Hydrogel microspheres for spatiotemporally controlled delivery of RNA and silencing gene expression within scaffold-free tissue engineered constructs.

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) aggregates may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell aggregates and thus shows great potential in tissue regeneration applications. STATEMENT OF SIGNIFICANCE: This work presents a new strategy to deliver RNA-nanocomplexes from photocrosslinked dextran microspheres for tunable presentation of bioactive RNA. These microspheres were embedded within scaffold-free, human mesenchymal stem cell (hMSC) aggregates for sustained gene silencing within three-dimensional cell constructs while maintaining cell viability. Unlike exogenous delivery of RNA within culture medium that suffers from diffusion limitations and potential need for repeated transfections, this strategy provides local and sustained RNA presentation from the microspheres to cells in the constructs. This system has the potential to inhibit translation of hMSC differentiation antagonists and drive hMSC differentiation toward desired specific lineages, and is an important step in the engineering of high-density stem cell systems with incorporated instructive genetic cues for application in tissue regeneration.

Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing.

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein-expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

Thiol-Epoxy "Click" Chemistry to Engineer Cytocompatible PEG-Based Hydrogel for siRNA-Mediated Osteogenesis of hMSCs.

Thiol-epoxy "click" chemistry is employed for the first time to engineer a new cytocompatible PEG-based hydrogel system in aqueous media with the ability to encapsulate human mesenchymal stem cells (hMSCs) and control their fate for tissue regeneration. Cells were easily encapsulated into the hydrogels and exhibited high cell viability over 4 weeks of culture regardless of the presence of siRNA, complexed with polyethylenimine (PEI) in the form of siRNA/PEI nanocomplexes, indicating the biocompatibility of the developed hydrogel. Loading pro-osteogenic siNoggin in the hydrogel significantly enhanced the osteogenesis of encapsulated hMSCs, demonstrating the potential application of this system in tissue engineering.

Cytocompatible Catalyst-Free Photodegradable Hydrogels for Light-Mediated RNA Release To Induce hMSC Osteogenesis.

Macroscopic hydrogels provide valuable platforms for controlling the release of genetic materials such as small interfering RNA (siRNA) and microRNA (miRNA) for biomedical applications. However, after these hydrogels are formed, it is challenging to alter the release rate of genetic materials. In this report, a Michael addition catalyst-free photodegradable poly(ethylene glycol) (PEG)-based hydrogel system has been developed that provides an active means of controlling the release of genetic materials postgelation using external UV light application. Photodegradation of photolabile linkages in the hydrogel network changes the hydrogel physiochemical properties such as swelling and degradation rate, augmenting the release rate of loaded genetic materials. In the absence of UV light, RNAs were released in a sustained fashion from both photodegradable and nonphotodegradable hydrogels. In contrast, RNA release rate from the photodegradable hydrogels was accelerated via UV light application, whereas it was not elevated with nonphotodegradable hydrogels. Regardless of the UV light exposure to the hydrogels, released siRNA against green fluorescent protein (siGFP) retained its bioactivity via effectively silencing GFP expression in destabilized GFP (deGFP)-expressing HeLa cells cultured in monolayer. Moreover, cells encapsulated in these hydrogels exhibited high cell viability, and loaded siGFP inhibited GFP expression of encapsulated deGFP-expressing HeLa cells with or without UV light application to the hydrogels. Importantly, released siRNA targeting noggin (siNoggin) and miRNA-20a from the hydrogels, with and without UV light application, induced osteogenic differentiation of human mesenchymal stem cells (hMSCs). This photodegradable hydrogel system may be a promising strategy for real-time, user-controlled release of genetic materials for tissue engineering and treatment of diseases such as cancer.

Light-triggered RNA release and induction of hMSC osteogenesis via photodegradable, dual-crosslinked hydrogels.

AIM: To engineer a photodegradable hydrogel system for actively controlled release of bioactive unmodified RNA at designated time points to induce hMSC osteogenesis. MATERIALS & METHODS: RNA/polyethylenimine complexes were loaded into dual-crosslinked photodegradable hydrogels to examine the capacity of UV light application to trigger their release. The ability of released RNA to drive hMSC osteogenic differentiation was also investigated. RESULTS & CONCLUSION: RNA release from photodegradable hydrogels was accelerated upon UV application, which was not observed in non-photodegradable hydrogels. Regardless of the presence of UV light, released siGFP exhibited high bioactivity by silencing GFP expression in HeLa cells. Importantly, siNoggin or miRNA-20a released from the hydrogels induced hMSC osteogenesis. This system provides a potentially valuable physician/patient-controlled 'on-demand' RNA delivery platform for biomedical applications.

Sulfamethazine-based pH-sensitive hydrogels with potential application for transcatheter arterial chemoembolization therapy.

UNLABELLED: Transcatheter arterial chemoembolization (TACE) is the most common palliative therapy for unresectable hepatocellular carcinoma (HCC). The conventional TACE technique, which employs the Lipiodol® emulsion, has been widely used for human cancer treatments. However, this delivery system seems to be inconsistent and unstable in maintaining a high concentration of drugs at tumor sites. An alternative approach for TACE is loading drugs into a liquid embolic solution that exists as an injectable solution and can exhibit a sol-to-gel phase transition to form a solidified state once delivered to the tumor site. Here, we develop a novel sulfamethazine-based anionic pH-sensitive block copolymer with potential application as a radiopaque embolic material. The copolymer, named PCL-PEG-SM, and comprised of poly(ε-caprolactone), sulfamethazine, and poly(ethylene glycol), was fabricated by free radical polymerization. An aqueous solution of the developed copolymer underwent a sol-to-gel phase transition upon lowering the environmental pH to create a gel region that covered the physiological condition (pH 7.4, 37°C) and the low pH conditions at tumor sites (pH 6.5-7.0, 37°C). The release of doxorubicin (DOX) from DOX-loaded copolymer hydrogels could be sustained for more than 4weeks in vitro, and the released DOX retained its fully bioactivity via inhibition the proliferation of hepatic cancer cells. The radiopaque embolic formulations that were prepared by mixing copolymer solutions at pH 8.0 with Lipiodol®, a long-lasting X-ray contrast agent, could exhibit the gelation inside the tumor after intratumoral injection or intraarterial administration using a VX2 carcinoma hepatic tumor rabbit model. These results suggest that a novel anionic pH-sensitive copolymer has been developed with a potential application as a liquid radiopaque embolic solution for TACE of HCC. STATE OF SIGNIFICANCE: Transcatheter arterial chemoembolization (TACE) has been widely used as a palliative treatment therapy for unresectable hepatocellular carcinoma (HCC). Conventional TACE technique, which usually employs emulsion of DOX-in-Lipiodol®, followed by an embolic agent, has significant limitation of inconsistency and lack of controlled release ability. To address these limitations of conventional TACE material system, we introduced a novel liquid radiopaque embolic material from our pH-sensitive hydrogel. The material has low viscosity that can be injected via a microcatheter, rather biocompatibility, and drug controlled release ability. Importantly, it can form gel in the tumor as well as tumoral vasculature in response to the lowered pH at the tumor site, which proved the potential for the use to treat HCC by TACE therapy.

Synthesis, Characteristics and Potential Application of Poly(ß-Amino Ester Urethane)-Based Multiblock Co-Polymers as an Injectable, Biodegradable and pH/Temperature-Sensitive Hydrogel System.

Physical polymeric hydrogels have significant potential for use as injectable depot drug/protein-delivery systems. In this study, a series of novel injectable, biodegradable and pH/temperature-sensitive multiblock co-polymer physical hydrogels composed of poly(ethylene glycol) (PEG) and poly(ß-amino ester urethane) (PEU) was synthesized by the polyaddition between the isocyanate groups of 1,6-diisocyanato hexamethylene and the hydroxyl groups of PEG and a synthesized monomer BTB (or ETE) in chloroform in the presence of dibutyltin dilaurate as a catalyst. The synthesized co-polymers were characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy and gel-permeation chromatography. Aqueous solutions of the co-polymers showed a sol-to-gel phase transition with increasing pH and a gel-to-sol phase transition with increasing temperature. The gel regions covered the physiological conditions (37°C, pH 7.4) and could be controlled by changing the molecular weight of PEG, PEG/PEU ratio and co-polymer solution concentration. A gel formed rapidly in situ after injecting the co-polymer solution subcutaneously into SD rats and remained for more than 2 weeks in the body. The cytotoxicity tests confirmed the non-cytotoxicity of this co-polymer hydrogel. The controlled in vitro release of the model anticancer drug, doxorubicin, from this hydrogel occurred over a 7-day period. This hydrogel is a potential candidate for biomedical applications and drug/protein-delivery systems.

pH-Sensitive pentablock copolymer nanocapsules as nontoxic and efficient gene carriers.

A biodegradable amphiphilic pentablock copolymer PAE-PCL-PEG-PCL-PAE with a pH-sensitive unit was synthesized for use as a nontoxic, biodegradable carrier for gene delivery by forming nanocapsules entrapping nucleic acid drugs. The PAE block can interact with plasmid DNA to form polyelectrolyte complexes in an acidic environment. At physiological pH, the PAE blocks are deprotonated and form an insoluble skin, resulting in the formation of nanocapsules that encapsulate plasmid DNA. The surface charges of the nanocapsules became almost neutral at pH = 7.4, and their size ranged from 210 to 280 nm. The nanocapsule maintained most of its transfection efficiency even in the presence of serum. These nanocapsules are therefore potential carriers for systemic gene therapy.