A better understanding of protein aggregation is bound to translate into critical advances in several areas, including the treatment of misfolded protein disorders and the development of self-assembling biomaterials for novel commercial applications. Because of its ubiquity and clinical potential, albumin is one of the best-characterized models in protein aggregation research; but its properties in different conditions are not completely understood. Here, we carried out all-atom molecular dynamics simulations of albumin to understand how electrostatics can affect the conformation of a single albumin molecule just prior to self-assembly. We then analyzed the tertiary structure and solvent accessible surface area of albumin after electrostatically triggered partial denaturation. The data obtained from these single protein simulations allowed us to investigate the effect of electrostatic interactions between two proteins. The results of these simulations suggested that hydrophobic attractions and counterion binding may be strong enough to effectively overcome the electrostatic repulsions between the highly charged monomers. This work contributes to our general understanding of protein aggregation mechanisms, the importance of explicit consideration of free ions in protein solutions, provides critical new insights about the equilibrium conformation of albumin in its partially denatured state at low pH, and may spur significant progress in our efforts to develop biocompatible protein hydrogels driven by electrostatic partial denaturation.

Oxidative stress in tissue can contribute to chronic inflammation that impairs wound healing and the efficacy of cell-based therapies and medical devices. We describe the synthesis and characterization of a biodegradable, thermoresponsive gel with intrinsic antioxidant properties suitable for the delivery of therapeutics. Citric acid, poly(ethylene glycol) (PEG), and poly-N-isopropylacrylamide (PNIPAAm) were copolymerized by sequential polycondensation and radical polymerization to produce poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN). PPCN was chemically characterized, and the thermoresponsive behavior, antioxidant properties, morphology, potential for protein and cell delivery, and tissue compatibility in vivo were evaluated. The PPCN gel has a lower critical solution temperature (LCST) of 26 °C and exhibits intrinsic antioxidant properties based on its ability to scavenge free radicals, chelate metal ions, and inhibit lipid peroxidation. PPCN displays a hierarchical architecture of micropores and nanofibers, and contrary to typical thermoresponsive polymers, such as PNIPAAm, PPCN gel maintains its volume upon formation. PPCN efficiently entrapped and slowly released the chemokine SDF-1α and supported the viability and proliferation of vascular cells. Subcutaneous injections in rats showed that PPCN gels are resorbed over time and new connective tissue formation takes place without signs of significant inflammation. Ultimately, this intrinsically antioxidant, biodegradable, thermoresponsive gel could potentially be used as an injectable biomaterial for applications where oxidative stress in tissue is a concern.

Biological hydrogels are fundamentally biocompatible and have intrinsic similarities to extracellular matrices in medical applications and drug delivery systems. Herein we demonstrate the ability to form drug-eluting protein hydrogels using a novel mechanism that involves the electrostatically triggered partial denaturation and self-assembly of the protein via changes in pH. Partial denaturation increases the protein’s solvent exposed hydrophobic surface area, which then drives self-assembly of the protein into a hydrogel within 10 min at 37 °C. We describe the properties of an albumin hydrogel formed by this mechanism. Intrinsic drug binding properties of albumin to all-trans retinoic acid (atRA) are conserved through the partial denaturation process, as confirmed by fluorescence quenching. atRA released from the hydrogel inhibited smooth muscle cell migration as per an in vitro scratch wound assay. Atomistic molecular dynamics and potential of mean force calculations show the preservation and potential creation of new atRA binding sites with a binding energy of −41 kJ/mol. The resulting hydrogel is also biocompatible and exhibits rapid postgelation degradation after its implantation in vivo. This interdisciplinary work provides a new tool for the development of biocompatible protein hydrogel drug delivery systems.

The field of orthopedic tissue engineering is quickly expanding with the development of novel materials and strategies designed for rapid bone regeneration. While autologous bone grafts continue to be the standard of care, drawbacks include donor-site morbidity and short tissue supplies. Herein we report a novel nanocomposite sponge composed of poly(1,8-octanediol-co-citrate) (POC) and the bioactive ceramic β-tricalcium phosphate (TCP). We show that these nanocomposite sponges can be used as a depot for bone-producing (a.k.a. osteogenic) growth factors. In vitro bioactivity is demonstrated by significant upregulation of osteogenic genes, osteopontin (∼3 fold increase), osteocalcin (∼22 fold increase), alkaline phosphatase (∼10 fold increase), and transcription factor, RUNX2 (∼5 fold increase) over basal expression levels in mesenchymal stem cells. In vivo osteogenicity and biocompatibility is demonstrated in a standard subcutaneous implant model in rat. Results show that the nanocomposite sponge supports complete cell infiltration, minimal adverse foreign body response, positive cellular proliferation, and cellular expression of osteogenic markers in subcutaneous tissue. The results shown herein are encouraging and support the use of this sponge for future bone tissue engineering efforts.

The study of biomaterials for gene delivery in tissue engineering and regenerative medicine is a growing area, necessitating the investigation of new biomaterials and gene delivery vectors. Poly(1,8-octanediol citrate) (POC) and poly(glycerol-sebacate) (PGS) are biodegradable, biocompatible elastomers that have tunable mechanical properties, surface characteristics, and degradation rate. The objective of this work was to investigate whether POC and PGS would support the immobilization and release of lentivirus to allow sustained and localized transgene expression. Porous biomaterials were prepared using salt as a porogen and in vitro and in vivo transgene expression from immobilized and released lentiviruses were assessed. Cells seeded onto biomaterials loaded with lentiviruses yielded titer-dependent transgene expression in vitro. Lentivirus activity on both biomaterials was maintained for at least 5 days. When implanted subcutaneously in rats, POC and PGS with immobilized lentivirus exhibited sustained and localized transgene expression for at least 5 weeks. This research demonstrates that lentivirus immobilization on POC and PGS is feasible and potentially useful for a variety of tissue engineering and regenerative medicine applications.