WO2017041609A1 - 固体脂质磁共振纳米粒子及其制备方法和应用 - Google Patents

固体脂质磁共振纳米粒子及其制备方法和应用 Download PDF

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WO2017041609A1
WO2017041609A1 PCT/CN2016/094834 CN2016094834W WO2017041609A1 WO 2017041609 A1 WO2017041609 A1 WO 2017041609A1 CN 2016094834 W CN2016094834 W CN 2016094834W WO 2017041609 A1 WO2017041609 A1 WO 2017041609A1
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magnetic resonance
solid lipid
lipid
nanoparticles
preparing
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French (fr)
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孙继红
袁弘
刘菲
陈少青
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes

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  • the invention relates to a solid lipid nanoparticle loaded with a magnetic resonance contrast agent, in particular gadopentetate, and a preparation method thereof, and relates to the optimization thereof and the drug release behavior in vitro, the cytopharmacological study and the in vitro MRI detection .
  • a magnetic resonance contrast agent in particular gadopentetate
  • MRI Compared with traditional imaging methods such as CT, PET, X-ray, etc., MRI has high spatial resolution, good soft tissue contrast, and no ionizing radiation, which can show the superiority of physiological and anatomical details to a certain extent. And can detect tumor diseases including breast cancer, liver cancer, and cholangiocarcinoma.
  • tumor diseases including breast cancer, liver cancer, and cholangiocarcinoma.
  • the relaxation times T1 and T2 overlap each other, and the signal intensity is not much different. Therefore, it is necessary to apply the magnetic resonance imaging contrast agent to improve the inter-tissue.
  • the contrast of the image which in turn increases the sensitivity and specificity of the MRI diagnosis.
  • MRI contrast agents used in clinical settings need to meet the following conditions: (1) good water solubility; (2) selective retention at the site to be tested; (3) high relaxation efficiency; (4) low toxicity.
  • T1 type contrast agents such as paramagnetic metal ions Gd 3+ , Mn 2+ , Fe 3+ chelate
  • T2 type contrast agents such as superparamagnetic and ferromagnetic substances.
  • Gd-DTPA the most widely used contrast agent, is the first MRI contrast agent to receive FDA-approved clinical application. It needs to form the water proton relaxation time and surrounding tissues by affecting local tissues in the body. Contrast produces contrast, but the blood circulation and retention time in the body is shorter after injection, and Gd-DTPA lacks targeting, which affects the application of Gd-DTPA in clinical MRI. Therefore, it is important to design a carrier-loaded Gd-DTPA to prolong the retention time of Gd-DTPA and shorten the T1 relaxation time and enhance the MRI signal.
  • Solid lipid nanoparticles are prepared from natural or synthetic lipid materials such as triglycerides and complex glycerides with particle sizes between 50 and 1000 nm.
  • SLN has a high oral availability and can be lowered Low drug nephrotoxicity, in addition to good stability, low toxicity, stable drug loading, low toxicity, large-scale production, etc., solid lipid nanoparticles are considered to be ideal carriers for drug loading.
  • the stability of the preparation can be improved and the relaxation efficiency can be improved.
  • Gd-DTPA Common methods for loading Gd-DTPA into SLN include solvent diffusion method, emulsification method, high-pressure emulsion homogenization method, etc., and the inventors used solvent diffusion in the previous work.
  • the method contains Gd-DTPA, and the encapsulation efficiency (encapsulated drug/drug total *100%) reaches 55%, but a short-term burst effect occurs in the release experiment because gadopentetate is a pro Aqueous drugs have weak affinity with SLN composed of lipid materials.
  • the first technical problem to be solved by the present invention is to provide a solid lipid magnetic resonance nanoparticle, which not only has the characteristics of high encapsulation efficiency of solid lipid magnetic resonance nanoparticles, but also has the advantages of prolonged release time.
  • the present invention adopts the following technical solutions:
  • a solid lipid magnetic resonance nanoparticle comprising a magnetic resonance imaging contrast agent, a lipid material, a fluorescent dye, characterized in that the lipid material contains 17.22%-56.47% cationic lipid material, the magnetic resonance
  • the imaging contrast agent is a water soluble paramagnetic contrast agent.
  • the content of the lipid material in the solid lipid magnetic resonance nanoparticles accounts for 17.22%-56.47%.
  • the magnetic resonance imaging contrast agent is a water-soluble paramagnetic magnetic resonance contrast agent, comprising Gd-DTPA (glucuronide), Gd-DTPA-BMA (decanediamine), Gd-DO 3 A-HP (Gentol), Gd-DOTA (iron carboxyglucamide), Gd-DO 3 A-butrol, etc.
  • Gd-DTPA glucuronide
  • Gd-DTPA-BMA decanediamine
  • Gd-DO 3 A-HP Gentol
  • Gd-DOTA iron carboxyglucamide
  • Gd-DO 3 A-butrol etc.
  • the fluorescent dye is stearylamine-isothiocyanatofluorescein (ODA-FITC), and the content in the solid lipid magnetic resonance nanoparticles is 1.72%-5.65%.
  • the selection of the lipid material other than the cationic lipid material includes glyceryl monostearate (monoglyceride), lecithin, triglyceride, preferably a single weight ratio of 85 to 95:5 to 15 Made up of glyceryl stearate and lecithin.
  • the cationic lipid material is selected from the group consisting of octadecylamine (ODA), hydrogenated soybean lecithin (HSPC), and (2,3-dioleoyl-propyl)trimethylammonium chloride (DOTAP).
  • ODA octadecylamine
  • HSPC hydrogenated soybean lecithin
  • DOTAP (2,3-dioleoyl-propyl)trimethylammonium chloride
  • Dipalmitoylphosphatidylcholine DPPC
  • trimethyl-2,3-dioleyloxypropylammonium chloride DOTMA
  • Spermine formylamino)ethylammonium DOSPA
  • trimethyldodecyl ammonium bromide DTAB
  • trimethyltetradecyl ammonium bromide TTAB
  • trimethylhexadecyl bromide Ammonium CTAB
  • dimethyldioctadecyl ammonium bromide DDAB
  • dimethyldioctadecyl ammonium bromide DORI
  • dimethyl-2-hydroxyethyl-2,3 -bisoctadecyloxypropylammonium DSRIE
  • the invention also provides the use of any of the above solid lipid magnetic resonance nanoparticles in the field of MRI detection.
  • Still another object of the present invention is to provide a method for preparing solid lipid magnetic resonance nanoparticles, which maintains the characteristics of high encapsulation efficiency and also has the advantage of prolonging the release time.
  • the present invention adopts the following technical solutions:
  • a method for preparing solid lipid magnetic resonance nanoparticles comprising the steps of:
  • the preparation method further includes the following steps
  • the ratio of the first surfactant to water is 1.8 to 2.5 g: 100 mL; and the ratio of the second surfactant to the organic solvent is 1.5 to 2.0 g: 100 mL.
  • the organic solvent is at least one of n-pentane, n-hexane and cyclohexane.
  • step (2) trehalose is added as a lyoprotectant during freeze-drying.
  • the range of surfactants used in preparing the solid lipid magnetic resonance nanoparticles includes spitting Tween-80, Tween-20, Span-80, sodium dodecyl sulfate (SDS), Pluronic-68 (F-68), Cetyltrimethylammonium bromide (CTMAB).
  • the mass concentration of the magnetic resonance imaging contrast agent in the aqueous solution is 5% to 8%; the mass concentration of the cationic lipid material in the ethanol solution is 0.45 to 0.7%,
  • the composite temperature is 55-65 ° C, and the compounding time is 30-60 min.
  • the lipid material other than the cationic lipid material is selected from one or more of glyceryl monostearate, lecithin, stearic acid, and glyceryl tristearate.
  • it consists of glyceryl monostearate and lecithin in a weight ratio of 85 to 95:5 to 15.
  • the cationic lipid material adopts a range including octadecylamine (ODA), hydrogenated soybean lecithin (HSPC), (2,3-dioleoyl-propyl)trimethylammonium chloride (DOTAP) and two Palmitoylphosphatidylcholine (DPPC), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), dimethyl-2,3-dioleoxypropyltrifluoroacetate -2-(2-arginylformylamino)ethylammonium (DOSPA), trimethyldodecyl ammonium bromide (DTAB), trimethyltetradecyl ammonium bromide (TTAB), bromide Methyl cetyl ammonium (CTAB), dimethyldioctadecyl ammonium bromide (DDAB), dimethyldioctadecyl ammonium bromide (DORI), oct
  • the invention can also adopt the following technical solutions:
  • a method for preparing solid lipid magnetic resonance nanoparticles comprises the following steps:
  • the solid lipid nanoparticles coated with the water-soluble paramagnetic contrast agent gadopentetate are applied to the drug.
  • the encapsulation rate is not high and will produce a burst of drug.
  • the invention adopts the solvent diffusion method in microemulsion to prepare solid lipid magnetic resonance nanoparticles, firstly compounding with Gd-DTPA by cationic lipid material such as octadecylamine, adding microemulsion, stirring, centrifuging and freeze-drying to improve solid fat.
  • the entrapment efficiency of the water-soluble drug in the magnetic resonance nanoparticle, and the sudden release phenomenon is effectively avoided.
  • the ratio of the amount of Tween-80 to water is 1.8 to 2.5 g: 100 mL; and the ratio of the amount of the Span-80 to the organic solvent is 1.5 to 2.0 g: 100 mL.
  • the organic solvent is a solvent which is immiscible with water at room temperature, such as a hydrocarbon solvent or an ester solvent.
  • the organic solvent is n-pentane. At least one of n-hexane and cyclohexane.
  • the lipid material (octadecylamine, etc.) in the ethanol solution is completely combined with gadopentetate, and the solvent is removed under the conditions of rotary evaporation at an appropriate temperature, and the solvent is removed after compounding.
  • the aqueous solution of the intermediate product has good solubility.
  • the mass percentage concentration of gadopentetate in the aqueous solution is 7.8%; the ethanol solution, the mass percentage concentration of octadecylamine in the solution is 0.6%; At 60 ° C, the time of recombination was 30 min.
  • the lipid material is selected from one or more of glyceryl monostearate, lecithin, stearic acid and glyceryl tristearate;
  • the drug loading amount of the amine is 4% to 35% (mass percent concentration), preferably 25% to 27.5%.
  • the lipid material is composed of glyceryl monostearate and lecithin in a weight ratio of 85 to 100:0 to 15.
  • the affinity of the solvent for dissolving the lipid material is less than the affinity of water, and preferably, the solvent selected is at least one of n-pentane, n-hexane and cyclohexane.
  • step (3) trehalose is added as a lyoprotectant during freeze-drying, and the stability of the product is improved by using trehalose as a lyoprotectant.
  • the invention also provides a solid lipid magnetic resonance nanoparticle prepared by the above preparation method.
  • the solid lipid magnetic resonance nanoparticles of the present invention have a particle diameter of 100 to 400 nm.
  • the solution of the lipid material described in the above step (2) or (3) is an organic solution, and the dissolved or dispersed expression described in the step (2) or (3) is considered in view of the self-properties of the intermediate product.
  • the same meaning is that the intermediate product is fully dissolved in the lipid material solution.
  • the invention also provides an application of the solid lipid magnetic resonance nanoparticle described in the field of MRI detection.
  • the invention also provides the in vitro pharmacodynamic application of the solid lipid nanoparticle loaded with the magnetic resonance contrast agent Gd-DTPA, and the solid lipid nanoparticle prepared by the above step scheme is compared with the breast cancer cell MCF-7 as a model cell.
  • the magnetic resonance solid lipid nano contrast agent prepared in the patent document CN 106491906 B is low in toxicity.
  • the concentration of the solid lipid nanoparticles was 4 mg/ml, the magnetic resonance imaging detection signal gradually increased.
  • the optimum mass ratio of gadopentetate, stearylamine and glyceryl monostearate is 31.3:9:50. At this time, the loading of gadopentetate in the obtained nanoparticles reaches or approaches The percentage of mass is 10%.
  • the solid lipid magnetic resonance nanoparticle of the invention can not only solve the problem of sudden release of the drug, but also the in vitro pharmacodynamic test results show that the cell survival rate is high and the toxicity is small; and the in vitro nuclear magnetic resonance test results show that The T1 relaxation rate is enhanced.
  • Example 1 is an in vitro drug release profile of the Gd-DTPA-loaded solid lipid nanoparticles prepared in Example 3 in Example 3.
  • Example 2 is a graph showing the in vitro cytotoxicity analysis of SLN-2-(1) and SLN-2-(2) prepared in Example 4 in Example 4.
  • Example 3 is a cell uptake-concentration-dependent flow pattern of SLN-2-(1) and SLN-2-(2) prepared in Example 4 in Example 4.
  • Figure 4 shows the magnetic resonance response of SLN-2-(1) and SLN-2-(2) prepared in Example 1 for MRI detection in Example 5.
  • Sex. The concentrations were 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, and 12 mg/mL, respectively.
  • Figure 5 is a TEM image of a loaded Gd-DTPA lipid nanoparticle prepared according to the preferred formulation SLN-2-(2) according to the Chinese patent document CN 106491906 B and Example 1, with a magnification of 100,000 times, A: according to the Chinese patent Document SLN-1, B prepared by document CN 106491906 B: SLN-2-(2) prepared in Example 1.
  • Example 6 is a comparison chart of the release ratio of SLN-loaded Gd-DTPA prepared by Example 6 to Chinese Patent Document CN 106491906 B and Example 1 preferred formulation SLN-2-(2).
  • Fig. 7 is a comparison diagram of cytotoxicity analysis of Gd-DTPA-loaded solid lipid nanoparticles prepared in Chinese Patent Document CN 106491906 B and Example 1 preferred formulation SLN-2-(2) in Example 7.
  • FIG. 8A is a comparative diagram of the cell uptake-time-dependent fluorescence pattern of the Gd-DTPA-loaded lipid nanoparticle SLN-2 prepared in Example 7 and the preferred formulation of SLN-2-(2) in Example 7; FIG. SLN-2-(2) cell uptake-concentration-dependent fluorescence map.
  • Example 9 is a comparative diagram of a cell uptake-concentration-dependent flow pattern in Example 7, A is SLN-1 in Chinese Patent Document CN 106491906 B, and B is SLN-2-(2) in Example 1.
  • the intermediate product prepared in the step (2) is added with 3 ml of ethanol and 100 mg of monoglyceride, and the mixture is injected into the microemulsion prepared in the step (1) under heating at 60 ° C, stirred at room temperature for 5 min; then centrifuged at 20000 rpm for 20 min, and centrifuged in n-hexane. Two times, 250 mg of trehalose was finally added as a lyoprotectant to freeze-dry, thereby obtaining solid lipid nanoparticles.
  • the specific cationic lipid names and amounts are shown in the following table:
  • Example 2 Physicochemical properties of solid lipid nanoparticles loaded with Gd-DTPA
  • the solid lipid nanoparticles prepared in the above Example 1 were taken, and ultrafine water was used as a double solvent, and the particle diameter was measured at a concentration of 0.01 mg/ml using a 3000HS particle size and a surface potential analyzer.
  • Gd-DTPA encapsulation rate (Wo-W free) / Wo * 100% (1)
  • the drug loading of Gd-DTPA is calculated according to formula (2):
  • Gd-DTPA drug loading (addition of drug * encapsulation rate) / (addition of drug * encapsulation rate + carrier material dosage) * 100%
  • Gd-DTPA is an excellent water-soluble cerium ion chelate. It is prepared by general solvent diffusion method to prepare solid lipid nanoparticles. It is difficult to leak into water due to the weak lipophilicity of drugs and lipid materials. Wrap it effectively In the lipid nanoparticle; by using the method provided by the invention, by controlling the ratio of octadecylamine to Gd-DTPA, the affinity of the lipid material and the drug is increased, the water solubility of the SLN is increased, and the release time of the drug is prolonged. Reduces cytotoxicity while enhancing the sensitivity of in vitro MRI assays.
  • Example 3 In vitro drug release behavior of solid lipid nanoparticles loaded with Gd-DTPA
  • a volume of the SLN solution of Example 1 was taken separately, placed in a dialysis bag (MWCO 3.5 KDa), and placed in a release tube containing 25 ml of a release medium (pH 7.2 PBS). In vitro release was carried out at 37 ° C, 65 rpm, and samples were taken at specific time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h and 72 h) while replacing the entire release medium.
  • Example 4 In vitro pharmacodynamic study of solid lipid nanoparticles loaded with Gd-DTPA
  • the effect of the solid lipid nanoparticles prepared by Example 1 (SLN-2(1), SLN-2(2)) on the survival rate of MCF-7 cells was quantitatively examined using a CCK-8 detection kit.
  • the cells were transferred to a 96-well cell plate at a cell density of 0.8-1 ⁇ 10 4 cells/mL, and placed in a 37 ° C 5% cell culture incubator overnight.
  • a series of polymer solutions with different concentrations are prepared.
  • the volume of the polymer solution added per well is 200 ⁇ L, parallel to 5 replicate wells, and the dilution medium is phenol red free 1640 high sugar medium.
  • the concentration of the polymer to be investigated is in turn 0, 50, 100, 200, 400, 500, 600, 800 in ⁇ g/mL.
  • the 96-well cell plate was taken out from the incubator, the culture solution was aspirated, and each well was washed once with 100 ⁇ L of PBS buffer solution, and then the phosphate buffer solution was aspirated, and a series of different concentrations of the polymer solution were sequentially added to the cell plate.
  • the cells were cultured in a 37 ° C 5% cell culture incubator for 48 hours.
  • 96-well cell plates were taken out from the incubator, 20 ⁇ L of LCCK-8 was added to each well, and the cells were further incubated in the incubator for 1-3 h, and then the cell plates were taken out, and the absorbance at 450 nm of the samples was measured using a multi-function microplate reader.
  • FITC labeled SLN Hoechst 33342 stained nuclei.
  • the cells were transferred to a 24-well cell plate at a cell density of 5 ⁇ 10 4 cells/mL, and cultured in a 37 ° C 5% cell culture incubator overnight.
  • a solution containing different concentrations of SLN 50 ug/ml, 100 ug/ml, 150 ug/ml, 200 ug/ml, 300 ug/ml
  • SLN-2 (1), SLN-2 (2) the results shown in Figure 3, the difference in cellular uptake between the two is not obvious.
  • Example 5 In vitro MRI detection of solid lipid nanoparticles loaded with Gd-DTPA
  • Example 1 A series of different concentrations (0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 3.0 mg/mL) of solid lipid nanoparticles prepared in Example 1 (SLN-2(1), SLN-) were set. 2(2)) SLN carrying Gd-DTPA, placed at a temperature of 20 ° C, MR scanning, MR field strength 3.0T, T1WI sequence (TR500, TE15), the result: SL1 of SLN-2-(2) The relaxation rate is higher than SLN-2-(1) as shown in FIG.
  • Example 6 In vitro drug release behavior of solid lipid nanoparticles loaded with Gd-DTPA
  • the SLN-1 prepared by the patent document CN 106491906 B and the preferred prescription SLN-2-(2) solution prepared in Example 2 were placed in a dialysis bag (MWCO 3.5KDa) and placed in a 25 ml release medium (pH 7.2). PBS) in the release tube. In vitro release was carried out at 37 ° C, 65 rpm, and samples were taken at specific time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h and 72 h) while replacing the entire release medium.
  • Example 7 In vitro pharmacodynamic study of solid lipid nanoparticles loaded with Gd-DTPA
  • SLN-1 prepared in Chinese patent document CN 106491906 B and the preferred formulation SLN-2-(2) of Example 1 on the survival rate of MCF-7 cells was quantitatively examined using a CCK-8 detection kit.
  • the cells were transferred to a 96-well cell plate at a cell density of 0.8-1 ⁇ 10 4 cells/mL, and placed in a 37 ° C 5% cell culture incubator overnight.
  • a series of polymer solutions with different concentrations are prepared.
  • the volume of the polymer solution added per well is 200 ⁇ L, parallel to 5 replicate wells, and the dilution medium is phenol red free 1640 high sugar medium.
  • the concentration of the polymer to be investigated is in turn 0, 50, 100, 200, 400, 500, 600, 800 in ⁇ g/mL.
  • the 96-well cell plate was taken out from the incubator, the culture solution was aspirated, and each well was washed once with 100 ⁇ L of PBS buffer solution, and then the phosphate buffer solution was aspirated, and a series of different concentrations of the polymer solution were sequentially added to the cell plate.
  • the cells were cultured in a 37 ° C 5% cell culture incubator for 48 hours.
  • 96-well cell plates were taken out from the incubator, 20 ⁇ L of LCCK-8 was added to each well, and the cells were further incubated in the incubator for 1-3 h, and then the cell plates were taken out, and the absorbance at 450 nm of the samples was measured using a multi-function microplate reader. Each sample was tested in parallel 5 times and the average was plotted (blank as a control group). Cell viability (percentage) is expressed as the absorbance value of the sample to be measured as compared to the absorbance value of normal cells. As a result, the cytotoxicity of the preferred formulation SLN-2-(2) was lower than that of SLN-1 as shown in FIG.
  • FITC labeled SLN Hoechst 33342 stained nuclei.
  • the cells were transferred to a 24-well cell plate at a cell density of 5 ⁇ 10 4 cells/mL, and cultured in a 37 ° C 5% cell culture incubator overnight.
  • the cells were collected for 4 hours, and the difference in uptake between SLN-1 and SLN-2-(2) was compared by flow cytometry.
  • the vector was ingested from 0.5 h, and The prolonged uptake at the time point was increased; the concentration of SLN-2-(2) in the concentration-dependent uptake assay was greater than that of SLN-1.

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Abstract

本发明公开了一种固体脂质磁共振纳米粒子及其制备方法和应用，该纳米粒子以单硬脂酸甘油酯为脂质材料，通过十八胺与磁共振对比剂钆喷替酸葡胺复合，海藻糖作为冻干保护剂，制备肠道易吸收的脂质纳米粒。本发明在原有固体脂质纳米粒制备方法的基础上进行优化，制备得到的纳米粒子的水溶性良好、粒径均一，延长了纳米粒中钆喷替酸葡胺的释放时间，并降低了材料对乳腺癌细胞的毒性，提高了MR成像的弛豫效率。

Description

固体脂质磁共振纳米粒子及其制备方法和应用 技术领域
本发明涉及负载磁共振对比剂尤其是钆喷酸葡胺的固体脂质纳米粒子、用途和此种粒子的制备方法，涉及其优化及其体外药物释放行为、细胞药效学研究、体外MRI检测。
背景技术
相较于传统的影像学检查方法如CT、PET、X射线等，MRI具有较高空间分辨率、良好的软组织对比度、无电离辐射特点，在一定程度上可以显示生理学和解剖学细节的优越性，并能够检测包括乳腺癌、肝癌、胆管癌等肿瘤疾病。但临床实践中发现良、恶性肿瘤组织之间，肿瘤组织与正常组织之间，弛豫时间T1、T2会互相重叠，信号强度相差不大，因此有必要应用磁共振成像对比剂、提高组织间的图像对比度，继而提高MRI诊断的敏感性与特异性。
应用于临床的MRI造影剂需要满足条件有：(1)良好水溶性；(2)选择性滞留在被检测部位；(3)弛豫效能高；(4)毒性低。
常用的磁共振造影剂分为两类：顺磁性金属离子Gd3+、Mn2+、Fe3+螯合物等T1类造影剂，超顺磁性和铁磁性物质等T2类造影剂。目前应用较为广泛的造影剂-钆喷酸葡胺(Gd-DTPA)是第一个获得美国FDA认证临床应用的MRI造影剂，它需要通过影响体内局部组织的水质子弛豫时间与周围组织形成对比产生造影作用，但注射后在体内血液循环和滞留时间较短，同时Gd-DTPA缺乏靶向性，从而影响了Gd-DTPA在临床MRI中的应用。因此设计一种载体包载Gd-DTPA，延长机体对Gd-DTPA的滞留时间并缩短T1弛豫时间、增强MRI信号具有重要的意义。
固体脂质纳米粒(solid lipid nanoparticles,SLN)是由甘油三酯、复合甘油酯等天然或合成的脂质材料制备，粒径在50-1000nm之间。SLN具有较高的口服利用度、可降 低药物的肾毒性，此外具有良好稳定性、毒性低、包载药物稳定、低毒、可大规模生产等优点，因此固体脂质纳米粒被认为是装载药物的理想载体。通过Gd-DTPA纳米化，可提高制剂的稳定性、提高弛豫效率常见Gd-DTPA载入SLN的方法有溶剂扩散法、乳化法、高压乳匀法等，发明人前期的工作中采用溶剂扩散法包载Gd-DTPA，包封率(包封的药物/药物总量*100％)达到55％，但是在释放实验中发生短时间突释效应，原因在于钆喷酸葡胺是一种亲水性药物，与由脂质材料构成的SLN亲和力弱。
发明内容
本发明首先所要解决的技术问题是提供一种固体脂质磁共振纳米粒子，不仅具备固体脂质磁共振纳米粒包封率高的特点，且还具有延长释放时间的优点。为此，本发明采用以下技术方案：
一种固体脂质磁共振纳米粒子，包括磁共振成像造影剂、脂质材料、荧光染料,其特征在于，所述脂质材料中含有17.22％-56.47％的阳离子脂质材料，所述磁共振成像造影剂为水溶性的顺磁性对比剂。
进一步地，所述脂质材料在固体脂质磁共振纳米粒子中的含量占17.22％-56.47％。
进一步地，所述磁共振成像造影剂为水溶性顺磁性磁共振对比剂，包含Gd-DTPA(钆喷酸葡胺)、Gd-DTPA-BMA(钆二胺)、Gd-DO3A-HP(钆替醇)、Gd-DOTA(铁羧葡胺)、Gd-DO3A-butrol等，本专利采用钆喷酸葡胺(Gd-DTPA)、在固体脂质磁共振纳米粒子中的含量10.78％-35.35％。
进一步地，所述荧光染料采用硬脂胺-异硫氰基荧光素(ODA-FITC)、在固体脂质磁共振纳米粒子中的含量占1.72％-5.65％。
进一步地，除阳离子脂质材料之外的脂质材料选择范围包括单硬脂酸甘油脂(单甘脂)、卵磷脂、甘油三酯，优选由重量比为85～95：5～15的单硬脂酸甘油酯和卵磷脂组成。
进一步地，所述阳离子脂质材料的选择范围包括为十八胺(ODA)、氢化大豆卵磷脂(HSPC)、(2,3-二油酰基-丙基)三甲基氯化铵(DOTAP)和二棕榈酰磷脂酰胆碱 (DPPC)、氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、溴化二甲基双十八烷基铵(DORI)、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵(DSRIE)、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺(DOGS)、脂质多聚-L-赖氨酸(LPLL)。
本发明还提供了上述任意一种固体脂质磁共振纳米粒子在MRI检测领域中的应用。
本发明再一个目的是提供一种固体脂质磁共振纳米粒子的制备方法，保持了包封率高的特点，且还具有延长释放时间的优点。为此，本发明采用以下技术方案：
一种固体脂质磁共振纳米粒子的制备方法，其特征在于，包括以下步骤：
将第一种表面活性剂溶于水中得到水相，将第二种表面活性剂溶于有机溶剂中得到有机相，然后将水相加入到有机相中，超声处理得到微乳，备用；
所述制备方法还包括以下步骤
(1)将磁共振成像造影剂溶解于水中得到水溶液，将阳离子脂质材料溶解于乙醇中得到乙醇溶液，然后将水溶液和乙醇溶液复合后除去溶剂得到中间产物，所述磁共振成像造影剂为水溶性的顺磁性对比剂；
(2)将步骤(1)得到的中间产物溶解到阳离子脂质材料之外的脂质材料溶液中，在加热条件下，注入到备用的微乳液中，经过搅拌、离心和冷冻干燥得到固体脂质磁共振纳米粒子。
进一步地，所述的第一种表面活性剂与水的用量比为1.8～2.5g：100mL；第二种表面活性剂与有机溶剂的用量比为1.5～2.0g：100mL。
进一步地，所述的有机溶剂为正戊烷、正己烷和环己烷中的至少一种。
进一步地，步骤(2)中，冷冻干燥时加入海藻糖作为冻干保护剂。
进一步地，制备固体脂质磁共振纳米粒子所采用的表面活性剂的选择范围包括吐 温-80(Tween-80)、吐温-20(Tween-20)、司盘-80(Span-80)、十二烷基硫酸钠(SDS)、普朗尼克-68(F-68)、十六烷基三甲基溴化铵(CTMAB)。
进一步地，步骤(1)中，所述的水溶液中磁共振成像造影剂的质量百分比浓度为5％～8％；所述的乙醇溶液中阳离子脂质材料的质量百分比浓度为0.45～0.7％，复合的温度为55～65℃，复合的时间为30～60min。
进一步地，步骤(1)中，所述阳离子脂质材料之外的脂质材料选用单硬脂酸甘油酯、卵磷脂、硬脂酸、三硬脂酸甘油酯中的一种或多种组合；优选由重量比为85～95：5～15的单硬脂酸甘油酯和卵磷脂组成。
作为进一步的优选，阳离子脂质材料采用范围包括十八胺(ODA)、氢化大豆卵磷脂(HSPC)、(2,3-二油酰基-丙基)三甲基氯化铵(DOTAP)和二棕榈酰磷脂酰胆碱(DPPC)、氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、溴化二甲基双十八烷基铵(DORI)、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵(DSRIE)、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺(DOGS)、脂质多聚-L-赖氨酸(LPLL)。
为实现上述目的，克服现有的磁共振对比剂中Gd-DTPA释放速度过快的现象。本发明还可采用以下技术方案：
一种固体脂质磁共振纳米粒子的制备方法，包括以下步骤：
(1)将吐温-80溶于水中得到水相，将司盘-80溶于有机溶剂中得到有机相，然后将水相加入到有机相中，超声处理得到微乳液；
(2)将钆喷酸葡胺溶解于水中得到水溶液，将十八胺溶解于乙醇中得到乙醇溶液，然后将水溶液和乙醇溶液复合后除去溶剂得到中间产物；
(3)将步骤(2)得到的中间产物分散到含有脂质材料的溶液中，然后加热条件下注入到步骤(1)得到的微乳液中，经过搅拌、离心和冷冻干燥得到所述的固体脂质 磁共振纳米粒子。
在现有的纳米粒制备过程中，由于脂质材料的疏水性及Gd-DTPA的亲水性，造成包载水溶性的顺磁性对比剂钆喷酸葡胺的固体脂质纳米粒对药物的包封率不高并且会产生药物的突释现象。本发明采用微乳中溶剂扩散法制备固体脂质磁共振纳米粒，首先通过十八胺等阳离子脂质材料与Gd-DTPA复合，加入微乳，再搅拌、离心并冷冻干燥，提高了固体脂质磁共振纳米粒子中水溶性药物的包载效率，并且有效地避免了突释现象。
作为优选，步骤(1)中，所述的吐温-80与水的用量比为1.8～2.5g：100mL；所述的司盘-80与有机溶剂的用量比为1.5～2.0g：100mL。
步骤(1)中，所述的有机溶剂为室温下与水互不混溶的溶剂，例如烃类溶剂或酯类溶剂，作为优选，步骤(1)中，所述的有机溶剂为正戊烷、正己烷和环己烷中的至少一种。
作为优选，步骤(2)中，所述乙醇溶液中脂质材料(十八胺等)，与钆喷酸葡胺复合完全，并在适当温度旋转蒸发条件下除去溶剂，复合后除去溶剂得到的中间产物的水溶液溶解性良好。
作为优选，步骤(2)中，所述的水溶液中钆喷酸葡胺的质量百分比浓度为7.8％；所述的乙醇溶液，溶液中十八胺的质量百分比浓度为0.6％；复合的温度为60℃，复合的时间为30min。
作为优选，步骤(3)中，所述的脂质材料选用单硬脂酸甘油酯、卵磷脂、硬脂酸、三硬脂酸甘油酯中的一种或多种组合；负载钆喷酸葡胺的载药量(包封药物的量/总重量*100％)为4％～35％(质量百分比浓度)，优选为25％～27.5％。
作为进一步的优选，步骤(3)中，所述的脂质材料由重量比为85～100：0～15的单硬脂酸甘油酯和卵磷脂组成。
步骤(3)中，用于溶解脂质材料的溶剂的亲和力小于水的亲和力，作为优选，所选用的溶剂为正戊烷、正己烷和环己烷中的至少一种。
步骤(3)中，冷冻干燥时加入海藻糖作为冻干保护剂，以海藻糖为冻干保护剂提高产物的稳定性。
本发明还提供了一种固体脂质磁共振纳米粒子，由上述的制备方法制备得到。
本发明固体脂质磁共振纳米粒子的粒径为100-400nm。
本发明中，上述步骤(2)或(3)所述的脂质材料的溶液为有机溶液，鉴于所述中间产物的自身性质，步骤(2)或(3)中所述的溶解、分散表达的是同一意思，均为中间产物充分溶解在脂质材料溶液中。
本发明还提供了一种所述的固体脂质磁共振纳米粒子在MRI检测领域中的应用。本发明还提供了负载磁共振对比剂Gd-DTPA的固体脂质纳米粒在体外药效学应用，以乳腺癌细胞MCF-7为模型细胞，通过上述步骤方案制备的固体脂质纳米粒较中国专利文献CN 106491906 B中制备的磁共振固体脂质纳米对比剂的毒性低。此外，当固体脂质纳米粒的浓度在4mg/ml时，磁共振成像检测信号逐渐增强。
本发明中，钆喷酸葡胺、十八胺和单硬脂酸甘油酯的最优质量比为31.3：9：50，此时，得到的纳米粒子中钆喷酸葡胺负载量达到或接近质量百分比10％。
同现有技术相比，本发明的固体脂质磁共振纳米粒子不仅可以解决药物的突释问题，而且体外药效学试验结果表明，细胞存活率高，毒性小；同时体外核磁共振试验结果表明，T1弛豫率增强。
附图说明
图1为实施例3中对实施例1制备的负载Gd-DTPA的固体脂质纳米粒的体外药物释放曲线。
图2为实施例4中对实施例1制备的SLN-2-(1)和SLN-2-(2)的体外细胞毒性分析图。
图3为实施例4中对实施例1制备的SLN-2-(1)和SLN-2-(2)的细胞摄取-浓度依赖性流式图片。
图4对实施例5中MRI检测实施例1制备的SLN-2-(1)和SLN-2-(2)的磁共振响应 性。浓度分别为2mg/mL、4mg/mL、6mg/mL、8mg/mL、12mg/mL。
图5为根据中国专利文献CN 106491906 B与实施例1优选处方SLN-2-(2)中制备的负载Gd-DTPA脂质纳米粒的透射电镜图片，放大倍数为100000倍，A：根据中国专利文献CN 106491906 B文件制备的SLN-1，B：实施例1制备的SLN-2-(2)。
图6为实施例6对中国专利文献CN 106491906 B与实施例1优选处方SLN-2-(2)制备的SLN包载Gd-DTPA随时间释放曲线对比图。
图7为实施例7中对中国专利文献CN 106491906 B与实施例1优选处方SLN-2-(2)制备的负载Gd-DTPA的固体脂质纳米粒的细胞毒性分析对比图。
图8A为实施例7中对实施例2优选处方SLN-2-(2)制备的负载Gd-DTPA的脂质纳米粒SLN-2的细胞摄取-时间依赖性荧光图的对比图；图8B为SLN-2-(2)细胞摄取-浓度依赖性荧光图。
图9为实施例7中细胞摄取-浓度依赖性流式图的对比图，A为中国专利文献CN 106491906 B中的SLN-1，B为实施例1中SLN-2-(2)。
具体实施方式
实施例1：负载Gd-DTPA的固体脂质纳米粒的制备
(1)首先称取54mg吐温-80溶于3ml水，组成水相，600mg司盘-80溶于30ml正己烷，组成有机相，在400rpm的室温搅拌条件下，将水相加入有机相，探头超声制得微乳液；
(2)然后称取62.6mg钆喷酸葡胺溶解于水中得到水溶液，将不同质量的阳离子脂质溶解在3ml乙醇中得到乙醇溶液，然后将两者于60℃下复合30min，并于60℃真空旋蒸除去溶剂得到中间产物；
(3)步骤(2)制备的中间产物加入3ml乙醇、100mg单甘脂，60℃加热条件下注入步骤(1)制备的微乳液中，室温搅拌5min；之后于20000rpm离心20min，正己烷离心沉淀2次，最终加入250mg海藻糖作为冻干保护剂进行冷冻干燥，从而得到固体脂质纳米粒。具体阳离子脂质名称及用量如下表所示：
Figure PCTCN2016094834-appb-000001
实施例2：负载Gd-DTPA的固体脂质纳米粒的理化性质考察
取上述实施例1制备的固体脂质纳米粒，以超纯水作为复溶剂，以0.01mg/ml浓度，使用3000HS粒度及表面电位分析仪测定其粒径。
采用间接法测定固体脂质纳米粒中Gd-DTPA的包封率。荧光分光光度法(Ex＝275nm,Em＝313nm,Slit＝5nm)测定荧光值，计算溶液中游离Gd-DTPA的量，按(1)式计算荧光嫁接物包封率：
Gd-DTPA包封率＝(Wo-W游离)/Wo*100％          (1)
Gd-DTPA载药量按照(2)式计算：
Gd-DTPA载药量＝(加入药物*包封率)/(加入药物*包封率+载体材料用量)*100％
具体见下表：
Figure PCTCN2016094834-appb-000002
Gd-DTPA是一种水溶性极好的钆离子螯合物，采用一般的溶剂扩散法制备固体脂质纳米粒，由于药物与脂质材料的亲脂性弱而极易向水中渗漏，很难将其有效地包裹 于脂质纳米粒中；而采用本发明提供的方法，通过控制十八胺与Gd-DTPA的投料比，以提高脂质材料与药物的亲和力，增加SLN的水溶性，延长药物的释放时间，降低细胞毒性，同时可以增强体外MRI检测的灵敏性。
实施例3：负载Gd-DTPA的固体脂质纳米粒的体外药物释放行为研究
分别取实施例1中一定体积的SLN溶液，置于透析袋(MWCO 3.5KDa)后放入装有25ml释放介质(pH 7.2PBS)的释放管中。在37℃、65rpm恒温震荡下进行体外释放，在特定时间点(0.5h，1h，2h，4h，6h，8h，12h，24h，36h，48h及72h)取样，同时更换全部释放介质。用荧光分光光度法测定样品中的药物浓度(Ex＝275nm，Em＝313nm，Slit＝5nm，工作电压＝700V)，计算药物的累计释放累积释放量及累计释放百分率。结果如图1所示。
实施例4：负载Gd-DTPA的固体脂质纳米粒的体外药效学考察
采用CCK-8检测试剂盒定量考察实施例1制备的固体脂质纳米粒(SLN-2(1)、SLN-2(2))对MCF-7细胞的生存率影响。首先，按照0.8-1×104个数/mL细胞液的细胞密度转96孔细胞板，置于37℃5％细胞培养箱里培养过夜。其次，配制一系列不同浓度的聚合物溶液，每孔加入聚合物溶液的体积是200μL，平行5个复孔，稀释介质是无酚红1640高糖培养基，所需考察聚合物的浓度依次为0，50，100，200，400，500，600，800，单位是μg/mL。接着，从培养箱中取出96孔细胞板，吸去培养液，每孔用100μL PBS缓冲溶液冲洗一次，再吸去磷酸盐缓冲溶液，将一系列不同浓度的聚合物溶液依次加入到细胞板中，置于37℃5％细胞培养箱里培养48小时。然后，从培养箱中取出96孔细胞板，每孔加入20μLCCK-8，再继续放入培养箱中孵育1-3h后取出细胞板，使用多功能酶标仪测定样品在450nm处的吸光度值。每个样品平行测试5次，取平均值作图(空白作为对照组)。细胞存活率(百分比)是以待测样的吸光度值相比正常细胞的吸光度值来表示。结果如图2所示，相比较于SLN-2-(1)，SLN-2-(2)对细胞毒性较小。
FITC标记SLN，Hoechst 33342染色细胞核。首先，按照5×104个/mL细胞液的细胞密度转24孔细胞板，置于37℃5％细胞培养箱里培养过夜。其次，将含有SLN不同浓度(50ug/ml、100ug/ml、150ug/ml、200ug/ml、300ug/ml)溶液于4h加至每孔并收集细胞，流式细胞计数仪比较实施例2制备的固体脂质纳米粒(SLN-2(1)、SLN-2(2))的摄取差异，结果如图3所示，二者细胞摄取差异并不明显。
实施例5：负载Gd-DTPA的固体脂质纳米粒的体外MRI检测
设置系列不同浓度(0.5mg/mL、1.0mg/mL、1.5mg/mL、2.0mg/mL、3.0mg/mL)实施例1制备的固体脂质纳米粒(SLN-2(1)、SLN-2(2))载有Gd-DTPA的SLN，置于温度20℃，行MR扫描，MR场强3.0T，T1WI序列(TR500，TE15)，得到的结果：SLN-2-(2)的T1弛豫率高于SLN-2-(1)如图4所示。
实施例6：负载Gd-DTPA的固体脂质纳米粒的体外药物释放行为研究
分别取专利号文件CN 106491906 B制备的SLN-1与实施例2制备的优选处方SLN-2-(2)溶液，置于透析袋(MWCO 3.5KDa)后放入装有25ml释放介质(pH 7.2PBS)的释放管中。在37℃、65rpm恒温震荡下进行体外释放，在特定时间点(0.5h，1h，2h，4h，6h，8h，12h，24h，36h，48h及72h)取样，同时更换全部释放介质。用荧光分光光度法测定样品中的药物浓度(Ex＝275nm，Em＝313nm，Slit＝5nm，工作电压＝700V)，计算药物的累计释放累积释放量及累计释放百分率。结果如图6所示。
实施例7：负载Gd-DTPA的固体脂质纳米粒的体外药效学考察
采用CCK-8检测试剂盒定量考察中国专利文献CN 106491906 B制备的SLN-1与实施例1的优选处方SLN-2-(2)对MCF-7细胞的生存率影响。首先，按照0.8-1×104个数/mL细胞液的细胞密度转96孔细胞板，置于37℃5％细胞培养箱里培养过夜。其次，配制一系列不同浓度的聚合物溶液，每孔加入聚合物溶液的体积是200μL，平行5个复孔，稀释介质是无酚红1640高糖培养基，所需考察聚合物的浓度依次为0，50， 100，200，400，500，600，800，单位是μg/mL。接着，从培养箱中取出96孔细胞板，吸去培养液，每孔用100μL PBS缓冲溶液冲洗一次，再吸去磷酸盐缓冲溶液，将一系列不同浓度的聚合物溶液依次加入到细胞板中，置于37℃5％细胞培养箱里培养48小时。然后，从培养箱中取出96孔细胞板，每孔加入20μLCCK-8，再继续放入培养箱中孵育1-3h后取出细胞板，使用多功能酶标仪测定样品在450nm处的吸光度值。每个样品平行测试5次，取平均值作图(空白作为对照组)。细胞存活率(百分比)是以待测样的吸光度值相比正常细胞的吸光度值来表示。结果如图7所示优选处方SLN-2-(2)的细胞毒性低于SLN-1的细胞毒性。
FITC标记SLN，Hoechst 33342染色细胞核。首先，按照5×104个/mL细胞液的细胞密度转24孔细胞板，置于37℃5％细胞培养箱里培养过夜。其次，分别按照不同时间点0.5h、1h、4h、8h、12h、24h加至每孔；以及不同浓度50ug/ml、100ug/ml、150ug/ml、200ug/ml、300ug/ml加至每孔并在摄取4h收集细胞，流式细胞计数仪比较SLN-1与SLN-2-(2)的摄取差异，结果如图8A、8B、9所示，载体从0.5h开始便有摄取，并且随着时间点的延长摄取随之增加；浓度依赖性摄取实验中对于不同浓度SLN-2-(2)的细胞摄取量多于SLN-1。

Claims (26)

  1. 一种固体脂质磁共振纳米粒子，包括磁共振成像造影剂、脂质材料、荧光染料,其特征在于，所述脂质材料中包含阳离子脂质材料，所述磁共振成像造影剂为水溶性的顺磁性对比剂。
  2. 如权利要求1所述的一种固体脂质磁共振纳米粒子，其特征在于，所述脂质材料在固体脂质磁共振纳米粒子中的含量占17.22％-56.47％，阳离子脂质材料在固体脂质磁共振纳米粒子中的含量占2.54％-70.27％。
  3. 如权利要求1所述的一种固体脂质磁共振纳米粒子，其特征在于，所述磁共振成像造影剂采用钆喷酸葡胺(Gd-DTPA)、在固体脂质磁共振纳米粒子中的含量10.78％-35.35％。
  4. 如权利要求1所述的一种固体脂质磁共振纳米粒子，其特征在于，所述荧光染料采用硬脂胺-异硫氰基荧光素(ODA-FITC)、在固体脂质磁共振纳米粒子中的含量占1.72％-5.65％。
  5. 如权利要求1所述的一种固体脂质磁共振纳米粒子，其特征在于，除阳离子脂质材料之外的脂质材料选择范围包括单硬脂酸甘油脂(单甘脂)、卵磷脂、甘油三酯。
  6. 如权利要求1所述的一种固体脂质磁共振纳米粒子，其特征在于，所述阳离子脂质材料的选择范围包括为十八胺(ODA)、氢化大豆卵磷脂(HSPC)、(2,3-二油酰基-丙基)三甲基氯化铵(DOTAP)和二棕榈酰磷脂酰胆碱(DPPC)、氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、溴化二甲基双十八烷基铵(DORI)、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵(DSRIE)、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺(DOGS)、脂质多聚-L-赖氨酸(LPLL)。
  7. 权利要求1、2、3、4、5或6任意一种固体脂质磁共振纳米粒子在MRI检测 领域中的应用。
  8. 一种固体脂质磁共振纳米粒子的制备方法，其特征在于，包括以下步骤：
    将第一种表面活性剂溶于水中得到水相，将第二种表面活性剂溶于有机溶剂中得到有机相，然后将水相加入到有机相中，超声处理得到微乳，备用；
    所述制备方法还包括以下步骤
    (1)将磁共振成像造影剂溶解于水中得到水溶液，将阳离子脂质材料溶解于乙醇中得到乙醇溶液，然后将水溶液和乙醇溶液复合后除去溶剂得到中间产物，所述磁共振成像造影剂为水溶性的顺磁性对比剂；
    (2)将步骤(1)得到的中间产物溶解到阳离子脂质材料之外的脂质材料的溶液中，在加热条件下，注入到备用的微乳液中，经过搅拌、离心和冷冻干燥得到固体脂质磁共振纳米粒子。
  9. 根据权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，所述的第一种表面活性剂与水的用量比为1.8～2.5g：100mL；第二种表面活性剂与有机溶剂的用量比为1.5～2.0g：100mL。
  10. 如权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，所述的有机溶剂为正戊烷、正己烷和环己烷中的至少一种。
  11. 如权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(2)中，冷冻干燥时加入海藻糖作为冻干保护剂。
  12. 如权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，制备固体脂质磁共振纳米粒子所采用的表面活性剂的选择范围包括吐温-80(Tween-80)、吐温-20(Tween-20)、司盘-80(Span-80)、十二烷基硫酸钠(SDS)、普朗尼克-68(F-68)、十六烷基三甲基溴化铵(CTMAB)。
  13. 如权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，其特征在于，步骤(1)中，所述的水溶液中磁共振成像造影剂的质量百分比浓度为5％～8％；所述的乙醇溶液中阳离子脂质材料的质量百分比浓度为0.45～0.7％，复合的温 度为55～65℃，复合的时间为30～60min。
  14. 如权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(1)中，所述阳离子脂质材料之外的脂质材料选用单硬脂酸甘油酯、卵磷脂、硬脂酸、三硬脂酸甘油酯中的一种或多种组合；
  15. 根据权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(1)中，所述阳离子脂质材料之外的脂质材料由重量比为85～95：5～15的单硬脂酸甘油酯和卵磷脂组成。
  16. 根据权利要求8所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，所述阳离子脂质材料的选择范围包括为十八胺(ODA)、氢化大豆卵磷脂(HSPC)、(2,3-二油酰基-丙基)三甲基氯化铵(DOTAP)和二棕榈酰磷脂酰胆碱(DPPC)、氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、溴化二甲基双十八烷基铵(DORI)、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵(DSRIE)、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺(DOGS)、脂质多聚-L-赖氨酸(LPLL)。
  17. 一种固体脂质磁共振纳米粒子的制备方法，其特征在于，包括以下步骤：
    (1)将第一种表面活性剂溶于水中得到水相，将第二种表面活性剂溶于有机溶剂中得到有机相，然后将水相加入到有机相中，超声处理得到微乳，第一种表面活性剂为吐温-80，第二种表面活性剂为司盘-80；
    (2)将钆喷酸葡胺溶解于水中得到水溶液，将十八胺溶解于乙醇中得到乙醇溶液，然后将水溶液和乙醇溶液复合后除去溶剂得到中间产物；
    (3)将步骤(2)得到的中间产物分散到含有脂质材料的溶液中，然后在加热条件下，注入步骤(1)得到的微乳液中，经过搅拌、离心和冷冻干燥得到所述的固体脂质磁共振纳米粒子。
  18. 根据权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(1)中，所述的第一种表面活性剂与水的用量比为1.8～2.5g：100mL；第二种表面活性剂与有机溶剂的用量比为1.5～2.0g：100mL。
  19. 如权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(1)中，所述的有机溶剂为正戊烷、正己烷和环己烷中的至少一种。
  20. 根据权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(2)中，所述的水溶液中钆喷酸葡胺的质量百分比浓度为5％～8％；所述的乙醇溶液中十八胺的质量百分比浓度为0.45～0.7％；复合的温度为55～65℃，复合的时间为30～60min。
  21. 根据权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(3)中，所述的脂质材料选用单硬脂酸甘油酯、卵磷脂、硬脂酸、三硬脂酸甘油酯中的一种或多种组合；负载钆喷酸葡胺的载药量为4％～35％。
  22. 根据权利要求21所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(3)中，所述的脂质材料由重量比为85～95：5～15的单硬脂酸甘油酯和卵磷脂组成。
  23. 根据权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(3)中，用于溶解脂质材料的溶剂为正戊烷、正己烷和环己烷中的至少一种。
  24. 根据权利要求17所述的一种固体脂质磁共振纳米粒子的制备方法，其特征在于，步骤(3)中，冷冻干燥时加入海藻糖作为冻干保护剂。
  25. 一种固体脂质磁共振纳米粒子，其特征在于，由权利要求17～24任一项所述的制备方法制备得到。
  26. 一种如权利要求25所述的固体脂质磁共振纳米粒子在MRI检测领域中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101496906A (zh) * 2009-02-19 2009-08-05 浙江大学 含磁共振对比剂的脂质纳米粒及其制备方法和应用
CN101637612A (zh) * 2008-07-31 2010-02-03 上海交通大学医学院附属仁济医院 呼吸道磁共振喷雾造影剂及其制备方法
CN103990151A (zh) * 2014-06-09 2014-08-20 北京大学 一种磁共振成像造影剂及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2046293A2 (en) * 2006-07-10 2009-04-15 Medigene AG Use of a cationic colloidal preparation for the diagnosis and treatment of ocular diseases
CN101129336B (zh) * 2007-07-24 2010-04-14 浙江大学 一种脂质纳米粒的制备方法
WO2009018332A1 (en) * 2007-08-01 2009-02-05 Alnylam Pharmaceuticals, Inc. Single-stranded and double-stranded oligonucleotides comprising a metal-chelating ligand
CN102078624B (zh) * 2010-12-23 2012-07-25 华东理工大学 一种高效载钆脂质体制剂及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101637612A (zh) * 2008-07-31 2010-02-03 上海交通大学医学院附属仁济医院 呼吸道磁共振喷雾造影剂及其制备方法
CN101496906A (zh) * 2009-02-19 2009-08-05 浙江大学 含磁共振对比剂的脂质纳米粒及其制备方法和应用
CN103990151A (zh) * 2014-06-09 2014-08-20 北京大学 一种磁共振成像造影剂及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, ZHIJIN: "Preliminary Study on Cationic Solid Lipid Nanoparticles/Gd-DTPA Complex as Molecular Imaging Agent", CHINESE JOURNAL OF NEW DRUGS, vol. 18, no. 15, 15 August 2009 (2009-08-15), pages 1443 - 1447, XP009157979, ISSN: 1003-3734 *
SUN, JIHONG: "Development of Solid Lipid Nanoparticle-Based Magnetic Resonance Colonography", MEDICINE & PUBLIC HEALTH, CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 October 2009 (2009-10-15), pages E060 - 6, ISSN: 1674-022X *

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