CN101606907A - Fotemustine solid lipid nanoparticle and preparation method thereof - Google Patents
Fotemustine solid lipid nanoparticle and preparation method thereof Download PDFInfo
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Abstract
The invention provides and a kind ofly can be used for injecting and oral fotemustine solid lipid nanoparticle.Its feature be with matrix material, emulsifying agent fotemustine is sealed make that particle diameter is little, envelop rate is high, good stability, fotemustine solid lipid nanoparticle that toxicity is low.The fotemustine solid lipid nanoparticle of the present invention's preparation has improved the dissolubility and the stability of fotemustine, and reduce its blood vessel irritation, the circulation time of prolong drug in blood, thereby improved the curative effect of medicine, the preparation that this solid lipid nanoparticle is made has the characteristics of hypotoxicity, low irritant and high efficiency in clinical practice.The invention still further relates to the preparation method of fotemustine solid lipid nanoparticle, this preparation technology is simple, with low cost, is fit to suitability for industrialized production.
Description
Technical field
The invention belongs to the pharmaceutics field, be specifically related to solid lipid nanoparticle that contains the anti-tumor active ingredient fotemustine and preparation method thereof.
Background technology
Glioblastoma is common brain tumor, and main Therapeutic Method is operation and postoperation radiotherapy.Operation itself is difficult to excise fully whole tumors, and be about 4 months the life cycle of surgery alone treatment malignant glioma.The radiation and chemotherapy comprehensive treatment can prolong malignant glioma patient's about 9～12 months of life span.Chemotherapy is replenished as glioblastoma operation and radiotherapy, for case some recurrence or that accepted radiotherapy, may be topmost Therapeutic Method particularly, is subjected to clinical gradually and the attention research worker.Fotemustine is the nitrosoureas antineoplastic agent of being developed by French Shi Weiya company, commodity are called " Muphoran ", belong to third generation nitrosoureas medicine, go on the market in France in November, 1989, its antitumor mechanism mainly is that its special construction is to have increased propionic acid amide. phosphoric acid fat, adds low-molecular-weight, fat-solubility by synthetic with DNA enzyme effect inhibition DNA and RNA in vivo, make it easier, thereby cerebroma is had good therapeutic effect through blood brain barrier.Through zoopery and clinical research confirmation, this product has broad-spectrum anti-tumor activity, and is especially evident in efficacy to glioblastoma, melanoma, thereby is listed in a kind of medicine of cerebral tumor chemotherapy first-selection.According to the literature, though this medicine has the good effect of passing through blood brain barrier, and can suppress tumor growth effectively, its tangible gastrointestinal reaction and retardance bone marrow depression have limited it and have further continued to use.
Because fotemustine is water insoluble, therefore when being prepared into preparation, there is certain difficulty.The fotemustine of listing is the lyophilized powder that French Les Laboratoires servier is produced at present, because this liquid medicine dissolubility is poor, needing increases the dissolubility of fotemustine with organic solvent, thereby freeze-drier is had specific (special) requirements, and this product stability is poor, can only preserve 9 months at 2-8 ℃.For addressing the above problem, Chinese patent literature (patent No.: ZL 200410024870.9) adopts solvent crystallization to prepare the sterile powder for injection pin, and stability of drug is improved; Other has patent documentation (application number: 200710126506.7) reported the preparation method of injection fotemustine freeze-dried powder, above preparation existence has problems such as specific (special) requirements, suitability for industrialized production difficulty to instrument and equipment, therefore, need seek a kind of effective preparation, can improve stability of drug, dissolubility and antitumous effect, reduce toxic and side effects, strengthen the clinical practice of fotemustine.
Solid lipid nanoparticle (solid lipid nanoparticles, SLN) be the new colloidal drug-supplying system of a kind of alternative Emulsion, liposome and the polymer nanoparticle that grow up in early 1990s, it is that matrix material with natural or synthetic is a carrier, with medicine absorption or be wrapped in the nanometer drug delivery system of making in the matrix material.The lipid components body that it contains can utilize, biodegradable, compare with polymer nanoparticle, its toxicity is lower.Compare with liposome,, do not have the leakage problem of medicine in storage process because medicine is encapsulated in the lipid skeleton.SLN also has slow release, controlled release, targeting.The aqueous dispersion system of SLN can carry out autoclaving or radiation sterilization, has secular physical and chemical stability, also can make solid preparation by lyophilization or spray drying, simultaneously, can adopt high pressure homogenizer to be prepared, and realizes suitability for industrialized production.
Summary of the invention
The fotemustine solid lipid nanoparticle that one object of the present invention is to provide a kind of stable in properties and can adopts the prior art suitability for industrialized production, the present invention is wrapped in fotemustine in the lactone nuclear, increased the water solublity of fotemustine, have advantages such as long-term physical chemistry is stable, may command drug release, targeting location release, the toxic reaction of fotemustine self can be reduced, the bioavailability and the therapeutic index thereof of fotemustine can be improved again.
Another object of the present invention provides the preparation method of above-mentioned fotemustine solid lipid nanoparticle, and preparation method is versatile and flexible, all adopts conventional process equipment, but commercial scale, high efficiency production, it is stable that product quality keeps, can be directly or the secondary operations preparation inject or oral formulations.
Another purpose of the present invention provides a kind of fotemustine solid lipid nanoparticle solid preparation, and this solid preparation is that above-mentioned fotemustine solid lipid nanoparticle adds caffolding agent and adopts lyophilization or spray-dired technology is made.
A further object of the present invention provides a kind of fotemustine injection or oral formulations, and it is to be prepared from by above-mentioned fotemustine solid lipid nanoparticle.
Fotemustine solid lipid nanoparticle of the present invention is formed by the feedstock production that comprises following weight portion:
Fotemustine: 1 part
Matrix material: 5-80 part
Emulsifying agent: 0.1-50 part.
Preferably, fotemustine solid lipid nanoparticle of the present invention is formed by the feedstock production that comprises following weight portion:
Fotemustine: 1 part
Matrix material: 5-50 part
Emulsifying agent: 0.4-30 part.
Above-mentioned matrix material is meant in SLN as carrier material, good biocompatibility, and be solid-state or liquid lipid materials under the room temperature, be selected from glyceride (glyceryl monostearate, glycerol distearate, glyceryl tristearate, myristin, monopalmitin, two tripalmitins, tripalmitin, single Glyceryl Behenate, two Glyceryl Behenates, three Glyceryl Behenates, trilaurin, glycerol trioleate and composition thereof), fatty acid (stearic acid, Palmic acid, mountain Yu acid, capric acid and composition thereof), steroid (cholesterol), wax fat (spermol cetylate, cetyl palmitate, microcrystalline wax and composition thereof), octadecanol and composition thereof.
The emulsifying agent of being narrated is the pharmaceutic adjuvant with emulsifying, stable, peptizaiton, fat-soluble emulsifier and water soluble emulsifier be can be, phospholipid, cholate, dexycholate, short chain alcohol, poloxamer, polysorbate, polyoxyethylene aliphatic alcohol ether, polyoxyethylene fatty acid ester and composition thereof are selected from.Wherein phospholipid is selected from phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, phosphatidyl glycerol, phosphatidic acid, two Laurel phosphatidyl cholines, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, the dioleoyl phospholipid phatidylcholine, the distearyl phosphatidyl glycerol, two palmityl phosphatidyl glycerols, two palmityl Phosphatidylserine, two palmityl PHOSPHATIDYL ETHANOLAMINE, DOPE, two myristoyl PHOSPHATIDYL ETHANOLAMINE and soybean phospholipids, lecithin and hydrolecithin and composition thereof; Cholate is selected from sodium cholate, sodium glycocholate and sodium taurocholate and composition thereof; Dexycholate is selected from NaTDC and deoxidation sodium taurocholate and composition thereof; Short chain alcohol is selected from glycerol and propylene glycol and composition thereof; The preferred Poloxamer-108 of poloxamer and Poloxamer-188 and composition thereof; The preferred tween 80 of polysorbate; The preferred Brij 78 of polyoxyethylene aliphatic alcohol ether, Brij 35 and Brij30 and composition thereof; The preferred Myrj 53 of polyoxyethylene fatty acid ester and Myrj 59 and composition thereof.
The particle diameter of described fotemustine solid lipid nanoparticle is less than 1000nm, and preferable particle size is less than 500nm.
The invention provides the preparation method of fotemustine solid lipid nanoparticle.
This preparation method can be used any one in the following several method:
Method one: emulsifying-sedimentation method preparation, the fotemustine, matrix material, the fat-soluble emulsifier that take by weighing recipe quantity are dissolved in organic solvent and get organic facies, take by weighing the water soluble emulsifier water that gets soluble in water; Organic facies and water are heated to uniform temp respectively, stir down organic facies and water are mixed, and reduction vaporization is removed organic solvent, carry out ultrasonic or the high pressure homogenize processing, can obtain solid lipid nanoparticle.Or
Method two: the preparation of emulsifying volatilization-low-temperature setting method, soon fotemustine, matrix material, fat-soluble emulsifier are dissolved in organic solvent in proportion and get organic facies; Take by weighing the water soluble emulsifier water that gets soluble in water; Organic facies and water are heated to uniform temp respectively, stir down organic facies and water are mixed, reduction vaporization is removed organic solvent, and system is concentrated, concentrated solution is mixed 0-4 ℃ of low temperature aqueous phase in 2-50 times of volume rapidly, the stirring cooling can be carried out ultrasonic then or the high pressure homogenize processing obtains fotemustine solid lipid nanoparticle.Or
Method three: thin film-dispersion method preparation, the fotemustine, matrix material, the fat-soluble emulsifier that take by weighing recipe quantity are dissolved in the organic solvent, evaporated under reduced pressure film forming in 20-60 ℃ of water-bath, add a certain amount of aqueous solution that contains the water solublity emulsifying agent, ultrasonic or high pressure homogenize promptly obtains fotemustine solid lipid nanoparticle.
Organic facies described in the above-mentioned preparation method and water temperature are 20-60 ℃, and optimum temperature is 20-40 ℃; Said organic solvent is selected from a kind of and mixed solvent in chloroform, ether, ethanol, methanol, ethyl acetate, dichloromethane, the acetone; For reducing the particle diameter of solid lipid nanoparticle, can adopt ultrasonic or high pressure homogenize technology.Wherein, ultrasonic power is 100-1000W, and ultrasonic time is 10-40min; High pressure homogenize pressure is 10000-25000psi, and the high pressure homogenize number of times is 3 times-10 times.
For prolonging the resting period of fotemustine solid lipid nanoparticle, be convenient to transportation, fotemustine solid lipid nanoparticle of the present invention can be made solid preparation by spray drying or freeze drying process; For the gathering that prevents solid lipid nanoparticle in the drying process need add caffolding agent, the caffolding agent that can select is aminoacid, maltose, lactose, mannitol, glucose, trehalose, arabic gum, xylitol, sorbitol, fructose and composition thereof, and its consumption is counted 1 part of gross weight by matrix material and emulsifying agent gross weight and added 0.05-4 part lyophilizing caffolding agent.
Fotemustine solid lipid nanoparticle of the present invention can be with injection or oral way administration, and wherein injection system can be venoclysis, intravenous injection, and preferred venoclysis mode gives the patient.
The present invention is that carrier material is made fotemustine the solid lipid nanoparticle with suitable particle diameter with the matrix material, not only increase stability of drug, improve degree of scatter and the bioavailability of medicine in carrier, and can change the physiological disposition of medicine, increase targeting.Select oral administration, fotemustine solid lipid nanoparticle can strengthen the affinity of intestinal endotheliocyte and can pass through approach absorptions such as cell endocytic, fusion and peyer's patches transhipment, effectively improves bioavailability; By drug administration by injection, fotemustine solid lipid nanoparticle can improve blood drug level, the circulation time of prolong drug in blood, and has targeting, can arrive diseased region, tissue or cell by selectivity, thereby improve the therapeutic index of medicine, reduce toxic and side effects.
Adopt the prepared fotemustine solid lipid nanoparticle of the inventive method can improve pharmaceutical chemistry stability and curative effect, thereby have certain novelty and practicality; Its mean diameter is below 1000nm, and envelop rate is greater than 80%; The gained better stability of preparation; And the said preparation mature preparation process, method is simple, and material source is wide, and cost is low, is convenient to suitability for industrialized production.
Description of drawings
Fig. 1 is a fotemustine solid lipid nanoparticle Chinese medicine release graphics.
The specific embodiment:
The invention will be further described with embodiment more below, provides implementation detail of the present invention.In preparation process, undeclared temperature, all expression is at room temperature carried out.
Embodiment 1:
Taking by weighing 208mg fotemustine, 1200mg stearic acid and 1000mg lecithin is dissolved in the dichloromethane of 20ml; 40 ℃ of following evaporated under reduced pressure; form lipid film; the tween 80 (2.5%) that adds 4ml; ultra-sonic dispersion 30min (ultrasonic power 600w); promptly get the solid lipid nanoparticle suspension, standby in 2 ℃ of preservations.Its mean diameter is 210nm, and all below 500nm, particle size distribution is narrow for all particles, shows comparatively homogeneous of solid lipid nanoparticle size; This solid lipid nanoparticle suspension can at room temperature be stablized a couple of days and stablize 12 months under 4 ℃, and lay up period does not observe precipitation.
Embodiment 2:
Take by weighing 624mg fotemustine, 6240mg glyceryl monostearate, 3000mg soybean phospholipid and add in the 50ml tool plug conical flask, add 10ml ethanol: acetone (1: 1) mixed solvent, ultrasonicly it is fully dissolved and be heated to 60 ℃, constitute organic facies.Other gets 500mg Poloxamer-188 and is dissolved in the 30ml redistilled water, constitutes water.With syringe organic facies is slowly injected 60 ℃ of aqueous phases that 1000r/min stirs, continue to be stirred to the translucent system of formation.40 ℃ of following reduction vaporizations are removed organic solvent, and make system be concentrated into 12ml, reduce to room temperature, obtain the solid lipid nanoparticle suspension.
Embodiment 3:
Get 0.624g fotemustine, 12.48g tripalmitin, 1.2g octadecanol, 6.24g dipalmitoyl phosphatidyl choline and be dissolved in ethanol 20ml, ultrasonic dissolution also is heated to (50 ± 2) and ℃ constitutes organic facies.Other gets 1g myrj 53 and adds in the 40ml distilled waters, ultrasonic it is fully dissolved, and constitutes water.The constant temperature aqueous phase that organic facies is slowly injected (50 ± 2) that 1000r/min stirs ℃ forms colostrum, continues stir about 2～3h, organic solvent is evaporated fully and makes system be concentrated into about 2ml.The translucent Emulsion of gained is mixed the 10ml ice aqueous phase that stirs in another 1000r/min of 0～2 ℃ fast, continue to stir 2 hours, breast even 5 times (homogenization pressure is 15000psi) in high pressure homogenizer promptly gets the solid lipid nanoparticle suspension then.
Embodiment 4
Get 0.624g fotemustine, the Yu acid of 12.48g mountain, 1.0g cholesterol, 3.12g distearyl phosphatidyl glycerol and place 50ml pyriform bottle, add the dissolving of 15ml chloroform and constitute organic facies; Other gets the 500mg sodium cholate and is dissolved in the 30ml water, constitutes water.Organic facies is injected aqueous phase, under 20000rpm, stir 30min, form O/W type colostrum.The gained colostrum is breast even 5 times (homogenization pressure is 15000psi) in high pressure homogenizer, and reduction vaporization is removed organic solvent then, and makes system be concentrated into 12ml, reduces to room temperature, promptly gets the solid lipid nanoparticle suspension.
Embodiment 5:
Claim 416mg fotemustine, 4160mg trilaurin, 4160mg two palmityl Phosphatidylserine to be dissolved in ether 20ml, in 30 ℃ of water-baths, be incubated behind the ultrasonic dissolution as organic facies.600mg NaTDC, 800mg glycerol are added injection water 40ml, in 30 ℃ of water-baths, be incubated after the stirring and dissolving as water.Under agitation organic facies is added drop-wise to synthermal aqueous phase while hot; use ultra-sonic dispersion instead behind the high-speed stirred 30min under the 20000rpm; room temperature; power 600W; ultrasonic time 20min; reduction vaporization is removed organic solvent then, and makes system be concentrated into 8ml, promptly makes the fotemustine solid lipid nanoparticle suspension.
Embodiment 6
Take by weighing 208mg fotemustine, 4160mg myristin, 1040mg dioleoyl phospholipid phatidylcholine and be dissolved in methanol 10ml, as organic facies.1000mg deoxidation sodium taurocholate, 1000mg propylene glycol are added injection water 40ml, and stirring and dissolving is as water.Under agitation water is added drop-wise in the organic facies, after continuing to stir 1h, goes in the high pressure homogenizer; spare 10 times through 20000psi pressure breast; reduction vaporization is removed organic solvent then, and makes system be concentrated into 4ml, can obtain the fotemustine solid lipid nanoparticle suspension.
Embodiment 7
Precision takes by weighing fotemustine 416mg, two Glyceryl Behenate 4160mg, hydrolecithin 3000mg, 600mg two Laurel phosphatidyl cholines in the 1000ml round-bottomed flask, add the dissolving of 50ml chloroform, 40 ℃ of following rotary evaporations are removed chloroform, form thin film on flask walls.Add 5% sodium glycocholate solution 10ml, the ultrasonic 20min of water-bath promptly gets fotemustine solid lipid nanoparticle under the 600W power.
Embodiment 8
Take by weighing fotemustine 416mg, glycerol trioleate 8320mg, cetyl palmitate 1000mg, distearoyl phosphatidylcholine 3000mg, be dissolved in ethyl acetate 20ml, as organic facies; Under stirring condition, add the aqueous solution 40ml that contains 0.5% glycerol and 0.5% sodium taurocholate then, make thick breast; Under the logical condition of nitrogen gas on the high pressure dispersing emulsification machine even 5 times of 25000psi pressure stimulating milk secretion, decompression is removed organic solvent and is made system be concentrated into 8ml then, cooling forms the solid lipid nanoparticle suspension rapidly.Preserve 4 ℃ of sealings, be for experiment.
Embodiment 9
Take by weighing DOPE 12.48g, Palmic acid 12.48g and fotemustine 0.416g and add that the 20ml ether is ultrasonic to make it dissolving fully, then under magnetic agitation with No. 6 syringe needles injection 40ml 0.5%brij78 solution.After 20000rpm stirs 30min, be transferred on the high pressure dispersing emulsification machine 20000psi pressure stimulating milk secretion even 5 times, decompression is removed organic solvent and is made system be concentrated into 8ml then, until forming the milky suspension, add sorbitol 4g, glycine 4g through supersound process (ultrasonic power 600w) 20min, medicinal liquid is dry in spray dryer, the spray drying condition is: 120 ℃ of inlet temperature, charging rate 50ml/min, cyclone separator pressure reduction 50mm water, atomizing disk rotating speed 2500r/min collects and obtains the fotemustine solid lipid nanoparticle spray dried formulations.
Precision takes by weighing Poloxamer-108 1.0g, glycerol 0.2g, under high speed magnetic agitation condition, joins in the 10ml distilled water that is heated to 60 ℃, treats to dissolve fully as water; Other claims glycerol distearate 5g, two palmityl phosphatidyl glycerol 4g, fotemustine 0.208g to add 10ml chloroform ultrasonic dissolution and be heated to 60 ℃ as organic facies; Organic facies is slowly dropped to aqueous phase; high speed shear (Fluko; 16000rpm) 5min; after making O/W type Emulsion; use water-bath type Ultrasound Instrument ultra-sonic dispersion (room temperature, power 600w, ultrasonic time 20min) instead; decompression is removed organic solvent and is made system be concentrated into 4ml then, promptly gets the tangible solid lipid nanoparticle suspension of opalescence.Add 0.5g mannitol and 0.6g glucose ultrasonic dissolution, behind-40 ℃ of pre-freeze 6h, with-40 ℃, 8h;-30 ℃, 8h;-20 ℃, 8h;-10 ℃, 6h; 20 ℃, the program lyophilization of 8h promptly gets the fotemustine solid lipid nanoparticle lyophilized formulations.
Embodiment 11
Precision takes by weighing monopalmitin 4160mg, two myristoyl PHOSPHATIDYL ETHANOLAMINE 8320mg, fotemustine 416mg, adds ethyl acetate 10ml, and ultrasonic making fully dissolved.The mixture solution of gained joined contain among the 1.0%brij 35 solution 30ml; behind high-speed stirred (20000rpm) 5min; use water-bath type Ultrasound Instrument ultra-sonic dispersion (room temperature instead; power 600w; ultrasonic time 30min); decompression is removed organic solvent and is made system be concentrated into 8ml then, promptly gets the tangible solid lipid nanoparticle suspension of opalescence.After adding 800mg lactose, 500mg glucose, 500mg trehalose ultrasonic dissolution, spray drying in the mini spray exsiccator, (inlet temperature 130-140 ℃, outlet temperature 50-60 ℃, air pressure 1.2Atm, charging rate 60ml/min), promptly get the fotemustine solid lipid nanoparticle spray dried formulations.
Embodiment 12
Get fotemustine 208mg, two tripalmitin 2080mg, capric acid 1000mg and methanol: acetone (1: 1) 20ml adds in the 25ml pyriform bottle, ultrasonic it is fully dissolved, and adds the 3000mg dimyristoyl phosphatidyl choline, and slight fever makes the melt into organic facies.Other gets among the 500mg mrij 59 water-soluble 20ml and constitutes water.Organic facies is stirred the aqueous phase that injects (40 ± 2) ℃ down at (1000r/min), continue stirring 4h, organic solvent is evaporated fully and make system be concentrated into about 4ml, promptly get the fotemustine solid lipid nanoparticle suspension.After this suspension added 400mg arabic gum, 500mg mannitol ultrasonic dissolution, in-40 ℃ of pre-freezes 6 hours, lyophilizing in freezer dryer, the lyophilizing program was-40 ℃, 8 hours;-30 ℃, 6 hours;-20 ℃, 6 hours;-10 ℃, 5 hours; 0 ℃, 5 hours; 15 ℃, 5 hours.
Embodiment 13
Take by weighing fotemustine 208mg, three Glyceryl Behenate 4000mg, two palmityl PHOSPHATIDYL ETHANOLAMINE 1000mg are dissolved in 10ml ethanol and make organic facies, add the aqueous solution 20ml contain 0.5% glycerol and 0.5% tween 80 then under stirring condition, make thick breast; (Avestin C5) goes up 15000psi pressure stimulating milk secretion even 5 times, and decompression is removed organic solvent and made system be concentrated into 4ml then, forms the solid lipid nanoparticle suspension at the high pressure dispersing emulsification machine under the logical condition of nitrogen gas.This suspension adds 400mg xylitol, 400mg fructose in-40 ℃ of pre-freezes 10 hours, and (the lyophilizing program is-40 ℃, 10 hours in lyophilizing in freezer dryer (labconco);-20 ℃, 8 hours; 0 ℃, 8 hours; 20 ℃, 5 hours), promptly get the fotemustine solid lipid nanoparticle lyophilized formulations.
Embodiment 14
Get the fotemustine solid lipid nanoparticle described in the embodiment 3 and carry out physicochemical property research.
It is an amount of to get the fotemustine solid lipid nanoparticle suspension, and thin up drips then and covering on the copper mesh of carbon film, observes the nanoparticle form and take pictures under transmission electron microscope.
Other gets the fotemustine solid lipid nanoparticle sample, and thin up uses the laser light scattering particle size analyzer 25 ℃ of temperature, the particle diameter under the wavelength 633.0nm condition to an amount of.
Result: observe solid lipid nanoparticle under the Electronic Speculum and be class spherical entity particle, and particle diameter is even, does not have and assembles and adhesion.
The particle size distribution of the fotemustine solid lipid nanoparticle that experiment records is narrower, and mean diameter is 240nm.
Embodiment 15
Fotemustine solid lipid nanoparticle entrapment efficiency determination among the embodiment 3
(10min) fotemustine of separated free is measured free fotemustine content with RP-HPLC to the fotemustine solid lipid nanoparticle suspension for retaining molecular weight 10000D, 4000rpm through the combining ultrafiltration supercentrifugal process.Chromatographic condition is as follows: chromatographic column: Eclipse XDP-C
18Post (250mm * 4.6mm, 5 μ m); Mobile phase: methanol-water (85: 15); Column temperature: 20 ℃; Detect wavelength: 230nm; Flow velocity: 1.0ml/min; Sample introduction 20 μ l are by following formula computational envelope rate.
Envelop rate=(1-M
Free/ M
Always) * 100%.
The result: the envelop rate that records the solid lipid nanoparticle suspension is 83.24%.
Embodiment 16
Fotemustine solid lipid nanoparticle entrapment efficiency determination among the embodiment 4
Envelop rate is measured in conjunction with HPLC by the polydextran gel column chromatography.Chromatography condition is: (30cm * 2cm), eluent is a distilled water to the Sephadex-G50 post, flow velocity 1ml/min, chromatography under the room temperature.Pipette solid lipid nanoparticle suspension 0.5ml upper prop, with column chromatography condition eluting, every 2ml collects 1 pipe, and encapsulated medication amount adopts high-performance liquid chromatogram determination.Chromatographic condition is as follows: chromatographic column: Eclipse XDP-C
18Post (250mm * 4.6mm, 5 μ m); Mobile phase: methanol-water (85: 15); Column temperature: 20 ℃; Detect wavelength: 230nm; Flow velocity: 1.0ml/min; Sample introduction 20 μ l are by following formula computational envelope rate.
Envelop rate=(M
Bag/ M
Always) * 100%
In the formula, M
BagThe medication amount that expression is sealed by solid lipid nanoparticle, M
AlwaysSum total dose in the expression solid lipid nanoparticle.
The result: the envelop rate that records the solid lipid nanoparticle suspension is 82.45%.
Embodiment 17
Fotemustine solid lipid nanoparticle physicochemical property research among the embodiment 5.
1, morphology research
The solid lipid nanoparticle suspension that takes a morsel drips on the copper mesh that covers carbon film, with 2.0% Sodium phosphotungstate solution-dyed, and observation size and form and taking pictures under transmission electron microscope, as seen its outward appearance is the sphere of rounding.
2, particle diameter and Zeta potential are measured
Get an amount of thin up of solid lipid nanoparticle suspension, under 632.8nm, measure particle diameter, particle size distribution and Zeta potential with multi-functional Zeta potential Particle Size Analyzer.The result: the mean diameter that records sample is 273nm; Average Zeta potential-26.7mV.
3, envelop rate is investigated
Get solid lipid nanoparticle suspension 1ml carry out the low temperature ultracentrifugation (4 ℃, 20000rpm, 30min), precision is measured supernatant 0.5ml, methanol dilution is settled to 10ml, the peak area that gets fotemustine with high effective liquid chromatography for measuring is A
Trip, be A with the peak area that records fotemustine after the solid lipid nanoparticle suspension dilution with method
Always
Envelop rate (%)=(1-A
Trip/ A
Always) * 100%
The result: the envelop rate that records fotemustine solid lipid nanoparticle is 82.50%.
Embodiment 18
Get fotemustine solid lipid nanoparticle suspension among the embodiment 6 and be positioned in room temperature and the 4 ℃ of refrigerators, whether the centrifugal 15min suspension of its outward appearance and 4000r/min layering or precipitation occur when observing 1 month, 3 months, 5 months, 6 months.
The result shows, is positioned over the sample in 4 ℃ of refrigerators, do not have precipitation in 6 months and drug crystallization is separated out, and lamination do not appear in the centrifugal 15min of 4000r/min, shows that this system has certain stability.And small amount of precipitate appears in the sample segment under the room temperature in the time of 5 months, and part is open to airborne sample surfaces and small pieces taupe mildew occurs.
Embodiment 19
The fotemustine solid lipid nanoparticle spray dried formulations of getting among the embodiment 9 adds water 10ml dispersion, go in the bag filter (molecular cut off 10000), place 250ml pH7.4 phosphate buffer, (37 ± 0.5) ℃, the 100rpm magnetic agitation, regularly draw medium 5ml, measure absorbance, and press standard curve Equation for Calculating cumulative release percentage rate.The results are shown in Figure 1.
The result shows that under release conditions, rate of releasing drug was very fast in preceding 10 hours, and rate of release slows down subsequently, through 36 hours drug releases 82.6%, shows after fotemustine is made solid lipid nanoparticle to have slow release effect.
The dried frozen aquatic products of getting among the embodiment 10 is an amount of, and sealing is in 4 ℃ of placements of refrigerator, in 0,1,2,3,6, December measures indexs such as particle diameter, envelop rate, content, estimates the stability of fotemustine solid lipid nanoparticle dried frozen aquatic products.The results are shown in Table 1.
Table 1
Table 1 explanation, the fotemustine solid lipid nanoparticle dried frozen aquatic products was placed 12 months for 4 ℃ at refrigerator, and quality index such as its particle diameter, envelop rate, content are constant substantially, show that the fotemustine solid lipid nanoparticle lyophilized formulations has good stability.
Embodiment 21
The fotemustine solid lipid nanoparticle preparation of getting in the foregoing description carries out irritant experiment.
(1) rabbit auricular vein vascular stimulation is tested:
With physiological saline solution fotemustine solid lipid nanoparticle and injection are mixed with the solution that concentration is 1mg/ml before the experiment.Select 6 of the healthy new zealand white rabbits of body weight 2.5-3.0kg for use, male and female have concurrently.After iodine tincture and ethanol disinfection were used in the injection site, 3 white rabbits were in auris dextra auricular vein injection 2ml fotemustine injection, and left ear injection equal volume physiological saline solution injection compares; 3 white rabbits are in auris dextra auricular vein injection 2ml fotemustine solid lipid nanoparticle in addition, and left ear injection equal volume physiological saline solution injection compares.Once a day, for three days on end, the last administration is after 24 hours, inject air by auricular vein and put to death white rabbit, the response situation of perusal injection site, and dissect rabbit ear blood vessel and surrounding tissue and make paraffin section (under the injection site entad section 1cm and 5cm place), dyeing, light microscopy checking.Perusal injection site reaction situation.The results are shown in Table 2.
Table 2
As a result, as shown in table 2, the fotemustine solid lipid nanoparticle blood vessel irritation obviously is weaker than the fotemustine injection.
(2) muscle irritation test
The dosage of fotemustine injection converts, the selection of medicine preparation, New Zealand white rabbit the same (totally 4, every kind dosage form 2).Cut off the rabbit hair at quadriceps femoris position, white rabbit both sides, behind iodine tincture and ethanol disinfection, respectively at right lateral thigh musculus quadriceps injection fotemustine solid lipid nanoparticle and fotemustine injection 2ml, left side quadriceps femoris injection equivalent physiological saline solution injection compares, and injects after 48 hours, injects air by auricular vein and puts to death white rabbit, dissect quadriceps femoris, vertically cut, observe the response situation of injection site muscular tissue, determine the order of reaction.
0 grade: no change
1 grade: mild hyperaemia, its scope is below 0.5cm * 1.0cm
2 grades: moderate hyperemia, its scope is more than 0.5cm * 1.0cm
3 grades: severe hyperemia, with myodegeneration
4 grades: necrosis occurs, the brown degeneration is arranged
5 grades: the popularity necrosis occurs
Calculate 4 quadriceps femoris order of reaction summations then, test again if the difference of the peak of the quadriceps femoris order of reaction and minimum, then should be got 2 white rabbits in addition greater than 2.After obtaining the result, if four quadriceps femoris order of reaction summations, think then that the local irritation experiment of test sample is up to specification less than 10.
Table 3
Result's (seeing Table 3) shows that the muscle irritation of fotemustine solid lipid nanoparticle obviously is weaker than the fotemustine injection
Embodiment 22 hypersensitive tests
Medication is got an amount of fotemustine injection and is diluted to the sample A that concentration is 8mg/ml with normal saline; Take from the system fotemustine solid lipid nanoparticle and be diluted to the sample B that concentration is 8mg/ml with normal saline.Get 12 of Cavia porcelluss, be divided into two groups of A, B, carry out sensitivity test usefulness for sample A and B respectively.6 of every group of Cavia porcelluss, body weight 250～350g.The next day intramuscular injection test sample 0.5ml, continuous 3 times.Then A, B group Cavia porcellus is divided into A1, A2 and B1, B2 group more respectively, wherein A1, B1 organize the test sample 2ml that after injection first the 14th day injects 2mg/ml by vein and attack, observing injection back animal has useless pawl to scratch reactions such as nose, sneeze, perpendicular hair, tic, dyspnea, gatism, shock, death, and A2, B2 organize after injection first the 21st day equally by the test sample 2ml of vein injection 2mg/ml and observe.
The result: fotemustine injection and fotemustine solid lipid nanoparticle all do not have tangible symptoms of allergic.
The experiment of embodiment 23 hemolytics
Get blood 20ml from the common carotid artery of New Zealand white rabbit, place in the flask, stir several minutes gently with Glass rod after, remove and to defibrinate, take out blood, add the equivalent normal saline solution, it is centrifugal that (1500rpm 10min), removes supernatant; Sedimentary erythrocyte adds normal saline solution again and cleans.Transparent up to supernatant so repeatedly, be made into 2% suspension with normal saline by erythrocytic capacity.
Get 7 of clean tube, numbering adds each liquid in the following table successively respectively, the 6th pipe does not add test sample and makes the blank pipe, and the 7th effective distilled water replaces normal saline, shakes up, whether place 37 ℃ of water-baths, observing respectively at 0.5,1,2,3 hour has haemolysis to take place.The results are shown in Table 4 and table 5.
Table 4
Table 5
The result shows that fotemustine injection and fotemustine solid lipid nanoparticle do not see that all haemolysis takes place, and the hemolytic experiment of two kinds of dosage forms is all qualified.
Claims (11)
1, a kind of fotemustine solid lipid nanoparticle is characterized in that, is formed by the feedstock production that comprises following weight portion:
Fotemustine: 1 part
Matrix material: 5-80 part
Emulsifying agent: 0.1-50 part.
2, fotemustine solid lipid nanoparticle according to claim 1 is characterized in that, is formed by the feedstock production that comprises following weight portion:
Fotemustine: 1 part
Matrix material: 5-50 part
Emulsifying agent: 0.4-30 part.
3, fotemustine solid lipid nanoparticle according to claim 1 and 2 is characterized in that, described matrix material is glyceride, fatty acid, steroid, wax fat, octadecanol or its mixture; Wherein glyceride is selected from glyceryl monostearate, glycerol distearate, glyceryl tristearate, myristin, monopalmitin, two tripalmitin, tripalmitin, single Glyceryl Behenate, two Glyceryl Behenate, three Glyceryl Behenates, trilaurin and triglyceride and composition thereof; Fatty acid is stearic acid, Palmic acid, mountain Yu acid, capric acid or its mixture; Steroid is a cholesterol; Wax fat is spermol cetylate, cetyl palmitate, microcrystalline wax or its mixture.
4, fotemustine solid lipid nanoparticle according to claim 1 and 2, it is characterized in that described emulsifying agent is phospholipid, cholate, dexycholate, short chain alcohol, poloxamer, polysorbate, polyoxyethylene aliphatic alcohol ether, polyoxyethylene fatty acid ester or its mixture; Wherein, phospholipid is selected from phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, phosphatidyl glycerol, phosphatidic acid, two Laurel phosphatidyl cholines, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, dioleoyl phospholipid phatidylcholine, distearyl phosphatidyl glycerol, two palmityl phosphatidyl glycerols, two palmityl Phosphatidylserine, two palmityl PHOSPHATIDYL ETHANOLAMINE, DOPE, two myristoyl PHOSPHATIDYL ETHANOLAMINE, soybean phospholipid, lecithin, hydrolecithin and composition thereof; Cholate is selected from sodium cholate, sodium glycocholate and sodium taurocholate and composition thereof; Dexycholate is selected from NaTDC and deoxidation sodium taurocholate and composition thereof; Short chain alcohol is selected from glycerol and propylene glycol and composition thereof; Poloxamer is selected from Poloxamer-108 and Poloxamer-188 and composition thereof; Polysorbate is selected from tween 80; Polyoxyethylene aliphatic alcohol ether is selected from Brij 78, Brij 35 and Brij 30 and composition thereof; Polyoxyethylene fatty acid ester is selected from Myrj 53 and Myrj 59 and composition thereof.
5, fotemustine solid lipid nanoparticle according to claim 1 and 2 is characterized in that, its particle diameter is less than 1000nm.
6, fotemustine solid lipid nanoparticle according to claim 5 is characterized in that, its particle diameter is less than 500nm.
7, the preparation method of claim 1 or 2 described fotemustine solid lipid nanoparticles is characterized in that, adopts in the following distinct methods any one:
1) emulsifying-sedimentation method: the fotemustine, matrix material, the fat-soluble emulsifier that take by weighing recipe quantity are dissolved in organic solvent and get organic facies, take by weighing the water soluble emulsifier water that gets soluble in water; Organic facies and water are heated to uniform temp respectively, stir down organic facies and water are mixed, and reduction vaporization is removed organic solvent then, carry out ultrasonic again or the high pressure homogenize processing, can obtain solid lipid nanoparticle; Or
2) emulsifying volatilization-low-temperature setting method: fotemustine, matrix material, fat-soluble emulsifier are dissolved in organic solvent in proportion get organic facies; Take by weighing the water soluble emulsifier water that gets soluble in water; Organic facies and water are heated to uniform temp respectively, stir down organic facies and water are mixed, reduction vaporization is removed organic solvent, and system is concentrated, concentrated solution is mixed 0-4 ℃ of low temperature aqueous phase in 2-50 times of volume rapidly, the stirring cooling is carried out ultrasonic then or the high pressure homogenize processing obtains fotemustine solid lipid nanoparticle; Or
3) thin film-dispersion method: the fotemustine, matrix material, the fat-soluble emulsifier that take by weighing recipe quantity are dissolved in the organic solvent, 20-60 ℃ of following evaporated under reduced pressure film forming, add the aqueous solution that contains the water solublity emulsifying agent, ultrasonic or high pressure homogenize is handled and is promptly got fotemustine solid lipid nanoparticle;
Described organic solvent is chloroform, ether, ethanol, methanol, ethyl acetate, dichloromethane or acetone or its mixed solvent; The temperature of described organic facies and water is 20-60 ℃; Pressure is 10000-25000psi during high pressure homogenize, and the number of times of high pressure homogenize is 3 times-10 times; Ultrasonic power is 100-1000W, and ultrasonic time is 10-40min.
8, the preparation method of fotemustine solid lipid nanoparticle according to claim 7 is characterized in that, the temperature of organic facies and water is 20-40 ℃.
9, a kind of fotemustine solid lipid nanoparticle solid preparation is characterized in that, described solid preparation is that the fotemustine solid lipid nanoparticle in claim 1 or 2 adds caffolding agent and adopts lyophilization or spray-dired technology is made.
10, fotemustine solid lipid nanoparticle solid preparation according to claim 9, it is characterized in that described caffolding agent is selected from aminoacid, maltose, lactose, mannose, glucose, trehalose, arabic gum, xylitol, sorbitol and fructose and composition thereof; And its consumption is counted 1 part of gross weight by matrix material and emulsifying agent gross weight and is added 0.05-4 part lyophilizing caffolding agent.
11, a kind of fotemustine injection or oral formulations is characterized in that, are to be prepared from by the fotemustine solid lipid nanoparticle in claim 1 or 2.
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