pH-temperature Responsive Hydrogel-Mediated Delivery of Exendin-4 Encapsulated Chitosan Nanospheres for Sustained Therapeutic Efficacy in Type 2 Diabetes Mellitus.

Type 2 Diabetes Mellitus (T2D) is a chronic, obesity-related, and inflammatory disorder characterize by insulin resistance, inadequate insulin secretion, hyperglycemia, and excessive glucagon secretion. Exendin-4 (EX), a clinically established antidiabetic medication that acts as a glucagon-like peptide-1 receptor agonist, is effective in lowering glucose levels and stimulating insulin secretion while significantly reducing hunger. However, the requirement for multiple daily injections due to EX's short half-life is a significant limitation in its clinical application, leading to high treatment costs and patient inconvenience. To address this issue, an injectable hydrogel system is developed that can provide sustained EX release at the injection site, reducing the need for daily injections. In this study, the electrospray technique is examine to form EX@CS nanospheres by electrostatic interaction between cationic chitosan (CS) and negatively charged EX. These nanospheres are uniformly dispersed in a pH-temperature responsive pentablock copolymer, which forms micelles and undergoes sol-to-gel transition at physiological conditions. Following injection, the hydrogel gradually degraded, exhibiting excellent biocompatibility. The EX@CS nanospheres are subsequently released, maintaining therapeutic levels for over 72 h compared to free EX solution. The findings demonstrate that the pH-temperature responsive hydrogel system containing EX@CS nanospheres can be a promising platform for the treatment of T2D.

Highly Prolonged Release of the Cancer Vaccine and Immunomodulator via a Two-Layer Biodegradable Microneedle for Prophylactic Treatment of Metastatic Cancer.

Simultaneous sustained release of cancer vaccines and immunomodulators may effectively trigger durable immune responses and avoid multiple administrations. Here, we established a biodegradable microneedle (bMN) based on a biodegradable copolymer matrix made of polyethylene glycol (PEG) and poly(sulfamethazine ester urethane) (PSMEU). This bMN was applied to the skin and slowly degraded in the epidermis/dermis layers. Then, the complexes composed of a positively charged polymer (DA3), cancer DNA vaccine (pOVA), and toll-like receptor 3 agonist poly(I/C) were synchronously released from the matrix in a pain-free manner. The whole microneedle patch was fabricated with two layers. The basal layer was formed using polyvinyl pyrrolidone/polyvinyl alcohol that could be rapidly dissolved upon applying the microneedle patch to the skin, whereas the microneedle layer was formed by complexes encapsulating biodegradable PEG-PSMEU, which was stuck at the injection site for sustained release of therapeutic agents. According to the results, 10 days is the time for the complexes to be completely released and express specific antigens in antigen-presenting cells in vitro and in vivo. It is noteworthy that this system could successfully elicit cancer-specific humoral immune responses and inhibit metastatic tumors in the lungs after a single shot of immunization.

Recent strategies to develop pH-sensitive injectable hydrogels.

"Smart" biomaterials that are responsive to pathological abnormalities are an appealing class of therapeutic platforms for the development of personalized medications. The development of such therapeutic platforms requires novel techniques that could precisely deliver therapeutic agents to the diseased tissues, resulting in enhanced therapeutic effects without harming normal tissues. Among various therapeutic platforms, injectable pH-responsive biomaterials are promising biomaterials that respond to the change in environmental pH. Aqueous solutions of injectable pH-responsive biomaterials exhibit a phase transition from sol-to-gel in response to environmental pH changes. The injectable pH-responsive hydrogel depot can provide spatially and temporally controlled release of various bioactive agents including chemotherapeutic drugs, peptides, and proteins. Therapeutic agents are imbibed into hydrogels by simple mixing without the use of toxic solvents and used for long-term storage or in situ injection using a syringe or catheter that could form a stable gel and acts as a controlled release depot in a minimally invasive manner. Tunable physicochemical properties of the hydrogels, such as biodegradability, ability to interact with drugs and mechanical properties, can control the release of the therapeutic agent. This review highlights the advances in the design and development of biodegradable and in situ forming injectable pH-responsive biomaterials that respond to the physiological conditions. Special attention has been paid to the development of amphoteric pH-responsive biomaterials and their utilization in biomedical applications. We also highlight key challenges and future directions of pH-responsive biomaterials in clinical translation.

Injectable Hydrogel Based on Protein-Polyester Microporous Network as an Implantable Niche for Active Cell Recruitment.

Despite the potential of hydrogel-based localized cancer therapies, their efficacy can be limited by cancer recurrence. Therefore, it is of great significance to develop a hydrogel system that can provoke robust and durable immune response in the human body. This study has developed an injectable protein-polymer-based porous hydrogel network composed of lysozyme and poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide (PCLA) (Lys-PCLA) bioconjugate for the active recruitment dendritic cells (DCs). The Lys-PCLA bioconjugates are prepared using thiol-ene reaction between thiolated lysozyme (Lys-SH) and acrylated PCLA (PCLA-Ac). The free-flowing Lys-PCLA bioconjugate sols at low temperature transformed to immovable gel at the physiological condition and exhibited stability upon dilution with buffers. According to the in vitro toxicity test, the Lys-PCLA bioconjugate and PCLA copolymer were non-toxic to RAW 263.7 cells at higher concentrations (1000 µg/mL). In addition, subcutaneous administration of Lys-PCLA bioconjugate sols formed stable hydrogel depot instantly, which suggested the in situ gel forming ability of the bioconjugate. Moreover, the Lys-PCLA bioconjugate hydrogel depot formed at the interface between subcutaneous tissue and dermis layers allowed the active migration and recruitment of DCs. As suggested by these results, the in-situ forming injectable Lys-PCLA bioconjugate hydrogel depot may serve as an implantable immune niche for the recruitment and modification of DCs.

Biodegradable and Injectable Hydrogels in Biomedical Applications.

Injectable hydrogels are a unique class of hydrogels that are formed upon injection into living bodies. They possess features of typical hydrogels such as softness, 3D network structures, large contents of water, the ability to load water-soluble substances, and so on. Furthermore, their injectability allows injectable hydrogels to be implanted into living bodies using a syringe in a minimally invasive way. After being loaded with different active substances (drugs, proteins, genes, viruses, cells, etc.), injectable hydrogels have been demonstrated to be potential in many different biomedical applications including controlled release and tissue engineering. However, biodegradability is also an important property of injectable hydrogels and allows removal of the hydrogels after accomplishment of their tasks. In this Perspective, we aim at introducing several different types of biodegradable and injectable hydrogels and compare their differences in properties and applications. Lastly, we also point out some remaining problems and future trends in the field of biodegradable and injectable hydrogels.

Therapeutic effects of boronate ester cross-linked injectable hydrogels for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma is the most common malignancy with a high incidence rate and is the leading cause of cancer-related deaths. Herein, we developed a thermo-responsive hydrogel comprising poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide (PCLA) that exhibits acidity-accelerated delivery of the tumor-targeting glucuronic acid-bearing doxorubicin (DOX-pH-GA) conjugate into tumor tissues. The PCLA copolymer was post-modified with boronic acid (BA-PCLA) to covalently cross-link with the pH-responsive DOX-pH-GA conjugate. The BA-PCLA copolymer effectively coordinated with the DOX-pH-GA conjugate through the boronate ester formation and showed a lower critical gelation temperature. The DOX conjugated via boronate ester exhibited a sustained release in vitro. Subcutaneous administration of PCLA copolymers formed in situ gels in the subcutaneous layers of Sprague-Dawley rats and degraded after 6 weeks. Similarly, BA-PCLA copolymers coordinated with DOX-pH-GA formed a stable in situ gel in vivo. In vivo imaging studies demonstrated that DOX-pH-GA was released in a sustained manner. The anti-tumor activity of the DOX releasing injectable hydrogel was examined using a HepG2 liver cancer xenograft model. The in vivo antitumor effect demonstrated that the DOX releasing hydrogel depot remarkably suppresses the tumor growth. These results demonstrate that the pH-responsive DOX releasing thermo-responsive hydrogel depot has great potential for application in localized anticancer therapy.

Temperature and pH-responsive in situ hydrogels of gelatin derivatives to prevent the reoccurrence of brain tumor.

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with a median survival time of approximately 12-16 months. Because of its highly aggressive and heterogeneous nature it is very difficult to remove by surgical resection. Herein we have reported dual stimuli-responsive and biodegradable in situ hydrogels of oligosulfamethazine-grafted gelatin and loaded with anticancer drug paclitaxel (PTX) for preventing the progress of Glioblastoma. The oligosulfamethazine (OSM) introduced to the gelatin backbone for the formation of definite and stable in situ hydrogel. The hydrogels transformed from a sol to a gel state upon changes in stimuli. pH and temperature and retained a distinct shape after subcutaneous administration in BALB/c mice. The viscosity of the sol state hydrogels was tuned by varying the feed molar ratio between gelatin and OSM. The porosity of the hydrogels was confirmed to be lower in higher degree OSM by SEM. Sustained release of PTX from hydrogels in physiological environments (pH 7.4) was further retarded up to 63% in 9th days in tumor environments (pH 6.5). While the empty hydrogels were non-toxic in cultured cells, the hydrogels loaded with PTX showed antitumor efficacy in orthotopic-GBM xenograft mice. Collectively, the gelatin-OSM formed porous hydrogels and released the cargo in a sustained manner in tumor environments efficiently suppressing the progress of GBM. Thus, gelatin-OSM hydrogels are a potential candidate for the direct delivery of therapeutics to the local areas in brain diseases.

Optimizing Active Tumor Targeting Biocompatible Polymers for Efficient Systemic Delivery of Adenovirus.

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.

CD44-Targeted and Enzyme-Responsive Photo-Cross-Linked Nanogels with Enhanced Stability for In Vivo Protein Delivery.

One of the biggest challenges of the protein delivery system is to realize stable and high protein encapsulation efficiency in blood circulation and rapid release of protein in the targeted tumor cells. To overcome these hurdles, we fabricated enzyme-responsive photo-cross-linked nanogels (EPNGs) through UV-triggered chemical cross-linking of cinnamyloxy groups in the side chain of PEGylation hyaluronic acid (HA) for CD44-targeted transport of cytochrome c (CC). The EPNGs showed high loading efficiency and excellent stability in different biological media. Notably, CC leakage effectively suppressed under physiological conditions but accelerated release in the presence of hyaluronidase, an overexpressed enzyme in tumor cells. Moreover, thiazolylblue tetrazolium bromide (MTT) results indicated that the vacant EPNGs showed excellent nontoxicity, while CC-loaded EPNGs exhibited higher killing efficiency to CD44-positive A549 cells than to CD44-negative HepG2 cells and free CC. Confocal images confirmed that CC-loaded EPNGs could effectively be internalized by CD44-mediated endocytosis pathway and rapidly escape from the endo/lysosomal compartment. Human lung tumor-bearing mice imaging assays further revealed that CC-loaded EPNGs actively target tumor locations. Remarkably, CC-loaded EPNGs also exhibited enhanced antitumor activity with negligible systemic toxicity. These results implied that these EPNGs have appeared as stable and promising nanocarriers for tumor-targeting protein delivery.

Development of pH-Responsive Polymer Coating as an Alternative to Enzyme-Based Stem Cell Dissociation for Cell Therapy.

Cell therapy usually accompanies cell detachment as an essential process in cell culture and cell collection for transplantation. However, conventional methods based on enzymatic cell detachment can cause cellular damage including cell death and senescence during the routine cell detaching step due to an inappropriate handing. The aim of the current study is to apply the pH-responsive degradation property of poly (amino ester) to the surface of a cell culture dish to provide a simple and easy alternative method for cell detachment that can substitute the conventional enzyme treatment. In this study, poly (amino ester) was modified (cell detachable polymer, CDP) to show appropriate pH-responsive degradation under mild acidic conditions (0.05% (w/v) CDP, pH 6.0) to detach stem cells (human adipose tissue-derived stem cells (hADSCs)) perfectly within a short period (less than 10 min). Compared to conventional enzymatic cell detachment, hADSCs cultured on and detached from a CDP-coated cell culture dish showed similar cellular properties. We further performed in vivo experiments on a mouse hindlimb ischemia model (1.0 × 106 cells per limb). The in vivo results indicated that hADSCs retrieved from normal cell culture dishes and CDP-coated cell culture dishes showed analogous therapeutic angiogenesis. In conclusion, CDP could be applied to a pH-responsive cell detachment system as a simple and easy nonenzymatic method for stem cell culture and various cell therapies.

A pH-activated charge convertible quantum dot as a novel nanocarrier for targeted protein delivery and real-time cancer cell imaging.

The rapid developments of nanocarriers based on quantum dots (QDs) have been confirmed to show substantial promise for drug delivery and bioimaging. However, optimal QDs-based nanocarriers still need to have their controlled behavior in vitro and in vivo and decrease heavy metal-associated cytotoxicity. Herein, a pH-activated charge convertible QD-based nanocarrier was fabricated by capping multifunctional polypeptide ligands (mPEG-block-poly(ethylenediamine-dihydrolipoic acid-2,3-dimethylmaleic anhydride)-L-glutamate, PEG-P(ED-DLA-DMA)LG) onto the surface of core/multishell CdSe@ZnS/ZnS QD by means of a ligand exchange strategy, followed by uploading of cytochrome C (CC) (CC-loaded QD-PEG-P(ED-DLA-DMA)LG) via electrostatic interactions, in which QDs that were water-soluble and protein-loading were perfectly integrated. That is, the CC-loaded QD-PEG-P(ED-DLA-DMA)LG inherited excellent fluorescence properties from CdSe@ZnS/ZnS QD for real-time imaging, as well as tumor-microenvironment sensitivities from PEG-P(ED-DLA-DMA)LG for enhanced cellular uptake and CC release. Experimental results verified that the QD-PEG-P(ED-DLA-DMA)LG showed enhanced internalization, rapid endo/lysosomal escape, and supplied legible real-time imaging for lung carcinoma cells. Furthermore, pH-triggered charge-convertible ability enabled the QD-PEG-P(ED-DLA-DMA)LG-CC to effectively kill cancer cells better than did the control groups. Hence, constructing smart nanocomposites by facile ligand-exchange strategy is beneficial to QD-based nanocarrier for tumor-targeting cancer therapy.

Advances in biodegradable and injectable hydrogels for biomedical applications.

In situ-forming injectable hydrogels are smart biomaterials that can be implanted into living bodies with minimal invasion. Due to pioneer work of Prof. Sung Wan Kim in this field, injectable hydrogels have shown great potentials in many different biomedical applications. Biodegradable and injectable hydrogels can be administered at room temperature as viscous polymer sols. They will degrade after accomplishing their tasks. Before injecting into living bodies, active substances can be loaded into viscous polymer sols with a high loading efficiency by simple mixing. After injecting into living bodies, active substances-loaded hydrogels can be formed and active substances can be released in a controlled manner upon diffusion or polymer degradation. Due to their outstanding properties and unique features, injectable hydrogels are very promising in many biomedical applications including drug/protein/gene delivery, tissue engineering, and regenerative medicine. In this review, we briefly introduce recent development of several important types of in situ-forming injectable hydrogels reported by our group during the last three years. Important properties and potential applications (such as cancer therapy, insulin release and wound healing) of these injectable hydrogels are reviewed. Challenges and perspectives in this research field are also discussed.

Intracellular delivery of cytochrome C using hypoxia-responsive polypeptide micelles for efficient cancer therapy.

To begin with, it is important to note that biodegradable polypeptides have been extensively applied as drug delivery carriers due to their excellent bioavailability, neglectful toxicity, good encapsulation and controlled release. Thus, a biodegradable and hypoxia-responsive polypeptide is a benefit when synthesized for the intracellular delivery of cytochrome c (CC). In its most positive context, this amphiphilic polypeptide can self-assemble into core/shell-structured micelles and encapsulate CC in their hydrophobic cores. Owing to the presence of hypoxia-responsive chemical bonds, the CC-loaded polymeric micelles (PMs) can potentially target hypoxic tissues (such as tumors) and release the proteins inside the cancer cells. For this reason, these PMs exhibit high protein loading content and efficiency and remain stable in several different kinds of cell culture media under normoxic condition. Moreover, the confocal microscopy indicates that CC-loaded PMs could be effectively uptaken by cancer cells and accelerate endo/lysosomal escape. Most importantly, the CC-loaded PMs show great killing effect to HepG2 liver cancer cells under hypoxic condition, which makes this nano-platform a promising candidate for use with efficient cancer therapy.

Dual activatable self-assembled nanotheranostics for bioimaging and photodynamic therapy.

Multifunctional nanosystems that can transport therapeutic and diagnostic agents into tumor sites and activate their respective functions via tumor-microenvironment recognition are highly desirable for clinical applications. We fabricated pH and redox dual-activatable self-assembled nanotheranostics (named as DA-SNs) via coordination-driven self-assembly of chlorin e6 (Ce6) disulfide-linked pH sensitive polymer ligand, poly (isobutylene-alt-maleic anhydride-graft-methoxy-poly (ethyleneglycol)-graft-imidazole-graft-Cystamine-Ce6) [PIMA-mPEG-API-SS-Ce6], and gadolinium ions (Gd3+). DA-SNs exhibited uniform particle size of ~48 nm, excellent stability, and inherent biosafety. Negatively charged DA-SNs could prolong blood circulation time (t1/2 = 2.91 h) and improve tumor accumulation. Moreover, DA-SNs could undergo surface charge switch from negative charge to positive one in a slightly acidic tumor extracellular environment (pH 6.8), thus enhancing cellular uptake. After entering tumor cells, fluorescence, photodynamic therapeutic activity, and T1MR contrast from DA-SNs could be activated within this intracellular environment with lowered pH and high level of GSH. Importantly, human tumors implanted in mice could be successfully visualized via distinct pH and redox dual-sensitive T1MR contrast and fluorescence imaging, indicating that DA-SNs could serve as a dual-modal MR/fluorescence imaging probe for tumor-targeting diagnosis. In addition, DA-SNs exhibited superior photodynamic therapeutic efficiency with negligible side effects. Therefore, this DA-SN shows great promise for synergistic photodynamic therapy and diagnostic imaging.

A novel injectable pH-temperature sensitive hydrogel containing chitosan-insulin electrosprayed nanosphere composite for an insulin delivery system in type I diabetes treatment.

A novel insulin composite delivery system was prepared and characterized. The composite consisted of a pH- and temperature-sensitive hydrogel, which is an oligomer serine-b-poly(lactide)-b-poly(ethylene glycol)-b-poly(lactide)-b-oligomer serine (OS-PLA-PEG-PLA-OS) pentablock copolymer, as matrix and chitosan-insulin electrosprayed nanospheres (CIN) as constituent materials. The properties of the OS-PLA-PEG-PLA-OS pentablock copolymer and the chitosan-insulin nanoparticles were characterized. The chitosan-insulin nanospheres uniformly distributed in the matrix had a reinforcing effect on the mechanical properties and prolonged the degradation time of the hydrogel depot under body conditions. The composite solutions accommodating different concentrations of the chitosan-insulin nanospheres were subcutaneously injected into induced diabetic BALB/c mice to study the in vivo insulin-release profile. The result showed that insulin concentrations in blood plasma were maintained at a steady-state level. Furthermore, the bio-properties of the insulin were retained and it showed a blood glucose level reducing effect for more than 60 hours after injection to a streptozotocin (STZ)-induced diabetic mouse model. The results suggested that this injectable pH-temperature sensitive hydrogel containing chitosan-insulin electrosprayed nanosphere composites has promising potential applications for type 1 diabetes treatment.

Recent Advances of pH-Induced Charge-Convertible Polymer-Mediated Inorganic Nanoparticles for Biomedical Applications.

The incorporation of functional polymers and inorganic nanoparticles into nanoplatforms has the potential to produce personalized nanomedicine systems for further biomedical applications. Polymers that endow inorganic nanoparticles with unique surface properties for prolonged blood circulation and improved tumor targeting and cellular uptake are especially desired. pH-induced charge-switchable polymers are sensitive to the pH of the tumor environment and maintain a negative or neutral charge in blood circulation, increasing their circulation time and enhancing tumor accumulation via the enhanced permeability and retention effect. This type of polymer further transforms its charge to positive in acidic tumor locations to promote cellular uptake. Furthermore, the combination of pH-induced charge-switchable polymers with various inorganic nanoparticles (e.g., magnetic nanoparticles, gold nanoparticles, quantum dots, and upconversion materials) activates their intrinsic functions in in situ diagnosis and disease therapy. This review briefly overviews the recent progress in the development and application of various pH-induced charge-convertible polymers functionalized with different types of inorganic nanoparticles for different biomedical applications. More importantly, future developments in this field are also discussed.

Albumin affibody-outfitted injectable gel enabling extended release of urate oxidase-albumin conjugates for hyperuricemia treatment.

Therapeutic proteins are attractive candidates for the treatment of human diseases. However, their short half-life often limits their clinical application. To overcome this problem, injectable hydrogels have been developed as depots for controlled release of therapeutic proteins, but these systems have not yet achieved the desired extended, sustained drug release profile. Our strategy herein was to implement selective and strong interactions between the hydrogels and therapeutic proteins. Specifically, we investigated whether strong and specific interactions between human serum albumin (HSA) and albumin-binding peptide (ABP) can be used to achieve extended release of urate oxidase (Uox), a therapeutic protein for hyperuricemia treatment, from pH- and temperature-sensitive injectable hydrogels consisting of poly(ethylene glycol)-poly(ß-amino ester urethane) (PEG-PAEU) copolymer. Thus, HSA was conjugated to Uox (Uox-HSA) and ABP was introduced in PEG-PAEU (PEG-PAEU-ABP). Polymers, conjugates, and hydrogels were extensively characterized for their physicochemical characteristics and in vivo efficacy in a hyperuricemia mouse model. Briefly, the hydrogels exhibited good injectability, in vitro biocompatibility and extended drug release, and in vivo gel formation and degradability. The serum half-life of the Uox-HSA loaded in PEG-PAEU-ABP hydrogels was ~96 h in mice, which was ~88, ~5.5, and ~2 times longer than that of free native Uox, free Uox-HSA, and Uox-HSA loaded in PEG-PAEU hydrogels, respectively. In the hyperuricemia mouse model, Uox-HSA loaded in PEG-PAEU-ABP hydrogels exhibited a substantially extended period of uric acid-lowering efficacy. These results clearly show that by applying ABP-HSA strong interaction to injectable hydrogels and therapeutic protein, the concentration of the therapeutic protein can be maintained for a long period in vivo, prolonging its therapeutic effect. Further, our approach can be tailored to accommodate other therapeutic proteins, which potentially expands the clinical applicability range of these systems.

Modularly engineered alginate bioconjugate hydrogel as biocompatible injectable scaffold for in situ biomineralization.

In the present study, a type of bioconjugate was synthesized by post modification of alginate by conjugating temperature-responsive poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) and O-phosphorylethanolamine as phosphorylation functional groups. Freely flowing bioconjugate sols at low temperature can transform to stable viscoelastic gels at the physiological temperature (37 °C). Subcutaneous administration of temperature-responsive bioconjugate sols into the dorsal region of Sprague-Dawley rats formed in situ hydrogel. in situ formation of bioconjugate gels in stimulated body fluids at 37 °C showed nucleation and hydroxyapatite mineral growth. Furthermore, hydroxyapatite growth was also found in in vivo gels, which suggested the potential of alginate-based bioconjugate gels as a scaffold for bone engineering. Bone morphogenetic protein 2 (BMP-2)-loaded bioconjugate formed stable gel in vivo, and demonstrated sustained release. BMP-2-loaded bioconjugates exhibited in situ biomineralization in vivo. These results imply that the in situ formation of injectable biomimetic materials has potential for bone tissue engineering applications.

Highly potent intradermal vaccination by an array of dissolving microneedle polypeptide cocktails for cancer immunotherapy.

Despite recent advances in cancer therapy using vaccines, the efficacy of vaccine regimens remains to be improved. Cutaneous transportation of biomolecules, particularly DNA vaccines, has potentially improved the therapeutic efficacy and has been found to be an appealing approach in cancer immunotherapy. Nevertheless, the effectiveness of transdermal vaccination is limited by the lack of efficacious immune stimulation. Here, to elicit strong immunogenicity in target cells, we propose an array of dissolving microneedle cocktails for pain-free implantation and triggered release of vaccines and adjuvants at cutaneous tissues. The microneedle cocktails comprising a bioresorbable polypeptide matrix with a nanopolyplex, which include cationic amphiphilic conjugates with ovalbumin-expressing plasmid OVA (pOVA) and immunostimulant-polyinosinic:polycytidylic acid (poly(I:C)), were prepared using a one-pot synthesis. The cationic nanopolyplex effectively transported pOVA and poly(I:C) into the intracellular compartments of dendritic cells and macrophages. Cutaneous implantation of microneedle cocktails on mice elicits a stronger antigen-specific antibody response than subcutaneous administration of the microneedle-free nanopolyplex. Compared with traditional vaccination, the dissolving microneedle cocktails enhanced the antibody recall memory after challenge; remarkably, the cocktail-based therapeutic vaccination also resulted in enhanced lung clearance of cancer cells. The dissolving microneedle cocktail therapy based on the triggered release of immunomodulators and adjuvants synergistically augmented the therapeutic effect in B16/OVA melanoma tumors.

Degradation-regulated architecture of injectable smart hydrogels enhances humoral immune response and potentiates antitumor activity in human lung carcinoma.

Cancer vaccines that elicit a robust and durable antitumor response show great promise in cancer immunotherapy. Nevertheless, low immunogenicity and weak immune response limit the application of cancer vaccines. To experience next generation cancer vaccines that elicit robust, durable, and anti-tumor T cell response, herein we design injectable smart hydrogels (ISHs) that self-assemble into a cellular microenvironment-like microporous network using a simple hypodermic needle injection, to localize the immune cells and program host cells. ISHs, composed of levodopa- and poly(ε-caprolactone-co-lactide)ester-functionalized hyaluronic acid (HA-PCLA), are loaded with immunomodulatory factor (OVA expressing plasmid, pOVA)-bearing nano-sized polyplexes and granulocyte-macrophage colony-stimulating factor (GM-CSF) as dendritic cell (DC) enhancement factor. Subcutaneous administration of ISHs effectively localized immune cells, and controlled the delivery of immunomodulatory factors to recruit immune cells. The microporous network allowed the recruitment of a substantial number of DCs, which was 6-fold higher than conventional PCLA counterpart. The locally released nano-sized polyplexes effectively internalized to DCs, resulting in the presentation of tumor-specific OVA epitope, and subsequent activation of CD4+ T cells and generation of OVA-specific serum antibody. By the controlled release of nano-sized polyplexes and GM-CSF through a single subcutaneous injection, the ISHs effectively eliminated B16/OVA melanoma tumors in mice. These ISHs can be administered using a minimal invasive technique that could bypass the need for extracorporeal training of cells ex vivo, and provide sustained release of cancer vaccines for immunomodulation. These important findings suggest that ISHs can serve as powerful biomaterials for cancer immunotherapy.