Fine-Tuning Crystallization-Induced Gelation in Amphiphilic Double-Brush Polymers.

A series of amphiphilic double-brush polymers based on itaconate diesters were synthesized with the objective of tailoring the thermal and mechanical properties of hydrogels formed by them; the amphiphilic itaconate diesters carried an MPEG350 segment and an alkyl chain, whose length was varied from C12 to C18. As was reported by us earlier (Macromolecules 2017, 50, 5004), the formation of the hydrogel was due to the crystallization of alkyl segments, as confirmed by the match of the rheological gel-to-sol transition with that of differential scanning calorimetry melting transition of the gel. In an effort to fine-tune the hydrogel-melting temperature and its strength, we varied the length of the alkyl chain length while keeping the hydrophilic segment length constant at MPEG350; apart from varying the alkyl chain length, an oxyethylene spacer was incorporated to examine the effect of decoupling the alkyl side-chain crystallization from the backbone. With these modifications, the melting temperature of the hydrogel was varied from 30 to 56 °C. Likewise, the strength of the hydrogel, as reflected by its storage modulus, varied from around 220 to 970 Pa; the softer gels typically exhibited a slightly larger critical shear strain beyond which the gel transformed into a sol. The thermal and shear-induced gel-to-sol transitions were reversible; however, the modulus after the shear-induced transition did not fully recover instantly (∼80%), suggesting that the formation of the extended gel network is slow. Further fine-tuning could be achieved by copolymerization of two different amphiphilic itaconate monomers, namely, those with C16 and C18, which provided an intermediate gel-melting temperature; however, co-gelation of the two preformed homopolymer gels yielded two distinct gel-melting transitions. Thus, this class of tuneable stimuli-responsive polymeric hydrogels prepared from biobenign components, namely, itaconic acid, 1-alkanols, and MPEGs, could serve as potential candidates for biomedical applications.

Biomaterials for topical and transdermal drug delivery in reconstructive transplantation.

Lifelong systemic immunosuppression remains the biggest challenge in vascularized composite allotransplantation (VCA) due to the adverse effects it causes. Since VCA is a life-enhancing procedure as compared with solid organ transplant which is life-saving; one needs to weigh the benefits and risks carefully. Thus, there is a huge unmet clinical need to design biomaterial-based vehicles that can deliver drugs more efficiently, topically and locally to eliminate adverse effects of systemic immune suppression. This review discusses several biomaterial-based systems that have been carefully designed, conceived and attempted to make VCA a more patient compliant approach. Variety of promising preclinical studies has shown the feasibility of the approaches, and clinical trials are required to bridge the gap. Several challenges for the future and new approaches have been discussed.

Chemical fuel-driven living and transient supramolecular polymerization.

Temporal control over self-assembly process is a desirable trait in the quest towards adaptable and controllable materials. The ability to devise synthetic ways to control the growth, as well as decay of materials has long been a property which only the biological systems could perform seamlessly. A common synthetic strategy which works on the biological principles such as chemical fuel-driven control over temporal self-assembly profile has not been completely realized synthetically. Here we show, we filled this dearth by showing that a chemical fuel driven self-assembling system can not only be grown in a controlled manner, but it can also result in precise control over the assembly and disassembly kinetics. Herein, we elaborate strategies which clearly show that once a chemical fuel driven self-assembly is established it can be made receptive to multiple molecular cues such that the inherent growth and decay characteristics are programmed into the ensemble.

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections.

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0-1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide 'proof of concept' for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

Graft-implanted, enzyme responsive, tacrolimus-eluting hydrogel enables long-term survival of orthotopic porcine limb vascularized composite allografts: A proof of concept study.

BACKGROUND: Currently, patients receiving vascularized composite allotransplantation (VCA) grafts must take long-term systemic immunosuppressive therapy to prevent immunologic rejection. The morbidity and mortality associated with these medications is the single greatest barrier to more patients being able to receive these life-enhancing transplants. In contrast to solid organs, VCA, exemplified by hand or face transplants, allow visual diagnosis of clinical acute rejection (AR), directed biopsy and targeted graft therapies. Local immunosuppression in VCA could reduce systemic drug exposure and limit adverse effects. This proof of concept study evaluated, in a large animal forelimb VCA model, the efficacy and tolerability of a novel graft-implanted enzyme-responsive, tacrolimus (TAC)-eluting hydrogel platform, in achieving long-term graft survival. METHODS: Orthotopic forelimb VCA were performed in single haplotype mismatched mini-swine. Controls (n = 2) received no treatment. Two groups received TAC hydrogel: high dose (n = 4, 91 mg TAC) and low dose (n = 4, 49 mg TAC). The goal was to find a dose that was tolerable and resulted in long-term graft survival. Limbs were evaluated for clinical and histopathological signs of AR. TAC levels were measured in serial blood and skin tissue samples. Tolerability of the dose was evaluated by monitoring animal feeding behavior and weight. RESULTS: Control limbs underwent Banff Grade IV AR by post-operative day six. Low dose TAC hydrogel treatment resulted in long-term graft survival time to onset of Grade IV AR ranging from 56 days to 93 days. High dose TAC hydrogel also resulted in long-term graft survival (24 to 42 days), but was not well tolerated. CONCLUSION: Graft-implanted TAC-loaded hydrogel delays the onset of Grade IV AR of mismatched porcine forelimb VCA grafts, resulting in long term graft survival and demonstrates dose-dependent tolerability.

Local release of tacrolimus from hydrogel-based drug delivery system is controlled by inflammatory enzymes in vivo and can be monitored non-invasively using in vivo imaging.

BACKGROUND: Local drug delivery systems that adjust the release of immunosuppressive drug in response to the nature and intensity of inflammation represent a promising approach to reduce systemic immunosuppression and its side effects in allotransplantation. Here we aimed to demonstrate that release of tacrolimus from triglycerol monostearate hydrogel is inflammation-dependent in vivo. We further report that by loading the hydrogel with a near-infrared dye, it is possible to monitor drug release non-invasively in an in vivo model of vascularized composite allotransplantation. MATERIALS AND METHODS: Inflammation was induced by local challenge with lipopolysaccharides in naïve rats 7 days after injection of tacrolimus-loaded hydrogel in the hind limb. Tacrolimus levels in blood and tissues were measured at selected time points. A near-infrared dye was encapsulated in the hydrogel together with tacrolimus in order to monitor hydrogel deposits and drug release in vitro and in vivo in a model of vascularized composite allotransplantation. RESULTS: Injection of lipopolysaccharides led to increased blood and skin tacrolimus levels (p = 0.0076, day 7 vs. day 12 in blood, and p = 0.0007 in treated limbs, 48 h after injection compared to controls). Moreover, lipopolysaccharides-injected animals had higher tacrolimus levels in treated limbs compared to contralateral limbs (p = 0.0003 for skin and p = 0.0053 for muscle). Imaging of hydrogel deposits and tacrolimus release was achieved by encapsulating near-infrared dye in the hydrogel for 160 days. The correlation of tacrolimus and near-infrared dye release from hydrogel was R2 = 0.6297 and R2 = 0.5619 in blood and grafts of transplanted animals respectively and R2 = 0.6066 in vitro. CONCLUSIONS: Here we demonstrate the inflammation-responsiveness of a tacrolimus-loaded hydrogel in vivo. Moreover, we show that encapsulating a near-infrared dye in the hydrogel provides a reliable correlation of tacrolimus and dye release from the hydrogel, and an accessible non-invasive method for monitoring drug release from hydrogel deposits.

Local Injections of Tacrolimus-loaded Hydrogel Reduce Systemic Immunosuppression-related Toxicity in Vascularized Composite Allotransplantation.

BACKGROUND: Routine application of vascularized composite allotransplantation is hampered by immunosuppression-related health comorbidities. To mitigate these, we developed an inflammation-responsive hydrogel for local immunosuppression. Here, we report on its long-term effect on graft survival, immunological, and toxicological impact. METHODS: Brown Norway-to-Lewis rat hindlimb transplantations were treated either systemically with daily injections of 1 mg/kg tacrolimus (TAC) or with subcutaneous intragraft injections of hydrogel containing 7 mg TAC, every 70 days. Animals were monitored for rejection or other pathology for 280 days. Systemic and graft TAC levels, regulatory T cells, and donor cell chimerism were measured periodically. At endpoint, markers for kidney, liver, and metabolic state were compared to naive age-matched rats. RESULTS: Both daily systemic TAC and subcutaneous intragraft TAC hydrogel at 70-day intervals were able to sustain graft survival longer than 280 days in 5 of 6 recipients. In the hydrogel group, 1 graft progressed to grade 3 rejection at postoperative day 149. In systemic TAC group, 1 animal was euthanized due to lymphoma on postoperative day 275. Hydrogel treatment provided stable graft and reduced systemic TAC levels, and a 4 times smaller total TAC dose compared with systemic immunosuppression. Hydrogel-treated animals showed preserved kidney function, absence of malignancies or opportunistic infections and increased hematopoietic chimerism compared with systemic immunosuppression. CONCLUSIONS: Our findings demonstrate that localized immunosuppression with TAC hydrogel is a long-term safe and reliable treatment. It may reduce the burden of systemic immunosuppression in vascularized composite allotransplantation, potentially boosting the clinical application of this surgical intervention.

A single localized dose of enzyme-responsive hydrogel improves long-term survival of a vascularized composite allograft.

Currently, systemic immunosuppression is used in vascularized composite allotransplantation (VCA). This treatment has considerable side effects and reduces the quality of life of VCA recipients. We loaded the immunosuppressive drug tacrolimus into a self-assembled hydrogel, which releases the drug in response to proteolytic enzymes that are overexpressed during inflammation. A one-time local injection of the tacrolimus-laden hydrogel significantly prolonged graft survival in a Brown Norway-to-Lewis rat hindlimb transplantation model, leading to a median graft survival of >100 days compared to 33.5 days in tacrolimus only-treated recipients. Control groups with no treatment or hydrogel only showed a graft survival of 11 days. Histopathological evaluation, including anti-graft antibodies and complement C3, revealed significantly reduced immune responses in the tacrolimus-hydrogel group compared with tacrolimus only. In conclusion, a single-dose local injection of an enzyme-responsive tacrolimus-hydrogel is capable of preventing VCA rejection for >100 days in a rat model and may offer a new approach for immunosuppression in VCA.