CN102406610B - Particle dosing system with long circulation performance and preparation method thereof - Google Patents
Particle dosing system with long circulation performance and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a particle dosing system for slightly soluble medicines and a preparation method thereof. A plurality of medicines for the present clinical application are slightly soluble medicines; in order to solve the problem of improving the solubility of the slightly soluble medicines to a certain degree and improving bioavailability, the invention provides a particle dosing system with a long circulation performance and a preparation method thereof, wherein the long circulation performance can be provided by PEG-chitosans. The medicines provided by the invention show slow release characteristics under in-vitro sink condition, and achieve the controlled release function. When entered into rabbit bodies through an intravenous fluid manner, the particle dosing system also shows a longer elimination half-life in comparison with the particle dosing system modified by pure medical solutions or chitosans, and the bioavailability is obviously improved.
Description
Technical field
The present invention relates to pharmaceutical technology field, a kind of particulate delivery system of insoluble drug and preparation method thereof is concretely related to.
Background technology
Many medicines of clinical practice are mostly insoluble drug.They are due to highly lipophilic, and solubility property is poor in water-soluble medium, and itself is limited by factors such as relatively large molecular weight, low small intestine transmitance, liver and gastrointestinal tract mucous first pass effects, make bioavilability that great individual difference is presented.Accordingly it is desirable to by modified form, including solubilized, hydrotropy and the solubility using the galenic pharmacy technologies such as particulate delivery system, to a certain extent improvement insoluble drug, aid in different methods of administration to improve the transhipment, distribution, absorption of medicines, improve drug effect.The bioavilability for improving insoluble drug is to realize that safe and efficient insoluble drug novel formulation needs one of key issue of solution, is the only way which must be passed of research and development with independent intellectual property right medical product.
Chitosan, is naturally occurring, hydrophilic, cation, biodegradable polysaccharide, with nontoxic, good biocompatibility, low immunological rejection, is suitable as the control slow-released carrier of medicine, is widely used in preparation research.However, due between chitosan molecule, the presence of interior hydrogen bond so that it does not dissolve in general organic solvent and water, could be dissolved only in acetum.This causes very big difficulty for its extensive use.In order to improve its dissolubility, people have carried out many modified work to the hydroxyl and amino on its glucosides, retain the advantage of chitosan, improve its shortcoming, such as to its tosylated, amination, esterification, carboxylated, aldehyde radical, sulfonation modification means, the soluble derivative of many chitosans is obtained.
Polyethylene glycol (PEG) is a kind of extremely wide high-molecular compound of purposes, it can be dissolved in water and many solvents, it with excellent biocompatibility, can be dissolved in vivo in tissue fluid, can rapidly be excluded external and not produce any toxic side effects by body.Research shows after microparticle surfaces grafting PEG, alternately stacked to be covered in microparticle surfaces, forms fine and close conformation cloud.It is this it is sterically hindered protect particulate not by blood opsonin identification, intake, slow down particulate supersession rate, residence time in blood, drug treating time extension.By the effect of hydrogen bond, PEGylation chitosan is attracted to phospholipid carrier surface, and the hydration shell that PEG long-chains are formed in microparticle surfaces reduces particulate delivery system by the possibility of the monocyte phagocytosis in human body reticuloendothelial system and blood.Moreover, PEGylation chitosan is wrapped in particulate delivery system surface, particulate can be made stably to be present in medium without assembling, stable effect is played.
Biomedical engineering magazine 2007；24 (4): page 941~945 Ma Chao, Anren of rectifying《The progress of long circulating liposome and its application in nuclear medicine》In disclose a kind of polyethyleneglycol modified liposome being connected by polyethylene glycol or with part, that is long circulating liposome, it is used as a kind of delivery system, but the delivery system is made up of liposome, liposome mainly constitutes skeleton by lecithin and cholesterol, PEG forms PEGylation phosphatide or PEGylation cholesterol by chemical reaction.The less stable of liposome, storage time is short, and storage condition is high.Commercial PEGylation phosphatide or PEGylation cholesterol price are high simultaneously.
The Chinese patent of Application No. 200610099318.5 discloses a kind of new long circulation alprostadil liposome drug feeding system for injection and preparation method thereof, medicine is wrapped in the bilayer lipid membrane to be formed by the invention, is prepared into the prostaglandin E 1 liposome delivery system for being available for injection.The polyethylene glycol long chain is by the polyethylene glycol of DSPE one（PEG-DSPE）There is provided.Formed after liposome; the surface that its polyethylene glycol long chain can be distributed in particulate forms hydrophilic protective films; on the one hand the mutual aggregation between particulate is prevented; on the other hand it can avoid by the identification of internal reticuloendothelial system, phagocytosis; extend particulate delivery system in sanguimotor retention time, reach macrocyclic effect.The pH of the liposome is citrate buffer solution, and pH is between 4.0-5.0, and the pH scopes are significantly less than blood pH7.4, although add anodyne --- tert-butyl hydroxy toluene（BHT）, after said preparation injection, still there may be do not accommodate pain by patient.In addition, substantial amounts of freeze drying protectant is added in the lyophilized formulations --- lactose and anodyne --- tert-butyl hydroxy toluene（BHT）Deng auxiliary material, toxicity is brought.
The content of the invention
Many medicines of current clinical practice are mostly insoluble drug, to solve the solubility for improving insoluble drug, improve the problems of bioavilability, the present invention proposes a kind of particulate delivery system with long circulating performance and preparation method thereof, the medicine of the present invention shows slow release characteristic under sink conditions in vitro, plays control-release function.When being entered in intravenous fluid mode in rabbit body, the particulate delivery system also shows the elimination half-life period longer than simple liposome or chitosan-modified particulate delivery system, and bioavilability is significantly increased.
The present invention is achieved by the following technical solutions：A kind of particulate delivery system with long circulating performance, the described particulate delivery system with long circulating performance is load adriamycin, the chitosan-modified particulate delivery system of PEGylation, long circulating performance is provided by PEGylation chitosan, and the representative general structure of PEGylation chitosan is：
Wherein n is the degree of polymerization；The molecular weight of chitosan is 1~100kDa, and deacetylation is 70-100%；Part free amino group on chitosan chain is by polyethylene glycol（PEG）Substitution, amino group substitution degree is 1~80%.
The hydrophilic segment of polyethylene glycol 5000 is introduced amino of chitosan glucose unit by us by the synthetic reaction of mild condition, synthesizes PEGylation chitosan, and the particulate delivery system for being loaded with insoluble drug is modified with it.The performance of the particulate delivery system in vivo, outer is characterized simultaneously.
A kind of particulate delivery system preparation method with long circulating performance, the preparation method of the described particulate delivery system with long circulating performance is following steps：
（1）Oil phase：Adriamycin, lecithin are weighed, is dissolved with dichloromethane and acetone mixture, the amount of mixed liquor is the quantity of solvent for being completely dissolved solute；
（2）Aqueous phase：Weigh PEGylation chitosan, Poloxmer（Poloxamer）, with water dissolves, the amount of water is the quantity of solvent for being completely dissolved solute；
（3）When oil phase is injected into aqueous phase, inject while stirring, add water and be settled to final concentration of 0.5 ~ 10mg/ml of adriamycin, 2 ~ 8 DEG C of ultrasonic disperses, room temprature evaporation（Room temperature is 18 ~ 32 DEG C）Remove to be washed with water after dichloromethane and wash, 6000 ~ 16000g of centrifugal speed, 2 ~ 8 DEG C centrifuge 15 ~ 50 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, and the film for crossing 0.3 ~ 0.6 μm removes constant volume after big microballoon and buildup, dries, as load adriamycin, the particulate delivery system of the chitosan-modified long circulating performance of PEGylation；Preferably, being washed twice with water or more than twice.
Wherein adriamycin：Lecithin：PEGylation chitosan：Poloxmer mass ratio is 1：0.5~2.0：0.1~1：0.3 ~ 2, the volume ratio of dichloromethane and acetone is 0.1 ~ 5：1.
The particulate that the particulate of the particulate delivery system with long circulating performance obtained by the present invention is 5~1000nm, zeta current potentials are -30mV~+30mV.The particulate delivery system prepared by emulsification-evaporation method, PEGylation chitosan is adsorbed on phospholipid carrier surface, using the soft long-chains of PEG in microparticle surfaces formation hydration shell, so as to reduce drug microparticles by the possibility of the monocyte phagocytosis in human body reticuloendothelial system and blood.PEGylation chitosan is wrapped in particulate delivery system surface simultaneously, particulate is stably present in medium without assembling, plays stable effect.
Particulate delivery system obtained by the present invention, envelop rate is between 30%-100%, and drugloading rate is between 1%-50%.Release shows sustained releasing character in vitro, plays control slow-release function.Also show in animal body than simple liposome and chitosan-modified particulate delivery system longer half-life period, bioavilability is significantly increased.
Particulate delivery system involved in the present invention, because of the stretching and flexibility of the PEG chains of microparticle surfaces, tight impermeable form cloud can be formed on surface, substantially increase the steric hindrance of particulate, make particulate that there is crypticity, reduce the interaction of plasma protein and microparticle surfaces, reduce by the identification and phagocytosis of mononuclear macrophage, the holdup time in the circulatory system is extended, bioavilability is substantially increased.Again because PEG is hydrophilic, this hydrated sheath can reduce the absorption of albumen, the intake of the netted phagocytosis system of endothelium be reduced, so as to reach macrocyclic purpose.
Compared with prior art, the beneficial effects of the invention are as follows：
（1）PEGylation chitosan in the present invention, combines PEG and the advantage both chitosan, and wherein PEG has " crypticity " that excellent hydrophily and soft long-chain are produced；Chitosan has cation, biocompatibility and biological degradability.Therefore, PEGylation chitosan is wrapped in particulate delivery system surface, and particulate can be made stably to be present in medium without assembling, stable effect is played.
（2）The chitosan-modified particulate delivery system of PEGylation of the present invention, preparation method is simple.
Brief description of the drawings
Fig. 1 is the TEM figures (Bar, 50nm) of the chitosan-modified particulate delivery system of PEGylation；
Fig. 2 is the zeta potential diagrams of the chitosan-modified particulate delivery system of PEGylation；
Fig. 3 discharges figure for medicine under release conditions in vitro from the chitosan-modified particulate delivery system of PEGylation；
Fig. 4 be the chitosan-modified particulate delivery system of PEGylation with intravenously administrable after, blood concentration of the different time points in rabbit body.
Embodiment
The present invention is described in further detail with reference to embodiment.
Embodiment 1
（a）Oil phase：Adriamycin 0.1g, lecithin 0.12g are weighed, is 3 with volume ratio：1 dichloromethane and acetone mixture 6ml dissolvings,
（b）Aqueous phase：0.05g PEGylations chitosan, 0.08g Poloxmer are weighed, 20ml water dissolves are used；
（c）Oil phase is slowly injected into the aqueous phase of magnetic agitation, add water and be settled to the final concentration of 1mg/ml of adriamycin, 4 DEG C of ultrasonic disperses, room temperature rotary evaporation, remove and use 20ml water washings twice again after dichloromethane, centrifugal speed 12,000g, 4 DEG C centrifuge 30 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.45 μm removes constant volume after big microballoon and buildup, as freeze-drying, the particulate delivery system of long circulating performance.
Embodiment 2
（a）Oil phase：Adriamycin 0.1g, lecithin 0.05g are weighed, is 0.5 with volume ratio：1 dichloromethane and acetone mixture 6ml dissolvings,
（b）Aqueous phase：0.01g PEGylations chitosan, 0.03g Poloxmer are weighed, 10ml water dissolves are used；
（c）Oil phase is slowly injected into the aqueous phase of magnetic agitation, add water and be settled to the final concentration of 1mg/ml of adriamycin, 4 DEG C of ultrasonic disperses, room temperature rotary evaporation, remove and used again after dichloromethane 10ml water washings three times, centrifugal speed 16,000g, 4 DEG C centrifuge 30 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.30 μm removes constant volume after big microballoon and buildup, as freeze-drying, the particulate delivery system of long circulating performance.
Embodiment 3
（a）Oil phase：Adriamycin 0.1g, lecithin 0.2g are weighed, is 5 with volume ratio：1 dichloromethane and acetone mixture 6ml dissolvings,
（b）Aqueous phase：0.1g PEGylations chitosan, 0.2g Poloxmer are weighed, 50ml water dissolves are used；
（c）Oil phase is slowly injected into the aqueous phase with magnetic agitation, add water and be settled to the final concentration of 1mg/ml of adriamycin, 4 DEG C of ultrasonic disperses, room temperature rotary evaporation, remove and used again after dichloromethane 50ml water washings three times, centrifugal speed 8,000g, 4 DEG C centrifuge 30 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.6 μm removes constant volume after big microballoon and buildup, as freeze-drying, the particulate delivery system of long circulating performance.
Embodiment 4
（a）Oil phase：Adriamycin 0.1g, lecithin 0.15g are weighed, is 1 with volume ratio：1 dichloromethane and acetone mixture 6ml dissolvings,
（b）Aqueous phase：0.02g PEGylations chitosan, 0.1g Poloxmer are weighed, 20ml water dissolves are used；
（c）Oil phase is slowly injected into the aqueous phase with magnetic agitation, add water and be settled to the final concentration of 3mg/ml of adriamycin, 5 DEG C of ultrasonic disperses, room temperature rotary evaporation, remove and use 20ml water washings twice again after dichloromethane, centrifugal speed 10,000g, 5 DEG C centrifuge 40 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.4 μm removes constant volume after big microballoon and buildup, as freeze-drying, the particulate delivery system of long circulating performance.
Embodiment 5
（a）Oil phase：Adriamycin 0.1g, lecithin 0.18g are weighed, is 4 with volume ratio：1 dichloromethane and acetone mixture 6ml dissolvings,
（b）Aqueous phase：0.08g PEGylations chitosan, 0.15g Poloxmer are weighed, 20ml water dissolves are used；
（c）Oil phase is slowly injected into the aqueous phase with magnetic agitation, add water and be settled to the final concentration of 7mg/ml of adriamycin, 3 DEG C of ultrasonic disperses, room temperature rotary evaporation, remove and use 20ml water washings twice again after dichloromethane, centrifugal speed 15,000g, 3 DEG C centrifuge 20 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.5 μm removes constant volume after big microballoon and buildup, as freeze-drying, the particulate delivery system of long circulating performance.
Embodiment is evaluated
（1）The measure of Yield of Mitoxantrone and drugloading rate
Precision weighs the particulate freeze-drying product prepared in embodiment 1, and phased soln is flowed with efficient liquid phase, and filter paper is filtered to remove insoluble matter, takes subsequent filtrate to be diluted to after the detectable concentration scope of standard curve, and concentration is determined with HPLC, calculates medicament contg.
Medication amount/prescription dosage × 100% for Yield of Mitoxantrone=measure.
Drugloading rate=medication amount/prescription dosage × 100%
Determined through HPLC, the entrapment efficiency of the chitosan-modified particulate delivery system of PEGylation is 69.22%, and drugloading rate is 37.04%.
2）Delivery of particulate form and particle size determination
It is quantitative that the chitosan-modified delivery of particulate freeze-drying product of PEGylation is scattered in quantitative physiological saline, suspension is obtained, 2.5% is diluted with water to, after being dipped with special copper mesh, a moment is dyed with 1% phosphotungstic acid, is placed under transmission electron microscope and observes its form and substantially particle diameter.The chitosan-modified drug microparticles delivery system dry product of quantitative PEGylation is dissolved under water, the HSa of ZETASIZE 3000 and measures particle diameter and zeta potential.
The TEM figures and Zeta-potential of delivery of particulate, such as Fig. 1 and Fig. 2.
From the above, the chitosan-modified delivery of particulate particle diameter of PEGylation is in 100nm or so, and the mutual adhesion of particulate, particle diameter distribution is uniform.
Knowable to the Zeta-potential figure of the chitosan-modified particulate delivery system of PEGylation, Zeta-potential is -8.5mv.The Zeta-potential of the chitosan-modified particulate delivery system prepared with same method is+15.2 mv, and the Zeta-potential in simple phosphatide administration body surface face is -17.5mv.These tables of data understand that chitosan is used as positive charge donor, and lecithin is negatively charged material, they can make microparticle surfaces electric charge change, simultaneously, may be because in this particulate prescription, the amount of lecithin be bigger than the amount of PEGylation chitosan, therefore, the chitosan-modified particulate delivery system surface charge of PEGylation is slightly negative, close to neutrality.This also shows that PEGylation chitosan has been wrapped in microparticle surfaces really from another side.
At room temperature, the chitosan-modified delivery of particulate freeze-drying product of a certain amount of PEGylation is suspended in 100mLPBS (pH7.4) dissolution medium, is placed in brown bottle and does release in vitro, make to meet sink conditions.Separated in time quantitative sampling, while supplementing the fresh dissolution medium of equivalent.The sample taken in different time points is centrifuged 30 minutes in 12,000g, 4 DEG C, takes supernatant, the concentration of the medicine in Supernatant samples is determined with HPLC methods, calculate cumulative release amount.
Delivery of particulate release in vitro containing PEGylation chitosan is shown in Fig. 3.From release figure, the no phenomenon of burst release of release of particulate in 48 hours, releases medicine about 30% from particulate.
With the chitosan-modified particulate delivery system dried frozen aquatic products of the PEGylation of quantitative aseptic normal saline dilution precise, the parenteral solution of the delivery of particulate is obtained.
2 kilograms or so of rabbit is taken, preceding fasting in 12 hours is tested, but can't help water.First blank blood is taken from rabbit auricular vein, by rabbit per kilogram of body weight drug containing 5mg PEGylation it is chitosan-modified particulate delivery system parenteral solution, respectively in 2min, 5min, 10min, 20min, 40min, 1h, 2h, 4h, 8h, 12h, 24h time point blood sampling, use Solvent Extract methods medicine, analyzed through HPLC, the blood concentration at each time point is obtained, plasma drug concentration data input medicine is moved into software, medicine is calculated and moves parameter.
The blood concentration at each time point is obtained by HPLC measure and sees Fig. 4.
Plasma drug concentration data input medicine is moved into software, medicine is calculated and moves parameter.Knowable to medicine moves parameter, the elimination half-life period of chitosan-modified delivery of particulate, AUC are 13 times of simple drug solution, 1.38 times；The elimination half-life period of the chitosan-modified delivery of particulate of PEGization, AUC are 21 times of simple drug solution, 25.8 times.Therefore, to eliminate half-life period more slightly longer than chitosan-modified particulate for the chitosan-modified particulate of PEGization, and bioavilability is 18.6 times of chitosan-modified particulate, there is significant difference.In summary, the chitosan-modified delivery of particulate solution of PEGylation is compared with chitosan-modified delivery of particulate solution, simple drug solution, eliminates half-life period t1/2betaExtension；AUC increases.
It these results suggest that, the chitosan-modified delivery of particulate of PEGylation has a long circulating performance, the Pharmacokinetic Characteristics of medicine and carrier there occurs obvious change.It is mainly shown as that drug metabolism and elimination slow down, bioavilability is greatly improved.These effects are all due to that PEG is grafted to the PEGylation particulate formed in chitosan skeletal chain, the PEG chains stretching and flexibility on surface, tight impermeable form cloud (conformational cloud) can be formed in microparticle surfaces, substantially increase the steric hindrance of particulate, make particulate that there is crypticity, reduce the interaction of plasma protein and microparticle surfaces, reduce by the identification and phagocytosis of mononuclear macrophage, the holdup time in the circulatory system is extended, bioavilability is substantially increased.Again because PEG is hydrophilic, this hydrated sheath can reduce the absorption of albumen, the intake of the netted phagocytosis system of endothelium be reduced, so as to reach macrocyclic purpose.
Claims (1)
1. a kind of particulate delivery system with long circulating performance, characterized in that, the described particulate delivery system with long circulating performance is load adriamycin, the chitosan-modified particulate delivery system of PEGylation, long circulating performance is provided by PEGylation chitosan, and the general structure of PEGylation chitosan is：
Wherein n is the degree of polymerization；The molecular weight of chitosan is 1~100kDa, and deacetylation is 70~100%；Part free amino group on chitosan chain is by polyethylene glycol（PEG）Substitution, amino group substitution degree is 1~80%；
The particulate that particulate delivery system particulate is 5~1000nm, zeta current potentials are -30mV~+30mV, forms the particulate delivery system envelop rate with long circulating performance between 30%~100%, drugloading rate is between 1%~50%；
The preparation method of the described particulate delivery system with long circulating performance is following steps：
（1）Oil phase：Adriamycin, lecithin are weighed, is dissolved with dichloromethane and acetone mixture, the amount of mixed liquor is the quantity of solvent for being completely dissolved solute；
（2）Aqueous phase：PEGylation chitosan, poloxamer are weighed, with water dissolves, the amount of water is the quantity of solvent for being completely dissolved solute；
（3）When oil phase is injected into aqueous phase, inject while stirring, add water and be settled to final concentration of 0.5~10mg/ml of adriamycin, 2~8 DEG C of ultrasonic disperses, room temprature evaporation, be washed with water and wash after removing dichloromethane, 6000~16000g of centrifugal speed, 2~8 DEG C centrifuge 15~50 minutes, abandoning supernatant, sediment after being centrifuged is resuspended with water, the film for crossing 0.3~0.6 μm removes constant volume after big microballoon and buildup, dries, obtains the particulate delivery system of long circulating performance；
Wherein adriamycin：Lecithin：PEGylation chitosan：The mass ratio of poloxamer is 1：0.5~2.0：0.1~1：0.3~2, the volume ratio of dichloromethane and acetone is 0.1~5：1.
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