1. Bibliographic Information

1.1. Title

Virus-inspired lipopeptide-derived nucleic acid delivery to cartilage for osteoarthritis therapy

1.2. Authors

Yu Fu, Yulan Huang, Yunjiao Wang, Zhenlan Fu, Wenyun Cai, Lu Wang, Yuchun Wu, Xing Zhou, Zhongyi Ma, Zhigang Xu, Yaqin Tang, Jing Xie, Jiayun Jiang, Robert J. Lee, Chong Li

The authorship includes a diverse group of researchers, indicating interdisciplinary collaboration. The presence of Robert J. Lee, a known expert in drug delivery systems, particularly lipid-based nanoparticles, and Chong Li, with affiliations to Southwest University and the State Key Laboratory of Molecular Engineering of Polymers, suggests expertise in nanomedicine, biomaterials, and therapeutic applications. Yu Fu is listed as the first author and likely contributed significantly to the experimental design and execution.

1.3. Journal/Conference

Published online: 16 October 2025 in Nature Communications.

Nature Communications is a highly reputable, peer-reviewed, open-access scientific journal published by Nature Portfolio (part of Springer Nature). It covers a wide range of natural sciences, including biology, chemistry, physics, and earth sciences. Its high impact factor and broad scope make it a prestigious venue for publishing significant research, suggesting the paper has undergone rigorous peer review and presents impactful findings.

1.4. Publication Year

2025

1.5. Abstract

The paper addresses the critical need for effective delivery vectors in cartilage-targeted gene therapy for osteoarthritis (OA). It introduces a modularly developed series of non-pathogenic, virus-inspired lipopeptide-based nanoparticles (VPN). These VPNs are designed for efficient nucleic acid delivery to cartilage. The optimized variant, VPN-2, featuring a specific cationic moiety with arginine and histidine residues, demonstrates a 2.5-fold improvement in transfection potency over conventional lipid nanoparticles (LNP) by facilitating sufficient endocytosis and effective lysosomal escape. To overcome the challenges of penetration and retention within articular cartilage, si-VPN-2 is formulated into a ROS-responsive nano-in-gel system. This system successfully alleviates cartilage degeneration in surgically induced anterior cruciate ligament transection (ACTL) mice and exhibits synergistic protective effects with methylprednisolone in post-traumatic osteoarthritis (PTOA) mice. The study highlights the significant potential of the VPN platform as a cartilage-targeted RNA delivery vector for innovative OA therapy.

1.6. Original Source Link

/files/papers/691763e0110b75dcc59ae089/paper.pdf (local link provided in the prompt), Published at (UTC): 2025-10-16T00:00:00.000Z. The paper is officially published and accessible via the provided PDF link.

2. Executive Summary

2.1. Background & Motivation

Osteoarthritis (OA) is a debilitating joint disease characterized by progressive degradation of articular cartilage. Current pharmaceutical treatments for OA are primarily palliative, meaning they only offer temporary relief from symptoms like inflammation and pain, without decelerating or reversing cartilage degeneration. This lack of disease-modifying OA drugs (DMOADs) represents a significant unmet medical need, as highlighted by the FDA.

A key factor in OA progression is the matrix metalloproteinase 13 (MMP-13) enzyme, which potently degrades type II collagen (Col2), a critical structural component of cartilage. Inhibiting MMP-13 is a promising therapeutic strategy. However, conventional small molecule inhibitors of MMP-13 have faced challenges in clinical translation due to safety concerns related to low selectivity, often causing off-target effects.

siRNA-based gene therapy offers an alternative by selectively silencing MMP-13 gene expression through a sequence-specific mechanism, potentially circumventing the selectivity issues of small molecule drugs. However, nucleic acids like siRNA and mRNA are intrinsically unstable and require efficient and safe delivery vectors to reach their target sites within cells.

Traditional viral vectors (e.g., adeno- and retroviruses) are highly effective at gene transport but carry inherent risks of immunogenicity and pathogenicity. Non-viral delivery vehicles, such as ionizable lipids formulated lipid nanoparticles (LNP), offer a safer and easier-to-manufacture alternative. While LNPs have seen success in liver diseases and vaccines, their application in extrahepatic tissues (tissues outside the liver) like cartilage is limited by insufficient transfection competency – the ability to deliver nucleic acids into cells and enable their function.

The core problem the paper aims to solve is the development of an effective, safe, and cartilage-targeted delivery system for nucleic acids (like siRNA and mRNA) to treat OA. This system needs to overcome the instability of nucleic acids, the limitations of viral vectors (safety), and the restricted transfection efficiency of non-viral vectors in cartilage, while also addressing the unique challenges of intra-articular delivery (balancing penetration and retention within dense cartilage).

2.2. Main Contributions / Findings

The paper makes several significant contributions to the field of OA gene therapy:

• Development of Virus-Inspired Lipopeptide-Based Nanoparticles (VPN): The authors modularly designed a novel series of non-pathogenic VPNs by combining a Col2-targeting peptide (WYRGRL), a cationic moiety with variable arginine and histidine residues (inspired by cell-penetrating peptides from viruses), and a hydrophobic tail. This rational design allows for flexible modulation of VPN properties, particularly transfection efficiency.

• Optimization of VPN-2 for Superior Transfection: Through systematic screening, VPN-2, with a cationic moiety of $$- [ ( \mathbf { R } ) _ { 5 } - ( \mathbf { H } ) _ { 4 } ] _ { 2 } -$$, was identified as the optimal formulation. It demonstrated superior endocytosis and potent lysosomal escape (likely via the proton sponge effect from histidine residues under acidic conditions), achieving approximately a 2.5-fold improvement in transfection potency over conventional MC3-LNP.

• pH-Sensitive and Cartilage-Targeted Delivery: The VPNs exhibit pH-sensitive abilities, with increased surface charge and enhanced cellular uptake under acidic conditions (relevant to the OA joint environment). The WYRGRL targeting head confers Col2-dependent cartilage-targeted ability, significantly enhancing uptake in Col2-expressing cells and cartilage.

• ROS-Responsive Nano-in-Gel System (si-VPN@HA) for Balanced Delivery: To address the tradeoff between penetration and retention in cartilage, si-VPN-2 was encapsulated within a ROS-responsive hyaluronic acid hydrogel (HA@gel). This nano-in-gel system allows for sustained release of si-VPN-2 specifically under high reactive oxygen species (ROS) conditions, typical of OA joints, enabling deep penetration while maintaining prolonged intra-articular retention (over 21 days).

• Effective Alleviation of Cartilage Degeneration in ACTL Mice: The si-VPN@HA system significantly alleviated cartilage degeneration, reduced inflammation, and relieved pain in a surgical ACTL mouse model. It achieved over 75% MMP-13 silencing in vivo and modulated macrophage polarization towards an anti-inflammatory M2 phenotype.

• Synergistic Joint Protection in PTOA Mice: In a post-traumatic osteoarthritis (PTOA) mouse model, co-loading si-VPN-2 with methylprednisolone (MP) into the HA@gel (MP&si-VPN@HA) demonstrated a superior synergistic effect in joint protection, showing better preservation of walking speed, reduced osteophyte formation, and improved subchondral bone microarchitecture compared to individual treatments.

• Versatility for mRNA Delivery: The VPN platform also proved functionally capable of intra-articular mRNA delivery, with m-VPN-2 showing significantly higher eGFP expression in vitro and in vivo compared to m-MC3-LNP.

  In summary, the paper provides a novel, tunable, and highly effective non-viral gene delivery platform that overcomes key challenges in OA therapy, demonstrating strong therapeutic potential in relevant animal models.

3. Prerequisite Knowledge & Related Work

3.1. Foundational Concepts

• Osteoarthritis (OA): A chronic, progressive joint disease characterized by the breakdown of articular cartilage, leading to pain, stiffness, and loss of joint function. It is the most common form of arthritis.
• Articular Cartilage: The smooth, slippery tissue that covers the ends of bones in a joint. It provides a low-friction surface for movement and acts as a shock absorber. It is an avascular (lacks blood vessels) and aneural (lacks nerves) tissue, making self-repair difficult. Its extracellular matrix (ECM) is dense and highly anionic.
• Gene Therapy: A therapeutic approach that involves introducing genetic material (like DNA, RNA) into a patient's cells to treat or prevent disease. In the context of OA, it aims to deliver genes that can protect cartilage, reduce inflammation, or promote regeneration.
• Nucleic Acids: The genetic material of living organisms. In this paper, two types are primarily discussed:
  • Small interfering RNA (siRNA): Short, double-stranded RNA molecules that silence (turn off) specific genes by degrading messenger RNA (mRNA) molecules before they can be translated into proteins. This is a sequence-specific mechanism.
  • Messenger RNA (mRNA): A single-stranded RNA molecule that carries genetic information from DNA to the ribosomes, where it serves as a template for protein synthesis. mRNA delivery aims to transiently express a therapeutic protein.
• Matrix Metalloproteinase 13 (MMP-13): An enzyme (a type of collagenase) that plays a critical role in the degradation of type II collagen in cartilage. Its overexpression is strongly associated with OA progression.
• Type II Collagen (Col2): The primary structural protein found in articular cartilage, responsible for its tensile strength and integrity.
• Lipopeptides: Molecules composed of a peptide (a short chain of amino acids) covalently linked to a lipid (fatty acid) chain. The lipid tail provides hydrophobicity (water-fearing) and allows for self-assembly, while the peptide portion can be designed for specific functions like targeting or cell penetration.
• Nanoparticles (NPs): Particles with dimensions typically between 1 and 100 nanometers. In drug delivery, nanoparticles can encapsulate therapeutic agents, protect them from degradation, and facilitate their targeted delivery to specific tissues or cells.
• Lipid Nanoparticles (LNP): A type of nanoparticle composed of lipids that encapsulate nucleic acids. Ionizable lipids are a key component, allowing the LNP to be positively charged under acidic conditions (for complexing with negatively charged nucleic acids) and more neutral at physiological pH (for reduced toxicity). MC3-LNP (DLin-MC3-DMA formulated LNP) is a commonly used benchmark LNP.
• Cell-Penetrating Peptides (CPPs): Short sequences of amino acids that can facilitate the cellular uptake of various cargo molecules, including nucleic acids, proteins, and nanoparticles. They are often rich in positively charged amino acids like arginine and lysine, which interact with the negatively charged cell membrane.
  • Arginine-rich CPPs: Many effective CPPs, such as TAT (derived from HIV) and VP22 (derived from herpes simplex virus), contain multiple arginine residues. These impart a positive charge, enabling electrostatic interactions with the cell membrane.
  • Polyhistidine-fused CPPs: The addition of histidine residues to CPPs (e.g., TAT-10H) can enhance their endosomolytic capacity. Histidine has an imidazole group with a pKa of approximately 6.0. Under the slightly acidic conditions of endosomes (pH 5.0-6.0), the histidine residues become protonated and positively charged. This protonation leads to an influx of protons and counter-ions, causing endosomal swelling and rupture, a process known as the proton sponge effect, which facilitates lysosomal escape.
• Endocytosis: The process by which cells engulf external substances by internalizing them in a vesicle (a small sac-like structure) formed from the cell membrane. Common pathways include macropinocytosis and clathrin-dependent endocytosis.
• Lysosomal Escape: After endocytosis, nanoparticles are often trapped in endosomes and subsequently delivered to lysosomes, which contain enzymes that degrade cargo. Lysosomal escape is crucial for nucleic acids to reach the cytoplasm and exert their therapeutic effect, as degradation in lysosomes would render them ineffective.
• Reactive Oxygen Species (ROS): Highly reactive molecules containing oxygen, such as hydrogen peroxide ($\mathrm{H_2O_2}$). Elevated ROS levels are often found in inflamed tissues, including OA joints, and contribute to pathology.
• Hyaluronic Acid (HA): A naturally occurring polysaccharide found in connective tissues, including cartilage and synovial fluid. It is biocompatible and biodegradable, making it suitable for biomedical applications.
• Hydrogels: Three-dimensional networks of hydrophilic polymers that can absorb large amounts of water. They are often used as drug delivery vehicles due to their biocompatibility and ability to control drug release.
• Host-Guest Interaction: A type of non-covalent molecular recognition where a "host" molecule forms a complex with a "guest" molecule. In this paper, beta-cyclodextrin (\beta-CD) (host) and ferrocene (Fc) (guest) are used.
  • $\beta$-cyclodextrin ($\beta$-CD): A cyclic oligosaccharide with a hydrophilic exterior and a hydrophobic interior cavity, capable of forming host-guest inclusion complexes with suitable "guest" molecules.
  • Ferrocene (Fc): An organometallic compound that can act as a "guest" molecule for

\beta`-CD`. `Fc` can be oxidized by `ROS` ($\mathrm{H_2O_2}$), leading to a change in its ability to bind

\beta-CD, thus enabling ROS-responsive dissociation.

3.2. Previous Works

• Viral vs. Non-Viral Vectors for Gene Delivery:

  • Viral Vectors (e.g., Adenoviruses, Retroviruses): Historically, viral vectors have been highly efficient in transfecting cells (delivering genetic material). They exploit the natural mechanisms of viruses to enter cells and deliver their genetic payload. However, their clinical use is limited by concerns regarding immunogenicity (triggering an immune response) and pathogenicity (causing disease).
  • Non-Viral Vectors (e.g., LNP): Developed as safer and easier-to-manufacture alternatives to viral vectors. Lipid nanoparticles (LNP) are a prominent example, having achieved clinical success for siRNA therapeutics (e.g., patisiran for hereditary transthyretin-mediated amyloidosis) and mRNA vaccines (e.g., COVID-19 vaccines). However, their transfection competency in extrahepatic tissues (like cartilage) remains limited, often requiring high doses or local administration.

• Virus-Inspired Delivery Systems: Recognizing the strengths and limitations of both viral and non-viral approaches, researchers have sought to create virus-resembling, non-pathogenic carriers. This involves incorporating elements of viral biology into non-viral systems to enhance transfection efficiency while maintaining safety.

  • Genetically Engineered Viral Proteins: Some strategies involve integrating genetically engineered viral proteins into LNPs to improve membrane fusion activity and thus transfection efficiency.
  • Virus-Derived Peptides: Virus-derived peptides are another avenue. Cell-penetrating peptides (CPPs) with arginine-rich residues, such as TAT (from HIV) and VP22 (from herpes simplex virus), are known for their ability to condense genetic materials and facilitate intracellular delivery.
  • Polyhistidine-Fused CPPs: Further enhancements have been achieved by fusing polyhistidine segments to CPPs (e.g., TAT-10H). The histidine imidazole group ($\mathrm{pKa}$ of 6.0) can protonate under acidic conditions found in endosomes, leading to endosomolytic capacity via the proton sponge effect, which boosts lysosomal escape and gene transfection.

• Cartilage-Targeted Delivery Challenges:

  • Avascular and Dense ECM: Cartilage's avascular nature and dense, anionic extracellular matrix (ECM) make it challenging for therapeutic agents to penetrate and remain localized.
  • Penetration vs. Retention Dilemma: Small delivery vectors might penetrate cartilage effectively but suffer rapid clearance from the synovial fluid. Larger carriers can achieve better retention but struggle to penetrate deep into the cartilage tissue to reach chondrocytes (the only resident cells in cartilage). Strategies like biofunctional polymer nanoparticles and microplates for prolonged intra-articular delivery have been explored to address this.

3.3. Technological Evolution

The field of gene therapy vectors has evolved from:

1. Early Viral Vectors: High efficiency, but safety concerns (immunogenicity, pathogenicity).

2. First-Generation Non-Viral Vectors (e.g., Cationic Liposomes, Polymers): Improved safety, but lower efficiency and issues with stability and targeting.

3. Advanced Non-Viral Vectors (e.g., Ionizable LNPs): Significant improvements in safety and efficiency, leading to clinical translation for specific diseases (e.g., liver-targeted siRNA, mRNA vaccines). However, tissue-specific delivery, especially to extrahepatic tissues like cartilage, remains a challenge.

4. Virus-Inspired Hybrid Systems: The current frontier, seeking to combine the high efficiency of viral entry/escape mechanisms with the safety and modularity of non-viral systems. This often involves incorporating specific viral components (like CPPs or viral proteins) into non-viral nanoparticles.

   This paper's work fits squarely into the fourth stage of this evolution. It leverages insights from virus-derived CPPs to design modular lipopeptides that self-assemble into nanoparticles for cartilage-targeted RNA delivery, aiming to overcome the transfection efficiency and tissue-specific delivery limitations of previous non-viral approaches.

3.4. Differentiation Analysis

The core innovations and differences of this paper's VPN approach compared to existing methods lie in:

• Modular, Virus-Inspired Design: Unlike conventional LNPs that rely on ionizable lipids for nucleic acid condensation, VPNs are built from lipopeptides with distinct functional moieties: a Col2-targeting head (WYRGRL), a cationic moiety with variable arginine and histidine residues (directly inspired by virally-derived CPPs for enhanced endocytosis and lysosomal escape), and a hydrophobic moiety. This modularity allows for precise tuning of biological properties.
• Enhanced Transfection Potency via Arginine/Histidine Optimization: The systematic screening of arginine and histidine combinations led to the identification of VPN-2 as an optimal vector. The specific arrangement $$- [ ( \mathbf { R } ) _ { 5 } - ( \mathbf { H } ) _ { 4 } ] _ { 2 } -$$ is crucial for balancing nucleic acid condensation, cellular uptake, and potent lysosomal escape through the proton sponge effect (driven by histidine protonation in acidic endosomes). This results in a 2.5-fold improvement over MC3-LNP, which typically has less efficient lysosomal escape in non-liver tissues.
• Cartilage-Specific Targeting: The incorporation of the WYRGRL Col2-targeting peptide provides active targeting to cartilage, enhancing cellular uptake in chondrocytes compared to non-modified counterparts. This is a significant advantage over untargeted LNPs.
• pH-Sensitive Properties: The histidine residues also confer pH-sensitive behavior to VPNs, leading to increased surface charge and boosted cellular uptake/penetration under the mildly acidic conditions found in OA joints (pH 6.8 or lower).
• ROS-Responsive Nano-in-Gel System: This is a novel strategy to resolve the penetration-retention dilemma in cartilage delivery. By encapsulating si-VPN-2 in a hyaluronic acid (HA) hydrogel that is ROS-responsive (dissolving in the presence of high H2O2 in OA joints), the system achieves long-term intra-articular retention while allowing si-VPN-2 to be released and penetrate the cartilage when therapeutic action is needed. Conventional LNPs would either be rapidly cleared or remain localized on the surface if too large.
• Broad Gene Expression Effects: The MMP-13 silencing by si-VPN-2 is shown to broadly affect gene expression profiles in OA chondrocytes, impacting various OA-related pathways (e.g., inflammation, cytokine-cytokine receptor interaction, PI3K-Akt, MAPK), suggesting a more comprehensive therapeutic effect than just MMP-13 inhibition.
• Synergy with Standard-of-Care: The demonstration of synergy between si-VPN-2 and methylprednisolone (a clinical standard steroid) in the PTOA model highlights its potential for combination therapies, which is often crucial in complex diseases.
• Versatility for both siRNA and mRNA Delivery: The VPN platform's ability to efficiently deliver both siRNA (for gene silencing) and mRNA (for protein expression) expands its therapeutic applicability for OA.

4. Methodology

4.1. Principles

The core idea behind the proposed methodology is to design virus-inspired lipopeptide-based nanoparticles (VPN) that mimic the efficient nucleic acid delivery mechanisms of viruses while avoiding their pathogenic downsides. This is achieved through a modular design of lipopeptides with three key functional moieties:

1. Cartilage Targeting Head (WYRGRL peptide): To specifically bind to type II collagen (Col2) within the cartilage extracellular matrix (ECM), ensuring localized delivery.

2. Cationic Moiety (Arginine and Histidine residues): Inspired by virally-derived cell-penetrating peptides (CPPs), this segment is rich in positively charged arginine and pH-sensitive histidine residues. Arginine facilitates strong electrostatic interaction with negatively charged nucleic acids for condensation and promotes endocytosis. Histidine residues, with their pKa around 6.0, become protonated in the acidic endosomal environment, leading to the proton sponge effect which mediates lysosomal escape and releases nucleic acids into the cytoplasm.

3. Hydrophobic Moiety (Docosanoic acid, $\mathrm{C_{22}}$ tail): To provide the amphiphilic property necessary for lipopeptide self-assembly into nanoparticles.

   These lipopeptides spontaneously self-assemble into unilamellar vesicle nanostructures. Upon packaging nucleic acids, they undergo a structural transformation into solid spherical nanoparticles, resembling the transition from liposomes to LNPs. To address the penetration-retention dilemma in intra-articular delivery (small carriers penetrate but clear quickly; large carriers retain but don't penetrate), the si-VPNs are further encapsulated into a ROS-responsive hyaluronic acid hydrogel (HA@gel). This hydrogel is designed to degrade and release the si-VPNs specifically in the ROS-rich microenvironment of OA joints, thus ensuring both prolonged retention and subsequent deep cartilage penetration.

4.2. Core Methodology In-depth (Layer by Layer)

4.2.1. Synthesis and Characterization of Lipopeptides

The lipopeptides are synthesized using a solid-phase peptide synthesis (SPPS) protocol.

• Solid Phase: Wang-resin is used as the solid support.
• Activators: N,N'-methanediylidenebis (DIC) and ethyl cyanohydroxyiminoacetate (Oxyma) are used as the activator and activator base, respectively, to facilitate peptide bond formation.
• Hydrophobic Moiety Attachment: Docosanoic acid (\mathrm{C_{22}}) is coupled to the peptides before deprotection and cleaving from the resin. This forms the hydrophobic tail.
• Deprotection and Cleavage: The lipopeptides are cleaved from the resin and protective groups are removed by incubation in a solution of trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5) at room temperature.
• Purification and Characterization: Crude lipopeptide products are purified using reverse-phase HPLC and characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to confirm molecular weights and sequences. The paper describes various lipopeptide designs (1-12, Supplementary Table 1) differing in their cationic moiety and hydrophobic tail configurations. For example, lipopeptides 1-3 have the WYRGRL targeting head and two $\mathrm{C_{22}}$ tails, but different cationic moieties:
  • Lipopeptide 1:

- ( \mathbf { R } ) _ { 5 } \mathsf { - } ( \mathbf { H } ) _ { 4 } \mathsf { - }
    *   `Lipopeptide 2`:
```
- [ ( \mathbf { R } ) _ { 5 } - ( \mathbf { H } ) _ { 4 } ] _ { 2 } -
```
*   `Lipopeptide 3`:
```
- ( \mathbf { R } ) _ { 1 0 } - ( \mathbf { H } ) _ { 8 } -
```
### 4.2.2. Construction and Characterization of VPNs and si-VPNs
*   **VPN Preparation**: `VPNs` are prepared by `self-assembly`. `3 mg` of `lipopeptides` (dissolved in `60 µL DMSO`) are slowly added into `2 mL DEPC water` and stirred (`400 g`) for `10 min`.
*   **Morphological Characterization**: `VPNs` morphologies are assessed using `transmission electron microscope (TEM)` and `field emission scanning electron microscopy (FE-SEM)`. Initial `VPNs` show `unilamellar vesicle structures`.
    The following are representative TEM images of VPN-1, VPN-2, VPN-3, and corresponding si-VPN-1, si-VPN-2, si-VPN-3, scale bar = 50 µm; and HR-TEM and FE-SEM images of VPN-2, scale bar = 50 µm.

    ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
    *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

*   **pH-Sensitivity Evaluation**: `VPNs` are incubated in `0.1 M PBS` at different pH values (7.4, 6.8, or 5.5) at `37°C`. Changes in `size` and `zeta potential` are monitored over time using a `particle size analyzer`. The expectation is an increase in positive surface charge and potentially size under acidic conditions due to `histidine` protonation.
    The zeta potential of VPN-2 under pH 7.4, pH 6.8, or pH 5.5 at different time points shows an increase in positive charge as pH decreases, indicating pH-sensitive behavior.

    ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
    *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

*   **si-VPN Preparation**: `siRNA-loaded VPNs (si-VPNs)` are prepared using a `microfluidic mixer`. Equal volumes of `VPNs` and `siRNA solution` (with appropriate concentration) are mixed. The mixture is stirred, then left for `2 h`.
    *   **N:P Ratio Optimization**: The `optimal nitrogen-to-phosphorus (N:P) ratio` (molar ratio of cationic amino groups from `lipopeptide` to phosphate groups from `siRNA`) for `siRNA condensation` is determined using `agarose gel electrophoresis`. A higher `N:P ratio` typically ensures better condensation and protection of `siRNA`.
        The siRNA loading efficacy of si-VPN-1, si-VPN-2 or si-VPN-3 at various N:P ratios by agarose gel electrophoresis shows complete retardation for si-VPN-2 and si-VPN-3 at N:P = 6:1.

        ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
        *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

    *   **Morphological Transition**: Upon `siRNA` packaging, `si-VPNs` transform into `solid spherical nanoparticles` from `unilamellar vesicles`, attributed to structural rearrangement due to charge interactions.
        The HAADF-STEM images of si-VPN-2 and the corresponding element mapping images of C, N, O, and P, scale bar = 50 µm, show uniform dispersion of siRNA within si-VPN-2.

        ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
        *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

*   **RNase Protection Assay**: `si-VPN formulations` are incubated with various `RNase cocktail enzyme mix`, `RNase A`, and `RNase I` at `37°C` for different durations. Residual `siRNA` is then displaced by `1% sodium dodecyl sulfate (SDS)`, and `electrophoresis` is used to visualize the protected `siRNA`. Free `siRNA` is used as a control. This assesses the `nanoparticle`'s ability to protect `siRNA` from enzymatic degradation.
    Stability of si-VPN-1, si-VPN-2 or si-VPN-3 against RNase cocktail, employing free siRNA as comparison, demonstrates excellent protection of siRNA by VPNs.

    ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
    *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

*   **Stability Testing**: `si-VPN-1, si-VPN-2, si-VPN-3` are incubated in `0.1 M PBS` at `4°C`. Changes in `size` and `zeta potential` are monitored. After `48 h`, `gel electrophoresis` (with and without `SDS`) is performed to check `siRNA` encapsulation integrity.
*   **MC3-LNP Preparation (Benchmark)**: A mixture of `DLin-MC3-DMA` (ionizable lipid), `DSPC` (helper lipid), `cholesterol`, and `DMG-PEG2000` (PEGylated lipid) at a molar ratio of `50:10:38.5:1.5` is dissolved in an organic phase. This is mixed with an acetate buffer (pH 4.0) containing `siRNA` or `mRNA` using a `microfluidic mixer`. The resulting `LNPs` are purified by `dialysis`.

### 4.2.3. Dissipative Particle Dynamics (DPD) Simulation
`Mesoscale dissipative particle dynamics (DPD)` simulations are employed to model the structural characteristics and assembly dynamics of `si-VPNs`.
*   **Coarse-Graining**: `siRNA` molecules are `coarse-grained` into beads representing phosphate (P), sugar (S), and bases (A, T, C, G, U). `Lipopeptides` are `coarse-grained` with `docosanoic acid (`\mathrm{C_{22}}`)` as `l1 D beads` and `peptide backbone` as `PB beads`, with side chains as `l2 beads`.
*   **Interaction Parameters**: The `blend module` in `Materials Studio 2016` and the `COMPASS force field` are used to calculate mixing energies between beads, which are then used to derive `DPD interaction parameters`.
*   **Simulation Setup**: `Coarse-grained` molecular units of `siRNA`, `lipopeptides`, and water are randomly distributed. `DPD force field` is constructed, and initial conformations are optimized.
*   **Dynamics Simulation**: A long-term `200 ns` simulation is conducted to analyze the stability and `self-assembly` process. The simulations indicate `si-VPNs` form solid spherical architectures with `lipopeptides-siRNA complexes` concentrated in the core, surrounded by a shell of `peptide backbones` and `hydrophilic groups`.
    DPD simulation snapshots depicting the front (upper) and cross-section view (bottom) of si-VPN-2 to illustrate the bead distribution and overall architectures. scale bar = 10 nm. Snapshots depicting the assembly dynamic of si-VPN-2 from a homogeneous mixture in long-term 200 ns dynamic simulation trajectory.

    ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
    *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

### 4.2.4. Fabrication and Characterization of HA@gel and si-VPN@HA
*   **ROS-Responsive HA@gel Construction**: The `hydrogel` is constructed based on `host-guest interaction` between
```
\beta`-cyclodextrin (`\beta`-CD)` (host) and `ferrocene (Fc)` (guest), both decorated onto a `hyaluronic acid (HA)` backbone.
    *   **HA-CD and HA-Fc Synthesis**: 
```
\beta`-CD` and `Fc` are conjugated to `HA` via `amidation reaction`.
    *   **Hydrogel Formation**: Equal volumes of `HA-CD` and `HA-Fc` solutions (1:1 wt%/wt%$) are mixed. The `host-guest interactions` lead to `crosslinking` and `hydrogel` formation.
        The schematic construction of ROS-responsive nano-in-gel system siVPN@HA.

        ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
        *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

*   **Rheological Properties**: The `storage modulus (G')` and `loss modulus (G'')` of `HA@gel` are measured using a `rotary rheometer` under stimulation with `0.1 mM` or `5.0 mM`\mathrm{H_2O_2}
```
. This assesses the `ROS-sensitive gel-sol transition`. `Thixotropic` experiments (cyclic stress `0.2    Rheology properties of HA@gel when stimulated with 0.1 or 5 mM$\mathrm{H_2O_2}$.

    ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
    *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

*   **Morphological Characterization of HA@gel**: The 3D network morphology of `HA@gel` is characterized using `SEM`.
    The representative photographs of gel-sol transition of HA@gel when stimulated with$\mathrm{H_2O_2}$(upper row), and the corresponding representative SEM images (bottom row), scale bar = 20 µm.

    ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
    *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

*   **si-VPN@HA Fabrication**: `25 µL` of `si-VPN-2 solution` and `25 µL` of `10 mg/mL HA-CD` are mixed (Mixture 1). Separately, `25 µL` of `si-VPN-2 solution` and `25 µL` of `10 mg/mL HA-Fc` are mixed (Mixture 2). Finally, Mixture 1 and Mixture 2 are combined to form `si-VPN@HA`.
*   **Release Kinetics**: The release of `Cy5-labeled siRNA` from `si-VPN-2`, `siRNA@HA` (free `siRNA` in `HA@gel`), and `si-VPN@HA` is investigated in `5 mM`\mathrm{H_2O_2}
```
solutions by detecting fluorescence intensity at `664 nm` using a `fluorescence spectrometer`. The morphologies of released `si-VPN-2` are checked by `TEM`.
    The in vitro release profiles of different formulations in 5 mM$\mathrm{H_2O_2}$solutions ($n=3 independent samples).

    ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
    *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

### 4.2.5. In Vitro and Ex Vivo Biological Assessment
*   **Hemolytic Activity**: Evaluated using rabbit erythrocytes. `VPNs` and `si-VPNs` at various concentrations (40-800 µg/mL$lipopeptide mass) are incubated with `4$\mathrm{dH_2O}$are used as minimal and maximal hemolytic controls.
*   **Cytotoxicity Study**: `ATDC5 cells` are seeded in 96-well plates. `VPNs` and `si-VPNs` at various concentrations are added, and cells are cultured for `24 h`. `Cell viability` is determined using the `MTT assay` by measuring absorbance at `570 nm`. Studies are conducted at different pH values (7.4, 6.8, 5.5).
*   **Cellular Uptake Study**:
    *   **Cell Model**: `Insulin-induced ATDC5 cells` (a chondrogenic cell line) and `patient-derived primary chondrocytes` are used.
    *   **Time Course**: `Cy5-labeled siRNA` formulations (free `siRNA`, `si-VPN-1/2/3`, `si-MC3-LNP`) are incubated with cells for varying times (`1 h, 2 h, 4 h, 8 h`).
    *   **pH Effect**: Uptake of `si-VPNs` is compared at `pH 7.4` and `pH 6.8`.
    *   **WYRGRL Targeting**: Uptake is compared in `non-induced ATDC5 cells`, `insulin-induced ATDC5 cells`, and$Col2α1 siRNA-knockdown ATDC5 cells$to validate `Col2-dependent targeting`.
    *   **Mechanism Study**: `ATDC5 cells` are pre-treated with inhibitors of specific `endocytosis pathways` (`4°C` for energy-dependence, `amiloride` for `macropinocytosis`, `chlorpromazine` for `clathrin-dependent endocytosis`, `filipin` for `caveolae-mediated endocytosis`, `Brefeldin A` and
```
\beta`-CD` for other pathways) before `si-VPN-2` incubation.
    *   **Detection**: Cellular uptake is quantified by `flow cytometry` (measuring `mean fluorescence intensity, MFI`) and visualized by `confocal microscopy`.
        The quantitative cellular uptake of different formulations by flow cytometry at 1 h or 2 h in insulin-induced ATDC5 cells, and confocal images of cellular uptake of WYRGRL-modified si-VPNs versus non-modified counterparts are presented.

        ![Fig. 2 | In vitro biological properties and RNAi efficacy of si-VPNs. A Quantitative cellular uptake of different formulations by flow cytometry at 1 h or$^ { 2 \\mathrm { h } }$in insulininduced AT…](/files/papers/691763e0110b75dcc59ae089/images/2.jpg)
        *该图像是图表，展示了si-VPNs的体外生物学特性及RNAi效能。A部分显示不同配方在1小时和2小时后通过流式细胞术测定的定量细胞摄取结果。B部分为WYRGRL修饰的si-VPNs与非修饰对照的共聚焦图像，绿色荧光表示Cy5标记的配方，红色荧光表示F-actin，蓝色荧光为DAPI染色的细胞核，比例尺为40μm。C部分展示了WYRGRL修饰肽与Col2的亲和力特征。*

*   **SPR Measurements (Binding Affinity)**: `Surface Plasmon Resonance (SPR)` is used to measure the binding kinetics between `WYRGRL-modified peptide` and `Col2`. `Col2 protein` is immobilized on a `COOH sensor chip`. Analytes (peptides) are injected, and `equilibrium dissociation constants (KD)` are calculated using `Tracedrawer software`.
    Characterization of affinity between WYRGRL-modified peptide and Col2 by surface plasmon resonance (SPR).

    ![Fig. 2 | In vitro biological properties and RNAi efficacy of si-VPNs. A Quantitative cellular uptake of different formulations by flow cytometry at 1 h or$^ { 2 \\mathrm { h } }$in insulininduced AT…](/files/papers/691763e0110b75dcc59ae089/images/2.jpg)
    *该图像是图表，展示了si-VPNs的体外生物学特性及RNAi效能。A部分显示不同配方在1小时和2小时后通过流式细胞术测定的定量细胞摄取结果。B部分为WYRGRL修饰的si-VPNs与非修饰对照的共聚焦图像，绿色荧光表示Cy5标记的配方，红色荧光表示F-actin，蓝色荧光为DAPI染色的细胞核，比例尺为40μm。C部分展示了WYRGRL修饰肽与Col2的亲和力特征。*

*   **In Vitro Protein Adsorption Evaluation**: `siVPNs` are incubated in `10*   **Multicellular Chondrocyte Spheroids Penetration Test**:
    *   **Spheroid Formation**: `Insulin-induced ATDC5 cells` are cultured in `ultra-low attachment 96-well plates` to form spheroids ($>200 µm$diameter).
    *   **Penetration Assay**: `Cy5-labeled siRNA` formulations are added to spheroids at `pH 7.4` or `pH 6.8` for `4 h, 8 h, or 12 h`.
    *   **Detection**: Spheroids are imaged using `Z-stack scanning confocal laser scanning microscopy (CLCSM)` to visualize penetration depth and fluorescence intensity.
*   **Lysosome Co-localization**: `Insulin-induced ATDC5 cells` or `primary human chondrocytes` are incubated with `Cy5-labeled` or `FAM-labeled siRNA` formulations (`si-VPN-2` or `si-MC3-LNP`). Cells are then stained with `Lyso Green` or `Lyso-Tracker Red`. `CLCSM` or `Operetta CLS` is used to visualize co-localization. `Pearson's Rr value` (a correlation coefficient) is used for quantitative analysis of co-localization.
*   **Cellular Immunofluorescent Staining**: After `transfection`, cells are fixed, permeabilized, blocked, and incubated with primary anti-MMP-13 antibody, followed by `Cy5-labeled secondary antibody`. `DAPI` (nuclear stain) and `rhodamine labeled phalloidine` (cytoskeleton stain) are used. Images are acquired with `Operetta CLS`.
*   **Quantitative RT-PCR (In Vitro)**:
    *   **Cell Models**: `Insulin-induced ATDC5 cells` (stimulated with$25 ng/mL TNFα$for `24 h` to mimic pro-inflammatory conditions) and `primary human chondrocytes` are used.
    *   **Transfection**: Formulations (`si-VPNs`, `si-MC3-LNP`) are introduced at `50 nM` or `100 nM siRNA`.
    *   **RNA Extraction and cDNA Synthesis**: Total RNA is extracted using `RNA Easy Fast Kit`, and `first-strand cDNA` is synthesized using `Quantscript RT Kit`.
    *   **PCR**: `Quantitative real-time PCR` is performed to measure relative `MMP-13 mRNA` levels, using `GAPDH` as an internal control.
*   **RNA-Seq Analysis**:
    *   **Sample Preparation**:$TNFα-activated primary human chondrocytes$are treated with `si-VPN-2` for `6 h`, then cultured for `24 h`.$TNFα-activated untreated cells$serve as control.
    *   **Sequencing**: Total RNA is extracted and sequenced using the `Novaseq 6000 platform`.
    *   **Data Analysis**: Raw `fastq` data are processed. `Differentially expressed genes (DEGs)` are identified (P value$\le 0.05$, fold Change$> 2$or$< 0.5). `Heat map` and `KEGG pathway enrichment analysis` are performed.
        The volcano plot of the DEGs in si-VPN-2 vs TNFα group (only stimulated with TNFα) and heat map showing representative DEGs between si-VPN-2 and TNFα group are presented.

        ![该图像是插图，展示了病毒启发的脂肽衍生的核酸在软骨递送中的作用，包括不同处理组的细胞表现和信号通路分析，如细胞增殖、炎症反应等，图中包含与 TNFα 信号通路相关的 KEGG 富集结果。](/files/papers/691763e0110b75dcc59ae089/images/3.jpg)
        *该图像是插图，展示了病毒启发的脂肽衍生的核酸在软骨递送中的作用，包括不同处理组的细胞表现和信号通路分析，如细胞增殖、炎症反应等，图中包含与 TNFα 信号通路相关的 KEGG 富集结果。*

### 4.2.6. Ex Vivo Trypsin-Damaged Cartilage Explants Penetration
*   **Cartilage Model**: `Fresh pig cartilage` explants are sliced and treated with `2.5% trypsin` for `30 min` at `37°C` to mimic `OA lesions`.
*   **Incubation**: Different `Cy5-labeled siRNA` formulations are incubated with the explants for `24 h`.
*   **Detection**: Explants are first imaged directly using `VISQUE in vivo Smart-LF System`. Then, they are embedded, sectioned (`15 µm` thick), stained with `DAPI`, and fluorescence images are acquired using `Operetta CLS` for quantitative analysis of penetration.

### 4.2.7. In Vivo Therapeutic Studies

#### 4.2.7.1. Intra-articular Retention (ACLT Mice)
*   **Animal Model**: Male `ACLT mice` (4 weeks post-surgery).
*   **Treatment**: `Intra-articular injection` of `Cy5-labeled siRNA` formulations (free `siRNA`, `si-MC3-LNP`, `si-VPN-2`, `si-VPN@HA`) at `0.5 mg/kg siRNA`. `si-VPN-2` is also assessed in healthy mice.
*   **Detection**: `Fluorescence images` of knee joints are acquired at predetermined time points over `28 days` using a `VISQUE in vivo Smart-LF System`. `Radiation efficiency-time curves` and `area under the curve (AUC)` are quantified.
    Representative IVIS images of OA mice knee joints after intra-articular injection of different formulations over 28 days.

    ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
    *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

#### 4.2.7.2. Surgical ACLT Model and In Vivo Therapeutic Study
*   **Animal Model**: Male `C57BL/6J mice (8 weeks)` undergo surgical `ACLT` to induce `OA`. A `sham operation` group is included.
*   **Treatment Regimen**: `Intra-articular injection` of different formulations (`Saline`, `si-MC3-LNP`, `si-VPN-2`, `si-VPN@HA`, `si-NEG@HA` (scrambled `MMP-13 siRNA`)) at `1 mg/kg siRNA`, once. Mice are euthanized after `4 weeks`.
*   **Outcome Measures**:
    *   **Body Weight**: Assessed weekly.
    *   **Histopathology**: Affected joints are stained with `H&E` and `Safranin O`. `OARSI histopathology initiative` scoring is used for quantitative assessment of cartilage degeneration.
    *   **Immunofluorescent (IF) and Immunohistochemical Staining**: Joint sections are stained for `MMP-13`, `nerve growth factor (NGF)`, `MMP-9`, `ADAMTS5`, F4/80, `CD206`, `CD80` to assess `MMP-13 silencing`, pain-related markers, catabolic enzymes, and `macrophage polarization`.
    *   **RT-qPCR (In Vivo)**: `Cartilage and synovial tissues` are mixed, and `total RNA` is extracted. `RT-qPCR` is performed to measure `MMP-13`, `TNF-α`, `IL-1β`, `NF-kB1`, `CCL2`, `CXCL10`, `ICAM-1`, and `COX-2 mRNA` levels (inflammation markers).
    *   **Biosafety**: `Serum biochemical indicators` and `H&E staining` of major organs are assessed.
        A schematic construction of surgical ACTL model with corresponding treatment regimen is depicted. Representative images of H&E and Safranin-O staining of mouse knee joint sections, and OARSI cartilage histopathology assessment scores are shown.

        ![该图像是示意图，展示了不同处理组在机械负荷下的行走速度（图B）、软骨损伤的HE和Safranin O染色结果（图C），以及三维重建的骨骼图像（图D）。图F列出了各组的BV/TV、Tb.N和Tb.Th等定量数据。结果显示si-VPN-2组在多个指标上均表现出显著改善。](/files/papers/691763e0110b75dcc59ae089/images/5.jpg)
        *该图像是示意图，展示了不同处理组在机械负荷下的行走速度（图B）、软骨损伤的HE和Safranin O染色结果（图C），以及三维重建的骨骼图像（图D）。图F列出了各组的BV/TV、Tb.N和Tb.Th等定量数据。结果显示si-VPN-2组在多个指标上均表现出显著改善。*

#### 4.2.7.3. Mechanical Loading PTOA Model and In Vivo Therapeutic Study
*   **Animal Model**: Female `C57BL/6J mice (8 weeks)` are subjected to `non-invasive repetitive mechanical loading` (`9.8 N, 500 cycles, 3 times/week for 6 weeks`) to induce `PTOA`.
*   **Treatment Regimen**: `Intra-articular injection` of formulations (`Saline`, `si-VPN-2`, `si-VPN@HA`, `si-NEG@HA`, `methylprednisolone (MP)`, `MP&si-VPN@HA`). Dosing is `1 mg/kg siRNA` and 4 mg/kg MP (if applicable). `MP` is given weekly; others every two weeks.
*   **Outcome Measures**:
    *   **Walking Speed**: Recorded weekly by `open field test`.
    *   **Histopathology**: `H&E`, `Safranin O`, and `MMP-13 immunohistochemical staining`.
    *   **Micro-CT Analysis**: `Micro-computed tomography` is used to visualize and quantify `osteophyte formation` and `subchondral bone microstructure` (e.g., `bone volume/total volume (BV/TV)`, `trabecular number (Tb.N)`, `trabecular thickness (Tb.Th)`, `trabecular separation (Tb.Sp)`).
    *   **Biosafety**: `Body weight` and `blood routine examination`.
        A schematic construction of non-invasive repetitive mechanical loading PTOA model with corresponding treatment regimen, and the walking speeds of different treated groups are presented.

        ![该图像是示意图，展示了不同处理组在机械负荷下的行走速度（图B）、软骨损伤的HE和Safranin O染色结果（图C），以及三维重建的骨骼图像（图D）。图F列出了各组的BV/TV、Tb.N和Tb.Th等定量数据。结果显示si-VPN-2组在多个指标上均表现出显著改善。](/files/papers/691763e0110b75dcc59ae089/images/5.jpg)
        *该图像是示意图，展示了不同处理组在机械负荷下的行走速度（图B）、软骨损伤的HE和Safranin O染色结果（图C），以及三维重建的骨骼图像（图D）。图F列出了各组的BV/TV、Tb.N和Tb.Th等定量数据。结果显示si-VPN-2组在多个指标上均表现出显著改善。*

### 4.2.8. mRNA Transfection
*   **eGFP mRNA**: `eGFP mRNA` (encoding green fluorescent protein) is used as a reporter `mRNA`.
*   **m-VPNs Preparation**: `mRNA-condensed VPNs (m-VPNs)` are prepared by mixing `VPNs` and `mRNA solution` using a `microfluidic mixer`.
*   **Binding Ratios**: `Agarose gel electrophoresis` is used to investigate `mRNA binding ratios`, with `SYBR Gold` stain for visualization.
*   **Morphology**: `TEM` is used to assess `m-VPN-2` morphology (solid spherical after `mRNA` loading).
*   **In Vitro mRNA Transfection**: `Insulin-induced ATDC5 cells` are transfected with `m-VPN-2` or `m-MC3-LNP` (at `500 ng mRNA/well`) for `6 h`, then cultured for `24 h`. `eGFP expression` is detected by `flow cytometry` and `Operetta CLS`.
*   **In Vivo mRNA Transfection**: `Surgical ACLT mice` receive `intra-articular injection` of `m-VPN-2` or `m-MC3-LNP` (`10 µg mRNA/mice`). Joints are harvested after `24 h`, `decalcified`, `sectioned`, stained with `DAPI`, and `eGFP fluorescence signals` are acquired using `Operetta CLS`.
    A schematic illustrating the fabrication process of m-VPNs, mRNA condensation of m-VPN-2 at various N:P ratio by agarose gel electrophoresis, TEM images of VPN-2 and mVPN-2, and flow cytometric analysis of eGFP expressions after different transfections are provided.

    ![Fig. 7 | VPN-2 functionally enable intra-articular mRNA delivery. A Schematic illustrating the fabrication process of m-VPNs. B mRNA condensation of m-VPN-2 a1 various N:P ratio by agarose gel electr…](/files/papers/691763e0110b75dcc59ae089/images/6.jpg)
    *该图像是图表，展示了VPN-2在mRNA递送中的性能，包括m-VPN-2的合成示意图（A），不同N:P比下的m-RNA凝聚及电泳结果（B），TEM图像（C），流式细胞术分析结果（D），eGFP表达的定量结果（E），及不同转染后eGFP表达的共聚焦图像（F）与荧光信号分析（G）。*

## 4.3. Statistical Analysis
`GraphPad Prism software (Version 7)` is used. Data are reported as `mean ± standard deviation (SD)`.
*   **Two-group comparisons**: `Two-sided unpaired Student's t-test`.
*   **Multiple comparisons**: `Unpaired one-way or two-way analysis of variance (ANOVA) test`.
*   `Statistical significance` is defined as P < 0.05.

# 5. Experimental Setup

## 5.1. Datasets
The study utilized a combination of cell lines, primary human cells, `ex vivo` tissue, and `in vivo` animal models.

*   **Cell Lines**:
    *   `ATDC5 cell line`: A mouse chondrogenic cell line obtained from `Sigma-Aldrich`. These cells undergo progressive chondrogenic differentiation and are used to model `chondrocytes` `in vitro`. They were cultured in `DMEM` medium with `10% FBS` and `penicillin-streptomycin`. For specific experiments, they were `insulin-induced` to promote chondrogenic differentiation and Col2α1 expression.
        *   **Purpose**: Used for initial screening of `VPN` properties (cytotoxicity, cellular uptake, `lysosomal escape`, `MMP-13 silencing`), `multicellular chondrocyte spheroid` formation, and `mRNA transfection` studies.
*   **Patient and Samples (Primary Human Cells)**:
    *   `Primary human chondrocytes`: Isolated from `knee cartilage samples` of `OA patients` (1 male, 2 females, age 56-83, Kellgren-Lawrence grade 4) undergoing total knee arthroplasty. Cartilage tissue was minced and digested with `0.2% collagenase II`.
        *   **Purpose**: Used to confirm `in vitro` efficacy (cellular uptake, `lysosomal escape`, `MMP-13 silencing`, `RNA-Seq analysis`) in a more physiologically relevant human cell model.
*   **Ex Vivo Tissue**:
    *   `Fresh pig cartilage explants`: Obtained from excised knee joints.
        *   **Purpose**: Used for the `ex vivo trypsin-damaged cartilage explants penetration` assay to evaluate the ability of `nanoparticles` to diffuse into cartilage tissue.
*   **In Vivo Animal Models**:
    *   `C57BL/6J mice (8 weeks)`: Used for both `surgical anterior cruciate ligament transection (ACTL)` model and `non-invasive repetitive mechanical loading post-traumatic osteoarthritis (PTOA)` model.
        *   **Purpose**:
            *   `ACTL model`: A well-established model for surgically induced `OA`, used to evaluate `intra-articular retention` and the therapeutic efficacy of `si-VPN@HA` in alleviating cartilage degeneration, inflammation, and pain.
            *   `PTOA model`: Induced by `non-invasive repetitive mechanical loading`, used to evaluate the long-term therapeutic efficacy and potential synergistic effects with `methylprednisolone`.

## 5.2. Evaluation Metrics

For every evaluation metric mentioned in the paper, here is a complete explanation:

*   **\mathrm{IC}_{50}$(Half Maximal Inhibitory Concentration)**:
    1.  **Conceptual Definition**:$\mathrm{IC}_{50}$represents the concentration of a substance (e.g., a drug or `nanoparticle`) that is required to inhibit a given biological process (like cell growth or viability) by 50    2.  **Mathematical Formula**: While the explicit formula for$\mathrm{IC}_{50} itself is not a direct calculation but rather derived from a dose-response curve, it typically involves fitting a sigmoid curve to the viability data. The general form of a dose-response curve (e.g., a four-parameter logistic model) is often used:
        \
        Y = \mathrm{Bottom} + \frac{\mathrm{Top} - \mathrm{Bottom}}{1 + \left(\frac{X}{\mathrm{IC}_{50}}\right)^{\mathrm{HillSlope}}}
        \$
        Where $Y$ is the observed response (e.g., cell viability percentage), $X$ is the concentration of the inhibitor, `Bottom` is the minimum response, `Top` is the maximum response, and `HillSlope` describes the steepness of the curve. $\mathrm{IC}_{50}$ is the $X$ value at which $Y$ is halfway between `Bottom` and `Top`.
    3.  **Symbol Explanation**:
        *   $Y$: Response (e.g., cell viability, often normalized from 0% to 100%).
        *   $\mathrm{Bottom}$: Minimum asymptote of the curve (response at very high inhibitor concentrations).
        *   $\mathrm{Top}$: Maximum asymptote of the curve (response at very low inhibitor concentrations).
        *   $X$: Concentration of the inhibitor.
        *   $\mathrm{IC}_{50}$: The concentration of inhibitor that produces 50% of the maximum possible inhibition.
        *   $\mathrm{HillSlope}$: The slope of the curve at its steepest point, indicating the cooperativity of the binding.

*   **Hemolysis Fraction (%)**:
    1.  **Conceptual Definition**: Measures the percentage of red blood cells (erythrocytes) that are ruptured, releasing hemoglobin, after exposure to a substance. It is a common `in vitro` assay to assess the `biocompatibility` and `cytotoxicity` of materials, particularly for intravenous applications, by evaluating their potential to damage blood cells.
    2.  **Mathematical Formula**:
        \$
        \mathrm{Hemolysis\, fraction\, (\%)} = \frac{A_T - A_{\mathrm{PBS}}}{A_{\mathrm{dH2O}} - A_{\mathrm{PBS}}} \times 100
        \$
    3.  **Symbol Explanation**:
        *   $A_T$: Absorbance of the tested `nanoparticle` sample supernatant at `570 nm` (wavelength for hemoglobin detection).
        *   $A_{\mathrm{PBS}}$: Absorbance of the negative control (erythrocytes in `PBS`, representing minimal hemolysis).
        *   $A_{\mathrm{dH2O}}$: Absorbance of the positive control (erythrocytes in `deionized water`, representing maximal hemolysis).

*   **Mean Fluorescence Intensity (MFI)**:
    1.  **Conceptual Definition**: The average intensity of fluorescence measured from a population of cells or particles. In `flow cytometry`, `MFI` is used to quantify the amount of fluorescently labeled substance (e.g., `Cy5-labeled siRNA`, `eGFP expression`) taken up by cells or expressed within them. Higher `MFI` indicates greater uptake or expression.
    2.  **Mathematical Formula**: `MFI` is typically calculated as the arithmetic mean of the fluorescence intensity values of all events (cells/particles) within a defined gate in a `flow cytometry` plot. No single universal formula, as it's an output of the `flow cytometer` software.
        \$
        \mathrm{MFI} = \frac{\sum_{i=1}^{N} \mathrm{FI}_i}{N}
        \$
    3.  **Symbol Explanation**:
        *   $\mathrm{FI}_i$: Fluorescence intensity of the $i$-th cell/particle.
        *   $N$: Total number of cells/particles analyzed in the population.

*   **$\mathrm{K_D}$ (Equilibrium Dissociation Constant)**:
    1.  **Conceptual Definition**: A measure of the affinity between a ligand (e.g., a peptide) and its receptor (e.g., a protein like `Col2`). It represents the concentration of ligand at which half of the binding sites on the receptor are occupied. A lower $\mathrm{K_D}$ value indicates a higher binding affinity (i.e., the ligand binds more tightly to the receptor).
    2.  **Mathematical Formula**: In `SPR` experiments, $\mathrm{K_D}$ is derived from the ratio of the dissociation rate constant ($\mathrm{k_d}$) to the association rate constant ($\mathrm{k_a}$):
        \$
        \mathrm{K_D} = \frac{\mathrm{k_d}}{\mathrm{k_a}}
        \$
        Alternatively, from binding equilibrium:
        \$
        \mathrm{K_D} = \frac{[\mathrm{R}][\mathrm{L}]}{[\mathrm{RL}]}
        \$
    3.  **Symbol Explanation**:
        *   $\mathrm{k_d}$: Dissociation rate constant (rate at which the ligand-receptor complex falls apart).
        *   $\mathrm{k_a}$: Association rate constant (rate at which the ligand binds to the receptor to form a complex).
        *   $[\mathrm{R}]$: Concentration of unbound receptor.
        *   $[\mathrm{L}]$: Concentration of unbound ligand.
        *   $[\mathrm{RL}]$: Concentration of ligand-receptor complex.

*   **Turbidity**:
    1.  **Conceptual Definition**: A measure of the cloudiness or haziness of a fluid caused by particles suspended within it. In this context, it's used to quantify `protein adsorption` onto `nanoparticles` after incubation in `FBS`. Increased `turbidity` can indicate aggregation or significant protein corona formation.
    2.  **Mathematical Formula**: Quantified by measuring the `optical density (OD)` or `absorbance` at a specific wavelength, typically `600 nm`, using a `spectrophotometer`.
        \$
        \mathrm{Turbidity} \propto \mathrm{OD}_{600}
        \$
    3.  **Symbol Explanation**:
        *   $\mathrm{OD}_{600}$: Optical density or absorbance measured at `600 nm` wavelength.

*   **Protein Concentration (Micro BCA Assay)**:
    1.  **Conceptual Definition**: Measures the total amount of protein present in a sample. The `Micro BCA assay` is a colorimetric method used for quantifying low concentrations of protein. In this study, it's used to quantify the amount of protein adsorbed onto `nanoparticles`.
    2.  **Mathematical Formula**: The `BCA assay` relies on the reduction of $\mathrm{Cu^{2+}}$ to $\mathrm{Cu^{1+}}$ by protein in an alkaline environment, followed by chelation of $\mathrm{Cu^{1+}}$ by bicinchoninic acid (BCA) to produce a purple colored product that absorbs light at `562 nm`. A standard curve of known protein concentrations is used to determine unknown concentrations.
        \$
        \mathrm{Absorbance}_{562} = m \cdot [\mathrm{Protein}] + b
        \$
    3.  **Symbol Explanation**:
        *   $\mathrm{Absorbance}_{562}$: Absorbance measured at `562 nm`.
        *   $[\mathrm{Protein}]$: Concentration of protein in the sample.
        *   $m$: Slope of the standard curve.
        *   $b$: Y-intercept of the standard curve.

*   **Pearson's Rr Value (Pearson's Correlation Coefficient)**:
    1.  **Conceptual Definition**: A statistical measure of the linear correlation between two sets of data. In microscopy, it is used to quantify the degree of co-localization (overlap) between two different fluorescent signals (e.g., `siRNA` and `lysosomes`). A value of `1` indicates perfect positive correlation (complete overlap), `0` indicates no linear correlation, and `-1` indicates perfect negative correlation.
    2.  **Mathematical Formula**: For two images (or channels) A and B, each with pixel intensities $\mathrm{a_i}$ and $\mathrm{b_i}$:
        \$
        \mathrm{Rr} = \frac{\sum_{i=1}^{N} (a_i - \bar{a})(b_i - \bar{b})}{\sqrt{\sum_{i=1}^{N} (a_i - \bar{a})^2 \sum_{i=1}^{N} (b_i - \bar{b})^2}}
        \$
    3.  **Symbol Explanation**:
        *   $\mathrm{Rr}$: Pearson's correlation coefficient.
        *   $N$: Total number of pixels.
        *   $a_i$: Intensity of pixel $i$ in image A.
        *   $b_i$: Intensity of pixel $i$ in image B.
        *   $\bar{a}$: Mean intensity of image A.
        *   $\bar{b}$: Mean intensity of image B.

*   **Relative mRNA Levels (RT-qPCR)**:
    1.  **Conceptual Definition**: Measures the amount of specific `mRNA` present in a sample, relative to a control gene (`housekeeping gene`) or a control sample. `Quantitative real-time PCR (RT-qPCR)` amplifies `DNA` targets and quantifies them as they are produced. In this study, it's used to assess the `silencing efficiency` of `MMP-13 mRNA` and the expression levels of various inflammation-associated genes.
    2.  **Mathematical Formula**: The relative quantification is typically performed using the $\Delta\Delta\mathrm{C_T}$ method:
        \$
        \mathrm{Relative\, Expression} = 2^{-\Delta\Delta\mathrm{C_T}}
        \$
        Where $\Delta\mathrm{C_T} = \mathrm{C_T}(\mathrm{Target}) - \mathrm{C_T}(\mathrm{Reference})$ and $\Delta\Delta\mathrm{C_T} = \Delta\mathrm{C_T}(\mathrm{Treated}) - \Delta\mathrm{C_T}(\mathrm{Control})$.
    3.  **Symbol Explanation**:
        *   $\mathrm{C_T}$: Cycle threshold, the PCR cycle number at which fluorescence signal crosses a defined threshold. Lower $\mathrm{C_T}$ indicates higher initial `mRNA` quantity.
        *   $\mathrm{Target}$: The gene of interest (e.g., `MMP-13`).
        *   $\mathrm{Reference}$: The `housekeeping gene` used for normalization (e.g., `GAPDH`), assumed to have stable expression.
        *   $\mathrm{Treated}$: Experimental sample.
        *   $\mathrm{Control}$: Control sample.

*   **Fluorescence Radiant Efficiency (In Vivo Imaging)**:
    1.  **Conceptual Definition**: A measure of the total radiant power emitted by a fluorescent source per unit area, divided by the incident excitation power per unit area. In `in vivo fluorescence imaging (IVIS)`, it quantifies the amount of fluorescent signal detected from a specific region of interest (e.g., a mouse knee joint), indicating the presence and concentration of the fluorescently labeled agent (`Cy5-siRNA`).
    2.  **Mathematical Formula**: Often expressed in units of $(p/s/cm²/sr) / (µW/cm²)$, where:
        *   `p/s`: photons per second.
        *   `cm²`: area.
        *   `sr`: steradian (unit of solid angle).
        *   $µW/cm²$: microwatts per square centimeter (incident excitation power).
            The software (e.g., `VISQUE in vivo Smart-LF System`) typically provides this quantitative output directly for defined regions of interest.
    3.  **Symbol Explanation**: The value directly represents the efficiency of light conversion and detection.

*   **Area Under the Curve (AUC)**:
    1.  **Conceptual Definition**: A mathematical integration of a curve, representing the total accumulation or exposure over a period. In `pharmacokinetics` or `in vivo imaging`, `AUC` of a `fluorescence radiant efficiency-time curve` indicates the total systemic or local exposure to the fluorescent agent over time. A larger `AUC` suggests greater or more prolonged retention.
    2.  **Mathematical Formula**: `AUC` is calculated using numerical integration methods (e.g., `trapezoidal rule`) applied to the data points of the curve. For a function `f(t)` from time $t_1$ to $t_N$:
        \$
        \mathrm{AUC} = \sum_{i=1}^{N-1} \frac{(f(t_{i+1}) + f(t_i))}{2} (t_{i+1} - t_i)
        \$
    3.  **Symbol Explanation**:
        *   $f(t_i)$: Fluorescence radiant efficiency at time $t_i$.
        *   $t_i$: Time point $i$.
        *   $N$: Total number of time points.

*   **OARSI Histopathology Assessment Scores**:
    1.  **Conceptual Definition**: A standardized scoring system developed by the `Osteoarthritis Research Society International (OARSI)` for quantitative evaluation of `OA`-related histological changes in joints. It assesses severity of cartilage degeneration based on features like cartilage surface integrity, proteoglycan loss, and cellularity. Higher scores indicate more severe `OA`.
    2.  **Mathematical Formula**: Not a single formula, but a sum of scores based on a detailed grading system for different aspects of cartilage and joint pathology (e.g., depth of lesion, extent, presence of osteophytes, inflammation). The scores are typically integers or discrete values.
    3.  **Symbol Explanation**: The final score is a composite value reflecting the overall pathological state.

*   **Micro-CT Parameters for Subchondral Bone**:
    1.  **Conceptual Definition**: `Micro-computed tomography (micro-CT)` provides high-resolution 3D images of bone structure. Several quantitative parameters are derived to characterize the `subchondral bone microarchitecture`.
    2.  **Mathematical Formulas and Symbol Explanations**:
        *   **Bone Volume/Total Volume (BV/TV)**:
            *   **Definition**: The ratio of the volume of bone tissue to the total volume of the region of interest. It is a measure of `bone density`.
            *   **Formula**: $\mathrm{BV/TV} = \frac{\mathrm{Bone\, Volume}}{\mathrm{Total\, Volume}}$
            *   **Symbols**:
                *   $\mathrm{Bone\, Volume}$: Volume of segmented bone tissue.
                *   $\mathrm{Total\, Volume}$: Total volume of the analyzed region.
        *   **Trabecular Number (Tb.N)**:
            *   **Definition**: The average number of `trabeculae` (small, rod-like or plate-like units of spongy bone) per unit length. Higher `Tb.N` indicates a denser `trabecular network`.
            *   **Formula**: Typically derived from standard `stereological analyses` of `micro-CT` images. For a cubic element, it can be approximated as $\mathrm{Tb.N} = \frac{1}{\mathrm{Tb.Sp} + \mathrm{Tb.Th}}$.
            *   **Symbols**:
                *   $\mathrm{Tb.N}$: Trabecular Number.
                *   $\mathrm{Tb.Sp}$: Trabecular Separation.
                *   $\mathrm{Tb.Th}$: Trabecular Thickness.
        *   **Trabecular Thickness (Tb.Th)**:
            *   **Definition**: The mean thickness of the `trabeculae`.
            *   **Formula**: Derived from standard `stereological analyses`, often calculated as $\mathrm{Tb.Th} = \frac{2 \times \mathrm{BV/TV}}{\mathrm{BS/TV}}$, where $\mathrm{BS/TV}$ is bone surface density.
            *   **Symbols**:
                *   $\mathrm{Tb.Th}$: Trabecular Thickness.
                *   $\mathrm{BV/TV}$: Bone Volume/Total Volume.
                *   $\mathrm{BS/TV}$: Bone Surface/Total Volume (surface area of bone tissue per unit total volume).
        *   **Trabecular Separation (Tb.Sp)**:
            *   **Definition**: The mean distance between adjacent `trabeculae`. Lower `Tb.Sp` indicates a more closely packed `trabecular network`.
            *   **Formula**: Derived from standard `stereological analyses`, often calculated as $\mathrm{Tb.Sp} = \frac{(1 - \mathrm{BV/TV})}{\mathrm{BS/TV}}$.
            *   **Symbols**:
                *   $\mathrm{Tb.Sp}$: Trabecular Separation.
                *   $\mathrm{BV/TV}$: Bone Volume/Total Volume.
                *   $\mathrm{BS/TV}$: Bone Surface/Total Volume.

## 5.3. Baselines
The paper compares its `VPN` system against several relevant baselines to demonstrate its superiority:

*   **Free siRNA / mRNA**: Unformulated `siRNA` or `mRNA` molecules. This serves as a control to show the absolute necessity of a delivery vector due to the inherent instability and poor cellular uptake of naked `nucleic acids`.
*   **siRNA loaded MC3-LNP (si-MC3-LNP) / mRNA loaded MC3-LNP (m-MC3-LNP)**: `MC3-LNP` (DLin-MC3-DMA formulated `LNP`) is a widely recognized and clinically translated `lipid nanoparticle` platform. It serves as the primary benchmark for `transfection efficiency` and delivery capability in both `in vitro` and `in vivo` settings, representing the "conventional" state-of-the-art non-viral `nanoparticle` delivery.
*   **Saline**: A vehicle control, typically sterile `0.9% NaCl` solution, representing no therapeutic intervention. Used in `in vivo` studies to observe the natural progression of `OA` in untreated animals.
*   **Sham**: A surgical control group in the `ACTL` model, where animals undergo the surgical procedure (incision, joint exposure) but without transection of the `anterior cruciate ligament`. This helps to differentiate effects caused by the `OA` pathology from those caused by the surgical intervention itself.
*   **si-NEG@HA (scrambled MMP-13 siRNA loaded HA@gel)**: A negative control for `siRNA` specificity. This formulation contains a `scrambled siRNA` sequence that does not target `MMP-13` (or any other known gene), delivered via the `HA@gel` system. It demonstrates that the observed therapeutic effects are due to specific `MMP-13 silencing` by `si-VPN-2`, not merely the presence of `siRNA` or the `HA@gel` carrier.
*   **Methylprednisolone (MP)**: A potent `corticosteroid` widely used clinically for `intra-articular injection` in `OA` to reduce inflammation and pain. It serves as a benchmark for comparing the therapeutic efficacy of `si-VPN@HA` and for evaluating potential synergistic effects (`MP&si-VPN@HA`) in the `PTOA` model.

# 6. Results & Analysis

## 6.1. Core Results Analysis

The study systematically evaluates the `VPN` platform, from its fundamental characteristics to its `in vivo` therapeutic efficacy in `OA` models and its versatility for `mRNA` delivery.

### 6.1.1. Rational Design and Fabrication of VPNs and si-VPNs

*   **Modular Design**: The `lipopeptides` were successfully synthesized with three functional moieties: a `Col2-targeting head` (WYRGRL), a `cationic moiety` (`arginine` and `histidine`), and a `hydrophobic docosanoic acid (`\mathrm{C_{22}}`)` tail (Fig. 1A). `MALDI-TOF mass spectrometry` confirmed the molecular weights of the synthetic `lipopeptides`.
*   **Self-Assembly and Morphology**: `Lipopeptides 1-9` spontaneously self-assembled into `nanoparticles (VPN)`. `VPN-1` and `VPN-2` had comparable `hydrodynamic diameters` (~50 nm), while `VPN-3` was slightly larger (~90 nm) (Supplementary Fig. 4A, B). All `VPNs` carried positive surface charges. `TEM` images showed that `VPNs` formed `unilamellar vesicle structures`, distinguishing them from common amphiphilic `peptide nanoparticles` that form `micelles`. This vesicle formation was dependent on the $\mathrm{C_{22}}$ hydrophobic moiety (Supplementary Fig. 10A-D).
    The following are representative TEM images of VPN-1, VPN-2, VPN-3, and corresponding si-VPN-1, si-VPN-2, si-VPN-3, scale bar = 50 µm; and HR-TEM and FE-SEM images of VPN-2, scale bar = 50 µm.

    ![该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。](/files/papers/691763e0110b75dcc59ae089/images/1.jpg)
    *该图像是示意图，展示了病毒启发的脂肽衍生的核酸递送系统，包括不同pH值下的Zeta电位变化、颗粒大小分布、透射电子显微镜图像及分子动态模拟。图中描述的VPN-2显示出优于其他载体的转染能力，适合骨关节炎基因治疗应用。*

*   **pH-Sensitivity**: The `zeta potentials` and `sizes` of `VPNs` progressively increased as pH decreased from 7.4 to 6.8 and 5.5 (Fig. 1B and Supplementary Fig. 4C-H). This `pH-sensitive` behavior, attributed to the protonation of `histidine` residues, is crucial for enhanced performance in the acidic `OA microenvironment`.
*   **siRNA Condensation and Protection**: `siRNA-loaded VPNs (si-VPNs)` were prepared via `microfluidics`. `Agarose gel electrophoresis` showed that `si-VPN-2` and `si-VPN-3` achieved sufficient `siRNA condensation` at an `N:P ratio` of 6:1, while `si-VPN-1` required a higher ratio (Fig. 1D). `HAADF-STEM` confirmed uniform dispersion of `siRNA` within `si-VPN-2` (Fig. 1E). `RNase protection assay` demonstrated that `si-VPNs` effectively protected `siRNA` from degradation (Fig. 1F and Supplementary Fig. 7A, B), indicating robust cargo encapsulation.
*   **Post-Loading Characteristics**: After `siRNA` condensation, `si-VPNs` showed slight increases in diameter and notable decreases in surface charge (Fig. 1G and Supplementary Fig. 8A, B). Crucially, `si-VPNs` transformed from `unilamellar vesicles` to `solid spherical nanoparticles` upon `siRNA` packaging, a structural change resembling that of `liposomes` to `LNPs` (Fig. 1H, I).
*   **Stability**: `si-VPNs` maintained reliable storage stability and `siRNA` encapsulation for at least `48 h` (Supplementary Fig. 9A-D).
*   **DPD Simulations**: `DPD simulations` confirmed that `si-VPNs` formed `solid spherical architectures` with `lipopeptide-siRNA complexes` concentrated in the core, surrounded by `peptide backbones` and `hydrophilic groups` (Fig. 1J). The assembly dynamics showed significant structural organization within `1000 ps` and stability over `200 ns` (Fig. 1K), validating the molecular interactions.

### 6.1.2. In Vitro Biological Assessment and Gene Silencing Efficacy of si-VPNs

*   **Cytocompatibility**: `si-VPNs` exhibited significantly reduced `cytotoxicity` compared to blank `VPNs`. The `IC50` of `si-VPN-2` was $4701 µg/mL$ at `pH 7.4`, far higher than $194 µg/mL$ for `VPN-2` (Supplementary Fig. 15). Hemolysis was less than `5%` below $300 µg/mL$ for `si-VPNs` (Supplementary Fig. 16A, B), suggesting good `cytocompatibility`.
*   **Cellular Uptake**: `si-VPN-2` consistently showed the highest `mean fluorescence intensity (MFI)` among all formulations (`si-VPN-1/2/3`, `si-MC3-LNP`) in `insulin-induced ATDC5 cells` across various time points (`1 h` to `8 h`) (Fig. 2A and Supplementary Fig. 17B).
*   **Cartilage Targeting**: `WYRGRL-modified si-VPNs` showed significantly elevated `MFI` compared to non-modified counterparts (Fig. 2B and Supplementary Fig. 17C). `SPR` confirmed high binding affinity between `WYRGRL-consisted peptide` and `Col2` ($\mathrm{K_D}$ of $2.05 × 10^-8 M$) (Fig. 2C and Supplementary Fig. 18). Uptake was higher in `insulin-induced ATDC5 cells` (high $Col2α1$) than in non-induced or `Col2α1-knockdown` cells (Supplementary Fig. 19A, B), validating `Col2-dependent targeting`.
    The following figure (Figure 2 from the original paper) shows the in vitro biological properties and RNAi efficacy of si-VPNs.

    ![Fig. 2 | In vitro biological properties and RNAi efficacy of si-VPNs. A Quantitative cellular uptake of different formulations by flow cytometry at 1 h or $^ { 2 \\mathrm { h } }$ in insulininduced AT…](/files/papers/691763e0110b75dcc59ae089/images/2.jpg)
    *该图像是图表，展示了si-VPNs的体外生物学特性及RNAi效能。A部分显示不同配方在1小时和2小时后通过流式细胞术测定的定量细胞摄取结果。B部分为WYRGRL修饰的si-VPNs与非修饰对照的共聚焦图像，绿色荧光表示Cy5标记的配方，红色荧光表示F-actin，蓝色荧光为DAPI染色的细胞核，比例尺为40μm。C部分展示了WYRGRL修饰肽与Col2的亲和力特征。*

*   **pH-Boosted Uptake**: Cellular uptake of `si-VPNs` increased approximately 2-fold at `pH 6.8` compared to `pH 7.4` (Fig. 2D and Supplementary Fig. 20), consistent with enhanced `polyhistidine ionization` and `pH-sensitivity`.
*   **Internalization Mechanism**: `si-VPN-2` internalization was significantly suppressed by `4°C` (energy-dependent), `amiloride` (`macropinocytosis`), and `chlorpromazine` (`clathrin-dependent endocytosis`) (Fig. 2E).
*   **Chondrocyte Spheroid Penetration**: `si-VPN-2` could penetrate deep into the center of `3D chondrocyte spheroids` with significantly higher fluorescence than `si-MC3-LNP` or `si-VPN-1` (Fig. 2F and Supplementary Fig. 21A, B). `si-VPN-3` showed poor penetration, localizing mostly on the outer layer. Penetration of `si-VPN-2` was substantially higher at `pH 6.8` than `pH 7.4`, confirming `acid-boosted penetration`.
*   **Lysosomal Escape**: `si-VPN-2` rapidly released `siRNA` into the cytoplasm, with a `Pearson's Rr` value of only `~0.2` at `12 h`, indicating excellent `lysosomal escape`. In contrast, `si-MC3-LNP` remained largely entrapped in `lysosomal vesicles` at `6 h` or `12 h` (Fig. 2G). This potent escape for `si-VPN-2` is attributed to the `proton sponge effect`.
*   **MMP-13 Silencing**: In $TNFα-stimulated ATDC5 cells$, `si-VPN-2` demonstrated the most efficient `MMP-13 silencing` in a dose-dependent manner, achieving over `85% knockdown` at `100 nM siRNA`, significantly outperforming `si-MC3-LNP` (Fig. 2H). `Immunofluorescent staining` showed minimal `MMP-13 expression` with `si-VPN-2` (Supplementary Fig. 22A, B).

### 6.1.3. MMP-13 Silencing Broadly Affects Gene Expression Profiles In Vitro

*   **Performance in Primary Chondrocytes**: In `patient-derived primary chondrocytes`, `si-VPN-2` again showed the highest `MFI` for cellular uptake (3.04-fold increase over `si-MC3-LNP`) (Fig. 3B and Supplementary Fig. 23A, B). `si-VPN-3` formed aggregates, likely due to `non-specific protein adsorption` (Supplementary Fig. 24A-D), explaining its poorer performance despite a similar number of `R/H` residues.
*   **Lysosomal Escape in Primary Cells**: `si-VPN-2` showed superior `lysosomal escape` in primary chondrocytes, with diminished `co-localization` of `FAM-labeled siRNA` and `lyso-tracker red` after `6 h` incubation, and a low `Pearson's Rr` value (Fig. 3C and Supplementary Fig. 25A, B).
*   **MMP-13 Silencing in Primary Cells**: `si-VPN-2` exerted the most potent `MMP-13 silencing efficacy` in primary chondrocytes, significantly reducing `MMP-13 mRNA` and protein expression to levels statistically equivalent to the $without TNFα$ group (Fig. 3D, E, and Supplementary Fig. 26).
    The following figure (Figure 3 from the original paper) shows the role of virus-inspired lipopeptide-derived nucleic acid delivery in cartilage.

    ![该图像是插图，展示了病毒启发的脂肽衍生的核酸在软骨递送中的作用，包括不同处理组的细胞表现和信号通路分析，如细胞增殖、炎症反应等，图中包含与 TNFα 信号通路相关的 KEGG 富集结果。](/files/papers/691763e0110b75dcc59ae089/images/3.jpg)
    *该图像是插图，展示了病毒启发的脂肽衍生的核酸在软骨递送中的作用，包括不同处理组的细胞表现和信号通路分析，如细胞增殖、炎症反应等，图中包含与 TNFα 信号通路相关的 KEGG 富集结果。*

*   **RNA-Seq Analysis**: `RNA-Seq` revealed `329 significantly downregulated` and `519 significantly upregulated genes` in the `si-VPN-2` group compared to the $TNFα$ group (Fig. 3F).
    *   **Downregulated Genes**: Included `chemokines` (`CCL2`, `CCL7`, `CXCL6`, `CXCL10`), `immunity-related markers` (`TLR2`), `pro-inflammatory markers` (`IL34`, `ICAM-1`, `NFkB2`), `negative regulator of cartilage matrix ADAMTS12`, and `pro-apoptosis genes` (`TP53`) (Fig. 3G).
    *   **Upregulated Genes**: Included `anti-inflammatory markers` (`IL11`, `IL33`) and `chondrocyte differentiation-related genes` (`GDF5`, `BMP6`).
*   **Biological Processes and Pathways**: `RT-qPCR` confirmed changes in these `DEGs`, involved in `chondrocyte differentiation`, `skeletal system development`, `cytokine-cytokine interaction`, `inflammation`, and `immune response` (Fig. 3H and Supplementary Fig. 27). `KEGG enrichment analysis` identified `nine highly affected pathways`, including `TNF`, `cytokine-cytokine receptor interaction`, `PI3K-Akt`, and `MAPK`, all linked to `OA pathogenesis` (Fig. 3I). These results demonstrate that `si-VPN-2`-mediated `MMP-13 silencing` has broad beneficial effects on the gene expression profile relevant to `OA`.

### 6.1.4. Construction and Release Kinetics of Nano-in-Gel si-VPN@HA

*   **HA@gel Properties**: The `ROS-responsive HA@gels` were successfully constructed based on `host-guest recognition` between 

\beta-CD and Fc (Supplementary Fig. 28A-C). The HA@gels exhibited rapid\mathrm{H_2O_2}-sensitive gel-sol transition (Fig. 4B and Supplementary Fig. 28D), with significantly lower storage modulus (G') in the presence of 5 mM\mathrm{H_2O_2}

 (Fig. 4C), indicating reduced `crosslinking density`. `Thixotropic` experiments confirmed `self-healing properties` (Supplementary Fig. 28E).
    The following figure (Figure 4 from the original paper) shows the construction and intra-articular retention of sustained-release siVPN@HA.

    ![Fig. 4 | Construction and intra-articular retention of sustained-release siVPN@HA. A Schematic construction of ROS-responsive nano-in-gel system siVPN@HA. B The representative photographs of gel-sol…](/files/papers/691763e0110b75dcc59ae089/images/4.jpg)
    *该图像是示意图，展示了ROS响应的nano-in-gel系统siVPN@HA的构建及其在关节内的持续释放机制。图中包括HA-CD、HA-Fc和si-VPN-2的示意结构及实验结果，涉及不同刺激下的荧光强度变化及释放曲线，表明该系统在OA治疗中的潜力。*

*   **Release Kinetics of si-VPN@HA**: `Fluorescence scanning` showed that `si-VPN-2` and `si-VPN@HA` did not exhibit dissociative `siRNA` in `5 mM`\mathrm{H_2O_2}

solutions, unlike free siRNA or siRNA@HA (Fig. 4D, E), confirming siRNA remained encapsulated within si-VPN-2 during HA@gel dissolution. Released si-VPN-2 maintained mono-dispersed spherical morphology (Supplementary Fig. 29A, B).

• Ex Vivo Cartilage Penetration: si-VPN-2 showed optimal penetration in trypsin-damaged cartilage explants, diffusing evenly with a uniform fluorescence profile. si-VPN-3 poorly penetrated, mostly adsorbing on the surface. si-VPN@HA had low fluorescence without H2O2, but achieved ~2-fold increase on explants and ~3-fold increase within cartilage sections under H2O2 stimulus (Fig. 4F-H and Supplementary Figs. 31, 32). This confirms its ROS-triggered release and subsequent penetration ability.
• Intra-articular Retention: In vivo IVIS imaging in ACLT mice showed free siRNA eliminated fast. si-VPN-2 maintained strong signals for 9 days, with superior retention in OA joints than healthy ones (Fig. 4I and Supplementary Fig. 33A-C). si-VPN@HA demonstrated the best performance, with signals extending over 21 days and AUC significantly increased (2.31-fold vs si-VPN-2, 3.41-fold vs si-MC3-LNP) (Fig. 4J, K). This successfully reconciles the penetration-retention dilemma.

6.1.5. Alleviating Cartilage Degeneration by si-VPN@HA Treatment in Surgical ACTL Model

• Therapeutic Efficacy: In the surgical ACTL mouse model, Saline group showed severe cartilage damage. While si-VPN-2 improved degeneration compared to si-MC3-LNP, si-VPN@HA effectively minimized cartilage damage, preserving joint structures with intact cartilage surface and perichondrium (Fig. 5B). OARSI scores for si-VPN@HA (1.4 ± 0.55) were significantly lower than si-VPN-2 or si-MC3-LNP and statistically equivalent to the Sham group (Fig. 5C). The following figure (Figure 5 from the original paper) illustrates the effective alleviation of cartilage damage and inflammation in the surgical ACTL model.

  该图像是示意图，展示了不同处理组在机械负荷下的行走速度（图B）、软骨损伤的HE和Safranin O染色结果（图C），以及三维重建的骨骼图像（图D）。图F列出了各组的BV/TV、Tb.N和Tb.Th等定量数据。结果显示si-VPN-2组在多个指标上均表现出显著改善。

• MMP-13 Silencing In Vivo: Immunofluorescent (IF) staining revealed over 75% reduction of MMP-13 expression in cartilage and meniscus in the si-VPN@HA group, the lowest among all treated groups (Fig. 5D, E).

• Pain Relief: NGF expression in synovium was substantially reduced by nearly 80% in the si-VPN@HA group compared to Saline, suggesting effective pain relief (Fig. 5D, E).

• Catabolic Enzyme Reduction: si-VPN@HA significantly down-regulated MMP-9 and ADAMTS-5 (other catabolic enzymes) expressions compared to si-VPN-2 or si-MC3-LNP (Fig. 5F, G), indicating a broader protective effect.

• Macrophage Polarization: si-VPN@HA treatment effectively increased synovial macrophage M2 polarization ($CD206+F4/80+$) and decreased M1 polarization ($CD80+F4/80+$) (Fig. 5H, I), shifting the immune response towards an anti-inflammatory and reparative phenotype.

• Inflammation Markers: RT-qPCR showed significant reduction in TNF-α, IL-1β, NF-kB1, CCL2, CXCL10, ICAM-1, and COX-2 mRNA levels in the si-VPN@HA group, with TNF-α, CCL2, and ICAM-1 reduced below 35% of the Saline group (Fig. 5J), confirming broad anti-inflammatory effects.

• Biosafety: si-VPN@HA was validated for biosafety by body weight, biochemical indexes, and H&E staining of major organs (Supplementary Figs. 34, 35).

6.1.6. Implementing Superior Joint Protection in Synergy with Steroid Treatment in PTOA Model

• Walking Speed: In the PTOA mouse model, MP group showed a rapid decrease in walking speed to 18.22% of the initial level after 6 weeks. In contrast, MP&si-VPN@HA preserved joint motion well, with walking speed at $606.2 cm/min$, significantly higher than si-VPN-2 ($292.6 cm/min$) and si-VPN@HA ($475 cm/min$) (Fig. 6B). The following figure (Figure 6 from the original paper) illustrates the implementing superior joint protection in synergy with steroid treatment in PTOA model.

  该图像是图表，展示了VPN-2在mRNA递送中的性能，包括m-VPN-2的合成示意图（A），不同N:P比下的m-RNA凝聚及电泳结果（B），TEM图像（C），流式细胞术分析结果（D），eGFP表达的定量结果（E），及不同转染后eGFP表达的共聚焦图像（F）与荧光信号分析（G）。

• Histopathology: MP, si-VPN-2, and si-VPN@HA groups showed typical PTOA characteristics (soft tissue mineralization, osteophyte formation, proteoglycan loss). MP&si-VPN@HA markedly improved these, showing well-preserved articular cartilage, minimal MMP-13 expression in cartilage and meniscus (~75% reduction vs. Saline) (Fig. 6C and Supplementary Fig. 36B). OARSI scores were lowest for MP&si-VPN@HA (Supplementary Fig. 36A).

• Osteophyte Formation and Subchondral Bone: Micro-CT analysis showed that MP&si-VPN@HA significantly blocked osteophyte formation and ectopic mineralization, reducing osteophyte outgrowth to ~10% of the Saline group (Fig. 6D, E). Subchondral bone density (BV/TV) was well-preserved, statistically equivalent to the Sham group (Fig. 6F). MP&si-VPN@HA was also more potent in increasing Tb.N and Tb.Th while decreasing Tb.Sp, indicating superior protection of subchondral bone microarchitecture.

• Biosafety: Body weight and blood routine examination indicated MP&si-VPN@HA was well-tolerated (Supplementary Fig. 37A-F).

6.1.7. VPN-2 Functionally Enabled Intra-articular mRNA Delivery

• m-VPNs Characterization: m-VPN2 achieved complete mRNA retardation at an N:P ratio of

\ge 2.304

(Fig. 7B). Blank VPN-2 vesicles transformed into solid spherical structures after mRNA loading (Fig. 7C), analogous to siRNA entrapment.

• In Vitro mRNA Transfection: m-VPN-2 yielded 59.98% eGFP-positive cell population in ATDC5 cells, 3.95-fold higher than m-MC3-LNP (Fig. 7D, E). MFI was also significantly higher (3.30- and 2.44-fold vs m-VPN-1 and m-MC3-LNP, respectively) (Supplementary Fig. 38). Confocal imaging confirmed elevated fluorescence (Fig. 7F and Supplementary Fig. 39).
• In Vivo mRNA Transfection: In ACLT mice, intra-articular injection of m-VPN-2 resulted in 4.30-fold higher eGFP fluorescence intensity within cartilage sections compared to m-MC3-LNP (Fig. 7G and Supplementary Fig. 40), demonstrating superior mRNA delivery to cartilage.

6.2. Data Presentation (Tables)

The original paper does not contain any tables in its main text that need to be transcribed. All result data is presented within figures or described in the text.

6.3. Ablation Studies / Parameter Analysis

While the paper doesn't present traditional "ablation studies" on the components of the final VPN-2 formulation (e.g., removing WYRGRL, R/H residues, or C22 tail from VPN-2 and re-testing), it performs a comprehensive comparative analysis that serves a similar purpose in optimizing the formulation:

• Comparison of Lipopeptide Designs (VPN-1, VPN-2, VPN-3): The initial design of lipopeptides 1-3 (and others 4-12, though 1-3 are the focus for cationic moiety comparison) represents a form of iterative design and selection.
  • VPN-1 (R5H4), VPN-2 ((R5H4)2), VPN-3 (R10H8) variations in the cationic moiety were directly compared for siRNA condensation (N:P ratio, Fig. 1D), cellular uptake (Fig. 2A, D), chondrocyte spheroid penetration (Fig. 2F), and MMP-13 silencing (Fig. 2H). This effectively acts as an ablation study to identify the optimal cationic moiety, with VPN-2 showing superior performance in most metrics.
  • The observation that si-VPN-3 formed aggregates and showed high-degree non-specific protein adsorption (Supplementary Fig. 24A-D) despite having the same total number of R/H residues as VPN-2 but in a different sequence ($(R5H4)2$ vs R10H8) highlights the critical importance of the specific sequence and arrangement of the cationic moiety, rather than just the raw number of R/H residues. This is a crucial finding from this comparative analysis.
• Role of WYRGRL Targeting Head: The WYRGRL-modified siVPNs were directly compared to non-modified counterparts for cellular uptake (Fig. 2B and Supplementary Fig. 17C). The significantly elevated MFI in the WYRGRL-modified group, coupled with SPR data (Fig. 2C) and differential uptake in Col2α1-expressing vs knockdown cells (Supplementary Fig. 19A, B), serves as an ablation study confirming the essential role of the targeting head.
• Role of Hydrophobic Moiety: The paper briefly notes that lipopeptides 4-6 (without WYRGRL head) still formed vesicles, while lipopeptides 7-9 (with one $\mathrm{C_{22}}$ tail) self-assembled into micelles (not vesicles), and peptide 10-12 (without $\mathrm{C_{22}}$ tail) couldn't form nanoparticles at all (Supplementary Fig. 10A-D). This comparison demonstrates the crucial role of the hydrophobic\mathrm{C_{22}}tail in lipopeptide self-assembly and the resulting nanostructure (vesicle vs. micelle).
• N:P Ratio Optimization: The agarose gel electrophoresis for siRNA condensation at various N:P ratios (Fig. 1D) is a parameter analysis to find the optimal ratio for effective siRNA encapsulation and protection.
• pH Effect Analysis: The extensive testing of VPN and si-VPN properties (zeta potential, size, cytotoxicity, cellular uptake, penetration) at different pH values (7.4, 6.8, 5.5) directly analyzes the impact of pH on their performance, validating the importance of their pH-sensitive design.
• ROS Responsiveness of HA@gel: The rheological assessment and release kinetics under 0.1 mM vs 5.0 mM\mathrm{H_2O_2}

 (Fig. 4C-E) explicitly demonstrate the `ROS-responsive` nature of the `HA@gel` and its ability to trigger `si-VPN-2` release, which is a key component of the overall delivery strategy.

    These comparative and parametric analyses effectively serve to validate the contribution of each design element to the overall success of the `VPN` platform.

# 7. Conclusion & Reflections

## 7.1. Conclusion Summary
This study successfully developed a novel `virus-inspired lipopeptide-based nanoparticle (VPN)` platform for targeted `nucleic acid delivery` to cartilage, offering a promising therapeutic strategy for `osteoarthritis (OA)`. The modular design of `lipopeptides`, incorporating a `Col2-targeting peptide` and a `cationic moiety` with optimized `arginine` and `histidine` residues, enabled the creation of `VPN-2`, which significantly outperformed conventional `lipid nanoparticles (LNP)` in `transfection potency` by enhancing `endocytosis` and `lysosomal escape`. To address the `penetration-retention dilemma` in `intra-articular delivery`, `si-VPN-2` was formulated into a `ROS-responsive hyaluronic acid hydrogel (HA@gel)` system (`si-VPN@HA`). This innovative `nano-in-gel system` demonstrated sustained intra-articular retention and `ROS-triggered release`, allowing for effective penetration into cartilage.

`In vivo` studies in `surgical ACTL mice` showed that `si-VPN@HA` effectively alleviated cartilage degeneration, reduced inflammation, and relieved pain, achieving substantial `MMP-13 silencing` and beneficial modulation of `macrophage polarization`. Furthermore, in a `PTOA mouse model`, a co-loaded formulation of `si-VPN-2` and `methylprednisolone` (`MP&si-VPN@HA`) exhibited superior synergistic joint protection. The `VPN` platform also proved versatile for `intra-articular mRNA delivery`, demonstrating high `eGFP expression` `in vitro` and `in vivo`. Overall, the study establishes `VPNs` as potent, cartilage-targeted `RNA delivery vectors` with significant potential for innovative `OA therapy`.

## 7.2. Limitations & Future Work
The paper highlights the "necessity and significance for further study," implying that while the results are promising, there are aspects that require more investigation or refinement before clinical translation. While the authors don't explicitly list limitations within the conclusion, several can be inferred from the study's scope and nature:

*   **Long-term Safety and Immunogenicity**: Although `VPNs` are described as "non-pathogenic" and `si-VPNs` show good `cytocompatibility` and `biosafety` in short-term animal studies, long-term `in vivo` safety, potential for chronic `immunogenicity` (even if low, repeated administration might trigger responses), and `biodegradation` products of the `lipopeptides` and `hydrogel` need thorough investigation for clinical application.
*   **Targeting Specificity and Off-target Effects**: While `Col2-targeting` is demonstrated, the precise distribution of `si-VPNs` to `chondrocytes` versus other joint tissues, and potential off-target gene silencing in other cell types within the joint, could be further elucidated.
*   **Scalability and Manufacturing**: The `solid-phase peptide synthesis` and `microfluidic mixing` methods used are well-established for research, but scaling up production of `lipopeptides` and `nano-in-gel systems` to clinical manufacturing standards can present challenges in terms of cost, purity, and batch-to-batch consistency.
*   **Precise ROS Triggering**: The `ROS-responsive` release mechanism relies on local `H2O2` levels. While `OA` joints are known to have elevated `ROS`, the exact concentration threshold and how reliably this translates to precise, localized release without premature degradation or insufficient release in varied `OA` conditions warrants further investigation.
*   **Universality of Gene Silencing**: While `MMP-13` is a key target, `OA` is multifactorial. The paper shows broad `gene expression effects`, but further studies on combined `siRNA` delivery (e.g., targeting multiple `MMPs` or inflammatory cytokines) could be explored.
*   **Clinical Translation Steps**: The `in vivo` studies are conducted in mouse models. Extensive preclinical studies in larger animal models (e.g., pigs, sheep) that more closely mimic human joint size and physiology would be essential before human trials. This includes optimizing dose, frequency of administration, and evaluating functional outcomes over longer periods.
*   **Mechanism of Synergy**: The synergy with `methylprednisolone` is promising. Future work could delve deeper into the molecular mechanisms underlying this synergistic joint protection.

## 7.3. Personal Insights & Critique
This paper presents a highly innovative and well-executed approach to a critical problem in `OA therapy`. The "virus-inspired" concept is a compelling paradigm for overcoming the limitations of both viral and non-viral gene delivery.

The **multi-modular design** of the `lipopeptide` is particularly insightful, allowing for independent optimization of targeting, cellular uptake, and `lysosomal escape`. The detailed investigation of `arginine` and `histidine` residue combinations, leading to the selection of `VPN-2`, highlights the importance of precise structural engineering over simply increasing cationic charge. The elegant solution to the `penetration-retention dilemma` using a `ROS-responsive nano-in-gel system` is a standout feature. This addresses a fundamental challenge for any `intra-articular` therapeutic, especially for diseases like `OA` where the diseased tissue itself provides the trigger for drug release. This strategy could be broadly applicable to other `ROS-driven pathologies`.

The comprehensive `in vitro` characterization, including `DPD simulations`, `SPR`, and advanced cellular assays (spheroid penetration, `lysosomal escape`), provides robust evidence for the `VPN`'s mechanisms of action. The `RNA-Seq` analysis is particularly strong, demonstrating that `MMP-13 silencing` by `si-VPN-2` does not just inhibit a single enzyme but broadly re-shapes the `transcriptomic landscape` toward a more favorable `anti-inflammatory` and `pro-chondrogenic` state, which is crucial for `disease modification`. The `in vivo` results in two distinct `OA` models, especially the synergy with `methylprednisolone`, offer compelling evidence for the platform's therapeutic potential. The validation for `mRNA delivery` further expands its utility.

A potential area for improvement or future exploration could involve a deeper understanding of the `degradation pathways` of the `lipopeptides` themselves `in vivo`. While `cytocompatibility` and `biosafety` are assessed, the long-term fate of the `lipopeptide` components and their metabolites is important. Also, investigating the `biodistribution` of the `si-VPNs` after release from the `hydrogel` (beyond just the joint) could provide further safety assurance. The `pH-sensitive` and `ROS-responsive` nature is excellent, but real-world `OA` joints exhibit varying degrees of acidity and `ROS` levels. Fine-tuning the responsiveness or developing `multifactorial responsive` systems could offer even greater control.

The methods and conclusions of this paper could be transferable to other therapeutic areas requiring targeted `nucleic acid delivery` to specific tissues with unique `microenvironments`. For instance, `tumor-targeted gene therapy` (leveraging tumor acidity or `hypoxia`), `inflammatory bowel disease` (local inflammation), or `wound healing` (high `ROS`) could potentially benefit from adaptations of this `modular lipopeptide` or `nano-in-gel` design strategy. The virus-inspired approach emphasizes learning from nature's highly evolved delivery systems, a powerful principle that will continue to drive innovation in `nanomedicine`.