Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Selective mycobacterial capture with ultraviolet-polymerized poly-dimethyldiallyl chloride functionalized surfaces.

Tuberculosis (TB) is the top cause of death from a single infectious pathogen after COVID-19. Despite molecular diagnostic advances, two-thirds of the 10 million annual TB cases are still diagnosed using direct smear microscopy which has ~50% sensitivity. To increase the analytical performance of smear microscopy, we developed and characterized a novel polymer (Polydiallyldimethylammonium chloride [PDADMAC]) engraftment on inexpensive polystyrene (PS) specifically functionalized for mycobacterial capture. Engraftment is achieved via UV photopolymerization of DADMAC monomer on plasma-activated PS. The platform was tested on sputum from presumptive TB cases in Kampala, Uganda (n = 50), with an increased overall sensitivity of 81.8% (27/33) vs. fluorescent smear microscopy 57% (19/33) compared to a molecular (Cepheid GeneXpert MTB/RIF) gold standard. Frugal smear diagnostic innovation that is rapid and does not require dedicated instrumentation may offer an important solution to bridge the TB diagnostic gap.

Quaternary nanoparticles enable sustained release of bortezomib for hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related mortality in the world. Over the past two decades, there has been minimal improvement in therapies as well as clinical outcomes for patients with Barcelona Clinic Liver Cancer (BCLC)-B. These patients are treated with local interventions, including transarterial chemoembolization. Current methodologies only allow sustained intratumoral release measured in hours. Methodologies to allow sustained local release of the drug cargo over days to weeks are acutely needed. We hypothesize that tumor response as well as outcomes of patients with BCLC-B can be improved through utilization of a highly cytotoxic agent delivered with a sustained release platform. APPROACH AND RESULTS: High-throughput drug screening across 40 HCC patient-derived organoids identified bortezomib (BTZ) as a highly cytotoxic small molecule for HCC. We designed and manufactured sustained release BTZ nanoparticles (BTZ-NP) using a flash nanocomplexation/nanoprecipitation process. We quantified the release profile and tested the anti-tumoral effects in vivo. The BTZ-NP formulation demonstrated a sustained release of BTZ of 30 days. This BTZ-NP formulation was highly effective in controlling tumor size and improved survival in vivo in three animal models of HCC, including when delivered via the hepatic artery, as we envision its delivery in patients. In addition, the BTZ-NP formulation was superior to treatment with doxorubicin-drug eluting beads. CONCLUSIONS: The BTZ-NP formulation provides a potent and safe treatment of HCC via a localized delivery approach. These results warrant additional preclinical studies to advance this technology to human clinical trials.

A Novel Patient-like Swine Model of Perianal Crohn's Disease.

BACKGROUND: Perianal Crohn's disease is associated with poor outcomes and high medical costs. It is notoriously difficult to treat despite therapeutic advancements for luminal disease. A large animal model that mimics human perianal disease is needed to test innovative therapies. OBJECTIVE: This study aimed to create a swine model that replicates the inflammatory component and therapeutic challenges found in patients with perianal Crohn's disease. DESIGN: This was an animal preclinical study. SETTINGS: The experiments were performed at the animal laboratory at the Johns Hopkins University. PATIENTS: Four sus scrufus female pigs were included in the study. INTERVENTIONS: Four female pigs underwent creation of 3 surgical perianal fistulas each, 1 rectovaginal and 2 perianal. Size 24 French setons were placed to maintain patency of the fistula tracts for 4 weeks. After removal of the setons, trinitrobenzene sulfonic acid was administered into the fistula tract to create and maintain local inflammation mimicking perianal Crohn's disease. MAIN OUTCOMES MEASURES: An MRI was obtained to assess the fistulas and the pigs were euthanized to review histopathology. RESULTS: Three inflammatory chronic fistula tracts were successfully created in each pig as confirmed by MRI and examination under anesthesia. This is the first report of maintaining patent fistulas in swine 2 weeks after removal of setons. For the first time, we reported that 2 pigs developed branching fistulas and small abscesses reminiscent of human perianal Crohn's disease. The corresponding histopathologic examination found significant chronic active inflammation on standard hematoxylin and eosin staining. LIMITATIONS: The fistulas were surgically induced and did not occur naturally. CONCLUSIONS: A chronic perianal fistula model in pigs that strongly resembles human perianal Crohn's disease was successfully created. This model can be used to test novel therapeutics and techniques to pave the path for human trials. See Video Abstract at http://links.lww.com/DCR/B969 . UN NUEVO MODELO PORCINO SIMILAR A UN PACIENTE DE LA ENFERMEDAD DE CROHN PERIANAL ANTECEDENTES: La enfermedad de Crohn perianal se asocia con malos resultados y altos costos médicos. Es notoriamente difícil de tratar a pesar de los avances terapéuticos para la enfermedad luminal. Se precisa de un modelo animal grande que imite la enfermedad perianal humana para probar terapias innovadoras.OBJETIVO:Nuestro objetivo de este estudio fue crear un modelo porcino que replique el componente inflamatorio y los desafíos terapéuticos que se encuentran en los pacientes con enfermedad de Crohn perianal.DISEÑO:Este fue un estudio preclínico en animales.AJUSTES:Los experimentos se realizaron en el laboratorio de animales de la Universidad Johns Hopkins.PACIENTES:Se incluyeron en el estudio cuatro cerdas sus scrofa.INTERVENCIONES:Cuatro cerdas fueron sometidas a la creación de 3 fístulas perianales quirúrgicas cada una: 1 recto vaginal y 2 perianales. Se colocaron sedales de 24 French para mantener la permeabilidad de los trayectos fistulosos durante 4 semanas. Tras el retiro de los sedales, se administró ácido trinitrobenceno sulfónico en el trayecto de la fístula para crear y mantener la inflamación local simulando la enfermedad de Crohn perianal.PRINCIPALES MEDIDAS DE RESULTADOS:Se obtuvo una resonancia magnética para evaluar las fístulas y los cerdos fueron sacrificados para revisar la histopatología.RESULTADOS:Se crearon de manera exitosa tres trayectos fistulosos inflamatorios crónicos en cada cerdo, confirmados por imágenes de resonancia magnética y examen bajo anestesia. Este es el primer informe de preservación de fístulas permeables en cerdos 2 semanas tras el retiro de los setones. Por primera vez, informamos que dos cerdos desarrollaron fístulas ramificadas y pequeños abscesos que recuerdan a la enfermedad de Crohn perianal humana. El examen histopatológico correspondiente encontró una significativa inflamación crónica activa en la tinción estándar de hematoxilina y eosina.LIMITACIONES:Las fístulas se indujeron quirúrgicamente y no se produjeron de forma natural.CONCLUSIONES:Se logro recrear con éxito un modelo de fístula perianal crónica en cerdos que se asemeja mucho a la enfermedad de Crohn perianal humana. Este modelo se puede utilizar para probar nuevas terapias y técnicas para allanar el camino para los ensayos en humanos. Consulte Video Resumen en http://links.lww.com/DCR/B969 . (Traducción-Dr Osvaldo Gauto).

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

Regenerative and durable small-diameter graft as an arterial conduit.

Despite significant research efforts, clinical practice for arterial bypass surgery has been stagnant, and engineered grafts continue to face postimplantation challenges. Here, we describe the development and application of a durable small-diameter vascular graft with tailored regenerative capacity. We fabricated small-diameter vascular grafts by electrospinning fibrin tubes and poly(ε-caprolactone) fibrous sheaths, which improved suture retention strength and enabled long-term survival. Using surface topography in a hollow fibrin microfiber tube, we enable immediate, controlled perfusion and formation of a confluent endothelium within 3-4 days in vitro with human endothelial colony-forming cells, but a stable endothelium is noticeable at 4 weeks in vivo. Implantation of acellular or endothelialized fibrin grafts with an external ultrathin poly(ε-caprolactone) sheath as an interposition graft in the abdominal aorta of a severe combined immunodeficient Beige mouse model supports normal blood flow and vessel patency for 24 weeks. Mechanical properties of the implanted grafts closely approximate the native abdominal aorta properties after just 1 week in vivo. Fibrin mediated cellular remodeling, stable tunica intima and media formation, and abundant matrix deposition with organized collagen layers and wavy elastin lamellae. Endothelialized grafts evidenced controlled healthy remodeling with delayed and reduced macrophage infiltration alongside neo vasa vasorum-like structure formation, reduced calcification, and accelerated tunica media formation. Our studies establish a small-diameter graft that is fabricated in less than 1 week, mediates neotissue formation and incorporation into the native tissue, and matches the native vessel size and mechanical properties, overcoming main challenges in arterial bypass surgery.

Size-Controlled and Shelf-Stable DNA Particles for Production of Lentiviral Vectors.

Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400-500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelf-stable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency.

Flash Technology-Based Self-Assembly in Nanoformulation: From Fabrication to Biomedical Applications.

Advances in nanoformulation have driven progress in biomedicine by producing nanoscale tools for biosensing, imaging, and drug delivery. Flash-based technology, the combination of rapid mixing technique with the self-assembly of macromolecules, is a new engine for the translational nanomedicine. Here, we review the state-of-the-art in flash-based self-assembly including theoretical and experimental principles, mixing device design, and applications. We highlight the fields of flash nanocomplexation (FNC) and flash nanoprecipitation (FNP), with an emphasis on biomedical applications of FNC, and discuss challenges and future directions for flash-based nanoformulation in biomedicine.

Ice-Inspired Superlubricated Electrospun Nanofibrous Membrane for Preventing Tissue Adhesion.

Inspired by the superlubricated surface (SLS) of ice, which consists of an ultrathin and contiguous layer of surface-bound water, we built a SLS on the polycaprolactone (PCL)/poly(2-methacryloxyethylphosphorylcholine) (PMPC) composite nanofibrous membrane via electrospinning under controlled relative humidity (RH). The zwitterionic PMPC on the nanofiber provided a surface layer of bound water, thus generating a hydration lubrication surface. Prepared under 20% RH, electrospun PCL/PMPC nanofibers reached a minimum coefficient of friction (COF) of about 0.12 when the weight ratio of PMPC to PCL was 0.1. At a higher RH, a SLS with an ultralow COF of less than 0.05 was formed on the composite nanofibers. The high stability of the SLS hydration layer on the engineered nanofibrous membrane effectively inhibited fibroblast adhesion and markedly reduced tissue adhesion during tendon repair in vivo. This work demonstrates the great potential of this ice-inspired SLS approach in tissue adhesion-prevention applications.

Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells.

T cells are critical players in disease; yet, their antigen-specificity has been difficult to identify, as current techniques are limited in terms of sensitivity, throughput, or ease of use. To address these challenges, we increased the throughput and translatability of magnetic nanoparticle-based artificial antigen presenting cells (aAPCs) to enrich and expand (E+E) murine or human antigen-specific T cells. We streamlined enrichment, expansion, and aAPC production processes by enriching CD8+ T cells directly from unpurified immune cells, increasing parallel processing capacity of aAPCs in a 96-well plate format, and designing an adaptive aAPC that enables multiplexed aAPC construction for E+E and detection. We applied these adaptive platforms to process and detect CD8+ T cells specific for rare cancer neoantigens, commensal bacterial cross-reactive epitopes, and human viral and melanoma antigens. These innovations dramatically increase the multiplexing ability and decrease the barrier to adopt for investigating antigen-specific T cell responses.

Flash Fabrication of Orally Targeted Nanocomplexes for Improved Transport of Salmon Calcitonin across the Intestine.

Salmon calcitonin (sCT) is a potent calcium-regulating peptide hormone and widely applied for the treatment of some bone diseases clinically. However, the therapeutic usefulness of sCT is hindered by the frequent injection required, owing to its short plasma half-life and therapeutic need for a high dose. Oral delivery is a popular modality of administration for patients because of its convenience to self-administration and high patient compliance, while orally administered sCT remains a great challenge currently due to the existence of multiple barriers in the gastrointestinal (GI) tract. Here, we introduced an orally targeted delivery system to increase the transport of sCT across the intestine through both the paracellular permeation route and the bile acid pathway. In this system, sCT-based glycol chitosan-taurocholic acid conjugate (GC-T)/dextran sulfate (DS) ternary nanocomplexes (NC-T) were produced by a flash nanocomplexation (FNC) process in a kinetically controlled mode. The optimized NC-T exhibited well-controlled properties with a uniform and sub-60 nm hydrodynamic diameter, high batch-to-batch reproducibility, good physical or chemical stability, as well as sustained drug release behaviors. The studies revealed that NC-T could effectively improve the intestinal uptake and permeability, owing to its surface functionalization with the taurocholic acid ligand. In the rat model, orally administered NC-T showed an obvious hypocalcemia effect and a relative oral bioavailability of 10.9%. An in vivo assay also demonstrated that NC-T induced no observable side effect after long-term oral administration. As a result, the orally targeted nanocomplex might be a promising candidate for improving the oral transport of therapeutic peptides.

Modified cell-electrospinning for 3D myogenesis of C2C12s in aligned fibrin microfiber bundles.

Electrospinning methods can generate scaffolds with alignment cues to guide the development of myogenic precursors into 3D skeletal muscle grafts. However, cells seeded onto these scaffolds adhere to the exterior resulting in regions of acellularity within the scaffold interior. To overcome this limitation, we modified an aqueous solution-electrospinning method to encapsulate C2C12s and electrospin them into fibrin/polyethylene oxide (PEO) microfiber bundles. We demonstrated that loading C2C12s as cellular aggregates (80-90 µm in diameter) and modifying several other electrospinning parameters dramatically increased cell viability following exposure to the 4.5 kV electric field. C2C12-seeded fibrin/PEO microfiber bundles were cultured for up to seven days. Uninduced and myogenically induced C2C12s proliferated, elongated and became multinucleated. Myogenic induction increased the number of myotube-associated nuclei (36.4 ±â€¯12% vs. 6.2 ±â€¯1.9%), myotube length (122.4 ±â€¯10.9 µm vs. 59.9 ±â€¯8.3 µm), and myotube diameter (16.76 ±â€¯2.06 µm vs. 12.49 ±â€¯0.93 µm). The data presented in this study demonstrates for the first time that cells can be loaded inside the aligned fibrin hydrogel 3D construct during aqueous solution electrospinning while retaining their potential for de novo tissue formation.

Scalable Manufacturing of Enteric Encapsulation Systems for Site-Specific Oral Insulin Delivery.

Oral drug delivery is a more favored mode of administration because of its ease of administration, high patient compliance, and low healthcare costs. However, no oral protein formulations are commercially available currently due to hostile gastrointestinal (GI) barriers resulting in insignificant oral bioavailability of macromolecular drugs. Herein, we used insulin as a model protein drug; insulin-loaded N-(2-hydroxy)-propyl-3-trimethylammonium chloride modified chitosan (HTCC)/sodium tripolyphosphate (TPP) nanocomplex (NC) as a nanocore was further encapsulated into enteric Eudragit L100-55 material, through a two-step flash nanocomplexation (FNC) process in a reliable and scalable manner, forming our NC-in-Eudragit composite particles (NE). Particle size and surface properties of our optimized NE were tailored to protect the loaded insulin from acidic degradation in the hostile stomach environment and to achieve intestinal site-specific drug release as well as the improvement of oral delivery efficiency of insulin. In addition, the oral administration of the optimized NE to type 1 diabetic rats could induce a very significant hypoglycemic effect with a relative oral bioavailability of 13.3%. Our results demonstrated that enteric encapsulation of nanotherapeutics using a FNC apparatus could cause drug formulations to possess better size controllability, batch-mode reproducibility, and homogeneous surface coating and then significantly enhance their oral bioavailability of insulin, indicating its great potential for clinical translation of oral protein therapeutics.

Subtle changes in surface-tethered groups on PEGylated DNA nanoparticles significantly influence gene transfection and cellular uptake.

PEGylation strategy has been widely used to enhance colloidal stability of polycation/DNA nanoparticles (NPs) for gene delivery. To investigate the effect of polyethylene glycol (PEG) terminal groups on the transfection properties of these NPs, we synthesized DNA NPs using PEG-g-linear polyethyleneimine (lPEI) with PEG terminal groups containing alkyl chains of various lengths with or without a hydroxyl terminal group. For both alkyl- and hydroxyalkyl-decorated NPs with PEG grafting densities of 1.5, 3, or 5% on lPEI, the highest levels of transfection and uptake were consistently achieved at intermediate alkyl chain lengths of 3 to 6 carbons, where the transfection efficiency is significantly higher than that of nonfunctionalized lPEI/DNA NPs. Molecular dynamics simulations revealed that both alkyl- and hydroxyalkyl-decorated NPs with intermediate alkyl chain length exhibited more rapid engulfment than NPs with shorter or longer alkyl chains. This study identifies a new parameter for the engineering design of PEGylated DNA NPs.

Polymeric nanofiber coating with tunable combinatorial antibiotic delivery prevents biofilm-associated infection in vivo.

Bacterial biofilm formation is a major complication of implantable medical devices that results in therapeutically challenging chronic infections, especially in cases involving antibiotic-resistant bacteria. As an approach to prevent these infections, an electrospun composite coating comprised of poly(lactic-coglycolic acid) (PLGA) nanofibers embedded in a poly(ε-caprolactone) (PCL) film was developed to locally codeliver combinatorial antibiotics from the implant surface. The release of each antibiotic could be adjusted by loading each drug into the different polymers or by varying PLGA:PCL polymer ratios. In a mouse model of biofilm-associated orthopedic-implant infection, three different combinations of antibiotic-loaded coatings were highly effective in preventing infection of the bone/joint tissue and implant biofilm formation and were biocompatible with enhanced osseointegration. This nanofiber composite-coating technology could be used to tailor the delivery of combinatorial antimicrobial agents from various metallic implantable devices or prostheses to effectively decrease biofilm-associated infections in patients.

Potency of a Scalable Nanoparticulate Subunit Vaccine.

Nanoparticulate vaccines can potentiate immune responses by site-specific drainage to lymph nodes (LNs). This approach may benefit from a nanoparticle engineering method with fine control over size and codelivery of antigen and adjuvant. Here, we applied the flash nanocomplexation (FNC) method to prepare nanovaccines via polyelectrolyte complexation of chitosan and heparin to coencapsulate the VP1 protein antigen from enterovirus 71, which causes hand-foot-mouth disease (HFMD), with tumor necrosis factor α (TNF) or CpG as adjuvants. FNC allows for reduction of the nanovaccine size to range from 90 to 130 nm with relatively narrower size distribution and a high payload capacity. These nanovaccines reached both proximal and distal LNs via subcutaneous injection and subsequently exhibited prolonged retention in the LNs. The codelivery induced strong immune activation toward a Th1 response in addition to a potent Th2 response, and conferred effective protection against lethal virus challenge comparable to that of an approved inactivated viral vaccine in mouse models of both passive and active immunization setting. In addition, these nanovaccines also elicited strong IgA titers, which may offer unique advantages for mucosal protection. This study addresses the issues of size control, antigen bioactivity retention, and biomanufacturing to demonstrate the translational potential of a subunit nanovaccine design.

Tuned Cationic Dendronized Polymer: Molecular Scavenger for Rheumatoid Arthritis Treatment.

Cell-free deoxyribonucleic acid (cfDNA) released from either dead or damaged cells serves as a key autoantigen in rheumatoid arthritis (RA). They can be recognized by nucleic acid (NA) sensors such as the toll-like receptor (TLR), leading to activation of the innate immune system and chronic inflammation. Developed here is a cationic molecular scavenger, by screening cationic dendronized polymers, which eliminates cfDNA and inhibits TLR recognition and nucleic-acid-induced inflammation. The structure-property study demonstrates that toxicity, NA binding capacity, and biodistribution could be balanced to achieve maximum therapeutic effect by exquisite control of the molecular structure. In addition, the optimized cationic polymer effectively inhibited joint swelling, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) rat models. The results offer support for synthetic polymers offering new paradigm in autoimmune disease treatment.

Effect of Humidity on Sporicidal Activity of Iodine Vapor on Bacillus thuringiensis.

The emergence of Bacillus anthracis as a potential bioterrorism and biological warfare agent points to the need for safe, effective, and economical sporicides for infection prevention and control. This work examined the efficacy of iodine vapor decontamination technologies to inactivate a surrogate for B. anthracis, Bacillus thuringiensis spores on glass materials. 106-107 colony-forming units of spores inoculated onto circular glass cover slips were treated with different concentrations of iodine vapor under various temperature and relative humidity. Only minimal spore killing activity was observed at low humidity. Higher humidity levels, as well as pre-hydration or post-hydration of the spores, increased the rate of inactivation as long as the contact between spores and iodine was maintained in a hydrated environment. Significant sporicidal activity of 3-log and 6-log spore reduction has been observed with 2.1 mg L-1 iodine vapor concentration at 90% relative humidity and 22 °C, with 1 and 24 h of exposure, respectively. The results showed that the relative humidity of the environment is of major importance in regulating the rate at which the spores are inactivated by iodine. The results of this study may provide insight into the parameters of effective decontamination procedures for Bacillus spores using gaseous iodine.

Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation.

Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.

Cross-Linking Approaches to Tuning the Mechanical Properties of Peptide π-Electron Hydrogels.

Self-assembling peptides are extensively exploited as bioactive materials in applications such as regenerative medicine and drug delivery despite the fact that their relatively weak noncovalent interactions often render them susceptible to mechanical destruction and solvent erosion. Herein, we describe how covalent cross-linking enhances the mechanical stability of self-assembling π-conjugated peptide hydrogels. We designed short peptide-chromophore-peptide sequences displaying different reactive functional groups that can form cross-links with appropriately modified bifunctional polyethylene glycol (PEG)-based small guest molecules. These peptides self-assemble into one-dimensional fibrillar networks in response to pH in the aqueous environment. The cross-linking reactions were promoted to create a secondary network locked in place by covalent bonds within the physically cross-linked (preassembled) π-conjugated peptide strands. Rheology measurements were used to evaluate the mechanical modifications of the network, and the chemical changes that accompany the cross-linking were further confirmed by infrared spectroscopy. Furthermore, we modified these cross-linkable π-conjugates by incorporating extracellular matrix (ECM)-derived Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Gly-Asp (RGD) bioactive epitopes to support human neural stem and progenitor cell (hNSCs) differentiation. The hNSCs undergo differentiation into neurons on IKVAV-derived π-conjugates while RGD-containing peptides failed to support cell attachment. These findings provide significant insight into the biochemical and electronic properties of π-conjugated peptide hydrogelators for creating artificial ECM to enable advanced tissue-engineering applications.