Faculty Bibliography

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute pain, fever, and inflammation and are being explored in a new indication in cancer. Side effects associated with long-term use of NSAIDs such as gastrointestinal damage and elevated risk of stroke, however, can limit their use and exploration in new indications. Here we report a facile method to prepare well-defined amphiphilic diblock copolymer NSAID prodrugs by direct reversible addition-fragmentation transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen (IBU), a widely used NSAID. The synthesis and self-assembling behavior of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic poly(ethylene glycol) block and a hydrophobic IBU-bearing prodrug block were investigated. Release profiles of IBU from the micelles by hydrolysis were evaluated. Furthermore, the antiproliferative action of the IBU-containing micelles in human cervical carcinoma (HeLa) and murine melanoma (B16-F10) cells was assessed.

Synergy in the downstream signaling pathways of the vascular endothelial growth factor receptor-2 (VEGFR-2) and the integrin αvβ3 is critical for blood vessel formation. Thus, agents that activate both receptors could possess efficient pro-angiogenic potential. Here, we created a fibrin-binding bi-functional protein (FNIII10-VEGF) consisting of the 10th type III domain of fibronectin (FNIII10) fused to a plasmin-resistant VEGF-A165 mutant (VEGF) that potentiated angiogenic processes when compared to the effect of the separate molecules. FNIII10-VEGF was able to bind both VEGFR-2 and integrin αvβ3. Intriguingly, cell attachment and spreading to immobilized FNIII10-VEGF was significantly enhanced compared to individual FNIII10 or VEGF proteins. Delivery of immobilized FNIII10-VEGF by covalent linkage to a fibrin matrix significantly enhanced the angiogenic response in an in vivo wound healing assay compared to soluble VEGF. Unexpectedly, the angiogenic response to fibrin-immobilized FNIII10-VEGF was reduced in comparison to the pro-angiogenic effect of fibrin-immobilized VEGF. Collectively, findings of this study corroborate a critical role for a subtle balance of the integrin-VEGF interplay in angiogenesis and provide insight in how engineered growth factors in concert with biomaterial matrices may offer a potent molecular/material approach to harness these interactions for therapeutic angiogenesis.

Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth.

Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS-bl-PEG polymersomes (PSs; watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8(+) T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4(+) or CD8(+) T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4(+) T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8(+) T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4(+) and CD8(+) T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses.

We report optimization of a serum- and feeder-free, three-dimensional (3D) niche created with a synthetic polyethylene glycol (PEG)-based extracellular matrix for self-renewal of human embryonic stem cells (hESCs). Three hESC lines (H9, H1 and Novo) were cultured in hydrogels of different mechanical properties, and cellular morphology and activity were compared to culture in feeder-free or feeder-containing two-dimensional (2D) niches. Significant effects of PEG concentration (5, 7.5, 10, 12.5 or 15%) and vinyl sulfone-functionalized PEG multiarm number (3, 4 or 8) on hESC morphology were detected in the H9 line. Cell growth was maximal with an 8 multiarm architecture of any PEG concentration, which yielded the highest expression of stemness-related genes. Alkaline phosphatase activity in cultured H9 cells was similar between the optimized feeder-free 3D and the feeder-containing 2D systems. However, increased expression of the KLF4, CDH1, TERT, SOX2, and UTF1 genes and expression of pluripotency-specific SSEA-4, Oct3/4, Nanog, Tra-1-60 and Tra-1-81 were detected in the 3D-cultured hESC clumps. H1 and Novo cell lines also expanded in the optimized 3D system, which maintain stemness properties. Although different proliferation activities were detected among three lines, the difference was decreased after the 3D culture. These results demonstrate that chemically defined, acellular niches created using PEG-based hydrogels have the potential to support hESC self-renewal. Modulation of 3D properties can create various models for cell transformation and differentiation.

Large-scale experimentation is becoming instrumental in enabling new discoveries in systems biology and personalized medicine. We developed a multiplexed high-throughput nanoimmunoassay chip capable of quantifying four biomarkers in 384 5 nL samples, for a total of 1536 assays. Our platform, compared to conventional methods, reduces volume and reagent cost by ~1000-fold. We applied our platform in the context of systems vaccinology, to assess the synergistic production of inflammatory cytokines from dendritic cells (DCs) stimulated with 10 different adjuvants that target members of the Toll-like receptor (TLR) family. We quantified these adjuvants both alone and in all pairwise combinations, for a total of 435 conditions, revealing numerous synergistic pairs. We evaluated two synergistic interactions, MPLA + Gardiquimod and MPLA + CpG-B, in a mouse model, where we measured the same inflammatory cytokines in bronchoalveolar lavage and in blood serum at 4 different time points using our chip, and observed similar synergistic effects in vivo, demonstrating the potential of our microfluidic platform to predict agonistic immunogenicity. More generally, a high-throughput, matrix-insensitive, low sample volume technology can play an important role in the discovery of novel therapeutics and research areas requiring large-scale biomarker quantitation.

By binding growth factors (GFs), the ECM tightly regulates their activity. We recently reported that the heparin-binding domain II of fibronectin acts as a promiscuous high-affinity GF-binding domain. Here we hypothesized that fibrin, the provisional ECM during tissue repair, also could be highly promiscuous in its GF-binding capacity. Using multiple affinity-based assays, we found that fibrin(ogen) and its heparin-binding domain bind several GFs from the PDGF/VEGF and FGF families and some GFs from the TGF-β and neurotrophin families. Overall, we identified 15 unique binding interactions. The GF binding ability of fibrinogen caused prolonged retention of many of the identified GFs within fibrin. Thus, based on the promiscuous and high-affinity interactions in fibrin, GF binding may be one of fibrin's main physiological functions, and these interactions may potentially play an important and ubiquitous role during tissue repair. To prove this role in a gain-of-function model, we incorporated the heparin-binding domain of fibrin into a synthetic fibrin-mimetic matrix. In vivo, the multifunctional synthetic matrix could fully mimic the effect of fibrin in a diabetic mouse model of impaired wound healing, demonstrating the benefits of generating a hybrid biomaterial consisting of a synthetic polymeric scaffold and recombinant bioactive ECM domains. The reproduction of GF-ECM interactions with a fibrin-mimetic matrix could be clinically useful, and has the significant benefit of a more straightforward regulatory path associated with chemical synthesis rather than human sourcing.

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

The main problem associated with the administration of anti-cancer medication is that the drug is delivered throughout the body causing undesirable side effects. Therefore, it is important to synthesize drug carriers capable of minimizing the adverse side effects of chemotherapy by preferentially targeting tumor cells both actively (e.g. a folate receptor) and using external stimulus (e.g. ultrasound). In this paper, we report the synthesis of Pluronic P105 micelles with a folate targeting moiety (with a yield of 48%) containing doxorubicin (Dox). We applied low frequency ultrasound as an external stimulus and measured the amount of release of Dox from these folated micelles. The results showed that the percent drug release increases as the power intensity of ultrasound increases. The maximum amount of release (14%) was measured at 5.4 W/cm(2). A power density threshold at approximately 0.55 W/cm(2) exists below which no statistically significant release was observed. This lower threshold suggests that cavitation plays an important role in triggering drug release from targeted micelles.

While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyaluronic acid (HA) hydrogel is a suitable carrier for the delivery of rhBMP-2, but a major limitation of this scaffold is its low cell adhesive properties. In this study, we have determined whether covalent grafting of an integrin-specific ligands into HA hydrogel could improve cell attachment and further enhance the osteogenic potential of rhBMP-2. A structurally stabilized fibronectin (FN) fragment containing the major integrin-binding domain of full-length FN (FN III9*-10) was engineered, in order to be incorporated into HA hydrogel. Compared to non-functionalized HA hydrogel, HA-FN hydrogel remarkably improved the capacity of the material to support mesenchymal stem cell attachment and spreading. In an ectopic bone formation model in the rat, delivery of rhBMP-2 with HA-FN hydrogel resulted in the formation of twice as much bone with better organization of collagen fibers compared to delivering the growth factor in non-functionalized HA hydrogel. This engineered hydrogel carrier for rhBMP-2 can be relevant in clinical bone repair.