WO2017152447A1 - 一种rgd肽与穿膜肽r8共修饰麦角甾醇联合顺铂主动载药脂质体 - Google Patents

一种rgd肽与穿膜肽r8共修饰麦角甾醇联合顺铂主动载药脂质体 Download PDF

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WO2017152447A1
WO2017152447A1 PCT/CN2016/078429 CN2016078429W WO2017152447A1 WO 2017152447 A1 WO2017152447 A1 WO 2017152447A1 CN 2016078429 W CN2016078429 W CN 2016078429W WO 2017152447 A1 WO2017152447 A1 WO 2017152447A1
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ergosterol
cisplatin
peptide
liposome
active drug
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PCT/CN2016/078429
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French (fr)
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黄绳武
黄挺
吴梅佳
赵丹丹
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浙江中医药大学
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Priority to US16/065,285 priority Critical patent/US20190083398A1/en
Publication of WO2017152447A1 publication Critical patent/WO2017152447A1/zh

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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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • 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 relates to a medicament for treating lung cancer, in particular to an RGD peptide and a transmembrane peptide R8 co-modified ergosterol combined with cisplatin active drug-loaded liposome.
  • Antrodia camphorata mainly contains polysaccharides, triterpenoids, proteins, vitamins, trace elements and other chemical components, in addition to superoxide dismutase (SOD), adenosine, accounting, lectin, amino acids, sterols, lignin , blood pressure stable substances, etc.
  • Triterpenoids are considered to be one of the sources of bitter taste of Antrodia camphorata, present in mycelium and fruiting bodies. So far, nearly 30 triterpenoids have been discovered, mainly two major parent structures, lanosterane and ergosterol.
  • Antrodia camphorata was first used by humans to alleviate hangovers and treat liver diseases. It has also been extensively studied in recent years, and further confirmed the anti-hepatocarcinoma and liver-protecting effects of Antrodia camphorata. Another hot spot in the research of Antrodia camphorata is the anti-tumor effect of Antrodia camphorata, in addition to liver cancer, including breast cancer, colon cancer, and oral cancer.
  • Xu Taihao et al. conducted a general analysis of the related papers of Taiwan Zhizhi in 1992-2010. It was found that the bioactivity studies of Antrodia camphorata can be divided into 24 categories, as shown in Table 1.
  • Antrodia camphorata on cardiovascular and cerebrovascular diseases, blood sugar lowering, lowering blood fat
  • Antrodia camphorata can resist liver cancer, breast cancer, colon cancer, oral cancer, etc., it has not been found to have a good anti-lung cancer effect.
  • Antrodia camphorata has an anticancer effect, it is due to the composition of Antrodia camphorata. Complex, people are not clear about which ingredients are playing a targeted anti-cancer effect, this is extremely It has greatly hindered the research of anticancer drugs.
  • Liposomes are lipid bilayer microvesicles that resemble biofilm structures.
  • the preparation methods mainly include passive drug loading method and active drug loading method.
  • the active drug loading method overcomes the early protrusion of the wrapped drug because of its high encapsulation efficiency and less leakage to the amphiphilic drug liposome. Release and leakage, especially with clinical value.
  • Cisplatin is a complex of heavy metal platinum. It is a difunctional alkylating agent. Its chemical name is cis-dichlorodiammonium chloride (II). It is a yellow powdery crystal. It is slightly soluble in water and insoluble. It is dissolved in dimethylformamide in a general organic solvent such as ethanol. It was first synthesized by M. Peyrone in 1845 and approved by the US FDA in 1978 for clinical treatment of cancer. The discovery of cisplatin has led to the development of metal complexes in the medical field and has revolutionized the treatment of cancer.
  • II cis-dichlorodiammonium chloride
  • the object of the present invention is to provide an RGD peptide and a transmembrane peptide R8 co-modified ergosterol combined with cisplatin active drug-loaded liposome, and the ergosterol moiety is used instead of cisplatin to effectively reduce the drug while ensuring anti-lung cancer effect.
  • the toxic side effect has little damage to the human body, and has the RGD peptide and the transmembrane peptide R8 as the target, the targeting is good, and the drug exerts a good effect.
  • An RGD peptide and a penetrating peptide R8 co-modified ergosterol combined with cisplatin active drug-loaded liposome characterized in that: ergosterol combined with cisplatin active drug-loaded liposome, RGD cyclic peptide and transmembrane peptide R8 in a water bath Prepared after incubation; the ergosterol combined with cisplatin active drug-loaded liposome is prepared from ergosterol liposome and cisplatin solution, wherein the mass ratio of ergosterol to cisplatin is controlled to 1: 1-4:1.
  • ergosterol has an obvious anti-lung cancer effect, and has low cytotoxicity and little damage to the human body.
  • ergosterol can not effectively reach the lesion after entering the human body. Therefore, ergosterol is encapsulated in the liposome to enable it to reach the lesion, and the targeting of the liposome makes the ergosterol better.
  • Anti-lung cancer effect Cisplatin, although anti-cancer effect The fruit is good, but it has great toxic and side effects, which greatly limits the application of cisplatin.
  • Ergosterol is a naturally occurring compound in plants, and has low cytotoxicity.
  • the present invention adopts a new discovery and has good anticancer effect and low toxicity.
  • the ergosterol partially replaces cisplatin, and the combined effect of cisplatin plays a role in ensuring the anti-lung cancer effect, while significantly reducing the side effects of the drug, and has little damage to the human body and is targeted.
  • the present invention is further modified on the ergosterol combined with cisplatin active drug-loaded liposome, and the RGD peptide and the membrane are attached.
  • the peptide R8 target makes the drug have excellent targeting and exerts good effects.
  • the ergosterol liposome is made up of ergosterol 8-15 wt% and liposomes 85-92%, the liposome consisting of lecithin and cholesterol, and the lecithin to cholesterol molar ratio is 3: 1-6:1.
  • the ergosterol liposome is made of ergosterol 10% by weight and liposome 90%, the liposome consisting of lecithin and cholesterol, and the lecithin to cholesterol molar ratio is 5:1.
  • the RGD cyclic peptide and the transmembrane peptide R8 are controlled in an amount such that the RGD cyclic peptide: transmembrane peptide R8:cholesterol molar ratio is 0.07:0.07:1.
  • the RGD cyclic peptide is specifically DSPE (distearoylphosphatidylethanolamine)-PEG3400-c; and the transmembrane peptide R8 is specifically DSPE-PEG1000-R8.
  • RGD cyclic peptide and transmembrane peptide R8 are commercially available or self-made, and the commercially available manufacturer is Shanghai Qiangyao Biotechnology Co., Ltd.
  • the RGD peptide and the transmembrane peptide R8 co-modified ergosterol combined with cisplatin active drug-loaded liposome from ergosterol combined with cisplatin active drug-loaded liposome, RGD cyclic peptide and transmembrane peptide R8 in a water bath at 55 ° C Prepared after incubation for 1 h.
  • the concentration of the cisplatin solution is from 0.03 to 0.3 mg/mL.
  • the concentration of the cisplatin solution is 0.15 mg/mL.
  • the specific preparation method of the ergosterol combined with cisplatin active drug-loaded liposome is:
  • Cisplatin is a weakly basic drug, and the invention is prepared by an ammonium chloride gradient method, and the encapsulation efficiency can reach 50% or more.
  • Active drug-loaded liposomes were prepared by ammonium chloride gradient method.
  • the main processes were: preparation of ergosterol liposome and formation of ammonium chloride gradient and drug loading.
  • ammonium chloride gradient formation is important.
  • the basic principle is that a certain concentration of ammonium chloride is encapsulated in the aqueous phase of the liposome, and the ammonium chloride of the outer aqueous phase is removed by dialysis.
  • the diffusion coefficient of ammonia molecules is much larger than that of ammonium chloride.
  • the liposomes gradually protonate, which indirectly forms a pH gradient from the ammonium chloride gradient.
  • cisplatin exists in the outer aqueous phase in a molecular state, and the transmembrane ability is strong, and the inner aqueous phase is separated.
  • the sub-state exists and is difficult to diffuse, thereby forming a stable encapsulation state.
  • the parameters of the probe sonication in the step (1) are: ultrasonic time 20 min, ultrasonic 2 s, stop 1 s, ultrasonic power 900 W, high pressure extrusion pressure 400-500 psi.
  • the amount of ergosterol liposome added in step (3) is such that the mass ratio of ergosterol to cisplatin is 2.5:1, the incubation temperature is 50 ° C, and the incubation time is 10 min.
  • the concentration of the ammonium chloride solution in the step (1) is from 0.1 to 1.5 mmol ⁇ L -1 .
  • Figure 2 is the cumulative release of ergosterol combined with cisplatin active drug-loaded liposomes.
  • Figure 3 is a 24 h MTT test of drug substance and liposome, **P ⁇ 0.01 compared to the same dilution factor of ergosterol cisplatin liposome group.
  • Figure 4 is a TEM image of modified and unmodified liposomes.
  • Figure 5 is the penetration of co-modified liposomes into tumor spheres at different pH.
  • Figure 7 is the results of a 2 h MTT assay of each targeted liposome.
  • Figure 8 is the results of a 24 h MTT assay of each targeted liposome.
  • the ratio (molar ratio) of lecithin to cholesterol when ergosterol was administered at 5% was investigated.
  • the ratio of the two was 1:1, 3:1, 5:1, 7:1, and the encapsulation efficiency was 71.59%, 89.15%, 92.58%, 96.62%.
  • the ratio of the two is 1:1, the rigidity of the liposome is enhanced due to the higher proportion of cholesterol, and when the ratio is 7:1, precipitation tends to occur after standing.
  • This experiment investigated the effect of different ergosterol loadings on the encapsulation efficiency.
  • the ergosterol drug loading was 5%, 10%, 15%, 20%, and the encapsulation efficiency was 90.76%, 85.81%, 69.79%, and 73.09%, respectively.
  • the results of single factor test showed that the ratio of lecithin to cholesterol, probe ultrasonic time and drug loading had significant effects on the encapsulation efficiency of ergosterol.
  • the ratio of lecithin to cholesterol, the ultrasonic time of probe and the drug loading amount are selected as independent variables, and each independent variable is determined by three levels, which are represented by codes -1, 0, 1, respectively, for a total of 15 test points ( 3 center points).
  • Each test was performed in parallel three times.
  • the encapsulation efficiency of ergosterol liposome was evaluated, and the process conditions were preferably prepared by star point design (Table 1).
  • the three-dimensional response surface of the relationship between the other two factors and the encapsulation efficiency of ergosterol is drawn by Design-Expert.V 8.0.6.1 software.
  • the response surface is a three-dimensional space graph composed of two-two interaction factors, and the steeper the effect surface curve, the more obvious the influence of each variable on the response value. Taking the maximum point of the regression model, the corresponding measured value is 5:1 for lecithin and cholesterol, 20 minutes for probe ultrasonic time, and 10% for ergosterol.
  • the lecithin 98mg, cholesterol 10mg, ergosterol 12mg, a total of 3 parts were accurately weighed and verified by the following optimal preparation conditions.
  • the average value of the encapsulation efficiency of the three batches of liposomes was 90.49%, RSD 2.64%, deviation from the predicted value of 90.40% 0.10%, indicating that the established mathematical model has good predictability, and the preferred process conditions are reproducible.
  • the optimal preparation process of ergosterol liposome is as follows: the molar ratio of lecithin to cholesterol is 5:1, the ergosterol is loaded with 10%, and the probe is ultrasonic for 20 min.
  • lecithin 98mg, cholesterol 10mg and ergosterol 12mg fully dissolved in 10mL chloroform, then placed on a rotary evaporator in a water bath of 40 ° C, spin dry, vacuum drying for 2h, add 10mL ammonium chloride solution (0.5mmol ⁇ L -1 ) hydrated on a horizontal shaker at room temperature for 30 min at a speed of 140 rpm ⁇ mL -1 . After hydration, the membrane was removed by ultrasonication, and the liposome was placed in an ice bath. The probe was sonicated for 20 min, sonicated for 2 s, stopped for 1 s, and the ultrasonic power was 900 W.
  • the mass ratio of ergosterol to cisplatin was investigated, which was equivalent to the drug loading of cisplatin in liposome.
  • the ergosterol liposome was added to the interception.
  • a dialysis bag with a molecular weight of 8000-14000 Da the dialysis bag was closed, dialyzed in distilled water for 2 h, the dialysate (distilled water) was replaced once, and dialysis was continued for 2 h.
  • the ergosterol liposome was mixed with different concentrations of cisplatin solution for incubation, and the mass ratio of the two was set to 0.313:1, 0.625:1, 1.25:1, 2.5:1, 5:1, and the results showed that when ergosterol and cis When the mass ratio of platinum is 2.5:1, the encapsulation efficiency of cisplatin is the largest, reaching 35.33%.
  • the three-factor and three-level orthogonal design was used to optimize the ergosterol combined with cisplatin active drug-loaded liposomes, and the L 9 (3 4 ) orthogonal table was used to arrange the experiment.
  • the incubation time, incubation temperature and cisplatin concentration were selected as the factors to determine the optimal preparation process of ergosterol combined with cisplatin active drug-loaded liposomes.
  • the orthogonal design factor level table the orthogonal design test plan and the experimental result analysis are shown in Table 3 and Table 4.
  • the ergosterol combined with cisplatin active drug liposome solution is milky white with uniform color.
  • the ergosterol and cisplatin active drug-loaded liposomes were diluted 20 times, injected into the sample cell, and the average particle size and its distribution were determined by laser particle size analyzer.
  • the results showed that the average particle size of the blank liposome was 145.8 nm, and the polydispersity coefficient PDI was 0.168, less than 0.3, and ergosterol liposome.
  • the average particle size is 131.4 nm, PDI is 0.152, less than 0.3.
  • the average particle size of ergosterol combined with cisplatin active drug-loaded liposomes is 112.5, PDI is 0.208, less than 0.3, and the particle size distribution of the two is concentrated.
  • ergosterol was combined with cisplatin active drug-loaded liposomes, diluted 20-fold, and Zeta potential was measured by Zeta potential meter. Results The zeta potential of blank liposome was -18.6mV, the zeta potential of ergosterol liposome was -23.4mV, and the zeta potential of ergosterol combined with cisplatin active drug-loaded liposome was -5.42mV. Liposomes were negatively charged. .
  • Phospholipid molecules contain unsaturated fatty acid chains, which are chemically unstable, easy to oxidize and hydrolyze, reduce membrane fluidity, accelerate drug leakage, and produce peroxidation products such as malondialdehyde and fatty acids, which may cause toxicity to human body.
  • Malondialdehyde (MDA) can react with thiobarbituric acid under acidic conditions, and the resulting red product (TBA-pigment) absorbs at 535 nm, and the absorption value is measured to obtain the malondialdehyde content.
  • TSA-pigment red product absorbs at 535 nm, and the absorption value is measured to obtain the malondialdehyde content.
  • the degree of oxidation of phospholipids can be investigated. In this experiment, the degree of oxidation of liposomes was examined by the malondialdehyde assay.
  • c 1 is the concentration of cisplatin released in each sampling point
  • V 0 is the volume of the release medium
  • V is the sampling volume
  • M 0 is the total amount of cisplatin contained in the active drug-loaded liposome of ergosterol combined with cisplatin.
  • the prepared FITC-labeled pH 7.4 co-modified liposome 1640 medium was quantitatively mixed so that the final dilution factor was 64, 96, 128.
  • A549 cells (commercially available, Institute of Life Sciences, Chinese Academy of Sciences) were inoculated in a 6-well plate. When the cells were fused to 80%, the old medium was aspirated, and ergosterol combined with cisplatin actively loaded with liposomes and A mixture of media.
  • the A549 lung cancer cells were cultured in vitro, and ergosterol, cisplatin, ergosterol combined with cisplatin and ergosterol were administered.
  • cisplatin active liposomal drug-stimulated cells was determined after administration of various concentrations of the growth inhibition rate was calculated liposomal IC 50 values for inhibition of A549 cell half.
  • the A549 cells in the logarithmic growth phase were adjusted to 1 ⁇ 10 5 ⁇ mL -1 after trypsinization. 100 ⁇ L of each well was seeded in a 96-well culture plate, cultured at 37 ° C in a 5% CO 2 incubator, and administrated as the cells were fused to 80%. Different dilutions of ergosterol combined with cisplatin active drug-loaded liposomes and corresponding ergosterol, cisplatin, ergosterol and cisplatin drug solution were added, and a normal control group was set. Five replicate wells were set for each concentration, and MTT assay was performed 24 hours after dosing.
  • the IC 50 value of ergosterol cisplatin liposome administration was 2.178 + 0.544 ⁇ g ⁇ mL -1 . It is indicated that after preparing ergosterol and cisplatin into a liposome preparation, the anti-lung cancer effect in vitro can be significantly increased.
  • FITC-labeled liposome For the FITC-labeled liposome, an appropriate amount of FITC methanol solution was added to the lipid material to form a thin film, and the effect of the concentration of different fluorescent substances FITC on the cell uptake rate was measured by a fluorescence spectrophotometer. The results are shown in Table 9. The final concentration of FITC in the liposome was determined to be 25 ⁇ g ⁇ mL -1 .
  • liposomes with no modification, RGD modification, R8 modification, RGD and R8 co-modification were diluted 20 times, injected into the sample cell, and the average particle size and its distribution were determined by laser particle size analyzer.
  • the results showed that the average particle size of the unmodified liposome was 153.4 nm, the polydispersity coefficient PDI was 0.156, less than 0.3, the average particle size of the RGD modified liposome was 156.7 nm, the PDI was 0.164, less than 0.3, and the R8 modified lipid.
  • the average particle size of the body is 154.3 nm
  • the PDI is 0.178, less than 0.3
  • the average particle size of the RGD and R8 co-modified liposomes is 155.2 nm
  • the PDI is 0.102, less than 0.3
  • the particle size distribution of each liposome is concentrated.
  • the present invention examined the ability of co-modified liposomes to penetrate the tumor sphere under different pH conditions.
  • 20 ⁇ g ⁇ mL -1 B27 serum - free medium, 20 ng ⁇ mL -1 EGF and bFGF (commercially available) were added to the culture solution, and the solution was changed every three days.
  • the mixed solution of the co-modified liposome and the medium at pH 6.0 and pH 7.4 was added to a 6-well plate containing tumor spheres, and after incubation with the tumor sphere for 2 hours, the tumor sphere was aspirated, and the tumor sphere was collected by centrifugation and rinsed with PBS.
  • Lyso-Tracker Red (commercially available) with a final concentration of 1 ⁇ g ⁇ mL -1 was added, and after incubation with the cells for 30 min, the medium was discarded by centrifugation, rinsed 3 times with PBS, and fixed with 4% paraformaldehyde for 30 min. After that, the supernatant was discarded by centrifugation, rinsed 3 times with PBS, stained with 1 ⁇ g ⁇ mL -1 of DAPI solution (commercially available) for 5 min, the supernatant was removed by centrifugation, PBS was rinsed 3 times, and the tumor sphere was added to polylysine. The treated slides were mounted on a 10% glycerol-PBS solution.
  • the ability of the co-modified liposome to penetrate the tumor sphere was observed by FV 1000 laser confocal microscopy. As can be seen from Figure 5, its ability to penetrate the tumor sphere is significantly pH dependent. Under incubation conditions of pH 6.0, the ability of the co-modified liposomes to penetrate the tumor sphere was significantly enhanced compared to the incubation conditions of pH 7.4.
  • the prepared FITC-labeled pH 7.4 co-modified liposomes were mixed with 1640 medium (commercially available) to give a final dilution factor of 64, 96, 128.
  • the A549 cells were seeded in a 6-well plate. When the cells were fused to 80%, the old medium was aspirated, and then a mixture of unmodified, RGD-modified, R8-modified, co-modified liposome and medium was added.
  • A549 was inoculated into a 6-well plate, and cultured at 37 ° C, 5% CO 2 until cell fusion was about 80%, and 500 ⁇ g ⁇ mL -1 of the cell uptake inhibitors clozapine, colchicine and sodium azide were added, respectively.
  • clozapine is an inhibitor of endocytic pathway mediated by trapping protein
  • colchicine is an inhibitor of giant cell drinking pathway
  • sodium azide is an energy-dependent endocytic pathway inhibitor.
  • the mixture of RGD and R8 co-modified liposomes and medium was added to the 6-well plate.
  • the A549 cells in the logarithmic growth phase were adjusted to 1 ⁇ 10 5 ⁇ mL -1 after trypsinization. 100 ⁇ L of each well was seeded in a 96-well culture plate, cultured at 37 ° C in a 5% CO 2 incubator, and administrated as the cells were fused to 80%. Co-modified liposomes with different dilution factors were added, and 5 replicate wells were set for each concentration in the normal control group, and MTT assay was performed at 2 h and 24 h after dosing.
  • transmembrane peptide R8 and RGD peptides can efficiently mediate the release of ergosterol and cisplatin from co-modified liposomes, thus making the co-modified liposomes more targeted, making ergosterol and cis.
  • Platinum drugs play a better role in inhibiting tumors.

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Abstract

一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，由麦角甾醇联合顺铂主动载药脂质体、RGD环肽及穿膜肽R8在水浴中孵育后制备而得。所述麦角甾醇联合顺铂主动载药脂质体由麦角甾醇脂质体、顺铂溶液为原料制成。所述麦角甾醇脂质体由麦角甾醇8-15wt%和脂质体85-92wt%制成，所述脂质体由卵磷脂与胆固醇组成。

Description

一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体 技术领域
本发明涉及一种治疗肺癌的药物，特别涉及一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体。
背景技术
牛樟芝中主要含有多糖、三萜类化合物、蛋白质、维生素、微量元素等化学成分，另外，还有超氧岐化酶(SOD)、腺苷、核算、凝集素、氨基酸、固醇类、木质素、血压稳定物质等。三萜类化合物被认为是牛樟芝苦味的只要来源之一，存在于菌丝体和子实体中。到目前为止，发现了近30种三萜类化合物，主要为羊毛甾烷和麦角甾烷两大母体结构。
牛樟芝最早被人食用是用来缓解宿醉和治疗肝部疾病，近年来也被广泛研究，进一步对牛樟芝抗肝癌、保肝作用进行了确认。牛樟芝研究的另一个热点是牛樟芝的抗肿瘤作用，除了肝癌，还包括乳腺癌、结肠癌、口腔癌等。徐泰浩等人对1992-2010年台湾樟芝相关硕博论文进行了概括分析发现，对牛樟芝的生物活性研究可分为24类，见表1，研究最多的前5类依次排序为：(1)抗肿瘤，(2)保肝，(3)抗氧化，(4)免疫调节，(5)抗发炎，显示了牛樟芝主要的药理活性，另外，牛樟芝对心脑血管疾病、降血糖、降血脂等方面也显示了一定的药理活性。虽然现有技术报道了牛樟芝可以抗肝癌、乳腺癌、结肠癌、口腔癌等，但是并未发现其具有良好的抗肺癌作用，此外，虽然认知到牛樟芝具有抗癌作用，但是，由于牛樟芝成分复杂，人们并未清楚具体何种成分在发挥针对性的抗癌作用，这极 大阻碍了抗癌药物的研究。
脂质体是一种类似生物膜结构的脂质双分子层微小囊泡。目前制备方法主要有被动载药法与主动载药法两大类，主动载药法因其对两亲性药物脂质体的包封率高、渗漏少，克服了被包裹药物的早期突释和泄露，特别具有临床价值。
顺铂(CDDP)是一种重金属铂的络合物，为双官能团烷化剂，化学名为：顺式-二氯二氨铂(II)，为黄色粉末状结晶，在水中微溶，不溶于乙醇等一般有机溶剂，溶于二甲基甲酰胺。它首次在1845年由M.Peyrone合成，1978年美国FDA批准其用于临床治疗癌症。顺铂的发现带动了金属配合物在医学领域的发展，对于癌症治疗具有革命性的意义。其抗癌作用特点有：(1)为高效广谱的抗肿瘤药，可与作用靶点DNA形成CDDP-DNA复合物而发挥作用，属细胞周期非特异性药物。对肿瘤抑制率为61％～98％，尤其对实体瘤和对一般化疗药不甚敏感的肿瘤疗效较为显著；(2)不仅能杀伤肿瘤细胞，抑制细胞修复，还具有较强的放疗增敏作用；(3)与多种抗肿瘤药物有协同作用，其毒性谱也与它们有不同，且无交叉耐药性，因此易与其它抗肿瘤药配伍，既有利于临床的联合用药，还可逆转联合化疗的毒性。因此，长期以来顺铂在抗癌药中地位显著。
但是，目前临床所用顺铂制剂，如中国药典2010版和欧洲药典2001收载的注射用顺铂，英国药典2000版收载的注射用顺铂和顺铂注射剂，均为对癌组织、癌细胞没有选择性的普通注射剂，药物的生物利用度低，毒副作用大。其存在的问题主要表现为：(1)严重毒副作用：顺铂及其代谢产物 主要从肾脏排泄，故肾毒性大。另有胃肠道毒性、耳毒性及神经毒性等也不可忽视；(2)对某些癌细胞活性较低，如乳腺癌、结肠癌等；(3)易产生耐药性；(4)仅微溶于水，性质不稳定，见光分解，其水溶液在室温中放置后会发生水解而失效，并转变为有毒却无抑瘤作用的反铂。
鉴于近年来肺癌发病率、死亡率均高居各类癌症之首，且比例仍在继续升高，如何减小顺铂的毒副作用成为亟需解决的问题。
发明内容
本发明的目的在于提供一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，采用麦角甾醇部分代替顺铂发挥功效，在保证抗肺癌效果的同时，显著降低药物的毒副作用，对人体的伤害小，同时具有RGD肽与穿膜肽R8作为靶头，靶向性好，药物发挥效果好。
本发明解决其技术问题所采用的技术方案是：
一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：由麦角甾醇联合顺铂主动载药脂质体、RGD环肽及穿膜肽R8在水浴中孵育后制备而得；所述麦角甾醇联合顺铂主动载药脂质体，由麦角甾醇脂质体、顺铂溶液为原料制成，其中，麦角甾醇与顺铂的质量比控制为1:1-4:1。
发明人经过长期研究，意外发现麦角甾醇具有明显的抗肺癌作用，且细胞毒性低，对人体的伤害小。而单纯的麦角甾醇在进入人体后并不能有效直达病灶，因此，将麦角甾醇包封于脂质体，使其能到达病灶发挥功效，脂质体的靶向性，使得麦角甾醇能更好发挥抗肺癌作用。顺铂虽然抗癌效 果较好，但是其存在极大的毒副作用，这大大限制了顺铂的应用，麦角甾醇为植物中天然存在的化合物，细胞毒性小，本发明采用新发现具有较好抗癌效果且低毒性的麦角甾醇部分代替顺铂，联合顺铂协同发挥作用，在保证抗肺癌效果的同时，显著降低药物的毒副作用，对人体的伤害小，且具有靶向性。
为了更好地发挥药物的效果，使其靶向性更佳，更准确的直达病灶，本发明在麦角甾醇联合顺铂主动载药脂质体上再进行修饰，接上了RGD肽与穿膜肽R8靶头，使得药物具有优异的靶向性，发挥效果好。
作为优选，所述麦角甾醇脂质体由麦角甾醇8-15wt％和脂质体85-92％制成，所述脂质体由卵磷脂与胆固醇组成，卵磷脂与胆固醇的摩尔比为3:1-6:1。作为优选，所述麦角甾醇脂质体由麦角甾醇10wt％和脂质体90％制成，所述脂质体由卵磷脂与胆固醇组成，卵磷脂与胆固醇的摩尔比为5:1。
作为优选，RGD环肽及穿膜肽R8用量控制为RGD环肽：穿膜肽R8：胆固醇的摩尔比为0.07:0.07:1。
作为优选，所述RGD环肽具体为DSPE(二硬脂酰磷脂酰乙醇胺)-PEG3400-c；所述穿膜肽R8具体为DSPE-PEG1000-R8。RGD环肽、穿膜肽R8可市售或自制，市售的厂商为上海强耀生物科技有限公司。
作为优选，所述RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体由麦角甾醇联合顺铂主动载药脂质体、RGD环肽及穿膜肽R8在55℃水浴中孵育1h后制备而得。
作为优选，所述顺铂溶液的浓度为0.03-0.3mg/mL。
作为优选，所述顺铂溶液的浓度为0.15mg/mL。
作为优选，所述麦角甾醇联合顺铂主动载药脂质体的具体制备方法为：
(1)麦角甾醇脂质体制备：称取卵磷脂、胆固醇、麦角甾醇，加入氯仿溶解，旋转蒸发成薄膜，真空干燥，以氯化铵溶液为水化液进行水化，超声脱膜，冰浴下探头超声处理，过滤，高压挤出得麦角甾醇脂质体；
(2)氯化铵梯度形成：将麦角甾醇脂质体加入截留分子量为8000-14000Da的透析袋中，封闭透析袋，投入蒸馏水中透析2h，更换一次蒸馏水，继续透析2h；
(3)载药：顺铂与水混合制成顺铂溶液，将上一步透析后的麦角甾醇脂质体加入顺铂溶液进行孵育，麦角甾醇脂质体的加入量以满足麦角甾醇与顺铂的质量比为1:1-4:1计算，孵育温度40-60℃，孵育时间5-40min，孵育结束后得到产品。
当顺铂溶液作为水化液通过水化进行包封，发现其包封率低于10％，说明薄膜分散法不适合包封顺铂这一药物。顺铂为弱碱性药物，本发明采用氯化铵梯度法制备，包封率可达到50％以上。
氯化铵梯度法制备主动载药脂质体，主要过程为：麦角甾醇脂质体制备及氯化铵梯度形成、载药。其中，氯化铵梯度形成至为重要。其基本原理是，一定浓度的氯化铵包于脂质体内水相，通过透析除去外水相的氯化铵。在内外浓度差的推动下，由于氨分子的扩散系数远大于氯化铵，随着氨分子的扩散，脂质体内逐步质子化，从而由氯化铵梯度间接形成pH梯度。在此梯度下，顺铂在外水相以分子状态存在，跨膜能力强，内水相中以离 子化状态存在，难以扩散，从而形成稳定的包封状态。
作为优选，步骤(1)中探头超声处理的参数为：超声时间20min，超声2s，停1s，超声功率900W，高压挤出的压力为400-500psi。
作为优选，步骤(3)中麦角甾醇脂质体的加入量以满足麦角甾醇与顺铂的质量比为2.5:1计算，孵育温度50℃，孵育时间10min。
作为优选，步骤(1)中氯化铵溶液的浓度为0.1-1.5mmol·L-1。
本发明的有益效果是：采用麦角甾醇部分代替顺铂发挥功效，在保证抗肺癌效果的同时，显著降低药物的毒副作用，对人体的伤害小，同时具有RGD肽与穿膜肽R8作为靶头，靶向性好，药物发挥效果好。
附图说明
图1是各脂质体透射电镜图。
图2是麦角甾醇联合顺铂主动载药脂质体累积释放度。
图3是原料药及脂质体的24h MTT试验结果，与相同稀释倍数的麦角甾醇顺铂脂质体组相比，**P<0.01。
图4是修饰及未修饰的脂质体的TEM图。
图5是不同pH下共修饰脂质体对肿瘤球的穿透。
图6是不同细胞摄取抑制剂的摄取抑制率，与对照组相比，**P<0.01,(n＝3)。
图7是各靶向脂质体的2h MTT试验结果。
图8是各靶向脂质体的24h MTT试验结果。
具体实施方式
下面通过具体实施例，并结合附图，对本发明的技术方案作进一步的 具体说明。
本发明中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
实施例：
一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，由麦角甾醇联合顺铂主动载药脂质体、RGD环肽(DSPE-PEG3400-c，市售)及穿膜肽R8(DSPE-PEG1000-R8，市售)在55℃水浴中孵育1h后制备而得；所述麦角甾醇联合顺铂主动载药脂质体，由麦角甾醇脂质体、顺铂溶液为原料制成，其中，麦角甾醇与顺铂的质量比控制为1:1-4:1；所述麦角甾醇脂质体由麦角甾醇8-15wt％和脂质体85-92％制成，所述脂质体由卵磷脂与胆固醇组成，卵磷脂与胆固醇的摩尔比为3:1-6:1；RGD环肽及穿膜肽R8用量控制为RGD环肽：穿膜肽R8：胆固醇的摩尔比为0.07:0.07:1。所述顺铂溶液的浓度为0.03-0.3mg/mL。
麦角甾醇联合顺铂主动载药脂质体的制备方法，包括以下步骤：
(1)麦角甾醇脂质体制备：称取卵磷脂、胆固醇、麦角甾醇，加入氯仿溶解，旋转蒸发成薄膜，真空干燥，以氯化铵溶液(浓度为0.1-1.5mmol·L-1)为水化液进行水化，超声脱膜，冰浴下探头超声处理，过滤，高压挤出得麦角甾醇脂质体；麦角甾醇脂质体的原料组分以总和100％计配比如下：麦角甾醇5-20wt％，余量为卵磷脂和胆固醇，卵磷脂与胆固醇的摩尔比为1:1-7:1。探头超声处理的参数为：超声时间20min，超声2s，停1s，超声 功率900W，高压挤出的压力为400-500psi。
(2)氯化铵梯度形成：将麦角甾醇脂质体加入截留分子量为8000-14000Da的透析袋中，封闭透析袋，投入蒸馏水中透析2h，更换一次蒸馏水，继续透析2h；
(3)载药：顺铂与水混合制成顺铂溶液，顺铂溶液的浓度为0.03-0.3mg/mL，将上一步透析后的麦角甾醇脂质体加入顺铂溶液进行孵育，麦角甾醇脂质体的加入量以满足麦角甾醇与顺铂的质量比为1:1-4:1计算，孵育温度40-60℃，孵育时间5-40min，孵育结束后得到产品。
一、麦角甾醇脂质体最佳制备工艺
1.1、单因素考察
1.1.1卵磷脂与胆固醇摩尔比考察
本实验考察了麦角甾醇载药5％时，卵磷脂与胆固醇的比例(摩尔比)，设置二者比例为1:1、3:1、5:1、7:1，其包封率分别为71.59％、89.15％、92.58％、96.62％。二者比例为1:1时，由于胆固醇的比例较高，脂质体的刚性增强，而二者为7:1时，放置后容易出现沉淀。
1.1.2探头超声时间考察
薄膜分散法制备脂质体后(称取卵磷脂、胆固醇、麦角甾醇，加入氯仿溶解，旋转蒸发成薄膜，真空干燥，水化，超声脱膜)，通过探头超声将大粒径的脂质体整粒成小粒径脂质体。本实验考察了不同探头超声时间对于麦角甾醇脂质体包封率的影响。设置探头超声时间为10min、20min、 30min、40min，其包封率分别为79.45％、91.73％、95.86％、95.94％。随着时间增加，包封率随之增大，30min时包封率不再变化，且探头超声时间过长，容易导致脂质体破裂。
1.1.3麦角甾醇载药量考察
本实验考察了不同麦角甾醇载药量对包封率的影响。麦角甾醇载药量为5％、10％、15％、20％，其包封率分别为90.76％、85.81％、69.79％、73.09％。
1.2响应面试验
1.2.1星点设计
单因素试验结果表明卵磷脂与胆固醇比例、探头超声时间和载药量3个因素对麦角甾醇包封率具有显著影响。根据星点设计原理，选择卵磷脂与胆固醇比例、探头超声时间和载药量为自变量，每个自变量确定3个水平，分别用代码－1，0，1表示，共15个试验点(3个中心点)。每个试验平行3次进行。以麦角甾醇脂质体包封率为评价指标，采用星点设计(表1)优选制备工艺条件。
表1 麦角甾醇脂质体制备工艺星点试验安排(n＝3)
Figure PCTCN2016078429-appb-000001
Figure PCTCN2016078429-appb-000002
1.2.2模型的建立与方差分析
运用Design-Expert.V 8.0.6.1软件对表中数据进行二次多元回归拟合，得自变量与因变量间的回归方程Y＝90.40+17.87X1－2.58X2+4.14X3－7.62X1X2+6.28X1X3+3.03X2X3－21.95X1 2－4.29X2 2－2.44X3 2。对该回归模型进行显著性检验。表2结果表明X1，X1 2对响应值的线性效应极显著，X2，X3，X2 2对响应值的曲面效应作用显著，交互项X1X2、X1X3作用显著。该模型的F＝82.84，P＜0.0001，表明该二次多元回归模型极显著，回归方程相关系数(r)0.9967，说明模型能解释99.67％响应值的变化，拟合情况良好。
表2 回归模型方差分析
Figure PCTCN2016078429-appb-000003
1.2.3响应面分析
根据回归方程，在保持1个因素编码值为0时，运用Design-Expert.V 8.0.6.1软件绘制另2个因素与麦角甾醇包封率关系的三维响应面图。响应面为响应值对两两交互因素所构成的三维空间曲线图，效应面曲线越陡峭，说明各自变量对响应值的影响越明显。取回归模型最大值点，对应的实测值为卵磷脂与胆固醇比例为5:1，探头超声时间为20min，麦角甾醇载药量为10％。
1.2.4验证试验
为验证模型方程的适用性，精密称取卵磷脂98mg，胆固醇10mg，麦角甾醇12mg，共3份，按以下最佳制备工艺条件进行验证试验，结果3批脂质体包封率的平均值为90.49％，RSD 2.64％，与预测值90.40％的偏差0.10％，表明建立的数学模型具有良好的预测性，优选的工艺条件重复性好。
麦角甾醇脂质体最佳制备工艺为：卵磷脂与胆固醇摩尔比5:1，麦角甾醇载药10％、探头超声20min。
具体工艺条件：卵磷脂98mg、胆固醇10mg以及麦角甾醇12mg，用10mL氯仿充分溶解后，置于40℃水浴的旋转蒸发仪上旋干，真空干燥2h，加入10mL氯化铵溶液(0.5mmol·L-1)于室温下置于水平摇床上水化30min，转速140rpm·mL-1。水化后，超声脱膜，将脂质体放置于冰浴下，探头超声20min，超声2s，停1s，超声功率900W。依次采用0.8μm、0.45μm、0.22μm微孔滤膜过滤，最后用0.1μm的聚碳酸酯膜高压挤出(压力 为400-500psi)，即得。
二、麦角甾醇联合顺铂主动载药脂质体最佳制备工艺考察
2.1单因素试验
2.1.1麦角甾醇/顺铂质量比的考察
本实验考察了麦角甾醇与顺铂的质量比，相当于顺铂在脂质体中的载药量，按1.2.4制备最佳工艺麦角甾醇脂质体后，将麦角甾醇脂质体加入截留分子量为8000-14000Da的透析袋中，封闭透析袋，投入蒸馏水中透析2h，更换一次透析液(蒸馏水)，继续透析2h。按麦角甾醇脂质体与不同浓度顺铂溶液混合进行孵育，设置二者的质量比为0.313:1、0.625:1、1.25:1、2.5:1、5:1，结果显示当麦角甾醇与顺铂的质量比为2.5:1时，顺铂的包封率最大，达到35.33％。
2.1.2孵育时间的考察
本实验考察了顺铂溶液孵育时间分别为5、10、20、40min时的包封率，结果显示当孵育时间为20min时，顺铂包封率为最大，31.07％。
2.1.3孵育温度的考察
本实验考察了40、50、60、80℃温度对于顺铂包封率的影响，结果显示50℃与80℃时的包封率最大，但是过高的温度会导致卵磷脂发生氧化而产生溶血性。
2.2正交试验
在单因素试验考察的基础上，采用三因素三水平正交设计优化麦角甾 醇联合顺铂主动载药脂质体，选用L9(34)正交表安排试验。选择孵育时间、孵育温度、顺铂浓度3个因素作为考察因素确定最佳麦角甾醇联合顺铂主动载药脂质体制备工艺。正交设计因素水平表见，正交设计试验方案及实验结果分析见表3，表4。
表3 L9(34)正交表
Figure PCTCN2016078429-appb-000004
表4 L9(34)实验计划表
Figure PCTCN2016078429-appb-000005
采用单指标正交设计试验结果的极差分析法。结果表明：各因素对顺 铂包封率的影响程度为C>A>B，最佳组合为麦角甾醇联合顺铂主动载药脂质体最佳工艺为A1B3C1，即顺铂浓度为0.150mg·mL-1，孵育温度70℃，孵育时间10min。考虑到70℃位于大豆卵磷脂的相变温度以上，且孵育温度这一因素对包封率的影响不大，因此将最佳工艺调整为顺铂浓度为0.150mg·mL-1，孵育温度50℃，孵育时间10min，对调整前后的最佳工艺进行验证。
2.3正交试验验证
按正交试验最佳工艺及调整后最佳工艺分别制备3批麦角甾醇联合顺铂主动载药脂质体，通过超滤法对顺铂包封率、平均粒径、Zeta电位进行测定。3批脂质体平均包封率结果分别为49.04％及52.24％。表5、表6结果表明，最佳工艺及调整后最佳工艺的包封率、平均粒径、Zeta电位均无显著性差异，因此，后续实验采用调整后最佳工艺，即顺铂浓度0.150mg·mL-1，孵育温度50℃，孵育时间10min进行麦角甾醇联合顺铂主动载药脂质体的制备。
表5 最佳工艺验证结果
Figure PCTCN2016078429-appb-000006
表6 调整后最佳工艺验证结果
Figure PCTCN2016078429-appb-000007
Figure PCTCN2016078429-appb-000008
三、麦角甾醇联合顺铂主动载药脂质体质量评价
3.1形态观察
3.1.1外观形态
麦角甾醇联合顺铂主动载药脂质体溶液呈乳白色，色泽均匀。
3.1.2显微形态
采用负染法制备样品。在室温条件下，取麦角甾醇联合顺铂主动载药脂质体，蒸馏水稀释至略显浑浊，滴至专用230目铜网上，滤纸吸干多余脂质体，静置1min。用1％磷钨酸负染，静置40s，使粒子在铜网上沉积，用滤纸吸取铜网边缘多余染液，自然挥干，电镜观察并照相。图1的结果表明，各脂质体形态圆整，粒径分布均匀。
3.1.3包封率与载药量
3批麦角甾醇联合顺铂主动载药脂质体中，其中麦角甾醇的平均包封率为90.49％，载药量为0.1401mg·mL-1；顺铂的平均包封率为52.24％，载药量为0.1382mg·mL-1。
3.1.4粒径及其分布
室温条件下，取麦角甾醇联合顺铂主动载药脂质体，稀释20倍，注入样品池，激光粒度仪测定平均粒径及其分布。结果表明，空白脂质体的平均粒径为145.8nm，多分散系数PDI为0.168，小于0.3，麦角甾醇脂质体 的平均粒径为131.4nm，PDI为0.152，小于0.3，麦角甾醇联合顺铂主动载药脂质体的平均粒径为112.5，PDI为0.208，小于0.3，可见两者的粒径分布较集中。
3.1.5Zeta电位测定
室温条件下，取麦角甾醇联合顺铂主动载药脂质体，稀释20倍，Zeta电位仪测定Zeta电位。结果空白脂质体的Zeta电位为-18.6mV，麦角甾醇脂质体的Zeta电位为-23.4mV，麦角甾醇联合顺铂主动载药脂质体的Zeta电位为-5.42mV，脂质体带负电。
3.1.6pH值测定
室温条件下，取麦角甾醇联合顺铂主动载药脂质体，稀释后，用酸度计测定pH值。测定3批样品的平均pH值为6.64。
3.1.7麦角甾醇联合顺铂主动载药脂质体过氧化值(POV)的测定
磷脂分子中含有不饱和脂肪酸链，化学性质不稳定，易氧化水解，使膜流动性降低，药物渗漏加快，生成过氧化产物，如丙二醛、脂肪酸等，会对人体产生毒性。丙二醛(MDA)在酸性条件下可与硫代巴比妥酸反应，生成的红色产物(TBA-pigment)，在535nm处有吸收，测定其吸收值即可得出丙二醛含量，从而可以考察磷脂的氧化程度。本实验采用丙二醛测定法考察脂质体的氧化程度。
精密吸取脂质体1mL，置于10mL离心管中，加入TTH试液(三氯醋酸30g，2-硫代巴比妥酸0.75g，加入0.25mol·L-1盐酸200mL，温热至溶解，放冷后滤过)5mL，混匀，置100℃水浴加热30min，冷却至室温， 加入4.0mL TTH试液，混匀后以4000rpm·min-1离心10min。吸取上清液，以TTH试液为空白对照，于535nm波长处测定吸光度值，记作过氧化值。平行测定3批样品，平均过氧化值为0.1095(表7)。
表7 脂质体过氧化值结果(POV)
Figure PCTCN2016078429-appb-000009
3.1.8释放度试验
由于麦角甾醇的脂溶性较强，在释放介质中的溶解度很小，因此本实验仅考察水溶性药物顺铂的释放度。分别精密吸取麦角甾醇联合顺铂主动载药脂质体1mL，及顺铂原料药溶液1mL，置于透析袋中，两端用透析夹夹紧。由于顺铂在无钠或低钠的溶液中不稳定，易水解成无抗癌作用成分反铂，因此释放介质选择100倍0.9％NaCl溶液，置于恒温水浴振荡器中，振摇速率为100rpm·min-1，释放温度为37℃，分别于第0.5、1、2、3、4、6、8、10、12、24h吸取透析液1mL，并补充1mL 37℃的0.9％NaCl新鲜透析介质。将各取样点样品采用0.45μm的微孔滤膜过滤后进样分析。通过HPLC进样，测定峰面积，代入线性回归方程计算各取样点下顺铂的释药浓度，计为c1，顺铂总药量记为M0。
按照如下公式进行计算：
Figure PCTCN2016078429-appb-000010
Figure PCTCN2016078429-appb-000011
其中，c1为各取样点中顺铂释放的浓度，V0为释放介质体积，V为取样体积，M0为麦角甾醇联合顺铂主动载药脂质体中含有的顺铂总量。《中国药典附录制剂通则》关于脂质体突释效应的要求：开始0.5h的释放量应≤40％，该麦角甾醇联合顺铂主动载药脂质体符合该要求，且24h的累积释放百分率超过80％，符合要求(图2)。
四、麦角甾醇联合顺铂主动载药脂质体细胞摄取试验
定量将所制备的FITC标记的pH为7.4共修饰脂质体1640培养基混合，使得最终的稀释倍数为64、96、128。将A549细胞(市售，中国科学院生命科学研究院细胞库)接种在6孔板内，待细胞融合至80％时，将吸出旧培养基后，麦角甾醇联合顺铂主动载药脂质体与培养基的混合液。在37℃、5％CO2培养箱中孵育2h后将培养基吸出，用PBS漂洗3次，胰酶消化收集细胞，加入PBS洗3遍，离心去除上清，最后加入0.5mL PBS重悬细胞，采用BD流式细胞仪检测细胞对不同给药浓度的共修饰脂质体的摄取强度。结果见表8，由结果表明，给予麦角甾醇联合顺铂主动载药脂质体，随着稀释倍数的增加，细胞摄取率随之降低。
表8 麦角甾醇联合顺铂主动载药脂质体细胞摄取
Figure PCTCN2016078429-appb-000012
Figure PCTCN2016078429-appb-000013
五、麦角甾醇联合顺铂主动载药脂质体体外细胞增殖抑制试验
本实验为了验证麦角甾醇、顺铂原料药制备成脂质体制剂后能够增强其抗肿瘤活性，采用体外培养A549肺癌细胞，给予麦角甾醇、顺铂、麦角甾醇联合顺铂原料药以及麦角甾醇联合顺铂主动载药脂质体制剂刺激后，测定不同浓度给药后的细胞增殖抑制率，并计算脂质体制剂对A549细胞的半数抑制率IC50值。
取对数生长期A549细胞，胰酶消化后调整细胞为1×105个·mL-1。以每孔加入100μL接种于96孔培养板，于37℃、5％CO2培养箱中培养，当细胞融合至80％后加药处理。加入不同稀释倍数的麦角甾醇联合顺铂主动载药脂质体及相应的麦角甾醇、顺铂、麦角甾醇联合顺铂原料药溶液，同时设置正常对照组。每个浓度各设置5个复孔，于加药后第24h后进行MTT检测。每孔加入MTT(5mg·mL-1)溶液20μL，37℃、5％CO2培养箱中孵育4h后，用酶标仪于492nm处测出各孔吸光度(OD)值。本实验平行3次测定。
图3看出，药物作用24h时，当稀释倍数为64、128、256倍时，相同稀释倍数下麦角甾醇联合顺铂主动载药脂质体组与其他三组相比，抑制 率均明显升高，差异有极显著性，**P<0.01(n＝3)。麦角甾醇顺铂脂质体给药的IC50值为2.178+0.544μg·mL-1。说明，将麦角甾醇及顺铂制备成脂质体制剂后，能够显著得增加其体外抗肺癌作用。
六、RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体制备
按照麦角甾醇联合顺铂主动载药脂质体最佳制备工艺制备麦角甾醇联合顺铂主动载药脂质体，采用后插入法按照穿膜肽R8(DSPE-PEG1000-R8，上海强耀生物科技有限公司合成)：胆固醇的摩尔比为0.07:1，在55℃水浴中孵育1h后制备单纯穿膜肽R8修饰的脂质体(R8-Lip)；按照RGD环肽(DSPE-PEG3400-c，上海强耀生物科技有限公司合成)：胆固醇的摩尔比为0.07:1，在55℃水浴中孵育1h后制备单纯RGD修饰的脂质体(RGD-Lip)；按照RGD环肽(DSPE-PEG3400-c，上海强耀生物科技有限公司合成)：穿膜肽R8(DSPE-PEG1000-R8，上海强耀生物科技有限公司合成)：胆固醇的摩尔比为0.07:0.07:1，在55℃水浴中孵育1h后制备共修饰脂质体(RGD with R8-Lip)。对于FITC标记的脂质体，将适量的FITC甲醇溶液加入至脂质材料中共同旋蒸形成薄膜，通过荧光分光光度计测定不同荧光物质FITC的浓度对细胞摄取率的影响，结果见表9。确定脂质体中FITC终质量浓度为25μg·mL-1。
表9 不同荧光物质FITC浓度对细胞摄取率的影响
Figure PCTCN2016078429-appb-000014
**P<0.01,各组间均有显著性差异。
七、RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体的质量评价
7.1显微形态
采用负染法制备样品。在室温条件下，取麦角甾醇联合顺铂主动载药脂质体，蒸馏水稀释至略显浑浊，滴至专用230目铜网上，滤纸吸干多余脂质体，静置1min。用1％磷钨酸负染，静置40s，使粒子在铜网上沉积，用滤纸吸取铜网边缘多余染液，自然挥干，电镜观察并照相(图4)。结果表明，各脂质体形态较圆整，具有双层结构，粒径分布均匀。
7.2粒径及其电位考察
室温条件下，取无修饰、RGD修饰、R8修饰、RGD与R8共修饰脂质体，稀释20倍，注入样品池，激光粒度仪测定平均粒径及其分布。结果表明，无修饰脂质体的平均粒径为153.4nm，多分散系数PDI为0.156，小于0.3，RGD修饰脂质体的平均粒径为156.7nm，PDI为0.164，小于0.3，R8修饰脂质体的平均粒径为154.3nm，PDI为0.178，小于0.3，RGD与R8共修饰脂质体的平均粒径为155.2nm，PDI为0.102，小于0.3，可见各脂质体的粒径分布较集中。
八、共修饰脂质体的肿瘤球穿透能力考察
由于体内肿瘤部位组织为多层细胞，为了模拟其体内微环境，本发明考查了不同pH条件下共修饰脂质体对肿瘤球的穿透能力。在培养液中加入 20μg·mL-1B27无血清培养基、20ng·mL-1EGF与bFGF(市售)，每隔3d半量换液。将pH 6.0及pH 7.4的共修饰脂质体与培养基的混合液加至含有肿瘤球的6孔板中，与肿瘤球共孵育2h后，吸出肿瘤球，离心收集肿瘤球，用PBS漂洗3次，加入终质量浓度为1μg·mL-1的Lyso-Tracker Red(市售)，使其与细胞共孵育30min后，离心弃去培养基，用PBS漂洗3次，4％多聚甲醛固定30min后，离心弃去上清，PBS漂洗3遍，加入1μg·mL-1的DAPI溶液(市售)染色5min，离心弃去上清，PBS漂洗3次，将肿瘤球加在多聚赖氨酸处理的载玻片上，10％甘油-PBS溶液封片。采用FV 1000激光共聚焦显微镜观察共修饰脂质体对肿瘤球的穿透能力。由图5可见，其对肿瘤球的穿透能力呈显著的pH依赖性。在pH 6.0的孵育条件下，相较于pH 7.4的孵育条件下共修饰脂质体对肿瘤球的穿透能力显著性增强。
九、A549细胞对共修饰脂质体摄取的考察
流式细胞仪测定摄取率
定量将所制备的FITC标记的pH为7.4共修饰脂质体与1640培养基(市售)混合，使得最终的稀释倍数为64、96、128。将A549细胞接种在6孔板内，待细胞融合至80％时，将吸出旧培养基后，加入未修饰、RGD修饰、R8修饰、共修饰脂质体与培养基的混合液。在37℃、5％CO2培养箱中孵育2h后将培养基吸出，用PBS漂洗3次，胰酶消化收集细胞，加入PBS洗3遍，离心去除上清，最后加入0.5mL PBS重悬细胞，采用BD流式细胞仪检测细胞对不同给药浓度的共修饰脂质体的摄取强度。结果见表10，由结果表明，给予共修饰脂质体，细胞摄取的荧光强度明显高于未修饰及 仅采用RGD或R8修饰脂质体。
表10 RGD、R8修饰麦角甾醇联合顺铂主动载药脂质体细胞摄取
Figure PCTCN2016078429-appb-000015
十、共修饰脂质体摄取机制的考察
将A549接种于6孔板中，在37℃、5％CO2下培养至细胞融合80％左右，分别加500μg·mL-1的细胞摄取抑制剂氯氮平、秋水仙素和叠氮钠，其中氯氮平为陷穴蛋白介导的内吞途径抑制剂，秋水仙素为巨胞饮途径抑制剂，叠氮钠为能量依赖型的内吞途径抑制剂。与细胞共孵育30min后再将RGD与R8共修饰脂质体与培养基的混合液加至6孔板中，2h后吸出培养基，用PBS漂洗3次，胰酶消化，离心后用0.5mL PBS重悬细胞，采用流式细胞仪检测加入不同抑制剂后A549细胞对共修饰脂质体摄取的程度，从而考察共修饰脂质体的摄取入胞机制。图6结果表明，共修饰脂 质体可通过多种途径入胞，其中主要为能量依赖型(叠氮钠)的内吞途径及巨胞饮途径(秋水仙素)。
十一、共修饰脂质体对A549细胞增殖抑制的影响
取对数生长期A549细胞，胰酶消化后调整细胞为1×105个·mL-1。以每孔加入100μL接种于96孔培养板，于37℃、5％CO2培养箱中培养，当细胞融合至80％后加药处理。加入不同稀释倍数的共修饰脂质体，同时设置正常对照组每个浓度各设置5个复孔，分别于加药后第2h及24h后进行MTT检测。每孔加入MTT(5mg·mL-1)溶液20μL，37℃、5％CO2培养箱中孵育4h后，用酶标仪于492nm处测出各孔吸光度(OD)值。药物作用2h时，R8修饰及RGD与R8共修饰脂质体在稀释2、4、8、16、32倍的药物浓度下的肿瘤细胞增殖抑制率高于未修饰及RGD修饰脂质体(图7)。当作用时间延长至24h时，稀释倍数为2、4、8倍时各组抑制率几乎无差别，均在90％以上。稀释倍数为16和32时，各组的抑制情况与2h时相同(图8)。
结果显示，穿膜肽R8和RGD肽可以高效的介导共修饰脂质体入胞释放麦角甾醇与顺铂药物，从而使共修饰脂质体具有更强的靶向性，使麦角甾醇与顺铂药物发挥更好的抑制肿瘤作用。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。

Claims (12)

  1. 一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：由麦角甾醇联合顺铂主动载药脂质体、RGD环肽及穿膜肽R8在水浴中孵育后制备而得；所述麦角甾醇联合顺铂主动载药脂质体，由麦角甾醇脂质体、顺铂溶液为原料制成，其中，麦角甾醇与顺铂的质量比控制为1:1-4:1。
  2. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述麦角甾醇脂质体由麦角甾醇8-15wt％和脂质体85-92％制成，所述脂质体由卵磷脂与胆固醇组成，卵磷脂与胆固醇的摩尔比为3:1-6:1。
  3. 根据权利要求2所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述麦角甾醇脂质体由麦角甾醇10wt％和脂质体90％制成，所述脂质体由卵磷脂与胆固醇组成，卵磷脂与胆固醇的摩尔比为5:1。
  4. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：RGD环肽及穿膜肽R8用量控制为RGD环肽：穿膜肽R8：胆固醇的摩尔比为0.07:0.07:1。
  5. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述RGD环肽具体为DSPE-PEG3400-c；所述穿膜肽R8具体为DSPE-PEG1000-R8。
  6. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述RGD肽与穿膜肽R8共修饰麦角 甾醇联合顺铂主动载药脂质体由麦角甾醇联合顺铂主动载药脂质体、RGD环肽及穿膜肽R8在55℃水浴中孵育1h后制备而得。
  7. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述顺铂溶液的浓度为0.03-0.3mg/mL。
  8. 根据权利要求7所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述顺铂溶液的浓度为0.15mg/mL。
  9. 根据权利要求1所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：所述麦角甾醇联合顺铂主动载药脂质体的具体制备方法为：
    (1)麦角甾醇脂质体制备：称取卵磷脂、胆固醇、麦角甾醇，加入氯仿溶解，旋转蒸发成薄膜，真空干燥，以氯化铵溶液为水化液进行水化，超声脱膜，冰浴下探头超声处理，过滤，高压挤出得麦角甾醇脂质体；
    (2)氯化铵梯度形成：将麦角甾醇脂质体加入截留分子量为8000-14000Da的透析袋中，封闭透析袋，投入蒸馏水中透析2h，更换一次蒸馏水，继续透析2h；
    (3)载药：顺铂与水混合制成顺铂溶液，将上一步透析后的麦角甾醇脂质体加入顺铂溶液进行孵育，麦角甾醇脂质体的加入量以满足麦角甾醇与顺铂的质量比为1:1-4:1计算，孵育温度40-60℃，孵育时间5-40min，孵育结束后得到产品。
  10. 根据权利要求9所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：步骤(1)中探头超声处理的参数为： 超声时间20min，超声2s，停1s，超声功率900W，高压挤出的压力为400-500psi。
  11. 根据权利要求9所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：步骤(3)中麦角甾醇脂质体的加入量以满足麦角甾醇与顺铂的质量比为2.5:1计算，孵育温度50℃，孵育时间10min。
  12. 根据权利要求9所述的一种RGD肽与穿膜肽R8共修饰麦角甾醇联合顺铂主动载药脂质体，其特征在于：步骤(1)中氯化铵溶液的浓度为0.1-1.5mmol·L-1。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1846691A (zh) * 2005-04-11 2006-10-18 北京大学 注射用的整合素配体修饰的载抗癌药的长循环脂质体
CN104000782A (zh) * 2014-05-14 2014-08-27 河南牧翔动物药业有限公司 泰拉菌素脂质体及其制备方法
CN105796592A (zh) * 2016-03-11 2016-07-27 浙江中医药大学 一种rgd肽与穿膜肽r8共修饰麦角甾醇联合顺铂主动载药脂质体的制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007064658A2 (en) * 2005-11-30 2007-06-07 Transave, Inc. Safe and effective methods of administering therapeutic agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1846691A (zh) * 2005-04-11 2006-10-18 北京大学 注射用的整合素配体修饰的载抗癌药的长循环脂质体
CN104000782A (zh) * 2014-05-14 2014-08-27 河南牧翔动物药业有限公司 泰拉菌素脂质体及其制备方法
CN105796592A (zh) * 2016-03-11 2016-07-27 浙江中医药大学 一种rgd肽与穿膜肽r8共修饰麦角甾醇联合顺铂主动载药脂质体的制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI, XIAOGANG ET AL.: "Antitumor Chemical Entities of Cordyceps taii Mycelia Powder from Guizhou", JOURNAL OF CHINESE MEDICINAL MATERIALS, vol. 38, no. 10, 31 October 2015 (2015-10-31), ISSN: 1001-4454 *
LV , XING ET AL.: "The Inhibition Effect of RGD and R8 Co-modified Paclitaxel Loaded Liposome on the Esophagus Carcinoma EC 9706 Cells", JOURNAL OF CANCER CONTROL AND TREATMENT, vol. 27, no. 5, 31 October 2014 (2014-10-31), ISSN: 1674-0904 *
ZHANG, LI ET AL.: "The Construction of Cell -Penetrating Peptide R8 and pH Sensitivecleavable Polyethylene Glycols Co-Modified Liposomes", ACTA PHARMACEUTICA SINICA, vol. 50, no. 6, 30 June 2015 (2015-06-30), pages 760 - 766, XP055421084, ISSN: 0513-4870 *

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