Thermoresponsive Chitosan Research Articles

A Thermoresponsive injectable drug delivery system of chitosan/β-glycerophosphate with gellan gum/alginate microparticles

The development of new Drug Delivery Systems (DDS) by incorporating microparticles within hydrogels can prolong the release rate of drugs and/or other bioactive agents. In this study, we combined gellan gum/alginate microparticles within a thermoresponsive chitosan (Ch) hydrogel with β-Glycerophosphate (β-GP), designing the system to be in the sol state at 21 °C and in the gel state at 37 °C to enable the injectability of the system. The system was in the sol state between 10 °C and 21 °C. Higher concentrations of β-GP (0, 2, 3, 4, 5 w/v%) and microparticles (0, 2 and 5 w/v%) allowed a faster sol-gel transition with higher mechanical strength at 37 °C. However, the sol-gel transition was not instantaneous. The release profile of methylene blue (MB) from the microparticles was significantly affected by their incorporation in Ch/β-GP hydrogels, only allowing the release of 60–70 % of MB for 6 days, while the microparticles alone released all the MB in 48 h. The proposed system did not present cytotoxicity to VERO cell lines as a preliminary assay, with the Ch/β-GP/GG:Alg having >90 % of cellular viability. The proposed Ch/β-GP system proved to have a delaying effect on drug release and biocompatible properties, being a promising future DDS.

Chitosan-Placental ECM Composite Thermos-Responsive Hydrogel as a Biomimetic Wound Dressing with Angiogenic Property.

Attempts are being made to develop an ideal wound dressing with excellent biomechanical and biological properties. Here, a thermos-responsive hydrogel is fabricated using chitosan (CTS) with various concentrations (1%, 2.5%, and 5% w/v) of solubilized placental extracellular matrix (ECM) and 20% β-glycerophosphate to optimize a smart wound dressing hydrogel with improved biological behavior. The thermo-responsive CTS (TCTS) alone or loaded with ECMs (ECM-TCTS) demonstrate uniform morphology using SEM. TCTS and ECM1%-TCTS and ECM2.5%-TCTS show a gelation time of 5min at 37°C, while no gel formation is observed at 4 and 25°C. ECM5%-TCTS forms gel at both 25 and 37°C. The degradation and swelling ratios increaseas the ECM content of the hydrogel increase. All the constructs show excellent biocompatibility in vitro and in vivo, however, the hydrogels with a higher concentration of ECM demonstrate better cell adhesion for fibroblast cells and induce expression of angiogenic factors (VEGF and VEGFR) from HUVEC. Only the ECM5%-TCTS has antibacterial activity against Acinetobacter baumannii ATCC 19606. The data obtained from the current study suggest the ECM2.5%-TCTS as an optimized smart biomimetic wound dressing with improved angiogenic properties now promises to proceed with pre-clinical and clinical investigations.

Nanoenzyme-chitosan hydrogel complex with cascade catalytic and self-reinforced antibacterial performance for accelerated healing of diabetic wounds.

The significant disability and fatality rate of diabetes chronic wounds necessitates the development of efficient diabetic wound healing techniques. The present oxygen treatments for wound healing is restricted by issues such as poor penetration, inadequate supply, and absorption difficulties as well as tanglesome diabetic wound microenvironment issues such as hyperglycemia, excessive reactive oxygen species (ROS), and hypoxia. Herein, we designed a multifunctional glucose oxidase (GOx) and catalase (CAT) nanoenzyme-chitosan (GCNC) hydrogel complex to improve the microenvironment of diabetic wounds and provide continuous oxygen delivery for efficient wound healing. By simultaneously forming the GOx-CAT nanoenzyme (GCNE) composite, the GCNC hydrogel complex could effectively reduce glucose and ROS (H2O2) concentrations in diabetic wounds through cascade catalytic reactions and achieve continuous oxygen supply, which promoted cell proliferation, migration, and angiogenesis, thereby accelerating diabetic wound healing. In addition, the byproduct gluconic acid produced by the cascade reaction can activate the amino group of chitosan to reinforce the antibacterial performance and prevent microbial infection. This multifunctional GCNC hydrogel complex with continuous oxygen supply, self-reinforcing antibacterial properties, and byproduct-free features provides a general strategy for repairing the extensive tissue damage in diabetes.

Polygonatum polysaccharide modified montmorillonite/chitosan/glycerophosphate composite hydrogel for bone tissue engineering

The stiffness and gelation temperature of thermo-responsive chitosan (CS), glycerophosphate (GP) hydrogel is not enough for differentiation of BMSCs and injecting use in bone defect repair. To improve these weaknesses, a kind of injectable hydrogel (PMCG) was prepared with Chitosan (CS), glycerophosphate (GP), polygonatum polysaccharide modified montmorillonite (PMMT). The gelation temperature of CS/GP (CG) hydrogel was 42 °C, the maximum mechanical strength was 1061 Pa. with the amount of PMMT increased, the gelation temperature decreased to 28 °C, and the maximum stiffness increased to 6789 Pa. At the same time, with the addition of PMMT, the swelling property of hydrogel was improved. The cytocompatibility of PMCG decreased than CG which maybe result from the poor cytocompatibility of montmorillonite, but it was improved with increasing amount of PMMT, which indicated the cell proliferation promoting effect coming from polygonatum polysaccharide. The SEM-EDX exhibited that these hydrogels possessed excellent morphological characteristics and could promote mineralization in simulated body fluid (SBF) solution, PMCG-2 Ca/P has a Ca/P ratio of 1.63 ± 0.04, which is similar to the ratio of human bone mineral (Ca/P ratio of 1.55–1.75). The cell viability confirmed the application availability of the hydrogel. In light of the effect on osteoblasts maturation promoting effect and decrease of osteoclast of polygonatum polysaccharide. We come to the conclusion that it is an effective strategy to improve the mechanic strength and gelation temperature, and keep the osteogenesis promoting effect at the same time.

Thermo-responsive chitosan hydrogel for healing of full-thickness wounds infected with XDR bacteria isolated from burn patients: In vitro and in vivo animal model

Treatment of non-healing skin wounds infected with extensively drug-resistant (XDR) bacteria remains as a big challenge. To date, different biomaterials have been applied for treatment of post-wound infections, nevertheless their efficacy for treatment of the wounds infected with XDR isolates has not been determined yet. In this study, the potential of the thermo-responsive chitosan (TCTS) hydrogel for protection of full-thickness wounds XDR bacteria isolated from burn patients was evaluated both in vitro and in vivo in a rat model. Antibacterial activity of the TCTS hydrogel against standard strain and clinical isolates of Acinetobacter baumannii, cytobiocompatibility for Hu02 fibroblast cells, degradation rate and swelling ratio were determined in vitro. MTT assay and disk diffusion test indicated no detectable cytotoxicity and antibacterial activity in vitro, respectively. In vivo study showed significant acceleration of wound healing, re-epithelialization, wound closure, and decreased colony count in the TCTS hydrogel group compared with control. This study suggests TCTS hydrogel as an excellent wound dressing for management of the wounds infected with XDR bacteria, and now promises to proceed with clinical investigations.

Studies on non-gelatinous & thermo-responsive chitosan with the N-isopropylacrylamide by RAFT methodology for control release of levofloxacin

The non-gelatinous and thermo-responsive properties were introduced in chitosan by incorporating the chain of poly(N-isopropylacrylamide) via reversible addition-fragmentation chain transfer (RAFT) polymerization. To achieve this, the reaction was carried out at 80 °C by modifying the chitosan(CS) with RAFT agent as a macroinitiator (CS-RAFT), where the amine group of CS was protected with phthalic anhydride and then reacted with 4-cyano-4-[(dodecyl sulfanyl thiocarbonyl)sulfanyl]-pentanoic acid (CDSTSP) to form CS-RAFT agent. Further, the addition of NIPAAm chains onto CS-RAFT was carried out in N,N′-dimethylformamide (DMF) solvent by using 2,2′-azobisisobutyronitrile (AIBN) as an initiator in N2 atmosphere. The controlled addition of NIPAAm chains on to CS was confirmed by 1H NMR spectroscopy, further, a kinetic study was performed to get the characteristic features of the RAFT reaction. The product was characterized by 1H NMR, FT-IR, UV–Visible spectroscopy, XRD, SEM, and TGA analyses. The product in aqueous solution showed LCST at 2.0 mg/mL on 33 ± 0.1 °C. Further, beads were prepared with the sodium alginate and loaded the water-soluble levofloxacin drug (60% w/w loading was achieved). The drug delivery process was studied in-vitro at 37 ± 0.1 °C & pH 7.4, which shown controlled release of drug up to 32 h and it was 71% of the loaded levofloxacin.

Antimicrobial peptides-loaded smart chitosan hydrogel: Release behavior and antibacterial potential against antibiotic resistant clinical isolates

In this study, we synthesized thermo-responsive chitosan (TCTS) hydrogels, and loaded with different concentrations of antimicrobial peptide (AMP) (0, 4, 8 and 16 μg·ml−1) to fabricate an antibacterial wound dressing against resistant clinical isolates. Physico-chemical properties, release behavior, cytobiocompatibility and antibacterial activity of the AMP-TCTS hydrogels against standard strain and resistant Acinetobacter baumannii were fully determined in vitro. The TCTS-40% β-glycerolphosphate hydrogels showed a gelation time of 15 min at 37 °C. 80% weight loss at day 35 with no changes in pH value was observed. AMP-TCTS hydrogels showed a burst release of AMP (around 40%) at day 1, and a controlled release up to day 7. A dramatic water uptake was observed at first 4 h, and then continued for 10 h in a steady manner. All the AMP-TCTS hydrogels showed excellent cytobiocompatibility for human fibroblasts. The TCTS showed no antibacterial activity against both standard strain and clinical isolates. All the AMP-TCTS hydrogels had strong antibacterial activity against standard strains, but only 16 μg·ml−1 showed antibacterial behavior against resistant A. baumannii. Our results strongly suggest the 16 μg·ml−1 AMP-TCTS hydrogel as an excellent antibacterial wound dressing against resistant A. baumannii, and now promises to proceed with pre-clinical investigations.

Inactivated Rabies Virus-Vectored Immunocontraceptive Vaccine in a Thermo-Responsive Hydrogel Induces High and Persistent Antibodies against Rabies, but Insufficient Antibodies against Gonadotropin-Releasing Hormone for Contraception.

Rabies is preventable through vaccination, but the need to mount annual canine vaccination campaigns presents major challenges in rabies control and prevention. The development of a rabies vaccine that ensures lifelong immunity and animal population management in one dose could be extremely advantageous. A nonsurgical alternative to spay/neuter is a high priority for animal welfare, but irreversible infertility in one dose has not been achieved. Towards this goal, we developed a rabies virus-vectored immunocontraceptive vaccine ERA-2GnRH, which protected against rabies virus challenge and induced >80% infertility in mice after three doses in a live, liquid-vaccine formulation (Wu et al., 2014). To improve safety and use, we formulated an inactivated vaccine in a thermo-responsive chitosan hydrogel for one-dose delivery and studied the immune responses in mice. The hydrogel did not cause any injection site reactions, and the killed ERA-2GnRH vaccine induced high and persistent rabies virus neutralizing antibodies (rVNA) in mice. The rVNA in the hydrogel group reached an average of 327.40 IU/mL, more than 200 times higher than the liquid vaccine alone. The Gonadotropin-releasing hormone (GnRH) antibodies were also present and lasted longer in the hydrogel group, but did not prevent fertility in mice, reflecting a possible threshold level of GnRH antibodies for contraception. In conclusion, the hydrogel facilitated a high and long-lasting immunity, and ERA-2GnRH is a promising dual vaccine candidate. Future studies will focus on rabies protection in target species and improving the anti-GnRH response.

Investigation of magnetic silica with thermoresponsive chitosan coating for drug controlled release and magnetic hyperthermia application

In this study, a drug delivery system for chemo-hyperthermia applications is proposed and fabricated. The delivery system consists of magnetic-silica (MagSi) particles being encapsulated within a pH/thermo-responsive chitosan‑g‑N‑isopropylacrylamide (Chi-g-NIPAAm) polymer matrix. The as-prepared MagSi@Chi-g-NIPAAm particles exhibit superparamagnetic behavior with a saturation magnetization (Ms) of 20.14 emu/g. In addition, the MagSi@Chi-g-NIPAAm particles can act as a heat source when subject to an alternating magnetic field (AMF) and have a specific absorptions rate (SAR) of 8.36 Wg−1. The release of the drug DOX from the synthesized particles is sensitive to both the pH and temperature of its environment. We have compared the drug release when the solution is externally heated up and when it is heated up by the AMF (internal heating). For external heating (when the pH/temperature is 4.0/45 °C), 83.30 ± 2.92% of the DOX were released within the first 5 h. The release of the DOX by the particles in pH 7.4 (temperature of 37 °C) was much slower (around 25.87 ± 1.30% after 25 h). The release of the DOX was much higher (under an acidic condition pH = 4.0) around 57.13 ± 2.36% within 1 h in the presence of AMF heating. The in vitro cytotoxicity tests of the of DOX-loaded MagSi@Chi-g-NIPAAm particles towards HeLa cancer cells. In general, the toxicities of the drug DOX as part of a MagSi@Chi-g-NIPAAm particles were less than those of the standalone DOX until the concentration of DOX-loaded particles reached 250 μg/mL, after which the toxicity of DOX in both forms were the same.

Chitosan hydrogels cross-linked with tris(2-(2-formylphenoxy)ethyl)amine: Swelling and drug delivery

Tris(2-(2-formylphenoxy)ethyl)amine was designed and synthesized by reaction of salicylaldehyde with tris(2-chloroethyl)amine hydrochloride and evaluated as a new multi-functional cross-linker for preparation of new pH- and thermo-responsive chitosan hydrogels through formation of covalent Schiff-base linkage. The structure and properties of the hydrogels were characterized by FT-IR, 1H NMR and scanning electron microscopy (SEM). The swelling behavior of prepared hydrogels at different pHs and temperatures was investigated. Also, in vitro controlled release behavior of the metronidazole model drug was studied with prepared hydrogels. The release profiles of metronidazole from the hydrogels were determined by UV–Vis absorption measurement. The results showed that the new hydrogels exhibit a pH and temperature-responsive swelling ratio. Also, the pH and temperature were found to strongly influence the drug release behavior of these swollen polymers. Due to the concurrent rapid and significant stimuli-response, these smart hydrogels prepared from chitosan as a natural polymer may expand the scope of hydrogel applications in various fields of research such as targeted (cellular or tissue) delivery of drugs. In addition, these new hydrogels can be used to improve bioavailability, sustain release of drugs or solubilize drugs for systemic delivery.

UV-crosslinkable and thermo-responsive chitosan hybrid hydrogel for NIR-triggered localized on-demand drug delivery

Innovative drug delivery technologies based on smart hydrogels for localized on-demand drug delivery had aroused great interest. To acquire smart UV-crosslinkable chitosan hydrogel for NIR-triggered localized on-demanded drug release, a novel UV-crosslinkable and thermo-responsive chitosan was first designed and synthesized by grafting with poly N-isopropylacrylamide, acetylation of methacryloyl groups and embedding with photothermal carbon. The UV-crosslinkable unit (methacryloyl groups) endowed chitosan with gelation via UV irradiation. The thermo-responsive unit (poly N-isopropylacrylamide) endowed chitosan hydrogel with temperature-triggered volume shrinkage and reversible swelling/de-swelling behavior. The chitosan hybrid hydrogel embedded with photothermal carbon exhibited distinct NIR-triggered volume shrinkage (∼42% shrinkage) in response to temperature elevation as induced by NIR laser irradiation. As a demonstration, doxorubicin release rate was accelerated and approximately 40 times higher than that from non-irradiated hydrogels. The UV-crosslinkable and thermal-responsive hybrid hydrogel served as in situ forming hydrogel-based drug depot is developed for NIR-triggered localized on-demand release.

Multifunctional Nanofibers for Specific Purification and Release of CTCs.

Recovering pure and viable circulating tumor cells (CTCs) from blood has been a challenging task for molecular characterization and functional analysis, which has attracted wide attention these days. Herein, we fabricate a thermoresponsive chitosan nanofiber substrate to effectively capture, purify, and release the target cancer cells, assisted by PNIPAAm brushes and DNA hybridization. The PNIPAAm brushes are designed to enable WBCs to detach from aptamer-PNIPAAm-chitosan-nanofiber (aptamer-P-CNFs) surfaces during the conformational transition. Meanwhile these specific captured CTCs are retained at a high purity. Moreover, effective and intact release of CTCs from the substrates without any foreign agents is realized by complementary sequences efficiently hybridizing with aptamers, and the specific cell release makes CTCs further purified. The present work provides a new strategy in the design of biointerface for recovering target CTCs from whole blood samples with high purity.

Preliminary evaluation of a thermosensitive chitosan hydrogel for Echinococcus granulosus vaccine delivery

The EG95 vaccine is effective in protecting grazing animals from infection with Echinococcus granulosus. Six male lambs were used in the study, two were each vaccinated subcutaneously with 50μg EG95/1mg Quil-A, two animals were each vaccinated with 50μg EG95/1mg Quil-A in 1% chitosan thermolabile gel subcutaneously, and two animals served as non-vaccinated controls. Two vaccinations were given at a 7 week interval. Two vaccinations induced a significantly higher antibody titre in the chitosan group compared with the Quil-A only group. The chitosan vaccine group also had a significantly higher antibody titre compared with a positive control sera from vaccinated and challenged sheep. Incorporating the EG95/Quil-A vaccine in a thermo-responsive chitosan sol–gel stimulated, after the second injection, a high level of antibody absorbance which remained high for at least one year. This response was significantly greater than the response to vaccine without the gel.

Characterization of a Thermoresponsive Chitosan Derivative as a Potential Draw Solute for Forward Osmosis.

A thermoresponsive chitosan derivative was synthesized by reacting chitosan (CS) with butyl glycidyl ether (BGE) to break the inter- and intramolecular hydrogen bonds of the polymer. An aqueous solution of the thermoresponsive CS derivative exhibits a lower critical solution temperature (LCST) than CS, and it undergoes a phase transition separation when the temperature changes. Successful incorporation of BGE into the CS was confirmed by FTIR and XPS analyses. Varying the BGE content and the concentration of the aqueous solution produced different LCST ranges, as shown by transmittance vs temperature curves. The particle size was observed by scanning electron microscopy, which revealed that the particles were smaller and well dispersed at 15 °C, whereas the particles became larger and tended to aggregate at 60 °C. A similar trend was observed with the mean particle size measured using dynamic light scattering. Positron annihilation lifetime spectroscopy data also revealed the reversibility of the particle properties as a function of temperature. Microstructure analysis showed that the particles had larger free-volume sizes at 15 °C than at 60 °C. The particles were also found to be nontoxic with 92% cell survival. A simple forward osmosis (FO) test for dye dehydration revealed the potential use of the thermoresponsive chitosan derivative as a draw solute with a flux of 8.6 L/m2 h and rejection of 99.8%.

Paclitaxel Loaded Nanoliposomes in Thermosensitive Hydrogel: A Dual Approach for Sustained and Localized Delivery

In an attempt to improve the localized paclitaxel delivery, carrier based thermoresponsive chitosan hydrogel was exploited in the present study. Nanoliposomes as carrier for paclitaxel were prepared and optimized in strength of 6 mg/ml similar to marketed paclitaxel formulation. The chitosan solution (2% w/v) mixed with different concentrations of dibasic sodium phosphate (DSP) was evaluated as thermoresponsive systems in terms of gelling temperature and time. Finally, the drug loaded nanoliposomes were incorporated in optimized chitosan- DSP hydrogel base to form nanoliposomal in situ thermosensitive hydrogel formulations having dual mechanism of protection and release. The optimal formulation containing DSP was selected on the basis of minimal gelation temperature (37±0.8 ºC) and time (6.7±0.3 min). In vitro drug release experiment illustrated that developed formulation manifested sustained release action in which drug release was extended for more than 72 h compared to marketed formulation. In addition, optimized nanoliposomal hydrogel demonstrated enhanced biological half-life of 15.7±1.5h, depicting maintenance of constant plasma concentration in contrast to marketed formulation that showed the half-life (t1/2) of 3.6±0.4h. The in vivo anti tumor activity tested using EAC model also corroborated the above findings that developed formulation was having significant higher anti-tumor activity and reduced toxicity than the marketed formulation. Tumor volume was found to reduce upto 89.1±3.5% by treatment with in situ hydrogel formulation. The histopathological study of tumor also demonstrated the better safety and efficacy of developed formulation in comparison to marketed paclitaxel formulation. Our results suggest that carrier based chitosan hydrogel could be an efficacious vehicle for sustained and localized delivery of paclitaxel.

Micropatterned fabrication of chitosan-based thermoresponsive membranes for improving cell adhesion and gene expression

A simple, rapid, and economical method to fabricate micropatterned thermoresponsive chitosan membranes was developed. Porous polystyrene films were prepared by liquid-induced phase separation. The size of pores on polystyrene films could be regulated by adjusting the composition of coagulation bath and changing the solvent evaporation rate. Subsequently, chitosan-based thermoresponsive membranes with island protrusions were fabricated using porous polystyrene films as templates. The effects of the micropatterns on the behaviors of mouse fibroblast L929 were investigated. The presence of micropatterns altered the cell cycle distribution and enhanced the gene expression of cyclin D1 and integrin β1. The micro-convex surface could promote the adhesion and proliferation of L929 cells. These results provided valuable guidance to design appropriate topographic surfaces for tissue engineering applications.

Thermoresponsive chitosan–agarose hydrogel for skin regeneration

Healing enhancement and pain control are critical issues on wound management. So far, different wound dressings have been developed. Among them, hydrogels are the most applied.Herein, a thermoresponsive hydrogel was produced using chitosan (deacetylation degree 95%) and agarose. Hydrogel bactericidal activity, biocompatibility, morphology, porosity and wettability were characterized by confocal microscopy, MTS assay and SEM. The performance of the hydrogel in the wound healing process was evaluated through in vivo assays, during 21 days.The attained results revealed that hydrogel has a pore size (90–400μm) compatible with cellular internalization and proliferation. A bactericidal activity was observed for hydrogels containing more than 188μg/mL of chitosan. The improved healing and the lack of a reactive or a granulomatous inflammatory reaction in skin lesions treated with hydrogel demonstrate its suitability to be used in a near future as a wound dressing.

Hyperthermia‐Induced Drug Delivery from Thermosensitive Liposomes Encapsulated in an Injectable Hydrogel for Local Chemotherapy

A novel drug delivery system, enabling an in situ, thermally triggered drug release is described, consisting of an injectable thermoresponsive chitosan hydrogel containing doxorubicin-loaded thermosensitive liposomes. The design, fabrication, characterization, and an assessment of in vitro bioactivity of this formulation is detailed. Combining on-demand drug delivery with in situ gelation results in a promising candidate for local chemotherapy.

Preparation of Thermoresponsive Chitosan Polymers and Its Application Controlling Release of Antifouling Agent

Thermoresponsive chitosan copolymers embed with antifouling agent paeonol in chitosan(CS) microparticles were prepared and the release dynamics was studied at different temperature. Chitosan microparticles have been formed based on ionic gelation process of CS and sodium tripolyphosphate (TPP). Paeonol was incorporated into the CS microparticles with the size about 0.1 μm. The physicochemical structure of samples was analyzed by FTIR and scanning electron microscopy (SEM). We investigated the influence of sodium tripolyphosphate (TPP) and paeonol on Encapsulation efficiency. Increasing TPP concentration from 1 to 3 mg/ml increased encapsulation efficiency of paeonol from 63% to 92%. Increasing peaonol concentration from 1.0 to 3.0 mg/ml increased peaonol encapsulation efficiency from 72% to 85%.

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia

Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/β-glycerophosphate (β-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33°C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100μM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.