Sustained Protein Release Research Articles

Two-phase matrices for the controlled release of therapeutic proteins.

Controlled and sustained delivery of therapeutic proteins is crucial for achieving desired effects in wound healing applications. Yet, this remains a challenge in growth factor delivery for bone tissue engineering. Current delivery systems can lead to negative side effects, such as ectopic bone growth and cancer, due to the over administration of growth inducing proteins. Here, we have developed a two-phase system for the controlled release of therapeutic proteins. The system consists of protein-loaded poly(methacrylic acid)-based nanoparticles conjugated to chitosan scaffolds. The effect of co-monomer hydrophilicity and crosslinking density on nanoparticle properties was evaluated. It was found that the release kinetics of model therapeutic proteins were dependent on nanoparticle hydrophilicity. The chitosan scaffold, chosen for its biocompatibility and osteogenic properties, provided additional barriers to diffusion and promoted nanoparticle retention, leading to more sustained protein delivery. Additionally, the ability of MC3T3-E1 pre-osteoblast cells to proliferate on scaffolds with and without conjugated nanoparticles was evaluated and all scaffolds were shown to promote cell growth. The results demonstrate that the two-phase scaffold system presents a superior strategy for the sustained and controlled release of therapeutic proteins for bone tissue engineering applications.

Charge Booster Tags for Controlled Release of Therapeutics from a Therapeutic Carrier

AbstractInjectable hydrogels are promising delivery vehicles for the sustained release of therapeutic proteins. Electrostatic interactions between proteins and hydrogels often increase affinity to decelerate protein release. However, this approach is not suitable for weakly charged proteins. The current study shows that the genetic fusion of a highly charged protein segment (charge booster tag) with proteins can control their interactions with injectable gels. A positive or negative charge booster tag is introduced into urate oxidase (UOX), a therapeutic protein for gout, to generate UOX variants with varying net charges. When a positively‐charged injectable hydrogel is used, both the in vitro release rate and in vivo serum half‐life of UOX are correlated with the net negative charge. This modified delivery approach results in a serum half‐life of over 106 h for the UOX variant, which is substantially longer than that of free UOX (3.3 h). Hence, charge booster tags can be used as a systematic strategy for controlling the release of therapeutic proteins.

Self-Initiated Photopolymerization of Anti-Inflammatory Zwitterionic Hydrogels with Sustained Release.

Hydrogels are three-dimensional networks of hydrophilic polymers that have garnered significant attention as wound-healing materials. Many synthetic hydrogels are fabricated using a radical polymerization approach that requires an initiator molecule that is often photo- or thermosensitive. Initiator-free hydrogels are an emerging area of research that focuses on hydrogel fabrication that occurs in the absence of an initiator or cross-linker molecule, making these hydrogels highly relevant in tissue engineering and regenerative medicine due to their excellent cytocompatibility and ease of scale-up. Here we present on the development of initiator-free zwitterionic hydrogels that photopolymerize without any initiator or cross-linker while under cytocompatible conditions. The hydrogels exhibit a wide range of mechanical characteristics that are dependent on their polymer composition. They resist nonspecific protein adsorption and exhibit a sustained release of proteins and small molecules. Additionally, these self-initiated hydrogels significantly mitigate inflammatory macrophage activation in vitro. Overall, the development of self-initiated photopolymerized zwitterionic hydrogels offers significant progress in the fields of biomaterials and materials science.

Controlled release of protein from gelatin/chitosan hydrogel containing platelet-rich fibrin encapsulated in chitosan nanoparticles for accelerated wound healing in an animal model

The physiological healing process is disrupted in many cases using the current wound healing procedures, resulting in delayed wound healing. Hydrogel wound dressings provide a moist environment to enhance granulation tissue and epithelium formation in the wound area. However, exudate accumulation, bacterial proliferation, and reduced levels of growth factors are difficulties of hydrogel dressings. Here, we loaded platelet-rich fibrin-chitosan (CH-PRF) nanoparticles into the gelatin-chitosan hydrogel (Gel-CH/CH-PRF) by solvent mixing method. Our goal was to evaluate the characteristics of hydrogel dressings, sustained release of proteins from the hydrogel dressing containing PRF, and reduction in the risk of infection by the bacteria in the wound area. The Gel-CH/CH-PRF hydrogel showed excellent swelling behavior, good porosity, proper specific surface area, high absorption of wound exudates, and proper vapor permeability rate (2023 g/m 2.day), which provided requisite moisture without dehydration around the wound area. Thermal behavior and the protein release from the hydrogels were investigated using simultaneous thermal analysis and the Bradford test, respectively. Most importantly, an excellent ability to control the release of proteins from the hydrogel dressings was observed. The high antimicrobial activity of hydrogel was confirmed using Gram-positive and Gram-negative bacteria. Due to the presence of chitosan in the hydrogels, the lowest scavenging capacity-50 value (5.82 μgmL−1) and the highest DPPH radical scavenging activity (83 %) at a concentration 25 μgmL−1 for Gel-CH/CH-PRF hydrogel were observed. Also, the hydrogels revealed excellent cell viability and proliferation. The wound healing process was studied using an in vivo model of the full-thickness wound. The wound closure was significantly higher on Gel-CH/CH-PRF hydrogel compared to the control group, indicating the highest epidermis thickness, and enhancing the formation of new granulation tissue. Our findings demonstrated that Gel-CH/CH-PRF hydrogel can provide an ideal wound dressing for accelerated wound healing.

Preparation and Characterization of PLG Microparticles by the Multiple Emulsion Method for the Sustained Release of Proteins.

Rapid release and diminished stability are two of the limitations associated with the growth factors that are essentially used in dental applications. These growth factors are employed to enhance the quality and quantity of tissue or bone matter during regeneration. Therefore, drug delivery devices and systems have been developed to address these limitations. In this study, bovine serum albumin (BSA), as a representative growth factor, was successfully sustained by encapsulation with the medium-absorbable copolymer, poly(L-lactide-co-glycolide) (PLG) 70:30% mol, via the multiple emulsion method. Different PLG, PVA, and BSA concentrations were used to investigate their effects on the BSA encapsulation efficiency. The suitable ratios leading to a better characterization of microparticles and a higher encapsulation efficiency in producing encapsulated PLG microparticles were 8% (w/v) of PLG, 0.25% (w/v) of PVA, and 8% (w/v) of BSA. Furthermore, an in vitro release study revealed a bursting release of BSA from the encapsulated PLG microsphere in the early phase of development. Subsequently, a gradual release was observed over a period of eight weeks. Furthermore, to encapsulate LL-37, different proteins were used in conjunction with PLG under identical conditions with regard to the loading efficiency and morphology, thereby indicating high variations and poor reproducibility. In conclusion, the encapsulated PLG microparticles could effectively protect the protein during encapsulation and could facilitate sustainable protein release over a period of 60 days. Importantly, an optimal method must be employed in order to achieve a high degree of encapsulation efficiency for all of the protein or growth factors. Accordingly, the outcomes of this study will be useful in the manufacture of drug delivery devices that require medium-sustained release growth factors, particularly in dental treatments.

Effects of immunoglobulin Y-loaded amorphous calcium phosphate on dentinal tubules occlusion and antibacterial activity.

Aim: This study aimed to evaluate the effects of immunoglobulin Y (IgY)-loaded amorphous calcium phosphate (ACP) (IgY@ACP) on dentinal tubule occlusion and antibacterial activity. Methodology: IgY@ACP was synthesized based on a biomimetic mineralization strategy. The structure was examined by transmission electron microscopy and Fourier transform infrared spectroscopy. The IgY release property was assessed in vitro. The cell biocompatibility of IgY@ACP was evaluated by CCK-8. The dentin disks were prepared using healthy human molars, and their dentinal tubules were exposed to EDTA. Subsequently, they were randomly selected and treated with or without IgY@ACP for 7 days. The tubule occlusion morphologies and newly formed layers were observed by scanning electron microscopy (SEM) and x-ray diffraction, respectively. To evaluate the acid resistance and abrasion resistance of IgY@ACP, dentin disks that were treated for 1 day were immersed in acid solution or subjected to a toothbrush. The antibacterial effects against Streptococcus mutans (S. mutans) were evaluated by colony-forming unit (CFU) counting, adhesion property assessment, and crystal violet and live/dead bacterial staining. Finally, the occlusion effect was evaluated in rat incisors in vivo. One-way analysis of variance (ANOVA) was performed for statistical analysis. The level of significance was set at 0.05. Results: IgY@ACP presented an amorphous phase with a nanosize (60–80 nm) and sustained release of protein within 48 h. The CCK-8 results showed that IgY@ACP had good biocompatibility. After treatment with IgY@ACP for 1 day, the majority of dentinal tubules were occluded by a 0.3-μm-thick mineralized layer. Seven days later, all dentinal tubules were occluded by mineralization with a thickness of 1.4 μm and a depth of 16 μm. The newly mineralized layer showed hydroxyapatite-like diffraction peaks. In addition, IgY@ACP had good acid and abrasion resistance. After treatment with IgY@ACP, the CFU counting and adhesion rate of S. mutans were significantly reduced, the crystal violet staining was lighter, and the S. mutans staining revealed more dead cells. Most importantly, IgY@ACP had a certain occluding property in rat incisors in vivo. Conclusion: IgY@ACP can effectively occlude dentinal tubules with acid-resistant stability and has prominent anti-S. mutans effects, rendering it a potentially suitable desensitization material in the clinic.

Surface modification of fibroblasts with peroxiredoxin-1-loaded polymeric microparticles increases cell mobility, resistance to oxidative stress and collagen I production

Modification of the cell surface with artificial nano- and microparticles (also termed “cellular backpacks”) containing biologically active payloads usually enables drug targeting via harnessing intrinsic cell tropism to the sites of injury. In some cases, using cells as delivery vehicles leads to improved pharmacokinetics due to extended circulation time of cell-immobilized formulations. Another rationale for particle attachment to cells is augmentation of desirable cellular functions and cell proliferation in response to release of the particle contents. In this study, we conjugated poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with multifunctional antioxidant enzyme peroxiredoxin-1 (Prx1) to the surface of fibroblasts. The obtained microparticles were uniform in size and demonstrated sustained protein release. We found that the released Prx1 maintains its signaling activity resulting in macrophage activation, as indicated by TNFα upregulation and increase in ROS generation. Functionalization of fibroblasts with PLGA/Prx1 microparticles via EDC/sulfo-NHS coupling reaction did not affect cell viability but increased cell migratory properties and collagen I production. Moreover, PLGA/Prx1 backpacks increased resistance of fibroblasts to oxidative stress and attenuated cell senescence. In summary, we have developed a novel approach of fibroblast modification to augment their biological properties, which can be desirable for wound repair, cosmetic dermatology, and tissue engineering.

Protein sustained release from isobutyramide-grafted stellate mesoporous silica nanoparticles

Proteins are great therapeutic candidates as endogenous biomolecules providing a wide range of applications. However, their delivery suffers from some limitations and specifically designed delivery systems having an efficient protein anchoring and delivery strategy are still needed. In this work, we propose to combine large pore stellate mesoporous silica (STMS) with isobutyramide (IBAM), as a “glue” molecule which has been shown promising for immobilization of various biomacromolecules at silica surface. We address here for the first time the ability of such IBAM-modified NPs to sustainably deliver proteins over a prolonged time. In this work, a quantitative loading study of proteins (serum albumin (HSA), peroxidase (HRP), immunoglobulin (IgG) and polylysine (PLL)) on STMS@IBAM is first presented using three complementary detection techniques to ensure precision and avoid protein quantification issues. The results demonstrated a high loading capacity for HSA and HRP (≥ ca. 350 μg.mg−1) but a moderate one for IgG and PLL. After evaluating the physicochemical properties of the loaded particles and their stability over scaling-up and washings, the ability of STMS@IBAM to release proteins over prolonged time was evaluated in equilibrium (static) and flow mimicking (dynamic) conditions and at different temperatures (25, 37, 45 °C). Results show not only the potential of such “glue” functionalized STMS to release proteins in a sustained way, but also the retention of the biological activity of immobilized and released HRP, used as an enzyme model. Finally, an AFM-force spectroscopy study was conducted to decipher the interactions between IBAM and proteins, showing the involvement of different interactions in the adsorption and release processes.

Real-time evaluation of a hydrogel delivery vehicle for cancer immunotherapeutics within embedded spheroid cultures.

Many protein immunotherapeutics are hindered by transport barriers that prevent the obtainment of minimum effective concentrations (MECs) in solid tumors. Local delivery vehicles with tunable release (infusion) rates for immunotherapeutics are being developed to achieve local and sustained release. To expedite their discovery and translation, in vitro models can identify promising delivery vehicles and immunotherapies that benefit from sustained release by evaluating cancer spheroid killing in real-time. Using displacement affinity release (DAR) within a hydrogel, we tuned the release of a CD133 targeting dual antigen T cell engager (DATE) without the need for further DATE or hydrogel modifications, yielding an injectable vehicle that acts as a tunable infusion pump. To quantify bioactivity benefits, a 3D embedded cancer spheroid model was developed for the evaluation of sustained protein release and combination therapies on T cell mediated spheroid killing. Using automated brightfield and fluorescent microscopy, the size of red fluorescent protein (iRFP670) expressing spheroids were tracked to quantify spheroid growth or killing over time as a function of controlled delivery. We demonstrate that sustained DATE release enhanced T cell mediated killing of embedded glioblastoma spheroids at longer timepoints, killing was further enhanced with the addition of anti-PD1 antibody (αPD1). The multi-cellular embedded spheroid model with automated microscopy demonstrated the benefit of extended bispecific release on T cell mediated killing, which will expedite the identification and translation of delivery vehicles such as DAR for immunotherapeutics.

Hydrogel microcapsules containing engineered bacteria for sustained production and release of protein drugs

Subcutaneous administration of sustained-release formulations is a common strategy for protein drugs, which avoids first pass effect and has high bioavailability. However, conventional sustained-release strategies can only load a limited amount of drug, leading to insufficient durability. Herein, we developed microcapsules based on engineered bacteria for sustained release of protein drugs. Engineered bacteria were carried in microcapsules for subcutaneous administration, with a production-lysis circuit for sustained protein production and release. Administrated in diabetic rats, engineered bacteria microcapsules was observed to smoothly release Exendin-4 for 2 weeks and reduce blood glucose. In another example, by releasing subunit vaccines with bacterial microcomponents as vehicles, engineered bacterial microcapsules activated specific immunity in mice and achieved tumor prevention. The engineered bacteria microcapsules have potential to durably release protein drugs and show versatility on the size of drugs. It might be a promising design strategy for long-acting in situ drug factory.

Injectable Acylhydrazone‐Linked RAFT Polymer Hydrogels for Sustained Protein Release and Cell Encapsulation (Adv. Healthcare Mater. 7/2022)

Injectable Hydrogels In article number 2101284 by Chien-Chi Lin and co-workers, a biocompatible and pH/thermo dual responsive injectable hydrogel is synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization and acylhdrazone click chemistry. Upon injection, sol-gel transition occurs rapidly at body temperature and the porous hydrogels degrade slowly under mild acidic condition due to pH-sensitive cleavages of acylhydrazone bonds. The slow hydrogel degradation may be leveraged for long-term sustained protein delivery.

Antibacterial polyelectrolyte coatings enable sustained release of rhBMP-2 from titanium alloy

The development of advanced technology to promote osteo-integration of metallic implants and to solve bacteria-associated infection is one of the urgent clinical requirements. A multilayer polyelectrolyte coating composed of phosphonate/quaternary amine block polymer (pDEMMP-co-pTMAEMA), polymethacrylic acid (PMAA) and polyethylenimine (PEI) was designed as the dual functional coating. Through a layer-by-layer technology, stable coatings were constructed on Ti alloy plate (TC4) substrates based on the strong covalent binding between phosphonate group and metallic substrate, and the electrostatic interaction between oppositely charged polymers. The polymeric coating enabled a high-efficiency sustained release of recombinant human bone morphogenetic protein 2 (rhBMP-2) from the TC4 substrates and promoted the trans-differentiation of C2C12 cells to osteoblast cells. Meanwhile, the polycationic component in the polyelectrolyte coating significantly improved the antibacterial properties of titanium alloy surface. The polyelectrolyte coating demonstrated here is a promising candidate to increase the osteo-integration and antibacterial property of metallic implants.

Photopolymerized Zwitterionic Hydrogels with a Sustained Delivery of Cerium Oxide Nanoparticle-miR146a Conjugate Accelerate Diabetic Wound Healing.

In the United States, $87 billion per year is spent on the care of diabetic ulcers alone. Although the pathophysiology of diabetic wound healing is multifaceted, high systemic levels of inflammation and increased reactive oxygen species are often implicated in the wound healing impairment. Zwitterionic materials have been demonstrated to reduce inflammation and increase extracellular matrix deposition in wound beds, and here, we demonstrate a fabrication method for photopolymerized zwitterionic hydrogels that also enables sustained drug delivery over time. A therapeutic molecule of interest that is examined in this work is cerium oxide nanoparticle tagged with microRNA-146a (CNP-miR146a) to combat both oxidative stress and inflammation. The hydrogels are composed of zwitterionic and nonzwitterionic monomers, and the hydrogel formation occurs in the absence of a crosslinker. The hydrogels exhibit a wide range of stiffness and mechanical properties depending on their monomer content. Additionally, these hydrogels exhibit sustained release of nanoparticles and proteins. Finally, when employed in an in vivo diabetic mouse wound healing model, the zwitterionic hydrogels alone and laden with the CNP-miR146a conjugate significantly improved the rate of diabetic wound healing. Overall, these materials have excellent potential to be used as a topical treatment for chronic diabetic wounds.

Nanoarchitectonics of a Microsphere-Based Scaffold for Modeling Neurodevelopment and Neurological Disease.

Three-dimensional cellular constructs derived from pluripotent stem cells allow the ex vivo study of neurodevelopment and neurological disease within a spatially organized model. However, the robustness and utility of three-dimensional models is impacted by tissue self-organization, size limitations, nutrient supply, and heterogeneity. In this work, we have utilized the principles of nanoarchitectonics to create a multifunctional polymer/bioceramic composite microsphere system for stem cell culture and differentiation in a chemically defined microenvironment. Microspheres could be customized to produce three-dimensional structures of defined size (ranging from >100 to <350 μm) with lower mechanical properties compared with a thin film. Furthermore, the microspheres softened in solution, approaching more tissue-like mechanical properties over time. With neural stem cells (NSCs) derived from human induced pluripotent stem cells, microsphere-cultured NSCs were able to utilize multiple substrates to promote cell adhesion and proliferation. Prolonged culture of NSC-bound microspheres under differentiating conditions allowed the formation of both neural and glial cell types from control and patient-derived stem cell models. Human NSCs and differentiated neurons could also be cocultured with astrocytes and human umbilical vein endothelial cells, demonstrating application for tissue-engineered modeling of development and human disease. We further demonstrated that microspheres allow the loading and sustained release of multiple recombinant proteins to support cellular maintenance and differentiation. While previous work has principally utilized self-organizing models or protein-rich hydrogels for neural culture, the three-dimensional matrix developed here through nanoarchitectonics represents a chemically defined and robust alternative for the in vitro study of neurodevelopment and nervous system disorders.

Controlled release of bioactive IL-2 from visible light photocured biodegradable elastomers for cancer immunotherapy applications

Biodegradable elastomeric controlled-release poly (decane-co-tricarballylate) (PDET) based matrices capable of maintaining the stability and bioactivity of Interleukin-2 (IL-2) through the utilization of visible-light curing and solvent-free loading of the cytokine are reported. The elastomeric devices were fabricated by intimately mixing lyophilized IL-2 powder with the acrylated prepolymer before photocrosslinking. The bioactivity of the released protein was assessed by its ability to stimulate the proliferation of the C57BL/6 mouse cytotoxic T lymphocyte, and its concentration was analysed using ELISA. The influence of changes in the polymer's physicochemical and mechanical properties on IL-2 release kinetics and bioactivity were also studied. The increase in the device's surface area and the incorporation of trehalose in the loaded lyophilized mix increased the IL-2 release rate with drug release proceeding via typical zero-order release kinetics. Moreover, the decrease in the degree of acrylation of the prepared devices increased the IL-2 release rate. The bioactivity assay showed that IL-2 retained over 94% of its initial bioactivity throughout 28 days of the release period. A new protein delivery vehicle composed of biodegradable PDET elastomers was demonstrated to be promising and effective for linear, constant, and sustained osmotic-driven release of bioactive IL-2 and other sensitive proteins and hormones.

Sustained Release of Gas6 via mPEG-PLGA Nanoparticles Enhances the Therapeutic Effects of MERTK Gene Therapy in RCS Rats.

Previous researches utilizing MER proto-oncogene tyrosine kinase (MERTK) gene therapy in Royal College of Surgeons (RCS) rats evidenced its effectiveness in treating MERTK-associated retinitis pigmentosa (RP). Specific ligands for receptor tyrosine kinases, such as growth arrest-specific 6 (Gas6), may enhance retinal phagocytosis via the MERTK receptor, and consequently, enhance the therapeutic effects of gene therapy. In order to overcome the short life effect of the injected Gas6 protein, we constructed a Gas6 loaded methoxy-poly (ethylene glyeol)-poly (lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles (Gas6 NPs) system which allowed for localized and sustained Gas6 protein release, and therefore, a prolonged biological effect. Our data demonstrated that Gas6 protein release from Gas6 NPs preserved the bioactivity and promoted retinal pigment epithelium (RPE) phagocytosis in vitro. In vivo studies showed that RCS rats in the hMERTK/Gas6 NPs group exhibiting the highest electroretinogram responses and more complete retinal structure than that in other groups, further demonstrating that the co-administration of AAV2-BEST1-hMERTK and Gas6 NPs could protect photoreceptors from degeneration. These findings strongly suggest that Gas6 NPs are a promising method to enable the sustained release of Gas6 protein and could therefore enhance the therapeutic effects of gene therapy for MERTK-associated RP.

Injectable Acylhydrazone-Linked RAFT Polymer Hydrogels for Sustained Protein Release and Cell Encapsulation.

A new class of temperature responsive polymer, termed PADO, is synthesized by reversible addition-fragmentation chain-transfer polymerization. Synthesized from copolymerization of diacetone acrylamide (DAAM), di(ethylene glycol) ethyl ether acrylate, and oligo(ethylene glycol) methyl ether acrylate, PADO polymer phase separates at temperature above its lower critical solution temperature (36-42°C) due to enhanced hydrophobic interactions between the short ethylene glycol side chains. Solution of PADO polymers exhibit injectable shear-thinning properties and reach sol-gel transition rapidly (<5min) at 37°C.Whenthe ketone moieties on DAAM are linked by adipic acid dihydrazdie, PADO polymers form crosslinked and injectable acylhydrazone hydrogels, which are hydrolytically degradable at a mild acidic environment owing to the pH sensitive acylhydrazone bonds. The pH-responsive degradation kinetics can be controlled by tuning polymer contents and ketone/hydrazide ratio. Importantly, the injectable PADO hydrogels are highly cytocompatible and can be easily formulated for pH-responsive sustained protein delivery.

Customizing biohybrid cryogels to serve as ready-to-use delivery systems of signaling proteins

Macroporous cryogels have recently gained increasing interest for the controlled administration of signaling proteins in tissue engineering due to an advantageous combination of material properties. However, most of the previously reported cryogel systems did not allow for tunable, sustained protein release. We therefore designed a set of ready-to-use multi-armed polyethylene glycol (starPEG)-heparin cryogel systems containing different amounts of the protein-affine glycosaminoglycan component heparin to enable systematically tunable long-term delivery of different signaling proteins without affecting other cell-instructive properties. Experimental data and mathematical modeling indicate that the macroporous structure causes local differences in the concentration of proteins released into the pores and in the surrounding of the cryogels. As a proof-of-concept for their ready-to-use potential, cryogels pre-functionalized with signaling proteins and cell adhesion-peptides were demonstrated to induce the neuronal differentiation of colonizing pheochromocytoma cells. The elaborated approach opens up new perspectives for cryogels as easily storable and applicable systems for the precision delivery of signaling proteins.

Injectable exosome-functionalized extracellular matrix hydrogel for metabolism balance and pyroptosis regulation in intervertebral disc degeneration

Exosome therapy is a promising therapeutic approach for intervertebral disc degeneration (IVDD) and achieves its therapeutic effects by regulating metabolic disorders, the microenvironment and cell homeostasis with the sustained release of microRNAs, proteins, and transcription factors. However, the rapid clearance and disruption of exosomes are the two major challenges for the application of exosome therapy in IVDD. Herein, a thermosensitive acellular extracellular matrix (ECM) hydrogel coupled with adipose-derived mesenchymal stem cell (ADSC) exosomes (dECM@exo) that inherits the superior properties of nucleus pulposus tissue and ADSCs was fabricated to ameliorate IVDD. This thermosensitive dECM@exo hydrogel system can provide not only in situ gelation to replenish ECM leakage in nucleus pulposus cells (NPCs) but also an environment for the growth of NPCs. In addition, sustained release of ADSC-derived exosomes from this system regulates matrix synthesis and degradation by regulating matrix metalloproteinases (MMPs) and inhibits pyroptosis by mitigating the inflammatory response in vitro. Animal results demonstrated that the dECM@exo hydrogel system maintained early IVD microenvironment homeostasis and ameliorated IVDD. This functional system can serve as a powerful platform for IVD drug delivery and biotherapy and an alternative therapy for IVDD.

A supramolecular host-guest interaction-mediated injectable hydrogel system with enhanced stability and sustained protein release

Injectable hydrogels have been studied as drug delivery systems because of their minimal invasiveness and sustained drug release properties. Pluronic F127, consisting of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers, exhibits thermo-responsive properties and hence is injectable due to its rapid sol-gel transition. Unmodified Pluronic F127-based hydrogels, however, have limited long-term stability and controllable release of drugs entrapped within them. In this study, host-guest interactions between adamantane-conjugated Pluronic F127 (F127-Ad) and polymerized β-cyclodextrin (CDP) were employed to develop a hydrogel-based protein delivery system. Single or multiple adamantane units were successfully introduced at the termini of Pluronic F127 with a 100% conversion yield, and the synthesized F127-Ad polymer produced a physically crosslinked micelle-packing structure when mixed with CDP. As the number of adamantanes at the terminal ends of Pluronic F127 increased, the critical gelation concentration of F127-Ad/CDP hydrogel decreased from 15 to 6% (w/v). The F127/CDP hydrogel was able to maintain its structure even with lower polymer content, and its injectability improved with a reduction of the hydrogel viscosity. The long-term stability of F127/CDP hydrogels was evaluated in vitro and in vivo, and it was demonstrated that the subcutaneously injected hydrogel did not disintegrate for up to 30 d. Throughout the drug release test using gelatin and insulin as model drugs, it was demonstrated that their release rates could be regulated via complexation between the protein drugs and the β-cyclodextrin molecules inside the hydrogel. In conclusion, the F127-Ad/CDP hydrogel is expected to be a versatile protein delivery system with controllable durability and drug release characteristics. Statement of significancePluronic F127 is one of the widely studied polymeric materials for thermo-sensitive injectable hydrogels due to its high biocompatibility and rapid sol-gel transition. Since the Pluronic F127-based hydrogel has some limitations in its long-term stability and mechanical property, it is inevitable to modify its structure for the application to drug delivery. In this study, mono- or multi- adamantane-conjugated Pluronic F127s were synthesized and mixed with β-cyclodextrin polymers to form hydrogels with host-guest interaction-mediated micelle-packing structures. The host-guest interaction introduced into the hydrogel system endowed it a sustained protein drug release behavior as well as high durability in vitro and in vivo. By increasing the number of adamantane molecules at the end of the Pluronic F127, both the stability and injectability of the hydrogel could be also modulated.