Bioinspired Lipid Nanocarriers for RNA Delivery.

RNA therapy is a disruptive technology comprising a rapidly expanding category of drugs. Further translation of RNA therapies to the clinic will improve the treatment of many diseases and help enable personalized medicine. However, in vivo delivery of RNA remains challenging due to the lack of appropriate delivery tools. Current state-of-the-art carriers such as ionizable lipid nanoparticles still face significant challenges, including frequent localization to clearance-associated organs and limited (1-2%) endosomal escape. Thus, delivery vehicles must be improved to further unlock the full potential of RNA therapeutics. An emerging strategy is to modify existing or new lipid nanocarriers by incorporating bioinspired design principles. This method generally aims to improve tissue targeting, cellular uptake, and endosomal escape, addressing some of the critical issues facing the field. In this review, we introduce the different strategies for creating bioinspired lipid-based RNA carriers and discuss the potential implications of each strategy based on reported findings. These strategies include incorporating naturally derived lipids into existing nanocarriers and mimicking bioderived molecules, viruses, and exosomes. We evaluate each strategy based on the critical factors required for delivery vehicles to succeed. Finally, we point to areas of research that should be furthered to enable the more successful rational design of lipid nanocarriers for RNA delivery.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation.

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Spatial delivery of immune cues to lymph nodes to define therapeutic outcomes in cancer vaccination.

Recently approved cancer immunotherapies - including CAR-T cells and cancer vaccination, - show great promise. However, these technologies are hindered by the complexity and cost of isolating and engineering patient cells ex vivo. Lymph nodes (LNs) are key tissues that integrate immune signals to coordinate adaptive immunity. Directly controlling the signals and local environment in LNs could enable potent and safe immunotherapies without cell isolation, engineering, and reinfusion. Here we employ intra-LN (i.LN.) injection of immune signal-loaded biomaterial depots to directly control cancer vaccine deposition, revealing how the combination and geographic distribution of signals in and between LNs impact anti-tumor response. We show in healthy and diseased mice that relative proximity of antigen and adjuvant in LNs - and to tumors - defines unique local and systemic characteristics of innate and adaptive response. These factors ultimately control survival in mouse models of lymphoma and melanoma. Of note, with appropriate geographic signal distributions, a single i.LN. vaccine treatment confers near-complete survival to tumor challenge and re-challenge 100 days later, without additional treatments. These data inform design criteria for immunotherapies that leverage biomaterials for loco-regional LN therapy to generate responses that are systemic and specific, without systemically exposing patients to potent or immunotoxic drugs.

Altering Antigen Charge to Control Self-Assembly and Processing of Immune Signals During Cancer Vaccination.

Biomaterial delivery systems offer unique potential to improve cancer vaccines by offering targeted delivery and modularity to address disease heterogeneity. Here, we develop a simple platform using a conserved human melanoma peptide antigen (Trp2) modified with cationic arginine residues that condenses an anionic toll-like receptor agonist (TLRa), CpG, into polyplex-like nanoparticles. We reasoned that these structures could offer several useful features for immunotherapy - such as tunable loading, co-delivery of immune cues, and cargo protection - while eliminating the need for synthetic polymers or other complicating delivery systems. We demonstrate that Trp2/CpG polyplexes can readily form over a range of Trp2:CpG ratios and improve antigen uptake by primary antigen presenting cells. We show antigen loading can be tuned by interchanging Trp2 peptides with defined charges and numbers of arginine residues. Notably, these polyplexes with greater antigen loading enhance the functionality of Trp-2 specific T cells and in a mouse melanoma model, decrease tumor burden and improve survival. This work highlights opportunities to control the biophysical properties of nanostructured materials built from immune signals to enhance immunotherapy, without the added complexity or background immune effects often associated with synthetic carriers.

Leveraging the modularity of biomaterial carriers to tune immune responses.

Biomaterial carriers offer modular features to control the delivery and presentation of vaccines and immunotherapies. This tunability is a distinct capability of biomaterials. Understanding how tunable material features impact immune responses is important to improve vaccine and immunotherapy design, as well as clinical translation. Here we discuss the modularity of biomaterial properties as a means of controlling encounters with immune signals across scales - tissue, cell, molecular, and time - and ultimately, to direct stimulation or regulation of immune function. We highlight these advances using illustrations from recent literature across infectious disease, cancer, and autoimmunity. As the immune engineering field matures, informed design criteria could support more rational biomaterial carriers for vaccination and immunotherapy.

An Optical Method for Quantitatively Determining the Surface Free Energy of Micro- and Nanoparticles.

Surface free energy (SFE) of micro- and nanoparticles plays a crucial role in determining the hydrophobicity and wettability of the particles. To date, however, there are no easy-to-use methods for determining the SFE of particles. Here, with the application of several inexpensive, easy-to-use, and commonly available lab procedures and facilities, including particle dispersion, settling/centrifugation, pipetting, and visible-light spectroscopy, we developed a novel technique called the maximum particle dispersion (MPD) method for quantitatively determining the SFE of micro- and nanoparticles. We demonstrated the versatility and robustness of the MPD method by studying nine representative particles of various chemistries, sizes, dimensions, and morphologies. These are triethoxycaprylylsilane-coated zinc oxide nanoparticles, multiwalled carbon nanotubes, graphene nanoplatelets, molybdenum(IV) sulfide flakes, neodymium(III) oxide nanoparticles, two sizes of zeolites, poly(vinylpolypyrrolidone), and polystyrene microparticles. The SFE of these micro- and nanoparticles was found to cover a range from 21 to 36 mJ/m2. These SFE values may find applications in a broad spectrum of scientific disciplines including the synthesis of these nanomaterials, such as in liquid-phase exfoliation. The MPD method has the potential to be developed into a standard, low-cost, and easy-to-use method for quantitatively characterizing the SFE and hydrophobicity of particles at the micro- and nanoscale.

Improving Vaccine and Immunotherapy Design Using Biomaterials.

Polymers, lipids, scaffolds, microneedles, and other biomaterials are rapidly emerging as technologies to improve the efficacy of vaccines against infectious disease and immunotherapies for cancer, autoimmunity, and transplantation. New studies are also providing insight into the interactions between these materials and the immune system. This insight can be exploited for more efficient design of vaccines and immunotherapies. Here, we describe recent advances made possible through the unique features of biomaterials, as well as the important questions for further study.

The toxin biliatresone causes mouse extrahepatic cholangiocyte damage and fibrosis through decreased glutathione and SOX17.

UNLABELLED: Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. CONCLUSION: Biliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).

Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.

Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions.

Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

Chitosan adsorption on hydroxyapatite and its role in preventing acid erosion.

Polymer adsorption onto an artificial saliva (AS) layer is investigated using quartz-crystal microbalance with dissipation (QCM-D) and chitosan as the model polymer. QCM-D is utilized in an innovative manner to monitor in situ adsorption of chitosan (CH) onto a hydroxyapatite (HA) coated crystal and to examine the ability of the adsorbed layer to "protect" the HA upon sequential exposure to acidic solutions. After deposition of a thin AS layer (16 nm), the total thickness on the HA substrate increases to 37 nm upon exposure to CH at pH 5.5 for 10 min. Correspondingly, the surface charge changes from negative (i.e., AS) to positive, consistent with the adsorption the polycationic CH onto or into the AS layer. Upon exposure to an oxidizing agent, the chitosan cross-links and collapses as noted by a decrease in thickness to 10 nm and an increase in the shear modulus by an order of magnitude. Atomic force microscopy (AFM) is used to determine the surface morphology and RMS roughness of the coated and HA surfaces after citric acid challenges. Both physisorbed and cross-linked chitosan are demonstrated to limit and prevent the erosion of HA, respectively.

Deciphering diatom biochemical pathways via whole-cell proteomics.

Diatoms play a critical role in the oceans' carbon and silicon cycles; however, a mechanistic understanding of the biochemical processes that contribute to their ecological success remains elusive. Completion of the Thalassiosira pseudonana genome provided 'blueprints' for the potential biochemical machinery of diatoms, but offers only a limited insight into their biology under various environmental conditions. Using high-throughput shotgun proteomics, we identified a total of 1928 proteins expressed by T. pseudonana cultured under optimal growth conditions, enabling us to analyze this diatom's primary metabolic and biosynthetic pathways. Of the proteins identified, 70% are involved in cellular metabolism, while 11% are involved in the transport of molecules. We identified all of the enzymes involved in the urea cycle, thereby describing the complete pathway to convert ammonia to urea, along with urea transporters, and the urea-degrading enzyme urease. Although metabolic exchange between these pathways remains ambiguous, their constitutive presence suggests complex intracellular nitrogen recycling. In addition, all C(4) related enzymes for carbon fixation have been identified to be in abundance, with high protein sequence coverage. Quantification of mass spectra acquisitions demonstrated that the 20 most abundant proteins included an unexpectedly high expression of clathrin, which is the primary structural protein involved in endocytic transport. This result highlights a previously overlooked mechanism for the inter- and intra-cellular transport of nutrients and macromolecules in diatoms, potentially providing a missing link to organelle communication and metabolite exchange. Our results demonstrate the power of proteomics, and lay the groundwork for future comparative proteomic studies and directed analyses of specifically expressed proteins and biochemical pathways of oceanic diatoms.