WO2023274105A1 - 一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法及应用 - Google Patents

一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法及应用 Download PDF

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WO2023274105A1
WO2023274105A1 PCT/CN2022/101401 CN2022101401W WO2023274105A1 WO 2023274105 A1 WO2023274105 A1 WO 2023274105A1 CN 2022101401 W CN2022101401 W CN 2022101401W WO 2023274105 A1 WO2023274105 A1 WO 2023274105A1
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pcm
kala
dna
rbcm
pro
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French (fr)
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陈一杰
陈梦纯
夏伟梁
诸葛德力
诸海燕
赵应征
姜琦
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温州医科大学附属第二医院（温州医科大学附属育英儿童医院）
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
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    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
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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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
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    • 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

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a preparation method and application of a bionic nano-protective agent for adriamycin cardiac and systemic toxicity detoxification.
  • DOX Doxorubicin
  • a classic chemotherapy drug is widely used in the treatment of different cancer types.
  • DOX exhibits good cancer therapeutic effects, its toxic side effects (including cardiotoxicity, liver toxicity, and gastrointestinal reactions) greatly limit its further clinical application.
  • the main mechanism of DOX killing cancer cells is to bind to double-stranded DNA in cancer cells to inhibit DNA replication in the nucleus and induce cell apoptosis.
  • DOX can be specifically inserted into the GC base pairs of DNA double strands. According to this feature, there have been studies on inserting DOX into double-stranded nucleic acids such as plasmids to achieve co-delivery of nucleic acid and DOX.
  • the existing patent application number is CN202010284531.3, which discloses the medical use of a CREG protein for the prevention or treatment of adriamycin myocardial injury. According to the description of the patent text, it can be seen that the invention is to treat the damaged myocardium. Timely treatment will still leave damage to the myocardium and/or liver, and there is still a large burden on the patient.
  • the purpose of the present invention is to provide a preparation method and application of a biomimetic nano-protective agent for the detoxification of the heart and systemic toxicity of adriamycin, which can overcome the damage to the myocardium and / or the problem of liver damage can reduce the physical burden of the patient.
  • the present invention provides the following technical scheme: a method for preparing a biomimetic nano-protective agent for adriamycin cardiac and systemic toxicity detoxification, comprising the following steps:
  • DNA&Pro@RBCM-PCM/KALA biomimetic nano-protective agent was prepared by mixing RBCM-PCM/KALA and DNA&Pro nanoparticles.
  • DNA&Pro@RBCM-PCM/KALA biomimetic nano-protective agent is prepared by Extrusion method after mixing RBCM-PCM/KALA and DNA&Pro nanoparticles.
  • the ratio of each component DNA:Protamine:RBCM:DSPE-PEG2000-MAL:PCM/KALA 6:5:150:3.75:7.5 (w/w).
  • the DNA used uses the green fluorescent protein particle as a DNA template, and is 630 bp in size and has a GC content of 65%.
  • DNA & Pro nanoparticles are formed by ultrasonication after DNA is mixed with protamine Protamine, and the ultrasonication time is 20s.
  • the ratio of heterofunctional polyethylene glycol DSPE-PEG2000-MAL to PCM/KALA is 1:2 (mol/mol), and they are all dissolved in ultrapure water, incubated at 37°C for 30-60min to synthesize DSPE -PEG2000-PCM/KALA.
  • DSPE-PEG2000-PCM/KALA is mixed with RBCM membrane protein at a mass ratio of 1:20 (w/w), and incubated at 37° C. for 30-60 min to prepare RBCM-PCM/KALA.
  • the Extrusion method used to prepare DNA&Pro@RBCM-PCM/KALA has a pore size of 400nm.
  • PCM/KALA has very good selectivity and can be enriched in normal organs such as the heart and liver, rather than tumor tissues;
  • nanoparticles composed of DNA and protamine to form a protective barrier to prevent doxorubicin from damaging myocardial and/or liver cells and causing harm to the human body;
  • the red blood cell membrane can bypass the immune system to ensure the construction efficiency and effect of the barrier
  • connection between PCM/KALA and the red blood cell membrane can wrap nanoparticles composed of DNA and protamine, and quickly deliver the barrier-forming nanoparticles to normal organs such as the heart and liver instead of tumor tissues.
  • PCM/KALA can quickly mediate the phagocytosis of nanoparticles to improve the efficiency of building a protective barrier.
  • the protective agent After the protective agent is phagocytized by normal cells such as cardiomyocytes, it escapes from the lysosome and disperses in the cytoplasm, effectively preventing DOX from entering the nucleus and preventing or alleviating cell apoptosis.
  • the protective agent does not or rarely enters the tumor tissue, and DOX can achieve the best tumor suppression effect.
  • Fig. 1 is the schematic diagram of collecting and purifying the erythrocyte membrane
  • Figure 2 is a schematic diagram of the verification of the size of the target DNA fragment
  • Figure 3 is a schematic diagram of the compression ability of Pro to DNA at different nitrogen and phosphorus ratios
  • Figure 4 is a schematic diagram of the particle size and potential of DNA&Pro nanoparticles
  • Figure 5 is a schematic diagram of the construction process of DNA&Pro@RBCM-PCM/KALA biomimetic nano protective agent
  • Figure 6 is a schematic diagram of the ratio screening of RBCM-PAM/KALA fully encapsulated DNA&Pro nanoparticles
  • Figure 7 is a schematic diagram of the co-localization of RBCM and DNA&Pro nanoparticles studied by CLSM imaging technology
  • Figure 8 is a schematic diagram of the particle size and surface potential of the DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector
  • Figure 9 is a schematic diagram of the targeting effect of DNA&Pro@RBCM-PCM/KALA on cardiomyocytes
  • Figure 10 is a schematic diagram of the lysosome escape ability of DNA&Pro@RBCM-PCM/KALA;
  • Figure 11 is a schematic diagram of different doses of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector protecting cardiomyocytes in a concentration-dependent manner;
  • Figure 12 is a schematic diagram of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protective agent against cardiomyocyte apoptosis;
  • Figure 13 is a schematic diagram of different doses of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector protecting liver cells in a concentration-dependent manner;
  • Figure 14 is a schematic diagram of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protective agent against liver cell apoptosis;
  • Figure 15 is a schematic diagram of the biodistribution of nanoparticles in different experimental groups in tumor-bearing B6 mice;
  • Figure 16 is a schematic diagram of the effect of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector protecting high dose DOX (20mg/kg) on blood routine at the time point of Day8;
  • Figure 17 is a schematic diagram of the effect of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector on blood biochemistry at the time point of Day 8 when protecting high-dose DOX (20mg/kg);
  • Figure 18 is a schematic diagram of the effect of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector protecting high dose DOX (20mg/kg) on the super long axis of the heart at the time point of Day8;
  • Figure 19 is a schematic diagram of the effect of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector on the short axis of the heart at the time point of Day 8 when protecting high-dose DOX (20mg/kg);
  • Figure 20 is a schematic diagram showing that DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector protects the body weight of tumor-bearing B6 mice without affecting the anti-tumor effect of DOX.
  • the erythrocytes are derived from C57BL/6 mice (B6 mice), and the process is as follows: collect whole blood of B6 mice, centrifuge at 3000-5000 rpm for 15 minutes to remove white blood cells, platelets and serum, and obtain erythrocytes.
  • DSPE-PEG2000-PCM and DSPE-PEG2000-PCM/KALA were prepared. Accurately weigh DSPE-PEG2000-MAL, PCM/KALA and PCM respectively, and dissolve them in ultrapure water. Subsequently, mix according to the ratio of DSPE-PEG2000-MAL:PCM/KALA 1:2 (mol/mol), DSPE-PEG2000-Mal:PCM 1:2 (mol/mol), and incubate at 37°C for 30-60min to synthesize DSPE-PEG2000-PCM/KALA and DSPE-PEG2000-PCM are ready for use.
  • DSPE-PEG2000-PCM/KALA or DSPE-PEG2000-PCM were mixed with RBCM according to the mass ratio of DSPE-PEG2000-MAL and RBCM (membrane protein content) at 1:20 (w/w), and incubated at 37°C for 30 -60min. After the reaction was completed, the above reactants were placed in a centrifuge and centrifuged at 14000rpm for 10 minutes to remove excess DSPE-PEG2000-PCM/KALA or DSPE-PEG2000-PCM to prepare RBCM-PCM/KALA and RBCM-PCM.
  • the prepared RBCM-PCM/KALA and Bio-DNA&Pro nanoparticles were mixed according to different mass ratios of RBCM membrane protein and DNA (0, 100:1, 25:1, 6.25:1, 1.56:1), and then passed the Extrusion method (400nm pore size) to obtain Bio-DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector. Subsequently, a certain amount of streptavidin was added, and then the particle size change of the nanoparticles was detected by a particle size potentiometer ( FIG. 6 ). The results showed that when the mass ratio of RBCM membrane protein to DNA was greater than 6.25:1, RBCM-PCM/KALA could completely wrap Bio-DNA/Pro nanoparticles.
  • the present invention determines the mass ratio of the two as 25:1 (w/w), and uses DiD dyes to label RBCM-PCM/KALA and FAM primers to prepare FAM -DNA, finally prepare double-labeled DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector with DiD label on RBCM and FAM label on DNA.
  • the mass ratio of RBCM membrane protein and DNA is determined to be 25:1 by the above method
  • the hydraulic size and surface potential of the constructed DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector were determined to be 400nm and -39.4mV, respectively ( Figure 8).
  • the DiD fluorescent dye was mixed with RBCM or RBCM-PCM or RBCM-PCM/KALA according to the ratio of DiD to RBCM mass (calibrated by membrane protein mass) 6:1000 (w/w), and incubated at 37°C for 30 min. Subsequently, the supernatant was removed by centrifugation at 14000 rpm for 10 min, and washed three times with PBS to ensure complete removal of free DiD dye. At the same time, DNA & Pro nanoparticles were synthesized for use.
  • DiD fluorescently labeled RBCM or RBCM-PCM or RBCM-PCM/KALA was wrapped with DNA&Pro nanoparticles at a ratio of RBCM:DNA of 25:1 (w/w) to construct DNA&Pro@RBCM, DNA&Pro@RBCM-PCM and DNA&Pro, respectively.
  • @RBCM-PCM/KALA was wrapped with DNA&Pro nanoparticles at a ratio of RBCM:DNA of 25:1 (w/w) to construct DNA&Pro@RBCM, DNA&Pro@RBCM-PCM and DNA&Pro, respectively.
  • NC group Negative Control, no treatment group
  • RBCM group only DiD-labeled DNA&Pro@RBCM
  • PCM group only DiD-labeled DNA&Pro@RBCM-PCM
  • PCM antagonistic group after pretreatment with free PCM for 4 hours, then add DiD-labeled DNA&Pro@RBCM-PCM
  • 5.PCM/KALA group only add DiD-labeled DNA&Pro@RBCM-PCM/KALA
  • 6.PCM/KALA antagonistic group After pretreatment with free PCM/KALA for 4 hours, add DiD-labeled DNA&Pro@RBCM-PCM/KALA).
  • FAM-DNA was used to prepare DNA&Pro@RBCM, DNA&Pro@RBCM-PCM and DNA&Pro@RBCM-PCM/KALA with FAM fluorescent label for use.
  • Spread H9C2 cardiomyocytes in a confocal cell culture dish and when the confluence rate reaches 70-80%, add three experimental groups of nanoparticles, remove free particles after 2 hours and wash three times with PBS. Subsequently, fresh complete medium was added again, and the lysosomal dye was added after continuing to culture for 24 hours. After incubation at 37°C for 60 min, wash with PBS three times, and add DAPI dye to mark the nuclei.
  • the present invention determines a dose of PCM/KALA-RBCM@DNA&Pro biomimetic nano-protector (NPs) to protect IC50 dose of DOX from damage to H9C2 cardiomyocytes.
  • NPs PCM/KALA-RBCM@DNA&Pro biomimetic nano-protector
  • three important apoptosis pathway signaling molecules including Bax, Bcl2, and Caspase-3) were selected for Western Blot (WB) detection (see Figure 12).
  • WB Western Blot
  • the results showed that PCM/KALA-RBCM@DNA&Pro nanoparticles significantly increased the expression of anti-apoptotic molecule Bcl2 and decreased the expression of pro-apoptotic molecule Bax after protection.
  • PCM/KALA-RBCM@DNA&Pro biomimetic nano-protectors (NPs) can also protect QSG-7702 liver cell damage caused by DOX through cell viability experiments ( Figure 13) and WB detection ( Figure 14).
  • TC1 mouse-derived cervical cancer cells were subcutaneously injected into the back of 6-8 week-old C57BL/6 mice (B6 mice) to construct a subcutaneous xenograft tumor model of cervical cancer.
  • DNA&Pro@RBCM-PCM/KALA abbreviated as RBCM-PCM/KALA
  • DNA&Pro@RBCM-PCM abbreviated as RBCM-PCM
  • DNA&Pro@RBCM abbreviated as RBCM with DiD fluorescent label in the RBCM membrane were prepared according to the above method.
  • the three kinds of granules were injected into the B6 mice through the tail vein with the dose of RBCM membrane protein at 50 mg/kg (the DNA dose was about 13 mg/kg).
  • the small animal in vivo imager detected the enrichment of nanoparticles in the heart, liver and tumor tissues of B6 mice in different experimental groups (see Figure 15).
  • the imaging results showed that, for the enrichment of heart and liver, RBCM-PCM/KALA group>RBCM-PCM group>RBCM group (Fig. 15A-B).
  • the RBCM-PCM/KALA group was much smaller than the other two groups, with almost no or very little access to the tumor site (Fig. 15C).
  • the heart, liver and tumor tissues of different experimental groups were homogenized, and the DiD fluorescence signal was quantitatively analyzed by a microplate reader ( FIG. 15D ).
  • the results showed that the total amount of fluorescence in different tissue homogenates in different experimental groups was consistent with the results in live imaging of small animals, indicating that the RBCM-PCM/KALA biomimetic nano-protective agent was effectively enriched in the heart and liver, and did not enter the tumor tissue.
  • DNA&Pro@RBCM-PCM/KALA biomimetic nanoprotector protects against cardiotoxicity and systemic toxicity induced by high doses of DOX in the treatment of tumor-bearing C57BL/6 mice
  • 1x10 6 TC1 cells were subcutaneously injected into the back of 6-8 week-old B6 mice to construct a subcutaneous xenograft tumor model of cervical cancer.
  • a certain dose of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector 80mg/kg DNA, corresponding to RBCM membrane protein concentration of 300mg/kg
  • NPs+DOX group 20 mg/kg DOX was injected again (Day 0, referred to as NPs+DOX group).
  • set DOX group alone and no treatment group (NC group) as the experimental control group.
  • Blood routine indicators include white blood cells (WBC: 10 9 /L), red blood cells (RBC: 10 12 /L), hemoglobin (Hb: g/L), platelets (PLT: 10 9 /L), red blood cells Volume (MCV: fL), hematocrit (HCT: %); blood biochemical indicators include serum creatine kinase isoenzyme (CKMB: IU/L), alanine aminotransferase (ALT: IU/L), aspartate aminotransferase (AST : IU/L), total protein (TP: g/L), albumin (ALB: g/L), globulin (GLO: g/L), blood glucose (GLU-S: mmol/L), blood urea nitrogen ( BUN: mmol/L), creatinine (CREA: mmol/L), lactate dehydrogenase (LDH: IU/L); echocardiographic indicators include stroke volume per minute (SV,
  • TC1 cells were subcutaneously injected into the back of 6-8 week-old B6 mice to construct a subcutaneous xenograft tumor model of cervical cancer.
  • the tumor volume reached 70-80mm 3
  • the NPs+DOX group, the DOX alone group and the NC group were performed respectively, and then the changes in tumor tissue size and body weight of B6 mice were observed and measured from Day 0 to Day 24.
  • the results showed that in terms of tumor suppressive effect, the growth trend of tumor tissue in the DOX group and the NPs+DOX group was significantly inhibited, and the volume at the end of Day 24 was only about 15 times the initial volume.
  • the volume of tumor tissue in the NC group grew to about 200 times the initial volume size on Day 24 ( FIG.
  • the body weight of the B6 mice in the NPs+DOX group was significantly different from that of the B6 mice in the NC group, the weight difference between the NC group and the NPs+DOX group basically came from the analysis of the weight data of the isolated tumor tissue. Tumor weight (Fig. 20C). Therefore, under the protection of DNA&Pro@RBCM-PCM/KALA biomimetic nano-protector (NPs), the body weight of B6 mice did not decrease but increased steadily. This further confirms that NPs are only distributed in normal organs rather than tumor tissues.
  • the present invention relates to the preparation of a biomimetic nano-protective agent with nanoscale and "chromatin-like" structure, which is used to protect the heart and systemic toxicity caused by DOX.
  • the biomimetic nano-protective agent is composed of biologically derived cell membranes modified with cardiomyocyte targeting groups and a "chromatin-like" structure formed by self-assembly of protamine (Protamine, Pro) and DNA fragments rich in GC base pairs.
  • the protective agent is dispersed in the cytoplasm after being phagocytized by cardiomyocytes and other normal cells, effectively preventing DOX from entering the nucleus, and preventing or alleviating cell apoptosis.
  • the cardiomyocyte targeting group is a fusion peptide PCM/KALA, and its sequence is (WSGTGRLARVTVVPGAESLW-CO-hydrazine hydrate-boc-WEAKLAKALAKALA-K(DDE)-HLAKALAKALKACEA).
  • PCM polypeptide sequence: WLSEAGPVVTVRALRGTGSW
  • KALA polypeptide sequence: WEAKLAKALAKALAKHLAKALAKALKACEA
  • the PCM/KALA fusion peptide not only targets cardiomyocytes, mediates the rapid phagocytosis of cardiomyocytes, but also realizes timely lysosome escape, and can quickly release the above-mentioned protective agents as a barrier into the cytoplasm to build a barrier for the nucleus. Block DOX from causing damage to the nucleus, thereby preventing damage to cardiac muscle and/or liver cells.
  • the specific action process is as follows. Firstly, through the targeting effect of PCM, DNA&Pro@RBCM-PCM/KALA is integrated into cardiac muscle and/or liver cells.
  • KALA can mediate the rapid phagocytosis of cells and the subsequent lysosomal escape, thereby allowing DNA&Pro to be quickly released into cells, providing The nucleus builds a protective barrier.
  • the present invention couples PCM/KALA polypeptide to the surface of red blood cell membrane (RBCM) to construct RBCM-PCM/KALA, and then wraps it with DNA&Pro nanoparticles formed by self-assembly of DNA and protamine (Pro) through the Extrusion method, and finally constructs about 400nm DNA&Pro@RBCM-PCM/KALA biomimetic nano protectant.
  • DNA&Pro@RBCM-PCM/KALA can efficiently adsorb DOX and reduce the damage of DOX to normal cells.
  • DNA&Pro@RBCM-PCM/KALA biomimetic nano protective agent is only enriched in heart, liver and other normal organs and rarely enters tumor tissue, so it protects normal organs from DOX damage (heart and other toxicity) and does not affect DOX tumor cells Killing effect.

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Abstract

一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂的制备方法，包括如下步骤：获取DNA与鱼精蛋白Protamine并混合制备获得DNA&Pro纳米颗粒；将异官能团聚乙二醇DSPE-PEG2000-MAL与PCM/KALA反应制备获得DSPE-PEG2000-PCM/KALA；将DSPE-PEG2000-PCM/KALA插入到红细胞膜RBCM表面制备获得PCM-KALA-RBCM；将PCM-KALA-RBCM和DNA&Pro纳米颗粒混合后制备获得DNA&Pro@PCM-KALA-RBCM仿生纳米保护剂。上述制备获得的仿生纳米保护剂可以应用在阿霉素解毒中，克服现有技术中在针对癌症治疗过程中对心肌和/或肝脏留下伤害的问题。

Description

一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法及应用 技术领域
本发明涉及生物医药技术领域，具体为一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法及应用。
背景技术
阿霉素(Doxorubicin，DOX)作为一种经典的化疗药物被广泛的应用到不同癌症类型的治疗。尽管DOX展现出良好的癌症治疗效果，但是它的毒副作用(包括心脏毒性、肝脏毒性和胃肠道反应)极大地限制了其进一步的临床应用。众所周知，DOX杀伤癌细胞的主要机理是与癌细胞中的双链DNA结合抑制细胞核内DNA复制，诱导细胞凋亡。另外，DOX能够特异性的插入到DNA双链的GC碱基对中，根据该特点，已经有研究将DOX插入到质粒等双链核酸中实现核酸与DOX的共递送。
现有的专利申请号为CN202010284531.3的发明专利公开了一种CREG蛋白用于预防或治疗阿霉素心肌损伤的医药用途，根据该专利文本的说明书可知，该发明是对损伤后的心肌进行及时的治疗，还是会在心肌和/或肝脏留下伤害，对于病人来说依旧存在较大的负担。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法及应用，能够克服现有技术中在针对癌症治疗过程中对心肌和/或肝脏留下伤害的问题，能够减少病人的身体负担。
为实现上述目的，本发明提供了如下技术方案：一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法，包括如下步骤：
获取DNA与鱼精蛋白Protamine并混合制备获得DNA&Pro纳米颗粒；
将异官能团聚乙二醇DSPE-PEG2000-MAL与PCM/KALA反应制备获得 DSPE-PEG2000-PCM/KALA；
将DSPE-PEG2000-PCM/KALA插入到红细胞膜RBCM表面制备获得PCM/KALA-RBCM；
将RBCM-PCM/KALA和DNA&Pro纳米颗粒混合后制备获得DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。
作为本发明的优选，RBCM-PCM/KALA和DNA&Pro纳米颗粒混合后利用Extrusion方法制备DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。
作为本发明的优选，各个组分比例DNA∶Protamine∶RBCM∶DSPE-PEG2000-MAL∶PCM/KALA＝6∶5∶150∶3.75∶7.5(w/w)。
作为本发明的优选，所采用的DNA以绿色荧光蛋白质粒为DNA模板，且为630bp、GC含量65％。
作为本发明的优选，DNA与鱼精蛋白Protamine混合后通过超声形成DNA&Pro纳米颗粒，超声时间为20s。
作为本发明的优选，混合的DNA与鱼精蛋白Protamine的氮磷比N/P＝2。
作为本发明的优选，异官能团聚乙二醇DSPE-PEG2000-MAL与PCM/KALA的比例为1∶2(mol/mol)，且均溶于超纯水中，37℃孵育30-60min合成DSPE-PEG2000-PCM/KALA。
作为本发明的优选，DSPE-PEG2000-PCM/KALA与RBCM膜蛋白质量的质量比为1∶20(w/w)混合，在37℃孵育30-60min，制备获得RBCM-PCM/KALA。
作为本发明的优选，制备DNA&Pro@RBCM-PCM/KALA是利用的Extrusion方法所采用的孔径为400nm。
本发明的有益效果，
1.PCM/KALA具有非常好的选择性，能够富集至心脏、肝脏等正常器官， 而非肿瘤组织；
2.利用DNA和鱼精蛋白构成的纳米颗粒组建防护屏障，避免阿霉素损伤心肌和/或肝脏细胞，对人体造成伤害；
3.红细胞膜能够绕过免疫系统，确保屏障的构建效率和作用效果；
4.PCM/KALA与红细胞膜的连接能够包裹DNA和鱼精蛋白构成的纳米颗粒，将组建屏障的纳米颗粒快速送达心脏、肝脏等正常器官而非肿瘤组织。
5.PCM/KALA能够快速介导细胞吞噬纳米颗粒，以提高组建防护屏障的效率。
6.该保护剂被心肌细胞等正常细胞吞噬后从溶酶体中逃逸并分散于细胞质中，有效阻止DOX进入细胞核，防止或缓解细胞凋亡。
7.该保护剂不进入或极少进入肿瘤组织内，DOX可以达到最佳的肿瘤抑制效果。
附图说明
图1为收集纯化的红细胞膜示意图；
图2为目的DNA片段大小验证示意图；
图3为不同氮磷比时Pro对DNA的压缩能力示意图；
图4为DNA&Pro纳米颗粒的粒径及电位示意图；
图5为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的构建流程示意图；
图6为RBCM-PAM/KALA完全包裹DNA&Pro纳米颗粒的比例筛选示意图；
图7为CLSM成像技术研究RBCM与DNA&Pro纳米颗粒的共定位示意图；
图8为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的粒径和表面电位示意图；
图9为DNA&Pro@RBCM-PCM/KALA对心肌细胞的靶向作用示意图；
图10为DNA&Pro@RBCM-PCM/KALA的溶酶体逃逸能力示意图；
图11为不同剂量DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂呈浓度依赖性保护心肌细胞示意图；
图12为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂抗心肌细胞凋亡示意图；
图13为不同剂量DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂呈浓度依赖性保护肝脏细胞示意图；
图14为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂抗肝脏细胞凋亡示意图；
图15为不同实验组纳米颗粒在荷瘤B6鼠体内的生物分布示意图；
图16为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护高剂量DOX(20mg/kg)在Day8时间点对血常规的影响示意图；
图17为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护高剂量DOX(20mg/kg)在Day8时间点对血生化的影响示意图；
图18为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护高剂量DOX(20mg/kg)在Day8时间点对心超长轴的影响示意图；
图19为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护高剂量DOX(20mg/kg)在Day8时间点对心短轴的影响示意图；
图20为DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护荷瘤B6鼠体重及不影响DOX抗肿瘤效果示意图。
具体实施方式
下面将结合附图所给出的实施例对本发明做进一步的详述。
参照图1-20所示，
实施例1
红细胞膜(RBCM)的制备
红细胞来源于C57BL/6鼠(B6鼠)，过程如下：收集B6鼠全血，3000-5000rpm离心15min去除白细胞、血小板和血清，获得红细胞。将250μL的红细胞加入950μL超纯水中，冰浴30-60min，加入20x PBS调渗透压至1x，混匀后，14000rpm离心10min，弃上清，加950μL超纯水重悬，再次冰浴，调渗透压，离心，以此循环直至上清无血红蛋白，并收集沉淀(图1)。收集的沉淀即为红细胞膜(RBCM)。通过BCA法测定RBCM中膜蛋白的浓度。
实施例2
富含65％GC碱基对的DNA制备
以绿色荧光蛋白质粒为DNA模板，设计前引物CGGCCACAAGTTCGTGAT以及后引物AATCCAGAGGTTGATTGTTCCA，拟获得630bp、GC含量约65％的DNA片段，其序列为CGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTCCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACG GTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGCCCTGAACGCGTCTGGAACAATCAACCTCTGGATT。随后，利用PCR仪扩增质粒DNA，纯化和分离目的DNA片段，并利用Nanodrop测定DNA浓度。通过琼脂糖凝胶验证DNA片段大小(图2)。图2中，左边条带为Marker，右边条带为目的DNA片段，其大小约在630bp左右，符合预期设计。
实施例3
DNA和Pro自组装形成DNA&Pro纳米颗粒的比例筛选
分别按照Pro与DNA的不同氮磷比(N/P＝0，0.5，1，1.5，2，2.5，3，mol/mol)将两者混合，超声20s形成DNA&Pro纳米颗粒待用。利用琼脂糖凝胶电泳方法检测Pro是否完全将DNA压缩包裹(图3)。结果显示，当N/P在2时，Pro能将DNA完全压缩成纳米颗粒，使DNA片段无法呈游离状态而在电流作用下发生位移。随后，利用粒度电位仪测量DNA&Pro纳米颗粒的水力学尺寸大小和表面电位(图4)。结果显示，该DNA&Pro纳米颗粒的粒径和电位分别为128nm和-50mV。
实施例4
RBCM-PCM/KALA仿生纳米颗粒的构建
首先，制备DSPE-PEG2000-PCM与DSPE-PEG2000-PCM/KALA。分别准确称取DSPE-PEG2000-MAL、PCM/KALA以及PCM，并溶于超纯水中。随后， 按照DSPE-PEG2000-MAL∶PCM/KALA为1∶2(mol/mol)、DSPE-PEG2000-Mal∶PCM为1∶2(mol/mol)的比例混合，37℃孵育30-60min分别合成DSPE-PEG2000-PCM/KALA和DSPE-PEG2000-PCM待用。其次，按照DSPE-PEG2000-MAL与RBCM(膜蛋白质量)质量比为1∶20(w/w)将DSPE-PEG2000-PCM/KALA或DSPE-PEG2000-PCM)与RBCM分别混合，37℃孵育30-60min。完成反应后，将上述反应物置于离心机中，14000rpm离心10min，去除多余DSPE-PEG2000-PCM/KALA或DSPE-PEG2000-PCM，制备RBCM-PCM/KALA和RBCM-PCM。
实施例5
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的构建与比例筛选
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的构建流程(见图5)以及筛选合适比例的RBCM-PCM/KALA与DNA&Pro纳米颗粒。首先，利用生物素化的前引物biotin-CGGCCACAAGTTCGTGAT以及后引物AATCCAGAGGTTGATTGTTCCA-biotin合成含有生物素标记的目标DNA片段(记作Bio-DNA)，再按照N/P＝2(mol/mol)的比例构建Bio-DNA&Pro纳米颗粒待用。将制备好的RBCM-PCM/KALA与Bio-DNA&Pro纳米颗粒按照RBCM膜蛋白和DNA的不同质量比(0，100∶1，25∶1，6.25∶1，1.56∶1)混合，再通过Extrusion方法(400nm孔径)获得Bio-DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。随后，加入一定量的链霉亲和素，再通过粒度电位仪检测纳米颗粒粒径大小变化(图6)。结果显示，当RBCM膜蛋白与DNA的质量比大于6.25∶1时，RBCM-PCM/KALA能完全包裹Bio-DNA/Pro纳米颗粒。当RBCM膜蛋白与DNA的质量比小于6.25∶1时，一部分Bio-DNA&Pro纳米颗粒将处 于游离状态，故能与加入的链霉亲和素结合发生团聚，出现微米级别的粒径分布。为了进一步确认RBCM-PCM/KALA完全包裹Bio-DNA&Pro纳米颗粒，本发明将两者质量比确定为25∶1(w/w)，同时利用DiD染料标记RBCM-PCM/KALA以及利用FAM引物制备FAM-DNA，最终制备RBCM上带有DiD标记以及DNA带有FAM标记的双标记DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。随后，利用CLSM成像技术观察RBCM-PCM/KALA(DiD)和DNA&Pro纳米颗粒(FAM)两者的共定位情况(图7)。结果显示，代表RBCM-PCM/KALA的DiD红色信号与代表DNA的FAM绿色信号均重叠，进一步说明在RBCM与DNA质量比为25∶1时，RBCM-PCM/KALA已经完全包裹DNA&Pro纳米颗粒形成DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。通过上述方法确定RBCM膜蛋白与DNA的质量比为25∶1时，仿生纳米保护剂各组成的质量比为DNA∶Pro∶RBCM∶PCM/KALA＝6∶5∶150∶7.5(w/w)。同时，测定构建的DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的水力学尺寸大小和表面电位分别为400nm和-39.4mV(图8)。
实施例6
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂对心肌细胞的靶向效果
将DiD荧光染料分别与RBCM或RBCM-PCM或RBCM-PCM/KALA按照DiD与RBCM质量(以膜蛋白质量标定)比6∶1000(w/w)混合，37℃孵育30min。随后，14000rpm离心10min去除上清，并利用PBS清洗3次，确保完全去除游离的DiD染料。同时，合成DNA&Pro纳米颗粒待用。最后，将DiD荧光标记的RBCM或RBCM-PCM或RBCM-PCM/KALA按照RBCM∶DNA为25∶1(w/w)的比例包裹DNA&Pro纳米颗粒分别构建DNA&Pro@RBCM、 DNA&Pro@RBCM-PCM及DNA&Pro@RBCM-PCM/KALA。
实验组分：NC组(Negative Control，无任何处理组)；RBCM组(仅加入DiD标记的DNA&Pro@RBCM)；3.PCM组(仅加入DiD标记的DNA&Pro@RBCM-PCM)；4.PCM拮抗组(利用free PCM预处理4h后，再加入DiD标记的DNA&Pro@RBCM-PCM)；5.PCM/KALA组(仅加入DiD标记的DNA&Pro@RBCM-PCM/KALA)；6.PCM/KALA拮抗组(利用free PCM/KALA预处理4h后，再加入DiD标记的DNA&Pro@RBCM-PCM/KALA)。大致实验流程：在6孔板中培养H9C2细胞(大鼠来源的心肌细胞)，待其汇合率至80％后分别加入上述不同实验组，每个组RBCM膜蛋白统一浓度为100μg/mL。2h后，去除上清，再利用PBS清洗3次。随后，利用胰酶消化细胞成单细胞悬液，再通过流式细胞术检测细胞内DiD荧光信号强弱。流式细胞术实验结果统计见图9。结果表明，PCM组和PCM/KALA组较单独RBCM组的心肌细胞靶向效率分别提高约2倍和10倍。在PCM组中，PCM仅提高颗粒与H9C2细胞之间的亲和力，并不能快速介导颗粒被H9C2细胞吞噬，因此靶向效果仅为RBCM组的2倍。对于PCM/KALA组，PCM在增加颗粒与H9C2细胞亲和力的同时，KALA能快速介导颗粒被H9C2细胞吞噬，故靶向效果达到RBCM组的10倍。尽管KALA在介导H9C2细胞快速吞噬颗粒的过程中起到关键作用，但PCM的靶向作用是前提。从拮抗实验结果可以得出，若利用freePCM/KALA预处理H9C2细胞(PCM/KALA拮抗组)，使H9C2细胞表面PCM受体提前封闭，该细胞不再有效吞噬PCM/KALA组颗粒，其靶向效果约为RBCM组的2倍。同样，经过freePCM预处理的PCM拮抗组也能显著降低颗粒被H9C2细胞吞噬。综上所述，DNA&Pro@RBCM-PCM/KALA先通过PCM靶向结合到H9C2细胞表面，同时再利用KALA实现快速的细胞吞噬。
实施例7
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂的溶酶体逃逸功能
按上述方法，利用FAM-DNA分别制备带有FAM荧光标记的DNA&Pro@RBCM、DNA&Pro@RBCM-PCM及DNA&Pro@RBCM-PCM/KALA待用。将H9C2心肌细胞铺于共聚焦细胞培养皿内，待其汇合率到达70-80％时分别加入3个实验组纳米颗粒，2h后去除游离颗粒并用PBS洗3次。随后，重新加入新鲜的完全培养基，继续培养至24h后加入溶酶体染料。37℃孵育60min后利用PBS洗3次，加入DAPI染料标记细胞核。最后，利用共聚焦激光扫描显微镜CLSM成像技术观察不同实验组细胞内颗粒荧光信号(绿色)与溶酶体荧光信号(红色)的共定位情况。结果显示，DNA&Pro@RBCM-PCM/KALA组中溶酶体荧光信号(红色)与颗粒荧光信号(绿色)几乎不重叠，说明颗粒已从溶酶体中逃逸。相反的，在相同时间点，DNA&Pro@RBCM-PCM或DNA&Pro@RBCM组中溶酶体荧光信号(红色)与颗粒荧光信号(绿色)大部分重叠，说明颗粒仍处于溶酶体内(见图10)。以上结果表明，KALA多肽的修饰能够有效介导颗粒穿透溶酶体进入细胞质。
实施例8
将不同剂量的DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂加入到H9C2心肌细胞中孵育4h，随后加入IC50(50％Inhibitory Concentration)剂量DOX，24h后利用CCK-8试剂检测H9C2心肌细胞的存活率，其中设置单独DOX组(DNA浓度为0)和无任何处理的NC组(Negative Control)为实验对照组。结果表明，单独DOX组导致H9C2细胞的存活率下降至约50％。然而，随着 PCM/KALA-RBCM@DNA&Pro仿生纳米保护剂剂量的不断提高，H9C2细胞的存活率也逐渐上升直至完全保护细胞活性，说明PCM/KALA-RBCM@DNA&Pro仿生纳米保护剂具有浓度依赖性的吸附DOX作用(见图11)。为了进一步阐述PCM/KALA-RBCM@DNA&Pro仿生纳米保护剂的保护机制，本发明确定一个PCM/KALA-RBCM@DNA&Pro仿生纳米保护剂(NPs)剂量用于保护IC50剂量DOX对H9C2心肌细胞的损伤，同时选择3个重要的凋亡通路信号分子(包括Bax，Bcl2，Caspase-3)进行Western Blot(WB)检测(见图12)。结果表明，PCM/KALA-RBCM@DNA&Pro纳米颗粒保护后显著提高抗凋亡分子Bcl2的表达，以及降低促凋亡分子Bax的表达。平行的，通过细胞存活率实验(图13)和WB检测(图14)证明PCM/KALA-RBCM@DNA&Pro仿生纳米保护剂(NPs)也能保护DOX导致的QSG-7702肝细胞细胞损伤。
实施例9
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂在荷瘤B6鼠中的生物分布
在6-8周龄的C57BL/6鼠(B6鼠)背部皮下注射1x10 6个TC1鼠源宫颈癌细胞，构建宫颈癌皮下移植瘤模型。按上述方法分别制备RBCM膜内带有DiD荧光标记的DNA&Pro@RBCM-PCM/KALA(简称RBCM-PCM/KALA)、DNA&Pro@RBCM-PCM(简称RBCM-PCM)及DNA&Pro@RBCM(简称RBCM)。待肿瘤体积至200-300mm 3时，将3种颗粒以RBCM膜蛋白剂量为50mg/kg(DNA剂量约13mg/kg)通过尾静脉分别注射至B6鼠体内，24h时间点解剖B6鼠，并利用小动物活体成像仪检测不同实验组纳米颗粒在B6鼠心脏、肝脏以及肿瘤组织中的富集情况(见图15)。影像结果显示，对于心脏和肝脏的富集，RBCM-PCM/KALA组＞RBCM-PCM组＞RBCM组(图15A-B)。相反的， 对于肿瘤部位的富集，RBCM-PCM/KALA组远小于其他两组，几乎没有或极少进入肿瘤部位(图15C)。在此基础上，将不同实验组的心脏、肝脏和肿瘤组织进行匀浆，并利用酶标仪对DiD荧光信号进行定量分析(图15D)。结果表明，不同实验组不同组织匀浆液中的荧光总量与小动物活体成像中的结果吻合，说明RBCM-PCM/KALA仿生纳米保护剂有效富集于心脏和肝脏，而不进入肿瘤组织。
实施例10
DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂保护高剂量DOX在治疗荷瘤C57BL/6鼠时产生的心脏及系统毒性
在6-8周龄的B6鼠背部皮下注射1x10 6个TC1细胞，构建宫颈癌皮下移植瘤模型。待肿瘤体积至70-80mm 3时，将一定剂量的DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂(80mg/kg DNA，对应RBCM膜蛋白浓度为300mg/kg)通过尾静脉注射至B6鼠体内，待24h后再注射20mg/kg DOX(Day0，简称NPs+DOX组)。同时设置单独DOX组和无任何处理组(NC组)为实验对照组。随后，每隔2天观察B6鼠的体重及测量肿瘤体积大小。到Day8时，通过血常规、血生化指标以及心超指标研究DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂对抗DOX心脏及系统毒性的保护作用。结果表明，在DOX注射后Day8时间点，NPs+DOX组能有效缓解或免除DOX对B6鼠血常规、血生化以及心脏的毒性，各项指标与NC组无明显差异。相反的，DOX组对B6鼠血常规、血生化及心脏基本功能产生严重的毒性(图16-19)。相关指标的信息如下：血常规指标包括白细胞(WBC：10 9/L)，红细胞(RBC：10 12/L)，血红蛋白(Hb：g/L)，血小板(PLT：10 9/L)，红细胞体 积(MCV：fL)，红细胞压积(HCT：％)；血生化指标包括血清肌酸激酶同工酶(CKMB：IU/L)，谷丙转氨酶(ALT：IU/L)，谷草转氨酶(AST：IU/L)，总蛋白(TP：g/L)，白蛋白(ALB：g/L)，球蛋白(GLO：g/L)，血糖(GLU-S：mmol/L)，尿素氮(BUN：mmol/L)，肌酐(CREA：mmol/L)，乳酸脱氢酶(LDH：IU/L)；心超指标包括每分钟搏出量(SV，Stroke Volume：μL)，射血分数(EF，Ejection Fraction：％)，左心室缩短分数(FS，Fractional Shortening：％)，心输出量(CO，Cardiac Output：mL/min)，左心室质量(LV Mass，LV Mass：mg)，心率(HR，Heart Rate：BeatPerMinute，BPM)。
平行的，在6-8周龄的B6鼠背部皮下注射1x10 6个TC1细胞，构建宫颈癌皮下移植瘤模型。待肿瘤体积至70-80mm 3时，分别执行NPs+DOX组、单独DOX组以及NC组，然后观察和测量从Day0至Day24时间段内肿瘤组织大小变化和B6鼠体重变化。结果表明，在肿瘤抑制效果方面，DOX组与NPs+DOX组的肿瘤组织生长趋势明显受到抑制，在Day24终点时体积大小仅为初始体积大小的约15倍。然而，NC组肿瘤组织体积在Day24生长至初始体积大小的约200倍(图20A)。在B6鼠体重变化方面，DOX组在抑制肿瘤生长的同时造成严重的心脏及系统毒性，表现为突然下降的体重。然而，NPs+DOX组由于NPs提前分布于心脏、肝脏等正常器官，从而有效保护正常器官免受DOX损伤，最终表现为体重的稳步上升。同时，NC组由于肿瘤组织的快速生长，其体重表现为大幅度提高(图20B)。过程中，虽然NPs+DOX组的B6鼠体重与NC组的B6鼠体重有明显差异，但是通过分析称重离体肿瘤组织重量数据可得，NC组与NPs+DOX组的体重差异基本来源于肿瘤重量(图20C)。因此，在DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂(NPs)的保护下，B6鼠的体重没有降低反而稳定上升，这说明NPs预处理能有效保护心脏、肝脏等重要器 官损伤，同时NPs不影响DOX对肿瘤组织的抑制效果，这也进一步确证NPs仅分布于正常器官而非肿瘤组织。
综上所述：
鉴于DOX对细胞核染色质DNA的高度亲和性，本发明涉及制备一种纳米级别、“类染色质”结构的仿生纳米保护剂，用于保护DOX引起的心脏及系统毒性。该仿生纳米保护剂由修饰心肌细胞靶向基团的生物体来源细胞膜以及由鱼精蛋白(Protamine，Pro)与富含GC碱基对DNA片段自组装形成的“类染色质”结构组成。该保护剂被心肌细胞等正常细胞吞噬后分散于细胞质中，有效阻止DOX进入细胞核，防止或缓解细胞凋亡。
心肌细胞靶向基团是一种融合肽PCM/KALA，其序列为(WSGTGRLARVTVVPGAESLW-CO-水合肼-boc-WEAKLAKALAKALA-K(DDE)-HLAKALAKALKACEA)。其中，PCM多肽(序列为WLSEAGPVVTVRALRGTGSW)与心肌细胞有较好的亲和力，具有靶向心肌细胞的功能；KALA多肽(序列为WEAKLAKALAKALAKHLAKALAKALKACEA)则能介导细胞快速吞噬以及后续的溶酶体逃逸。因此，PCM/KALA融合肽不仅靶向心肌细胞、介导心肌细胞的快速吞噬，而且能实现及时的溶酶体逃逸，能够将作为屏障的上述保护剂快速释放到细胞质中，为细胞核构建屏障，阻挡DOX对细胞核造成损伤，进而防止心肌和/或肝脏细胞受损。
其具体作用过程如下，首先通过PCM的靶向作用，将DNA&Pro@RBCM-PCM/KALA整体靶向结合到心肌和或/肝脏细胞中，KALA多肽在pH＝7.4环境中维持二级结构，KALA还具有蜷缩的疏水性氨基酸和暴露 的氨基酸，暴露的氨基酸能够介导KALA被细胞快速吞噬，在KALA进入到溶酶体后，随着pH的下降，KALA多肽的二级结构被破坏，KALA的疏水性氨基酸暴露并与溶酶体膜融合，此时介导KALA多肽从溶酶体中逃逸，因此KALA能够介导细胞快速吞噬以及后续的溶酶体逃逸，进而让DNA&Pro快速释放到细胞内，为细胞核构建防护屏障。
本发明将PCM/KALA多肽偶联至红细胞膜(RBCM)表面构建RBCM-PCM/KALA，再通过Extrusion方法将其包裹由DNA和鱼精蛋白(Pro)自组装形成的DNA&Pro纳米颗粒，最终构建约400nm的DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。DNA&Pro@RBCM-PCM/KALA能高效吸附DOX，减少DOX对正常细胞的损伤。DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂仅富集至心脏、肝脏等正常器官而极少进入肿瘤组织，故在保护正常器官免受DOX损伤(心脏等毒性)的同时不影响DOX的肿瘤细胞杀伤效果。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种针对阿霉素心脏及系统毒性解毒的仿生纳米保护剂制备方法，其特征在于，包括如下步骤：
    获取DNA与鱼精蛋白Protamine并混合制备获得DNA&Pro纳米颗粒；
    将异官能团聚乙二醇DSPE-PEG2000-MAL与PCM/KALA反应制备获得DSPE-PEG2000-PCM/KALA；
    将DSPE-PEG2000-PCM/KALA插入到红细胞膜RBCM表面制备获得PCM/KALA-RBCM；
    将RBCM-PCM/KALA和DNA&Pro纳米颗粒混合后制备获得DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。
  2. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，RBCM-PCM/KALA和DNA&Pro纳米颗粒混合后利用Extrusion方法制备DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂。
  3. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，各个组分比例DNA∶Protamine∶RBCM∶DSPE-PEG2000-MAL∶PCM/KALA＝6∶5∶150∶3.75∶7.5(w/w)。
  4. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，所采用的DNA以绿色荧光蛋白质粒为DNA模板，且为630bp、GC含量65％。
  5. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，DNA与鱼精蛋白Protamine混合后通过超声形成DNA&Pro纳米颗粒，超声时间为20s。
  6. 根据权利要求5所述的仿生纳米保护剂制备方法，其特征在于，混合的DNA与鱼精蛋白Protamine的氮磷比N/P＝2。
  7. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，异官能团聚乙二醇DSPE-PEG2000-MAL与PCM/KALA的比例为1∶2(mol/mol)，且均溶于 超纯水中，37℃孵育30-60min合成DSPE-PEG2000-PCM/KALA。
  8. 根据权利要求1所述的仿生纳米保护剂制备方法，其特征在于，DSPE-PEG2000-PCM/KALA与RBCM膜蛋白质量的质量比为1∶20(w/w)混合，在37℃孵育30-60min，制备获得RBCM-PCM/KALA。
  9. 根据权利要求2所述的仿生纳米保护剂制备方法，其特征在于，制备DNA&Pro@RBCM-PCM/KALA是利用的Extrusion方法所采用的孔径为400nm。
  10. 一种根据权利要求1～9任意一项所述的仿生纳米保护剂制备方法制备获得的DNA&Pro@RBCM-PCM/KALA仿生纳米保护剂对阿霉素解毒的应用。
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WANG XIN: "The Study of PCM and TAT Co-modified Liposome: Myocardium Targeting Delivery System", CHINA MASTER'S' THESES FULL-TEXT DATABASE (ELECTRONIC JOURNAL)-INFORMATION & TECHNOLOGY), TIANJIN POLYTECHNIC UNIVERSITY, CN, vol. 2018, no. 1, 15 January 2018 (2018-01-15), CN , XP093019652, ISSN: 1674-0246 *
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