells to the hydrogel. Such a platform is expandable, where lactate and other immunogenic moieties can be investigated together. Moreover, the isolated effects of lactate on immune cells in vivo can provide evidence for targeting this molecule in immunotherapies.

The transport of fluids, biomolecules and cells from the tumor microenvironment to draining lymph nodes and into the circulation is facilitated by the concerted influence of the blood and lymphatic vascular systems. Our efforts to characterize the impact of these transport processes on disease progression, in particular by regulating both metastasis and adaptive immunity, as well as to develop therapeutic approaches for tumor therapy that mitigate these effects by leveraging engineered biomaterials, will be described.

Immunotherapy can be made safer and more effective by using materials approaches to control the spatial and temporal exposure to immuno-stimulants. We previously described a polymer-nanoparticle (PNP) based hydrogel that is biocompatible, long lasting, and injectable<!--[endif]---->^{1, 2}, traits that we hypothesized would improve controlled local delivery of immunotherapy. Here, we report that PNP hydrogels prolong the local exposure to CD40 agonist antibody (CD40 mAb), improving tolerability and therapeutic efficacy in the B16 model of melanoma.<!--![endif]---->
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We used positron emission tomography (PET) imaging to confirm hydrogel delivery altered CD40 mAb pharmacokinetics over the course of 12 days. Compared to dose-matched local bolus injections, hydrogels redistributed antibody exposure to the injection site and the tumor draining lymph node, increasing CD40 mAb AUC by 96% and 35%, respectively. As anticipated, hydrogel delivery reduced CD40 mAb loss to off-target antigen sinks such as the liver and the spleen, where CD40 mAb AUC decreased by 15% and 23%, respectively.

Consistent with these findings, we observed reduced toxicity in mice treated with hydrogels. In particular, mice treated with CD40 hydrogels had attenuated weight loss and recovered their weight more quickly than bolus controls. Serum cytokine analyses confirmed that hydrogels mitigated cytokine storm compared to dose-matched local bolus controls. Toxic side effects were further diminished by reducing the dose of CD40 mAb in the hydrogels, but reduced dose did not impair antitumor efficacy. Overall, these results indicate that delivery via PNP gel improves safety and maintains efficacy of CD4 mAb at low doses.

This approach did not require modifying cargo in any way, thus we anticipate hydrogel delivery will also improve the safety of other potent biologics, including cytokines and bispecific antibodies. Overall, PNP hydrogels may provide a highly translatable solution to the problem of immune-related adverse effects.

References

1. Appel, E.A. et al. Self-assembled hydrogels utilizing polymer-nanoparticle interactions. Nat Commun 6, 6295 (2015).
2. Stapleton, L.M. et al. Use of a supramolecular polymeric hydrogel as an effective post-operative pericardial adhesion barrier. Nat Biomed Eng 3, 611-620 (2019).
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Cancer immunotherapy is fast emerging as the most promising treatment approach for a number of cancers, fueled by the breakthrough success of immune checkpoint inhibitors which promote immune-mediated clearance of tumors. However, the response rates to checkpoint inhibitors remain limited to 10-40% of patients, and it is a significant clinical challenge to identify candidates for treatment to minimize immunotherapy-related adverse events. Studies have shown that the presence of tumor-infiltrating lymphocytes (such as CD8+ T-cells, which drive tumor-cell killing) is one of the strongest prognostic factors predicting overall survival across a large number of cancers. Tumor biopsies have shown that during immunotherapy, responders exhibit an influx of activated CD8+ T-cells to the tumor site, while non-responding tumors continue to exclude them. Thus, the ability to visualize the distribution of tumor-infiltrating lymphocytes before and during treatment will allow us to observe and monitor the response status to therapy. However, it is a significant challenge to image and track immune cells in-vivo because they are relatively sparse and highly mobile. There is a need to develop new tools that can non-invasively assess the evolving immune composition of the tumor microenvironment, allowing evaluation of therapy-induced responses in real time.

Imaging in the near-infrared window (NIR-II 1000nm-1700nm) has generated growing excitement in recent years for deep-tissue optical imaging approaches. Biological tissue in this spectral range exhibits reduced scattering of photons and autofluorescence, enabling for high resolution imaging of deep tissue structures. Rare-earth nanoparticles (RENPs) have emerged as superior imaging agents due to their emission bands in regions of near-zero tissue autofluorescence (1300-1700nm), high photostability, and low intrinsic toxicity in a variety of animal models. We leverage advances in near-infrared imaging technologies using rare-earth nanoparticles to target and image immune cells in tumors as markers for therapeutic response. To achieve this, we synthesized small but ultra-bright rare-earth nanoparticles (sodium ytterbium fluoride doped with erbium, cerium and zinc), and tailored the size and brightness during synthesis to enable observation of microscale features in-vivo. We then used a custom PEG-rich lipid formulation to make the nanoparticles serum-stable, and conjugated targeting antibodies on the surface to target immune cells of interest. We then performed both ex-vivo and in-situ labeling of immune cells in a murine melanoma tumor model, known for its poor response to immunotherapy. We demonstrate the ability of these nanoparticles to non-invasively image trafficking of labeled immune cells to the tumor in subsets of mice undergoing checkpoint inhibitor therapy (anti-PD1), indicating immune-mediated anti-tumor activity. Our work focuses on one key immune-cell sub-type (CD8+ T-cells), but the modularity of rare-earth nanoparticles makes them ideal tools for multiplexing, allowing future studies that simultaneously image multiple cell types in tumors for a more complex understanding of tumor-immune dynamics.

Although immunotherapies have been highly effective for some forms of cancer, it has generally worked best in tumors in which there are significant numbers of leukocytes present that enable immune activation. Unfortunately, there are numerous aggressive cancer types which do not present with significant immune cell infiltration. These tumors tend to show little or no response to check point inhibitors or other therapies that block the regulatory response of the immune system; furthermore, such tumors are often also poor responders to Car-T and other more established immuno-therapeutic approaches. Cytokines can be potent immune stimulators that upregulate the innate immune response within a tumor microenvironment, causing a cascade of inflammatory signaling that can lead to activation of antigen presenting cells, and ultimately, the infiltration of tumor-specific CD8+ T cells and NK cells to the tumor site. This effect of creating a localized cytokine storm within the tumor can lead to a much more effective immune response against tumor cells; however delivery of cytokines, particularly highly potent and effective ones such as IL-12 can be difficult due to patient safety concerns. Release of cytokine to the blood stream can lead to harmful off-target effects, and these kinds of effects have made these proteins difficult to introduce as promising immunotherapies for patients. We have developed a modular nanoparticle approach using liposomal core particles and layering them with an electrostatic layer-by-layer (LBL) process in a simple and elegant method of constructing highly tailored ultrathin polymer coatings. The resulting LbL nanoparticles (NPs) have negatively charged outer layers that present polyelectrolytes such as dextran sulfate or hyaluronic acid in a hydrated brush arrangement that enables hydration, steric repulsion, colloidal and serum stability, and specific or non-specific targeting. We have determined a subset of polyanion outer layers that have high affinity and selectivity for ovarian cancer cells and, based on the polyanion composition, will cause trafficking either to the outer surface or to intracellular compartments in ovarian cancer cells. We have used this unique ability to control trafficking to create LbL NPs that can deliver IL-12 from the outer surfaces of ovarian cancer cells, thus generating highly localized depots that efficiently release cytokine and upregulate the immune response in high grade serous ovarian cancer, a cancer which has not previously benefitted from immunotherapeutic approaches. In vitro and in vivo results will be discussed, as well as release mechanisms, toxicity studies and clinical outlook for these targeted systems.

Cancer immunotherapy represented by immune checkpoint blockades has achieved remarkable clinical success. However, an outstanding challenge is that a great majority of patients fail to respond to this therapy. This is in part due to the fact that tumor infiltrating lymphocytes (TILs) become exhausted and eventually incapable to control tumor progression. Among exhausted CD8⁺ TILs, a subpopulation, termed terminally exhausted CD8⁺ T-cells, have superior cytotoxicity but largely reduced proliferation capacity, and are known to be the direct contributors to the anti-tumor cytotoxicity. However, this subpopulation does not respond to immune checkpoint blockades or most current immunotherapies and are difficult to expand and maintain with their cytotoxicity. In the first part of the talk, I will discuss a new metabolic immunoengineering approach that expands the terminally exhausted CD8⁺ TILs and sustains their cytotoxic functions leading to eradication of established solid tumors and durable cures in a majority of treated mice when combined with adoptive T-cell transfer immunotherapies. Next, I will discuss a new cancer vaccine platform we developed recently for personalized neoantigen vaccine delivery, termed polycondensate neoepitope (PNE). PNE is a redox-responsive vaccine made through a reversible polycondensation reaction of peptide neoantigens and adjuvants together with a tracelessly responsive linker-monomer. Peptide-based neoantigens with diverse sequences and structures could be co-polymerized with molecular adjuvants to form PNEs of high loading capacity for vaccine delivery without adding any carriers. The redox-responsive PNEs with controlled molecular weights and sizes efficiently targeted and accumulated in draining lymph nodes, and greatly promoted the antigen capture and cross-presentation by professional antigen presenting cells. Mice immunized with PNEs showed markedly enhanced antigen-specific T cell response and the protective immunity against the tumor cell challenge.

Hypoxia, a state of low oxygen tension, is one of the major obstacles in the treatment of solid tumors due to its inhibitory potential on cytotoxic immune cells. Targeting hypoxia-driven immunosuppression is a promising approach to prevent tumor escape from immunosurveillance. Biomaterials with a capacity to produce oxygen and be delivered near a tumor bed represent a viable strategy for reversing local hypoxia. This approach could boost effector functions of antitumor T cells, and potentially reinforce current cancer immunotherapies. Recently, we engineered injectable oxygen-generating biomaterials, known as cryogels. Oxygen-generating cryogels (O₂-cryogels), with their unique physical features, including mechanical robustness and syringe injectability, were designed to release oxygen in a controlled and sustained fashion. We found that local oxygenation from O₂-cryogels downregulated hypoxia-inducible gene expression in hypoxic murine melanoma and reduced the accumulation of immunosuppressive metabolites such as extracellular adenosine. Strikingly, O₂-cryogels released the break on the tumoricidal function of hypoxic T cells against melanoma in a very short time (< 4h). Mechanistically, O₂-cryogels i) enhanced T cell–tumor cell interactions; ii) increased T cell-mediated production of cytotoxic proteins (perforin and granzyme B) fatal to tumor cells; and, as a result, iii) restored the killing ability of antigen-specific T cells. Collectively, O₂-cryogels provide a unique platform to expand our understanding on how hypoxia affects immune cells and may leverage emergent cancer immunotherapies to circumvent hypoxia-driven immunosuppressive mechanisms and achieve more predictable and favorable outcomes.

Acknowledgement: This work was supported by Northeastern University Seed Grant/Proof of Concept Tier 1 Research Grant and NSF CAREER award (DMR 1847843).

Increased tumor stiffness and alterations in viscoelasticity has been implicated in cancer progression. Viscoelastic materials exhibit stress relaxation, which results in resistance to a deformation relaxing over time.Emerging evidence indicates that macrophage immune cells play a key role in regulating cancer progression and can constitute up to 50% of the tumor mass. Macrophages migrate into the tumor microenvironment where they can polarize to a cancer supporting (anti-inflammatory) or cancer inhibiting (pro-inflammatory) phenotype. Current work has demonstrated the impact of 2D elastic substrates on macrophage phenotype. However, the impact of viscoelasticity on macrophage polarization and migration in 3D is unknown.
Here, we develop an alginate-collagen based matrix that has tunable viscoelastic properties to examine how matrix viscoelasticity impacts macrophage migration and phenotype. The choice of collagen as the biochemical ligand is to mimic the stromal matrix where immune cells are typically found. Importantly, the viscoelastic properties are tuned independent of polymer concentration and Young’s modulus by varying the molecular weight and crosslinking of the gels, allowing for the impact of matrix viscoelasticity to be specifically demonstrated. Specifically, we can tune the characteristic stress relaxation times from ~100 to 5,000 seconds while keeping the initial Young’s modulus of all the materials at ~2kPa. U937 pro-monocytic cells lines were encapsulated in the 3D alginate-collagen matrix and cell migration was assessed using confocal time-lapse microscopy. The results demonstrate enhanced cell migration with faster matrix stress relaxation. Ongoing studies are examining the impact of matrix viscoelasticity on U937 polarization as well as elucidate the biophysical mechanisms of U937 immune cell migration.

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody-mediated effector killing by innate immune cells through complement activation (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP). As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody-recruiting polymers (ARmacro) as a novel class of immunotherapy. ARmacro consist of a cell-binding motif linked to a polymer that contains multiple small molecule antibody-binding motifs such as dinitrophenol and rhamnose along its backbone or at dangling chain ends. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane or a ctclooctyne that covalently conjugates to the glycocalyx of metabolically azide-labeled cells. We demonstrate that macro allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that macro-treated cancer cells are prone to antibody-mediated killing through phagocytosis by macrophages.

Introduction: The interactions between platelets and circulating tumor cells (CTCs) facilitate the survival and dissemination of these cells during the metastatic cascade by cushioning the CTCs and thus protecting them from both shear stress and the immune response [1-3]. We recently demonstrated that drug free-platelet decoys display an anti-metastatic effect on disseminating cancer cells in vivo by providing an anti-platelet effect [4]. The goal of this current study is to design a platelet-based drug delivery system capable of targeting CTC clusters, which are multicellular assemblies of CTCs and platelets, consolidated by fibrin. Developing novel therapeutic strategies to abate the enhanced metastatic potential of these clusters (as opposed to single CTCs) is essential to limit metastatic dissemination and increase CTC vulnerability in the circulation. A system leveraging the effect of decoy platelets while incorporating a drug delivery component could effectively target and dissociate CTC clusters.
Materials and Methods: For the development of this cellular engineering system, the FDA-approved therapeutic agent Tissue Plasminogen Activator (TPA) was immobilized on the surface of a Lyophilized Platelet (LP) carrier. The LP carrier was chosen because it preserves platelet surface structures while inhibiting platelet function. This system was characterized by flow cytometry and a fluorescence assay to determine the carrier decoration level and drug loading. The loading method was optimized for incubation time and component concentration. Flow cytometry was used to determine the availability of GP IIb/IIIa receptors, integral to the system's interaction with CTCs. Finally, fibrinolytic and clot lysis activity of the system was measured using a fluorescent assay and an absorbance assay, respectively.
Results and Discussion: The results demonstrated that TPA was successfully loaded onto the LP carrier with a high level of decoration (97.8% decorated LP) and loading efficiency (38.5%). Flow cytometry confirmed that the GP IIb/IIIa receptors needed for the system to interact with cancer cells were available for CTC binding. The fibrinolytic assessment demonstrated that TPA retained its effectiveness when loaded onto the system. The fluorescent assay indicated that the rate of fibrinolysis was maintained for loaded TPA and the clot lysis assay showed almost identical curves for absorbance. These results demonstrate that the loading was successful, and the system components retained their functionality.
Conclusions: Through an optimized loading procedure, a platelet-based delivery system for TPA targeting CTC clusters was created. This is an important step in developing a method to delay metastasis by targeting CTC clusters. Moreover, this system can be adapted toward other conditions requiring a fibrinolytic response. With minor alterations, this system could treat strokes by targeting and lysing dangerous clots. To create a viable system, the effectiveness of the system will need to be assessed in vitro and in vivo, however, these results demonstrate the viability of this approach for multiple medical applications.
Acknowledgments: This work has been supported by the George Washington University startup funds and the Department of Defense Breast Cancer Research Program Expansion Award (W81XWH-19-1-0667).
References:
[1] L. Erpenbeck and M. P. Schön, “Deadly allies: the fatal interplay between platelets and
metastasizing cancer cells,” Blood, vol. 115, no. 17, pp. 3427–3436, Apr. 2010.
[2] N. M. Bambace and C. E. Holmes, “The platelet contribution to cancer progression,” J. Thromb.
Haemost., vol. 9, no. 2, pp. 237–249, Feb. 2011.
[3] L. J. Gay and B. Felding-Habermann, “Contribution of platelets to tumour metastasis,” Nat. Rev.
Cancer, vol. 11, no. 2, pp. 123–134, Feb. 2011.
[4] A.-L. Papa et al., “Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in
preclinical models,” Sci Transl Med, vol. 11, no. 479, 13 2019.

Adjuvants play critical roles in shaping the immune response to vaccines. Here we describe a novel nanoparticle adjuvant, saponin-MPLA nanoparticles (SMNP), formed by the immunostimulatory compound saponin, the Toll like receptor (TLR)-4 agonist monophosphoryl lipid A (MPLA), cholesterol, and phospholipids. These components self-assemble into striking ~40 nm diam. cage-like structures reminiscent of ISCOMs (Immunostimulatory complexes) previously reported for saponin/phospholipid complexes, but have synergistic immunostimulatory properties from the combined action of saponins and MPLA. SMNP exhibited striking potency in both mouse and non-human primate (NHP) preclinical animal models of vaccination with a variety of subunit vaccines. Protein vaccines adjuvanted with SMNP elicited robust germinal center B cell and Tfh responses, leading to strong serum IgG responses and (in NHPs) HIV tier 2 neutralizing antibody responses. Strikingly, SMNP exhibited distinctive mechanisms of action, altering lymph flow and promoting antigen entry into draining lymph nodes, in a manner dependent on co-assembly of MPLA and saponin together in the same nanoparticle. Altogether, these findings suggest SMNP may be a promising novel adjuvant for subunit vaccines in settings of HIV, COVID-19, and other infectious diseases.

A critical element of the immune system is tissue-level spatial organization. Cells and extracellular matrix elements are all highly organized in the lymph node, spleen, and thymus, as well as in the peripheral organs where immune cells infiltrate. Furthermore, secreted molecules are arranged in local patterns that depend on diffusion and interstitial flow. To begin to probe the functional outcomes of this intricate organizational structure, our laboratory has focused on combining live tissues and engineered biomaterials with microfluidic and micropatterning for fluidic and spatial control. We have developed microsystems to deliver drugs locally to specific regions of spatially structured tissues, to co-culture multiple structured organs under recirculating flow, and to begin to build a micropatterned lymph node. Here we will discuss our recent progress towards modeling complex immune function outside the body, to gain insight into the spatiotemporal dynamics of inflammation and immunity.