Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable Bioactivity and Controllable Cell Adhesion

Injectable hydrogels have been widely investigated for applications in biomedical fields, for instance, as biomimetic scaffolds mimicking the extracellular matrix (ECM). In addition to as scaffolds for mechanical support and transferring of nutrients, the dynamic bioactivity of ECM is another critical factor that affects cell behavior. In this work, a novel injectable poly­(l-glutamic acid)-based hydrogel decorated with RGD was fabricated. The presentation of RGD significantly enhanced the cell-matrix interaction and promoted cell adhesion and proliferation. Moreover, the cell-adhesive RGD was conjugated to the network via a disulfide bond, so that the density of RGD and the bioactivity of hydrogel can be well controlled by tuning the RGD content through treating with glutathione. As a result, the cell behaviors on the hydrogel can be tuned on demand. The injectable hydrogel with controllable bioactivity may provide an interesting strategy to develop a scaffold mimicking ECM that can regulate cell adhesion dynamically

Injectable, Biomolecule-Responsive Polypeptide Hydrogels for Cell Encapsulation and Facile Cell Recovery through Triggered Degradation

Injectable hydrogels have been widely investigated in biomedical applications, and increasing demand has been proposed to achieve dynamic regulation of physiological properties of hydrogels. Herein, a new type of injectable and biomolecule-responsive hydrogel based on poly­(l-glutamic acid) (PLG) grafted with disulfide bond-modified phloretic acid (denoted as PLG-<i>g</i>-CPA) was developed. The hydrogels formed in situ via enzymatic cross-linking under physiological conditions in the presence of horseradish peroxidase and hydrogen peroxide. The physiochemical properties of the hydrogels, including gelation time and the rheological property, were measured. Particularly, the triggered degradation of the hydrogel in response to a reductive biomolecule, glutathione (GSH), was investigated in detail. The mechanical strength and inner porous structure of the hydrogel were influenced by the addition of GSH. The polypeptide hydrogel was used as a three-dimensional (3D) platform for cell encapsulation, which could release the cells through triggered disruption of the hydrogel in response to the addition of GSH. The cells released from the hydrogel were found to maintain high viability. Moreover, after subcutaneous injection into rats, the PLG-<i>g</i>-CPA hydrogels with disulfide-containing cross-links exhibited a markedly faster degradation behavior in vivo compared to that of the PLG hydrogels without disulfide cross-links, implying an interesting accelerated degradation process of the disulfide-containing polypeptide hydrogels in the physiological environment in vivo. Overall, the injectable and biomolecule-responsive polypeptide hydrogels may serve as a potential platform for 3D cell culture and easy cell collection

Injectable Polypeptide Hydrogels with Tunable Microenvironment for 3D Spreading and Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells

Bone-marrow-derived mesenchymal stem cells (BMSCs) possess vast potential for tissue engineering and regenerative medicine. In this study, an injectable hydrogel comprising poly­(l-glutamic acid)-<i>graft</i>-tyramine (PLG-<i>g</i>-TA) with tunable microenvironment was developed via enzyme-catalyzed cross-linking and used as an artificial extracellular matrix (ECM) to explore the behaviors of BMSCs during three-dimensional (3D) culture. It was found that the mechanical property, porous structure as well as degradation process of the hydrogels could be tuned by changing the copolymer concentration. The PLG-<i>g</i>-TA hydrogels showed good cytocompatibility in vitro. After being subcutaneously injected into the back of rats, the hydrogels degraded gradually within 8 weeks and exhibited good biocompatibility in vivo. BMSCs were then encapsulated in the polypeptide-based hydrogels with different copolymer concentration to investigate the influence of 3D matrix microenvironment on stem cell behaviors. It is intriguing to note that the BMSCs within the 2% hydrogel showed a well-spread morphology after 24 h and a higher proliferation rate during 7 days of culture, in contrast to a rounded morphology and lower proliferation rate of BMSCs in the 4% hydrogel. Furthermore, the hydrogels with different microenvironment also regulated the matrix biosynthesis and the gene expression of BMSCs. After incubation in the 2% hydrogel for 4 weeks, the BMSCs produced more type II collagen and expressed higher amounts of chondrogenic markers, compared to the cells in the 4% hydrogel. Therefore, the PLG-<i>g</i>-TA hydrogels with tunable microenvironment may serve as an efficient 3D platform for guiding the lineage specification of BMSCs

Dual Stimuli-Responsive Nanoparticle-Incorporated Hydrogels as an Oral Insulin Carrier for Intestine-Targeted Delivery and Enhanced Paracellular Permeation

For enhanced oral insulin delivery, a strategy of acid-resistant and enteric hydrogels encapsulating insulin-loaded nanoparticles was developed. The nanoparticles were prepared by the formation of an anionic insulin/heparin sodium (Ins/HS) aggregate, followed by coating of chitosan (CS) on the surface. The nanoparticles, tagged as CS/Ins/HS NPs, exhibited excellent mucosa affinity, effective protease inhibition, and marked paracellular permeation enhancement. Moreover, to improve the acid-stability of CS/Ins/HS NPs and impart the capacity of intestine-targeted delivery, a pH- and amylase-responsive hydrogel was synthesized via free radical copolymerization, using methacrylic acid as the monomer and acrylate-<i>grafted</i>-carboxymethyl starch as the cross-linker. The resulting hydrogel exhibited sharp pH-sensitivity in the gastrointestinal tract and rapid enteric behavior under intestinal amylase. The additional protection for insulin in artificial gastric fluid was confirmed by packaging CS/Ins/HS NPs into the hydrogel. The obtained nanoparticle-incorporated hydrogel was named as NPs@Gel-2. The release of insulin from NPs@Gel-2 was evidently accelerated in artificial intestinal fluid containing α-amylase. Furthermore, the hypoglycemic effects were evaluated with type-1 diabetic rats. Compared to subcutaneous injection of insulin solution, the relative pharmacological availability (rPA) for oral intake of NPs@Gel-2 (30 IU/kg) was determined to be 8.6%, along with rPA of 4.6% for oral administration of unpackaged CS/Ins/HS NPs (30 IU/kg). Finally, the two-week therapeutic outcomes in diabetic rats were displayed after twice-daily treatments by oral intake of NPs@Gel-2, showing the relief of diabetic symptoms and suppression of weight loss in the rats. Therefore, this dual stimuli-responsive nanoparticle-incorporated hydrogel system could be a promising platform for oral insulin delivery

Localized Co-delivery of Doxorubicin, Cisplatin, and Methotrexate by Thermosensitive Hydrogels for Enhanced Osteosarcoma Treatment

Localized cancer treatments with combination drugs have recently emerged as crucial approaches for effective inhibition of tumor growth and reoccurrence. In this study, we present a new strategy for the osteosarcoma treatment by localized co-delivery of multiple drugs, including doxorubicin (DOX), cisplatin (CDDP) and methotraxate (MTX), using thermosensitive PLGA–PEG–PLGA hydrogels. The release profiles of the drugs from the hydrogels were investigated in vitro. It was found that the multidrug coloaded hydrogels exhibited synergistic effects on cytotoxicity against osteosarcoma Saos-2 and MG-63 cells in vitro. After a single peritumoral injection of the drug-loaded hydrogels into nude mice bearing human osteosarcoma Saos-2 xenografts, the hydrogels coloaded with DOX, CDDP, and MTX displayed the highest tumor suppression efficacy in vivo for up to 16 days, as well as led to enhanced tumor apoptosis and increased regulation of the expressions of apoptosis-related genes. Moreover, the monitoring on the mice body change and the ex vivo histological analysis of the key organs indicated that the localized treatments caused less systemic toxicity and no obvious damage to the normal organs. Therefore, the approach of localized co-delivery of DOX, CDDP, and MTX by the thermosensitive hydrogels may be a promising approach for enhanced osteosarcoma treatment

Intracellular pH-Sensitive PEG-<i>block</i>-Acetalated-Dextrans as Efficient Drug Delivery Platforms

Intracellular pH-sensitive micelles of PEG-<i>block</i>-acetalated-dextran (PEG-<i>b</i>-AC-Dex) were prepared and used for acid-triggered intracellular release of anticancer drug. The hydrodynamic radii (<i>R</i>_h) of PEG-<i>b</i>-AC-Dex micelles could increase after incubation in PBS solution at pH 5.5. Based on the pH-responsive <i>R</i>_h variation behavior, it was expected that the PEG-<i>b</i>-AC-Dex micelles should be interesting for intracellular drug delivery. Thus, doxorubicin (DOX), a wide-spectrum anticancer drug, was loaded into the micelles and the pH-dependent release of the payload DOX was tested <i>in vitro</i>. The <i>in vitro</i> drug release profiles showed that only a small amount of the loaded DOX was released in PBS solution at pH 7.4, while up to about 90% of the loaded DOX could be quickly released in PBS solution at pH 5.5. Compared to pH-insensitive PEG-PLA micelles, the PEG-<i>b</i>-AC-Dex micelles displayed a faster drug release behavior in tumor cells. Moreover, higher cellular proliferation inhibition efficacy was achieved toward tumor cells. These features suggested that DOX could be efficiently loaded and delivered into tumor cells <i>in vitro</i> by the intracelluar pH-sensitive micelles, leading to enhanced inhibition of tumor cell proliferation. Therefore, the pH-sensitive micelles may provide a promising carrier for acid-triggered drug release for cancer therapy

Versatile Biofunctionalization of Polypeptide-Based Thermosensitive Hydrogels via Click Chemistry

In this study, we report thermosensitive hydrogels based on poly­(ethylene glycol)-<i>block</i>-poly­(γ-propargyl-l-glutamate) (PEG-PPLG). ¹³C NMR spectra, DLS, and circular dichroism spectra were employed to study the mechanism of the sol–gel phase transition. Mouse fibroblast L929 cells were encapsulated and cultured within the hydrogel matrices, and the encapsulated cells were shown to be highly viable in the gel matrices, suggesting that the hydrogels have excellent cytocompatibilities. The mass loss of the hydrogels in vitro was accelerated by the presence of proteinase K compared to the control group. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ∼21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. The presence of alkynyl side groups in the PEG-PPLG copolymers allowed convenient further functionalization with azide-modified bioactive molecules, such as biotin and galactose. The biofunctionalized PEG–polypeptide block copolymers showed sol–gel phase transitions similar to the parent copolymers. Interestingly, the incorporation of galactose groups into the hydrogels was found to improve cell adhesion, likely due to the adsorption of fibronectin (FN) in cell–extracellular matrix (ECM). Because bioactive materials have shown unique advantages in biomedical applications, especially tissue engineering and regenerative medicine applications, we believe our novel functionalizable thermosensitive hydrogels have potential to serve as a versatile platform for the development of new biofunctional materials, for example, bioadhesive and bioresponsive hydrogels

pH-Responsive Poly(ethylene glycol)/Poly(l‑lactide) Supramolecular Micelles Based on Host–Guest Interaction

pH-responsive supramolecular amphiphilic micelles based on benzimidazole-terminated poly­(ethylene glycol) (PEG-BM) and β-cyclodextrin-modified poly­(l-lactide) (CD-PLLA) were developed by exploiting the host–guest interaction between benzimidazole (BM) and β-cyclodextrin (β-CD). The dissociation of the supramolecular micelles was triggered in acidic environments. An antineoplastic drug, doxorubicin (DOX), was loaded into the supramolecular micelles as a model drug. The release of DOX from the supramolecular micelles was clearly accelerated as the pH was reduced from 7.4 to 5.5. The DOX-loaded PEG-BM/CD-PLLA supramolecular micelles displayed an enhanced intracellular drug-release rate in HepG2 cells compared to the pH-insensitive DOX-loaded PEG-<i>b</i>-PLLA counterpart. After intravenous injection into nude mice bearing HepG2 xenografts by the tail vein, the DOX-loaded supramolecular micelles exhibited significantly higher tumor inhibition efficacy and reduced systemic toxicity compared to free DOX. Furthermore, the DOX-loaded supramolecular micelles showed a blood clearance rate markedly lower than that of free DOX and comparable to that of the DOX-loaded PEG-<i>b</i>-PLLA micelles after intravenous injection into rats. Therefore, the pH-responsive PEG-BM/CD-PLLA supramolecular micelles hold potential as a smart nanocarrier for anticancer drug delivery

Intracellular pH-Sensitive Metallo-Supramolecular Nanogels for Anticancer Drug Delivery

For drug delivery systems, the most important factors are biocompatibility and stability. To achieve excellent biocompatibility, learning from naturally occurring systems may be the best choice. Herein, a series of pH-sensitive metallo-supramolecular nanogels (MSNs) were prepared by the metallo-supramolecular coordinated interaction between histidine and iron-<i>meso</i>-tetraphenylporphin, which mimicks the way that hemoglobin carries oxygen. With the excellent biocompatibility and special supramolecular pH sensitivity, MSNs had been exploited to load and release anticancer drug doxorubicin (DOX). In vitro drug release profiles showed that only a small amount of the loaded DOX was released in PBS solution at pH 7.4, while up to about 80% of the loaded DOX could be quickly released at pH 5.3 due to the pH-dependent disassembly of MSNs. Confocal laser scanning microscopy (CLSM) and flow cytometry were used to verify the cellular uptake and intracellular drug release behaviors of DOX-loaded MSNs toward MCF-7. Efficient cellular proliferation inhibition against MCF-7 and HeLa cells was also observed by a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. These features suggested that MSNs could be of great potential as intelligent drug delivery systems