CN111920768A - Entrapped molecular targeted drug liposome and application thereof in preparation of tumor treatment drug - Google Patents
Entrapped molecular targeted drug liposome and application thereof in preparation of tumor treatment drug Download PDFInfo
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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Abstract
The invention belongs to the field of biological medicines, and relates to a liposome for encapsulating a molecular targeted drug and application thereof in preparing a drug for treating tumor. The molecular targeted drug liposome of the invention is composed of molecular targeted drugs, liposome components such as phospholipid, cholesterol, mPEG-DSPE and/or targeted molecule modified PEG-DSPE. The invention is prepared by loading the molecular targeted drug into the liposome internal water phase or phospholipid bilayer in an active or passive drug loading mode, can realize the targeted drug delivery to the tumor, and obviously enhances the anti-tumor effect, especially the anti-brain tumor effect of the molecular targeted drug. The molecular targeted drug liposome is further combined with pharmaceutically acceptable medicinal components and/or diluents to prepare various dosage forms for targeted therapy of tumors, and has potential clinical application value.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a liposome for encapsulating a molecular targeted drug and application thereof in preparing a drug for treating tumor.
Background
The prior art discloses that the molecular targeted drug mainly aims at the key target of the generation and development of malignant tumor for therapeutic intervention, and has better tolerance and small toxic reaction because the molecular targeted drug can selectively kill tumor cells, thereby having more advantages compared with the traditional cytotoxic antitumor drugs. Some molecular targeted drugs have been reported to show better efficacy in the corresponding tumor treatment. Molecularly targeted drugs can be divided into single-target inhibitors and multi-target inhibitors, and currently known single-target inhibitors include BCr-Abl kinase inhibitors (e.g., imatinib, dasatinib, ponatinib, etc.), Epidermal Growth Factor (EGFR) inhibitors (e.g., gefitinib, erlotinib, ocitinib, etc.), Vascular Endothelial Growth Factor (VEGF) inhibitors (e.g., apacept, etc.), Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitors (e.g., apatinib, furoquintinib, tositunib, cediranib, etc.), cyclin-dependent kinase 4/6(CDK4/6) inhibitors (e.g., paucib, ribociclovir, abemaciclib, etc.), Poly ADP Ribose Polymerase (PARP) inhibitors (e.g., tarazol panib, olaparib, inipaib, nilaparili, etc.), Hedgehog inhibitors (e.g., virgine, guid, daismo, etc.), and multi-target inhibitors, Rapamycin target protein (mTOR) inhibitors (e.g., everolimus, AZD8055, AZD2014, temsirolimus, SCC-31, etc.), and the like. Known multi-target inhibitors such as AL3180, sunitinib, regoratinib, sorafenib, vandetanib, cabozantinib, lenvatinib, pazopanib, vemurafenib, axitinib, dabrafenib, rilivanib, nintedanib, etc.; research reports that the bioavailability of most molecular targeted drugs is influenced due to poor solubility of the drugs, or the therapeutic effect on brain tumors and partial solid tumors is influenced due to the difficulty in penetrating biological barriers (such as blood-brain barrier BBB, blood-tumor barrier BBTB, and the like).
Liposomes (lipopomes) are small vesicles of phospholipid bilayers with similar biological membrane structures and are prepared from materials such as phospholipid, cholesterol and the like. The liposome can embed the drug in a phospholipid bilayer or an internal water phase of the liposome, so as to achieve the purposes of improving the solubility of the drug, protecting active groups of the drug, prolonging the half-life period of the drug, improving the therapeutic index of the drug, reducing the toxic and side effects of the drug and the like. In addition, targeted molecular modification of liposomes can provide active targeted drug delivery capabilities to liposomes, such as: the liposome modified by the tumor targeting molecules can realize the targeted drug delivery to tumor cells, thereby enhancing the anti-tumor curative effect of the drug and simultaneously reducing the toxic and side effects to normal tissues; this active targeting strategy is particularly important for the treatment of brain tumors. Clinical practice shows that brain tumors are in situ brain tumors (such as brain glioma) and metastatic brain tumors (such as brain metastatic tumors of lung cancer and breast cancer), are one of the most challenging malignant tumors, and because BBB and BBTB exist in the brain tumors, most medicaments are difficult to permeate, so that the drug effect of the brain tumors is greatly weakened; other studies show that liposome modified by BBB or/and BBTB targeting molecules entrap anti-tumor drugs such as adriamycin, paclitaxel and the like for treating brain glioma, and have shown significant advantages.
The liposome has wide application field, relates to medicines, gene therapy, medical materials, vaccines, biological agents, pesticides, cosmetics, health care products and the like, and has wide market prospect. At present, there are many liposome drugs which have been approved to be on the market at home and abroad, such as doxorubicin hydrochloride liposome injection (Doxil or Caelyx), irinotecan hydrochloride liposome injection (Onivyde), amphotericin B liposome (ambiome), paclitaxel liposome (lipocalin), bupivacaine liposome (Exparel), vincristine liposome (Marqibo), daunorubicin liposome (Daunoxome) and the like.
Based on the foundation and the current situation of the prior art, in view of the successful application of liposome technology in other medicines, the inventor of the application intends to prepare a molecular targeted medicine into a liposome preparation to help the molecular targeted medicine to expand clinical indications and improve curative effect, and particularly provides a liposome encapsulating the molecular targeted medicine and application thereof in preparing a medicine for treating tumors.
Disclosure of Invention
The invention aims to provide a liposome for encapsulating a molecular targeted drug and application thereof in preparing a drug for treating tumor based on the foundation and the current situation of the prior art. In the application, the molecular targeted drug is prepared into the liposome preparation, so that the assisted molecular targeted drug develops clinical indications and improves curative effect.
The invention comprises the following steps: 1) the prepared molecular targeted drug liposome has good solubility and/or sustained release effect under simulated physiological conditions; 2) the prepared molecular targeted drug liposome realizes the targeted drug delivery to the tumor, and obviously enhances the effect of the molecular targeted drug on resisting the tumor, especially the brain tumor; experiments prove that the molecular targeted drug liposome has better sustained and controlled release performance and tumor targeted treatment effect compared with free molecular targeted drugs.
The purpose of the invention is realized by the following specific technical scheme:
the invention discloses a liposome carrying a molecular targeted drug, which is an anti-tumor molecular targeted drug.
Preferably, the anti-tumor molecule targeted drug is a hydrophilic molecule targeted drug or a hydrophobic molecule targeted drug.
Preferably, the hydrophilic or hydrophobic anti-tumor molecule targeting drug may be one or any combination of imatinib, dasatinib, ponatinib, gefitinib, erlotinib, oxitinib, apacept, apatinib, furotinib, tositunib, cediranib, palbociclib, rabecinib, abemaciclib, tarazolabib, olaparib, Iniparib, nilapari, nilapali, vismodegib, sondega, Daurismo, everolimus, AZD8055, AZD2014, temsirolimus, AL3810, SCC-31, sunitinib, regoratinib, sorafenib, delavatinib, cabozantinib, lenvatinib, pazopanib, vemurafenib, axitinib, rilatinib, rilivatinib, and danivatinib.
It is to be understood that the molecular targeted drug of the present invention is not limited to the above-listed molecular targeted drugs, and those skilled in the art can select any suitable molecular targeted drug as needed to complete the present invention and fall within the scope of the present invention.
The second aspect of the invention discloses a basic prescription and a preparation method of a liposome carrying a molecular targeted medicament, wherein the liposome is a tumor active targeted liposome or a tumor passive targeted liposome.
Preferably, the targeted or non-targeted liposomes are composed of phospholipids, cholesterol, methoxypolyethylene glycol-distearoylethanolamine (mPEG-DSPE) and/or targeting molecule-modified polyethylene glycol-distearoylethanolamine (PEG-DSPE).
Preferably, the targeting molecule modified PEG-DSPE, the targeting molecule for modifying PEG-DSPE is a small molecule compound, such as: folic acid, p-hydroxybenzoic acid (pHA) and its derivatives, fatty acids, wherein the fatty acid is preferably myristic acid (MC).
Preferably, the targeting molecule modified PEG-DSPE is a polypeptide molecule or a protein molecule, wherein the polypeptide molecule is: VAP, cVAP,SVAP、DVAP，pHA-VAP、pHA-SVAP and pHA-DVAP，MC-VAP、MC-SVAP and MC-DVAP，D8、D8-VAP、D8-SVAP and D8-DVAP，WSW、DWSW、WSW-VAP、DWSW-SVAP andDWSW-DVAP，TGN、DTGN、TGN-VAP、DTGN-SVAP andDTGN-DVAP；A7R、cA7R、DA7R, pHA-A7R and pHA-DA7R, MC-A7R and MC-DA7R, D8-A7R and D8-DA7R, WSW-A7R andDWSW-DA7R, TGN-A7R andDTGN-DA7R; RGD polypeptide, staged-RGD polypeptide, pHA-RGD polypeptide, MC-RGD polypeptide, D8-RGD polypeptide, WSW-RGD polypeptide, and TGN-RGD polypeptide; RW, mn, pHA-RW and pHA-mn, MC-RW and MC-mn, D8-RW and D8-mn, WSW-RW andDWSW-mn, TGN-RW andDTGN-mn; t7 andDt7; RAP12 andD RAP 12. Wherein the protein molecule is transferrin, lactoferrin, etc., and one or any combination of the polypeptide molecule or the protein molecule can be used.
Table 1 is a sequence table of the polypeptide amino acids of the present invention.
TABLE 1
Preferably, the preparation method of the molecular-targeted drug-loaded liposome is a film dispersion method or an ethanol injection method.
Preferably, the drug entrapment method of the molecular targeted drug-loaded liposome is active drug loading or passive drug loading.
It should be understood that the basic formulation of the liposome of the present invention is not limited to phospholipid, cholesterol, mPEG-DSPE and/or PEG-DSPE modified with targeting molecules, and the targeting molecules of the modified PEG-DSPE of the present invention are not limited to the above-mentioned small molecule compounds, polypeptide molecules and protein molecules, and those skilled in the art can select any suitable formulation and suitable ratio of liposome to complete the present invention as required, and all that fall within the protection scope of the present invention.
The third aspect of the invention discloses the application of the molecular targeted drug liposome in the preparation of antitumor drugs.
The molecular targeted drug liposome can be further combined with other medicinal components and/or diluents to form a drug combination to prepare various preparations for targeted therapy of tumors, and preferably, the preparation is an injection.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
the molecular targeted drug liposome disclosed by the invention can increase the solubility and/or sustained and controlled release performance of hydrophilic or hydrophobic molecular targeted drugs under physiological conditions, can meet the requirements of liquid preparations, improves the flexibility of the design and administration mode of the molecular targeted drug dosage form, and compared with free drugs, the molecular targeted drug liposome modified by targeted polypeptide can realize the targeted drug delivery to tumors, particularly the targeted drug delivery to tumors penetrating biological barrier membranes, obviously enhances the effect of the targeted drug on treating the tumors, further expands the clinical indications of the molecular targeted drugs, and better exerts the medicinal value, thereby providing a basis for the design and future clinical application of a liposome drug delivery system composition based on the molecular targeted drugs.
Description of the figures
FIG. 1: Liposome/SCC-31 andDparticle size, PDI, entrapment efficiency, drug loading capacity and TEM (transmission electron microscope) image of VAP-liposome/SCC-31.
FIG. 2: Liposome/SCC-31 andDin vitro release profile of VAP-liposome/SCC-31.
FIG. 3: Liposome/SCC-31 andDin vitro U87 cell inhibitory effect of VAP-liposome/SCC-31.
FIG. 4: Liposome/SCC-31 andDin vivo efficacy test survival curves for VAP-liposomes/SCC-31
FIG. 5: Liposome/SCC-31 andDVAP-liposome/SCC-31 in vivo efficacy test body weight change curve
FIG. 6: particle size characterization of each loaded AL3810 liposome.
FIG. 7: characterization of particle size stability at 37 ℃ for each loaded AL3810 liposome.
FIG. 8: characterization of particle size stability of each loaded AL3810 liposome in 50% rat serum at 37 ℃.
FIG. 9: in vitro release assay of each AL 3810-loaded liposome in PBS buffer.
FIG. 10: in vitro release assay of each loaded AL3810 liposome in 30% rat serum.
FIG. 11: HUVEC cell uptake assay in vitro with each AL 3810-loaded liposome.
FIG. 12: in vitro U87 cytostatic assays each loaded with AL3810 liposomes.
Table 1: polypeptide amino acid sequence table.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1 active drug Loading method for preparation of liposomes/SCC-31 and characterization
The embodiment discloses a method for preparing a liposome/molecular targeted drug SCC-31 by an active drug loading method and characterization, and the method specifically comprises the following steps:
the membrane material of liposome/SCC-31 was formulated as HSPC/Chol/mPEG2000-DSPE (50:45:5, molar ratio). Respectively weighing the membrane materials, dissolving in chloroform, performing reduced pressure rotary evaporation to remove organic solvent to obtain uniform lipid membrane, and vacuum drying for 24 h. Adding a certain volume of 0.2M citric acid solution, and shaking in water bath at 60 ℃ for 2h to obtain liposome suspension. And (3) carrying out ultrasonic treatment on the cell by an ultrasonic cell crusher for 10min (80w, 2s ultrasonic treatment and 1s interval) to obtain the blank liposome. Eluting with normal saline, and passing through Sephadex G-50 gel column to replace blank liposome external water phase. Adding SCC-31 free base solid powder according to the medicine-fat ratio of 1:5 (molar ratio), and carrying out water bath at 55 ℃ for 1 h. And (3) eluting with normal saline through a Sephadex G-50 gel column to remove free drugs to obtain the SCC-31 liposome. The particle size of the liposome/SCC-31 is 112.6nm and the PDI is 0.141 as determined by a Malvern laser scattering particle sizer, and the morphology of the liposome under a transmission electron microscope is round (the particle size and the electron microscope results are shown in figure 1). The entrapment rate of the liposome is 97.04% measured by HPLC, the drug loading rate is 11.27%, the concentration of the drug contained in the liposome suspension is 4.12mg/mL, and the solubility of the free base of the SCC-31 in 0.9% physiological saline is only about 0.15mg/mL measured at the same time, which proves that the solubility of the SCC-31 in the physiological saline can be remarkably increased by the liposome SCC-31 prepared by the active drug loading method. The results of in vitro release of the liposome/SCC-31 in 50% rat serum by the dialysis bag method are shown in figure 2, which proves that the liposome/SCC-31 prepared by the active drug loading method has slow release characteristics.
This example discloses active drug-loading methodDThe VAP-liposome/molecular targeted drug SCC-31 and the characterization thereof specifically comprise the following steps:
Dthe VAP-liposome/SCC-31 membrane material is formulated as HSPC/Chol/mPEG2000-DSPE/DVAP-PEG3400DSPE (50:45:3:2, molar ratio). Respectively weighing the membrane materials, dissolving in chloroform, performing reduced pressure rotary evaporation to remove organic solvent to obtain uniform lipid membrane, and vacuum drying for 24 h. Adding a certain volume of 0.2M citric acid solution, and shaking in a water bath at 60 ℃ for 2h to obtain liposome suspension. And (3) carrying out ultrasonic treatment on the cell by an ultrasonic cell crusher for 10min (80w, 2s ultrasonic treatment and 1s interval) to obtain the blank liposome. Eluting with normal saline, and passing through Sephadex G-50 gel column to replace blank liposome external water phase. Adding SCC-31 free base solid powder according to the medicine-fat ratio of 1:5 (molar ratio), and carrying out water bath at 55 ℃ for 1 h. Eluting with normal saline solution through Sephadex G-50 gel column to remove free drug to obtainDVAP-Liposome SCC-31. Determination by Malvern laser scattering particle sizerDThe particle size of the VAP-liposome SCC-31 is 122.2nm, the PDI is 0.090, the shape of the liposome under a transmission electron microscope is round (the particle size and the electron microscope result are shown in figure 1), the entrapment rate of the liposome is 95.32% and the drug loading rate is 10.43% as measured by HPLC, the concentration of the drug contained in the liposome suspension is 3.68mg/mL, and the solubility of SCC-31 free alkali in 0.9% physiological saline is only about 0.15mg/mL, which proves that the active drug loading method is used for preparing the drug-loaded liposomeDVAP-liposome SCC-31 can significantly increase the solubility of SCC-31 in physiological saline. Examination by dialysis bag methodDThe in vitro release result of VAP-liposome SCC-31 in 50% rat serum is shown in figure 2, which proves that the drug prepared by the active drug loading methodDVAP-liposome SCC-31 has a slow release characteristic.
Example 3 preparation of liposomes/SCC-31 by Passive drug Loading and characterization
The embodiment discloses a method for preparing a liposome/molecular targeted drug SCC-31 by a passive drug loading method and characterization, and the method specifically comprises the following steps:
the membrane material of the liposome/SCC-31 is formulated as HSPC/Chol/mPEG2000DSPE (50:45:5, molar ratio)) The membrane materials are respectively weighed and dissolved in ethanol, and the solution is dropwise added into 0.9 percent of physiological saline under the condition of magnetic stirring. And (3) carrying out ultrasonic treatment on the cell by an ultrasonic cell crusher for 10min (80w, 2s ultrasonic treatment and 1s interval) to obtain the liposome/SCC-31. The particle size of the liposome/SCC-31 was 124.27nm and the PDI was 0.201 as determined by a Malvern laser scattering particle sizer. The entrapment rate of the liposome is 85.56% measured by HPLC, the drug loading rate is 8.91%, the concentration of the drug contained in the liposome suspension is 2.15mg/mL, and the solubility of the free base of the SCC-31 in 0.9% physiological saline is only about 0.15mg/mL measured at the same time, which proves that the liposome/SCC-31 prepared by the passive drug loading method can obviously increase the solubility of the SCC-31 in the physiological saline. The dialysis bag method inspects that the SCC-31 is completely released after the liposome/SCC-31 is released in 50% of rat serum in vitro for 4h, and proves that the liposome/SCC-31 prepared by the passive drug loading method releases drug rapidly in a simulated in vivo environment.
Examples 4,DIn vitro anti-glioma assay for VAP-liposomes/SCC-31
Prepared by active drug loading method for MTT method investigationDInhibition of U87 cells in vitro by VAP-liposomes/SCC-31. Taking U87 cells in logarithmic growth phase, digesting and preparing to density of 2 × 104U87 single cell suspension per mL and seeded in 96-well plates at 100. mu.L per well volume, transferred into CO2Incubator (37 ℃, 5% CO)2Saturated humidity) for 24h, discarding the original culture medium, and replacing with 200 μ L of culture medium containing or not containing drug. The test groups had 5 groups: 1) normal saline, 2) free SCC-31, 3) liposomes/SCC-31, 4)DVAP-liposome/SCC-31, 5) drug-free medium. Each formulation was HPLC-quantified and diluted to the same concentration (2mg/mL) as SCC-31, re-filtered in a clean bench, and the medium diluted to 9 doses of each formulation, with a maximum concentration of 200. mu.g/mL (350. mu. mol), and the remaining concentrations were diluted 5-fold in sequence, 3 duplicate wells per concentration. 37 ℃ and 5% CO2After incubation for 4h in the incubator, the drug-containing culture solution is discarded from each 4h drug administration group, PBS is washed for three times, and new drug-free culture medium is added for continuous culture for 72 h. And after 72h, adding 20 mu L of 5mg/mL MTT solution into each well, continuously incubating for 4h at 37 ℃, discarding the supernatant, adding 150 mu L of LDMSO into each well, shaking for 10min at 37 ℃ in an air shaking table, and measuring the OD value of each well at the wavelength of 570nm of a microplate reader. Test repetition3 times. Cell viability was calculated as follows: cell survival (%) ═ (OD)s-ODblank)/(ODcontrol-ODblank) X 100%. In the formula: ODsIs the absorbance, OD, of the administered groupcontrolAbsorbance, OD, of blank controlblankAbsorbance in blank wells. Growth curves were plotted by Graph PadPrism 7.0 software for various drug concentrations with respect to cell viability and IC50 was calculated (as shown in figure 3), indicating that,DVAP-liposome/SCC-31 can exert better killing effect of U87 cells in vitro than free drugs and non-target liposomes.
Examples 5,DVAP-Liposome/SCC-31 in vivo anti-glioma assay
(1) In situ glioma model mouse construction
Collecting U87 cells in logarithmic growth phase, digesting, counting, suspending with appropriate amount of PBS buffer solution, inoculating 5 × 10 cells to each nude mouse5Individual cells (5 μ L volume). A micro-injector is fixed on a brain stereotaxic apparatus, sterilized by 75 percent ethanol, rinsed by sterile PBS and absorbed with 5 mu LU87 cell suspension for later use. The mouse is anesthetized, fixed in a brain stereotaxic apparatus, a sterilized surgical instrument dissects the top skin of the head of the mouse, and the scalp is digested by 10 percent hydrogen peroxide to expose white cross-shaped bregma. The micro-syringe needle is aligned to the center of bregma and is used as the origin, the position of the micro-syringe needle is adjusted by the brain stereotaxic apparatus, the micro-syringe needle moves to the right by 1.8mm and moves up by 0.6mm, namely the position which is opposite to the striatum of the mouse. The three-edged needle is used for drilling a hole in the skull part opposite to the needle head, the micro-injector is downwards adjusted through the brain stereotaxic apparatus, the counting is started when the needle head of the injector enters the small hole of the skull, and the downward adjustment of the needle head is 4mm, and the upward adjustment of the needle head is 1 mm. Slowly injecting the cells within 1min, waiting for 3min, slowly lifting up for 1mm within 1min, waiting for 2min, completely lifting out the needle after 2min, sealing bone wax, performing surgical suture, continuously culturing the mice after operation in an SPF environment, observing the state of the mice every day, and performing related experiments after the state is stable.
(2) In situ glioma resistance drug effect test
Mice after tumor inoculation are randomly divided into 4 groups (9 mice/group), administration is started on day 7 (the dose of each group is 24 mg/kg/time in SCC-31), administration is carried out every other day for 16 times, and each administration is carried outThereafter, the mice were weighed, and the survival time of the mice was recorded, and a survival curve was drawn. The specific grouping is as follows: 1) normal saline, 2) free SCC-31, 3) liposomes/SCC-31, 4)DVAP-liposome/SCC-31. Wherein the liposome is prepared by an active drug loading method. The results of the survival time and body weight of the mice are shown in fig. 4 and 5, and the results show that,DVAP-liposome/SCC-31 can significantly enhance the in vivo anti-glioma effect of SCC-31.
Example 6 active drug delivery method for preparing liposomes/AL 3810 and characterization
The embodiment also discloses a method for preparing a liposome/molecular targeted medicament AL3810 by an active medicament carrying method and a characterization method, and the method specifically comprises the following steps:
the formulation of liposome/AL 3810 membrane material is HSPC/Chol/mPEG2000-DSPE (52:43:5, molar ratio). Weighing the membrane material, dissolving in chloroform, performing reduced pressure rotary evaporation to remove the chloroform, and vacuum drying the obtained uniform lipid membrane overnight. Adding 0.32M ammonium sulfate solution, hydrating at 63 deg.C for 1h, ultrasonic treating with probe ultrasonic instrument, and passing through G50 gel column with physiological saline. Adding citric acid solution of AL3810, adjusting pH to about 6.1 with 1M disodium hydrogen phosphate, incubating in shaking table in water bath at 63 deg.C for 2 hr, separating with normal saline as eluent through Sephadex G-50 column to remove free drug, and removing citric acid solution to obtain AL3810 liposome. The particle size of the liposome/AL 3810 measured by a Malvern laser scattering particle size analyzer is about 110nm and is uniformly distributed (the particle size result is shown in figure 6A), the stability of the liposome is good at 37 ℃ (shown in figure 7), the particle size and PDI of the liposome/AL 3810 do not change significantly even if the liposome/AL 3810 is incubated with equal volume of rat serum at 37 ℃ (shown in figure 8), the in-vitro release results of the liposome/AL 3810 in pH 7.4PBS buffer solution and 30% of rat serum simulating physiological conditions are respectively shown in figures 9 and 10 by a dialysis bag method, and the liposome/AL 3810 prepared by the active drug loading method is proved to have slow release characteristics.
Example 7 active drug delivery method preparation of c (RGDyK) -Liposome/AL 3810 and characterization
The embodiment also discloses a method for preparing c (RGDyK) -liposome/molecular targeted medicament AL3810 by an active medicament loading method and a characterization method, and the method specifically comprises the following steps:
the prescription of the (RGDyK) -liposome/AL 3810 membrane material is HSPC/Chol/mPEG2000-DSPE/c (RGDyK) -PEG3400-DSPE (52:43:3:2, molar ratio). Weighing the membrane material, dissolving in chloroform, performing reduced pressure rotary evaporation to remove the chloroform, and vacuum drying the obtained uniform lipid membrane overnight. Adding 0.32M ammonium sulfate solution, hydrating at 63 deg.C for 1h, ultrasonic treating with probe ultrasonic instrument, and passing through G50 gel column with physiological saline. Adding citric acid solution of AL3810, adjusting pH to about 6.1 with 1M disodium hydrogen phosphate, incubating in water bath at 63 deg.C for 2h, separating with dextran gel G-50 column using physiological saline as eluent to remove free drug, and removing citric acid solution to obtain c (RGDyK) -liposome/AL 3810. The particle size of c (RGDyK) -liposome/AL 3810 is about 110nm and is uniformly distributed (the particle size result is shown in figure 6B) measured by a Malvern laser scattering particle sizer, the liposome stability is good under the condition of 37 ℃ (shown in figure 7), the particle size and PDI of c (RGDyK) -liposome/AL 3810 are not significantly changed even if the c (RGDyK) -liposome/AL 3810 is incubated with equal volume of rat serum at 37 ℃ (shown in figure 8), the in-vitro release results of c (RGDyK) -liposome/AL 3810 in PBS buffer solution with the pH of 7.4 and 30% rat serum simulating physiological conditions are respectively examined by a dialysis bag method and are shown in figures 9 and 10, and the c (RGDyK) -liposome/AL 3810 prepared by the active drug loading method is proved to have the slow-release characteristic.
Example 8 active drug Loading RW-Liposome/AL 3810 preparation and characterization
The embodiment also discloses an active drug loading method for preparing RW-liposome/molecular targeted drug AL3810 and characterization, and the method specifically comprises the following steps:
the RW-liposome/AL 3810 membrane material was formulated as HSPC/Chol/mPEG2000-DSPE/RW-PEG3400-DSPE (52:43:3:2, molar ratio). Weighing the membrane material, dissolving in chloroform, performing reduced pressure rotary evaporation to remove the chloroform, and vacuum drying the obtained uniform lipid membrane overnight. Adding 0.32M ammonium sulfate solution, hydrating at 63 deg.C for 1h, ultrasonic treating with probe ultrasonic instrument, and passing through G50 gel column with physiological saline. Adding citric acid solution of AL3810, adjusting pH to about 6.1 with 1M disodium hydrogen phosphate, incubating in shaking bath at 63 deg.C for 2 hr, separating with dextran gel G-50 column using physiological saline as eluent to remove free drug, and removing citric acid solution to obtain RW-liposome/AL 3810. The particle size of RW-liposomes/AL 3810 was approximately 110nm and was uniformly distributed as measured by malvern laser scattering particle sizer (particle size results are shown in fig. 6C), liposome stability was good at 37 ℃ (shown in fig. 7), and there was no significant change in particle size and PDI even when RW-liposomes/AL 3810 were co-incubated with equal volume of rat serum at 37 ℃ (shown in fig. 8). The results of the dialysis bag method for examining the in vitro release of RW-liposome/AL 3810 in PBS buffer solution with pH 7.4 and 30% rat serum simulating physiological conditions are respectively shown in fig. 9 and fig. 10, and the RW-liposome/AL 3810 prepared by the active drug loading method is proved to have slow release characteristics.
Example 9 active drug Loading preparation of mn-liposomes/AL 3810 and characterization
The embodiment also discloses an active drug loading method for preparing an mn-liposome/molecular targeted drug AL3810 and characterization, and the method specifically comprises the following steps:
the prescription of mn-liposome/AL 3810 membrane material is HSPC/Chol/mPEG2000-DSPE/mn-PEG3400-DSPE (52:43:3:2, molar ratio). Weighing the membrane material, dissolving in chloroform, performing reduced pressure rotary evaporation to remove the chloroform, and vacuum drying the obtained uniform lipid membrane overnight. Adding 0.32M ammonium sulfate solution, hydrating at 63 deg.C for 1h, ultrasonic treating with probe ultrasonic instrument, and passing through G50 gel column with physiological saline. Adding citric acid solution of AL3810, adjusting pH to about 6.1 with 1M disodium hydrogen phosphate, incubating in water bath at 63 deg.C for 2 hr, separating with dextran gel G-50 column using physiological saline as eluent to remove free drug, and removing citric acid solution to obtain mn-liposome/AL 3810. The particle size of mn-liposome/AL 3810 measured by a Malvern laser scattering particle size analyzer is about 110nm and is uniformly distributed (the particle size result is shown in figure 6D), the liposome stability at 37 ℃ is good (shown in figure 7), the particle size and PDI of mn-liposome/AL 3810 are not significantly changed even if the mn-liposome/AL 3810 is incubated with equal volume of rat serum at 37 ℃, the in-vitro release results of mn-liposome/AL 3810 in pH 7.4PBS buffer and 30% of rat serum simulating physiological conditions are respectively shown in figures 9 and 10 by a dialysis bag method, and the mn-liposome/AL 3810 prepared by the active drug loading method is proved to have slow release characteristics.
Example 10 in vitro targeting and in vitro inhibition of brain glioma cell growth assays of entrapped AL3810 liposomes (1) HUVEC uptake of AL3810 liposomes
Culturing and preparing at density of 2 × 105HUVEC of one/mLSingle cell suspension was inoculated into 12-well plates, 1mL per well volume, and CO was transferred2Incubator (37 ℃, 5% CO)2Saturated humidity) for 24h, discarding the original culture medium, and respectively replacing with 1mL of the drug-containing culture medium for cell culture. The experimental groups included 4 groups (n ═ 3): 1) LS/AL3810, 2) RW-LS/AL3810, 3) mn-LS/AL3810, 4) c (RGDyK) -LS/AL 3810. Each formulation was HPLC quantified, filtered, and diluted with media to dose 180 μ g/mL of AL3810, 1mL per well. 37 ℃ and 5% CO2After incubation for 4h in the incubator, the culture solution containing the drug is discarded, PBS is washed for three times, and cells in each hole are collected by trypsinization. Centrifuging at 1000rpm, discarding supernatant, diluting the cells in each well to 1mL, and counting the cells with a cell counter to obtain the number of cells in each well (N)cell) The cells were centrifuged again at 1000rpm, the supernatant was discarded, and the volume of the cells was adjusted to 0.1 mL. Ultrasonically crushing cells in a water bath, adding 0.2mL of acetonitrile to precipitate proteins in the cells, centrifuging at 12000rpm for 15min, and taking supernatant liquid for HPLC sample injection to detect the concentration C of the medicament. Every 10 th4The cellular drug uptake was calculated as: cellular drug uptake/104Each (ng) ═ C × 3 × 0.1)/(Ncell×104). The results (as shown in FIG. 11) show that target liposomes RW-LS/AL3810, mn-LS/AL3810 and c (RGDyK) -LS/AL3810 are well taken up by HUVEC, and the intracellular drug concentration is obviously higher than that of the non-target group LS/AL3810, and the significant difference exists.
(2) Inhibition of U87 cell growth by AL3810 liposomes
Culturing and preparing at density of 2 × 104U87 single cell suspension per mL and seeded in 96-well plates at 100. mu.L per well volume, transferred into CO2Incubator (37 ℃, 5% CO)2Saturated humidity) for 24h, and then adding 100 mu L of culture medium containing or not containing medicine for cell culture. Experimental group 4 (n ═ 3): 1) LS/AL3810, 2) RW-LS/AL3810, 3) mn-LS/AL3810, 4) c (RGDyK) -LS/AL 3810. HPLC (high performance liquid chromatography) quantification of each liposome preparation is performed, the liposome preparation is diluted to the same concentration measured by AL3810, the liposome preparation is filtered in an ultra-clean bench, the liposome preparation is diluted to gradient concentration by using a culture medium for administration, 20 mu L of 5mg/mL MTT solution is added into each hole after 4 hours or 72 hours of culture, the supernatant is discarded after continuous incubation for 4 hours at 37 ℃, 150 mu L of LDMSO is added into each hole, the shaking table is vibrated at 37 ℃ for 10min, and the OD (optical density) value of each hole is measured at the wavelength of 570nm of an enzyme labeling instrument. Cell viability was calculated as follows: cell survival (%) ═ (OD)s-ODblank)/(ODcontrol-ODblank) X 100%. In the formula: ODsIs the absorbance, OD, of the administered groupcontrolAbsorbance, OD, of blank controlblankAbsorbance in blank wells. Growth curves were plotted by Graph Pad Prism 7.0 software for different drug concentrations using cell viability and IC50 was calculated (as shown in figure 12) and the results showed that AL3810 with each target liposome entrapped could exert better killing of U87 cells in vitro than the free drug, non-target liposomes.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The liposome carrying the molecular targeted drug is characterized in that the molecular targeted drug is an anti-tumor molecular targeted drug, and the liposome is a tumor active targeted liposome or a tumor passive targeted liposome; the tumor active targeting liposome or the tumor passive targeting liposome consists of phospholipid, cholesterol, mPEG-DSPE and/or PEG-DSPE modified by targeting molecules.
2. The liposome of claim 1, wherein the targeting molecule modified PEG-DSPE comprises a small molecule compound selected from the group consisting of: folic acid, p-hydroxybenzoic acid (pHA) and its derivatives or myristic acid (MC).
3. The liposome of claim 1, wherein the targeting molecule-modified PEG-DSPE comprises a polypeptide molecule or a protein molecule as the targeting molecule, and the polypeptide molecule or the protein molecule is: VAP, cVAP,SVAP、DVAP，pHA-VAP、pHA-SVAP and pHA-DVAP，MC-VAP、MC-SVAP and MC-DVAP，D8、D8-VAP、D8-SVAP and D8-DVAP，WSW、DWSW、WSW-VAP、DWSW-SVAP andDWSW-DVAP，TGN、DTGN、TGN-VAP、DTGN-SVAP andDTGN-DVAP；A7R、cA7R、DA7R, pHA-A7R and pHA-DA7R, MC-A7R and MC-DA7R, D8-A7R and D8-DA7R, WSW-A7R andDWSW-DA7R, TGN-A7R andDTGN-DA7R; RGD polypeptide, staged-RGD polypeptide, pHA-RGD polypeptide, MC-RGD polypeptide, D8-RGD polypeptide, WSW-RGD polypeptide, and TGN-RGD polypeptide; RW, mn, pHA-RW and pHA-mn, MC-RW and MC-mn, D8-RW and D8-mn, WSW-RW andDWSW-mn, TGN-RW andDTGN-mn; t7 andDt7; RAP12 andDRAP 12; transferrin or lactoferrin, or any combination thereof.
4. The molecularly targeted drug-loaded liposome of claim 1, wherein said anti-tumor molecularly targeted drug is a hydrophilic molecularly targeted drug or a hydrophobic molecularly targeted drug.
5. The molecularly targeted drug-loaded liposome of claim 4, wherein the antineoplastic molecularly targeted drug is imatinib, dasatinib, ponatinib, gefitinib, erlotinib, oxitinib, apacet, apatinib, furazitinib, tositunib, cedanib, palbociclib, ribciclib, abemaciclib, talazolarib, olaparib, Iniparib, nilapari, vinimod, sondegel, Daurismo, everolimus, D8055, AZD2014, temsirolimus, AL3810, SCC-31, sunitinib, regoranib, phenanthrenib, vandetanib, cabotenib, ibrinib, pazopanib, vemurafenib, axitinib, rilatinib, and azxib, or any combination thereof.
6. The liposome of claim 1, wherein the liposome of the targeted drug is prepared by membrane dispersion or ethanol injection.
7. The liposome of claim 6, wherein the liposome is prepared by active drug loading or passive drug loading to encapsulate the targeted drug.
8. The molecularly targeted drug-loaded liposome of claim 1, wherein said molecularly targeted drug-loaded liposome is combined with other pharmaceutical ingredients and/or diluents to form a pharmaceutical composition.
9. The liposome carrying targeted drugs of claim 8, wherein the drug combination is formulated as a tumor targeted therapy formulation.
10. The liposome carrying targeted drugs of claim 9, wherein the tumor targeted therapy preparation is an injection.
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