Enhanced intra-articular therapy for rheumatoid arthritis using click-crosslinked hyaluronic acid hydrogels loaded with toll-like receptor antagonizing peptides.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that results in the deterioration of joint cartilage and bone. Toll-like receptor 4 (TLR4) has been pinpointed as a key factor in RA-related inflammation. While Toll-like receptor antagonizing peptide 2 (TAP2) holds potential as an anti-inflammatory agent, its in vivo degradation rate hinders its efficacy. We engineered depots of TAP2 encapsulated in click-crosslinked hyaluronic acid (TAP2+Cx-HA) for intra-articular administration, aiming to enhance the effectiveness of TAP2 as an anti-inflammatory agent within the joint cavity. Our data demonstrated that FI-TAP2+Cx-HA achieves a longer retention time in the joint cavity compared to FI-TAP2 alone. Mechanistically, we found that TAP2 interacts with TLR4 on the cell membranes of inflammatory cells, thereby inhibiting the nuclear translocation of NF-κB and maintaining it in an inactive cytoplasmic state. In a rat model of RA, the TAP2+Cx-HA formulation effectively downregulated the inflammatory cytokines TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This led to a more rapid restoration of cartilage thickness, increased deposition of glycosaminoglycans, and new bone tissue formation in the regenerated cartilage, in comparison to a single TAP2 treatment after a six-week period. Our results suggest that TAP2+Cx-HA could serve as a potent intra-articular treatment for RA, offering both symptomatic relief and promoting cartilage regeneration. This innovative delivery system holds significant promise for improving the management of RA and other inflammatory joint conditions. STATEMENT OF SIGNIFICANCE: In this study, we developed a therapy by creating toll-like receptor 4 (TLR4)-antagonizing peptide (TAP2)-loaded click-crosslinked hyaluronic acid (TAP2+Cx-HA) depots for direct intra-articular injection. Our study demonstrates that FI-TAP2+Cx-HA exhibits a more than threefold longer lifetime in the joint cavity compared to FI-TAP2 alone. Furthermore, we found that TAP2 binds to TLR4 and masks the nuclear localization signals of NF-κB, leading to its sequestration in an inactive state in the cytoplasm. In a rat model of RA, TAP2+Cx-HA effectively suppresses inflammatory molecules, specifically TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This resulted in faster regeneration of cartilage thickness, increased glycosaminoglycan deposits in the regenerated cartilage, and a twofold increase in new bone tissue formation compared to a single TAP2 treatment.

Injectable click-crosslinked hydrogel containing resveratrol to improve the therapeutic effect in triple negative breast cancer.

Triple-negative breast cancer (TNBC) patients are considered intractable, as this disease has few effective treatments and a very poor prognosis even in its early stages. Here, intratumoral therapy with resveratrol (Res), which has anticancer and metastasis inhibitory effects, was proposed for the effective treatment of TNBC. An injectable Res-loaded click-crosslinked hyaluronic acid (Res-Cx-HA) hydrogel was designed and intratumorally injected to generate a Res-Cx-HA depot inside the tumor. The Res-Cx-HA formulation exhibited good injectability into the tumor tissue, quick depot formation inside the tumor, and the depot remained inside the injected tumor for extended periods. In vivo formed Res-Cx-HA depots sustained Res inside the tumor for extended periods. More importantly, the bioavailability and therapeutic efficacy of Res remained almost exclusively within the tumor and not in other organs. Intratumoral injection of Res-Cx-HA in animal models resulted in significant negative tumor growth rates (i.e., the tumor volume decreased over time) coupled with large apoptotic cells and limited angiogenesis in tumors. Therefore, Res-Cx-HA intratumoral injection is a promising way to treat TNBC patients with high efficacy and minimal adverse effects.

Wavelength-Selective Softening of Hydrogel Networks.

Photoresponsive hydrogels hold key potential in advanced biomedical applications including tissue engineering, regenerative medicine, and drug delivery, as well as intricately engineered functions such as biosensing, soft robotics, and bioelectronics. Herein, the wavelength-dependent degradation of bio-orthogonal poly(ethylene glycol) hydrogels is reported, using three selective activation levels. Specifically, three chromophores are exploited, that is, ortho-nitrobenzene, dimethyl aminobenzene, and bimane, each absorbing light at different wavelengths. By examining their photochemical action plots, the wavelength-dependent reactivity of the photocleavable moieties is determined. The wavelength-selective addressability of individual photoreactive units is subsequently translated into hydrogel design, enabling wavelength-dependent cleavage of the hydrogel networks on-demand. Critically, this platform technology allows for the fabrication of various hydrogels, whose mechanical properties can be fine-tuned using different colors of light to reach a predefined value, according to the chromophore ratios used. The softening is shown to influence the spreading of pre-osteoblastic cells adhering to the gels as a demonstration of their potential utility. Furthermore, the materials and photodegradation processes are non-toxic to cells, making this platform attractive for biomaterials engineering.

Amphiphilic copolymers in biomedical applications: Synthesis routes and property control.

The request of new materials, matching strict requirements to be applied in precision and patient-specific medicine, is pushing for the synthesis of more and more complex block copolymers. Amphiphilic block copolymers are emerging in the biomedical field due to their great potential in terms of stimuli responsiveness, drug loading capabilities and reversible thermal gelation. Amphiphilicity guarantees self-assembly and thermoreversibility, while grafting polymers offers the possibility of combining blocks with various properties in one single material. These features make amphiphilic block copolymers excellent candidates for fine tuning drug delivery, gene therapy and for designing injectable hydrogels for tissue engineering. This manuscript revises the main techniques developed in the last decade for the synthesis of amphiphilic block copolymers for biomedical application. Strategies for fine tuning the properties of these novel materials during synthesis are discussed. A deep knowledge of the synthesis techniques and their effect on the performance and the biocompatibility of these polymers is the first step to move them from the lab to the bench. Current results predict a bright future for these materials in paving the way towards a smarter, less invasive, while more effective, medicine.

An injectable click-crosslinked hyaluronic acid hydrogel modified with a BMP-2 mimetic peptide as a bone tissue engineering scaffold.

An injectable, click-crosslinking (Cx) hyaluronic acid (HA) hydrogel scaffold modified with a bone morphogenetic protein-2 (BMP-2) mimetic peptide (BP) was prepared for bone tissue engineering applications. The injectable click-crosslinking HA formulation was prepared from HA-tetrazine (HA-Tet) and HA-cyclooctene (HA-TCO). The Cx-HA hydrogel scaffold was prepared simply by mixing HA-Tet and HA-TCO. The Cx-HA hydrogel scaffold was stable for a longer period than HA both in vitro and in vivo, which was verified via in-vivo fluorescence imaging in real time. BP acted as an osteogenic differentiation factor for human dental pulp stem cells (hDPSCs). After its formation in vivo, the Cx-HA scaffold provided a fine environment for the hDPSCs, and the biocompatibility of the hydrogel scaffold with tissue was good. Like traditional BMP-2, BP induced the osteogenic differentiation of hDPSCs in vitro. The physical properties and injectability of the chemically loaded BP for the Cx-HA hydrogel (Cx-HA-BP) were nearly identical to those of the physically loaded BP hydrogels and the Cx-HA-BP formulation quickly formed a hydrogel scaffold in vivo. The chemically loaded hydrogel scaffold retained the BP for over a month. The Cx-HA-BP hydrogel was better at inducing the osteogenic differentiation of loaded hDPSCs, because it prolonged the availability of BP. In summary, we successfully developed an injectable, click-crosslinking Cx-HA hydrogel scaffold to prolong the availability of BP for efficient bone tissue engineering.

In Vivo Imaging of Click-Crosslinked Hydrogel Depots Following Intratympanic Injection.

In this study, we developed injectable intratympanic hyaluronic acid (HA) depots for the treatment of hearing loss. We prepared an injectable click-crosslinking formulation by modifying HA with tetrazine (HA-TET) and trans-cyclooctene (HA-TCO), which crosslinked to form an HA depot (Cx-HA). Preparation of the click-crosslinking HA formulation was facile, and Cx-HA depot formation was reproducible. Additionally, the Cx-HA hydrogel was significantly stiffer than HA hydrogel. To monitor the degradation pattern of hydrogels, we mixed a zwitterionic near-infrared (NIR) fluorophore (e.g., ZW800-1C) in the click-crosslinking HA formulation. Then, HA-TET and HA-TCO solutions containing ZW800-1C were loaded separately into the compartments of a dual-barrel syringe for intratympanic injection. The Cx-HA depots formed quickly, and an extended residence time in the tympanic cavity was confirmed by performing NIR fluorescence imaging. We have successfully prepared an injectable click-crosslinking HA formulation that has promise as an intratympanic drug depot.

Injectable Click-Crosslinked Hyaluronic Acid Depot To Prolong Therapeutic Activity in Articular Joints Affected by Rheumatoid Arthritis.

The aim of this study was to design a click-crosslinked hyaluronic acid (HA) (Cx-HA) depot via a click crosslinking reaction between tetrazine-modified HA and trans-cyclooctene-modified HA for direct intra-articular injection into joints affected by rheumatoid arthritis (RA). The Cx-HA depot had significantly more hydrogel-like features and a longer in vivo residence time than the HA depot. Methotrexate (MTX)-loaded Cx-HA (MTX-Cx-HA)-easily prepared as an injectable formulation-quickly formed an MTX-Cx-HA depot that persisted at the injection site for an extended period. In vivo MTX biodistribution in MTX-Cx-HA depots showed that a high concentration of MTX persisted at the intra-articular injection site for an extended period, with little distribution of MTX to normal tissues. In contrast, direct intra-articular injection of MTX alone or MTX-HA resulted in rapid clearance from the injection site. After intra-articular injection of MTX-Cx-HA into rats with RA, we noted the most significant RA reversal, measured by an articular index score, increased cartilage thickness, extensive generation of chondrocytes and glycosaminoglycan deposits, extensive new bone formation in the RA region, and suppression of tumor necrosis factor-α or interleukin-6 expression. Therefore, MTX-Cx-HA injected intra-articularly persists at the joint site in therapeutic MTX concentrations for an extended period, thus increasing the duration of RA treatment, resulting in an improved relief of RA.