Synthesis of Poly(allyl glycidyl ether)-Derived Polyampholytes and Their Application to the Cryopreservation of Living Cells.

Through the postpolymerization modification of poly(allyl glycidyl ether) (PAGE), a functionalizable polyether with a poly(ethylene oxide) backbone, we engineered a new class of highly tunable polyampholyte materials. These polyampholytes can be synthesized to have several useful properties, including low cytotoxicity and pH-responsive coacervate formation. In this study, we used PAGE-based polyampholytes (PAGE-PAs) for the cryopreservation of mammalian cell suspensions. Typically, dimethyl sulfoxide (DMSO) is the cryoprotectant used for preserving mammalian cells, but DMSO suffers from key drawbacks including toxicity and difficult post-thaw removal that motivates the development of new materials and methods. Toxicity and post-thaw survival were dependent on PAGE-PA composition with the highest immediate post-thaw survival for normal human dermal fibroblasts occurring for the least toxic PAGE-PA at a cation/anion ratio of 35:65. With low toxicity, the PAGE-PA concentration could be increased in order to increase immediate post-thaw survival of the immortalized mouse embryonic fibroblasts (NIH/3T3). While immediate post-thaw viability was achieved using only the PAGE-PAs, long-term cell survival was low, highlighting the challenges involved with the design of cryoprotective polyampholytes. An environment utilizing both PAGE-PAs and DMSO in a cryoprotective solution offered promising post-thaw viabilities exceeding 70%, with long-term metabolic activities comparable to unfrozen cells.

Engineering hyaluronic acid-based cryogels for CD44-mediated breast tumor reconstruction.

Breast cancer is a major health concern worldwide and is the leading cause of cancer-related death among American women. Traditional therapies, such as surgery, chemotherapy, and radiotherapy, are usually ineffective. Furthermore, cancer recurrence following targeted therapy often results from acquired drug resistance. Therefore, more realistic tumor models than monolayer cell culture for drug screening and discovery in an in vitro setting would facilitate the development of new therapeutic strategies. Toward this goal, we first developed a simple, rapid, low-cost, and high-throughput method for generating uniform multi-cellular tumor spheroids (MCTS) with controllable size. Next, biomimetic cryogel scaffolds fabricated from hyaluronic acid (HA) were utilized as a platform to reconstruct breast tumor microtissues with aspects of the complex tumor microenvironment in three dimensions. Finally, we investigated the interactions between the HA-based cryogels and CD44-positive breast tumor cells, individually or as MCTS. We found that incorporating the adhesive RGD peptide in cryogels led to the formation of a monolayer of tumor cells on the polymer walls, whereas MCTS cultured on RGD-free HA cryogels resulted in the growth of large and dense microtumors, more similar to native tumor masses. As a result, the MCTS-laden HA cryogel system induced a highly aggressive and chemotherapy drug-resistant tumor model. RGD-free HA-based cryogels represent an effective starting point for designing tumor models for preclinical research, therapeutic drug screening, and early cancer diagnosis.

Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots.

Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

Oxygen-Generating Cryogels Restore T Cell Mediated Cytotoxicity in Hypoxic Tumors.

Solid tumors are protected from antitumor immune responses due to their hypoxic microenvironments. Weakening hypoxia-driven immunosuppression by hyperoxic breathing of 60% oxygen has shown to be effective in unleashing antitumor immune cells against solid tumors. However, efficacy of systemic oxygenation is limited against solid tumors outside of lungs and has been associated with unwanted side effects. As a result, it is essential to develop targeted oxygenation alternatives to weaken tumor hypoxia as novel approaches to restore immune responses against cancer. Herein, we report on injectable oxygen-generating cryogels (O2-cryogels) to reverse tumor-induced hypoxia. These macroporous biomaterials were designed to locally deliver oxygen, inhibit the expression of hypoxia-inducible genes in hypoxic melanoma cells, and reduce the accumulation of immunosuppressive extracellular adenosine. Our data show that O2-cryogels enhance T cell-mediated secretion of cytotoxic proteins, restoring the killing ability of tumor-specific CTLs, both in vitro and in vivo. In summary, O2-cryogels provide a unique and safe platform to supply oxygen as a co-adjuvant in hypoxic tumors and have the potential to improve cancer immunotherapies.

Needle-injectable microcomposite cryogel scaffolds with antimicrobial properties.

Porous three-dimensional hydrogel scaffolds have an exquisite ability to promote tissue repair. However, because of their high water content and invasive nature during surgical implantation, hydrogels are at an increased risk of bacterial infection. Recently, we have developed elastic biomimetic cryogels, an advanced type of polymeric hydrogel, that are syringe-deliverable through hypodermic needles. These needle-injectable cryogels have unique properties, including large and interconnected pores, mechanical robustness, and shape-memory. Like hydrogels, cryogels are also susceptible to colonization by microbial pathogens. To that end, our minimally invasive cryogels have been engineered to address this challenge. Specifically, we hybridized the cryogels with calcium peroxide microparticles to controllably produce bactericidal hydrogen peroxide. Our novel microcomposite cryogels exhibit antimicrobial properties and inhibit antibiotic-resistant bacteria (MRSA and Pseudomonas aeruginosa), the most common cause of biomaterial implant failure in modern medicine. Moreover, the cryogels showed negligible cytotoxicity toward murine fibroblasts and prevented activation of primary bone marrow-derived dendritic cells ex vivo. Finally, in vivo data suggested tissue integration, biodegradation, and minimal host inflammatory responses when the antimicrobial cryogels, even when purposely contaminated with bacteria, were subcutaneously injected in mice. Collectively, these needle-injectable microcomposite cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

A bioinspired, photostable UV-filter that protects mammalian cells against UV-induced cellular damage.

While commercially available suncare products are effective at absorbing ultraviolet (UV)-light, recent studies indicate systemic toxicities associated with many traditional chemical and physical UV-filters. We demonstrate the application of xanthommatin, a biochrome present in arthropods and cephalopods, as an alternative chemical UV-filter that is cytocompatible while maintaining its photostability and photoprotective properties.

Autoclavable and Injectable Cryogels for Biomedical Applications.

Prior to any clinical application, terminal sterilization of biomaterials is a critical process imposed by the Food and Drug Administration. Of all the methods available for sterilization, high-pressure steam sterilization such as autoclaving is the most widely used. While autoclave sterilization minimizes pathogen contamination, it can dramatically impact both structural and biological properties of biomaterials. It has recently been reported that injectable cryogels with shape memory properties hold great promises as 3D macroporous biomimetic scaffolds for biomedical applications including tissue engineering. In this study, the impact of autoclave sterilization on properties of a series of cryogels is measured. Unlike conventional hydrogels, cryogels made of natural polymers demonstrate a strong resilience to autoclave sterilization. This process does not alter either their macrostructural or unique physical properties including syringe injectability. The scaffolds' bioactive sites are preserved and autoclaved cryogels retain their excellent cytological compatibility post-autoclaving. Furthermore, autoclaved cryogels do not trigger a notable activation of primary murine bone marrow-derived dendritic cells suggesting a minimal risk for biomaterial-induced inflammation, which is further confirmed by an in vivo histologic analysis. In summary, these results further demonstrate the huge potential of cryogels in the biomedical field and their capacity to be translated into clinical applications.

Injectable Hyaluronic Acid-co-Gelatin Cryogels for Tissue-Engineering Applications.

Polymeric scaffolds such as hydrogels can be engineered to restore, maintain, or improve impaired tissues and organs. However, most hydrogels require surgical implantation that can cause several complications such as infection and damage to adjacent tissues. Therefore, developing minimally invasive strategies is of critical importance for these purposes. Herein, we developed several injectable cryogels made out of hyaluronic acid and gelatin for tissue-engineering applications. The physicochemical properties of hyaluronic acid combined with the intrinsic cell-adhesion properties of gelatin can provide suitable physical support for the attachment, survival, and spreading of cells. The physical characteristics of pure gelatin cryogels, such as mechanics and injectability, were enhanced once copolymerized with hyaluronic acid. Reciprocally, the adhesion of 3T3 cells cultured in hyaluronic acid cryogels was enhanced when formulated with gelatin. Furthermore, cryogels had a minimal effect on bone marrow dendritic cell activation, suggesting their cytocompatibility. Finally, in vitro studies revealed that copolymerizing gelatin with hyaluronic acid did not significantly alter their respective intrinsic biological properties. These findings suggest that hyaluronic acid-co-gelatin cryogels combined the favorable inherent properties of each biopolymer, providing a mechanically robust, cell-responsive, macroporous, and injectable platform for tissue-engineering applications.