CN103263388B - Stealthy vesicle of a kind of modified with folic acid norcantharidin and preparation method thereof - Google Patents
Stealthy vesicle of a kind of modified with folic acid norcantharidin and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to stealthy vesicle of a kind of modified with folic acid norcantharidin and preparation method thereof.This vesicle is using poloxamer188-cholesterol cpds and FA-PEG-chol as main carriers material, and bag obtains surface hydrophilic after carrying norcantharidin and has the stealthy vesicle of modified with folic acid, and it is not containing free cholesterol.The envelop rate of this vesicle is (52.30 ± 2.16) %, and mean diameter is (100.87 ± 0.23) nm, zeta current potential is-25.96mV, and tablets in vitro is significantly slow in crude drug, the release t in simulation normal structure pH7.4 environment
1/2be 1.98 times in simulation tumor cell pH5.0 environment, slow releasing function significantly and be conducive to discharging in target cell.And this vesicle can significantly strengthen the growth inhibition ratio of tumor cell and take the photograph dose, produce the active targeting effect to tumor cell at molecular level, thus improve efficacy of drugs, be conducive to high-efficiency low-toxicity Therapeutic cancer.
Description
Technical field
The present invention relates to a kind of drug-supplying system, be specifically related to a kind of stealthy vesicle of medicine of modified with folic acid, the invention still further relates to the preparation method of vesicle.
Background technology
Targeting drug delivery system adopts special carrier material, be prepared from by special process and technology, drug selectivity can be made to concentrate in the drug-supplying system of diseased region (target site), play a role for target organ, target tissue, target cell or intracellular targets, thus it is large to overcome the ubiquitous anticarcinogen dosage of current clinical treatment, metabolism is fast, and the dose entering tumor cell is few, killing tumor cell lacks selectivity, and infringement normal cell causes the problems such as serious toxicity.Usually require that the characteristic that targeting drug delivery system has is: A. can evade reticuloendothelial system in body and remove, tool stealth characteristics.General particle diameter <200nm, surface is modified by hydrophilic chain polymer, can evade reticuloendothelial system (RES) interception in vivo, and have long circulating action; B. optionally in target site accumulation also release.In addition, carrier material should nontoxic or low toxicity.
The modified with folic acid particulate delivery system of large quantity research at present, designing for the folacin receptor in all kinds of tumor cell overexpressions such as cervical cancer, ovarian cancer, breast carcinoma, gastric cancer, pulmonary carcinoma and nasopharyngeal carcinoma, is with the active targeting drug-supplying system of ligand-receptor specific effect for guiding.Be low expression due to folacin receptor at normal tissue cell or do not express, modified with folic acid medicine carrying microgranule can with folacin receptor specific bond, namely realize the active targeting administration to tumor cell by molecular targeted effect.
The stealthy vesicle of modified with folic acid, both long circulating action was possessed, also there is the active targeting feature of modified with folic acid microgranule, carrier material is in the solution by self assembly bag medicine carrying thing, form particulate delivery system, its surface hydrophilic, similar are in biomembrane, and inside and outside character is similar to liposome, but because not containing oxidizable rotten phospholipid, and more stable.And it all can wrap hydrophilic drugs, hydrophobic drug, insoluble drug and carry, can medicine stability be improved, the distribution in vivo of adjustment medicine and release characteristics, evade RES and remove, thus prolongation circulation time in vivo, be conducive to transmission medicine to leakage strong tumor tissues; Can be combined with the folacin receptor of tumor cell surface specifically and enter tumor cell again, and the environment of pH5.0 in tumor cell (normal structure is pH7.4) can be utilized to discharge medicine, play antitumor action.
The characteristic of vesicle depends on the lypohydrophilic character (HLB value) of the carrier material non-ionic surface active agent preparing vesicle to a great extent.Commercial vector material can be selected, also can synthesize voluntarily as required.At present conventional macromolecular material poloxamer has 407,188(trade name: pluronic F127, F68) two kinds, be Pluronic F68, general formula is HO-(C
2h
4o)
a-(C
3h
6o)
b-(C
2h
4o)
c-H.In its molecule, polyoxyethylene moities increases, and water solublity increases; Polyoxypropylene portion ratio increases, then water solublity declines, and lipophile increases.The HLB value of PLURONICS F87 (FW:8350) is 29, and the HLB value of poloxamer188 (FW:12000) is 22.The poloxamer188 comparatively large with molecular weight, HLB value is slightly little and the conjugate of cholesterol, bag carries the better effects if of polar molecule medicine, and the particle diameter of gained vesicle is even more ideal.
Anticarcinogen norcantharidin (NCTD) is colorless crystalline powder, odorless, slightly zest, and slightly water-soluble, ethanol is soluble in hot water, acetone.It can tumoricidal form, Inhibit proliferaton, and interference is divided and suppresses its DNA to synthesize.The clinical treatment for hepatocarcinoma, the esophageal carcinoma, stomach and carcinoma of gastric cardia and low leukocyte counts disease etc.And it is to medullary cell unrestraint effect, and have the effect of unique leukocyte increasing, therefore in anticarcinogen, status is remarkable for a long time.But, clinically usedly be mainly Norcantharidin tablet and demethylcantharidin essence injecta at present, be ordinary preparation, selectivity is not had to tumor tissues, tumor cell, the bioavailability of medicine is low, thus there is serious Liver and kidney toxicity, easily produce drug resistance, and very easily hydrolysis becomes the problems such as the lower demethylcantharidin acid of drug effect.
Summary of the invention
For improving the deficiency of the existing preparation of anticarcinogen norcantharidin in clinical practice, high-efficiency low-toxicity ground Therapeutic cancer, the invention provides a kind of stealthy vesicle Novel Drug Delivery Systems of modified with folic acid being different from ordinary preparation.It is for the characteristic of folacin receptor overexpression tumor cell, and will have the medicine carrying microgranule of pH sensitivity, long circulating and ligand-receptor active targeting characteristic, directional transmissions plays antitumaous effect to cancerous cell.The present invention also provides the preparation method of the stealthy vesicle of this modified with folic acid.
For achieving the above object, the present invention adopts following technical scheme:
A kind of bag carries the stealthy vesicle of modified with folic acid of norcantharidin, as main carriers material in order to poloxamer188-cholesterol cpds and FA-PEG-chol, bag obtains surface hydrophilic after carrying norcantharidin and has the stealthy vesicle of modified with folic acid, and it is not containing free cholesterol.The present invention's research shows after poloxamer188 is combined with cholesterol, and the better effects if of bag medicine carrying thing, the particle diameter of gained vesicle is even more ideal.
Vesicle every milliliter suspension of the present invention is specifically made up of following material:
Above-mentioned FA-PEG-chol is preferably the folic acid-PEG-cholesterol cpds that folic acid and PEG and cholesterol are formed; Described surfactant can be selected from that fatty acid Pyrusussuriensis is smooth, in Polysorbate one or both; Described buffer solution can be selected from the borate of pH3.6 ~ 9.5, phosphate or carbonate one or more.
The present invention also provides the preparation method of above-mentioned vesicle, it comprises step: by poloxamer188-cholesterol, FA-PEG-chol, the surfactant of recipe quantity, after complete with appropriate organic solvent dissolution, ultrasonic mixing is incubated at 20 ~ 70 ° of C with recipe quantity medicine, injection method is adopted to mix with buffer solution again, insulated and stirred is also ultrasonic, lipophilic moieties is assembled automatically because of hydrophobic interaction, simultaneously hydrophilic parts outwards diastole and self assembly is shaped automatically, obtains the stealthy vesicle suspension of medicine of modified with folic acid after organic solvent volatilization.
Above-mentioned organic solvent to be selected from methanol, ethanol, isopropyl alcohol, ethyl acetate, acetone, dichloromethane one or more.
Poloxamer188-cholesterol of the present invention, its structural formula is as shown in following formula (I):
（I）
The present invention also provides the preparation method of described poloxamer188-cholesterol cpds.The method is dissolved in DMSO by poloxamer188, poloxamer188-aldehyde is obtained with acetic anhydride under room temperature, electrophilic addition poloxamer188-aldehyde and cholesterol is occurred in acid condition again and generates hemiacetal, then hemiacetal continues to obtain described poloxamer188-cholesterol with cholesterol generation substitution reaction.
Vesicle drug-supplying system of the present invention has long circulating characteristic, pH sensitivity, active targeting anti-tumor activity simultaneously, effect of medicine can be made significantly to improve, active anticancer strengthens, and be conducive to reducing the Normocellular toxicity to the low expression of folacin receptor, thus play the effect of high-efficiency low-toxicity Therapeutic cancer.
The stealthy vesicle Novel Drug Delivery Systems of modified with folic acid norcantharidin of the present invention, adopt the polymer of poloxamer188-cholesterol, modified with folic acid and other carrier material (but not containing free cholesterol) to prepare the stealthy vesicle suspension of gained modified with folic acid norcantharidin in self assembly mode, can directly intravenous injection use, or make freeze-dried powder further, injecting before use with aqueous dispersion is use after suspension.The mean diameter of this Novel Drug Delivery Systems is (100.87 ± 0.23) nm, zeta current potential is-25.96mV, and envelop rate is (52.30 ± 1.28) %, and medicine fat ratio is 8.07%; Tablets in vitro t
1/2=1.31h(free drug 30min discharges completely), in the pH7.4 environment of simulation normal structure, discharge t
1/2be 1.98 times in simulation tumor cell pH5.0 environment, slow releasing function significantly and be conducive to discharging in target cell.And, confirmed by the growth inhibition test to folacin receptor overexpression tumor cell, cellular uptake quantitative test and fluidic cell test, folic acid vesicle significantly can improve the growth inhibited of tumor cell and the picked-up to medicine, to take the initiative targeting at molecular level, improve medicine active anticancer, thus have and can reduce the Normocellular toxicity of the low expression of folacin receptor, play the outstanding advantage of high-efficiency low-toxicity Therapeutic cancer effect.
Accompanying drawing explanation
Fig. 1 be poloxamer188-cholesterol (P407-chol) and the infared spectrum of raw material be called for short:,
Wherein, Fig. 1 .A. cholesterol (chol);
Fig. 1 .B. poloxamer188 (P407);
Fig. 1 .C. poloxamer188-cholesterol.
Fig. 2 is the X-ray diffracting spectrum of poloxamer188-cholesterol and raw material thereof,
Wherein, Fig. 2 .A. is the x-ray diffraction pattern of cholesterol;
Fig. 2 .B. is the x-ray diffraction pattern of poloxamer188;
Fig. 2 .C. is the x-ray diffraction pattern of poloxamer188-cholesterol conjugate.
Fig. 3 is differential scanning calorimetry collection of illustrative plates (DSC) collection of illustrative plates of poloxamer188-cholesterol and raw material thereof,
Wherein, Fig. 3 .A. is the differential scanning calorimetry collection of illustrative plates of cholesterol;
Fig. 3 .B. is the differential scanning calorimetry collection of illustrative plates of poloxamer188;
Fig. 3 .C. is the differential scanning calorimetry collection of illustrative plates of poloxamer188-cholesterol conjugate.
Fig. 4 is the stealthy vesicle of modified with folic acid norcantharidin (being called for short: folic acid vesicle) particle size distribution and scanning electron microscope (SEM) photograph,
Wherein, Fig. 4 .A. is the grain size distribution of folic acid vesicle;
Fig. 4 .B. is the scanning electron microscope (SEM) photograph of folic acid vesicle.
Fig. 5 is the release profiles of folic acid vesicle in the artificial cellular environment of difference.
Fig. 6 is the Toxicity test result of the test of folic acid vesicle to Hela cell,
Wherein, Fig. 6 .A. is that folic acid vesicle different action time is on the impact of cell survival rate;
Fig. 6 .B. is the impact of folic acid vesicle different pharmaceutical concentration versus cell survival rate.
Fig. 7 is the investigation result of the test that Hela cell variable concentrations folic acid vesicle takes the photograph dose,
Wherein, Fig. 7 .A. is that Hela cell takes the photograph dose to 19 μ g/ml folic acid vesicles;
Fig. 7 .B. is that Hela cell takes the photograph dose to 38 μ g/ml folic acid vesicles.
Fig. 8 is that cells were tested by flow cytometry Hela cell is to folic acid vesicle ' picked-up result of the test.
Fig. 9 is the synthesis path of poloxamer188-cholesterol.
Detailed description of the invention
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.
Preparation process of the present invention, not only the norcantharidin of recipe quantity and the organic facies containing carrier material can be mixed, the stealthy vesicle of modified with folic acid norcantharidin (being called for short: folic acid vesicle) is obtained by self assembly, also adjustable prescription must contrast with the blank stealthy vesicle of modified with folic acid (being called for short: blank vesicle) with legal system, the stealthy vesicle of norcantharidin (being called for short: stealthy vesicle), or bag carries norcantharidin and rhodamine B (RdB) simultaneously, the stealthy vesicle of obtained modified with folic acid norcantharidin/RdB (be called for short: folic acid vesicle '), the vesicle suspensions such as the stealthy vesicle of norcantharidin/RdB (be called for short: stealthy vesicle '), be stored in 4 DEG C, for relevant research.
The preparation of embodiment 1 poloxamer188-cholesterol and checking
1, the preparation and characterization of poloxamer188-cholesterol:
1.1. the preparation of poloxamer188-cholesterol
There is end-OH according to the macromolecular substances such as PEG, poloxamer188, can synthetic route be adopted: PEG-OH+ (CH
3cO)
2o+CH
3sOCH
3→ PEGOCH
2cHO obtains polymer acetal (title polymer-aldehyde).That is: 5.123g poloxamer188 is taken in 50ml round-bottomed flask, add 0.4g acetic anhydride and 15ml dimethyl sulfoxine (DMSO), electromagnetic agitation RT reacts 30h, in instillation 150ml absolute ether, shake up, dripping dichloromethane makes solution become homogeneous phase, then adds appropriate ether and make it precipitate, and 4 DEG C are placed to precipitation and separate out completely, decompress filter, vacuum drying, to constant weight, obtains white loose shape solid: poloxamer188-aldehyde 1.3590g, and yield is 26.5%.
By poloxamer188-aldehyde and cholesterol, electrophilic addition occurs in acid condition again and generate hemiacetal, then hemiacetal continues to become acetal with cholesterol generation substitution reaction.Take 3.5g poloxamer188-aldehyde in 50ml round-bottomed flask, add 1g cholesterol, 5ml dehydrated alcohol, 0.1ml concentrated hydrochloric acid, 80 DEG C of water-bath backflow 2.5h, cooling obtains solid-state or semi-solid products.Sucking filtration, obtains white waxy solid, and after being washed with water to neutrality, in the NaOH solution of 50ml0.1mol/L, soak 2h, filter, drying under reduced pressure, to constant weight, obtains white solid, and (be called for short a) in prescription, yield is 22.95% to be poloxamer188-cholesterol.
1.2. the sign of poloxamer188-cholesterol
Instrument etc.:
Cold field emission scanning electron microscope S-4700(Hitachi, Ltd, Japan)
High-resolution-ration transmission electric-lens TecnaiG220(FEI, the U.S.)
NICOMP380zls particle diameter potentiometric analyzer (NICOMP, the U.S.)
Infrared spectrophotometer ProStarLC240(Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A., the U.S.)
NMR spectrometer with superconducting magnet UNITYINOVA400(Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A., the U.S.)
Bag filter (molecular cut off is respectively 1500,14000)
1.2.1 the infrared spectrum measurement of poloxamer188-cholesterol and raw material thereof
Get appropriate raw material cholesterol, poloxamer188 and product poloxamer188-cholesterol respectively, after vacuum drying, with KBr tabletting, with infrared spectrophotometer ProStarLC240(Varian Associates, Inc. (US) 611 Hansen Way, Palo Alto, California 94303, U.S.A., the U.S.) measure, obtain infared spectrum, see Fig. 1 (A), (B), (C).Main characteristic absorption has: figure A. cholesterol, at 1603cm
-1neighbouring absorption is the C=C stretching vibration absworption peak on its own ring.Figure B. poloxamer188, at 1112.395cm
-1the stretching vibration absworption peak of the C-O-C at place.Figure C. poloxamer188-cholesterol, at 1588.139cm
-1near be C=C stretching vibration absworption peak on the own ring of cholesterol, 1055.430cm
-1place is the stretching vibration absworption peak of the C-O-C of poloxamer188.Prove that synthesis gained is target product poloxamer188-cholesterol.
1.2.2. the X-ray diffraction analysis of poloxamer188-cholesterol
Get appropriate raw material cholesterol respectively, after poloxamer188 and product poloxamer188-cholesterol grind, adopt X-ray diffraction instrument CuK-AIPHA1/40kV/100mA(MERCURYCCD, Japan), scanning 3 ~ 60 measures, obtain its X-ray diffracting spectrum, see Fig. 2 (A), (B), (C), wherein Fig. 2 (C) is the diffraction curve of poloxamer188-cholesterol, 19.6 and 23.0(unit 2 θ °) there is very strong diffraction maximum at place, for poloxamer188 segment produces, show that poloxamer188 still remains its crystalline texture in poloxamer 407-cholesterol.In poloxamer188-cholesterol, the diffraction maximum of cholesterol segment is also obvious, and just peak intensity weakens to some extent, and the diffraction maximum of high angle disappears, cholesterol segment still exists with the state of aggregation of folded chain fragment, crystal layer thickness reduces, and degree of crystallinity declines to some extent, proves that synthesis gained is target product.
1.2.3 differential scanning calorimetric analysis (DSC)
Take appropriate raw material cholesterol, poloxamer188 and product poloxamer188-cholesterol respectively, measure with differential calorimetric scan instrument (SDT2960, DSC2010, U.S. TAInstruments).Probe temperature is 50 ~ 300 DEG C, programming rate 10K/min.The results are shown in Figure 3(A), (B), (C).As shown in Figure 3, in poloxamer188-cholesterol, the fusing point of poloxamer188 segment rises to 122.15 DEG C by 54.67 DEG C of cholesterol, proves that poloxamer188 segment and cholesterol segment have very strong interaction.Cholesterol has very strong melting endothermic peak at 149 DEG C, but this peak appears at 139 DEG C on the DSC curve of poloxamer 407-cholesterol, illustrate that cholesterol also maintains the crystalline texture of homopolymer in poloxamer 407-cholesterol, this is just in time consistent with the measurement result of X-ray diffraction.In addition, with the melting endothermic peak area of poloxamer188 segment, the crystallization situation of poloxamer188 is compared (the contrast longitudinal axis is observed), the larger then degree of crystallinity of area is higher, and the endothermic peak area of curve C is much smaller than the endothermic peak area of A, B curve, namely the crystallizing power of product declines, and proves the formation of poloxamer188-cholesterol novel carriers material.
2. the preparation of FA-PEG-Chol:
The folic acid of 0.15mmol is dissolved in the dimethyl sulfoxine of 1.3ml, again the N-N-dicyclohexyl carbodiimide (DCC) of 0.3mmolN-hydroxy-succinamide (NHS), 0.15mmol and the triethylamine of 33 μ L are added in the lump, lucifuge stirred overnight at room temperature, the insoluble by-product of filtering, obtaining solution is FA-NHS(intermediate product 1).Again by 0.15mmolPEG (NH
2)
2be dissolved in 5mlDMSO and make solution, dropwise added by intermediate product 1, lucifuge stirred overnight at room temperature obtains FA-PEG(intermediate product 2).In addition, the cholesterol of 0.15mmol is dissolved in 500 μ L dichloromethane, and add the 4-dimethylamino pyridine (DMAP of 0.45mmol, catalyst), and the N of 0.45mmol, N-carboxyl diimidazole (CDI), room temperature for overnight, liquid becomes yellow from colourless, obtains the cholesterol (intermediate product 3) of activation.
By in intermediate product 3 slowly instillation intermediate product 2 solution, after stirring at room temperature reacts 24 hours, it to be dialysed in 4 DEG C of pure water 48h(1000ml × 2), by the solution lyophilization in bag filter, obtain product FA-PEG-Chol (that is: FA-PEG-chol, be called for short b) in prescription, lucifuge stores.Overall yield of reaction is 45.59%.
The preparation of the stealthy vesicle Novel Drug Delivery Systems of embodiment 2 modified with folic acid norcantharidin and analysis
3. the preparation of the stealthy vesicle Novel Drug Delivery Systems of modified with folic acid norcantharidin
The preparation of the stealthy vesicle suspension of 3.1 modified with folic acid norcantharidin (being called for short: folic acid vesicle)
By every milliliter of suspension containing norcantharidin 3.8mg, poloxamer188-cholesterol (a) 20.0mg, FA-PEG-chol (b) 1.2mg, surfactant (c) 29.4mg, after carrier material a, b, c of recipe quantity is complete with organic solvent dissolution, mix ultrasonic with norcantharidin, injection method is adopted to mix with the PBS of 60 DEG C of preheatings again, 20 ~ 70 DEG C stir and ultrasonic, be stirred to organic solvent volatilization after and get final product.
The preparation of the stealthy vesicle suspension of 3.2 modified with folic acid norcantharidin/RdB (be called for short: folic acid vesicle ')
" 3.1 " method of employing, but add fluorescein rhodamine B(RdB with norcantharidin simultaneously.Consumption: 10% norcantharidin) preparation, to obtain final product.
The preparation of the stealthy vesicle suspension of 3.3 norcantharidin (being called for short: stealthy vesicle)
" 3.1 " method of employing, but no carrier added material b, to obtain final product.
The preparation of the stealthy vesicle suspension of 3.4 norcantharidin/RdB (be called for short: stealthy vesicle ')
" 3.2 " method of employing, but no carrier added material b, to obtain final product.
The preparation of the blank stealthy vesicle suspension (blank vesicle) of 3.5 modified with folic acid
" 3.1 " method of employing, but do not add norcantharidin, to obtain final product.
4. the physicochemical of Novel Drug Delivery Systems
4.1 morphologic observation
The mode of appearance of folic acid vesicle and particle size distribution after measured, finds that it is circular substantially, good evenness.Its particle size distribution and scanning electron microscope (SEM) photograph are shown in Fig. 4 A, Fig. 4 B respectively.
4.2 mean diameters and surperficial Zeta potential measure
The active targeting effect checked for making the test neoplastic cell of folic acid vesicle is more reliable, get rid of positive charge to the trend effect of electronegative tumor cell, vesicle prepared by the present invention, all with the elecrtonegativity consistent with tumor cell, investigates the active targeting effect of ligand-receptor specific binding better.
Record folic acid vesicle mean diameter for (100.87 ± 0.23) nm, zeta current potential be-25.96mV.Illustrate that folic acid vesicle (particle diameter <200nm) is conducive to evading RES interception, and because not easily assembling with electric charge on surface.
But according to same preparation technology, prescription almost remains unchanged, only wherein will change poloxamer F68-cholesterol into by poloxamer188-cholesterol, obtained vesicle particle diameter is 310nm; If increase cholesterol again in prescription, obtained vesicle particle diameter is at more than 500nm.
4.3 surperficial intrinsic hydrated sheath thickness measurements
The intrinsic hydrated sheath in surface is relevant with vesicle hidden property.According to Gu Yi-Cha Puman diffuse double layer model, the charged particle surface logarithm value of Zeta potential and the pass of the intrinsic hydrated sheath thickness (L) of particle surface are: ㏑ (ζ)=㏑ A-κ L, and wherein A is constant,
c is electrolytical molar concentration.Respectively folic acid vesicle concentration is respectively 0,5,10, after the NaCl solution dilution dispersion of 20mM, measure Zeta potential, bringing above-mentioned equation into, namely to obtain surperficial intrinsic hydrated sheath thickness be 1.63nm (n=3), and having exceeded can the intrinsic hydrated sheath thickness (1.00nm) in macrocyclic Mycocet surface in bibliographical information body.Therefore, particle diameter and the surface intrinsic hydrated sheath thickness of folic acid vesicle all can meet common long circulating requirement.
4.4 entrapment efficiency determination
Respectively recipe quantity folic acid vesicle and stealthy vesicle suspension are all proceeded in the bag filter of anticipating, dialysis medium (0.9% normal saline is suspended under room temperature, pH7.4) in, 150 turns/min stirs, dialysis solution is changed every 20min, stop (now the medicine dialysance of the mixture of blank formulation and recipe quantity norcantharidin is more than 83%) after 40min, measure norcantharidin amount sum in dialysis solution, be the amount of free drug; Bag filter content is all proceeded in volumetric flask respectively, destroys vesicle with 30% hydrochloric acid-isopropyl alcohol, measure medication amount in bag.The envelop rate calculating folic acid vesicle is (52.30 ± 2.16) %, and the envelop rate of stealthy vesicle is (44.67 ± 0.92) %.
The release rule of 4.5 different artificial cellular environments is in vitro investigated
For investigating the folic acid vesicle release rule at normal cellular environment (pH7.4) and tumor cell environment (pH5.0), on the basis of prerun, dialysis medium is made with the sodium chloride solution of pH7.4 and pH5.0, compare with equivalent free drug solution, stealthy vesicle, investigate the release characteristics of folic acid vesicle.Dialyse in 37 DEG C of 100ml sodium chloride solutions, stir 100 turns/min, get dialysis solution 1ml in each time point: 0.5,1.0,2.0,3.0,5.0,7.0,10.0,15.0,23.0,33.0h, add 1ml release medium simultaneously, maintain sink conditions and continue dialysis to terminal.Sample calculates after measured, obtains drug accumulation release percentage rate (Q%), is (Q%-t) figure, obtains Cumulative release profile (n=3 is shown in Fig. 5) with drug accumulation release percentage rate to percentage rate sample time.
As shown in Figure 5, free drug discharges completely in 30min, and the release of folic acid vesicle, stealthy vesicle is obviously slow.Each bar release profiles carries out matching according to zero level, one-level, Higuchi, Weibulldistribution pharmacokinetics model, and calculates t respectively
1/2, the results are shown in Table 1.
The release profiles fit equation (n=3) of table 1 different artificial cellular environment in vitro
* between each group, t1/2 difference is P<0.01, and each group difference and is significantly described.
Because the tablets in vitro of folic acid vesicle is significantly slower than crude drug, and the release t in the pH7.4 environment of simulation normal structure
1/2be 1.98 times in the pH5.0 environment of simulation tumor cell, therefore the slow releasing function of folic acid vesicle significantly and be conducive to discharging in target cell.
5. cell assay in vitro is investigated
5.1 pairs of s toxicity tests
For checking folic acid vesicle enters cells play effect through folacin receptor mediated approach, that selects the substrate without folic acid to get rid of substrate Folic Acid affects cultured cell, using stealthy vesicle, in folic acid vesicle, add 0.5mM dissociate folic acid as positive controls, blank vesicle, as negative control group, investigates folic acid vesicle to the growth inhibition ratio of tumor cell.
By Hela cell at 37 DEG C, 5%CO
2cultivate in incubator.To take the logarithm the cell of trophophase, be diluted to every hole 5 × 10 with containing 10% hyclone without the RPMI-1640 of folic acid
4individual cell adds 96 well culture plates, every hole 100 μ L, after cultivating 24h, adds the solution of same drug level respectively: folic acid vesicle, positive control solution: stealthy vesicle, folic acid+folic acid vesicle, drug solution, negative controls: blank vesicle, PBS is benchmark, often group establishes 6 concentration: 7.8, 15.6, 31.2, 62.5, 125, 250 μ g/ml, cultivate 3h respectively, 6h, 12h, after changing 1640 culture medium continuation cultivation 24h into, every hole adds MTT solution (5mg/ml) 10uL, liquid is discarded after hatching 4h in incubator, add 100 μ l/ hole DMSO, put in microplate reader and detect, determined wavelength is 490nm, with formula: " suppression ratio=(1-medicine group absorbance/PBS group absorbance) × 100% " calculates suppression ratio, respectively with each concentration of different time for abscissa, survival rate (=100%-suppression ratio) is vertical coordinate, obtain when fixed drug concentration is 250 μ gml
-1the tumor cell survival (see Fig. 6 A) of Shi Butong action time, the cell survival rate curve chart (see Fig. 6 B) of 12h different pharmaceutical concentration.
As shown in Figure 6, respectively organize all to growth inhibition ratio presentative time dependency and the dose dependent of Hela cell.Further, the cell survival rate from Fig. 6 A, 12h: stealthy vesicle is 74.0% of free drug; Folic acid vesicle is then only 35.3% of free drug, but is then 58.8% of free drug when there being free folic acid to exist.Illustrate that folic acid vesicle improves drug effect the most remarkable, and free folic acid can reduce its drug effect.Difference between folic acid vesicle and each positive controls, enlarges markedly along with the prolongation of time, and the effect of killing off tumor cells is the most effective, and the time dependence of drug effect is also the strongest.Median lethal dose(LD 50) IC is calculated by Fig. 6 B curve
50: folic acid vesicle is 46 μ g/ml, significantly lower than each positive controls: stealthy vesicle 148.00 μ g/ml, folic acid+folic acid vesicle 91.00 μ g/ml, free drug 261.00 μ g/ml.Farther lower than negative control group: blank vesicle 1470.00 μ g/ml.That is, each positive controls IC
50for its 1.98 ~ 5.67 times, the IC of the blank vesicle of negative control
50for its 31.96 times.And when there being free folic acid to exist, having occupied folacin receptor competitively, folic acid vesicle utilizes ligand-receptor mediation influenced, makes the IC of folic acid vesicle
5091 μ g/ml are increased to by 46 μ g/ml, prove that free folic acid can disturb the performance of folic acid vesicle drug effect, prompting norcantharidin Novel Drug Delivery Systems folic acid vesicle Bao Zaihou improves the ability of killing off tumor cells, utilizes folic acid-folacin receptor specific active targeting action effect remarkable.And the cytotoxicity of blank formulation is very little, and, namely reach constant (Fig. 6 B) at low concentration in measured 3h ~ 12h almost constant (Fig. 6 A), point out its toxicity may from experimental system itself instead of blank formulation.
5.2 cellular uptake quantitative tests
For investigating folic acid vesicle to the impact entering cell dose, to study for sample containing equivalent medicine folic acid vesicle, stealthy vesicle, drug solution.To take the logarithm the Hela cell of trophophase, be diluted to every hole 3 × 10 with the RPMI-1640 containing 10% new-born calf serum
5individual cell is added on 24 well culture plates, every hole 1ml, after 37 DEG C of cultivation 24h are adherent, add respectively and dilute with containing the RPMI-1640 of 10% hyclone without folic acid, obtain two groups of drug level (A.19 μ g/ml, B.38 μ g/ml) sample liquid 1ml, continue cultivation 1, 2, after 4h, draw culture fluid, with brine, 0.5ml/ time, wash 3 times, the trypsin adding 200 μ l again digests, add 0.5ml normal saline again, carry out cell counting, thereafter-20 DEG C of freeze overnight are carried out, melt, ultrasonic with 200W1 second/time, Probe Ultrasonic Searching instrument, every minor tick 1 second, ultrasonic 15 times.In PBS and smudge cells liquid, add 0.2ml acid isopropyl alcohol respectively destroy vesicle, get appropriate smudge cells liquid 0.22 μm of membrane filtration, get subsequent filtrate and carry out efficient Liquid Detection, and calculate medicament contg, the results are shown in Figure 7A, Fig. 7 B.
As seen from Figure 7: the picked-up presentative time of tumor cell to medicine and the dependency of dosage, and to the intake of folic acid vesicle and stealthy vesicle apparently higher than drug solution.Wherein, the taken amount being shot of each time point of folic acid vesicle under 19 μ g/ml drug level, after improving 1 times to 38 μ g/ml than drug solution raising 84.04% ~ 227.85%(drug level, picked-up raising ratio is larger, for: 190.73% ~ 399.61%), after improving 1 times than stealthy vesicle raising 22.52% ~ 33.55%(drug level, improve ratio similar: 20.67% ~ 30.85%).Proved invention drug-supplying system folic acid vesicle can utilize folacin receptor mediated, and significantly increase cellular uptake dose, active targeting effect is remarkable.
5.3 fluidic cell picked-up tests
Cell situation is entered by cytophagy for investigating folic acid vesicle further, with with the Novel Drug Delivery Systems of the in kind obtained bag medicine carrying thing introduced above and fluorescein and each matched group for sample: folic acid vesicle ', stealthy vesicle ' (drug level: 0.76mg/ml, RdB concentration: 0.076mg/ml), blank vesicle and the mixture (blank vesicle+RdB, RdB concentration: 0.076mg/ml) with RdB thereof.
To take the logarithm the Hela cell of trophophase, be diluted to every hole 3 × 10 with the RPMI-1640 containing 10% new-born calf serum
5individual cell is added on 6 well culture plates, every hole 2ml, cultivates 24h for 37 DEG C, after cell attachment, be changed to containing the RPMI-1640 of 10% hyclone without folic acid, add above-mentioned sample liquid respectively, make medicine ultimate density be 40 μ g/ml, discard culture fluid after 37 DEG C of cultivation 2h, add appropriate trypsinization liquid by cell dissociation, centrifugally obtain cell, wash 3 times with PBS, by cell suspension in the cold PBS of 1ml, proceed in fluidic cell measuring tube, flow cytometer gathers cell, counting 1 × 10
4individual cell, measures the fluorescence intensity of cell, obtains the situation of cytophagy RdB.The results are shown in Figure 8.By Fig. 8 to each group by cytophagic fluorescence intensity, and deduction blank formulation revised result is as table 2.
Table 2 fluidic cell picked-up result of the test
| Folic acid vesicle ' | Stealthy vesicle ' | Blank vesicle+RdB | Blank vesicle | |
| Fluorescence intensity | 94.2 | 70.3 | 52.0 | 15.3(benchmark) |
| Fluorescence intensity after revising | 78.9 | 55.0 | 36.7 |
Therefore, after getting rid of the impact of blank vesicle, folic acid vesicle increases by 2.15 times than blank vesicle+RdB cytophagy, increases by 1.43 times, further demonstrate the active targeting of folic acid vesicle than stealthy vesicle.
Claims (3)
1. bag carries the stealthy vesicle of modified with folic acid of norcantharidin, it is characterized in that: every milliliter of vesicle suspension is made up of following material:
The stealthy vesicle of described modified with folic acid obtains by the following method: by poloxamer188-cholesterol, FA-PEG-chol, the surfactant of recipe quantity, after complete with appropriate organic solvent dissolution, ultrasonic mixing is incubated at 20 ~ 70 DEG C with recipe quantity norcantharidin, injection method is adopted to mix with buffer solution again, insulated and stirred is also ultrasonic, lipophilic moieties is assembled automatically because of hydrophobic interaction, simultaneously hydrophilic parts outwards diastole and self assembly is shaped automatically, obtains the stealthy vesicle suspension of medicine of modified with folic acid after organic solvent volatilization;
Wherein, described poloxamer188-cholesterol, its structural formula is such as formula shown in (I):
The preparation method of described poloxamer188-cholesterol is: be dissolved in by poloxamer188 in DMSO, carry out being obtained by reacting poloxamer188-aldehyde with acetic anhydride under room temperature, again poloxamer188-aldehyde and cholesterol be there is in acid condition electrophilic addition and generate hemiacetal, then hemiacetal continues and cholesterol generation substitution reaction, namely obtains described poloxamer188-cholesterol;
Described FA-PEG-chol is the folic acid-PEG-cholesterol cpds that folic acid and PEG and cholesterol are formed; Its preparation method is: be dissolved in the dimethyl sulfoxine of 1.3ml by the folic acid of 0.15mmol, again the triethylamine of the N-N-dicyclohexyl carbodiimide of 0.3mmolN-hydroxy-succinamide, 0.15mmol and 33 μ L is added in the lump, lucifuge stirred overnight at room temperature, the insoluble by-product of filtering, obtaining solution is FA-NHS, i.e. intermediate product 1; Again by 0.15mmolPEG (NH
2)
2be dissolved in 5mlDMSO and make solution, dropwise added by intermediate product 1, lucifuge stirred overnight at room temperature obtains FA-PEG, i.e. intermediate product 2; The cholesterol of 0.15mmol is dissolved in 500 μ L dichloromethane, and adds the 4-dimethylamino pyridine of 0.45mmol, and the N of 0.45mmol, N-carboxyl diimidazole, room temperature for overnight, liquid becomes yellow from colourless, obtains the cholesterol of activation, i.e. intermediate product 3; By in intermediate product 3 slowly instillation intermediate product 2 solution, after stirring at room temperature reacts 24 hours, dialyse it in 4 DEG C of pure water 48h, by the solution lyophilization in bag filter, obtains product FA-PEG-Chol;
Described surfactant is selected from that fatty acid Pyrusussuriensis is smooth, in Polysorbate one or both;
Described buffer solution to be selected from the borate of pH3.6 ~ 9.5, phosphate or carbonate one or more.
2. the preparation method of vesicle described in claim 1, it comprises step: by poloxamer188-cholesterol, FA-PEG-chol, the surfactant of recipe quantity, after complete with appropriate organic solvent dissolution, ultrasonic mixing is incubated at 20 ~ 70 DEG C with recipe quantity norcantharidin, injection method is adopted to mix with buffer solution again, insulated and stirred is also ultrasonic, lipophilic moieties is assembled automatically because of hydrophobic interaction, simultaneously hydrophilic parts outwards diastole and self assembly is shaped automatically, obtains the stealthy vesicle suspension of medicine of modified with folic acid after organic solvent volatilization.
3. preparation method according to claim 2, is characterized in that, described organic solvent to be selected from methanol, ethanol, isopropyl alcohol, ethyl acetate, acetone, dichloromethane one or more.
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| CN101099866A (en) * | 2006-07-07 | 2008-01-09 | 张文芳 | Cholesterol copolymer decorated by poloxamer and its application |
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| CN101099866A (en) * | 2006-07-07 | 2008-01-09 | 张文芳 | Cholesterol copolymer decorated by poloxamer and its application |
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