Coiled Coil Crosslinked Alginate Hydrogels Dampen Macrophage-Driven Inflammation.

Alginate hydrogels are widely used for tissue engineering and regenerative medicine due to their excellent biocompatibility. A facile and commonly used strategy to crosslink alginate is the addition of Ca2+ that leads to hydrogelation. However, extracellular Ca2+ is a secondary messenger in activating inflammasome pathways following physical injury or pathogenic insult, which carries the risk of persistent inflammation and scaffold rejection. Here, we present graft copolymers of charge complementary heterodimeric coiled coil (CC) peptides and alginate that undergo supramolecular self-assembly to form Ca2+ free alginate hydrogels. The formation of heterodimeric CCs was confirmed using circular dichroism spectroscopy, and scanning electron microscopy revealed a significant difference in crosslink density and homogeneity between Ca2+ and CC crosslinked gels. The resulting hydrogels were self-supporting and display shear-thinning and shear-recovery properties. In response to lipopolysaccharide (LPS) stimulation, peritoneal macrophages and bone marrow-derived dendritic cells cultured in the CC crosslinked gels exhibited a 10-fold reduction in secretion of the proinflammatory cytokine IL-1ß compared to Ca2+ crosslinked gels. A similar response was also observed in vivo upon peritoneal delivery of Ca2+ or CC crosslinked gels. Analysis of peritoneal lavage showed that macrophages in mice injected with Ca2+ crosslinked gels display a more inflammatory phenotype compared to macrophages from mice injected with CC crosslinked gels. These results suggest that CC peptides by virtue of their tunable sequence-structure-function relationship and mild gelation conditions are promising alternative crosslinkers for alginate and other biopolymer scaffolds used in tissue engineering.

Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines.

De novo designed peptides that self-assemble into cross-ß rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-ß peptide KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-ß-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. STATEMENT OF SIGNIFICANCE: Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-ß rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.

Peptide-based supramolecular vaccine systems.

Currently approved replication-competent and inactivated vaccines are limited by excessive reactogenicity and poor safety profiles, while subunit vaccines are often insufficiently immunogenic without co-administering exogenous adjuvants. Self-assembling peptide-, peptidomimetic-, and protein-based biomaterials offer a means to overcome these challenges through their inherent modularity, multivalency, and biocompatibility. As these scaffolds are biologically derived and present antigenic arrays reminiscent of natural viruses, they are prone to immune recognition and are uniquely capable of functioning as self-adjuvanting vaccine delivery vehicles that improve humoral and cellular responses. Beyond this intrinsic immunological advantage, the wide range of available amino acids allows for facile de novo design or straightforward modifications to existing sequences. This has permitted the development of vaccines and immunotherapies tailored to specific disease models, as well as generalizable platforms that have been successfully applied to prevent or treat numerous infectious and non-infectious diseases. In this review, we briefly introduce the immune system, discuss the structural determinants of coiled coils, ß-sheets, peptide amphiphiles, and protein subunit nanoparticles, and highlight the utility of these materials using notable examples of their innate and adaptive immunomodulatory capacity. STATEMENT OF SIGNIFICANCE: Subunit vaccines have recently gained considerable attention due to their favorable safety profiles relative to traditional whole-cell vaccines; however, their reduced efficacy requires co-administration of reactogenic adjuvants to boost immune responses. This has led to collaborative efforts between engineers and immunologists to develop nanomaterial-based vaccination platforms that can elicit protection without deleterious side effects. Self-assembling peptidic biomaterials are a particularly attractive approach to this problem, as their structure and function can be controlled through primary sequence design and their capacity for multivalent presentation of antigens grants them intrinsic self-adjuvanticity. This review introduces the various architectures adopted by self-assembling peptides and discusses their application as modulators of innate and adaptive immunity.

Direct lineage tracing reveals Activin-a potential for improved pancreatic homing of bone marrow mesenchymal stem cells and efficient ß-cell regeneration in vivo.

BACKGROUND: Despite the potential, bone marrow-derived mesenchymal stem cells (BMSCs) show limitations for beta (ß)-cell replacement therapy due to inefficient methods to deliver BMSCs into pancreatic lineage. In this study, we report TGF-ß family member protein, Activin-a potential to stimulate efficient pancreatic migration, enhanced homing and accelerated ß-cell differentiation. METHODS: Lineage tracing of permanent green fluorescent protein (GFP)- tagged donor murine BMSCs transplanted either alone or in combination with Activin-a in diabetic mice displayed potential ß-cell regeneration and reversed diabetes. RESULTS: Pancreatic histology of Activin-a treated recipient mice reflected high GFP+BMSC infiltration into damaged pancreas with normalized fasting blood glucose and elevated serum insulin. Whole pancreas FACS profiling of GFP+ cells displayed significant homing of GFP+BMSC with Activin-a treatment (6%) compared to BMSCs alone transplanted controls (0.5%). Within islets, approximately 5% GFP+ cells attain ß-cell signature (GFP+ Ins+) with Activin-a treatment versus controls. Further, double immunostaining for mesenchymal stem cell markers CD44+/GFP+ in infiltrated GFP+BMSC deciphers substantial endocrine reprogramming and ß-cell differentiation (6.4% Ins+/GFP+) within 15 days. CONCLUSION: Our investigation thus presents a novel pharmacological approach for stimulating direct migration and homing of therapeutic BMSCs that re-validates BMSC potential for autologous stem cell transplantation therapy in diabetes.

Amyloid Fibrils with Positive Charge Enhance Retroviral Transduction in Mammalian Cells.

Amyloid fibrils are cross-ß-sheet-rich protein/peptide fibrils that are typically associated with neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Recently, functional amyloids have been discovered where amyloids are implicated in performing normal physiological functions of the host organism rather than creating diseases. The ability of amyloids to interact with the cell membrane and other small biomolecules exhibits its great potential to be used as a biomaterial for cell adhesion and gene delivery system. Given the established ability of semen-derived amyloids to concentrate HIV in semen and that of charged polymers as an enhancer of retroviral gene transfer, we hypothesized that charged amyloid fibrils can augment virus-mediated delivery system. We show that amyloids of α-synuclein formed in the presence and absence of cationic polymers chitosan and amyloid of poly-l-lysine can interact with lentiviral particles and enhance transduction efficiency in cells. The amyloid nanofibrils increase transduction efficiency up to ∼4 fold similar to widely used cationic polymer Polybrene. This study shows that amyloid nanofibril scaffolds may be used as targeted gene delivery systems.

Efficient in situ gene delivery via PEG diacrylate matrices.

For diseases related to genetic disorders or cancer, many cellular therapies rely on the ex vivo modification of cells for attaining a desired therapeutic effect. The efficacy of such therapies involving the genetic modification of cells relies on the extent of gene expression and subsequent persistence of modified cells when infused into the patient's body. In situ gene delivery implies the manipulation of cells in their in vivo niche such that the effectiveness can be improved by minimizing post manipulation effects like cell death, lack of persistence, etc. Furthermore, material-based in situ localized gene delivery can reduce the undesired side effects caused by systemic modifications. Here, we have used polyethylene (glycol) diacrylate (PEGDA) based cryogels to genetically modify cells in vivo with a focus on immunotherapy. PEGDA cryogels were either blended with gelatin methacrylate (GELMA) or surface modified with poly-l-lysine (PLL) in order to improve cell adhesion and/or retain viruses for localized gene delivery. On using the lentiviruses encoding gene for green fluorescent protein (GFP) in in vitro experiments, we found higher transduction efficiency in HEK 293FT cells via PEGDA modified with poly-l-lysine (PEGDA-PLL) and PEGDA-GELMA cryogels compared to PEGDA cryogels. In vitro release experiments showed improved retention of GFP lentiviruses in PEGDA-PLL cryogels, which were then employed for in vivo gene delivery and were demonstrated to perform better than the corresponding bolus delivery of lentiviruses through an injection. Both physical and biological characterization studies of these cryogels show that this material platform can be used for gene delivery as well as other tissue engineering applications.