WO2023178531A1 - 偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用 - Google Patents

偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用 Download PDF

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WO2023178531A1
WO2023178531A1 PCT/CN2022/082356 CN2022082356W WO2023178531A1 WO 2023178531 A1 WO2023178531 A1 WO 2023178531A1 CN 2022082356 W CN2022082356 W CN 2022082356W WO 2023178531 A1 WO2023178531 A1 WO 2023178531A1
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indocyanine green
oxaliplatin
oxp
albumin
platinum
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French (fr)
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杨红
陈亮
陈华兵
邓益斌
柯亨特
李明
董知妤
吴优璇
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to drug preparation technology, and specifically relates to albumin nanoparticles coupled with indocyanine green and oxaliplatin and their preparation and application.
  • the third generation platinum anti-tumor drug oxaliplatin can treat colon cancer, rectal cancer, etc. It is a product of the interaction between chloroplatinous acid and trans-cyclohexanediamine. It is heated and suspended in water and mixed with silver nitrate. It is obtained by reacting with potassium oxalate and is a commonly used drug in clinical practice.
  • Phototherapy is another tumor treatment method.
  • Indocyanine green (ICG) is a photosensitizer approved by the US FDA. When it is delivered by nanoparticles and accumulated at the tumor site, and is irradiated by laser of a specific wavelength, it can generate heat and singlet oxygen that can kill tumor cells, and has the advantages of safe and minimally invasive tumor treatment.
  • Bovine serum albumin (BSA) is used as a common drug carrier, which can promote drug treatment of tumors with high efficiency and low toxicity.
  • Anticancer drugs are often made into drug-loaded nanopreparations using physical encapsulation methods.
  • oxaliplatin is a water-soluble drug, the encapsulation rate is often not ideal.
  • Another report uses the principle of biomineralization to induce the binding of metal ions to amino acid residues on albumin, allowing the drug to nucleate and grow in the albumin cavity through precipitation reaction or redox. It has the characteristics of mild conditions, high safety, and nanometer Controllable particle size and other advantages.
  • the present invention uses potassium chloroplatinite as raw material, reacts with cyclohexanediamine to prepare a suspension through a new process, reacts with silver nitrate, combines it with BSA, and then adds indocyanine green to prepare a simultaneous coupling Austrian Albumin nanoparticles of thaliplatin and indocyanine green have both phototherapy and chemotherapy effects, and their research has laid the foundation for further development and utilization.
  • An albumin nanoparticle coupled with indocyanine green and oxaliplatin including albumin and indocyanine green and oxaliplatin inside, preferably composed of albumin and indocyanine green and oxaliplatin inside.
  • the composition of thaliplatin; the hydrated particle size of the albumin nanoparticles coupled with indocyanine green and oxaliplatin ranges from 10 to 100 nm, and further ranges from 30 to 70 nm.
  • the preparation method of the above-mentioned albumin nanoparticles coupled with indocyanine green and oxaliplatin is as follows: adding cyclohexanediamine dihydrate platinum ion solution to the albumin solution, then adjusting the system to weak acidity, and then stirring the reaction, Centrifuge again, take the supernatant and perform ultrafiltration to obtain albumin nanoparticles coupled with indocyanine green and oxaliplatin.
  • the molar ratio of platinum and indocyanine green is 1: (1-5), preferably, the molar ratio of platinum and indocyanine green is 1: (1.5-2.5); weak acidity means that the pH is 4.5-6.5, preferably 5.5 ⁇ 6.5; the concentration of albumin solution is 10 ⁇ 30 mg/mL; in the cyclohexanediamine dihydrate platinum ion solution, the concentration of platinum is 55 ⁇ 65 mM.
  • the stirring reaction is carried out at room temperature for 2 to 4 hours, and the stirring is performed using conventional techniques; the centrifugation treatment is performed at 1500 to 4000 centrifuge at r/min for 1 to 10 minutes; for ultrafiltration, centrifuge at 1500 to 5000 r/min for 5 to 15 minutes.
  • the centrifugation process is at 2500 to 3500 r/min for 3 to 6 minutes; for ultrafiltration, centrifuge at 1500 to 2500 r/min for 8 ⁇ 12min.
  • potassium chloroplatinite is reacted with trans-cyclohexanediamine to obtain dichlorocyclohexanediamine platinum; and then dichlorocyclohexanediamine platinum is reacted with silver nitrate in the dark to obtain dihydrate cyclohexanediamine. Hexamethylenediamine platinum ion.
  • the reaction time of potassium chloroplatinite and trans-cyclohexanediamine is 2 to 5 hours; mix platinum dichlorocyclohexanediamine and absolute ethanol and then add water to obtain dichlorocyclohexanediamine.
  • the present invention does not require toxic solvents. It only uses water or water and a small amount of ethanol at room temperature, without heating, and the time is greatly shortened. It can effectively prepare albumin nanoparticles coupled with indocyanine green and oxaliplatin. granules, which facilitates subsequent drug preparation and application.
  • the invention discloses the application of the above-mentioned albumin nanoparticles coupled with indocyanine green and oxaliplatin in the preparation of chemotherapy-phototherapy drugs and anti-cancer drugs.
  • the invention discloses for the first time that dichlorocyclohexanediamine platinum is mixed with absolute ethanol and then water is added to obtain a suspension that achieves excellent suspension effect in a very short time at room temperature, and is mixed with 10% ethanol/water. Under the solvent, 10-50 mg/mLBSA aqueous solution will not appear turbid, indicating that the added ethanol will not affect the preparation of protein nanoparticles; using the role of OXP intermediates and BSA, indocyanine green (ICG) is added, and its carboxyl group replaces OXP.
  • ICG indocyanine green
  • the water molecules in the intermediate molecules form OXP-ICG conjugates (OXP-ICG@BSA) in the BSA cavity, and further grow to obtain OXP-ICG albumin nanoparticles (OXP-ICG) encapsulated by the chemical coupling method.
  • OXP-ICG@BSA OXP-ICG conjugates
  • OXP-ICG albumin nanoparticles OXP-ICG
  • the hydrated particle size is 30-70nm, which is much smaller than the particle size of existing drug delivery systems containing oxaliplatin.
  • the preparation process of the drug-conjugated albumin nanoparticles of the present invention requires normal temperature reaction, mild conditions, simple method, and easy operation.
  • the nanoparticles have been characterized by preparation-related characteristics, and the results show that they meet the requirements of a passive targeted drug delivery system.
  • the in vitro release results show that the nanoparticles have obvious sustained release, and the responsive release of OXP, which needs to be released within tumor cells to exert its effect under simulated tumor cell lysosome conditions, as well as the good temperature-raising properties of the nanoparticles, are all beneficial to subsequent tumor development.
  • Treatment research provides the basis.
  • the new method for preparing oxaliplatin of the present invention is used to prepare drug-conjugated albumin nanoparticles OXP-ICG NPs, which fully exerts the efficient synergistic anti-tumor effect of chemotherapy-phototherapy and provides a basis for anti-tumor research and further development. Utilization lays the foundation; and, with an efficient and rapid suspension preparation process, the level of intermediate preparation is improved.
  • Figure 1 is a schematic diagram of the reaction equation for the preparation of oxaliplatin intermediate.
  • Figure 2 is a schematic diagram of the preparation of oxaliplatin-indocyanine green albumin nanoparticles.
  • Figure 3 is a schematic diagram of preparing oxaliplatin albumin nanoparticles.
  • Figure 4 shows the TEM image of OXP NPs (A) and the hydrated particle size images of OXP NPs and OXP&ICGNPs (B, C).
  • Figure 5 shows the cumulative release curve of OXP.
  • Figure 6 shows the photothermal temperature rise curves of free ICG (A, C) and OXP-ICG NPs (B, D).
  • Figure 7 shows the cytotoxicity of samples from the non-illuminated group (A, C) and the illuminated group (B, D) to CT26 cells.
  • Figure 8 shows the drug uptake by CT26 cells.
  • Figure 9 shows the changes in mitochondrial membrane potential of CT26 cells (A) and the green light/red light intensity ratio (B).
  • the present invention utilizes the effect of OXP intermediate and BSA, and then adds indocyanine green (ICG), whose carboxyl group replaces the water molecules in the OXP intermediate molecule, forming an OXP-ICG conjugate (OXP-ICG@ BSA), and further grown to obtain OXP-ICG albumin nanoparticles (OXP-ICG NPs) encapsulated by chemical coupling method.
  • ICG indocyanine green
  • OXP-ICG@ BSA OXP-ICG conjugate
  • OXP-ICG NPs OXP-ICG albumin nanoparticles
  • OXP@BSA By replacing indocyanine green with potassium oxalate during preparation, OXP@BSA can be formed in the BSA cavity, and further grown to obtain OXP albumin nanoparticles (OXP NPs) encapsulated by the chemical coupling method for control experiments.
  • OXP NPs OXP albumin nanoparticles
  • BSA124S-CW analytical balance (Starious Company, USA); 81-2 constant temperature magnetic stirrer (Shanghai Sile Instrument Factory); 710-OES inductively coupled plasma optical emission spectrometer (Varian Company, USA); Zetasizer Nano ZS90 particle size potential analyzer (Malvern Company, UK); 2700 UV-visible spectrophotometer (Shimadzu Company, Japan); HT7700 transmission electron microscope (Hitachi Company, Japan); ZHWY-100 multi-amplitude orbital shaker (Shanghai Intelligent Technology Co., Ltd.
  • Oxaliplatin (ArkPharm, USA); indocyanine green (ICG, Dalian Meilun Biotechnology Co., Ltd.); potassium chloroplatinite (Bide Pharmaceuticals); cyclohexanediamine, dimethyl sulfoxide (Shanghai McLean Biochemical) Technology Company); elemental platinum standard solution (National Nonferrous Metals and Electronic Materials Analysis and Testing Center, 1000 ⁇ g ⁇ mL -1 ); disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid (Jiangsu Qiangsheng Functional Chemical Company); sodium acetate, concentrated nitric acid ( Sinopharm Chemical Reagent Company); Rhodamine B isothiocyanate (Shanghai Dibai Biotechnology Co., Ltd.); Tetramethylazolium blue (MTT, Shanghai Jianglai Biotechnology Co., Ltd.); Hoechst 33342, Lysotracker, mitochondrial membrane potential Detection kit, trypsin cell digestion solution (Shanghai Bey
  • Synthesis example Dissolve 1g of potassium chloroplatinite (K 2 PtCl 4 , about 2.4 mmol) in 15 mL of water, add 273.6 mg of trans-cyclohexanediamine (about 2.4 mmol) dropwise while stirring, and continue stirring for 3 hours. , filter to collect the precipitate, wash and dry at 35°C to obtain a yellow solid, which is intermediate A (dichlorocyclohexanediamine platinum, about 2.4 mmol).
  • Example 1 Take 916.5 mg of Intermediate A, add 3.9 mL of absolute ethanol, shake in a water bath at 20°C (20Hz) for 2 minutes, add 35.1 mL of water, and obtain a suspension of Intermediate A.
  • the ratio is 1:2, stir the reaction at room temperature for 3 hours, centrifuge at 3000 r ⁇ min -1 for 5 minutes to remove the bottom precipitate, take the supernatant and place it in an ultrafiltration tube, centrifuge at 2000 r ⁇ min -1 for 10 minutes, ultrafiltrate 5 times and remove the excess solution.
  • FIG. 1 is Schematic diagram of the preparation of oxaliplatin-indocyanine green albumin nanoparticles (OXP-ICG NPs).
  • K 2 C 2 O 4 1:2 (mmol/mmol)
  • FIG. 1 is a schematic diagram of the preparation of oxaliplatin albumin nanoparticles (OXP NPs).
  • RhB -OXP NPs solution In addition, take the newly prepared OXP NPs solution, add 5 mg ⁇ mL -1 RhB solution (solvent is DMSO) at 1000:5 (v/v), stir at room temperature for 2 hours, remove free RhB by ultrafiltration, and obtain RhB -OXP NPs solution. For development and tracing experiments.
  • ICG-NPs Preparation of ICG-NPs: Prepare 20 mL of BSA aqueous solution with a concentration of 20 mg ⁇ mL -1 . After adjusting to pH 7.0 with 2 M NaOH aqueous solution, add 5.0 mL of ICG solution (2 mg ⁇ mL -1 ) during the stirring process. After stirring for 1 min, Add hydrochloric acid to adjust the reaction pH to about 5.0, and react at room temperature for 3 hours. After the reaction is complete, centrifuge at 3000 r ⁇ min -1 for 5 minutes to remove the bottom precipitate. Place the supernatant in an ultrafiltration tube and perform ultrafiltration at 2000 r ⁇ min -1 . When the lower filtrate becomes colorless, ICG-NPs are finally obtained. Store in the dark at 4°C. , for control experiments.
  • Control example Take 916.5 mg of Intermediate A, add 3.9 mL of water, shake in a 50°C water bath (20Hz) for 1 hour, add 35.1 mL of water, and obtain a suspension of Intermediate A.
  • Table 1 shows that after using absolute ethanol instead of water, the suspension of Intermediate A can be slightly shaken at room temperature, which greatly shortens the suspension time and greatly improves the efficiency. For pure water, shaking for 1 hour will improve the suspension effect. Still not good.
  • the morphology of the nanoparticles was observed using a transmission electron microscope, and the particle size distribution and Zeta potential of the nanoparticles were measured using a dynamic light scattering particle sizer. See Figure 4 and Table 2.
  • the platinum content was determined using inductively coupled plasma optical emission spectrometer (ICP-OES).
  • Drug loading capacity DL% mass of drug in nanoparticles/mass of freeze-dried powder ⁇ 100%.
  • Figure 4 and Table 2 show that OXP NPs and OXP-ICGNPs have the characteristics of small particle size, good uniformity, and high encapsulation rate, and meet the requirements of passive targeting.
  • Figure 5 shows that free OXP is rapidly released in the lysosomal environment of simulated blood and tumor cells, while the drug release of OXP NPs is significantly slowed down, and the release in the simulated tumor cell environment is faster than that in blood, suggesting targeted delivery and improvement. therapeutic potential.
  • the release is basically complete; however, the release of OXP NPs under two pH conditions conforms to the Weibull equation, and the release is slow (T 1/2 : 13.00 h for pH 5.5, 83.80 h for pH 7.4), and T 1/2 is higher than free OXP Increased by 30.95 ⁇ 178.30 times, the sustained release effect is significant. And after OXP NPs reach the target site of tumor cell lysosomes from the simulated blood, the drug release speed increases by 6.45 times. It is suggested that OXP NPs have the potential to promote the entry and release of drugs into the target site, exert their therapeutic effect, and reduce the metabolism of drugs in the blood.
  • Panels A and B of Figure 6 show that the photothermal heating properties of ICG are concentration-dependent regardless of whether it is in nanoparticles or not. However, when the illumination is the same, the temperature-raising effect of OXP-ICG NPs is significantly greater than that of free ICG. When the concentration is 5 ⁇ g ⁇ mL -1 , the temperature-raising difference of OXP-ICG NPs is more significant than that of free ICG, increasing by about 54.8%.
  • Pictures C and D of Figure 6 show the photothermal stability at the same concentration (5 ⁇ g ⁇ mL -1 ). The temperature rise of free ICG is small and the attenuation is significant.
  • Table 4 IC 50 of samples from the non-illuminated group and the illuminated group.
  • Figure 7 and Table 4 show: 1) The cytotoxicity of the photosensitizer ICG, coupled to the nanoparticles under light, is significantly higher than that of the free group; 2) The cytotoxicity of the chemotherapy drug OXP, coupled to the nanoparticles, is significantly higher In the free group, light has little effect; 3) The free OXP&ICG mixture has good cytotoxicity under light, but after it is made into OXP-ICG NPs, it is significantly improved to the strongest cytotoxicity.
  • IC 50 decreased from 12.23 to 28.56 to 4.77; 4)
  • the calculated synergistic index (CI) of OXP-ICG NPs is 0.56, which meets the existing technology requirement that CI ⁇ 0.8 means a significant synergistic therapeutic effect, indicating that the present invention combines 2
  • FIG. 8 shows that the amount of OXP NPs taken up by tumor cells was significantly higher than free OXP, and it was time-dependent. Among them, the uptake of nanoparticles (2.84 ⁇ g ⁇ 10 -6 cells) in 24 h was 1.45 times that of free OXP (1.96 ⁇ g ⁇ 10 -6 cells). This shows that the superiority of OXP NPs in inhibiting tumor cell growth is related to the significantly increased cell drug uptake.
  • Green light (low matrix membrane potential) was significantly enhanced, increasing green light intensity by 30% and 58.3% respectively (drugs in OXP NPs can increase apoptosis by 94.33%).
  • Statistics on the green and red light intensity ratio show that OXP NPs can convert red light into green light significantly better than OXP. Free OXP is 1.6 times that of PBS, while OXP NPs are 2.4 times that of PBS. This shows that the superiority of OXP among OXP NPs in inhibiting tumor cell growth comes from its ability to enable drugs to better induce tumor cell apoptosis.
  • the present invention uses potassium chloroplatinite as raw material, reacts with trans-cyclohexanediamine, adopts a new process to prepare a suspension, reacts with silver nitrate, combines with BSA, and then adds potassium oxalate or indocyanine green, OXP NPs and OXP-ICG were obtained respectively.
  • NPs and carry out formulation-related characterization and in vitro anti-tumor activity research, which can effectively solve the problems of low bioavailability, poor efficacy, and high toxic and side effects of oxaliplatin.
  • the intermediate A In order to ensure that the intermediate A prepared by potassium chloroplatinite and trans-cyclohexanediamine can react smoothly with silver nitrate in the next step, the intermediate A needs to be dispersed in the solution as much as possible.
  • the existing technology uses heating at 50°C for 1 hour, suspends it in water, and then adds potassium oxalate to prepare oxaliplatin.
  • This invention starts from the same chemical raw materials, adopts a new process to first disperse with a small amount of ethanol at room temperature, and then adds water to achieve suspension within 2 minutes. This not only greatly improves the efficiency, reduces the risk of side reactions, but also provides the basis for subsequent breakthroughs.
  • the usual practice of preparing nanoparticles by physically encapsulating drugs is to prepare drug-conjugated nanoparticles with high efficiency and high quality: OXP NPs, OXP-ICG NPs, played a key role.
  • the preparation process of the drug-conjugated albumin nanoparticles of the present invention requires normal temperature reaction, mild conditions, simple method, and easy operation.
  • the nanoparticles have been characterized by preparation-related characteristics, and the results show that they meet the requirements of a passive targeted drug delivery system.
  • In vitro release results show that the nanoparticles have obvious sustained release and can be released responsively under simulated tumor cell lysosome conditions, which provides a basis for subsequent tumor treatment research.
  • MTT test nanoparticles combined with chemotherapy and phototherapy have significant anti-tumor and anti-tumor synergistic effects on CT26 colon cancer cells. They can increase tumor cells to engulf OXP and enter the cells to exert chemotherapy effects, and significantly increase cell apoptosis. death; thus confirming that OXP-ICG NPs can effectively inhibit the growth of tumor cells and have a synergistic therapeutic effect of chemotherapy and phototherapy.
  • Biomineralization requires metal ions to interact with amino acid residues on proteins to form a nucleation center, which then induces the nucleation and growth of nanoparticles through precipitation reactions or reduction reactions.
  • oxaliplatin is a non-ionic metal complex, which requires The biomimetic synthesis of oxaliplatin albumin nanoparticles can only be achieved by opening up new paths.
  • New method for preparing oxaliplatin nanomedicine of the present invention used for preparing albumin nanoparticles OXP-ICG coupled to the drug NPs give full play to the highly efficient synergistic anti-tumor effect of chemotherapy-phototherapy, laying the foundation for anti-tumor research and further development and utilization; and, with an efficient and rapid suspension preparation process, the level of intermediate preparation is improved.

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Abstract

本发明公开了偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用，其采用新工艺并由生物矿化方法成功制备获得共偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒(OXP-ICG NPs)。本发明纳米粒的制备条件温和，简单易行；经过制剂学相关表征，纳米粒符合被动靶向给药系统要求。本发明的OXP-ICG NPs能够高效抑制肿瘤细胞生长，其具有化疗-光疗的协同治疗效果。

Description

偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用 技术领域
本发明属于药物制备技术，具体涉及偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用。
 背景技术
当前在面对健康大敌癌症时，化疗仍是最主要的治疗手段之一[Siegel Rebecca L,Miller Kimberly D,Fuchs Hannah E, et al. Cancer Statistics[J]. 2021 CA Cancer J Clin, 2021, 71: 7-33]。第三代铂类抗肿瘤药奥沙利铂（OXP），可治疗结肠癌、直肠癌等，由氯亚铂酸与反式环己二胺作用的产物，在水中加热混悬后与硝酸银作用，再与草酸钾作用而得，是临床常用药物。但因其与血浆蛋白结合度高，生物利用度低，毒副作用大，应用受限[RottenbergSven,DislerCarmen,PeregoPaola,The rediscovery of platinum-based cancer therapy[J]. Nat Rev Cancer, 2021, 21: 37-50]。有关奥沙利铂聚合物胶束、脂质体、壳聚糖或白蛋白纳米粒等研究报道，说明研发奥沙利铂新型给药系统的重要性和必要性。光疗为另一种肿瘤治疗方法，吲哚菁绿(ICG)是美国FDA批准上市的光敏剂。当其由纳米粒等递送并蓄积于肿瘤部位，受到特定波长的激光照射时，可产生能杀死肿瘤细胞的热量和单线态氧，具有安全、微创地治疗肿瘤等优势。
牛血清白蛋白（BSA）作常见药物载体，可促进药物高效低毒治疗肿瘤。多以物理包载的方法，将抗癌药制成载药纳米制剂，但是因奥沙利铂属水溶性药物而包封率往往不理想。另有报道利用生物矿化原理诱导金属离子与白蛋白上的氨基酸残基结合，通过沉淀反应或氧化还原，使药物在白蛋白空腔中成核、生长，具有条件温和、安全性高、纳米粒尺寸可控等优点。
 技术问题
本发明以氯亚铂酸钾为原料，与环己二胺反应后以新工艺制得混悬液，与硝酸银作用后，与BSA结合，再加入吲哚菁绿，制得同时偶联奥沙利铂和吲哚菁绿的白蛋白纳米粒，兼具光疗及化疗作用，并对其进行研究，为进一步开发利用奠定基础。
 技术解决方案
一种偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒，包括白蛋白及其内部的吲哚菁绿和奥沙利铂，优选由白蛋白及其内部的吲哚菁绿和奥沙利铂组成；所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的水合粒径为10～100nm，进一步为30～70nm。
上述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法为，向白蛋白溶液中加入二水环己二胺合铂离子溶液，再调节体系为弱酸性，然后搅拌反应，再离心处理，取上清进行超滤，得到偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒。铂、吲哚菁绿的摩尔比为1∶（1～5），优选的，铂、吲哚菁绿的摩尔比为1∶（1.5～2.5）；弱酸性指pH为4.5～6.5，优选为5.5～6.5；白蛋白溶液的浓度为10～30 mg/mL；二水环己二胺合铂离子溶液中，铂的浓度为55～65 mM。优选的，搅拌反应为室温搅拌反应2～4小时，搅拌为常规技术；离心处理为1500～4000 r/min离心1～10min；超滤为1500～5000 r/min离心5～15min，优选的，离心处理为2500～3500 r/min离心3～6min；超滤为1500～2500 r/min离心8～12min。
本发明中，将氯亚铂酸钾与反式环己二胺反应，得到二氯环己二胺合铂；再将二氯环己二胺合铂与硝酸银避光反应，得到二水环己二胺合铂离子。优选的，氯亚铂酸钾与反式环己二胺反应的时间为2～5小时；将二氯环己二胺合铂与无水乙醇混合后再加水，得到二氯环己二胺合铂混悬液，然后加入硝酸银溶液进行避光反应0.5～3小时优选0.5～1.5小时，再过滤，收集滤液为二水环己二胺合铂离子溶液。进一步优选的，将二氯环己二胺合铂与无水乙醇混合0.1～15分钟优选0.5～5分钟后再加水，可以在短时间内形成混悬效果好且稳定的混悬液，优选的，无水乙醇的体积为水体积的5～20%，优选8～15%。与现有技术不同，本发明无需毒性溶剂，仅室温下采用水或者水与少量乙醇，却无需加热，时间也大大缩短，可有效制备偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒，利于后续药物制备及应用。
 有益效果
本发明公开了上述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒在制备化疗-光疗药物、抗癌药物中的应用。
本发明首次公开了将二氯环己二胺合铂与无水乙醇混合后再加水，得到混悬液实现了室温下极短时间下混悬效果优异的效果，且在10%乙醇/水混合溶剂下，10～50mg/mLBSA水溶液都不会出现浑浊，说明加入的乙醇不会影响制备蛋白纳米粒；利用OXP中间体与BSA的作用，再加入吲哚菁绿（ICG），其羧基取代OXP中间体分子中的水分子，在BSA空腔内形成OXP-ICG偶联物（OXP-ICG@BSA），并进一步生长得到化学偶联方法包载的OXP-ICG白蛋白纳米粒（OXP-ICG NPs），水合粒径为30～70nm，较现有包载奥沙利铂的给药系统粒径小得多。
本发明偶联药物的白蛋白纳米粒的制备工艺，常温反应，条件温和，方法简单，易于操作，纳米粒通过制剂相关表征，结果表明符合被动靶向给药系统的要求。体外释放结果表明，纳米粒缓释明显，且在模拟肿瘤细胞溶酶体条件下需要在肿瘤细胞内释放才能发挥作用的OXP可响应性释放，以及纳米粒良好的升温性能，均为后续的肿瘤治疗研究提供了依据。细胞试验中，MTT试验，化疗-光疗结合的纳米粒，对CT26结肠癌细胞，具有显著的抑瘤作用和抗肿瘤协同作用（协同指数CI为0.56，现有技术认为CI＜0.8即有显著的协同治疗效果），可增加肿瘤细胞吞噬OXP进入细胞发挥化疗作用，并大幅增加细胞凋亡；从而证实了OXP-ICG NPs高效抑制肿瘤细胞生长，具有化疗-光疗的协同治疗效果。
总之，本发明制备奥沙利铂的新方法，用于制备偶联药物的白蛋白纳米粒OXP-ICG NPs，充分发挥了化疗-光疗的高效协同抗肿瘤作用，为抗肿瘤研究和进一步的开发利用奠定基础；并且，以高效快速的混悬液制备工艺，实现了中间体制备水平的提升。
 附图说明
图1为奥沙利铂中间体的制备反应方程式示意图。
图2为制备奥沙利铂-吲哚菁绿白蛋白纳米粒示意图。
图3为制备奥沙利铂白蛋白纳米粒示意图。
图4为OXP NPs的TEM图(A)及OXP NPs和OXP&ICGNPs的水合粒径图(B、C)。
图5为OXP的累计释放曲线。
图6为游离ICG(A、C)、OXP-ICG NPs(B、D)光热升温曲线图。
图7为非光照组（A、C）和光照组（B、D）样品对CT26细胞的细胞毒性。
图8 为CT26细胞对药物的摄取量。
图9为CT26细胞线粒体膜电位变化情况(A)及绿光/红光强度比值(B)。
 本发明的实施方式
本发明利用OXP中间体与BSA的作用，再加入吲哚菁绿（ICG），其羧基取代OXP中间体分子中的水分子，在BSA空腔内形成OXP-ICG偶联物（OXP-ICG@BSA），并进一步生长得到化学偶联方法包载的OXP-ICG白蛋白纳米粒（OXP-ICG NPs），水合粒径为30～70nm，较现有包载奥沙利铂的给药系统粒径小得多。制备时通过用草酸钾替代吲哚菁绿，可在BSA空腔内形成OXP@BSA，并进一步生长得到化学偶联方法包载的OXP白蛋白纳米粒（OXP NPs），供对照实验用。
BSA124S-CW分析天平（美国Starious公司）；81-2型恒温磁力搅拌器（上海司乐仪器厂）；710-OES电感耦合等离子体发射光谱仪（美国瓦里安公司）；Zetasizer Nano ZS90粒径电位分析仪（英国马尔文公司）；2700型紫外-可见分光光度计（日本岛津公司）；HT7700透射电子显微镜（日本日立公司）；ZHWY-100多振幅轨道摇床（上海智诚分析仪器公司）；710型共聚焦荧光扫描显微镜（德国卡尔蔡司公司）；多功能酶标仪（瑞士，TACAN）；透析袋（MWCO: 3.5 kDa，国药集团化学试剂公司）；超滤离心管（Millipore YM-10，MWCO: 100 kDa，苏州科赛恩生物科技公司）。
奥沙利铂（美国ArkPharm）；吲哚菁绿（ICG，大连美仑生物技术公司）；氯亚铂酸钾（毕徳医药）；环己二胺、二甲基亚砜（上海麦克林生化科技公司）；单质铂标准品溶液（国家有色金属及电子材料分析测试中心，1000μgŸmL -1）；磷酸氢二钠、磷酸二氢钠、乙酸（江苏强盛功能化学公司）；乙酸钠、浓硝酸（国药集团化学试剂公司）；罗丹明B异硫氰酸酯（上海笛柏生物科技公司）；四甲基偶氮唑蓝（MTT，上海江莱生物科技公司）；Hoechst 33342、Lysotracker、线粒体膜电位检测试剂盒、胰酶细胞消化液（上海碧云天生物技术公司）；DMEM高糖培养基（Hyclone，美国Thermo公司）。
小鼠结肠癌细胞（CT26，苏州大学）。
合成例：取1g氯亚铂酸钾（K 2PtCl 4，约2.4 mmol）溶于15 mL水，搅拌下，滴加反式环己二胺273.6 mg（约2.4mmol），继续搅拌反应3h后，过滤取沉淀，洗涤后35℃烘干得黄色固体，即中间体A（二氯环己二胺合铂，约2.4 mmol）。
实施例一：取916.5 mg中间体A，加无水乙醇3.9 mL，20℃水浴振荡（20Hz）2min，加水35.1mL，得到中间体A混悬液。
向所得中间体A混悬液中，加入1mL硝酸银水溶液（820 mgŸmL -1，约4.8 mmol），避光反应1h，过滤，收集滤液（约40mL）。取0.5mL滤液加5%氯化钠水溶液不产生沉淀，说明其中不含游离Cl -。滤液为OXP中间体溶液（二水环己二胺合铂离子，以Pt计约60 mM），备用，反应方程式示意如图1。
称取200mg BSA，加水10mL溶解制得BSA溶液（20 mgŸmL -1），常规搅拌下向BSA溶液中加入1 mL所得OXP中间体溶液（约0.06 mmol），再用2 M NaOH水溶液调节至pH 6.0后，加入ICG溶液（称取93.40mgICG，约0.12 mmol，配成2mgŸmL -1水溶液），使体系中OXP中间体∶ICG=1∶2（mmol/mmol），即铂、吲哚菁绿的摩尔比为1∶2，室温搅拌反应3h，3000 rŸmin -1离心5min去除底部沉淀，取上清置于超滤管中，2000 rŸmin -1离心10 min，经5次超滤并除去多余的溶液，保留溶液体积为10 mL，并收集于离心管中，即为OXP-ICG NPs溶液A（0.2 mgŸmL -1，以Pt计；1 mgŸmL -1，以ICG计），4℃保存备用；图2为制备奥沙利铂-吲哚菁绿白蛋白纳米粒（OXP-ICG NPs）示意图。
另外，采用本发明的中间体A混悬新工艺后，不会影响以浓度为20 mg/mL的BSA溶液（溶剂成分10%乙醇/水）制备白蛋白纳米粒的结果，向BSA溶液（20 mgŸmL -1）中加入1 mL所得OXP中间体溶液，形成的体系稳定，没有浑浊。
对比例： 称取800 mg BSA，加水40mL溶解制得BSA溶液（20 mgŸmL -1）。搅拌下向其中加入20 mL所得OXP中间体溶液（约1.2 mmol），再用2 M NaOH水溶液调节至pH 6.0后，加入1mL草酸钾溶液（K 2C 2O 4 ，440 mgŸmL -1，约2.4 mmol），使体系中OXP中间体∶K 2C 2O 4=1∶2（mmol/mmol），室温搅拌反应3h后，3000 rŸmin -1离心5min去除底部沉淀，取上清置于超滤管中，2000 rŸmin -1离心10 min，经5次超滤并除去多余溶液，保留溶液体积为6 mL，并收集于离心管中，即为OXP NPs溶液（5.85 mgŸmL -1，以Pt计约30 mM），4℃保存备用；图3为制备奥沙利铂白蛋白纳米粒（OXP NPs）示意图。
另外，取新制备的OXP NPs溶液，以1000∶5（v/v）向其中加入5 mgŸmL -1RhB溶液（溶剂为DMSO），室温搅拌2 h后，超滤法除去游离RhB，即得RhB-OXP NPs溶液。供显影示踪实验用。
ICG-NPs制备：配制20 mL浓度为20 mgŸmL -1 BSA水溶液，用2 M NaOH水溶液调节至pH 7.0后，在搅拌过程中加入5.0 mL ICG溶液（2 mgŸmL -1），搅拌反应1 min后，加入盐酸调节反应pH值为5.0左右，常温反应3 h。待反应完全后，3000 rŸmin -1离心5min去除底部沉淀，取上清置于超滤管中，2000 rŸmin -1超滤，待下层滤液呈无色，最终得到ICG-NPs，4℃避光保存，供对照实验用。
对照例：取916.5 mg中间体A，加水3.9 mL，50℃水浴振荡（20Hz）1h，加水35.1mL，得到中间体A混悬液。
取916.5 mg中间体A，加水3.9 mL， 20℃水浴振荡（20Hz）1h，加水35.1mL，得到中间体A混悬液。
观察各中间体A的混悬情况，结果如表1，混悬情况的判断，通过观察表象，混悬差的是仍有块状物贴在容器底部，且不能与水浸润，而好的混悬情况是固体物不结块，底部无沉淀，均匀分布在体系中。
表1 混悬中间体A条件比较结果： 。
表1显示，用无水乙醇替代水后，中间体A的混悬，室温稍稍振荡即可，大大缩短了混悬时间，极大地提高了效率；而纯水的话，震荡1小时，混悬效果仍然不佳。
纳米粒的形态、粒径及Zeta电位、包封率和载药量考察。
分别采用透射电镜观察纳米粒形态，用动态光散射粒径仪测定纳米粒的粒径分布及Zeta电位，参见图4、表2。采用电感耦合等离子体发射光谱仪（ICP-OES）测定铂的含量。
取浓度为1000 μgŸmL -1单质铂标准品溶液，用3%硝酸溶液配成0、1、2、5、10 μgŸmL -1的系列铂标准溶液（n=3），测定并利用ICP-OES软件得到结果。
采用分光光度法建立测定ICG的标准曲线。称取ICG粉末1.00 mg，溶于10.0 mL的DMSO中配成储备液，再用DMSO稀释成浓度为0.2、0.5、1.0、2.0、4.0、8.0μgŸmL -1的ICG标准溶液（n=3），在785 nm测定其吸光度值并计算。建立检测ICG的标准曲线为y=0.1771x+0.0039，R²=0.9999，线性关系良好。
包封率测定，超滤新制OXP NPs、OXP-ICG NPs溶液，分别收集超滤管上层、下层液体，并精密量取其体积，取上层、下层液体各20μL，加入180 μL浓硝酸，超声硝解破坏纳米粒的蛋白结构，用水稀释至6 ml，过0.22μm滤膜，ICP-OES测定溶液中的铂浓度，计算溶液中药物的浓度及纳米粒的包封率。包封率=M1/(M1+M2)×100%（其中，M1是超滤管上层液体药物量，M2是超滤管下层液体药物量）。
载药量测定，将新制定量过的OXP NPs溶液冻干后，精密称取0.30g用5.0mL水分散，ICP-OES测定铂浓度，并计算纳米粒的载药量。载药量DL% = 纳米粒中药物质量/冻干粉质量´ 100%。
表2 纳米粒的表征结果（n=3）。
。
图4、表2显示，OXP NPs、OXP-ICGNPs具有粒径小且均一性好、包封率高等特性，符合被动靶向要求。
纳米粒的体外释放行为考察。以游离药物OXP为对照，采用透析法考察。将OXP NPs溶液、同浓度游离OXP溶液分别置于透析袋中，置于模拟的血液环境的pH 7.4 PBS、肿瘤细胞溶酶体内环境的pH 5.5 PBS中，37℃、120 r·min -1透析，分别在0.5、1、2、4、8、12、24h取出释放液，同时补充等体积的缓冲液，用ICP-OES测定铂的浓度，并计算OXP的累积释放量、考察释放规律和T 1/2。
表3 OXP的释放规律拟合方程。
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图5显示，游离OXP在模拟血液、肿瘤细胞的溶酶体环境中均快速释放，而OXP NPs释药显著减缓，在模拟肿瘤细胞环境中较血液中的释放加快，提示具有靶向传输和提高疗效的潜力。
分别采用零级方程、一级方程、Higuchi方程和Weibull方程对测定结果进行拟合，得到的释放规律，结果见表3。游离 OXP在模拟的血液、肿瘤细胞溶酶体环境中，释放均符合一级方程，且突释明显，T 1/2相似（pH 5.5为0.42 h，pH 7.4为0.47 h），2 h左右均基本释放完全；然而，OXP NPs在2种pH条件下的释放均符合Weibull方程，且释放缓慢（T 1/2：pH 5.5为13.00 h，pH 7.4为83.80 h），T 1/2比游离OXP增加30.95~178.30倍，缓释效果显著。并且OXP NPs从模拟的血液中到达靶部位肿瘤细胞溶酶体后，释药速度提高6.45倍。提示OXP NPs具有促进药物进入靶部位并释放、发挥疗效，减少药物在血液中代谢的潜力。
纳米粒光热升温及光热稳定性考察。分别取游离ICG和OXP-ICG NPs溶液，用超纯水，配制ICG浓度为0、1、2、5、10、20 μgŸmL -1的溶液避光备用。用超纯水作为对照，取每个浓度的溶液0.5mL，用0.5 W cm -2的785nm激光照射5min，同时用数字温度计监测溶液的温度，每隔30s记录一次。
纳米粒的光热稳定性考察取ICG浓度为5μgŸmL -1的游离ICG和OXP-ICG NPs溶液0.5 mL，以785 nm激光器(0.5 W cm -2)光照5 min，每30 s记录一次溶液温度。关闭激光器，待溶液冷却到室温后，以相同条件再次照射5 min，如此反复操作5次并记录。
图6的A图、B图显示，ICG无论是否处于纳米粒中，光热升温性质均具有浓度依赖性。然而，当光照相同时，OXP-ICG NPs的升温效果显著大于游离ICG，当浓度为5μgŸmL -1时，OXP-ICG NPs较游离ICG的升温差异更加显著，提高约54.8%。图6的C图、D图显示，在同一浓度（5μgŸmL -1）下的光热稳定性，游离ICG升温幅度小且衰减显著。而OXP-ICG NPs前3次升温幅度大，且相对稳定，由初始温度20℃升到31.8±0.5℃，说明ICG与纳米粒偶联后使光稳定性显著提高。
纳米粒对CT26结肠癌细胞的毒性考察。采用噻唑蓝（MTT）法考察OXP-ICG NPs对小鼠结肠癌CT26细胞的毒性作用。取对数生长期的CT26细胞，以细胞密度5000个/孔接种于96孔板中，每孔中加入100mL培养基，孵育（37 oC，5% CO 2）24h，用新鲜培养基稀释游离药物ICG、OXP、OXP&ICG溶液，以及白蛋白纳米粒ICGNPs、OXPNPs、OXP-ICG NPs溶液，浓度依次为 0、2、4、8、16、32 mM（以ICG浓度计，ICG:OXP=5:4w/w；0浓度用PBS替代纳米粒溶液），加入100mL/孔含药培养基孵育24 h后，更换培养基，1）非光照组，继续孵育24 h；2）光照组，785 nm激光器（0.5 W cm -2，3 min），继续孵育24 h。然后，更换培养基，避光下加入100 mL0.5 mgŸmL -1MTT溶液，孵育4 h，弃去液体，加入100mL /孔DMSO，振荡混匀，用酶标仪测490 nm处吸光度值Abs，计算细胞存活率（%）=Abs 实验组/Abs 对照组。将各组细胞存活率、给药浓度输入IC 50计算器，算得IC 50值。
表4 非光照组和光照组样品的IC 50。
。
图7、表4显示：1）光敏剂ICG，光照下，偶联于纳米粒中的细胞毒性，显著高于游离组；2）化疗药OXP，偶联于纳米粒中的细胞毒性，显著高于游离组，而光照影响甚微；3）游离OXP&ICG混合物，光照下的细胞毒性尽管不错，但将其制成OXP-ICG NPs后，显著提升为最强细胞毒性，与各对照组相比，IC 50由12.23~28.56降低为4.77；4）经计算OXP-ICG NPs的协同指数（CI）为0.56，满足现有技术认为CI＜0.8即有显著的协同治疗效果的要求，说明本发明联合2种治疗手段，同时偶联ICG和OXP的白蛋白纳米粒，具有显著的协同抗肿瘤效果，本发明纳米药物具有优异的细胞毒性。
细胞摄取量考察。将CT26细胞与药物共孵育，考察不同时间点的摄取情况。取对数生长的CT26细胞（密度5´10 5个/孔）接种于6孔细胞培养板中，孵育（条件同上述MTT毒性考察）24 h至细胞完全贴壁后，分别提前6、12、24 h向各孔中加入游离OXP和OXP NPs（均以Pt浓度计：5mgŸmL -1，n=3），孵育时间到后，细胞用PBS洗涤并重悬，测定计数 [9]。超声（500 W，5min）破碎细胞，加入硝酸硝解过夜，离心，取上清过滤后用ICP-OES检测Pt浓度，并计算细胞摄取量。图8显示，肿瘤细胞摄取OXP NPs的量显著高于游离OXP，且呈现时间依赖性。其中，24 h纳米粒（2.84 μgŸ10 -6cells）摄取量为游离OXP（1.96 μgŸ10 -6cells）的1.45倍。说明OXP NPs在抑制肿瘤细胞生长上的优越性，与显著增加的细胞摄药量有关。
线粒体膜电位变化考察。肿瘤细胞能量中枢线粒体的膜电位下降是细胞早期凋亡的标志之一。考察通过改变线粒体的膜电位，研究药物诱导肿瘤细胞凋亡而发挥杀伤作用。在其膜电位较高时，JC-1聚集在线粒体的基质中，形成聚合物（产生红色荧光）；在其膜电位较低时，JC-1不能在基质中聚集（JC-1为单体，产生绿色荧光）。故可用共聚焦显微镜观察给药前后颜色变化，考察其膜电位的。变化和纳米粒诱导细胞凋亡的能力。取对数生长期的CT26细胞，以细胞密度1´10 5个/孔接种于confocal玻底培养皿中，加入1 mL/孔新配培养基，孵育（条件同上述MTT毒性考察），设置PBS、游离OXP、OXP NPs三组，加入1 mL/孔培养基稀释的样品溶液（Pt浓度同3.2），孵育24 h后，弃去液体，用PBS洗涤后，加入线粒体膜电位探针JC-1染色工作液（10 mgmL -1），混匀，避光孵育20 min，弃去液体，用JC-1染色缓冲液洗涤2次，加入1 mL PBS稀释的细胞核染色液Hoechst 33342，避光孵育5 min，移除染色液，洗涤后，立刻用激光共聚焦显微镜观察。图9显示，与PBS相比，游离OXP、OXP NPs可使线粒体的红光（基质膜电位高）明显减弱（基质膜电位由高到低，出现凋亡），二者分别使红光强度减弱19.8%、35%（药物在OXP NPs中可增加凋亡76.77%）。绿光（基质膜电位低）显著增强，分别使绿光强度增加30%、58.3%（药物在OXP NPs中可增加凋亡94.33%）。统计绿色与红光强度比值，可知OXP NPs使红光转变为绿光显著高于OXP，游离OXP为PBS的1.6倍，而OXP NP为PBS的2.4倍。说明OXP在OXP NPs中，对肿瘤细胞生长抑制的优越性，源自其能够使药物更好地诱导肿瘤细胞凋亡。
本发明以氯亚铂酸钾为原料，与反式环己二胺反应后，采用新工艺制得混悬液，与硝酸银作用，并与BSA结合，再加入草酸钾或吲哚菁绿，分别得到OXP NPs、OXP-ICG NPs，并对其进行制剂相关表征和体外抗肿瘤活性研究，能有效解决奥沙利铂生物利用度低，疗效差，毒副作用大等问题。
为保证氯亚铂酸钾与反式环己二胺制得的中间体A，下一步顺利与硝酸银反应，需要将中间体A尽量分散在溶液中。现有技术采用50℃加热1h将其混悬于水中，然后加入草酸钾，制得奥沙利铂。本发明以同样的化学原料出发，采用新工艺在室温下以少量乙醇先分散、再加水，实现了在2min内完成混悬，不仅大大提高了效率，降低了副反应风险，而且为接下来突破通常以物理包载药物制备纳米粒的惯例，高效率、高质量地制备偶联药物的纳米粒：OXP NPs、OXP-ICG NPs，发挥了关键性作用。
本发明偶联药物的白蛋白纳米粒的制备工艺，常温反应，条件温和，方法简单，易于操作，纳米粒通过制剂相关表征，结果表明符合被动靶向给药系统的要求。体外释放结果表明，纳米粒缓释明显，且在模拟肿瘤细胞溶酶体条件下可响应性释放，为后续的肿瘤治疗研究提供了依据。细胞试验中，MTT试验，化疗-光疗结合的纳米粒，对CT26结肠癌细胞，具有显著的抑瘤作用和抗肿瘤协同作用，可增加肿瘤细胞吞噬OXP进入细胞发挥化疗作用，并大幅增加细胞凋亡；从而证实了OXP-ICG NPs高效抑制肿瘤细胞生长，具有化疗-光疗的协同治疗效果。
生物矿化需金属离子与蛋白质上的氨基酸残基作用形成成核中心，进而通过沉淀反应或还原反应等诱导纳米粒的成核与生长，然而奥沙利铂为非离子金属配合物，要实现奥沙利铂白蛋白纳米粒的仿生合成，必须开辟新的路径的方法才能实现。本发明制备奥沙利铂纳米药物的新方法，用于制备偶联药物的白蛋白纳米粒OXP-ICG NPs，充分发挥了化疗-光疗的高效协同抗肿瘤作用，为抗肿瘤研究和进一步的开发利用奠定基础；并且，以高效快速的混悬液制备工艺，实现了中间体制备水平的提升。

Claims (10)

  1. 一种偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒，其特征在于，包括白蛋白及其内部的吲哚菁绿和奥沙利铂。
  2. 根据权利要求1所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒，其特征在于，所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的水合粒径为10～100nm。
  3. 权利要求1所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，向白蛋白溶液中加入二水环己二胺合铂离子溶液，再调节体系为弱酸性，然后搅拌反应，再离心处理，取上清进行超滤，得到偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒。
  4. 根据权利要求3所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，铂、吲哚菁绿的摩尔比为1∶（1～5）。
  5. 根据权利要求3所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，白蛋白溶液的浓度为10～30 mg/mL；二水环己二胺合铂离子溶液中，铂的浓度为55～65 mM。
  6. 根据权利要求3所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，离心处理为1500～4000 r/min离心1～10min；超滤为1500～5000 r/min离心5～15min。
  7. 根据权利要求3所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，将氯亚铂酸钾与反式环己二胺反应，得到二氯环己二胺合铂；再将二氯环己二胺合铂与硝酸银避光反应，得到二水环己二胺合铂离子。
  8. 根据权利要求7所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒的制备方法，其特征在于，将二氯环己二胺合铂与无水乙醇混合后再加水，得到二氯环己二胺合铂混悬液，然后加入硝酸银溶液进行避光反应0.5～3小时，再过滤，收集滤液为二水环己二胺合铂离子溶液。
  9. 权利要求1所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒在制备化疗-光疗药物中的应用。
  10. 权利要求1所述偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒在制备抗癌药物中的应用。
PCT/CN2022/082356 2022-03-22 2022-03-22 偶联吲哚菁绿和奥沙利铂的白蛋白纳米粒及其制备与应用 WO2023178531A1 (zh)

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