Browsing by Subject "Polyethylene glycol"

Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP regeneration, the development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration.

Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels.

A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 - 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 hydrogel carrier, as compared to cells in liquid suspension.

The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.

Biologics are potent, highly specific, and well-tolerated and have become an important class of drugs. Despite their promise, most of them have a short half-life due to rapid renal elimination and in vivo degradation. Their short in vivo half-life hence necessitates frequent injections, resulting in a peak-and-valley profile in drug concentration that is pharmacologically suboptimal and incurs a high treatment cost and suboptimal patient compliance because of the need for frequent drug administration and the side-effects associate with a peak-and-valley drug concentration profile.One of the most common approaches to overcome these challenges is the covalent attachment of biologics to polyethylene glycol (PEG), a technology that is colloquially termed PEGylation. PEGylated drug conjugates have a much longer plasma half-life than the native drug due to their larger size leading to slower renal clearance, reduced opsonization that reduces clearance by the reticuloendothelial system (RES) organs, and improved solubility. Unfortunately, PEGylation has several significant limitations, some of which have come to the forefront recently. First, PEG was initially believed to be non-immunogenic. However, it is now well accepted that PEGylated therapeutics induce PEG antibodies upon treatment. Pre-existing PEG antibodies have also been reported in up to 70% of the population who are naïve to PEGylated therapeutics, possibly due to chronic exposure to PEG in consumer products and because PEG is used as an excipient in many drug formulations. Both induced and pre-existing PEG antibodies have caused a severe allergic reaction and accelerated clearance in some patients, reducing the drugs’ clinical efficacy. These issues have collectively led to the early termination of several clinical trials of PEGylated drug candidates and the withdrawal of several PEGylated therapeutics from the market. Second, attempts to improve the pharmacokinetics (PK) of PEG have focused on synthesizing branched and star-shaped PEGs. However, these architectures have a modest effect on PK and have antigenic and immunogenic profiles similar to linear PEG. Third, PEG forms vacuoles in major organs due to its non-biodegradable structure and clearance by the RES. Clearly, optimization of PEG is now at an asymptote, and new architectures that radically depart from linear PEG are needed to address these limitations. Motivated by these needs, we have developed a “next-gen” PEG-like polymer, poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). POEGMA is an amphiphilic, hyperbranched polymer that breaks up the long ethylene glycol sequences in PEG and presents them as much shorter oligomeric ethylene glycol (OEG) side-chains along a hydrophobic backbone. It has been previously shown that POEGMA with three EG unit long side chains improves the PK of exendin, a peptide drug used in the clinic to treat type 2 diabetes (T2D). The POEGMA conjugate of exendin (Ex-POEGMA) also showed no in vitro binding to human-derived PEG antibodies, presumably because the short OEG side-chains in POEGMA lack the anti-PEG epitope. Although these results were encouraging, the soluble Ex-POEGMA conjugate reported previously only showed a modest improvement in the PK of the drug. This study also did not report how POEGMA conjugates compare with PEG in the PK and efficacy and the intrinsic immunogenicity of POEGMA that could occur upon repeated administration of POEGMA-drug conjugates, which are critical for its clinical translation. This doctoral dissertation had three goals. The first goal was to devise a strategy to further enhance the half-life of exendin beyond the four days of therapeutic action of a soluble POEGMA conjugate in mice and do so without compromising its lack of reactivity to pre-existing PEG antibodies. Improving the PK of POEGMA beyond the duration exhibited by soluble POEGMA conjugates is important because recent long-acting delivery technologies such as Fc fusions have been shown to work for 4-6 day in mice and 7 days in humans so that a next-generation PEGylation technology must match—at a minimum— the performance of Fc conjugates. To address this goal, we report on the design and optimization of a gel-like injectable depot of an Ex-POEGMA conjugate that achieves sustained-release from the depot into the bloodstream while retaining its lack of reactivity towards PEG antibodies. We identify an optimal Ex-POEGMA depot that maximizes blood glucose control in diabetic mice, followed by investigating its PK and efficacy benefits. The second goal was to investigate the intrinsic immunogenicity and toxicity of a POEGMA-drug conjugate and compare it to PEG. We report the somewhat remarkable result that Ex-POEGMA conjugates have very little if any humoral immunogenicity in mice, as seen by the lack of an IgM and IgG response to repeated doses of Ex-POEGMA. Furthermore, this lack of immunogenicity is highly robust, as confirmed by the lack of Immunoglobulin (Ig) G and IgM against POEGMA observed with a POEGMA conjugate of the highly immunogenic ovalbumin (OVA) protein, even with co-administered Freund’s adjuvant. While POEGMA was non-immunoreactive, PEG induced a persistent anti-PEG immune response, leading to its subsequent doses' early clearance and loss of efficacy. Fortuitously, POEGMA did not induce vacuolization. The third goal was to apply the POEGMA technology to solve the limitations of two PEGylated drugs that failed at the late-stage clinical trials (Pegnivacogin) or was withdrawn from the market (Pegloticase) due to the life-threatening infusion reactions deriving from the reactivity to pre-existing PEG antibodies and induction of a strong anti-PEG immune response, respectively. Remarkably, the POEGMA formulated drugs showed no in vitro reactivity to pre-existing PEG antibodies and did not induce an anti-POEGMA immune response while showing efficacy at least comparable to the PEG conjugates. Overall, solving immunogenicity problems of PEG and improving upon its half-life benefits by creating injectable POEGMA conjugates that form a drug depot under the skin and provide sustained efficacy breathe new life into an established and valuable drug delivery technology that is facing an impasse.

Tumor growth, survival, and metastasis depend upon interactions with the matrix and cells which compose the tumor microenvironment (TME). Tumor cell interactions with transformed extracellular matrix (ECM) and immune cells, such as tumor associated neutrophils (TANs) have been shown to affect tumor progression both clinically and in animal models. Unfortunately, while the complexity of the TME is difficult to recapitulate in standard cell culture, it is also difficult to analyze and to influence in vivo. Researchers have sought to circumvent these challenges by developing 3D models in naturally derived matrices like collagen and Matrigel, but these scaffolds often sacrifice biochemical tunability and fail to meet the stiffness regimes often seen in malignant ECM. Thus, there is a need for highly controlled 3D culture systems which mimic the biophysical properties and cell-cell interactions of the TME to better investigate how these interactions direct cancer development and progression. To meet this goal, researchers have employed synthetic hydrogel systems for 3D in vitro cell culture. Our lab has previously engineered a synthetic scaffold to serve as an ECM by incorporating a matrix metalloproteinase cleavable peptide into a biocompatible poly(ethylene glycol) (PEG) backbone and grafting in an integrin-binding peptide. This system allows independent tuning of adhesivity and matrix stiffness and supports tumor cell growth and spheroid formation. Here, we present two applications of this system to model cell-matrix interactions in the TME. First, we orthogonally tuned the adhesion ligand concentration and stiffness of our PEG-based hydrogels to investigate the individual and interactive impact of these matrix properties in a physiologically relevant regime. We assessed the tumor progression of a fibrosarcoma cell line (derived from mesenchymal cells) and a triple-negative breast carcinoma cell line (TNBC, derived from epithelial cells) cultured in and on these hydrogels. We observed that the cell proliferation, invasion, and focal complex formation in the fibrosarcoma cells responded to changes in matrix stiffness, while the same behaviors in the TNBC cell line occurred in response to changes in matrix adhesivity. to discern the differential behavior of these broad classes of tumors. We found no interactive effect between the two matrix properties within the conditions tested. These results helped to reiterate the importance of independently tunable systems in assessing cell response to specific matrix properties and established our system’s ability to discern differential tumorigenic behavior across diseases. Next, we incorporated neutrophil extracellular traps (NETs) in our system to study their impact on tumorigenesis in TNBC cells in a highly controlled environment. We observed that NETs helped to increase cell survival, proliferation, and pro-metastatic morphological phenotype. We also showed that the presence of NETs influenced the secretion of IL-8, a pro-NETosis factor, and that conditioned media from cells cultured in these gels influenced NETosis in an IL-8 dependent manner. The results observed in this system correlate with murine models and clinical studies in the literature and help to provide additional insight of the individual factors at play in the NET-mediated promotion of TNBC progression and metastasis. To expand upon current models of cell-matrix interactions within the TME, we applied an existing model to assess the differences in behavior between a fibrosarcoma and TNBC cell line and developed a new model for assessing NETs as a matrix biomolecule in TME models. These two reductionist models help to advance our understanding of the roles specific aspects of the TME play in tumor progression, ultimately helping to drive better rational design of TME-targeted therapies to improve clinical outcomes of cancer patients.