Major limitations of current tissue regeneration approaches using artificial scaffolds are fibrous encapsulation, lack of host cellular infiltration, unwanted immune responses, surface degradation preceding biointegration, and artificial degradation byproducts. Specifically, for scaffolds larger than 200 500 μm, implants must be accompanied by host angiogenesis in order to provide adequate nutrient/waste exchange in the newly forming tissue. In the current work, we design a peptide-based self-assembling nanofibrous hydrogel containing cell-mediated degradation and proangiogenic moieties that specifically address these challenges. This hydrogel can be easily delivered by syringe, is rapidly infiltrated by cells of hematopoietic and mesenchymal origin, and rapidly forms an extremely robust mature vascular network. scaffolds show no signs of fibrous encapsulation and after 3 weeks are resorbed into the native tissue. These supramolecular assemblies may prove a vital paradigm for tissue regeneration and specifically for ischemic tissue disease.

Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.

Delivery of small molecules and drugs to tissues is a mainstay of several tissue engineering strategies. Next generation treatments focused on localized drug delivery offer a more effective means in dealing with refractory healing when compared to systemic approaches. Here we describe a novel multidomain peptide hydrogel that capitalizes on synthetic peptide chemistry, supramolecular self-assembly and cytokine delivery to tailor biological responses. This material is biomimetic, shows shear stress recovery and offers a nanofibrous matrix that sequesters cytokines. The biphasic pattern of cytokine release results in the spatio-temporal activation of THP-1 monocytes and macrophages. Furthermore, macrophage-material interactions are promoted without generation of a proinflammatory environment. Subcutaneous implantation of injectable scaffolds showed a marked increase in macrophage infiltration and polarization dictated by cytokine loading as early as 3 days, with complete scaffold resorption by day 14. Macrophage interaction and response to the peptide composite facilitated the (i) recruitment of monocytes/macrophages, (ii) sustained residence of immune cells until degradation, and (iii) promotion of a pro-resolution M2 environment. Our results suggest the potential use of this injectable cytokine loaded hydrogel scaffold in a variety of tissue engineering applications.

The recent and precipitous increase in opioid analgesic abuse and overdose has inspired investigation of the dopamine D3 receptor (D3R) as a target for therapeutic intervention. Metabolic instability or predicted toxicity has precluded successful translation of previously reported D3R-selective antagonists to clinical use for cocaine abuse. Herein, we report a series of novel and D3R crystal structure-guided 4-phenylpiperazines with exceptionally high D3R affinities and/or selectivities with varying efficacies. Lead compound 19 was selected based on its in vitro profile: D3R Ki = 6.84 nM, 1700 fold D3R versus D2R binding selectivity, and its metabolic stability in mouse microsomes. Compound 19 inhibited oxycodone-induced hyperlocomotion in mice and reduced oxycodone-induced locomotor sensitization. In addition, pretreatment with 19 also dose-dependently inhibited the acquisition of oxycodone-induced conditioned place preference (CPP) in rats. These findings support the D3R as a target for opioid dependence treatment and compound 19 as a new lead molecule for development.

Self-assembly of multidomain peptides (MDP) can be tailored to carry payloads that modulate the extracellular environment. Controlled release of growth factors, cytokines, and small-molecule drugs allows for unique control of in vitro and in vivo responses. In this study, we demonstrate this process of ionic cross-linking of peptides using multivalent drugs to create hydrogels for sustained long-term delivery of drugs. Using phosphate, heparin, clodronate, trypan, and suramin, we demonstrate the utility of this strategy. Although all multivalent anions result in good hydrogel formation, demonstrating the generality of this approach, suramin led to the formation of the best hydrogels per unit concentration and was studied in greater detail. Suramin ionically cross-linked MDP into a fibrous meshwork as determined by scanning and transmission electron microscopy. We measured material storage and loss modulus using rheometry and showed a distinct increase in G′ and G″ as a function of suramin concentration. Release of suramin from scaffolds was determined using UV spectroscopy and showed prolonged release over a 30 day period. Suramin bioavailability and function were demonstrated by attenuated M1 polarization of THP-1 cells compared to positive control. Overall, this design strategy has allowed for the development of a novel class of polymeric delivery vehicles with generally long-term release and, in the case of suramin, cross-linked hydrogels that can modulate cellular phenotype.

Tissue engineered vascular grafts require long fabrication times, in part, due to the requirement of cells from a variety of cell sources to produce a robust load bearing, extracellular matrix. Herein, we propose a design strategy for the fabrication of tubular conduits comprised of collagen fiber networks and elastin-like protein polymers to mimic native tissue structure and function. Dense fibrillar collagen networks exhibited an ultimate tensile strength (UTS) of 0.71 ± 0.06 MPa, strain to failure of 37.1 ± 2.2%, and Young’s modulus of 2.09 ± 0.42 MPa, comparing favorably to an UTS and a Young’s modulus for native blood vessels of 1.4 – 11.1 MPa and 1.5 ± 0.3 MPa, respectively. Resilience, a measure of recovered energy during unloading of matrices, demonstrated that 58.9 ± 4.4% of the energy was recovered during loading-unloading cycles. Rapid fabrication of multilayer tubular conduits with maintenance of native collagen ultrastructure was achieved with internal diameters ranging between 1 to 4 mm. Compliance and burst pressures exceeded 2.7 ± 0.3%/100 mmHg and 830 ± 131 mmHg, respectively, with a significant reduction in observed platelet adherence as compared to ePTFE (6.8 ± 0.05 × 105 vs. 62 ± 0.05 × 105 platelets/mm2, p < 0.01). Using a rat aortic interposition model, early in vivo responses were evaluated at 2 weeks via Doppler ultrasound and CT angiography with immunohistochemistry confirming a limited early inflammatory response (n=8). Engineered collagen-elastin composites represent a promising strategy for fabricating synthetic tissues with defined extracellular matrix content, composition, and architecture.

For a proangiogenic therapy to be successful, it must promote the development of mature vasculature for rapid reperfusion of ischemic tissue. Whole growth factor, stem cell, and gene therapies have yet to achieve the clinical success needed to become FDA-approved revascularization therapies. Herein, we characterize a biodegradable peptide-based scaffold engineered to mimic VEGF and self-assemble into a nanofibrous, thixotropic hydrogel, SLanc. We found that this injectable hydrogel was rapidly infiltrated by host cells and could be degraded while promoting the generation of neovessels. In mice with induced hind limb ischemia, this synthetic peptide scaffold promoted angiogenesis and ischemic tissue recovery, as shown by Doppler-quantified limb perfusion and a treadmill endurance test. Thirteen-month-old mice showed significant recovery within 7 days of treatment. Biodistribution studies in healthy mice showed that the hydrogel is safe when administered intramuscularly, subcutaneously, or intravenously. These preclinical studies help establish the efficacy of this treatment for peripheral artery disease due to diminished microvascular perfusion, a necessary step before clinical translation. This peptide-based approach eliminates the need for cell transplantation or viral gene transfection (therapies currently being assessed in clinical trials) and could be a more effective regenerative medicine approach to microvascular tissue engineering.

Progress in self-assembly and supramolecular chemistry has been directed toward obtaining macromolecular assemblies with higher degrees of complexity, simulating the highly structured environment in natural systems. One approach to this type of complexity are multistep, multicomponent, self-assembling systems that allow approaches comparable to traditional multistep synthetic organic chemistry; however, only a few examples of this approach have appeared in the literature. Our previous work demonstrated nanofibrous mimics of the extracellular matrix. Here we demonstrate the ability to create a unique hydrogel, developed by stepwise self-assembly of multidomain peptide fibers and liposomes. The two-component system allows for controlled release of bioactive factors at multiple time points. The individual components of the self-assembled gel and the composite hydrogel were characterized by TEM, SEM, and rheometry, demonstrating that peptide nanofibers and lipid vesicles both retain their structural integrity in the composite gel. The rheological robustness of the hydrogel is shown to be largely unaffected by the presence of liposomes. Release studies from the composite gels loaded with different growth factors EGF, MCP-1, and PlGF-1 showed delay and prolongation of release by liposomes entrapped in the hydrogel compared to more rapid release from the hydrogel alone. This bimodal release system may have utility in systems where timed cascades of biological signals may be valuable, such as in tissue regeneration.