Stochastic Particle Barcoding for Single-Cell Tracking and Multiparametric Analysis

This study presents stochastic particle barcoding (SPB), a method for tracking cell identity across bioanalytical platforms. In this approach, single cells or small collections of cells are co-encapsulated within an enzymatically-degradable hydrogel block along with a random collection of fluorescent beads, whose number, color, and position encode the identity of the cell, enabling samples to be transferred in bulk between single-cell assay platforms without losing the identity of individual cells. The application of SPB is demonstrated for transferring cells from a subnanoliter protein secretion/phenotyping array platform into a microtiter plate, with re-identification accuracies in the plate assay of 96±2%. Encapsulated cells are recovered by digesting the hydrogel, allowing subsequent genotyping and phenotyping of cell lysates. Finally, a model scaling is developed to illustrate how different parameters affect the accuracy of SPB and to motivate scaling of the method to thousands of unique blocks.Ragon Institute of MGH, MIT and HarvardNational Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F32CA180586

Enhancing Humoral Responses Against HIV Envelope Trimers via Nanoparticle Delivery with Stabilized Synthetic Liposomes

An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.National Institute of Allergy and Infectious Diseases (U.S.) (Award UM1AI100663)National Institutes of Health (U.S.) (Award P01-AI104715)National Institutes of Health (U.S.) (Award P01-AI048240)National Cancer Institute (U.S.) (Grant P30-CA14051

Synthetic Nanoparticles for Vaccines and Immunotherapy

The immune system plays a critical role in our health. No other component of human physiology plays a decisive role in as diverse an array of maladies, from deadly diseases with which we are all familiar to equally terrible esoteric conditions: HIV, malaria, pneumococcal and influenza infections; cancer; atherosclerosis; autoimmune diseases such as lupus, diabetes, and multiple sclerosis. The importance of understanding the function of the immune system and learning how to modulate immunity to protect against or treat disease thus cannot be overstated. Fortunately, we are entering an exciting era where the science of immunology is defining pathways for the rational manipulation of the immune system at the cellular and molecular level, and this understanding is leading to dramatic advances in the clinic that are transforming the future of medicine.1,2 These initial advances are being made primarily through biologic drugs– recombinant proteins (especially antibodies) or patient-derived cell therapies– but exciting data from preclinical studies suggest that a marriage of approaches based in biotechnology with the materials science and chemistry of nanomaterials, especially nanoparticles, could enable more effective and safer immune engineering strategies. This review will examine these nanoparticle-based strategies to immune modulation in detail, and discuss the promise and outstanding challenges facing the field of immune engineering from a chemical biology/materials engineering perspectiveNational Institutes of Health (U.S.) (Grants AI111860, CA174795, CA172164, AI091693, and AI095109)United States. Department of Defense (W911NF-13-D-0001 and Awards W911NF-07-D-0004

Design and Characterization of Porous Hyaluronic Acid Hydrogels for in vitro and in vivo Non-Viral DNA Delivery

Natural wound healing and angiogenesis are a result of a cascade of bioactive signals being delivered at specified times in response to local biological cues. However, for ischemic wounds which cannot heal naturally, external therapies are required. We are interested in engineering hydrogel scaffolds for cell-demanded release of non-viral DNA nanoparticles to more efficiently guide blood vessel formation in such tissues. Vascularization within tissue-engineered constructs still remains the primary cause of construct failure following implantation. While a myriad of approaches to enhance the vascularization of implants are being investigated none have completely solved the problem. We investigated two hypotheses to enhance scaffold vascularization, both long-term mechanical support and DNA delivery. Our preliminary in vivo studies showed that after subcutaneous implantation for three weeks enzymatically degradable hydrogels had cellular infiltration only at the periphery of the hydrogel, while hydrogels with micron sized interconnected pores (µ-pore) were extensively infiltrated. Significant positive staining for endothelial markers (PECAM) was also found for µ-pore implants and not for nano-pore implants, even in the absence of pro-angiogenic factors. We hypothesized that an open pore structure will increase the rate of vascularization through enhanced cellular infiltration and that the added delivery of DNA encoding for angiogenic growth factors would result in long lasting angiogenic signals. To test these hypotheses, two approaches to make DNA-loaded enzymatically degradable µ-pore hydrogel scaffolds were developed. In the first approach, polystyrene nanoparticles, similar in size to DNA polyplexes, were immobilized to the hydrogel pore surface through protease sensitive peptide tethers. Enzymatically degradable tethers have been utilized for the immobilization and release of growth factors and small drugs, which are only liberated by cleavage caused by cell secreted proteases, such as matrix metalloproteinases (MMPs) or plasmins, during local tissue remodeling. These proteases are known to be up-regulated during wound healing, microenvironment remodeling, and in diseased states and can, therefore, serve as triggers for bioactive signal delivery. The goal was to use peptide sequences that have been shown to degrade at different rates through the action of MMPs to achieve temporally controlled nanoparticle internalization by cells that overexpress MMPs. Cellular internalization of the peptide-immobilized nanoparticles was shown to be a function of the peptide sensitivity to proteases, the number of tethers between the nanoparticle and the biomaterial and the MMP expression profile of the seeded cells. By immobilizing nanoparticles through protease sensitive peptide tethers, release was tailored specifically for an intended cellular target, which over-expresses such proteases. Alternatively, in the second approach, µ-pore hyaluronic acid-MMP (HA-MMP) hydrogels were used to encapsulate a high concentration of DNA/poly(ethylene imine) polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique. Porous hydrogels provide the additional advantages of being able to effectively seed cells in vitro post scaffold fabrication and allow for cell spreading and proliferation without requiring extensive degradation. Thus, release of encapsulated DNA polyplexes was assessed in the presence of mMSCs in hydrogels of various pore sizes (30, 60, and 100 µm). Steady release was observed starting by day four for up to ten days for all investigated pore sizes. Likewise, transgene expression in seeded cells was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. Combined these results suggested that DNA nanoparticle internalization and subsequent transgene expression could be controlled by both the protease expression profile of the seeded or infiltrating cells as well as the structural properties of the hydrogel. Using the knowledge acquired through these in vitro models, 100 and 60 µm porous and nano-pore HA-MMP hydrogels were used to study scaffold-mediated gene delivery for local gene therapy in both a subcutaneous implant and wound healing mouse models. Hydrogels with encapsulated pro-angiogenic (pVEGF) or reporter (pGFPluc) plasmids were tested for their ability to induce an enhanced angiogenic response by transfecting infiltrating cells in vivo. GFP expression in control hydrogels was used to track transfection at one, three, and six weeks in the subcutaneous implant study. While GFP-expressing transfected cells were present inside all hydrogel samples over the course of the study, transfection levels peaked around week three for 100 and 60 µm porous hydrogels. Transfection in nano-pore hydrogels continued to increase over time corresponding with continued gel degradation. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis by increasing vessel number, maturity, or size. Only in 60 µm porous hydrogels did the VEGF expression play a role in preventing vessel regression and helping to sustain the number of vessels present from three to six weeks. Regardless, pore size seemed to be the dominant factor in determining the angiogenic response with 60 µm porous hydrogels having more vessels present per area than 100 µm porous hydrogels at the initial onset of angiogenesis at three weeks. Increased pore rigidity may have been a key factor. The effect of porosity on wound healing was even more pronounced than what was observed in the subcutaneous implants. 100 and 60 µm porous hydrogels allowed for significantly faster wound closure than nano-pore hydrogels, which did not degrade and essentially provided a mechanical barrier to closure. Interestingly, total porosity and not specific pore size seemed to be the dominant factor in determining the wound closure rates. GFP-expressing transfected cells were present throughout the newly formed granulation tissue surrounding all hydrogel samples at two weeks. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis within the granulation tissue by statistically increasing vessel number, maturity, or size. Although the anticipated enhancement in angiogenesis in either model was not shown, the presence of transfected cells shows promise for the use of polyplex loaded porous hydrogels to produce a response in vivo. With further optimization we believe the proposed hydrogel system(s) has applications for controlled release of various DNA particles and other gene delivery vectors for in vivo tissue engineering and blood vessel formation

Design and Characterization of Porous Hyaluronic Acid Hydrogels for in vitro and in vivo Non-Viral DNA Delivery

Natural wound healing and angiogenesis are a result of a cascade of bioactive signals being delivered at specified times in response to local biological cues. However, for ischemic wounds which cannot heal naturally, external therapies are required. We are interested in engineering hydrogel scaffolds for cell-demanded release of non-viral DNA nanoparticles to more efficiently guide blood vessel formation in such tissues. Vascularization within tissue-engineered constructs still remains the primary cause of construct failure following implantation. While a myriad of approaches to enhance the vascularization of implants are being investigated none have completely solved the problem. We investigated two hypotheses to enhance scaffold vascularization, both long-term mechanical support and DNA delivery. Our preliminary in vivo studies showed that after subcutaneous implantation for three weeks enzymatically degradable hydrogels had cellular infiltration only at the periphery of the hydrogel, while hydrogels with micron sized interconnected pores (µ-pore) were extensively infiltrated. Significant positive staining for endothelial markers (PECAM) was also found for µ-pore implants and not for nano-pore implants, even in the absence of pro-angiogenic factors. We hypothesized that an open pore structure will increase the rate of vascularization through enhanced cellular infiltration and that the added delivery of DNA encoding for angiogenic growth factors would result in long lasting angiogenic signals. To test these hypotheses, two approaches to make DNA-loaded enzymatically degradable µ-pore hydrogel scaffolds were developed. In the first approach, polystyrene nanoparticles, similar in size to DNA polyplexes, were immobilized to the hydrogel pore surface through protease sensitive peptide tethers. Enzymatically degradable tethers have been utilized for the immobilization and release of growth factors and small drugs, which are only liberated by cleavage caused by cell secreted proteases, such as matrix metalloproteinases (MMPs) or plasmins, during local tissue remodeling. These proteases are known to be up-regulated during wound healing, microenvironment remodeling, and in diseased states and can, therefore, serve as triggers for bioactive signal delivery. The goal was to use peptide sequences that have been shown to degrade at different rates through the action of MMPs to achieve temporally controlled nanoparticle internalization by cells that overexpress MMPs. Cellular internalization of the peptide-immobilized nanoparticles was shown to be a function of the peptide sensitivity to proteases, the number of tethers between the nanoparticle and the biomaterial and the MMP expression profile of the seeded cells. By immobilizing nanoparticles through protease sensitive peptide tethers, release was tailored specifically for an intended cellular target, which over-expresses such proteases. Alternatively, in the second approach, µ-pore hyaluronic acid-MMP (HA-MMP) hydrogels were used to encapsulate a high concentration of DNA/poly(ethylene imine) polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique. Porous hydrogels provide the additional advantages of being able to effectively seed cells in vitro post scaffold fabrication and allow for cell spreading and proliferation without requiring extensive degradation. Thus, release of encapsulated DNA polyplexes was assessed in the presence of mMSCs in hydrogels of various pore sizes (30, 60, and 100 µm). Steady release was observed starting by day four for up to ten days for all investigated pore sizes. Likewise, transgene expression in seeded cells was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. Combined these results suggested that DNA nanoparticle internalization and subsequent transgene expression could be controlled by both the protease expression profile of the seeded or infiltrating cells as well as the structural properties of the hydrogel. Using the knowledge acquired through these in vitro models, 100 and 60 µm porous and nano-pore HA-MMP hydrogels were used to study scaffold-mediated gene delivery for local gene therapy in both a subcutaneous implant and wound healing mouse models. Hydrogels with encapsulated pro-angiogenic (pVEGF) or reporter (pGFPluc) plasmids were tested for their ability to induce an enhanced angiogenic response by transfecting infiltrating cells in vivo. GFP expression in control hydrogels was used to track transfection at one, three, and six weeks in the subcutaneous implant study. While GFP-expressing transfected cells were present inside all hydrogel samples over the course of the study, transfection levels peaked around week three for 100 and 60 µm porous hydrogels. Transfection in nano-pore hydrogels continued to increase over time corresponding with continued gel degradation. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis by increasing vessel number, maturity, or size. Only in 60 µm porous hydrogels did the VEGF expression play a role in preventing vessel regression and helping to sustain the number of vessels present from three to six weeks. Regardless, pore size seemed to be the dominant factor in determining the angiogenic response with 60 µm porous hydrogels having more vessels present per area than 100 µm porous hydrogels at the initial onset of angiogenesis at three weeks. Increased pore rigidity may have been a key factor. The effect of porosity on wound healing was even more pronounced than what was observed in the subcutaneous implants. 100 and 60 µm porous hydrogels allowed for significantly faster wound closure than nano-pore hydrogels, which did not degrade and essentially provided a mechanical barrier to closure. Interestingly, total porosity and not specific pore size seemed to be the dominant factor in determining the wound closure rates. GFP-expressing transfected cells were present throughout the newly formed granulation tissue surrounding all hydrogel samples at two weeks. Transfection levels of pVEGF, however, did not seem to be high enough to enhance angiogenesis within the granulation tissue by statistically increasing vessel number, maturity, or size. Although the anticipated enhancement in angiogenesis in either model was not shown, the presence of transfected cells shows promise for the use of polyplex loaded porous hydrogels to produce a response in vivo. With further optimization we believe the proposed hydrogel system(s) has applications for controlled release of various DNA particles and other gene delivery vectors for in vivo tissue engineering and blood vessel formation

Design and Characterization of Micro-Porous Hyaluronic Acid Hydrogels for \u3ci\u3ein vitro\u3c/i\u3e Gene Transfer to mMSCs

The effective and sustained delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is to use porous hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the affected sites. We have designed and characterized micro-porous (μ-pore) hyaluronic acid hydrogels which allow for effective cell seeding in vitro post scaffold fabrication and allow for cell spreading and proliferation without requiring high levels of degradation. These factors, coupled with high loading efficiency of DNA polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique, then allowed for long-term sustained transfection and transgene expression of incorporated mMSCs. In this study, we examined the effect of pore size on gene transfer efficiency and the kinetics of transgene expression. For all investigated pore sizes (30, 60, and 100 μm), encapsulated DNA polyplexes were released steadily starting by day 4 for up to 10 days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. The knowledge acquired through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model where host cells infiltrate the scaffold over time

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation. There is limited experience with recombinant Env trimer immunogens in nonhuman primates. Pauthner et al. compare multiple Env trimer designs and immunization strategies for generating HIV neutralizing antibodies. They identify protocols for rapid and consistent generation of tier 2 nAbs, providing a framework for future pre-clinical and clinical vaccine studies.Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (NIAIDUM1AI100663

Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery

© 2020 American Chemical Society. Although cytokine therapy is an attractive strategy to build a more robust immune response in tumors, cytokines have faced clinical failures due to toxicity. In particular, interleukin-12 has shown great clinical promise but was limited in translation because of systemic toxicity. In this study, we demonstrate an enhanced ability to reduce toxicity without affecting the efficacy of IL-12 therapy. We engineer the material properties of a NP to meet the enhanced demands for optimal cytokine delivery by using the layer-by-layer (LbL) approach. Importantly, using LbL, we demonstrate cell-level trafficking of NPs to preferentially localize to the cell's outer surface and act as a drug depot, which is required for optimal payload activity on neighboring cytokine membrane receptors. LbL-NPs showed efficacy against a tumor challenge in both colorectal and ovarian tumors at doses that were not tolerated when administered carrier-free

Gold-Nanocrystal-Enhanced Bioluminescent Nanocapsules

Metal-enhanced bioluminescence presents a great opportunity to achieve ultrasensitive analysis and imaging with low bioluminescent background and enhanced luminescence. We hereby report metal-enhanced bioluminescence based on bioluminescent protein nanocapsules conjugated with gold nanocrystals. Such gold-nanocapsule complexes exhibit near 10-fold enhancement in bioluminescent intensity and are effectively delivered into the cells with outstanding stability. This work offers a class of bioluminescent nanoparticles for imaging and other applications