| 摘要: | 背景：膝關節炎是一種造成失能的退化性關節，其特徵是關節軟骨持續性的退化，主要是發炎過程所致。山奈酚（KM）是一種存在於多種水果和蔬菜中的黃酮類化合物，因其抗發炎特性而受到認可，使其成為膝關節炎治療的可行選擇。本研究評估了利用玻尿酸（HA）包覆的明膠奈米粒子（GNP）封裝KM（HA-KM GNP）在膝關節內注射以治療膝關節炎。該研究聚焦於HA-KM GNP的開發，和在膝關節炎大鼠模型中的治療效果，突顯了在膝關節炎治療中的重大進展。
材料與方法：HA-KM GNP的配方過程需要謹慎以確保最佳的封裝和塗層。首先，使用兩步驟去溶劑化方法將KM封裝在明膠奈米粒子 (KM GNP），該方法能生產出粒徑均一、載藥效率高的粒子。封裝後，這些KM GNP被HA包覆塗層，形成HA-KM GNP。通過動態光散射分析儀和透射電子顯微鏡對HA-KM GNP的物理性質（包括粒徑、表面電位、和形態）進行表徵。這些評估確保奈米粒子處於理想的尺寸範圍並具有穩定性和細胞相互作用所需的表面正電荷。
利用大鼠膝蓋軟骨細胞在體外評估 HA-KM GNP 的生物相容性和細胞攝取。使用 CCK-8 測定評估細胞毒性，同時以流式細胞儀分析細胞攝取。通過對IL-1β刺激的大鼠軟骨細胞進行治療，對 HA-KM GNP、缺少KM的HA包覆奈米粒子（HA GNP）和單獨的 KM進行比較，進而研究證實HA-KM GNP的抗炎和促軟骨生成效果。
為了評估藥物保留效果，將紅色螢光染料TAMRA偶聯到KM GNP和HA-KM GNP上，以便能夠在大鼠膝關節內進行追蹤。在體內研究中，通過前十字韌帶切斷誘導的膝關節炎大鼠模型評估了HA-KM GNP的臨床前有效性。大鼠接受HA-KM GNP膝關節內注射，並透過微型電腦斷層掃描分析和軟骨的組織病理學檢查來評估治療結果。
結果：本研究合成的HA-KM GNP呈現清晰的球形形態，直徑為88.62 ± 3.90 nm。這些奈米粒子具有帶正電的表面，表面電位為32.94 ± 1.66 mV。HA-KM GNP中的KM封裝率，達到約 98.34%，負載率約為4.92%。此外，HA-KM GNP 在48小時內表現出KM的逐漸釋放率為 18%。HA-KM GNP中的無毒 KM 濃度維持在 2.5 μg/mL。利用流式細胞儀，與 TAMRA 標記的 KM GNP 相比，大鼠軟骨細胞內螢光TAMRA標記的HA-KM GNP的螢光累積明顯更高。在IL-1β誘導的膝關節炎大鼠軟骨細胞中，HA-KM GNP成為一種有效的治療候選物。它證明關鍵發炎和分解代謝介質的表達顯著減少，包括 IL-1β、TNF-α、COX-2、MMP-9 和 MMP-13，同時顯示SOX9表現增加，表明有促進軟骨形成的效果。這些觀察結果明顯優於使用HA GNP和單獨KM的結果。體內影像顯示，與KM GNP相比，注射 HA-KM GNP的3小時後，大鼠膝關節內的螢光強度明顯較高（185.2% ± 34.1%對比45.0% ± 16.7%）。這種藥物動力學優勢有助於實現持續治療的效果。此外，HA-KM GNP在膝退化關節炎的大鼠模型中顯示出在減少軟骨下硬化、減輕發炎、抑制基質降解、恢復軟骨厚度和降低關節炎的嚴重程度方面的顯著功效。
結論：本研究的綜合結果為 HA-KM GNP 在膝關節炎治療中的治療潛力提供了令人信服的證據。這種創新的藥物傳輸方法利用了生物相容性奈米粒子系統中KM的抗發炎特性，為未來的臨床應用帶來了巨大的希望。這項研究的結果不僅凸顯了解決膝關節炎治療緊迫醫療需求的潛力，也為再生醫學領域的進一步探索和發展開闢了途徑。
Background: Knee osteoarthritis (OA) is a debilitating degenerative joint condition characterized by the continuous degradation of joint cartilage, primarily driven by inflammatory processes. Kaempferol (KM), a potent flavonoid present in a variety of fruits and vegetables, has been recognized for its strong anti-inflammatory properties, making it a viable option for OA therapy. This research evaluates the use of hyaluronic acid (HA)-coated gelatin nanoparticles (GNPs) with KM loading (HA-KM GNPs) for targeted drug delivery within the knee joint for OA treatment. The study focuses on the development, characterization, and therapeutic efficacy of HA-KM GNPs in a rat model of knee OA, highlighting a significant advancement in OA therapy.
Materials and methods: The formulation of HA-KM GNP involved a meticulous process to ensure optimal encapsulation and coating. Initially, KM was encapsulated within gelatin nanoparticles (KM GNPs) using a two-step desolvation method, which is known for producing particles with uniform size and high drug loading efficiency. Following the encapsulation, these KM GNPs were coated with HA to form HA-KM GNPs. The physical properties of the HA-KM GNPs, including particle size, zeta potential, and morphology, were characterized using a dynamic light scattering analyzer and a transmission electron microscope. These assessments ensured the nanoparticles were within the desired size range and had appropriate surface charge for stability and cellular interaction.
Biocompatibility and cellular uptake of the HA-KM GNPs were evaluated in vitro using rat chondrocytes. Cytotoxicity was assessed using the CCK-8 assay, while cellular uptake was analyzed via flow cytometry. The anti-inflammatory and chondrogenic effects of the HA-KM GNPs were investigated by treating IL-1β-stimulated rat chondrocytes and measuring the expression levels of inflammatory cytokines and cartilage matrix components. Comparisons were made between HA-KM GNPs, HA-coated nanoparticles lacking KM (HA GNPs), and KM alone to determine the specific contributions of the nanoparticle delivery system and the active compound.
To assess drug retention, TAMRA, a red fluorescent dye, was conjugated to KM GNPs and HA-KM GNPs to enable tracking within the rat knee joints. For in vivo therapeutic evaluation, the preclinical effectiveness of HA-KM GNPs was evaluated in a rat model of knee OA induced by anterior cruciate ligament transection (ACLT). Rats received intra-articular injections of HA-KM GNPs, and the therapeutic outcomes were assessed through micro-CT scanning analysis and histopathological examination of cartilage.
Results: The HA-KM GNP synthesized in this study exhibited a well-defined spherical morphology with a diameter measuring 88.62 ± 3.90 nm. These nanoparticles possessed a positively charged surface with a zeta potential of 32.94 ± 1.66 mV. KM encapsulation within HA-KM GNPs demonstrated impressive efficiency, reaching around 98.34%, with a loading rate of approximately 4.92%. Furthermore, HA-KM GNPs exhibited a gradual release rate of 18% for KM over 48 hours. A non-toxic KM concentration of 2.5 μg/mL was maintained in HA-KM GNPs. Utilizing flow cytometry, there was a notably higher accumulation of fluorescent TAMRA-labeled HA-KM GNPs within rat chondrocytes compared to TAMRA-labeled KM GNPs. In treating IL-1β-induced osteoarthritic rat chondrocytes, HA-KM GNPs emerged as a potent therapeutic candidate. It demonstrated a significant reduction in the expression of key inflammatory and catabolic mediators, including IL-1β, TNF-α, COX-2, MMP-9, and MMP-13, while simultaneously showing an increase in SOX9 levels, indicating potential chondrogenic benefits. These observations were notably superior to those seen with HA GNPs, and KM alone. In vivo imaging demonstrated significantly higher fluorescence intensity within rat knee joints for 3 hours post HA-KM GNPs injection compared with KM GNPs (185.2% ± 34.1% vs. 45.0% ± 16.7%). This pharmacokinetic advantage contributes to the potential for sustained therapeutic action. Furthermore, HA-KM GNPs demonstrated significant effectiveness in reducing subchondral sclerosis, attenuating inflammation, inhibiting matrix degradation, restoring cartilage thickness, and reducing the severity of OA in the ACLT rat model.
Conclusion: The comprehensive findings presented in this study offer compelling evidence for the therapeutic potential of HA-KM GNPs in the treatment of knee OA. This innovative approach to drug delivery, harnessing the anti-inflammatory properties of KM within a biocompatible nanoparticle system, holds significant promise for future clinical applications. The results of this study not only highlight the potential to address an urgent medical need for osteoarthritis treatment, but also open avenues for further exploration and development in the field of regenerative medicine. |
