Faculty Bibliography

With proper control over copolymer design and solvation conditions, self-assembled materials display impressive morphological variety that encompasses nanoscale colloids as well as bulk three-dimensional architectures. Here we take advantage of both hydrophobicity and crystallinity to mediate supramolecular self-assembly of spherical micellar, linear fibrillar, or hydrogel structures by a family of highly asymmetric poly(ethylene glycol)-b-oligo(ethylene sulfide) (PEG-OES) copolymers. Assembly structural polymorphism was achieved with modification of PEG-OES topology (linear versus multiarm) and with precise, monomer-by-monomer control of OES length. Notably, all three morphologies were accessed utilizing OES oligomers with degrees of polymerization as short as three. These exceptionally small assembly forming blocks represent the first application of ethylene sulfide oligomers in supramolecular materials. While the assemblies demonstrated robust aqueous stability over time, oxidation by hydrogen peroxide progressively converted ethylene sulfide residues to increasingly hydrophilic and amorphous sulfoxides and sulfones, causing morphological changes and permanent disassembly. We utilized complementary microscopic and spectroscopic techniques to confirm this chemical stimulus-responsive behavior in self-assembled PEG-OES colloidal dispersions and physical gels. In addition to inherent stimulus-responsive behavior, fibrillar assemblies demonstrated biologically relevant molecular delivery, as confirmed by the dose-dependent activation of murine bone marrow-derived dendritic cells following fibril-mediated delivery of the immunological adjuvant monophosphoryl lipid A. In physical gels composed of either linear or multiarm PEG-OES precursors, rheologic analysis also identified mechanical stimulus-responsive shear thinning behavior. Thanks to the facile preparation, user-defined morphology, aqueous stability, carrier functionality, and stimuli-responsive behaviors of PEG-OES supramolecular assemblies, our findings support a future role for these materials as injectable or implantable biomaterials.

Antigen-specific immune responses to protein drugs can hinder efficacy and compromise safety because of drug neutralization and secondary clinical complications. We report a tolerance induction strategy to prevent antigen-specific humoral immune responses to therapeutic proteins. Our modular, biomolecular approach involves engineering tolerizing variants of proteins such that they bind erythrocytes in vivo upon injection, on the basis of the premise that aged erythrocytes and the payloads they carry are cleared tolerogenically, driving the deletion of antigen-specific T cells. We demonstrate that binding the clinical therapeutic enzyme Escherichia coli l-asparaginase to erythrocytes in situ antigen-specifically abrogates development of antibody titers by >1000-fold and extends the pharmacodynamic effect of the drug 10-fold in mice. Additionally, a single pretreatment dose of erythrocyte-binding asparaginase tolerized mice to multiple subsequent doses of the wild-type enzyme. This strategy for reducing antigen-specific humoral responses may enable more effective and safer treatment with therapeutic proteins and drug candidates that are hampered by immunogenicity.

The use of peptide engineering to develop self-assembling membranes that are responsive to cellular enzyme activities is reported. The membranes are obtained by combining hyaluronan (HA) and a rationally designed peptide amphiphile (PA) containing a proteolytic domain (GPQGIWGQ octapeptide) sensitive to matrix metalloproteinase-1 (MMP-1). Insertion of an octapeptide in a typical PA structure does not disturb its self-assembly into fibrillar nanostructures neither the ability to form membranes with HA. In vitro enzymatic degradation with hyaluronidase and MMP-1 shows that membranes containing the MMP-1 substrate exhibit enhanced enzymatic degradation, compared with control membranes (absence of MMP-1 cleavable peptide or containing a MMP-1 insensitive sequence), being completely degraded after 7 days. Cell viability and proliferation is minimally affected by the enzymatically cleavable functionality of the membrane, but the presence of MMP-1 cleavable sequence does stimulate the secretion of MMP-1 by fibroblasts and interfere with matrix deposition, particularly the deposition of collagen. By showing cell-responsiveness to biochemical signals presented on self-assembling membranes, this study highlights the ability of modulating certain cellular activities through matrix engineering. This concept can be further explored to understand the cellular remodeling process and as a strategy to develop artificial matrices with more biomimetic degradation for tissue engineering applications.

To minimize the adverse side effects of conventional chemotherapy, a targeted micellar drug carrier was investigated that retains hydrophobic drugs in its core and then releases the drug via ultrasonic activation. This paper compares the percent drug release from folated versus non-folated micelles by insonation at 70 kHz and different acoustic power densities. The encapsulated drug is Doxoru- bicin (Dox). A physical model of zero-order release with first-order re-encapsulation was used to fit the experimental kinetic data. Additionally, the acoustic activation power density and Gibbs free energy were introduced and calculated for folated and non-targeted micelles. The data suggests an important role of inertial cavitation in drug release and the presence of a power density threshold for inertial cavitation.

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)-based hydrogels, creating a three-dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2 week-old mice were cultured in a crosslinked four-arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7-15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60-80%; p = 0.76). Significant (p < 0.04) model effects, however, were detected in the maturation and developmental competence of the follicle-derived oocytes. A swelling ratio of > 21.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel-based, 3D microenvironment.

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8(+) T cell responses in two murine cancer models.

Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.

Regenerative therapies that use allogeneic cells are likely to encounter immunological barriers similar to those that occur with transplantation of solid organs and allogeneic hematopoietic stem cells (HSCs). Decades of experience in clinical transplantation hold valuable lessons for regenerative medicine, offering approaches for developing tolerance-induction treatments relevant to cell therapies. Outside the field of solid-organ and allogeneic HSC transplantation, new strategies are emerging for controlling the immune response, such as methods based on biomaterials or mimicry of antigen-specific peripheral tolerance. Novel biomaterials can alter the behavior of cells in tissue-engineered constructs and can blunt host immune responses to cells and biomaterial scaffolds. Approaches to suppress autoreactive immune cells may also be useful in regenerative medicine. The most innovative solutions will be developed through closer collaboration among stem cell biologists, transplantation immunologists and materials scientists.

Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600-1,000 μm in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture.