WO2023109456A1 - 骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法及应用 - Google Patents

骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法及应用 Download PDF

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WO2023109456A1
WO2023109456A1 PCT/CN2022/134004 CN2022134004W WO2023109456A1 WO 2023109456 A1 WO2023109456 A1 WO 2023109456A1 CN 2022134004 W CN2022134004 W CN 2022134004W WO 2023109456 A1 WO2023109456 A1 WO 2023109456A1
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synovial
aptamer
nucleic acid
nanoparticles
cells
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郭保生
陈响
张蕾
蒋青
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南京大学
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to the screening and construction of bone joint synovial fibroblast targeting aptamer nanoparticles.
  • Osteoarthritis is a joint degenerative disease that occurs with aging. It is a joint disease of articular cartilage, synovium and subchondral bone, which mainly causes progressive degeneration of articular cartilage and synovial inflammation. Clinical Manifested as joint stiffness and pain, eventually leading to movement disorders of the body. Epidemiological studies have found that the global prevalence of knee and hip osteoarthritis is as high as 3.8% and 0.85%4. Osteoarthritis is a common and disabling musculoskeletal disease with a significantly increased incidence, especially in the elderly population over the age of 50.
  • hip and knee OA is the 11th leading risk factor for disability in the world, causing a huge burden on the health care system, economy and society.
  • There are various factors affecting the repair of articular cartilage in OA including local joint factors, including the degree of joint damage and deformity, and overall organism factors, including gender, age, menopause, genes, nutrition, body weight/body mass index, and bone density.
  • the degeneration process of OA articular cartilage is intricate and regulated by a variety of cells and cytokines around the joint, including bone marrow mesenchymal stem cells, chondrocytes, synoviocytes, immune cells, and many inflammatory cytokines.
  • OA is an senile disease
  • body cells has become an important reason for the occurrence and development of OA.
  • drugs that can specifically remove senescent cells.
  • these drugs are difficult to be widely used in clinical practice.
  • targeted nano-drug delivery systems have been widely used clinically due to their high efficiency and low side effects. Therefore, if it is possible to design targeted removal of senescent cells for specific cells or tissues, it may bring good news to the treatment of senile diseases in the future.
  • the present invention provides a construction method and application of bone-joint synovial fibroblast-targeted aptamer nanoparticles to realize targeted removal of aging fibrous synoviocytes in OA synovium, thereby inhibiting the occurrence of OA develop.
  • a method for constructing bone and joint synovial fibroblast-targeted aptamer nanoparticles comprising the following steps:
  • S2 synthetic random nucleic acid aptamer library, its sequence is as shown in SEQ ID NO.1;
  • step S3 Screening: the synovial fibroblasts constructed in step S1 are used as positive screen cells, and chondrocytes are used as negative screen cells.
  • the random nucleic acid aptamer library synthesized in step S2 is repeatedly screened to enrich the The nucleic acid aptamer CX3 that specifically binds to synovial fibroblasts and does not bind to chondrocytes has a sequence as shown in SEQ ID CX3;
  • the mouse synovial fibroblasts are isolated from the mouse synovial tissue, and the mouse chondrocyte line is ATDC5 cells.
  • step S4 is: synthesize CX3-PEG2000-DSPE by Michael addition reaction, then add CX3-PEG2000-DSPE to mixture A composed of HSPC, DSPE-PEG2000 and cholesterol and mix uniformly to obtain mixture B, and then Dasatinib and quercetin were added to mixture B, and then evaporated to dryness under reduced pressure, hydrated, and sonicated, and then filtered with a microporous membrane to obtain the aptamer CX3 modified with dasatinib and quercetin.
  • the liposomal nanoparticles CX3-LS-DQ The liposomal nanoparticles CX3-LS-DQ.
  • the mass ratio of HSPC, DSPE-PEG2000 and cholesterol in mixture A is 80-150:25-50:1, and the amount of CX3-PEG2000-DSPE added accounts for 1-3% of the molar ratio of mixture B.
  • the final concentration of dasatinib is 0.5-2 mg/mL, and the final concentration of quercetin is 2.5-7.5 mg/mL.
  • liposome nanoparticle constructed by any of the above construction methods in the preparation of a drug carrier targeting to clear the synovial membrane of osteoarthritis.
  • the targeted elimination of the application is the senescent fibrous synoviocytes in the synovial membrane of osteoarthritis.
  • the present invention constructs a drug that can target synovial fibroblasts to remove senescent fibroblasts, and uses pharmacological experiments to prove the use of synovium-targeted nanoparticle-encapsulated senescent cell-scavenging drugs in anti-osteoarthritis. It is verified by experiments that the present invention exhibits better synovium-targeting properties. In vivo and in vitro experiments confirmed that: 1. The surface-modified CX3 nucleic acid aptamer liposome nanoparticles can specifically bind synovial fibroblasts, and the intra-articular injection of the nanoparticles can effectively accumulate in the synovial tissue and reduce its accumulation in other tissues. Distribution of organs and tissues. 2. Encapsulating senescent cell-removing drugs dasatinib and quercetin in the aptamer nanoparticles can effectively remove senescent synovial fibroblasts and effectively inhibit the occurrence and development of arthritis.
  • Figure 1 is a flow chart of synovial fibroblast-targeted aptamer screening
  • Figure 2 is flow cytometric detection of nucleic acid aptamers with different screening times combined with synovial fibroblasts and chondrocytes respectively;
  • Fig. 3 is flow cytometric detection nucleic acid aptamer CX1, CX2, CX3 combine with synovial fibroblast and chondrocyte respectively;
  • Figure 4 is a two-dimensional structural simulation of nucleic acid aptamers CX1, CX2, and CX3;
  • Fig. 5 is the synthesis route of nucleic acid aptamer CX3 modified phospholipid DSPE-PEG2000;
  • Fig. 6 is flow cytometric detection CX3 modified liposome nanoparticle and synovial fibroblast and chondrocyte binding situation respectively;
  • Figure 7 shows the fluorescence distribution of different organs and tissues after injection of CX3-modified liposome nanoparticles into the knee joint
  • Figure 8 shows the fluorescence distribution in the knee joint after injection of CX3-modified liposome nanoparticles
  • Fig. 9 is CCK8 and SA- ⁇ -Gal method detection CX3 modified encapsulated DQ drug liposome nanoparticles clearing the situation of aging synovial fibroblasts;
  • Fig. 10 is a flowchart of intra-articular injection of synovium-targeted nano-medicine
  • Figure 11 shows the cell senescence in the synovial tissue after the injection of nano-medicine into the joint cavity by immunofluorescence
  • Figure 12 shows the degeneration of cartilage after the injection of nano-medicine into the joint cavity by safranin fast green staining
  • Figure 13 is the immunohistochemical detection of the expression of chondrocyte apoptosis proteins and inflammatory factors in synovial tissue after the injection of nano-medicine into the joint cavity.
  • the materials used in the examples of the present invention are all commercially available products.
  • mouse chondrocyte line ATDC5 cells were purchased from ATCC;
  • the random nucleic acid aptamer library was synthesized in Sangon Bioengineering (Shanghai) Co., Ltd.
  • SEQ ID NO.1 is:
  • n is any base in A, T, C, G
  • centrifuge the cells at 150g remove the supernatant, heat the cells at 95°C for 10min, and obtain the bound synovium in the supernatant
  • Nucleic acid aptamers for fibroblasts after co-incubating the obtained nucleic acid aptamers binding synovial fibroblasts with chondrocyte ATDC5 for 1 h, the cells were centrifuged at 150 g, and the supernatant was collected to obtain specific binding synovial fibroblasts.
  • Nucleic acid aptamers that bind to chondrocytes repeat the above steps, as shown in Figure 1, through 14 rounds of enrichment screening, obtain a group of nucleic acid aptamers that can specifically bind to synovial fibroblasts and do not bind to chondrocytes; Flow cytometry was used to verify that the screened aptamers could specifically bind to synovial fibroblasts, as shown in Figure 2; the two-dimensional structure simulations of the screened aptamers CX1, CX2, and CX3 were shown in Figure 4.
  • nucleic acid aptamer CX3 capable of specifically binding to synovial fibroblasts was obtained, the sequence of which is shown in SEQ ID CX3,
  • SEQ ID CX3 is:
  • the sulfhydryl-modified CX3 was recorded as HS-CX3 and DSPE-PEG2000-MAL to obtain the target compound CX3-PEG2000-DSPE through nucleophilic substitution reaction, as shown in Figure 5.
  • the experimental method is as follows, HS-CX3 and DSPE-PEG2000-MAL are dissolved in freshly distilled chloroform and methanol according to the molar ratio of 1:1.2, wherein the volume ratio of chloroform and methanol is 4:1, and the pH value is adjusted using triethylamine to 10. After stirring at room temperature for 48 h, the reaction mixture was dialyzed against deionized water for 48 h using a dialysis bag with a molecular weight cut-off (MW) of 3000 to remove unreacted aptamer. Store at -20°C after being lyophilized by a lyophilizer.
  • MW molecular weight cut-off
  • the mixed solvent is composed of chloroform and methanol, prepared according to the volume ratio of 2:1. After the solution was evaporated to dryness under reduced pressure at 30°C to form a film, it was hydrated with 5 mL of Phosphate Buffer Saline (PBS), and then sonicated (10 min, 10%) with an ultrasonic cell disruptor in an ice-water bath.
  • PBS Phosphate Buffer Saline
  • the aptamer-modified liposome (liposome, LS) nanoparticle CX3-LS-DQ loaded with D and Q was obtained by successively filtering through 0.45 ⁇ m and 0.22 ⁇ m microporous membranes.
  • the fluorescent dye Coumarin 6, abbreviated as C6, was encapsulated in the liposome nanoparticle CX3-LS-DQ constructed in Example 1, and incubated in synovial fibroblasts and chondrocytes respectively, and detected by flow cytometry Fluorescence intensity in the cells was used to determine whether synovial fibroblast-targeted nanoparticles could specifically bind to synovial fibroblasts, as shown in Figure 6 .
  • the results showed that the amount of liposome nanoparticles C6-CX3-LS with surface modified aptamer CX3 entering synovial fibroblasts was significantly higher than that of liposome nanoparticles C6-LS with unmodified aptamer CX3.
  • the chondrocytes were treated with these two kinds of liposome nanoparticles, there was no significant difference in the amount of them entering into the chondrocytes.
  • the nano-particle Dir dye-CX3-LS wrapped with the fluorescent dye Dir dye was injected into the joint cavity of the mouse, and the in vivo imaging and laser confocal technology were used to detect the changes in the mouse knee joint and synovial tissue.
  • the fluorescence intensity is shown in Figure 7 and Figure 8. The results showed that the fluorescence intensity in the synovial tissue of the mouse joint cavity in the Dir dye-CX3-LS group was significantly higher than that in the Dir dye group and the Dir dye-LS group, indicating that liposomal nanoparticles CX3-LS can specifically bind to the synovial tissue .
  • Chondrocytes, synovial fibroblasts, and senescent synovial fibroblasts were treated with different concentrations of nanoparticles loaded with senescent cell-clearing drugs (dasatinib and quercetin).
  • the CX3-modified nanoparticle-encapsulated senescence-removing drug can more effectively remove senescent cells, and reduces the toxic and side effects of the drug on normal chondrocytes and synovial fibroblasts.
  • the present invention has the functions of targeting and removing senescent synoviocytes and inhibiting the occurrence and development of osteoarthritis.
  • the present invention screens nucleic acid aptamers targeting synovial fibroblasts, and constructs liposome nanoparticles modified by the aptamers, so as to deliver senescent cell-clearing drugs to synovial tissue in a targeted manner, thereby clearing synovial tissue. Senescent cells, thus having an anti-osteoarthritis effect.

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Abstract

本发明公开了骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法及应用，包括以下步骤：S1、分别构建小鼠滑膜成纤维细胞和软骨细胞的培养体系，备用；S2、合成随机核酸适配体文库，其序列如SEQ ID NO.1所示；S3、筛选：以步骤S1构建的滑膜成纤维细胞为正筛细胞，软骨细胞为负筛细胞，采用CELL-SELEX技术，对步骤S2合成的随机核酸适配体文库经过重复筛选，富集能够特异性结合滑膜成纤维细胞，不结合软骨细胞的核酸适配体CX3，其序列如SEQ ID CX3所示；S4、构建步骤S3筛选出的核酸适配体CX3修饰的脂质体纳米粒。本发明适配体与靶细胞结合力更强，能够实现靶向清除骨性关节炎滑膜中衰老的纤维状滑膜细胞，进而抑制骨性关节炎的发生发展。

Description

骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法及应用 技术领域
本发明属于生物技术领域，具体涉及一种骨关节滑膜成纤维细胞靶向适配体纳米粒的筛选与构建。
背景技术
骨性关节炎(OA)是一种伴随增龄发生的关节退化性疾病，是关节软骨、滑膜及软骨下骨共同参与，主要造成关节软骨进行性退化及滑膜炎症的全关节疾病，临床表现为关节僵硬和疼痛，最终导致机体的运动障碍。流行病学研究发现，全球膝关节和髋关节骨性关节炎患病率高达3.8％和0.85％4。特别在50岁以上的老年人群中，骨性关节炎的发病率显著增加，是一种常见的具有致残性的肌肉骨骼疾病。2010年全球疾病负担研究报告指出，髋关节和膝关节OA是导致全球残疾的第11大高危因素，对医疗保健系统、经济和社会均造成巨大的负担。影响OA关节软骨修复的因素多种多样，既有局部关节因素，包括关节损伤和畸形程度，也有机体整体因素，包括性别、年龄、更年期、基因、营养、体重/身体质量指数及骨密度等。OA关节软骨退变过程错综复杂，受到关节周围多种细胞及细胞因子的调控，包括骨髓间充质干细胞、软骨细胞、滑膜细胞、免疫细胞及众多炎症细胞因子等。
OA作为一种老年性疾病，机体细胞老化成为OA发生发展的重要原因。有研究发现，清除衰老细胞能够有效抑制疾病的发生发展。目前，能够特异性清除衰老细胞的药物多种多样，但是，由于其对机体造成的多种副作用，使得这些药物很难广泛应用于临床。近年来，靶向纳米药物递送系统因其高效性、低副作用在临床上得到了广泛应用。因此，如果能够设计针对特定细胞或组织衰老细胞的靶向清除，可能会对未来老年性疾病治疗带来福音。
目前，针对OA的靶向治疗研究还很匮乏。由于关节腔内的细胞类型复杂多样，针对特定病变细胞的个性化治疗，实现对关节滑膜FLS衰老的原位调控一直都是OA临床治疗的一个重要难点。
发明内容
针对上述技术问题，本发明提供一种骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法及应用，实现靶向清除OA滑膜中衰老的纤维状滑膜细胞，进而抑制OA的发生发展。
为解决现有技术问题，本发明采取的技术方案为：
骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，包括以下步骤：
S1、分别构建小鼠滑膜成纤维细胞和软骨细胞的培养体系，备用；
S2、合成随机核酸适配体文库，其序列如SEQ ID NO.1所示；
S3、筛选：以步骤S1构建的滑膜成纤维细胞为正筛细胞，软骨细胞为负筛细胞，采用CELL-SELEX技术，对步骤S2合成的随机核酸适配体文库经过重复筛选，富集能够特异性结合滑膜成纤维细胞，不结合软骨细胞的核酸适配体CX3，其序列如SEQ ID CX3所示；
S4、构建步骤S3筛选出的核酸适配体CX3修饰的脂质体纳米粒。
优选的，所述步骤S1中小鼠滑膜成纤维细胞分离于小鼠滑膜组织，小鼠软骨细胞系为ATDC5细胞。
优选的，步骤S4的具体过程为：通过迈克尔加成反应合成CX3-PEG2000-DSPE，然后将CX3-PEG2000-DSPE加到HSPC、DSPE-PEG2000和胆固醇组成的混合物A中混合均匀得到混合物B，然后向混合物B中加入达沙替尼和槲皮素，再经过减压蒸干、水合、超声后，用微孔滤膜过滤，获得载有达沙替尼和槲皮素的适配体CX3修饰的脂质体纳米粒CX3-LS-DQ。
优选的，混合物A中HSPC、DSPE-PEG2000和胆固醇的质量比为80～150:25～50:1，CX3-PEG2000-DSPE的添加量占混合物B的摩尔比的1～3％。
优选的，达沙替尼的终浓度为0.5～2mg/mL，槲皮素的终浓度为2.5～7.5mg/mL。
上述任一构建方法构建的脂质体纳米粒在制备靶向清除骨性关节炎滑膜的药物载体上的应用。
优选的，所述应用靶向清除的是骨性关节炎滑膜中衰老的纤维状滑膜细胞。
有益效果：
本发明构建一种可以靶向到滑膜成纤维细胞，清除衰老的成纤维细胞，并借助药理实验以证明滑膜靶向纳米粒包裹衰老细胞清除药物在抗骨性关节炎中的用途。经实验验证本发明表现出较好的滑膜靶向特性。体内外实验证实：1.表面修饰CX3核酸适配体脂质体纳米粒能够特异性结合滑膜成纤维细胞，关节腔注射该纳米粒能够有效的在滑膜组织中蓄积，并减少其在其他器官组织的分布。2.在该适配体纳米粒中包裹衰老细胞清除药物达沙替尼和槲皮素可以有效清除衰老的滑膜成纤维细胞，并有效地抑制了关节炎的发生发展。
附图说明
图1是滑膜成纤维细胞靶向适配体筛选流程图；
图2是流式细胞检测不同筛选次数的核酸适配体分别与滑膜成纤维细胞和软骨细胞结合情况；
图3是流式细胞检测核酸适配体CX1、CX2、CX3分别与滑膜成纤维细胞和软骨细胞结合 情况；
图4是核酸适配体CX1、CX2、CX3的二维结构模拟；
图5是核酸适配体CX3修饰磷脂DSPE-PEG2000的合成路线；
图6是流式细胞检测CX3修饰的脂质体纳米粒分别与滑膜成纤维细胞和软骨细胞结合情况；
图7是膝关节注射CX3修饰的脂质体纳米粒后，不同器官组织的荧光分布情况；
图8是膝关节注射CX3修饰的脂质体纳米粒后，膝关节中的荧光分布情况；
图9是CCK8及SA-β-Gal法检测CX3修饰的包裹DQ药物脂质体纳米粒清除衰老的滑膜成纤维细胞情况；
图10是关节腔注射滑膜靶向纳米药物流程图；
图11是免疫荧光检测关节腔注射纳米药物后，滑膜组织中的细胞衰老情况；
图12是番红固绿染色检测关节腔注射纳米药物后，软骨退变情况；
图13是免疫组化检测关节腔注射纳米药物后，软骨细胞凋亡蛋白及滑膜组织中炎症因子的表达情况。
具体实施方式
下面结合附图和具体实施例对本发明作详细说明：
本发明实施例中所用的材料均为市售产品。
其中，小鼠软骨细胞系ATDC5细胞购买于ATCC公司；
随机核酸适配体文库在生工生物工程(上海)股份有限公司合成。
实施例1
(1)滑膜成纤维细胞靶向适配体文库筛选
将核酸适配体随机序列文库与滑膜成纤维细胞共同孵育1h后，其中核酸适配体随机序列文库的序列如SEQ ID NO.1所示，
SEQ ID NO.1为：
5’-ATACCAGCTTATTCAATTnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnAGATAGTAAGTGCAATCT-3’，其中n为A、T、C、G中任意的碱基，然后150g离心细胞，去除上清后，在95℃加热细胞10min，在上清中获得结合滑膜成纤维细胞的核酸适配体；将获得的结合滑膜成纤维细胞的核酸适配体与软骨细胞ATDC5共同孵育1h后，150g离心细胞，收集上清，获得特异性结合滑膜成纤维细胞，不结合软骨细胞的 核酸适配体；重复上述步骤，如图1所示，通过14轮富集筛选，获得一群能够特异性结合滑膜成纤维细胞，不结合软骨细胞的核酸适配体文库；采用流式细胞技术，验证所筛选的适配体能够特异性结合滑膜成纤维细胞，如图2所示；所筛选的适配体CX1、CX2、CX3的二维结构模拟如图4所示。
(2)滑膜成纤维细胞靶向适配体测序及靶向性验证
采用二代测序技术，对上述筛选的适配体文库进行测序分析，并获得一条能够特异性结合滑膜成纤维细胞的核酸适配体CX3，其序列如SEQ ID CX3所示，
SEQ ID CX3为：
Figure PCTCN2022134004-appb-000001
采用流式细胞技术验证该适配体能够特异性结合滑膜成纤维细胞，如图3所示。
(3)构建核酸适配体CX3修饰的脂质体纳米粒
为了制备靶向适配体修饰的脂质体，将巯基修饰的CX3记为HS-CX3与DSPE-PEG2000-MAL通过亲核取代反应得到目标化合物CX3-PEG2000-DSPE，如图5所示。
实验方法如下，HS-CX3与DSPE-PEG2000-MAL按照摩尔比为1:1.2的比例溶解在新蒸馏氯仿和甲醇，其中氯仿和甲醇的体积比为4:1，使用三乙胺将pH值调整到10。在室温下搅拌48小时后，将反应混合物用截留分子量为3000的透析袋(MW)在去离子水中透析48小时，以除去未反应的适配体。通过冻干机冻干后，保存在-20℃。然后，称取质量比为100:30:10:3的HSPC、DSPE-PEG2000、胆固醇和CX3-PEG2000-DSPE，以及质量比为1:10的达沙替尼(Dasatinib Tablets，D)和槲皮素(Quercetin，Q)，称量完毕后溶解在混合溶剂中。混合溶剂组成为氯仿和甲醇，按照体积比2:1配制。溶液在30℃下减压蒸干成膜后，用5mL磷酸盐缓冲溶液(Phosphate Buffer Saline，PBS)水合，之后在冰水浴中用超声波细胞破碎仪超声(10min，10％)。而后，依次用0.45μm和0.22μm的微孔滤膜过滤，即得到载有D和Q的适配体修饰的脂质体(liposome，LS)纳米粒CX3-LS-DQ。
实施例2
确定核酸适配体CX3修饰的脂质体纳米粒CX3-LS-DQ是否具有滑膜成纤维细胞靶向特异性。
首先，在实施例1所构建的脂质体纳米粒CX3-LS-DQ中包裹荧光染料Coumarin 6，简写为C6，并分别于滑膜成纤维细胞和软骨细胞孵育，通过流式细胞技术，检测细胞中的荧光强度，明确滑膜成纤维细胞靶向纳米粒是否能够特异性与滑膜成纤维细胞结合，如图6 所示。结果显示，表面修饰适配体CX3的脂质体纳米粒C6-CX3-LS进入滑膜成纤维细胞的量明显高于未修饰适配体CX3的脂质体纳米粒C6-LS。而用这两种脂质体纳米粒处理软骨细胞后，其进入软骨细胞的量无明显差别。
其次，通过关节腔注射技术，将包裹荧光染料Dir染料的纳米粒Dir染料-CX3-LS注射到小鼠关节腔中，采用活体成像和激光共聚焦技术，检测小鼠膝关节及滑膜组织中的荧光强度如图7、图8所示。结果显示，Dir染料-CX3-LS组小鼠关节腔滑膜组织中的荧光强度明显高于Dir染料组和Dir染料-LS组，表明脂质体纳米粒CX3-LS能够特异性结合滑膜组织。
实施例3
为进一步明确包裹衰老细胞清除药物的靶向纳米粒是否能够更为有效的清除衰老细胞。
将不同浓度的装载衰老细胞清除药物(达沙替尼和槲皮素)的纳米粒分别处理软骨细胞、滑膜成纤维细胞和衰老的滑膜成纤维细胞。
如图9所示，根据细胞活力检测结果显示，CX3修饰的纳米粒包裹衰老清除药物能够更为有效的清除衰老细胞，并降低了药物对正常软骨细胞和滑膜成纤维细胞毒副作用。
实施例4
为了确认包裹衰老清除药物的靶向纳米粒是否具有抑制骨性关节炎的作用。我们通过离断小鼠内侧半月板，构建小鼠膝关节炎模型，在造模28天后关节腔注射该纳米粒，如图10所示，造模8周后检测小鼠关节组织切片进行番红固绿和免疫组化染色结果显示，从而明确其是否具有治疗骨性关节炎地作用。
结果显示，关节腔注射CX3-LS-DQ能够有效抑制软骨磨损，如图12所示，抑制滑膜组织中验证因子的表达和老化细胞的数量，如图11、图13所示。因此，本发明具有靶向清除衰老滑膜细胞，抑制骨性关节炎发生发展的作用。
本发明通过筛选靶向滑膜成纤维细胞的核酸适配体，并构建该适配体修饰的脂质体纳米粒，将衰老细胞清除药物靶向递送到滑膜组织中，清除滑膜组织中衰老的细胞，从而具有抗骨性关节炎的作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Figure PCTCN2022134004-appb-000002
Figure PCTCN2022134004-appb-000003

Claims (7)

  1. 骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，其特征在于，包括以下步骤：
    S1、分别构建小鼠滑膜成纤维细胞和软骨细胞的培养体系，备用；
    S2、合成随机核酸适配体文库，其序列如SEQ ID NO.1所示；
    S3、筛选：以步骤S1构建的滑膜成纤维细胞为正筛细胞，软骨细胞为负筛细胞，采用CELL-SELEX技术，对步骤S2合成的随机核酸适配体文库经过重复筛选，富集能够特异性结合滑膜成纤维细胞，不结合软骨细胞的核酸适配体CX3，其序列如SEQ ID CX3所示；
    S4、构建步骤S3筛选出的核酸适配体CX3修饰的脂质体纳米粒。
  2. 根据权利要求1所述的骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，其特征在于：所述步骤S1中小鼠滑膜成纤维细胞分离于小鼠滑膜组织，小鼠软骨细胞系为ATDC5细胞。
  3. 根据权利要求1所述的骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，其特征在于：步骤S4的具体过程为：通过迈克尔加成反应合成CX3-PEG2000-DSPE，然后将CX3-PEG2000-DSPE加到HSPC、DSPE-PEG2000和胆固醇组成的混合物A中混合均匀得到混合物B，然后向混合物B中加入达沙替尼和槲皮素，再经过减压蒸干、水合、超声后，用微孔滤膜过滤，获得载有达沙替尼和槲皮素的适配体CX3修饰的脂质体纳米粒CX3-LS-DQ。
  4. 根据权利要求3所述的骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，其特征在于：混合物A中HSPC、DSPE-PEG2000和胆固醇的质量比为80～150:25～50:1，CX3-PEG2000-DSPE的添加量占混合物B的摩尔比的1～3％。
  5. 根据权利要求3所述的骨关节滑膜成纤维细胞靶向适配体纳米粒构建方法，其特征在于：达沙替尼的终浓度为0.5～2mg/mL，槲皮素的终浓度为2.5～7.5mg/mL。
  6. 基于权利要求1-5任一项构建方法构建的脂质体纳米粒在制备靶向清除骨性关节炎滑膜的药物载体上的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述应用靶向清除的是骨性关节炎滑膜中衰老的纤维状滑膜细胞。
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