WO2022016555A1 - 载铂类药物/光敏剂的蛋白纳米粒及其制备方法和应用 - Google Patents

载铂类药物/光敏剂的蛋白纳米粒及其制备方法和应用 Download PDF

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WO2022016555A1
WO2022016555A1 PCT/CN2020/104637 CN2020104637W WO2022016555A1 WO 2022016555 A1 WO2022016555 A1 WO 2022016555A1 CN 2020104637 W CN2020104637 W CN 2020104637W WO 2022016555 A1 WO2022016555 A1 WO 2022016555A1
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platinum
nanoparticles
photosensitizer
protein
based drug
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PCT/CN2020/104637
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French (fr)
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杨红
徐涛
陈华兵
张米娅
陈亮
邓益斌
姚佳璐
罗嘉丽
翟艳华
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苏州大学
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Priority to US18/012,295 priority Critical patent/US20230364239A1/en
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Publication of WO2022016555A1 publication Critical patent/WO2022016555A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0061—5-aminolevulinic acid-based PDT: 5-ALA-PDT involving porphyrins or precursors of protoporphyrins generated in vivo from 5-ALA
    • 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
    • A61K31/409—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 having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61K47/69—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
    • A61K47/6921—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 the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • 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 present invention relates to the preparation of albumin nanoparticles with dual therapeutic effects encapsulating platinum drugs and photosensitizers, which can be used as a new dosage form of antitumor drugs to realize combined treatment of tumors and inhibit tumor metastasis.
  • Nano-materials are prepared by using biological macromolecular proteins as carriers, which have the advantages of good biocompatibility, safety and non-toxicity.
  • drug carrier materials drugs can be selectively targeted Therefore, it has an important position and research value in the pharmaceutical research of nano-drug carriers and anti-tumor.
  • Albumin also known as albumin, is the most abundant protein in plasma.
  • Human serum albumin Human Serum Albumin, HSA
  • HSA Human Serum Albumin
  • Non-glycosylated single-chain polypeptide contains 585 amino acids, acidic protein with an isoelectric point of 4.7 ⁇ 4.9, easily soluble in water, and its concentration in plasma is 42 g/L, accounting for about 60% of total plasma protein.
  • Platinum drugs have been studied for more than 50 years. In 1965, American scholar Rosenberg and others accidentally discovered that cisplatin complexes can inhibit cell proliferation and can be used as antitumor drugs. Since then, platinum-based drugs have quickly attracted people's attention, and research on platinum-based anti-tumor drugs has also become a hot spot. Today, the research on platinum drugs has developed to the fourth generation, among which cisplatin, carboplatin and oxaliplatin have been used in clinical practice. The important precursor for the synthesis of these platinum drugs is dihydrate diamine platinum nitrate. , so: dihydrate diamine platinum ion is also known as cisplatin precursor ion.
  • Cisplatin also known as cis-dichlorodiammine, Cis-Dichlorodiammine platinum(II), CDDP for short
  • Cisplatin plays an important role in tumor chemotherapy.
  • Cisplatin has a wide spectrum of anticancer properties and its application. It is mainly used in the treatment of testicular cancer, ovarian cancer, bladder cancer, head and neck cancer, esophageal cancer and small cell lung cancer.
  • doxorubicin, paclitaxel, 5-fluorouracil (5-Fu), etc. it can treat head and neck cancer and gastric cancer.
  • clinical high-dose and long-term use are limited by its severe toxicity, low solubility and drug resistance.
  • Photodynamic therapy (PDT) in tumor treatment is a new technology for disease diagnosis and disease treatment using photodynamic reactions after the tumor tissue takes up photosensitizers.
  • PDT has 3 important factors: excitation light source, photosensitizer and reactive oxygen species.
  • the mechanism of action is as follows: the photosensitizer molecule absorbs the photon energy of the corresponding wavelength light and changes from the ground state to the singlet excited state, and the excited state photosensitizer molecule can return to the ground state through the physical de-excitation process, and generate fluorescence for clinical diagnosis, that is, fluorescence. It can also be converted into a triplet excited state, transferring energy to adjacent molecular oxygen to generate reactive oxygen species (ROS) to induce autophagy, apoptosis and necrosis of target organ cells.
  • ROS reactive oxygen species
  • Ce6 is commonly used in PDT, which is activated by near-infrared light (NIR) light, can be eliminated relatively quickly from the body, and has high reactive oxygen species generation efficiency, and Ce6 can also be used for NIR fluorescence imaging, with a wavelength range of 650 ⁇ 900 nm to avoid interference from endogenous chromophores in vivo.
  • NIR near-infrared light
  • Ce6 has poor stability under physiological conditions and poor fluorescence quantum yield ( ⁇ f ) and photosensitivity.
  • the purpose of the present invention is to provide a platinum drug/photosensitizer-loaded protein nanoparticle and its preparation method and application.
  • the present invention prepares HSA nanoparticles under mild reaction conditions, especially through suitable prescription and process, realizes effective control of particle size, and improves tumor targeting and curative effect.
  • the invention Based on the protein template, the invention prepares the protein nanoparticle with the combined treatment effect of chemotherapy and photodynamics through biomimetic synthesis for research.
  • the chemotherapeutic drug cisplatin precursor ion and the photosensitizer Ce6 were co-precipitated in the inner cavity of albumin, and further nucleated and grown to prepare bifunctional small-sized albumin with chemotherapeutic/photodynamic therapy effects. Nanoparticles can improve the delivery efficiency of two poorly water-soluble drugs, enhance the drug uptake ability of tumor cells and the accumulation of drugs in cells.
  • Ce6 After the protein nanoparticle enters the lysosome of tumor cells, Ce6 generates significant photodynamic force under the excitation of near-infrared light, and can also induce lysosome rupture through ROS, and at the same time promote the transport of platinum drugs into the cytoplasm and then into the nucleus, and use
  • the active form of divalent platinum exists, which enhances the effect of chemotherapy, so as to achieve a synergistic effect in the combined treatment of tumors; when used in vivo, it shows better tumor targeting in vivo, and it exerts the chemotherapy effect of platinum drugs to inhibit tumors.
  • the effect of completely eliminating tumors and inhibiting tumor metastasis can be achieved; especially nanoparticles can be affected by small particle size ( ⁇ 10% in vivo)
  • the renal clearance effect produced by nm) is efficiently metabolized with good biosafety.
  • the present invention adopts the following technical solutions.
  • Platinum-based drug/photosensitizer-loaded protein nanoparticles including platinum-based drug/photosensitizer complexes, and proteins encapsulating platinum-based drug/photosensitizer complexes.
  • the protein is albumin, preferably human serum albumin; the platinum drug is dihydrate platinum ion, and the photosensitizer is chlorin e6.
  • the quantitative determination of platinum element is adopted, and cisplatin is used as a control, and the result is reliable; in cell and in vivo experiments, cisplatin is used as a control, and reliable results are obtained.
  • the prior art cannot directly prepare protein nanoparticles encapsulating diamine platinum ions, nor can prepare nanoparticles encapsulating cisplatin and Ce6 at the same time.
  • the present invention adopts safe HSA to co-encapsulate diamine platinum ion and photosensitizer Ce6, and has the advantages of mild conditions, simple process, small and controllable particle size, good biocompatibility, and tumor targeting. It is a new type of albumin nanoparticles that integrates chemotherapy and photodynamic therapy, and realizes the preparation and application of albumin nanoparticles that can treat tumors with high efficiency and low toxicity and inhibit tumor metastasis.
  • the invention discloses a method for preparing the above-mentioned platinum-based drug/photosensitizer-loaded protein nanoparticle, comprising the following steps: adding a chlorin e6 solution into a mixed solution of diamine platinum ion and protein, and performing ultrafiltration after the reaction. Centrifuge to obtain platinum-based drug/photosensitizer-loaded protein nanoparticles.
  • the invention selects protein nano-carriers, and simultaneously encapsulates platinum drugs and photosensitizers, so as to realize the dual-targeted therapeutic effect on tumors combining chemotherapy toxicity and phototoxicity in one nano-drug-loading platform.
  • the particle size of the platinum drug/photosensitizer-loaded protein nanoparticles is 2 ⁇ 50 nm, and the hydrated particle size is 20 ⁇ 150 nm. nm.
  • the dosage ratio of protein, diammine platinum ion and chlorin e6 is 100 mg: (20-50 ⁇ mol): (3-15 ⁇ mol), preferably 100 mg: (25-40 ⁇ mol) ): (5 ⁇ 10 ⁇ mol); add the chlorin e6 solution to the mixed solution of diammine platinum ion and protein, adjust the pH to 4.0 ⁇ 8.0, preferably 5.0 ⁇ 6.5, and then adjust the pH to 4.0 ⁇ 6.5 at 25 ⁇ 60°C.
  • the reaction is carried out for 1 ⁇ 8 h; the rotation speed of ultrafiltration and centrifugation is 1500 ⁇ 4000 r ⁇ min -1 .
  • the protein nanoparticle prepared by adopting safe HSA in the present invention is ideal in particle size, chemical stability, light stability and active oxygen generation ability, and has a great application prospect in tumor treatment.
  • the invention discloses the application of the above-mentioned platinum-based drug/photosensitizer-loaded protein nanoparticles in the preparation of drugs, specifically anti-tumor drugs, and further dual anti-tumor drugs for chemotherapy/photodynamic therapy.
  • the nanoparticle prepared by the invention has good tumor targeting, good retention in the tumor, and shows strong toxicity to tumor cells, and has a synergistic effect when combined with chemotherapy/photodynamic therapy under the condition of near-infrared illumination. It enhances the effect of chemotherapy, can effectively eliminate tumors, significantly inhibit tumor growth and metastasis, and exhibits high-efficiency, low-toxicity and anti-tumor effects with both chemotherapy and photodynamic therapy. It is a safe and effective new nano preparation.
  • HSA is an endogenous substance, does not produce toxicity and inflammatory response, has good stability and unique spatial structure, increases the solubility of poorly soluble drugs, has a good protective effect on easily oxidized drugs, and can significantly Extend drug half-life, improve targeting, significantly reduce drug toxicity, and because the energy and nutrient sources required for tumor growth depend on albumin, endogenous albumin has aggregation effect in tumor tissue, and HSA is used as drug carrier to prepare drug delivery system
  • the protein nano-drug delivery system designed in the present invention encapsulates platinum dihydrate dihydrate ions to form adducts with DNA, thereby inhibiting DNA replication and transcription, leading to DNA fragmentation and miscoding, and inhibiting cell mitosis, so the development of relevant new preparations is very important. significance.
  • Combination therapy is an effective anti-tumor strategy that combines different anti-cancer drugs or different treatment methods to achieve synergistic anti-cancer efficacy in a multi-pronged approach to overcome and reduce drug resistance and toxic side effects and improve drug efficacy.
  • the current clinical platinum combination therapy strategies are mostly the combined application of different chemotherapy drugs. However, in many cases, the combined therapy effect is not good. Although the direct combination of existing drugs can adjust the dose during administration, its metabolism Different results are less effective and sometimes more toxic.
  • the nanomedicine of the present invention has less toxicity and adverse reaction, less trauma, definite curative effect, no drug resistance, and the photosensitizer (cytotoxic agent in PDT) can absorb light of a specific wavelength and convert it into useful energy, Strong absorbance with high extinction coefficient at longer wavelengths (600 ⁇ 850 nm), large tissue penetration of light, and sufficient activity to generate ROS, excellent photochemical reactivity, high triplet yield and long triplet It has a long lifespan and is able to efficiently generate ROS, has minimal dark toxicity, and is only cytotoxic in the presence of light, is preferentially retained by target tissues, and is rapidly excreted from the body, causing low systemic toxicity.
  • the photosensitizer cytotoxic agent in PDT
  • the prior art protein encapsulates organic molecules, and the particle size is difficult to control within the ideal range, and the protein nanoparticles reported in the prior art need to use strong alkaline conditions (such as pH 12), such as bovine serum albumin BSA to prepare encapsulated Gd 2 O 3 /Ce6 nanoparticles integrated research on diagnosis and treatment.
  • strong alkaline conditions such as pH 12
  • bovine serum albumin BSA bovine serum albumin BSA
  • HSA can be used to prepare and encapsulate two kinds of compounds for chemotherapy and photodynamic under near-neutral conditions.
  • the therapeutic protein nanoparticles can achieve the effect of combined treatment of tumors and inhibition of metastasis.
  • Fig. 1 Morphological characterization of nanoparticles: A, transmission electron microscope image of nanoparticles; B, dynamic particle size distribution of nanoparticles; C, negative staining electron microscope image of nanoparticles.
  • FIG. 2 Structure characterization diagram of nanoparticles: A. XPS analysis pattern; B. EDX analysis pattern; C. FTIR analysis pattern.
  • Fig. 3 Normalized absorption change graph of reactive oxygen species: A. Nanoparticles; B. Free photosensitizers.
  • Fig. 4 The results of in vitro stability investigation of nanoparticles: A. Chemical stability; B. Light stability.
  • Figure 7 The results of the nanoparticle inhibition experiment on subcutaneous tumors in mice.
  • Fig. 8 Results of nanoparticles inhibiting lung metastases from orthotopic breast cancer in mice.
  • Figure 10 (A) ALT, AST, ALP and (B) Urea, Crea levels in mouse serum at different times after intravenous injection of nanoparticles (C) ALT, AST, ALP and (D) Levels of Urea and Crea.
  • FIG. 11 Schematic diagram of nanoparticle preparation.
  • the invention relates to the preparation of albumin nanoparticles encapsulating the dual therapeutic effects of platinum drugs (such as cisplatin precursor ion dihydrate diammine platinum ion) and photosensitizers (such as chlorphene e6), and used as antitumor
  • platinum drugs such as cisplatin precursor ion dihydrate diammine platinum ion
  • photosensitizers such as chlorphene e6
  • the new dosage form of the drug enhances the toxicity and targeting of the drug to the tumor, exerts a synergistic effect, and reduces the systemic side effects, thereby realizing the combined treatment of the tumor and inhibiting the metastasis of the tumor.
  • the chlorin e6 solution is added into the mixed solution of diammine platinum ion and protein, and after the reaction, ultrafiltration and centrifugation are performed to obtain protein nanoparticles carrying platinum drugs/photosensitizers. details as follows.
  • step (2) adding the Ce6 solution to the mixed solution in step (1), adjusting to pH 4.0 ⁇ 8.0, the concentration of the Ce6 solution being 2 ⁇ 8 mmol ⁇ L -1 , and then reacting the mixed solution at 25 ⁇ 60°C 1 to 8 hours.
  • step (3) Put the mixture obtained by the reaction in step (1) into an ultrafiltration centrifuge tube, and perform ultrafiltration centrifugation at a rotational speed of 1500-4000 r ⁇ min -1 to remove free small molecules until the lower filtrate is colorless.
  • the platinum dihydrate dihydrate ion interacts with Ce6 to form a complex, which is co-precipitated in the protein cavity to obtain nanoparticles.
  • the chemical structural formula of the platinum drug/photosensitizer complex is as follows.
  • the electron around the platinum atom is 4f14 5d10 6s1, the divalent platinum ion is 4f14 5d9, and the tetravalent platinum ion is 4f14 5d7; the divalent platinum 4f14 5d9 interacts with 1 -COO - , and the outer layer of the monovalent platinum ion is 4f14 5d10;
  • the reaction of photosensitizer with diamine platinum ion is shown as follows.
  • nanoparticles carrying platinum drugs/photosensitizers of the present invention are referred to as "nanoparticles" for short.
  • Example 1 Weigh 720 mg of cisplatin (Pt(NH 3 ) 2 Cl 2 , 2.4 mmol ⁇ L -1 ) and 800 mg of silver nitrate (AgNO 3 , 4.7 mmol ⁇ L -1 ) and dissolve them in 6.0 mL of distilled water After stirring for 3 h in a water bath (60 °C) in the dark, and then stirring at room temperature for 20 h; after the reaction, take out the reaction solution (the reaction solution is clear and no turbidity) and centrifuge at 14,000 r min-1 for 15 min to remove the AgCl precipitate formed.
  • the supernatant was filtered with a 0.22 ⁇ m membrane to obtain a dihydrate diammine platinum nitric acid ([Pt(NH 3 ) 2 (H 2 O) 2 ](NO 3 ) 2 ) solution; an inductively coupled plasma emission spectrometer (ICP- OES) to determine its concentration and add water to adjust the final concentration to 320 mmol ⁇ L -1 , which is the stock solution concentration.
  • ICP- OES inductively coupled plasma emission spectrometer
  • the purified reaction solution was filtered through a filter membrane with a pore size of 0.22 ⁇ m to obtain platinum-based drug dihydrate diammine platinum/photosensitizer Ce6 albumin nanoparticles (Pt/Ce6@HSA NPs) solution, referred to as nanoparticle solution.
  • platinum-based drug dihydrate diammine platinum/photosensitizer Ce6 albumin nanoparticles Pt/Ce6@HSA NPs
  • Morphological characterization of nanoparticles (1) Transmission electron microscope characterization of nanoparticles: Take 20 ⁇ L of the nanoparticle solution and drop it on the copper mesh carbon support film, put it in a desiccator to evaporate the water, and use 120 The morphology was observed by kV transmission electron microscope (TEM). The results are shown in Figure 1-A. The results show that the prepared nanoparticles are regular circles with a particle size of 4.9 ⁇ 1.0 nm.
  • TEM In the presence of heavy metals, TEM only showed heavy metals in the core of nanoparticles, and albumin was not imaged; through protein staining experiments, TEM determined that the particle size of nanoparticles plus the protein layer was 7.9 ⁇ 0.4 nm.
  • the nanoparticles prepared by the present invention are regular circles, relatively uniformly dispersed, with a particle size of less than 10 nm, can be cleared by the kidneys, and have in vivo EPR effect and tumor targeting.
  • Singlet oxygen generation ability of nanoparticles The absorbance change after singlet oxygen capture was measured using 1,3-diphenylisobenzofuran (DPBF).
  • DPBF 1,3-diphenylisobenzofuran
  • the prepared nanoparticle solution and free Ce6 solution were diluted into samples with Ce6 concentrations of 0.5, 1.0, 2.0, and 5.0 ⁇ mol ⁇ L -1 , respectively, and 2.97 mL were taken into a quartz cuvette to measure the initial UV absorption value.
  • a 30 ⁇ mol ⁇ L -1 DPBF solution was prepared with DMSO, 30 ⁇ L was added to a quartz cuvette, and the solution was irradiated with a 660 nm laser (0.15 W ⁇ cm -2 ) for 3 min while stirring, at intervals of 30 s.
  • a and B are the normalized graphs of the absorbance values of DPBF in the nanoparticle and free Ce6 solutions as a function of illumination time, respectively. It can be seen from Figure A that the absorbance of DPBF decreases rapidly with the prolongation of illumination time at low concentration of the nanoparticle solution, and it has a concentration dependence, indicating that a large amount of singlet oxygen is generated, and the nanoparticles have a strong singlet The ability to generate state oxygen has potential in PDT applications; while free Ce6 in Figure B only generates a small amount of singlet oxygen at the same concentration.
  • In vitro stability of nanoparticles (1) Chemical stability of nanoparticles: take 0.1 mL of nanoparticles with Ce6 concentration of 20 ⁇ mol ⁇ L -1 and 20 ⁇ mol ⁇ L -1 free Ce6 solution, respectively, and add them to 2.9 mL of purified water, pH 5.5 phosphate buffer, pH 7.4 phosphate buffer, and RPMI 1640 medium containing 10% fetal bovine serum. Store at room temperature away from light, and prepare 3 copies in parallel for each environment. The absorbance changes of Ce6 within 72 h were measured by UV-Vis spectrophotometer.
  • the encapsulation of nanoparticles will be beneficial to improve the stability and play a PDT role in the acidic environment of tumors.
  • Cytotoxicity of nanoparticles Take single cell suspension of 4T1 mouse breast cancer cells in logarithmic growth phase and inoculate in 96-well cell culture plate (1.0 ⁇ 10 5 cells/well, 1640 medium), 100 ⁇ L per well, in Cultivate overnight in a 37°C constant temperature cell incubator to make the cells completely adherent and deformed. After the cells grow to 80%, the original medium is removed.
  • Example 1 of CN110368374A The nanoparticles prepared in Example 1 of CN110368374A were taken and subjected to the same cytotoxicity test as above. Calculated according to the concentration of Pt element, the non-illuminated IC 50 was 87.86 ⁇ mol ⁇ L -1 .
  • Nanoparticles subcutaneous tumor inhibitory effects in mice Using the above method, constructing mice bearing subcutaneous tumor model, tumor volume to be 60 mm 3, the administration according to the following design: Subcutaneous tumor-bearing mice were divided into groups PBS, PBS Light group (PBS-irradiation), free CDDP/Ce6, free CDDP/Ce6 light group, nanoparticle group, nanoparticle light group, nanoparticle pre-injection Vc light group. The mice were given tail vein administration on the 0th, 2nd and 5th day respectively, and the injection dose was 5 mg ⁇ kg-1 (calculated according to the concentration of Pt element). nm, 0.15 W ⁇ cm -2 ), and the illumination time was 5 min.
  • Vc solution (25.0 ⁇ mol ⁇ kg -1 ) was injected into the tumor half an hour before illumination each time.
  • the tumor inhibition time was 30 days, during which the body weight and tumor volume changes of the mice were measured and recorded. After the experiment, the mice were sacrificed, and the tumors were collected and fixed with 4% formaldehyde and photographed. The results are shown in Figure 7.
  • the tumor growth was not only completely inhibited, but also the effect of tumor ablation was achieved, confirming the excellent advantages of the encapsulation of the nanoparticles of the present invention for combined chemotherapy/photodynamic therapy; and intratumoral injection of reducing reactive oxygen species After the dose of Vc, the tumor-inhibitory effect was weakened, which confirmed the synergistic contribution of platinum drugs and Ce6 in the combined chemotherapy/photodynamic therapy.
  • the tumor can be eliminated and the metastasis of the tumor to the lung can be effectively inhibited after three treatments, which proves that the nanoparticle of the present invention has significant advantages in inhibiting tumor growth and inhibiting tumor metastasis after combining chemotherapy and PDT; and Subcutaneous tumor experiment results are the same, Vc can weaken the anti-tumor effect of nanoparticles.
  • the nanoparticles were mainly distributed in the liver and spleen after injection, and the platinum content had decreased by about 90% by 42 days. However, the distribution of platinum in the heart, lung, kidney, and intestine gradually decreased. At 35 days, it was found that the content of Pt in each organ was very little or even zero, indicating that the nanoparticles can be gradually removed from the tissue and will not accumulate heavy metals in the body. , so as not to cause long-term toxic and side effects, and it has better biological safety.
  • Detection and investigation of biochemical indicators after the action of nanoparticles In order to determine the toxicity of the drug during metabolism in the body, the biochemical indicators of liver and kidney function were investigated: detection of liver indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and renal indicators urea (Urea), creatinine (Crea), and compared with normal values to determine the damage to liver and kidney function by drugs.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • Re renal indicators urea
  • Urea renal indicators urea
  • Crea creatinine
  • Healthy Balb/c mice were randomly divided into 3 groups: PBS group, Pt/Ce6@HSA nanoparticle group, free CDDP group, three PBS group, 12 mice in each group, nanoparticle group and free CDDP group, respectively.
  • the granules and free CDDP solution were 200 ⁇ L, and the injection dose was 5 mg ⁇ kg -1 (calculated according to the concentration of Pt element), administered once every other day, for a total of 3 administrations.
  • blood samples were collected from the retro-orbital venous plexus of mice by removing the eyeballs at 7, 14, 21, and 28 days after administration, respectively. In the PBS group, blood was also collected from the eyeballs on 28 days.
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • Urea renal function indicators urea
  • Urea renal function indicators urea
  • Crea creatinine
  • Example 2 When preparing protein nanoparticles in Example 1, the pH was adjusted to 5.0 and 6.0 respectively (in Example 1, the pH was 5.5), and other steps were the same as in Example 1, and the TEM size was 4.5 ⁇ 5.3. Nanoparticles of nm (due to the action of heavy atoms, only reflect the size of the area where the platinum atoms in the core of the nanoparticle are located, the same below).
  • Example 3 During the preparation of protein nanoparticles in Example 1, the molar ratios of platinum element and Ce6 were adjusted to 1:1 and 8:1 respectively (in Example 1, the molar ratio of Pt:Ce6 was 4:1), Other conditions are the same as in Example 1, and Pt/Ce6 protein nanoparticles with good stability can be prepared, and the TEM size is between 4.2 and 4.9 nm.
  • Example 1 In the preparation process of protein nanoparticles in Example 1, the molar ratio of platinum element and Ce6 was adjusted to 1:2 (in Example 1, the molar ratio of Pt:Ce6 was 4:1), other conditions were the same as in Example 1, and the reaction After the solution was turbid, Pt/Ce6 protein nanoparticles with good stability could not be prepared.
  • Example 4 During the preparation of protein nanoparticles in Example 1, the reaction temperature was adjusted to 25°C and 37°C respectively (in Example 1, the reaction temperature was 55°C), and other conditions were the same as those of Example 1, and the obtained
  • the Pt/Ce6 protein nanoparticles with good stability have a TEM size ranging from 4.2 nm to 5 nm.
  • Example 5 The reaction time in the preparation process of platinum sulfide protein nanoparticles in Example 1 was adjusted to 1 h and 8 h (in Example 1, the reaction time was 4 h), and other conditions were the same as in Example 1; Reaction 1 h can prepare Pt/Ce6 protein nanoparticles with good stability, and the TEM size is about 5 nm; if the reaction is performed for 8 hours, the solution is cloudy after the reaction, and Pt/Ce6 protein nanoparticles with good stability cannot be prepared.
  • HSA is used as the protein template, and the platinum-based chemotherapeutic drugs that are difficult to be directly encapsulated and the photosensitizer Ce6 are co-precipitated in the inner cavity of albumin to prepare nanoparticles Pt/Ce6@HSA, see Fig. 11, thereby realizing the co-encapsulation
  • the combined application of chemotherapeutic action/photodynamic action in tumor treatment lays a foundation for further research and clinical drug use.
  • drug-loaded protein nanoparticles with smaller particle size, better dispersion and regular morphology were successfully obtained.
  • the existence of divalent platinum element and the successful coordination of platinum and carboxyl groups were proved by XPS, EDX and FTIR characterization.
  • the nanoparticle has good photostability and chemical stability; at the same time, the nanoparticle has a good ability to generate singlet oxygen.
  • the results of the release law study showed that the nanoparticles have a certain sustained release effect and are beneficial to reach the tumor site to play a role.
  • the present invention ingeniously designs and successfully prepares the protein nanoparticles encapsulating platinum-based drugs and photosensitizers that combine chemotherapeutic action/photodynamic action, resulting in a synergistic anti-tumor effect and a remarkable tumor-inhibiting effect.

Abstract

一种载铂类药物/光敏剂的蛋白纳米粒及其制备方法和应用，包括铂类药物/光敏剂配合物，以及包裹铂类药物/光敏剂配合物的蛋白质。制备的纳米粒粒径较小、分散均匀、形态圆整，具有良好的化学稳定性、光照稳定性，在近红外光的照射下具有较高的活性氧产生能力，在细胞实验和动物实验中，验证了对肿瘤细胞具有较强的细胞毒性和良好的体内肿瘤靶向性，发挥协同增效的作用，降低毒副作用，实现了化学疗法和光动力疗法联合治疗肿瘤，并抑制肿瘤的转移。

Description

载铂类药物/光敏剂的蛋白纳米粒及其制备方法和应用 技术领域
本发明涉及包载铂类药物和光敏剂的具有双重治疗效果的白蛋白纳米粒的制备，并作为抗肿瘤药新剂型，实现肿瘤的联合治疗，并抑制肿瘤的转移。
 背景技术
恶性肿瘤是威胁人类健康的严重疾病，临床治疗中需要治疗和抑制转移并重。
目前作为纳米载药系统研究的材料多种多样，以生物大分子蛋白质为载体制备纳米材料，具有生物相容性好，安全无毒的优点，作为药物的载体材料可将药物选择性的靶向病变部位，有效降低其对正常组织的毒副作用，因此在纳米药物载体和抗肿瘤方面的药剂学研究中具有重要地位和研究价值。
白蛋白（Albumin）又称清蛋白，是血浆中含量最丰富的蛋白质。人血清白蛋白（Human Serum Albumin，HSA）是目前结构最清晰的白蛋白，分子量为66 kD。非糖基化的单链多肽包含585个氨基酸，等电点为4.7~4.9的酸性蛋白质，易溶于水，在血浆中其浓度为42 g/L，约占血浆总蛋白的60%。
铂类药物的研究已有50多年历史。1965年，美国学者Rosenberg等偶然发现顺铂类配合物可以抑制细胞繁殖，能够用作抗肿瘤药物。此后，铂类药物迅速引起人们的关注，铂类抗肿瘤药物的研究也成为热点。时至今日，铂类药物的研究已发展到第四代，其中顺铂、卡铂和奥沙利铂已用于临床，合成这些铂类药物的重要前体为二水二氨合铂硝酸盐，故：二水二氨合铂离子亦称为顺铂前体离子。顺铂（又称顺-二氯二氨合铂，Cis-Dichlorodiammine platinum(II)，简称CDDP），作为经典的铂类抗肿瘤药物，在肿瘤化疗中占有重要地位。顺铂抗癌谱及其应用范围很广，临床主要用于治疗睾丸癌、卵巢癌、膀胱癌、头颈部癌、食管癌以及小细胞肺癌。当和阿霉素，紫杉醇，5-氟尿嘧啶（5-Fu）等联合用药时可治疗头颈部癌以及胃癌。但临床的大剂量和长期使用受限于其严重的毒副反应、低溶解性及耐药性。
肿瘤治疗中的光动力疗法（Photodynamic therapy, 简称PDT）是在肿瘤组织摄取光敏剂后，利用光动力反应进行疾病诊断与疾病治疗的新技术。PDT有3个重要因素：激发光源、光敏剂和活性氧。其作用机制为：光敏剂分子吸收相应波长光的光子能量，由基态变为单重激发态，激发态光敏剂分子可通过物理退激过程回到基态，并产生荧光用于临床诊断，即荧光诊断；也可转化为三重激发态，将能量转移给邻近的分子氧产生活性氧簇（Reactive oxygen species, 简称ROS）诱导靶器官细胞发生自噬、凋亡与坏死。
由于“叶绿素a”的衍生物，在红光区域具有高消光系数和高单重态氧量子产率，所以已被广泛用作光敏剂。其中，PDT常用Ce6，它通过近红外光(NIR)光激活，能相对快速地从体内消除，以及具有高的活性氧产生效率，且Ce6还可用作NIR荧光成像，波长范围为650~900 nm，以避免内源性发色团在体内的干扰。但是，Ce6在生理条件下的稳定性差，且荧光量子产率（Φ f）和光敏效果较差。因此，为增强光敏剂的稳定性和对肿瘤组织的靶向性，除开发新一代光敏剂外，为了提高PDT效率开发各类纳米载体为Ce6提供新平台，以及有效整合不同治疗方式，改善肿瘤治疗效果是非常必要的。
技术问题
为解决以上技术问题，本发明的目的是提供一种载铂类药物/光敏剂的蛋白纳米粒及其制备方法和应用。本发明在温和的反应条件下制备HSA纳米粒，尤其是通过合适的处方和工艺，实现粒径的有效控制，提高肿瘤靶向性和疗效。本发明基于蛋白质模板，通过仿生合成制备具有化疗/光动力联合治疗效果的蛋白纳米粒进行研究。以HSA为蛋白质模板，将化疗药顺铂前体离子和光敏剂Ce6在白蛋白内腔中共沉淀，并进一步成核生长，制备得到具有化疗/光动力治疗作用的双功能的小粒径白蛋白纳米粒，改善两种水溶性不强的药物的递送效率，增强肿瘤细胞的药物摄取能力和药物在细胞内的累积。此蛋白纳米粒进入肿瘤细胞溶酶体后，在近红外光激发下Ce6产生显著的光动力，还可通过ROS诱导溶酶体破裂，同时促进铂类药物转运进入胞浆进而进入细胞核，并以二价铂的活性形式存在，增强化疗作用，从而实现肿瘤的联合治疗起到协同增效作用；而在体内运用时呈现出较好体内肿瘤靶向性，且在发挥铂类药物化疗作用抑制肿瘤生长的同时，联合光动力治疗，达到完全消除肿瘤和抑制肿瘤转移的效果；尤其是纳米粒在生物体内可因小粒径（<10 nm）产生的肾清除效应被有效代谢，具有良好的生物安全性。
 技术解决方案
本发明采用如下技术方案。
载铂类药物/光敏剂的蛋白纳米粒，包括铂类药物/光敏剂配合物，以及包裹铂类药物/光敏剂配合物的蛋白质。进一步的，蛋白质为白蛋白，优选人血清白蛋白；铂类药物为二水二氨合铂离子，光敏剂为二氢卟吩e6。
本发明包载二水二氨合铂离子的纳米粒中，采用铂元素定量测定，用顺铂作对照，结果可靠；细胞和体内实验，用顺铂作对照，得到可靠的结果。现有技术无法直接制成包载二水二氨合铂离子的蛋白纳米粒，也未能制得同时包载顺铂与Ce6的纳米粒。经过创造性的劳动，本发明采用安全的HSA将二水二氨合铂离子和光敏剂Ce6共包载，具有条件温和、工艺简单、粒径小且可控、生物相容性好、肿瘤靶向性和滞留性好等优势，是整合了化疗和光动力治疗手段的新型白蛋白纳米粒，实现了肿瘤高效低毒治疗并抑制肿瘤转移的白蛋白纳米粒的制备和应用。
本发明公开了上述载铂类药物/光敏剂的蛋白纳米粒的制备方法，包括以下步骤，将二氢卟吩e6溶液加入二水二氨合铂离子与蛋白的混合溶液中，反应后超滤离心，得到载铂类药物/光敏剂的蛋白纳米粒。本发明选用蛋白纳米载体，同时包载铂类药物和光敏剂，实现在一个纳米载药平台结合化疗毒性和光毒性对肿瘤的双重靶向治疗作用。载铂类药物/光敏剂的蛋白纳米粒的粒径为2~50 nm，水合粒径20~150 nm。
本发明中，蛋白、二水二氨合铂离子、二氢卟吩e6的用量比为100 mg：(20~50 μmol)∶(3~15 μmol)，优选为100 mg∶(25~40 μmol)∶(5~10 μmol)；将二氢卟吩e6溶液加入二水二氨合铂离子与蛋白的混合溶液中，调节至pH为 4.0~8.0，优选5.0~6.5，然后于25~60℃反应1~8 h；超滤离心的转速为1500~4000 r·min -1。
本发明采用安全的HSA制备的蛋白纳米粒，粒径、化学稳定性、光照稳定性及活性氧产生能力，均较为理想，在肿瘤治疗中具有较大的应用前景。
本发明公开了上述载铂类药物/光敏剂的蛋白纳米粒在制备药物中的应用，具体为抗肿瘤药物，进一步为化疗/光动力治疗双重抗肿瘤药物。
本发明制备的纳米粒肿瘤靶向性好，在肿瘤的滞留好，并显示出对肿瘤细胞的强毒性，在近红外光照条件下联合化疗/光动力治疗具有协同作用，光动力治疗损伤细胞并增强了化疗效果，可有效消除肿瘤，显著抑制肿瘤生长及转移，展示了兼具化疗和光动力治疗的高效低毒抗肿瘤作用，是安全有效的纳米新制剂。
本发明中，HSA是内源性物质，不会产生毒性和炎症反应，具有良好的稳定性和独特的空间结构，增加难溶性药物的溶解度，对易氧化药物具有较好的保护作用，可显著延长药物半衰期，提高靶向性，显著降低药物毒性，并且由于肿瘤生长所需能量和营养源依赖白蛋白，所以内源性白蛋白在肿瘤组织中有聚集作用，HSA作为药物载体制备给药系统优势显著：与药物结合能力高，稳定性好（在pH 4~9，60℃条件下10 h不产生有害物质），具有很好的生物相容性和生物可降解性，良好的肿瘤靶向性，高效低毒治疗肿瘤，故研究价值极高。
本发明设计蛋白纳米给药系统包载二水二氨合铂离子，与DNA形成加合物，从而抑制DNA复制和转录，导致DNA断裂和错码，抑制细胞有丝分裂，故研发相关新制剂很有意义。
联合治疗是将不同的抗癌药物或不同治疗方式联合，多管齐下达到协同抗癌功效，以克服、减轻耐药性和毒副作用，提高药物疗效的有效抗肿瘤策略。目前临床已有的铂类联合治疗策略，多是不同化疗药物的联合应用，但是不少情况下，联合治疗效果不佳，现有药物的直接联合虽然能在给药时调整剂量，但其代谢不同导致效果不尽如人意，有时产生的毒性更大。本发明的纳米药物有较小的毒性和不良反应，创伤小、疗效确切、无耐药性，光敏剂（在PDT中的细胞毒性剂）能够吸收特定波长的光并将其转化为有用能量，在较长波长处（600~850 nm）具有高消光系数的强吸光度，光的组织穿透性大，并有足够活力以产生ROS，具有优异的光化学反应性，具有高三重态产率和长三重态寿命，并能够有效地产生ROS，具有最小的暗毒性，并且在光的存在下仅为细胞毒性，优先被靶组织保留，从身体迅速排泄，引起的全身毒性低。
 有益效果
现有技术蛋白包载有机分子，粒径难以控制在理想范围，而且现有报道的蛋白纳米粒，需要采用强碱性条件（比如pH 12），比如牛血清白蛋白BSA制备包载Gd 2O 3/Ce6纳米粒的诊疗一体化研究。目前尚未见有制备白蛋白纳米粒同时包载铂类药物和光敏剂的报道，尤其是现有技术还未见有采用HSA在近中性条件，可以制备包载2种化合物用于化疗和光动力治疗的蛋白纳米粒，实现联合治疗肿瘤并抑制转移的效果。
附图说明
图1 纳米粒的形态表征图：A、纳米粒的透射电镜图；B、纳米粒的动态粒径分布图；C、纳米粒的负染电镜图。
图2 纳米粒的结构表征图：A. XPS分析图谱；B. EDX分析图谱；C. FTIR分析图谱。
图3 活性氧产生的归一化吸收变化图：A.纳米粒；B.游离光敏剂。
图4 纳米粒的体外稳定性考察结果图：A. 化学稳定性；B. 光照稳定性。
图5 纳米粒对4T1细胞的细胞毒性结果图。
图6 纳米粒小鼠体内组织分布结果图。
图7 纳米粒对小鼠皮下瘤抑瘤实验结果图。
图8 纳米粒抑制小鼠原位乳腺癌的肺转移肿瘤结果图。
图9尾静脉注射纳米粒后铂元素在小鼠体内各组织中的长期分布。
图10（A）静脉注射纳米粒后不同时间小鼠血清中ALT、AST、ALP以及（B）Urea、Crea的水平（C）静脉注射顺铂后不同时间小鼠血清中ALT、AST、ALP以及（D）Urea、Crea的水平。
图11纳米粒制备示意图。
图12化疗/光动力协同作用机理。
 本发明的实施方式
本发明涉及包载铂类药物（如顺铂前体离子二水二氨合铂离子）和光敏剂（如二氢卟吩e6）的双重治疗效果的白蛋白纳米粒的制备，并作为抗肿瘤药新剂型，增强药物对肿瘤的毒性和靶向性，发挥协同增效作用，降低全身毒副作用，从而实现肿瘤的联合治疗，并抑制肿瘤的转移。
本发明将二氢卟吩e6溶液加入二水二氨合铂离子与蛋白的混合溶液中，反应后超滤离心，得到载铂类药物/光敏剂的蛋白纳米粒。具体如下。
（1）顺铂和硝酸银溶液反应，合成得到二水二氨合铂硝酸盐水溶液，然后将二水二氨合铂离子的溶液与蛋白溶液混合，所述二水二氨合铂溶液的浓度为8~32 mmol·L -1；所述蛋白溶液的浓度为5~20 mg·mL -1。
（2）将Ce6溶液加入步骤（1）中的混合溶液中，调节至pH 4.0~8.0，所述Ce6溶液的浓度为2~8 mmol·L -1，然后将混合溶液于25~60℃反应1~8 h。
（3）将步骤（1）反应得到的混合物置于超滤离心管中，以1500~4000 r·min -1的转速，超滤离心除去游离小分子至下层滤液无色。
本发明中二水二氨合铂离子与Ce6作用形成配合物，并共沉淀在蛋白空腔中，得到纳米粒。
本发明中，铂类药物/光敏剂配合物的化学结构式如下。
Figure 44547dest_path_image001
。
铂原子外围的电子是4f14 5d10 6s1，二价铂离子为4f14 5d9，四价铂离子为4f14 5d7；二价铂4f14 5d9与1个-COO -作用，得一价铂离子外层为4f14 5d10；光敏剂与二水二氨合铂离子反应示意如下。
Figure 788381dest_path_image002
。
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不局限于此。本发明载铂类药物/光敏剂的蛋白纳米粒，简称“纳米粒”。
实施例一：称取720 mg顺铂（Pt(NH 3) 2Cl 2，2.4 mmol·L -1）和800 mg 硝酸银（AgNO 3，4.7 mmol·L -1），溶于6.0 mL的蒸馏水中，水浴（60℃）避光搅拌3 h后，然后室温搅拌20小时；反应结束后，取出反应液（反应液清澈无浑浊）14000 r ·min -1离心15 min，除去生成的AgCl沉淀，上清以0.22 μm滤膜过滤后得到二水二氨合铂硝酸（[Pt(NH 3) 2(H 2O) 2](NO 3) 2）溶液；用电感耦合等离子发射光谱仪（ICP-OES）确定其浓度并加水调整终浓度为320 mmol·L -1，为储备液浓度，制备纳米粒时用水稀释成32 mmol·L -1。
采用10 mg·mL -1的HAS水溶液10 mL，加入浓度为32 mmol·L -1的二水二氨合铂前体离子溶液1 mL，搅拌下加入1 mL浓度为 8 mmol·L -1的Ce6溶液，用氢氧化钠水溶液（0.1M）调节体系pH至5.5后，在55℃水浴中搅拌反应4 h。反应结束后将反应液离心（3000 r ·min -1，10 min）、超滤（MW:100 kD，2000 r ·min -1，10 min）纯化，以除去游离的二水二氨合铂前体离子和Ce6；最后，将纯化后的反应液用孔径为0.22 μm的滤膜过滤，即得载铂类药物二水二氨合铂/光敏剂Ce6的白蛋白纳米粒（Pt/Ce6@HSA NPs）溶液，简称纳米粒溶液。
纳米粒的形态表征：（1）纳米粒的透射电镜表征：取纳米粒溶液20 μL滴到铜网碳支持膜上，放入干燥器中挥干水分后用120 kV透射电镜（TEM）观察形态。结果如图1-A。结果表明，制备的纳米粒为规整的圆形，粒径为4.9±1.0 nm。
由于在重金属存在时，TEM仅显示纳米粒核心的重金属，白蛋白没有成像；通过蛋白染色实验，TEM测得加上蛋白层的纳米粒粒径为7.9±0.4 nm。
（2）纳米粒的粒径及其分布表征：取纳米粒溶液1 mL采用激光散射粒径仪对其粒径及分布进行分析。结果如图1-B。结果表明制得的蛋白纳米粒为单峰分布，平均水合粒径为33.1 nm（此粒径包含表面水化层），多分散系数（PDI）为0.198，有利于避免内皮网状细胞拦截，形成血中长循环，发挥被动靶向作用。
（3）纳米粒的负染电镜图：取纳米粒溶液滴在铜网上，5 min后吸去多余液体，滴加0.2%的醋酸铀水溶液染色3 min，而后用滤纸吸去染色液，待干燥后用120 kV电镜观察。结果如图1-C。结果表明，负染后纳米粒蛋白分布在纳米粒外层，为颜色较浅的白色，四周和内核背景较深，纳米粒局部放大后可看到蛋白外层粒径为8 nm左右，内核粒径为5 nm，而单分子空腔的大小一般为6~8 nm，说明本发明制备纳米粒的核心存在Ce6与二水二氨合铂离子配合物。
以上结果表明，本发明制备的纳米粒为规整的圆形，分散比较均匀，粒径小于10 nm，可被肾清除，具有体内EPR效应和肿瘤靶向性。
游离CDDP/Ce6溶液的制备，将顺铂、Ce6分别用生理盐水和DMSO溶解后，按照Pt∶Ce6=1.5∶1的摩尔比混合，在用水稀释至所需浓度，得到游离CDDP/Ce6溶液。
纳米粒的结构表征：（1）纳米粒Pt 配体的内层电子结合能：将制得的纳米粒溶液于-80 ℃冰箱预冻后放入真空冷冻干燥机中冻干48 h，然后在研钵中研磨成细腻的粉末，以X射线光电子能谱（XPS）分析纳米粒和游离物理混合物的原子价态。结果如图2-A。根据Pt元素 4 f轨道在其X射线光电子能谱（XPS）中的峰拟合过程，发现纳米粒在72.9和76.4 eV处出现峰，表明纳米粒子中存在二价铂，与游离二水二氨合铂离子/Ce6混合物的对照组结果一致。
（2）纳米粒的元素分析：取纳米粒溶液滴于超薄碳膜上，挥干水分后进行能量色散X射线（EDX）分析，判断其元素组成。结果如图2-B。结果表明纳米粒中有铂元素的存在。
（3）纳米粒的配位分析：取冻干后的纳米粒粉末、游离二水二氨合铂离子/Ce6混合物、Ce6粉末通过傅里叶红外光谱（FTIR）分析，结果如图2-C。
纳米粒的单线态氧产生能力：利用1,3-二苯基异苯并呋喃（DPBF）对单线态氧捕获后的吸光度变化进行测定。将制备好的纳米粒溶液和游离Ce6溶液稀释成Ce6浓度分别为0.5、1.0、2.0、5.0 μmol·L -1的样品，分别取2.97 mL到石英比色皿中，测定初始紫外吸收值。用DMSO配制30 μmol·L -1的DPBF溶液，取30 μL加入到石英比色皿中，边搅拌边用660 nm激光器（0.15 W·cm -2）照射溶液3 min，期间每隔30 s时测量DPBF在418 nm处的紫外吸收值的变化，并作归一化后的DPBF变化折线图。结果如图3。A、B分别是纳米粒和游离Ce6溶液中DPBF 吸光度值随光照时间变化的归一化图。从图A中可以看出，纳米粒溶液在低浓度时随光照时间的延长，即可使DPBF吸光度迅速下降，并且具有浓度依赖性，说明有大量单线态氧产生，纳米粒具有较强的单线态氧产生能力，在PDT应用中具有潜力；而图B中游离Ce6同浓度下仅产生少量单线态氧。
纳米粒的体外稳定性：（1）纳米粒的化学稳定性：分别取Ce6浓度为20 μmol·L -1的纳米粒及20 μmol·L -1游离Ce6溶液各0.1 mL，分别加入到2.9 mL的纯水、pH 5.5的磷酸缓冲液、pH 7.4的磷酸缓冲液，以及含10%胎牛血清的RPMI 1640培养基中。置于室温避光保存，每种环境平行配置3份。采用紫外-可见分光光度计测定72 h内Ce6的吸光度变化。结果如图4A，纳米粒在去离子水、pH为5.5、7.4的磷酸缓冲液、以及含10%胎牛血清的培养基中，72 h之内各时间点紫外吸收基本不变，说明纳米粒有着更优越的稳定性，有助于纳米粒药效的发挥；而游离Ce6组随着时间的变化，其紫外吸收下降20%~40%。
（2）纳米粒的光稳定性：分别取Ce6浓度为20 μmol·L -1的纳米粒及20 μmol·L -1游离Ce6溶液各0.1 mL，分别加入到2.9 mL的纯水，pH 5.5和pH 7.4的磷酸缓冲液，采用660 nm激光器，功率0.15 W·cm-2，每隔30 s照射一次并立即用紫外-可见分光光度计测定其紫外吸收值，照射直至满5 min。结果如图4 B，结果表明Ce6被包裹进蛋白以后，与铂形成配合物，对Ce6具有一定的稳定作用，因而使纳米粒在去离子水中及酸性和中性环境中均具有更好的光照稳定性；而游离Ce6在光照条件下，光漂白较快，有其是在酸性环境中120 s内就光漂白完全，溶解度低、稳定性差。
因此纳米粒的包载将有利于稳定性提高并在肿瘤酸性环境中发挥PDT作用。
纳米粒的细胞毒性：取对数生长期的4T1小鼠乳腺癌细胞单细胞悬液接种于96孔细胞培养板中（1.0×10 5细胞/孔、1640培养基），每孔100 μL，于37℃恒温细胞培养箱中培养过夜使细胞完全贴壁变形，待细胞长到80%后，去除原培养基。
分成游离药物组和纳米粒组，进一步分成非光照组和光照组，分别加入100 μL相同浓度稀释好的纳米粒以及游离CDDP/Ce6溶液替换原培养基，浓度设置为0.1，0.2，0.5，1.0，2.0，5.0，10.0，20.0，50.0，100.0 μmol·L -1（按Pt元素的浓度计算），每个浓度4个复孔。
为考察纳米粒的细胞化疗毒性，非光照组分别培养4、12、24、48 h后，弃去带药培养基后每孔加入 5 mg·mL -1 的MTT溶液 10 μL和1640培养基90 μL，继续培养 4 h 后弃去液体，每孔加入150 μL DMSO，用酶标仪设置震荡30 s，紫外吸收490 nm，测定吸光度A，取复孔平均值计算得到细胞存活率：细胞存活率 = 实验组A /阴性对照组A 0 × 100%（以未加药细胞作为阴性对照组）。结果如图5a。
为了探索化疗光动力联合治疗效果，选取孵育24 h时间点进行光照。光照组加药后培养24 h后每孔用激光照射（660 nm，0.15 W·cm -2，5 min）后继续培养24 h继续加MTT等相同的操作过程。结果如图5b。
图5结果显示，在仅有化疗效果存在时纳米粒中的药物在细胞内缓慢释放，产生长久的细胞毒性，说明具有高效低毒的潜力；而光照条件下，相较于游离药物组，按Pt元素的浓度计算，纳米粒组的细胞存活率显著下降，IC 50 从非光照条件下的40.91 µmol·L -1下降至1.91 µmol·L -1，降低了21.5倍，而游离组的暗毒性IC 50为9.52 µmol·L -1，光毒性IC 50为6.18 µmol·L -1，仅降低1.5倍。表明该药物发挥的光动力作用对杀死肿瘤细胞具有重大作用，反映了本发明将化学疗法与光动力疗法相结合的优点。
取CN110368374A实施例一制备的纳米粒，进行如上同样的细胞毒性测试，按Pt元素的浓度计算，非光照IC 50 为87.86 µmol·L -1。
纳米粒小鼠体内组织分布：（1）皮下肿瘤模型的建立：培养4T1肿瘤细胞，收集对数生长期的肿瘤细胞将其消化离心后，加入不含血清的培养基洗一次，离心后加入预冷的PBS制成细胞悬液，浓度1x10 7个/mL ，放在冰盒中备用。小白鼠右腿后肢脱毛后，在肌肉处以注射器挑起表皮，注射50 µL细胞悬浮液，待肿瘤体积长到60-100 mm 3左右时可进行实验。（瘤体积=长×宽 2÷2）。
（2）组织分布实验：取皮下荷瘤小鼠18只，分为两组，分别尾静脉注射纳米粒和游离CDDP/Ce6溶液200 µL，注射剂量为5 mg·kg -1（按Pt元素的浓度计算），注射后分别于6、12、24 h后在每组取出3只用生理盐水心脏灌流后脱颈处死并将心、肝、脾、肺、肾及肿瘤解剖取下。取出的组织先通过小动物成像系统观察各时间点的Ce6荧光分布情况。结果如图6A。
随后将组织称重、剪碎，分别放入100 mL锥形瓶中，做上标记，加入王水进行高温硝解，待组织硝解完全之后取出 100 μL 稀释至一定体积过膜之后用ICP-MS对各组织中的铂元素进行定量。结果如图6B。
图6结果表明，静脉注射不同时间后，纳米粒在肿瘤组织的蓄积显著高于游离药物具有良好的肿瘤靶向性，因此具有肿瘤治疗的潜力。
纳米粒的小鼠皮下瘤抑瘤效果：采用如上方法，构建小白鼠皮下荷瘤模型，待肿瘤体积至 60 mm 3时，按照以下设计给药 ：将皮下荷瘤小鼠随机分成PBS组、PBS光照组（PBS-irradiation），游离CDDP/Ce6、游离CDDP/Ce6光照组、纳米粒组、纳米粒光照组、纳米粒预注射Vc光照组。分别在第0、2、5天对小鼠进行尾静脉给药，注射剂量为5 mg·kg-1（按Pt元素的浓度计算），光照组在每次给药后12 h进行光照（660 nm，0.15 W·cm -2），光照时长为5 min，纳米粒/Vc光照组每次在光照前半小时瘤内注射Vc溶液（25.0 μmol·kg -1）。抑瘤时间30天，期间测量记录小鼠体重和肿瘤体积变化。实验结束后，处死小鼠，收集肿瘤并用4%甲醛固定后拍照。结果如图7。
从图7，A、C、D结果可以看出，PBS组光照前后肿瘤生长情况相差不大，与初始体积相比增加了约37倍，说明光照本身并不会抑制肿瘤生长；而游离CDDP/Ce6组，在非光照时仅有化疗作用，但光照后，应兼具化疗/光动力治疗作用，但抑瘤效果却与非光照相似；纳米粒组光照前后所产生的肿瘤抑制作用差异大，3次给药光照后，不仅完全抑制肿瘤生长，而且达到肿瘤消融的效果，证实了本发明纳米粒的包载对于联合化疗/光动力治疗的卓越优势；并且瘤内注射了清除活性氧的还原剂Vc后，抑瘤效果减弱，证实了化疗/光动力联合治疗中，铂类药物与Ce6协同的贡献。
从图7，B、D结果可以看出，由于化疗药物毒性较大，导致小鼠体重比初始体重下降约8 g，严重影响小鼠生存健康，且游离药物加入光动力治疗后也并不能完全消除肿瘤；而纳米粒+光照组，不仅可以完全消除肿瘤，对小鼠体重几乎无影响，说明疗效好、毒性低。综上，纳米粒有着高效低毒的肿瘤治疗效果。
纳米粒抑制小鼠原位乳腺癌的肺转移肿瘤的效果：（1）建立原位乳腺癌肺转移肿瘤模型：培养4T1-Luciferasee肿瘤细胞，收集对数生长期的肿瘤细胞将其消化制备成不含血清的预冷的PBS细胞混悬液，浓度1x10 7个/mL ，放在冰盒中备用。在小鼠乳房垫中注射50 µL细胞悬浮液，接种后通过小动物活体成像系统检测荧光信号强度。
（2）抑制肿瘤肺转移的效果：待肿瘤部位荧光信号大约为3×10 5 p/s/cm 2/sr时，将原位荷瘤小鼠按照如上皮下抑瘤实验分组、给药、光照。给药后分别在0、2、6、12、18 d使用小动物活体成像系统进行生物发光成像来检测肿瘤部位信号强度，检测前每只小鼠腹腔注射水合氯醛麻醉（剂量为35 mg·kg -1），同时腹腔注射荧光素纳盐（剂量为150 mg·kg -1），10 min后进行扫描成像，观察肿瘤的生长情况，定量分析各时间点的荧光值，绘制肿瘤生长曲线。最后一天小鼠扫描完成后解剖出肺组织进行生物发光成像并拍摄白光照片。结果如图8。
图8结果表明，PBS处理的小鼠的肿瘤生长较快，肿瘤部位的荧光信号最强，肺部转移灶最多。游离药物非光照组与光照组、纳米粒非光照组，抗肿瘤功效和抑制肿瘤肺转移效果有限，与PBS组相比荧光信号减弱，说明仅有化疗不能完全抑制肿瘤生长和转移，游离化疗药/光敏剂+光照同样效果有限。而纳米粒+光照组，经过三次治疗即可消除肿瘤并有效抑制肿瘤向肺的转移，证明了本发明纳米粒将化疗和PDT结合后，在抑制肿瘤生长以及抑制肿瘤转移方面的显著优势；与皮下瘤实验结果一样，Vc可消弱纳米粒的抑瘤效果。
纳米粒的长期组织分布：将健康Balb/c小白鼠随机分成8组，每组3只，尾静脉注射纳米粒溶液，注射剂量为5 mg·kg -1（按Pt元素的浓度计算）。给药后分别在1、3、7、14、21、28、42 d处死小鼠解剖出心、肝、脾、肺、肾、肠，各脏器称重后加入王水和高氯酸进行高温硝解，定容后采用ICP-MS检测各器官中Pt元素含量。结果如图9，注射纳米粒后主要分布在肝和脾中，到42 d时铂的含量已下降约90%。而心、肺、肾、肠中铂的分布逐渐降低，到35 d时发现各器官中Pt元素含量均很少甚至为零，说明纳米粒可以从组织内逐渐清除，不会将重金属蓄积在体内，以免造成长期的毒副反应，具有较好的生物安全性。
纳米粒作用后的生化指标检测考察：为确定药物在体内代谢期间内的毒性，考察了肝肾功能的生化指标：检测肝指标丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）和碱性磷酸酶（ALP）和肾指标尿素（Urea）、肌酐（Crea），并与正常值比较，确定药物对肝肾功能的损伤。
将健康Balb/c小白鼠随机分成3组：PBS组、Pt/Ce6@HSA 纳米粒组、游离CDDP组，PBS组三只，纳米粒组和游离CDDP组每组12只，分别尾静脉注射纳米粒和游离CDDP溶液200 µL，注射剂量为5 mg·kg -1（按Pt元素的浓度计算），每隔一天给药一次，总共给药3次。全部给药完毕后，分别在给药后的7、14、21、28 d采取摘取眼球法从小鼠眼眶后静脉丛收集血液样品。PBS组也在28 d眼球取血。将收集的血样置于4℃冰箱过夜，然后用离心机1000 r·min -1离心5 min，取出上层血清，用血生化仪分析血清中肝功能指标天门冬氨酸氨基转移酶（AST）、碱性磷酸酶（ALP）、丙氨酸氨基转移酶（ALT）以及肾功能指标尿素（Urea）和肌酐（Crea）的水平。结果如图10，纳米粒给药组肝肾指标与对照组（PBS组）无显著性差异，说明肝肾功能无明显损伤，而游离CDDP组肝指标变化较大且Urea和Crea肾指标升高迅速，表明CDDP对肾具有一定毒性。综上，从生化指标的测量结果来看，本发明纳米粒药物没有明显的肝肾毒性副作用，具有广阔的临床应用前景。
实施例二：在实施例一中制备蛋白纳米粒时，分别将pH调节为5.0、6.0（实施例一中，pH为5.5），其他步骤同实施例一，可制得TEM尺寸为4.5~5.3 nm（因重原子作用，仅反映纳米粒核心的铂原子所在区域大小，下同）的纳米粒。
实施例三：将实施例一中蛋白纳米粒制备过程中，分别调节铂元素与Ce6的摩尔比例为1:1、8:1（实施例一中，Pt:Ce6摩尔比为4:1），其他条件与实施例一相同，可制得稳定性良好的Pt/Ce6蛋白纳米粒，TEM尺寸在4.2~4.9 nm之间。
将实施例一中蛋白纳米粒制备过程中，调节铂元素与Ce6的摩尔比例为1:2（实施例一中，Pt:Ce6摩尔比为4:1），其他条件与实施例一相同，反应后溶液较浑浊，不可制得稳定性良好的Pt/Ce6蛋白纳米粒。
实施例四：将实施例一中蛋白纳米粒制备过程中，分别调节反应温度为25℃、37℃（实施例一中，反应温度为55℃），其他条件与实施例一相同，可制得稳定性良好的Pt/Ce6蛋白纳米粒，TEM尺寸为4.2 nm~5 nm。
实施例五：将实施例一中硫化铂蛋白纳米粒的制备过程中反应时间调整为1 h、8 h（实施例一中，反应时间为4 h），其他条件与实施例一相同；反应1 h可制得稳定性良好的Pt/Ce6蛋白纳米粒，TEM尺寸在5 nm左右；如果反应 8 h，反应后溶液较浑浊，不可制得稳定性良好的Pt/Ce6蛋白纳米粒。
本发明以HSA为蛋白质模板，将难以直接包载的铂类化疗药与光敏剂Ce6通过在白蛋白内腔中共沉淀，制得纳米粒Pt/Ce6@HSA，参见图11，从而实现了共包载以及化疗作用/光动力作用联合应用于肿瘤治疗，为进一步研究为临床用药的奠定了基础。在纳米粒的制备中，成功获得粒径较小、分散性较好、形态规整的载药蛋白纳米粒。通过XPS、EDX和FTIR等表征证明了二价铂元素的存在以及铂和羧基的成功配位。此纳米粒的光稳定性、化学稳定性良好；同时，该纳米粒具有良好的单线态氧产生能力。此外，释放规律研究结果表明，纳米粒具有一定的缓释作用且有利于到达肿瘤部位发挥作用。
在纳米粒的抗肿瘤效果研究中，细胞水平的实验结果表明：肿瘤细胞对纳米粒的摄取显著增加且具有时间依赖性；摄取的纳米粒主要定位在溶酶体中，光照后细胞内产生大量ROS导致溶酶体破裂，从而促进药物的进一步转运进入胞浆及细胞核，实现了化疗与光动力治疗的协同作用，参见图12；MTT实验说明，纳米粒联合治疗有效地提高了抗肿瘤效力，且药物发挥的光动力作用、化疗作用强，体现了化疗与光动力治疗结合的优势；细胞增殖EdU染色实验结果表明，纳米粒能够显著地抑制肿瘤细胞增殖，并且光照后细胞增殖抑制效果显著增强，进一步佐证了纳米粒的抗肿瘤效果；细胞凋亡、线粒体膜电位染色、铂-DNA加合物及Western blot实验结果发现纳米粒治疗后线粒体膜电位下降，光照后电位下降显著，凋亡水平显著增加，加合物生成增多，凋亡相关蛋白表达变化显著，进一步证明了纳米粒联合两种治疗方法的优势。
小鼠体内的抗肿瘤效果评价结果表明：纳米粒消除半衰期延长，具有更好的长循环能力；纳米粒具有良好的肿瘤靶向性；纳米粒靶向到肿瘤部位后能够有效地产生活性氧，具有良好的PDT效果；纳米粒联合化疗作用/光动力作用治疗后，具有显著的小鼠肿瘤的生长抑制作用和转移抑制作用；切片染色结果表明，纳米粒能够显著损伤肿瘤细胞，抑制肿瘤增殖，证明了联合治疗的优势；纳米粒在小鼠体内能够经过生物代谢排出体外，无长期毒性，对肝肾无显著损伤，生物安全性高。
综上，本发明巧妙设计并成功制备了包载铂类药物和光敏剂的将化疗作用/光动力作用结合的蛋白纳米粒，产生协同抗肿瘤作用肿瘤抑制效果显著。

Claims (10)

  1. 载铂类药物/光敏剂的蛋白纳米粒，包括铂类药物/光敏剂配合物，以及包裹铂类药物/光敏剂配合物的蛋白质。
  2. 根据权利要求1所述载铂类药物/光敏剂的蛋白纳米粒，其特征在于，蛋白质为白蛋白，铂类药物为二水二氨合铂离子，光敏剂为二氢卟吩e6。
  3. 权利要求1所述载铂类药物/光敏剂的蛋白纳米粒的制备方法，包括以下步骤，将二氢卟吩e6溶液加入二水二氨合铂离子与蛋白的混合溶液中，反应后超滤离心，得到载铂类药物/光敏剂的蛋白纳米粒。
  4. 根据权利要求3所述载铂类药物/光敏剂的蛋白纳米粒的制备方法，其特征在于，蛋白、二水二氨合铂离子、二氢卟吩e6的用量比为100mg∶（20～50μmol）∶（3～15μmol）。
  5. 根据权利要求4所述载铂类药物/光敏剂的蛋白纳米粒的制备方法，其特征在于，蛋白、二水二氨合铂离子、二氢卟吩e6的用量比为100mg∶（25～40μmol）∶（5～10μmol）。
  6. 根据权利要求3所述载铂类药物/光敏剂的蛋白纳米粒的制备方法，其特征在于，将二氢卟吩e6溶液加入二水二氨合铂离子与蛋白的混合溶液中，调节至pH为 4.0～8.0，然后于25～60℃反应1～8 h；超滤离心的转速为1500～4000 r·min -1。
  7. 根据权利要求6所述载铂类药物/光敏剂的蛋白纳米粒的制备方法，其特征在于，调节至pH为 5.0～6.5；超滤离心时截留分子量为100 kD。
  8. 权利要求1所述载铂类药物/光敏剂的蛋白纳米粒在制备药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，药物为抗肿瘤药物。
  10. 根据权利要求8所述的应用，其特征在于，载铂类药物/光敏剂的蛋白纳米粒的粒径为2～50 nm。
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