Thermoresponsive Gel Research Articles

Fabrication and characterization of novel thermoresponsive emulsion gels and oleogels stabilizied by assembling nanofibrous from dual natural triterpenoid saponins.

Novel thermoresponsive emulsion gels and oleogels were fabricated by assembling nanofibrous from natural triterpenoid Quillaja saponin (QS) and glycyrrhizic acid (GA). The viscoelasticity of QS-coated emulsion was observed to be remarkably improved by GA and thus obtain the advantages of excellent gelatinous, thermoresponsive and reversible manner due to the viscoelastic texture from GA nanofibrous as scaffolds in continuous phase. In the gelled emulsions, the phase transition of the GA fibrosis network structure upon heating and cooling was attributed to a thermal sensitivity, whereas interface-induced fibrosis assembly of amphiphilic QS endowed the formation of stable emulsion droplets. Then these emulsion gels were further used as an effective template to fabricate soft-solid oleogels with high oil content of 96%. These findings open up new opportunities for the use of all-natural and sustainable ingredients to develop smart soft materials for replace trans and saturated fats in food industry and other fields.

Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors

Immuno-oncology therapies have been of great interest with the goal of inducing sustained tumor regression, but clinical results have demonstrated the need for improved and widely applicable methods. An antigen-free method of cancer immunotherapy can stimulate the immune system to recruit lymphocytes and produce immunostimulatory factors without prior knowledge of neoantigens, while local delivery reduces the risk of systemic toxicity. To improve the interactions between tumor cells and cytotoxic lymphocytes, a gene delivery nanoparticle platform was engineered to reprogram the tumor microenvironment (TME) in situ to be more immunostimulatory by inducing tumor-associated antigen-presenting cells (tAPCs) to activate cytotoxic lymphocytes against the tumor. Biodegradable, lipophilic poly (beta-amino ester) (PBAE) nanoparticles were synthesized and used to co-deliver mRNA constructs encoding a signal 2 co-stimulatory molecule (4-1BBL) and a signal 3 immuno-stimulatory cytokine (IL-12), along with a nucleic acid-based immunomodulatory adjuvant. Nanoparticles are combined with a thermoresponsive block copolymer for gelation at the injection site for local NP retention at the tumor. The reprogramming nanoparticle gel synergizes with immune checkpoint blockade (ICB) to induce tumor regression and clearance in addition to resistance to tumor rechallenge at a distant site. In vitro and in vivo studies reveal increases in immunostimulatory cytokine production and recruitment of immune cells as a result of the nanoparticles. Intratumoral injection of nanoparticles encapsulating mRNA encoding immunostimulatory agents and adjuvants via an injectable thermoresponsive gel has great translational potential as an immuno-oncology therapy that can be accessible to a wide range of patients.

Optimization of hydrophobic nonresponsive sol-ketal acrylate gel film to hydrophilic thermo-responsive gel by graft-polymerization

This work has been done in three steps. First, the preparation of acrylate monomers; they are [dimethyl-1,3-dioxoylan-4-yl-methylacrylate (sol-ketal acrylate) (SKA)], [4-acetylphenyl acrylate (APHA)], and [4-formyl-2-methoxyphenylacrylate (VA)]. All monomers were evaluated using 1H, 13C-NMR, and FT-IR. In the next step, two kinds of polymers were prepared. Two series of copolymers and terpolymers were carried out via the free-radical polymerization; SKA with the photo-cross-linker for poly (SKA-co-DMIAm) photo-cross-linker polymer and VA and APHA with N-isopropylacrylamide for poly (NIPA-co-VA-co-APHA) functional-thermo-responsive terpolymer. All fabricated polymers were investigated by (1H-NMR, FT-IR, UV, GPC, and DSC). The phase separation temperature of polymer solutions has been measured through the turbidity and the change in transmittance to the temperatures using UV–Vis spectroscopy. Eventually, the UV was used to form the gel layer after the deposition of the gold layer. The nonresponsive gel layer was grafted with poly (NIPA-co-VA-co-APHA) to optimize the upper layer to the thermo-responsive functional layer. SPR/OW measured the swelling properties of the gel layers. The active layer will immobilize biological molecules with the primary amine group.Graphical abstractThe schematic diagram shows the steps of gel formation: The cross-linking initiated by UV; SPR/OW for film thickness; grafting for gel optimization.

PH Modulated Formation of Complexes with Various Stoichiometry between Polymer Network and Fe(III) in Thermosensitive Gels Modified with Gallic Acid.

Thermoresponsive gels based on N-isopropylacrylamide functionalized with amino groups were modified with gallic acid, with gallate (3,4,5-trihydroxybenzoic) groups being introduced into the polymer network. We investigated how the properties of these gels were affected at varying pH, by the formation of complexes between the polymer network of the gels and Fe3+ ions (which form stable complexes with gallic acid, exhibiting 1:1, 1:2, or 1:3 stoichiometry, depending on pH). The formation of complexes with varying stoichiometry within the gel was confirmed using UV-Vis spectroscopy, and the influence of such complexes on swelling behavior and volume phase transition temperature were investigated. In the appropriate temperature range, complex stoichiometry was found to strongly affect the swelling state. Changes in the pore structure and mechanical properties of the gel caused by the formation of complexes with varying stoichiometry were investigated using scanning electron microscopy and rheological measurements, respectively. The volume changes exhibited by p(NIPA-5%APMA)-Gal-Fe gel were found to be greatest at close to human body temperature (~38 °C). Modification of thermoresponsive pNIPA gel with gallic acid opens new opportunities for the development of pH- and thermosensitive gel materials.

Correction: Jaik et al. Photomotion of Hydrogels with Covalently Attached Azo Dye Moieties—Thermoresponsive and Non-Thermoresponsive Gels. Gels 2022, 8, 541

In the original publication [...].

Multimodal Wearable Ionoskins Enabling Independent Recognition of External Stimuli Without Crosstalk.

Wearable ionoskins are one of the representative examples of the many useful applications offered by deformable stimuli-responsive sensory platforms. Herein, ionotronic thermo-mechano-multimodal response sensors are proposed, which can independently detect changes in temperature and mechanical stimuli without crosstalk. For this purpose, mechanically robust, thermo-responsive ion gels composed of poly(styrene-ran-n-butyl methacrylate) (PS-r-PnBMA, copolymer gelator) and 1-butyl-3-methylimidazolium bis(trifluoromethyl sulfonyl)imide ([BMI][TFSI], ionic liquid) are prepared. The optical transmittance change arising from the lower critical solution temperature (LCST) phenomenon between PnBMA and [BMI][TFSI] is exploited to track the external temperature, creating a new concept of the temperature coefficient of transmittance (TCT). The TCT of this system (-11.5% °C-1 ) is observed to be more sensitive to temperature fluctuations than the conventional metric of temperature coefficient of resistance. The tailoring molecular characteristics of gelators selectively improved the mechanical robustness of the gel, providing an additional application opportunity for strain sensors. This functional sensory platform, which is attached to a robot finger, can successfully detect thermal and mechanical environmental changes through variations in the optical (transmittance) and electrical (resistance) properties of the ion gel, respectively, indicating the high practicality of on-skin multimodal wearable sensors.

Thermoresponsive Ozone-Enriched Spray Gel for Postsurgical Treatment of Hepatocellular Carcinoma.

Surgical resection of hepatocellular carcinoma suffers from a high recurrence rate. Ozone directly kills tumor cells by generating reactive oxygen species in vitro, but its high reactivity and short half-life severely limit its tumor accumulation and penetration for the treatment of tumors in vivo. Herein, a thermoresponsive ozone-enriched spray gel is developed to suppress the tumor recurrence of hepatocellular carcinoma (Huh-7 tumors). Briefly, a perfluorocarbon nanoemulsion (PFTBA@LIP) consisting of a perfluorotributylamine core and a lipid monolayer is fabricated, which is encapsulated in the thermoresponsive hydrogel. Ozone is then dissolved in the nanoemulsion owing to its high affinity to PFTBA (O3/PFTBA@LIP@Gel), which effectively improves its stability. Of note is that O3/PFTBA@LIP@Gel induces both ferroptosis and apoptosis by regulating the expression of relevant genes (GPX4, ACSL4, CDKN1A, etc.) and inducing considerable lipid peroxidation, which significantly reduces the tumor recurrence of the Huh-7 tumor by spraying the gel in the surgical cavity and prolongs the survival of tumor-bearing mice.

Potential Hemostatic and Wound Healing Effects of Thermoresponsive Wound Dressing Gel Loaded with Lignosus rhinocerotis and Punica granatum Extracts.

Biologically active compounds in Lignosus rhinocerotis and Punica granatum are found to facilitate wound healing and exhibit hemostatic activity, making them a good combination as bioactives for wound dressings. This study, therefore, aimed to evaluate the potential of thermoresponsive gels loaded with L. rhinocerotis (HLRE) and P. granatum (PPE) extracts as dressings for wound treatment. The gels were prepared using Pluronic PF127 polymer and mixed with PEG 400 and/or starch prior to incorporation with both extracts (0.06 to 1 mg/mL). The gelation temperature (Tgel) at the skin temperature was achieved when Pluronic PF127 was mixed with 22% w/v PEG 400 and reduced to 25.7 ± 0.3-26.7 ± 1.2 °C after adding HLRE and PPE. The gels exhibited satisfactory hardness (2.02 ± 0.19-6.45 ± 0.53 N), cohesiveness (0.9 ± 0.07-2.28 ± 0.4 mJ), adhesiveness (5.07 ± 2.41-19.6 ± 1.1 mJ), and viscosity (0.15 ± 0.04-0.95 ± 0.03 Pa.s), suitable for wound dressings. The optimized gels displayed high thrombin activity and cell migration rate (wound closure of 74% ± 12-89% ± 2 within 24 h), demonstrating hemostatic and healing effects. The thermoresponsive gels demonstrated advantages to be used as dressings for treating acute and open wounds.

Formulation of biobased soap gels from waste-derived feedstocks

Novel thermoresponsive biobased soap gels are formulated from waste fats and oils to prevent their disposal and mitigate their environmental impacts.

Effect of small amounts of akaganeite (β-FeOOH) nanorods on the gelation, phase behaviour and injectability of thermoresponsive Pluronic F127.

Pluronic F127 (PF127) is a copolymer with an amphiphilic nature and can self-assemble to form micelles and, beyond 20% (w/v), form a thermoresponsive physical gel state. However, they are mechanically weak and easily dissolve in physiological environments, which limits their use in load-bearing in specific biomedical applications. Therefore, we propose a pluronic-based hydrogel with enhanced stability by incorporating small amounts of paramagnetic nanorods, akaganeite (β-FeOOH) nanorods (NRs) of aspect ratio ∼7, with PF127. Due to their weak magnetic properties, β-FeOOH NRs have been used as a precursor for preparing stable iron-oxide states (e.g., hematite and magnetite), and the studies on β-FeOOH NRs to be used as a primary component in hydrogels are at the nascent stage. Here we report a method to synthesize β-FeOOH NRs on a gram scale using a simple sol-gel process and characterize the NRs with various techniques. A phase diagram and thermoresponsive behaviour based on rheological experiments and visual observations are proposed for 20% (w/v) PF127 with low concentrations (0.1-1.0% (w/v)) of β-FeOOH NRs. We observe a unique non-monotonous behaviour in the gel network represented by various rheological parameters like storage modulus, yield stress, fragility, high-frequency modulus plateau, and characteristic relaxation time as a function of nanorod concentration. A plausible physical mechanism is proposed to fundamentally understand the observed phase behaviour in the composite gels. These gels show thermoresponsiveness and enhanced injectability, and could find applications in tissue engineering and drug delivery.

Thermo-responsive and mucoadhesive gels for the treatment of cystinosis

Mucoadhesive thermogels were developed by crosslinking poly(n-isopropylacrylamide) based polymers with chitosan and incorporating disulfide bridges, capable of releasing cysteamine upon interaction with mucin, for the treatment of cystinosis. Through crosslinking with chitosan and incorporating varying concentrations of the disulfide monomer into the polymer backbone, the extent of how mucoadhesive the developed thermogels were could be controlled. Through disulfide bridging with mucin, the thermogels released 6 to 10 μg of the conjugate model 2-mercaptopyridine over five days. Utilizing chitosan as the crosslinker, the developed thermogels were shown to degrade to a statistically higher extent following incubation with lysozyme, the highest concentration tear enzyme, by gravimetric and rheologic analysis. The developed thermogels were extensively tested in vivo utilizing a rat model in which materials were applied directly to the corneal surface and a rabbit model in which thermogels were applied to the inferior fornix. With the developed models, there was no adverse reactions or visual discomfort incurred following application of the thermogels. It has been demonstrated that the thermogels produced can be applied to the inferior fornix and release the stable conjugated payload over several days. The developed thermogel was designed to improve upon the current clinical treatment options for ocular cystinosis which are acidic topical formulations that require reapplication multiple times a day.

Rheology and microstructure of thermoresponsive composite gels of hematite pseudocubes and Pluronic F127.

Stimuli-responsive materials or smart materials are designed materials whose properties can be changed significantly by applying external stimuli, such as stress, electric or magnetic fields, light, temperature, and pH. We report the linear and nonlinear rheological properties of thermoresponsive composite gels based on submicron-sized hematite pseudocube-shaped particles and a triblock copolymer Pluronic F127 (PF127). These novel composites form hard gels at an elevated temperature of 37°C. For certain concentrations (<20 w/v. %) of hematite pseudocubes in 17.5 w/v. % of PF127, the gel strength is enhanced and the brittleness of the gels decreases. Higher concentrations (>20 w/v. %) of hematite pseudocubes in PF127 result in weaker and fragile gels. We develop an extensive rheological fingerprint using linear and nonlinear rheological studies. Adsorption of PF127 copolymer molecules on the hematite cube surfaces would further assist the formation of particle clusters along with magnetic interactions to be held effectively in the PF127 micellar network at elevated temperatures. The microscopic structure of these composite gels is visualized through a confocal microscope. Our experiments show that addition of hematite cubes up to 20 w/v. % does not change the rapid thermal gelation of PF127 solutions; hence, the hematite-PF127 composite, which transforms into a hard gel near human body temperature of 37°C, could be suitable for use in smart drug delivery systems.

Design and evaluation of rufinamide nanocrystals loaded thermoresponsive nasal in situ gelling system for improved drug distribution to brain.

Rufinamide (Rufi) is an antiepileptic drug used to manage Lennox-Gastaut Syndrome and partial seizures. The oral bioavailability of Rufi is less due to its poor solubility and low dissolution rate in the gastrointestinal fluids. This results in less amount of drug reaching the brain following the oral administration of drug. Oral formulations of Rufi are prescribed at a high dose and dosing frequency to increase its distribution to the brain. A Rufi loaded thermoresponsive nasal in situ gel which showed significantly high brain concentrations compared to aqueous suspension of Rufi administered through nasal route was developed by our research group and published. In the current work, we have formulated nanocrystals of Rufi and suspended them in a xyloglucan based thermoresponsive gel to improve the nose-to-brain distribution. The particle size, polydispersity index, and yield (%) of the optimized Rufi nanocrystals were 261.2 ± 2.1 nm, 0.28 ± 0.08, and 89.6 ± 2.0 respectively. The narrow PDI indicates that the manufacturing process is reproducible and reliable. Higher % yield suggested that the method of preparation is efficient. The sol-to-gel transition of in situ gel loaded with Rufi nanocrystals was at 32°C which suggested that the formulation transforms into gel at nasal epithelial temperatures. The nasal pharmacokinetic studies showed that Rufi nanocrystals loaded in situ gel produced higher concentration of the drug in brain (higher brain Cmax) and maintained the drug concentrations for longer duration (higher mean residence time) compared to aqueous suspension of Rufi nanocrystals as well aqueous suspension of Rufi and Rufi loaded in situ gel, reported previously. Nanometric size of the Rufi nanocrystals combined with the in situ gelling properties helped the optimized formulation achieve higher brain distribution and also sustain the drug concentrations in brain for longer duration compared to any of the formulations studied by our research group.

Thermoresponsive Dual-Structured Gel Emulsions Stabilized by Glycyrrhizic Acid Nanofibrils in Combination with Monoglyceride Crystals.

Responsive dual-structured emulsions and gel emulsions have attracted more and more attention due to their complex microstructures, on-demand responsive properties, and controlled release of active cargoes. In this work, the effect of monoglyceride (MG)-based oil phase structuring on the formation and stability, structural properties, and thermoresponsive and cargo release behavior of gel emulsions stabilized by glycyrrhizic acid (GA) nanofibrils were investigated. Owing to the formation of GA fibrillar networks in the aqueous phase and MG crystalline networks in the oil phase, a stable dual-structured gel emulsion can be successfully developed. The microstructure of the dual-structured gel emulsions largely depended on the concentration of MG in the oil phase. At low MG concentrations (1–2 wt%), the larger formed and lamellar MG crystals may pierce the interfacial fibrillar film, inducing the formation of partially coalesced droplets. In contrast, at high MG concentrations (4 wt% or above), the smaller MG crystals with enhanced interfacial activity can lead to the formation of a bilayer shell of GA nanofibrils and MG crystals, thus efficiently inhibiting the interfacial film damage and forming a jamming structure with homogeneously distributed small droplets. Compared to pure GA nanofibril gel emulsions, the GA−MG dual-structured gel emulsions showed significantly improved mechanical performance as well as good thermoresponsive behavior. Moreover, these stable GA−MG gel emulsions can be used as food-grade delivery vehicles for encapsulating and protecting hydrophobic and hydrophilic bioactive cargoes. They also have great potential as novel and efficient aroma delivery systems showing highly controlled volatile release. The dual-structured emulsion strategy is expected to broaden the applications of natural saponin GA-based gel emulsions in the food, pharmaceutical, and personal care industries.

Correlating rheology with 3D printing performance based on thermo-responsive κ-carrageenan/Pleurotus ostreatus protein with regard to interaction mechanism

Accurate 3D printing of thermo-responsive gel inks is a great challenge as the sol-gel transition and rheological properties change during extrusion are not easy to precisely regulate with 3D printing behavior has not been sufficiently studied. The effect of Pleurotus ostreatus protein (POP) correlated with the rheological properties and thermo-responsive behavior of the system on 3D printing performance of the thermo-responsive gel, namely κ-carrageenan (KG). Afterwards, their interaction mechanism was further clarified and final printed samples were evaluated in terms of antioxidant performance. Results indicated that POP addition increased the gelatinization temperature (Tg), significantly affecting the selection of optimum printing temperature. POP addition also improved viscosity and consistency index (K) of the printing ink, ultimately affecting the extrusion behavior. Temperature recovery property was facilitated by POP incorporation and was critical to the formability during recovery stage in 3D printing. Mechanical strength was improved with POP addition, which played an important role for the printed structures to be self-supportive. A lower concentration of POP (10%∼40%) was dispersed in a continuous KG-rich gel network and visible phase separation occurred when POP level was increased to 50%. Molecular interaction analysis showed that electrostatic interactions, hydrophobic interactions, ion on the correlation between the rheological properties and thermo-responsive behavior of gum-based 3D printing ink. In addition, POP presented a certain antioxidant activity. This study not only provided useful information on the correlation between the rheological and thermo-responsive behavior of gum-based 3D printing ink, but also provided a way to regulate 3D printing behavior of gum-based gel by protein incorporation. Moreover, it also showed the way to improve the nutritional value of the 3D printed sample. • 3D printing behavior was correlated with thermo-responsive and rheology properties. • 3D printed KG based gel rich in protein was created. • The interactive mechanism between POP and KG was investigated.

Thermoresponsive Azithromycin-Loaded Niosome Gel Based on Poloxamer 407 and Hyaluronic Interactions for Periodontitis Treatment

Azithromycin (AZM) is a potential antimicrobial drug for periodontitis treatment. However, a potential sustained-release system is needed for intra-periodontal pocket delivery. This study focused on the development and evaluation of a thermoresponsive azithromycin-loaded niosome gel (AZG) to search for a desirable formulation for periodontitis treatment. AZG was further developed from an AZM-loaded niosomal formulation by exploiting the advantages of poloxamer 407 (P407) and hyaluronic acid (HA) interactions. The results showed that the addition of HA decreased the gelation temperature and gelation time of AZG. HA was found to increase the viscosity as well as mucoadhesive and tooth-root surface adhesive properties. The AZG solution state was injectable and exhibited pseudoplastic shear-thinning behavior. P407–HA interactions in AZG could contribute to gel strength. AZG showed 72 h of continuous drug release following the Korsmeyer–Peppas model and potentially enhanced drug permeation. The formulations apparently presented more efficient antibacterial activity against major periodontal pathogens than the standard AZM solution. AZM intra-periodontal pocket formulation and the remarkable properties of niosomes exhibited potential characteristics, including ease of administration, bioadhesion to the anatomical structure of the periodontal pocket, and sustained drug release with competent antimicrobial activity, which could be beneficial for periodontitis treatment.

Vasorin-containing small extracellular vesicles retard intervertebral disc degeneration utilizing an injectable thermoresponsive delivery system

Intervertebral disc degeneration (IDD) is the pathological reason of back pain and the therapeutic approaches are still unsatisfactory. Recently, mesenchymal stem cell-derived small extracellular vesicles (EVs) have emerged as the novel regenerative method for IDD. In this study, we intensively investigated the therapeutic mechanism of small EVs, and found that vasorin protein enriched in EVs promoted the proliferation and extracellular matrix anabolism of nucleus pulposus cells via the Notch1 signaling pathway. Then, we fabricated a thermoresponsive gel which composed of Pluronic F127 and decellularized extracellular matrix (FEC) for the delivery and sustained release of EVs. Besides, ex vivo and in vivo results showed that EVs embedded in FEC (EVs@FEC) ameliorate the disc degeneration efficiently and achieve better therapeutic effects than one-off EVs delivery. Collectively, these findings deepen the understanding of EVs mechanism in treating intervertebral disc degeneration, and also illustrate the promising capacity of sustained EVs release system for intervertebral disc regeneration.

Photomotion of Hydrogels with Covalently Attached Azo Dye Moieties-Thermoresponsive and Non-Thermoresponsive Gels.

The unique photomotion of azo materials under irradiation has been in the focus of research for decades and has been expanded to different classes of solids such as polymeric glasses, liquid crystalline materials, and elastomers. In this communication, azo dye-containing gels are obtained by photocrosslinking of non-thermoresponsive and lower critical solution temperature type thermoresponsive copolymers. These are analysed with light microscopy regarding their actuation behaviour under laser irradiation. The influences of the cloud-point temperature and of the laser power are investigated in a series of comparative experiments. The thermoresponsive hydrogels show more intense photoactuation when the cloud-point temperature of the non-crosslinked polymer is above, but closer to, room temperature, while higher laser powers lead to stronger motion, indicating a photothermal mechanism. In non-thermoresponsive gels, considerably weaker photoactuation occurs, signifying a secondary mechanism that is a direct consequence of the optical field-azo dye interaction.

Swelling of Thermo-Responsive Gels in Aqueous Solutions of Salts: A Predictive Model.

The equilibrium degree of swelling of thermo-responsive (TR) gels is strongly affected by the presence of ions in an aqueous solution. This phenomenon plays an important role in (i) the synthesis of multi-stimuli-responsive gels for soft robotics, where extraordinary strength and toughness are reached by soaking of a gel in solutions of multivalent ions, and (ii) the preparation of hybrid gels with interpenetrating networks formed by covalently cross-linked synthetic chains and ionically cross-linked biopolymer chains. A model is developed for equilibrium swelling of a TR gel in aqueous solutions of salts at various temperatures T below and above the critical temperature at which collapse of the gel occurs. An advantage of the model is that it involves a a small (compared with conventional relations) number of material constants and allows the critical temperature to be determined explicitly. Its ability (i) to describe equilibrium swelling diagrams on poly(N-isopropylacrylamide) gels in aqueous solutions of mono- and multivalent salts and (ii) to predict the influence of volume fraction of salt on the critical temperature is confirmed by comparison of observations with results of numerical simulation.

Sulfonated Thermoresponsive Injectable Gel for Sequential Release of Therapeutic Proteins to Protect Cardiac Function after Myocardial Infarction.

Myocardial infarction causes cardiomyocyte death and persistent inflammatory responses, which generate adverse pathological remodeling. Delivering therapeutic proteins from injectable materials in a controlled-release manner may present an effective biomedical approach for treating this disease. A thermoresponsive injectable gel composed of chitosan, conjugated with poly(N-isopropylacrylamide) and sulfonate groups, was developed for spatiotemporal protein delivery to protect cardiac function after myocardial infarction. The thermoresponsive gel delivered vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), and platelet-derived growth factor (PDGF) in a sequential and sustained manner in vitro. An acute myocardial infarction mouse model was used to evaluate polymer biocompatibility and to determine therapeutic effects from the delivery system on cardiac function. Immunohistochemistry showed biocompatibility of the hydrogel, while the controlled delivery of the proteins reduced macrophage infiltration and increased vascularization. Echocardiography showed an improvement in ejection fraction and fractional shortening after injecting the thermal gel and proteins. A factorial design of experimental study was implemented to optimize the delivery system for the best combination and doses of proteins for further increasing stable vascularization and reducing inflammation using a subcutaneous injection mouse model. The results showed that VEGF, IL-10, and FGF-2 demonstrated significant contributions toward promoting long-term vascularization, while PDGF's effect was minimal.