Gene Delivery Research Articles

Nanotechnology in healthcare, and its safety and environmental risks.

Nanotechnology holds immense promise in revolutionising healthcare, offering unprecedented opportunities in diagnostics, drug delivery, cancer therapy, and combating infectious diseases. This review explores the multifaceted landscape of nanotechnology in healthcare while addressing the critical aspects of safety and environmental risks associated with its widespread application. Beginning with an introduction to the integration of nanotechnology in healthcare, we first delved into its categorisation and various materials employed, setting the stage for a comprehensive understanding of its potential. We then proceeded to elucidate the diverse healthcare applications of nanotechnology, spanning medical diagnostics, tissue engineering, targeted drug delivery, gene delivery, cancer therapy, and the development of antimicrobial agents. The discussion extended to the current situation surrounding the clinical translation and commercialisation of these cutting-edge technologies, focusing on the nanotechnology-based healthcare products that have been approved globally to date. We also discussed the safety considerations of nanomaterials, both in terms of human health and environmental impact. We presented the in vivo health risks associated with nanomaterial exposure, in relation with transport mechanisms, oxidative stress, and physical interactions. Moreover, we highlighted the environmental risks, acknowledging the potential implications on ecosystems and biodiversity. Lastly, we strived to offer insights into the current regulatory landscape governing nanotechnology in healthcare across different regions globally. By synthesising these diverse perspectives, we underscore the imperative of balancing innovation with safety and environmental stewardship, while charting a path forward for the responsible integration of nanotechnology in healthcare.

Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies.

Ribozymes are small catalytic RNA sequences capable of nucleotide-specific self-cleavage found widespread in nature. Ribozyme cleavage generates distinct 2',3'-phosphate and 5'-hydroxyl termini that resemble substrates for recently characterized RNA repair pathways in cells. We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for Stitch RNA). Optimization of StitchR activity in mammalian cells resulted in a ~900-fold increase in protein expression that approached levels observed for genes expressed from single vectors. We demonstrate that StitchR can be harnessed for effective dual adeno-associated virus gene therapies to correct muscular dystrophies by restoring large functional muscle proteins to endogenous levels in vivo.

Multifunctional Microneedle Patches for Perivascular Gene Delivery and Treatment of Vascular Intimal Hyperplasia.

Gene therapy has emerged as a promising approach to address challenging cardiovascular diseases. Extensive efforts have been focused on developing highly efficient gene vectors with precise delivery techniques to enhance its effectiveness. In this study, we present multifunctional dopamine-gelatin microneedle patches with gene therapy capabilities to achieve perivascular gene delivery for intimal hyperplasia treatment. These patches that were fabricated through freeze-drying of gelatin are with recombinant adeno-associated virus (rAAVs)-carrying tips and dopamine coating backing layers. The lyophilized gelatin could not only effectively preserve the therapeutic activity of rAAVs but could also demonstrate the capability to penetrate the adventitia for efficient delivery. The incorporation of dopamine facilitated patch adhesion and extended the release duration. Based on these advantages, we have demonstrated that the rAAVs-loaded microneedle patches (AMNPs) behave satisfactorily in perivascular gene delivery to inhibit carotid artery restenosis in rats. These features indicate that the AMNPs are clinically valuable in the treatment of vascular intimal hyperplasia diseases.

Navigating the intricate in-vivo journey of lipid nanoparticles tailored for the targeted delivery of RNA therapeutics: a quality-by-design approach.

RNA therapeutics, such as mRNA, siRNA, and CRISPR-Cas9, present exciting avenues for treating diverse diseases. However, their potential is commonly hindered by vulnerability to degradation and poor cellular uptake, requiring effective delivery systems. Lipid nanoparticles (LNPs) have emerged as a leading choice for in vivo RNA delivery, offering protection against degradation, enhanced cellular uptake, and facilitation of endosomal escape. However, LNPs encounter numerous challenges for targeted RNA delivery in vivo, demanding advanced particle engineering, surface functionalization with targeting ligands, and a profound comprehension of the biological milieu in which they function. This review explores the structural and physicochemical characteristics of LNPs, in-vivo fate, and customization for RNA therapeutics. We highlight the quality-by-design (QbD) approach for targeted delivery beyond the liver, focusing on biodistribution, immunogenicity, and toxicity. In addition, we explored the current challenges and strategies associated with LNPs for in-vivo RNA delivery, such as ensuring repeated-dose efficacy, safety, and tissue-specific gene delivery. Furthermore, we provide insights into the current clinical applications in various classes of diseases and finally prospects of LNPs in RNA therapeutics.

In vivo gene editing and in situ generation of chimeric antigen receptor cells for next-generation cancer immunotherapy.

Chimeric antigen receptor (CAR) cell therapy has achieved groundbreaking success in treating hematological malignancies. However, its application to solid tumors remains challenging due to complex manufacturing processes, limited in vivo persistence, and transient therapeutic effects. In vivo CAR-immune cells induced by gene delivery systems loaded with CAR genes and gene-editing tools have shown efficiency for anti-tumor immunotherapy. In situ programming of autologous immune cells avoids the safety concerns of allogeneic immune cells, and the manufacture of gene delivery systems could be standardized. Therefore, the in vivo editing and in situ generation of CAR-immune cells might potentially overcome the abovementioned limitations of current CAR cell therapy. This review mainly focuses on CAR structures, gene-editing tools, and gene delivery techniques applied in anti-tumor immunotherapy to help design and develop in situ CAR-immune cell therapy. The recent applications of in vivo CAR-immune cell therapy in both hematologic malignancies and solid tumors are investigated. To sum up, the in vivo editing and in situ generation of CAR therapy holds promise for offering a practical, cost-effective, efficient, safe, and widely applicable approach to the next-generation anti-tumor immunotherapy.

Fine-Tuned "Click" Functionalization of PAMAM Dendrimers with a Linear Fluorinated Guanidino Linker: Synthesis, Characterization, and Applications.

This study presents the synthesis, characterization, and application of multifunctional PAMAM G2 and G4 dendrimers decorated with a linear fluorinated guanidino linker designed to improve gene delivery efficiency while minimizing cytotoxicity. For the first time, we were able to fine-tune the degree of grafting (DG) during the functionalization process through efficient "click" Michael addition, achieving the synthesis of a collection of six PAMAM conjugates that showed a significant enhancement in transfection efficiency (TE), surpassing the performance of traditional nonviral vectors. The incorporation of fluorinated moieties not only facilitated better deoxyribonucleic acid (DNA) condensation and TE but also introduced potential applications in 19F magnetic resonance imaging thanks to the sharp and intense fluorine nuclear magnetic resonance signals and favorable relaxation parameters. The new dendrimer conjugates demonstrated a promising balance between low cytotoxicity and high TE, with the low-generation PAMAM G2 with lower DG being the best-performing conjugate, making them strong candidates for further development in gene therapy. These findings highlight the potential of these multifunctional PAMAM dendrimers as efficient, nontoxic, and trackable gene delivery vectors.

Alkylated RALA-Derived Peptides for Efficient Gene Delivery.

RALA is an amphipathic cationic peptide demonstrated to be a low-toxicity and high-efficiency delivery platform for the systemic delivery of nucleic acid therapeutics. This work reports three RALA-derived peptides modified with N-terminal palmitic acid, engineered through amino acid substitutions and truncated sequences. All three peptides have good nucleic acid encapsulation, release and uptake, biocompatibility, and endolysosome escape. The siRNA transfection efficiency is about 90%, and the silencing rate of GA (C16-GLFWHHHARLARALARHLARALRA) exceeds that of lipofectamine 2000 (siRNA concentration = 50 nM). Truncating the peptide chain while retaining a certain amount of arginine ensures an effective particle size. Replacing glutamic acid with three histidines ensures an effective zeta potential and accelerates the endosome escape process through the proton sponge phenomenon. Introducing phenylalanine enhances the carrier-cell interaction. We believe that they are powerful carriers of siRNA therapy and may have good application prospects in treating various diseases.

The Adeno-Associated Virus Replication Protein Rep78 Contains a Strictly C-Terminal Sequence Motif Conserved Across Dependoparvoviruses

Adeno-Associated Viruses (AAVs, genus Dependoparvovirus) are the leading gene therapy vector. Until recently, efforts to enhance their capacity for gene delivery had focused on their capsids. However, efforts are increasingly shifting towards improving the viral replication protein, Rep78. We discovered that Rep78 and its shorter isoform Rep52 contain a strictly C-terminal sequence motif, DDx3EQ, conserved in most dependoparvoviruses. The motif is highly negatively charged and devoid of prolines. Its wide conservation suggests that it is required for the life cycle of dependoparvoviruses. Despite its short length, the motif’s strictly C-terminal position has the potential to endow it with a high recognition specificity. A candidate target of the DDx3EQ motif might be the DNA-binding interface of the origin-binding domain of Rep78, which is highly positively charged. Published studies suggest that this motif is not required for recombinant AAV production, but that substitutions within it might improve production.

Gene Therapy in Cardiovascular Disease: Recent Advances and Future Directions in Science: A Science Advisory From the American Heart Association.

Cardiovascular disease remains the foremost cause of morbidity and mortality globally, affecting millions of individuals. Recent discoveries illuminate the substantial role of genetics in cardiovascular disease pathogenesis, encompassing both monogenic and polygenic mechanisms and identifying tangible targets for gene therapies. Innovative strategies have emerged to rectify pathogenic variants that cause monogenic disorders such as hypertrophic, dilated, and arrhythmogenic cardiomyopathies and hypercholesterolemia. These include delivery of exogenous genes to supplement insufficient protein levels caused by pathogenic variants or genome editing to correct, delete, or modify mutant sequences to restore protein function. However, effective delivery of gene therapy to specified cells presents formidable challenges. Viral vectors, notably adeno-associated viruses and nonviral vectors such as lipid and engineered nanoparticles, offer distinct advantages and limitations. Additional risks and obstacles remain, including treatment durability, tissue-specific targeting, vector-associated adverse events, and off-target effects. Addressing these challenges is an ongoing imperative; several clinical gene therapy trials are underway, and many more first-in-human studies are anticipated. This science advisory reviews core concepts of gene therapy, key obstacles, patient risks, and ongoing research endeavors to enable clinicians to understand the complex landscape of this emerging therapy and its remarkable therapeutic potential to benefit cardiovascular disease.

Cationic lipid transfection induces nuclear actin filaments.

Cationic lipids are widely used for gene delivery. Here, we report the transient formation of nuclear actin filaments in mammalian cells transfected with commercially available transfection reagents regardless of the proteins transfected. Readily detectable with phalloidin, nuclear actin ranges from short filaments to a fully developed network. Nuclear actin filaments persist for hours, peak 20 h after transfection, and may be involved in DNA damage repair.

CNSC-22. GLIOBLASTOMA CELLS EXHIBIT NEURON-LIKE EXCITABILITY IN BOTH ACUTE AND ORGANOTYPIC HUMAN BRAIN SLICES

Abstract Glioblastomas (GBM) are known for their significant intratumor heterogeneity, featuring a variety of plastic cell types that make effective treatment challenging. Recent studies have shown that neuronal-progenitor-like transcriptomic cell states at the leading edge of the tumor receive synaptic input from nearby neurons, which drives disease proliferation. However, the excitability of GBM cells remains controversial, with observations ranging from non-excitable to neuron-like excitability, complicating our understanding of their pathophysiology. In this study, we developed a novel experimental approach to study glioblastoma cells in both acute and cultured brain slices infiltrated with cancer from GBM patients. Using an adeno-associated virus for gene delivery, we selectively expressed fluorescent proteins in specific cell types. Fluorescence-guided whole-cell patch-clamp recordings were then used to analyze the electrophysiological properties of GBM cells and neurons within the tumor microenvironment. Our findings show that GBM cells have distinct electrophysiological properties, including a depolarized resting membrane potential (-31.95 ± 2.18 mV, n = 55 cells) and high membrane resistance (1.27 ± 0.19 GΩ, n = 62 cells). Approximately 56% of GBM cells at the tumor’s leading edge exhibited neuron-like excitability, generating aberrant action potentials (aAPs) upon depolarization. Collectively, our study provides direct evidence of the intrinsic excitability of GBM cells and a detailed characterization of their electrophysiological properties in the cancer-infiltrated human cortex. These insights shed new light on tumor heterogeneity and cell-cell interactions in glioblastoma, potentially guiding the development of targeted therapies.

Preparation and characterization of calcium-doped graphene oxide-chitosan Nanocarrier to enhance the gene delivery in MCF-7 cell line

Gene delivery has emerged as a novel and effective method in the treatment of malignancies within medical interventions by applying nanotechnology. Consequently, the development of appropriate nanocarriers is a key focus of this research. Dynamic light scattering (DLS), fourier transform infrared (FT-IR) spectroscopy, x-ray diffraction (XRD), and thermal gravimetric analysis (TGA) were employed for the characterization of the synthesized nanocarrier. Furthermore, to assess the gene transfer capability of the nanocarrier, various techniques such as gel retardation assay, nuclease resistance assay, cytotoxicity assay, flow cytometry, and transfection were employed. The average particle size and zeta potential of the GO-CS@Ca nanocarrier were obtained as 319.8 nm and + 92.8 mv, respectively. In the gel retardation test, it was observed that pDNA was effectively condensed by the GO-CS@Ca nanocarrier. The results of the MTT assay indicated that both GO-CS@Ca nanocarrier and the GO-CS@Ca/pDNA nanoplex with low toxicity. In flow cytometry analysis, it was observed that the complexation of pDNA with the GO-CS@Ca nanocarrier resulted in effective gene delivery to the MCF-7 cell line and consequently increased apoptosis induction.

Nano Plasma Membrane Vesicle-Lipid Nanoparticle Hybrids for Enhanced Gene Delivery and Expression.

Lipid nanoparticles (LNPs) have emerged as the leading nonviral nucleic acid (NA) delivery system, gaining widespread attention for their use in COVID-19 vaccines. They are recognized for their efficient NA encapsulation, modifiability, and scalable production. However, LNPs face efficacy and potency limitations due to suboptimal intracellular processing, with endosomal escape efficiencies (ESE)below 2.5%. Additionally, up to 70% of NPs undergo recycling and exocytosis after cellular uptake. In contrast, cell-derived vesicles offer biocompatibility and high-delivery efficacy but are challenging to load with exogenous NAs and to manufacture at large-scale. To leverage the strengths of both systems, a hybrid system is designed by combining cell-derived vesicles, such as nano plasma membrane vesicles (nPMVs), with LNPs through microfluidic mixing and subsequent dialysis. These hybrids demonstrate up to tenfold increase in ESE and an 18-fold rise in reporter gene expression in vitro and in vivo in zebrafish larvae (ZFL)and mice, compared to traditional LNPs. These improvements are linked to their unique physico-chemical properties, composition, and morphology. By incorporating cell-derived vesicles, this strategy streamlines the development process, significantly enhancing the efficacy and potency of gene delivery systems without the need for extensive screening.

Engineering an Ultrasound-Responsive Glycopolymersome for Hepatocyte-Specific Gene Delivery.

The ability to design liver-targeted gene delivery vectors is plagued with difficulties ranging from carrier-mediated cellular toxicity to challenges in encapsulating sensitive nucleic acids. Herein, we present an ultrasound-responsive glycopolymersome strategy for in situ loading of nucleic acids and achieving hepatocyte-specific gene delivery. This glycopolymersome is self-assembled from a block copolymer, N-acetylgalactosamine-grafted poly(glutamic acid)-block-poly(ε-caprolactone) (PGAGalNAc-b-PCL). GalNAc is introduced to afford liver targeting through the selective binding to the asialoglycoprotein receptor overexpressed on hepatocytes. External ultrasound is utilized to assist in encapsulating nucleic acids within the hydrophilic lumen of glycopolymersomes by exploiting their ultrasound responsiveness nature. Biological studies confirmed the successful encapsulation of plasmid DNA (pDNA) and small interfering RNA (siRNA), rapid nuclear internalization, and efficient gene transfection. These findings collectively demonstrated that this ultrasound-responsive glycopolymersome could be exploited as a novel safe and efficient gene vector targeting hepatocytes.

Enhancing Gene Delivery in NB-4 Cells: Overcoming Transduction and Selection Challenges

Efficient gene transduction and cell viability are critical factors in genetic manipulation for research and therapeutic purposes. In this study, we explored the challenges associated with transducing the NB-4 cell line, a well-established model for acute promyelocytic leukemia (APL), using lentiviral vectors. While the initial transduction efficiency in NB-4 cells reached approximately 30%, we observed a significant decrease in cell viability, a phenomenon not observed in other acute leukemia cell lines such as THP-1 cells. We identified that this toxicity could be mitigated by purifying viral particles through ultracentrifugation or polyethylene glycol (PEG) precipitation, indicating that toxic substances, potentially secondary metabolites released by HEK293, could be responsible for the cell death. Nevertheless, cell selection by puromycin was still ineffective; crucially, we discovered that the human phosphoglycerate kinase (hPGK) promoter, commonly used in the PLKO1 vector, may become silenced in NB-4 cells, preventing effective selection with puromycin. By replacing the hPGK promoter with the elongation factor-1 alpha (EF1α) promoter, we successfully achieved high transduction efficiency and robust selection, demonstrating the potential for this modified vector system to facilitate genetic studies in APL models. These findings provide important insights into optimizing gene transduction protocols not only for NB-4 cells but also for other challenging cell lines, offering a refined approach for gene delivery and selection in cell models.

Advancements in Colloidosome Fabrication, Functionalization, and Applications: A Comprehensive Review

Abstract: Among the recent developments in the field of microcapsules, colloidosomes microcapsules made of colloidal particles have elicited much interest in promising perspectives in microencapsulation technologies. First reported by Dinsmore et al., these structures hold a lot of potential in different areas of application, which include gene delivery, targeting the brain, and tumor treatment. However, the challenges associated with their permeability and stability limit the use of these coatings. Nevertheless, these problems define colloidosomes as possessing some features of protocells that include metabolic activities and response to stimuli, which make them significant in synthetic biology and biomimetic systems. In drug delivery, colloidosomes have the potential of providing solubility, stability, and a controlled rate of drug release, which will have an impact on the therapeutic use of drugs. Fabrication of colloidosomes is done in many ways, and the common ones include the mulsion strategy, layer adsorption, and microfluidics strategy. Some of these fabrication techniques have been investigated in the recent past to determine how their application can lead to enhanced colloidosome characteristics. Some of these methods include spray drying, sol-gel processes, and electrostatic assembly. For example, the study by Payizila Zulipiker et al. showed that different levels of permeability are controllable through the use of spray drying technology. Another study by Jia Jia et al. presented the preparation of pH-responsive colloidosomes and the high efficiency of encapsulation. Consequently, improvements in the manufacturing process and new self-assembly technologies that include microfluidics systems have also improved the creation of colloidosomes. New advancements in the field of colloidosome synthesis, as well as their surface modification, will open up numerous application prospects in the fields of pharmaceutics, regenerative medicine, and bioinspired materials science. New trends range from 3D printing to stimuli-responsive materials and design, as well as hybrid systems that are application- specific. Despite the difficulties that remain to be faced, colloidosomes represent an extraordinary opportunity for the growth of biotechnology and materials science to open the path to new generations of drugs and bioinspired applications.

Multifunctional Neural Probes Enable Bidirectional Electrical, Optical, and Chemical Recording and Stimulation In Vivo.

Recording and modulation of neuronal activity enables the study of brain function in health and disease. While translational neuroscience relies on electrical recording and modulation techniques, mechanistic studies in rodent models leverage genetic precision of optical methods, such as optogenetics and fluorescent indicator imaging. In addition to electrical signal transduction, neurons produce and receive diverse chemical signals which motivate tools to probe and modulate neurochemistry. Although the past decade has delivered a wealth of technologies for electrophysiology, optogenetics, chemical sensing, and optical recording, combining these modalities within a single platform remains challenging. This work leverages materials selection and convergence fiber drawing to permit neural recording, electrical stimulation, optogenetics, fiber photometry, drug and gene delivery, and voltammetric recording of neurotransmitters within individual fibers. Composed of polymers and non-magnetic carbon-based conductors, these fibers are compatible with magnetic resonance imaging, enabling concurrent stimulation and whole-brain monitoring. Their utility is demonstrated in studies of the mesolimbic reward pathway by interfacing with the ventral tegmental area and nucleus accumbens in mice and characterizing the neurophysiological effects of a stimulant drug. This study highlights the potential of these fibers to probe electrical, optical, and chemical signaling across multiple brain regions in both mechanistic and translational studies.

Identifying Multiomic Signatures of X-Linked Retinoschisis-Derived Retinal Organoids and Mice Harboring Patient-Specific Mutation Using Spatiotemporal Single-Cell Transcriptomics.

X-linked retinoschisis (XLRS) is an inherited retinal disorder with severe retinoschisis and visual impairments. Multiomics approaches integrate single-cell RNA-sequencing (scRNA-seq) and spatiotemporal transcriptomics (ST) offering potential for dissecting transcriptional networks and revealing cell-cell interactions involved in biomolecular pathomechanisms. Herein, a multimodal approach is demonstrated combining high-throughput scRNA-seq and ST to elucidate XLRS-specific transcriptomic signatures in two XLRS-like models with retinal splitting phenotypes, including genetically engineered (Rs1emR209C) mice and patient-derived retinal organoids harboring the same patient-specific p.R209C mutation. Through multiomics transcriptomic analysis, the endoplasmic reticulum (ER) stress/eukryotic initiation factor 2 (eIF2) signaling, mTOR pathway, and the regulation of eIF4 and p70S6K pathways are identified as chronically enriched and highly conserved disease pathways between two XLRS-like models. Western blots and proteomics analysis validate the occurrence of unfolded protein responses, chronic eIF2α signaling activation, and chronic ER stress-induced apoptosis. Furthermore, therapeutic targeting of the chronic ER stress/eIF2α pathway activation synergistically enhances the efficacy of AAV-mediated RS1 gene delivery, ultimately improving bipolar cell integrity, postsynaptic transmission, disorganized retinal architecture, and electrophysiological responses. Collectively, the complex transcriptomic signatures obtained from Rs1emR209C mice and patient-derived retinal organoids using the multiomics approach provide opportunities to unravel potential therapeutic targets for incurable retinal diseases, such as XLRS.

Gene Delivery via Octadecylamine-Based Nanoparticles for iPSC Generation from CCD1072-SK Fibroblast Cells

This study presents a novel biotechnological approach using octadecylamine-based solid lipid nanoparticles (OCTNPs) for the first-time reprogramming of human CCD1072-SK fibroblast cells into induced pluripotent stem cells (iPSCs). OCTNPs, with an average size of 178.9 nm and a positive zeta potential of 22.8 mV, were synthesized, thoroughly characterized, and utilized as a non-viral vector to efficiently deliver reprogramming factors, achieving a remarkable transfection efficiency of 82.0%. iPSCs were characterized through immunofluorescence, flow cytometry, and RT-qPCR, confirming the expression of key pluripotency markers such as OCT4, SOX2, and KLF4, with alkaline phosphatase activity further validating their pluripotent state. Following this comprehensive characterization, the iPSCs were successfully differentiated into cardiomyocyte-like cells using 5-azacytidine. Our research highlights the innovative application of OCTNPs as a safe and effective alternative to viral vectors, addressing key limitations of iPSC reprogramming. The novel application of OCTNPs for efficient gene delivery demonstrates a powerful tool for advancing stem cell technologies, minimizing risks associated with viral vectors. These findings pave the way for further innovations in biotechnological applications, particularly in tissue engineering and personalized medicine.

Self-Healing Hyaluronic Acid-based Hydrogel with miRNA140-5p Loaded MON-PEI Nanoparticles for Chondrocyte Regeneration: Schiff Base Self-Assembly Approach.

Articular cartilage defects present a significant therapeutic challenge due to the inherent avascular and aneural characteristics of cartilage tissue. Gene therapy has emerged as a promising strategy for cartilage regeneration, particularly through the use of functional RNA and biomaterial-assisted frameworks. In this study, an innovative gene-activated self-healing hydrogel is developed and fabricated for the controlled release of miR140-5p, a key regulator of cartilage regeneration. The hydrogel, crosslinked via UV radiation, is composed of aminated hyaluronic acid and a modified photosensitizer (NB). To enhance the scaffold's structural integrity and gene delivery efficiency, mineralized silk fibroin and miR140-5p-loaded MON-PEI nanoparticles are incorporated. These findings demonstrate that this novel hydrogel (miR140-5p-CaP@mSF-HA-NB) effectively encapsulates and releases miR140-5p, exhibits excellent biocompatibility, and promotes enhanced cartilage regeneration in both in vitro and in vivo models. Therefore, this gene-activated hydrogel holds significant potential for clinical applications in the treatment of articular cartilage defects.