RESUMO
The conventional synthesis of methacryloyl hyaluronic acid (HA-MA) requires an extremely high amount of modification reagents, the organic solvents, and strenuous purification steps. Herein, a new green synthetic approach for the methacryloyl hyaluronic acid preparation with a tailorable substitution degree (SD) is reported, in which methacryloyl hydrazide is used as a more reactive reagent and only water is used as the solvent. The new method significantly reduces the amount of functionalization reagents (as low as only 0.3 equiv) and avoids the use of any organic solvents. The substitution degree can be tailored from 26% to 86% in a facile controllable manner. The new HA-MA (termed as HA-MA-H) can be UV-cross-linked to form a biocompatible hydrogel.
Assuntos
Ácido Hialurônico , Hidrogéis , Materiais Biocompatíveis , Reagentes de Ligações CruzadasRESUMO
Delivery of functional genetic materials into fibroblast cells to manipulate the transgene expression is of great significance in skin gene therapy. Despite numerous polymeric gene delivery systems having been developed, highly safe and efficient fibroblast gene transfection has not yet been achieved. Here, through a new linear oligomer combination strategy, linear poly(ß-amino ester) oligomers are connected by the branching units, forming a new type of poly(ß-amino ester). This new multifunctional linear-branched hybrid poly(ß-amino ester) (LBPAE) shows high-performance fibroblast gene transfection. In human primary dermal fibroblasts (HPDFs) and mouse embryo fibroblasts (3T3s), ultrahigh transgene expression is achieved by LBPAE: up to 3292-fold enhancement in Gaussia luciferase (Gluc) expression and nearly 100% of green fluorescence protein expression are detected. Concurrently, LBPAE is of high in vitro biocompatibility. In depth mechanistic studies reveal that versatile LBPAE can navigate multiple extra- and intracellular barriers involved in the fibroblast gene transfection. More importantly, LBPAE can effectively deliver minicircle DNA encoding COL7A1 gene (a large and functional gene construct) to substantially upregulate the expression of type VII collagen (C7) in HPDFs, demonstrating its great potential in the treatment of C7-deficiency related genodermatoses such as recessive dystrophic epidermolysis bullosa.
Assuntos
Técnicas de Transferência de Genes , Transfecção , Transgenes/genética , Animais , Ésteres/química , Fibroblastos/metabolismo , Expressão Gênica/genética , Terapia Genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Queratinócitos/metabolismo , CamundongosRESUMO
The rapid development of additive manufacturing techniques in the field of tissue regeneration offers unprecedented success for artificial tissue and organ fabrication. However, some limitations still remain for current bioinks, such as the compromised cell viability after printing, the low cross-linking efficiency leading to poor printing resolution and speed due to the relatively slow gelation rate, and the requirement of external stimuli for gelation. To address these problems, herein, a biocompatible and printable instant gelation hydrogel system has been developed based on a designed hyperbranched poly(ethylene glycol) (PEG)-based multihydrazide macro-cross-linker (HB-PEG-HDZ) and an aldehyde-functionalized hyaluronic acid (HA-CHO). HB-PEG-HDZ is prepared by the postfunctionalization of hyperbranched PEG-based multivinyl macromer via thiol-ene chemistry. Owing to the high functional group density of HB-PEG-HDZ, the hydrogel can be formed instantly upon mixing the solutions of two components. The reversible cross-linking mechanism between the hydrazide and aldehyde groups endows the hydrogel with shear-thinning and self-healing properties. The minimally toxic components and cross-linking chemistry allow the resulting hydrogel to be a biocompatible niche. Moreover, the fast sol-to-gel transition of the hydrogel, combining all of the advanced characteristics of this platform, protects the cells during the printing procedure, avoids their damage during extrusion, and improves the transplanted cell survival.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Hidrogéis/química , Tinta , Células 3T3 , Animais , Materiais Biocompatíveis/farmacologia , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular , Ácido Hialurônico/química , Camundongos , Polietilenoglicóis/química , Impressão TridimensionalRESUMO
Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O2) and compared to normoxia (21% O2). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively. Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro.
RESUMO
Conventional wound healing materials suffer from low efficiency, poor mechanical strength, and nontunable properties. Responsive hydrogels are appealing candidates for tissue engineering. Herein, we developed a double-cross-linked hydrogel system composed of hyperbranched PEG-based polymer, comprising pre-cross-linked acetal structure and numerous terminal acrylate groups, which can form hydrogels in situ and can be further strengthened by UV irradiation. The hyperbranched glycidyl methacrylate-co-poly(ethylene glycol) diacrylate polymers (HB-GMA-PEGs) were first synthesized via in situ deactivation enhanced atom transfer radical polymerization (DE-ATRP). A series of pre-cross-linked materials were achieved after postfunctionalization. The material can absorb a high amount of water to form hydrogels, and the gel stiffness was evaluated in detail before and after UV irradiation. The in vitro cytotoxicity experiments were conducted with the resulting materials and have demonstrated their good biocompatibility. The results indicate that this type of hydrogel with high water uptake capacity has appealing potential as a responsive biomaterial for wound closure.
RESUMO
Synthetic reactive oxygen species (ROS)-responsive biomaterials have emerged as a useful platform for regulating critical aspects of ROS-induced pathologies and can improve such hostile microenvironments. Here, we report a series of new hyperbranched poly(ß-hydrazide ester) macromers (HB-PBHEs) with disulfide moieties synthesized via an "A2 + B4" Michael addition approach. The three-dimensional structure of HB-PBHEs with multiacrylate end groups endows the macromers with rapid gelation capabilities to form (1) injectable hydrogels via cross-linking with thiolated hyaluronic acid and (2) robust UV-cross-linked hydrogels. The disulfide-containing macromers and hydrogels exhibit H2O2-responsive degradation compared with the counterparts synthesized by a dihydrazide monomer without disulfide moieties. The cell viability under a high ROS environment can be well-maintained under the protection of the disulfide containing hydrogels.
Assuntos
Antioxidantes/química , Azidas/química , Ésteres/química , Hidrogéis/química , Células 3T3 , Adipócitos/citologia , Animais , Compostos de Bifenilo/química , Sobrevivência Celular , Técnicas de Cocultura , DNA/química , Dissulfetos/química , Sequestradores de Radicais Livres/química , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Picratos/química , Conformação Proteica , Espécies Reativas de Oxigênio/química , Reologia , Espectrofotometria Ultravioleta , Células-Tronco/citologia , Engenharia TecidualRESUMO
Adjusting biomaterial degradation profiles to match tissue regeneration is a challenging issue. Herein, biodegradable hyperbranched poly(ß-amino ester)s (HP-PBAEs) were designed and synthesized via "A2 + B4" Michael addition polymerization, and displayed fast gelation with thiolated hyaluronic acid (HA-SH) via a "click" thiol-ene reaction. HP-PBAE/HA-SH hydrogels showed tunable degradation profiles both in vitro and in vivo using diamines with different alkyl chain lengths and poly(ethylene glycol) diacrylates with varied PEG spacers. The hydrogels with optimized degradation profiles encapsulating ADSCs were used as injectable hydrogels to treat two different types of humanized excisional wounds - acute wounds with faster healing rates and diabetic wounds with slower healing and neo-tissue formation. The fast-degrading hydrogel showed accelerated wound closure in acute wounds, while the slow-degrading hydrogel showed better wound healing for diabetic wounds. The results demonstrate that the new HP-PBAE-based hydrogel in combination with ADSCs can be used as a well-controlled biodegradable skin substitute, which demonstrates a promising approach in the treatment of various types of skin wounds.
RESUMO
The injectable hydrogel with desirable biocompatibility and tunable properties can improve the efficacy of stem cell-based therapy. However, the development of injectable hydrogel remains a great challenge due to the restriction of crosslinking efficiency, mechanical properties, and potential toxicity. Here, we report that a new injectable hydrogel system was fabricated from hyperbranched multi-acrylated poly(ethylene glycol) macromers (HP-PEGs) and thiolated hyaluronic acid (HA-SH) and used as a stem cell delivery and retention platform. The new HP-PEGs were synthesized via in situ reversible addition fragmentation chain transfer (RAFT) polymerization using an FDA approved anti-alcoholic drug-Disulfiram (DS) as the RAFT agent precursor. HP-PEGs can form injectable hydrogels with HA-SH rapidly via thiol-ene click reaction under physiological conditions. The hydrogels exhibited stable mechanical properties, non-swelling and anti-fouling properties. Hydrogels encapsulating adipose-derived stem cells (ADSCs) have demonstrated promising regenerative capabilities such as the maintenance of ADSCs' stemness and secretion abilities. The ADSCs embedded hydrogels were tested on the treatment of diabetic wound in a diabetic murine animal model, showing enhanced wound healing. STATEMENT OF SIGNIFICANCE: Diabetic wounds, which are a severe type of diabetes, have become one of the most serious clinical problems. There is a great promise in the delivery of adipose stem cells into wound sites using injectable hydrogels that can improve diabetic wound healing. Due to the biocompatibility of poly(ethylene glycol) diacrylate (PEGDA), we developed an in situ RAFT polymerization approach using anti-alcoholic drug-Disulfiram (DS) as a RAFT agent precursor to achieve hyperbranched PEGDA (HP-PEG). HP-PEG can form an injectable hydrogel by crosslinking with thiolated hyaluronic acid (HA-SH). ADSCs can maintain their regenerative ability and be delivered into the wound sites. Hence, diabetic wound healing process was remarkably promoted, including inhibition of inflammation, enhanced angiogenesis and re-epithelialization. Taken together, the ADSCs-seeded injectable hydrogel may be a promising candidate for diabetic wound treatment.