CN101669909B - Biological adhesive liposome preparation for eyes and preparation method thereof - Google Patents
Biological adhesive liposome preparation for eyes and preparation method thereof Download PDFInfo
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- CN101669909B CN101669909B CN2008100429420A CN200810042942A CN101669909B CN 101669909 B CN101669909 B CN 101669909B CN 2008100429420 A CN2008100429420 A CN 2008100429420A CN 200810042942 A CN200810042942 A CN 200810042942A CN 101669909 B CN101669909 B CN 101669909B
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Abstract
The invention belongs to the field of medicinal preparations, and relates to liposome for eyes and a preparation method thereof. In order to overcome the defects that common eye drops have short residence time in the conjunctival sac to cause low bioavailability at the eyes and the semi-solid dosage form has poor compliance and is not easy to be accepted by patients and the like in the prior art, the invention provides a biological adhesive liposome preparation for eyes. The biological adhesive liposome preparation for the eyes consists of the liposome, a liposome membrane modification material and a medicament wrapped in the liposome, wherein the surface of the liposome is modified with free mercapto, and a covalent binding disulfide bond can be formed by the free mercapto and a mucoprotein subdomain rich in cysteine on the surface of the eye to anchor the liposome on the surface of a mucous membrane and serve as a medicament store to slowly release the medicament in the conjunctival sac and provide permanent driving force for the absorption of the medicament. The preparation is helpful for promoting the absorption of the medicament at the eyes, and can improve the bioavailability of the medicament.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, relate to a kind of with Liposomal formulation and preparation method thereof.Described surface of liposome is modified with free sulfydryl, can form covalently bound disulfide bond with the mucoprotein subdomain that is rich in cysteine in eye surface, liposome is anchored on mucous membrane surface, as drug depot slowly release in conjunctival sac, for drug absorption provides persistent driving force.
Background technology
Eyes are extremely sensitive and have the organ of multiple protective mechanism, so the design of ocular drug transmission system has challenge.Because the medicinal liquid that splashes in the conjunctival sac is eliminated rapidly, the biological barrier effect of cornea in addition, conventional eye drop only have the medicine of less than 10% can see through cornea arrive ophthalmic performance therapeutical effect (N.M.Davies, Clin.Exp.Pharmacol.Physiol.2000,27:558-562).Many ophthalmic diseases adopt eye drop to treat the effect that is difficult to obtain satisfaction, must increase the absorption of medicine by semisolid as eye ointment, gel and intercalating agent or solid dosage forms, or can arrive the target site onset to guarantee the effective dose of medicine thing by the mode administration of intraocular injection at eye.Though but these dosage forms and administering mode can improve the curative effect of medicine be difficult for being accepted by the patient, use the important trend that comfortable novel efficiently drug-supplying system treatment ophthalmic disease has become current ophthalmic remedy development.
Ideal dosing eyes system should have following several respects characteristics: 1. safe, long-term prescription can not produce eye and general toxicity; 2. good effect is sustainable at the eye positioning release medicine, and target site drug level height need not frequent drug administration; 3. compliance is good, not damaged after the administration, and no foreign body sensation, the patient is acceptant.Liposome is as a kind of ophthalmic preparation of liquid type, have easy to use comfortable, compliance good, can increase the cornea permeability, delay advantages such as release and reduction drug toxicity, weak point is that the eye holdup time is shorter relatively, for example, it is close with common eye drops that neutral eye with the elecrtonegativity liposome is eliminated speed, is unfavorable for that medicine is in ocular absorption.Therefore, be necessary further to research and develop that the eye retentivity is stronger, the Liposomal formulation of better efficacy.
Cornea and scleral surface are covered with the mucus that a layer thickness is 3～40 μ m, and its main component is mucoprotein.The approximately per 15～20h of this slime layer upgrades 1 time.Utilize this physiological feature that the dosing eyes system of research design mucosa adhesion is arranged, as macromolecular solution and various microgranule, semisolid and solid ophthalmic preparation (A.Ludwig, Adv.DrugDeliv.Rev.2005,57:1595-1639).Because eyes can be removed foreign body effectively, therefore based on mucosa adhesion drug-supplying system, the especially liquid dosage form of noncovalent interaction, the effect of its prolong drug holdup time is unsatisfactory.
Sulfhydrylation polymer (Thiomers) is the novel mucosa adhesion macromolecular material of a class, its design philosophy is to introduce the part that contains sulfydryl in the molecule of existing adjuvant, as cysteine, homocysteine or mercaptoethylmaine, utilize these sulfydryls to form covalently bound disulfide bond at agents area and the mucoprotein subdomain that is rich in cysteine, high polymer adjuvant is anchored on mucous membrane surface forms the three dimensional gel network, and then the time of contact of prolong drug and mucosa, for its transmembrane transport creates favorable conditions.Finally, covalently bound pasting material will be eliminated along with mucoprotein renewal.The sulfhydrylation polymer of bibliographical information can be divided into anionic and cationic two big classes up to now, polyacrylic acid (carbomer (the A.Bernkop-Schn ü rch that comprises sulfhydrylation altogether, et al, Drug Dev.Ind.Pharm.2004,30:1-8) and Polycarbophil (A.Bernkop-Schn ü rch, et al, Int.J.Pharm.2001,226:185-194)), carboxymethyl cellulose (A.Bernkop-Schn ü rch, S.Steininger.Int.J.Pharm.2000,194:239-247), alginate (A.Bernkop-Schn ü rch, et al, J.Control.Release2001,71:277-285) and chitosan (A.Bernkop-Schn ü rch, et al, Int.J.Pharm.2003,260:229-237) four base polymers relate to gel, three kinds of dosage forms of microgranule and tablet, but the report of the relevant sulfhydrylation liposome of Shang Weijian.
Summary of the invention
The objective of the invention is common eye drops holdup time weak point in conjunctival sac for overcoming prior art, cause its eye bioavailability low, and the compliance of semisolid dosage form is bad, is difficult for by defectives such as patient's acceptance, provides a kind of with bio-adhesive Liposomal formulation and preparation method thereof.
The present invention is with the liposome of the sulfydryl modification carrier as dosing eyes, the time of contact of coming prolong drug and cornea by the covalent bond between sulfydryl and the mucosa albumen, and then improve the eye bioavailability of medicine.
Eye of the present invention is made up of medicine three parts of sealing in liposome, liposome membrane decorative material and the liposome with the bio-adhesive Liposomal formulation.Described liposome is made of phospholipid and cholesterol.The phospholipid that is used to prepare this liposome can be soybean phospholipid and lecithin, and the weight ratio of itself and cholesterol is 2:1 to 16:1.In order to regulate the flowability of lipid film, in the liposome prescription also selectivity add an amount of vitamin E.
Described liposome membrane decorative material is the sulfhydrylation polymer.This polymer can be selected from polyacrylic acid, carboxymethyl cellulose, alginate and chitosan, wherein optimization polypropylene acid.Polyacrylic acid (PAA) molecular weight can be between 5,000～5, and 000,000, preferred molecular weight ranges is 100,000～5,000,000.Covalently bound a certain proportion of cysteine (Cys) and PHOSPHATIDYL ETHANOLAMINE (PE) form (Cys-PAA-PE) complex on the free carboxy of PAA, wherein the free sulfhydryl groups of Cys can with mucoprotein interaction, form disulfide bond, PE then can be embedded in the liposome membrane, and then sulfhydrylation is polymer-modified to surface of liposome.The amount that connects Cys on the PAA molecule can be between 50～5000 μ mol/g, preferred 500～2000 μ mol/g.The consumption of this sulfhydrylation polymer in prescription accounts for 1～5% of liposome membrane material gross weight.
Described medicine can be the various medicines that are used for the treatment of ocular disease, and the consumption in prescription accounts for 0.1～10% of liposome membrane material gross weight, and is preferred 0.1～5%, and described medicine is selected from a kind of in the following medicine or their compound recipe:
1) anti-glaucoma medicine is as pilocarpine, beta-blocker etc.;
2) antibiotic is as beta-lactam, Tetracyclines, aminoglycosides, Macrolide, chloromycetin and derivant thereof etc.;
3) antibacterials are as quinolones, imidazoles etc.;
4) antiviral drugs is as ucleosides etc.;
5) steroidal anti-inflammatory drugs thing is as adrenocortical hormone etc.;
6) polypeptide protein class medicine is as alexin, interferon, cyclosporin and epidermal growth factor etc.
Described bio-adhesive liposome prepares by following step:
(1) preparation of liposome,
(2) the 2nd, the finishing of liposome.
Described liposome can adopt the method preparation of pharmaceutics field routine, becomes embrane method, alcohol injection, reverse evaporation, multi-emulsion method etc. as aquation.Becoming embrane method with aquation below is the example brief description: take by weighing phospholipid, cholesterol and the vitamin E of recipe quantity, with the chloroform dissolving, reduction vaporization is removed chloroform on Rotary Evaporators, obtains immobilized artificial membrane; The medicine dissolution of recipe quantity in an amount of water for injection or buffer salt, under oscillating condition, with this drug solution hydrated phospholipid film, is obtained the liposome crude product, obtained the satisfactory drug-loaded liposome of particle diameter through pushing film, freeze thawing or supersound process.Also can be with blank buffer solution hydrated phospholipid film, the method that adopts medicine carrying initiatively again with drug encapsulation in liposome.After treating that liposome preparation is good, can adopt the back imbedding method that sulfhydrylation is polymer-modified to surface of liposome.Concrete way is that liposome and sulfhydrylation polymer are bathed 30min altogether under the phase transition temperature (being generally 60 ℃) of phospholipid, the PE molecule that connects on the polymer is embedded in the liposome membrane spontaneous, forms the bioadhesive liposome that the surface connects the sulfhydrylation polymer.
Eye of the present invention is modified with free sulfydryl with the bio-adhesive surface of liposome, can form covalently bound disulfide bond with the mucoprotein subdomain that is rich in cysteine in eye surface, liposome is anchored on mucous membrane surface, as drug depot slowly release in conjunctival sac, for drug absorption provides persistent driving force.This bio-adhesive liposome helps lend some impetus to medicine in ocular absorption, can improve bioavailability of medicament.
Description of drawings
Fig. 1 is the bioadhesion liposome release in vitro curve that embodiment 9 bags carry fluorescein,
Wherein :-◆-contrast liposome ,-■-PAA-PE liposome ,-▲-the Cys-PAA liposome ,-●-calcein solution.
The specific embodiment
Below be embodiments of the invention.The purpose that provides specific embodiment is in order further to illustrate the present invention, but does not limit its protection domain.
Embodiment 1
The preparation of cysteine-polyacrylic acid (Cys-PAA) complex with separate, purification: get three parts of 1g PAA and add in the 100ml water, regulate between pH value to 7.2～7.5, it is disperseed in water with 2M NaOH.Each adds 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) 3.8g, and making its concentration is 200mM, and room temperature stirred 45 minutes down.Regulating pH value with 5M HCl is 4～5, and inflated with nitrogen 15 minutes adds Cys inflated with nitrogen stirring at normal temperature reaction 3 hours again.Three parts respectively to add the cysteine amount as shown in table 1.
Table 1
The dialysis separated product.Reactant liquor is transferred in the bag filter, in the 2000ml dialysis solution, dialyses under the lucifuge, 4 ℃.Dialysis solution is 1mM HCl for the first time, contains 2 μ M EDTA.Dialysis solution is 1mM HCl for the second time, contains 2 μ MEDTA, and comprises 1%NaCl, twice of same medium dialysis.At last once with 0.5mM HCl dialysis.Obtain reference substance D, reference substance E, reference substance F with identical preparation and isolating method simultaneously.
Table 2 is PAA:Cys mass ratioes of reference substance D, reference substance E, reference substance F.
Table 2
With product and reference substance lyophilization, obtain the white solid powder of Cys-PAA complex.
Embodiment 2
The mensuration of sulfhydryl content in the Cys-PAA complex: the quantity of sulfydryl can utilize the DTNB method to measure.Getting each 2mg of product and reference substance uses 10m10.5M phosphate buffer (PH8.0) to disperse respectively, respectively get 0.5ml, add 0.5ml0.03%5,5-dimercapto-2,2-dinitrobenzoic acid (DTNB) solution, room temperature was placed 2 hours, got 0.3ml and moved into 96 orifice plates, put into microplate reader and surveyed absorbance (absorption value is set 405nm).Obtain cysteine solution standard curve Y=163.38X+28.456, calculate sulfhydryl content among the Cys-PAA with this.The sulfhydryl content of gained is as shown in table 3 behind deduction blank product (D, E, the F) absorbance:
Table 3
Embodiment 3
The bioadhesive evaluation of PAA-Cys complex: get adult rat, put to death the anesthesia back, dissects, and gets small intestinal mucosa, and-20 ℃ of preservations are stand-by.
Get an amount of PAA-Cys (1:1) respectively, PAA-Cys (1:2), PAA-Cys (1:4), PAA are made into and contain 0.2%PAA solution.Be divided into two parts then, a extremely neutral with 2M NaOH adjusting pH value, it is 3 that another part regulated pH value with 5M HCl.Equally the pure water pH value is also transferred to neutral and 3.The above-mentioned solution that will prepare is respectively respectively got 0.2ml and is dripped in the small intestinal mucosa surface, measures its 10 minutes and 30 minutes adhesive force (mg) with torsion balance, each sample replication 6 times.The gained adhesive force the results are shown in Table 4:
Table 4 (neutrality)
The result shows, behind the introducing Cys, can improve its bio-adhesive performance in the PAA molecule, and the degree of bio-adhesive performance raising, and is relevant with the amount of its contained Cys.
Embodiment 4
Cysteine-polyacrylic acid-PHOSPHATIDYL ETHANOLAMINE (Cys-PAA-PE) complex prepares and separates: get 0.08g PAA and be scattered among the 12ml DMSO, be stirred to dissolving fully, and add initiator DMAP0.002g and DCC0.04g, and be heated to 60 ℃, be stirred to dissolving fully.Get 0.01g PE and add 0.5ml CHCl
3In, be heated to 60 ℃ and add to after it is fully dissolved and stir among the DMSO and keep 60 ℃ of reactions 8 hours.Reaction is spent the night under the room temperature.
Separated product in second day.Divide precipitation, flush away solvent and initiator DMAP and DCC with reactant three times with 50ml acetone.Centrifugal 10 minutes of 4000r/min obtains white thick semisolid.Rotary evaporation is removed acetone as far as possible.Water will precipitate dispersion once more.0.1N NaOH regulate the pH value of aqueous systems, it is remained between 7.2～7.5, drips NaOH while stirring and dissolve substantially to spawn, and system is better mobile, till solution is milky.It is transferred to lyophilization in the round-bottomed flask.Obtain the white powder product after the lyophilizing, productive rate is respectively 87.7%.
Get the PAA-PE complex, prepare liposome membrane decorative material Cys-PAA-PE according to the method for embodiment 1.And measure its sulfhydryl content according to embodiment 2 methods, in the product sulfhydryl content be 1.106mmol/g.
The preparation of humanized's alexin liposome: with phospholipid: the ratio of cholesterol (2:1) takes by weighing an amount of phospholipid and cholesterol, the chloroform dissolving, and the rotation film forming places vacuum drying oven, and room temperature condition is transferred the usefulness of purchasing; In above-mentioned flask, add certain density alexin solution, be higher than phospholipid phase transition temperature water-bath vibration 2 hours, through water-bath/frozen-thawed 10 times, promptly get the human alpha-defensin liposome again.
Embodiment 6
The BCA method is measured liposome encapsulation: according to the explanation of BCA protein content test kit, be abscissa with alexin concentration, the absorbance A value is that vertical coordinate is drawn the human alpha-defensin standard curve.The human alpha-defensin liposome adds an amount of Triton x-100 and destroys liposome membrane after the high speed centrifugation abandoning supernatant, and with alexin content in the kit measurement liposome, calculating its envelop rate is 40.22 ± 8.93%.
Embodiment 7
Bag carries the preparation of the bioadhesion liposome of fluorescein: take by weighing phosphatidylcholine (PC) 0.02g and cholesterol 0.005g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing an amount of calcein, the adding distil water dissolving, stirring adds 4M NaOH down and regulates pH value to 7.4, and being diluted with water to calcein solution final concentration is 50mM.
The calcein solution 4ml for preparing is added in the round-bottomed flask of liposome membrane, and making phospholipid concentration wherein is 5mg/ml.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.Solution is dark brown red.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the orange red part that about 1/3 column volume eluting comes out, promptly get and wrap the liposome that carries calcein.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 0.5mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 2ml wraps the liposome (crossing post) that is loaded with calcein hatched under 60 ℃ 0.5 hour jointly.Obtain orange red suspension, be the bioadhesion liposome that bag carries fluorescein.
Embodiment 8
Bag carries the mensuration of the bioadhesion surface of liposome sulfhydryl content of fluorescein: get the bioadhesion liposome that bag carries fluorescein, centrifugal 10 minutes of 5000r/min, get supernatant and cross gel column, collect the orange red liposome part that 1/3 column volume comes out, get the 1ml iodometric titrationiodimetry titration.Get precipitation simultaneously, use iodometric titrationiodimetry titration after the phosphate buffer dispersion with pH7.4.Measurement result substitution standard curve y=20.8176x+34.8922 is calculated, obtain that sulfydryl concentration is 1.206 μ M in the supernatant, sulfydryl concentration is 3.128 μ M in the precipitation.
Embodiment 9
Bag carries the release in vitro of the bioadhesion liposome of fluorescein: adopt dialysis to investigate the release in vitro that bag carries the bioadhesion liposome of calcein.Liposome, the calcein solution that the liposome that the liposome of unmodified, PE-PAA complex are modified, Cys-PAA-PE complex are modified is respectively got 1ml, and to be added to molecular cut off be to tighten in 12000～14000 the bag filter, puts 50ml pH7.4 physiology etc. is housed oozes in the tool plug conical flask of PBS.2 parts of parallel tests, 37 ℃ of water-bath concussions respectively at 0.5,1,2,4,6,8,10,12,24 hour sampling 1ml, replenish the equal-volume blank medium simultaneously.Every hole adds the sample of 300 each time point of μ l on 96 hole white background fluorescent screens, and each sample is done 3 multiple holes, is that 572nm, emission wavelength are that 536nm measures fluorescent value (F) with the absorbing wavelength on LS-55 type spectrofluorophotometer.From the release in vitro curve (Fig. 1) of each sample as can be seen, the bioadhesion liposome has the good slow release performance.
Bag carries the preparation of the bioadhesion liposome of pilocarpine: take by weighing phosphatidylcholine (PC) 0.02g and cholesterol 0.01g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing an amount of pilocarpine, add the dissolving of phosphoric acid buffer solution.The pilocarpine solution 5ml for preparing is added in the round-bottomed flask of liposome membrane.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the part that about 1/3 column volume eluting comes out, promptly get and wrap the liposome that carries pilocarpine.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 1.5mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 5ml wraps the liposome (crossing post) that is loaded with pilocarpine hatched under 60 ℃ 0.5 hour jointly.Obtain wrapping the bioadhesion liposome that carries pilocarpine, wherein pilocarpine accounts for 1% of liposome gross weight.
Embodiment 11
The preparation of the bioadhesion liposome of bag oxygen carrier Flucloxacillin: take by weighing phosphatidylcholine (PC) 0.64g and cholesterol 0.04g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing an amount of ofloxacin, add the dissolving of phosphoric acid buffer solution.The Ofloxacin solution 40ml for preparing is added in the round-bottomed flask of liposome membrane.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the part that about 1/3 column volume eluting comes out, promptly get the liposome that wraps the oxygen carrier Flucloxacillin.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 6.8mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 40ml wraps the liposome (crossing post) that is loaded with ofloxacin hatched under 60 ℃ 0.5 hour jointly.Obtain wrapping the bioadhesion liposome of oxygen carrier Flucloxacillin, wherein ofloxacin accounts for 10% of liposome gross weight.
Embodiment 12
Bag carries the preparation of the bioadhesion liposome of acyclovir: take by weighing phosphatidylcholine (PC) 0.4g and cholesterol 0.05g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing an amount of acyclovir, add the sodium bicarbonate buffer solution dissolving.The acyclovir solution 8ml for preparing is added in the round-bottomed flask of liposome membrane.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the part that about 1/3 column volume eluting comes out, promptly get and wrap the liposome that carries acyclovir.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 18mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 8ml wraps the liposome (crossing post) that is loaded with acyclovir hatched under 60 ℃ 0.5 hour jointly.Obtain wrapping the bioadhesion liposome that carries acyclovir, wherein acyclovir accounts for 5% of liposome gross weight.
Embodiment 13
Bag carries the preparation of the bioadhesion liposome of econazole: take by weighing phosphatidylcholine (PC) 0.5g and cholesterol 0.05g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing an amount of econazole, add the buffer solution dissolving.The econazole solution 10ml for preparing is added in the round-bottomed flask of liposome membrane.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the part that about 1/3 column volume eluting comes out, promptly get and wrap the liposome that carries econazole.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 16.5mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 10ml wraps the liposome (crossing post) that is loaded with econazole hatched under 60 ℃ 0.5 hour jointly.Obtain wrapping the bioadhesion liposome that carries econazole, wherein econazole accounts for 8% of liposome gross weight.
Embodiment 14
Bag carries the preparation of the bioadhesion liposome of ground thiophene rice pine: take by weighing phosphatidylcholine (PC) 0.48g and cholesterol 0.04g (CHOL) in the 50ml round-bottomed flask, add an amount of chloroform and make its dissolving.40 ℃ of water-baths rotation down are evaporated to solvent and volatilize fully, put that dried overnight promptly gets liposome membrane in the vacuum drying oven.
Take by weighing thiophene rice pine in right amount, add the buffer solution dissolving.The ground thiophene rice pine solution 6ml for preparing is added in the round-bottomed flask of liposome membrane.In 65 ℃ of water-baths with 50 shake/minute frequency oscillation 2 hours.
The liposome solutions that aquation is good is crossed the 100nm film, collects liposome after pushing 20 times.The liposome of collecting is crossed Sepharose CL-4B silicagel column, and eluent is a 0.9%NaCl solution.Collect the part that about 1/3 column volume eluting comes out, promptly get and wrap the liposome that carries ground thiophene rice pine.
Take by weighing embodiment 4 prepared Cys-PAA-PE complex 5.2mg and join 0.5ml, pH7.4 waits in the phosphatizing acid buffer, and drips the NaOH adjusting pH value of 0.1N, makes it fully disperse the liquid that is creamy white.Adding 6ml wraps the liposome (crossing post) that is loaded with ground thiophene rice pine hatched under 60 ℃ 0.5 hour jointly.Obtain wrapping the bioadhesion liposome that carries ground thiophene rice pine, wherein thiophene rice pine accounts for 0.1% of liposome gross weight.
Claims (11)
1. eye bio-adhesive Liposomal formulation, it is characterized in that liposome membrane is made of phospholipid and cholesterol, the two weight ratio is 2: 1 to 16: 1, described surface of liposome is modified with cysteine-polyacrylic acid-PHOSPHATIDYL ETHANOLAMINE complex, its consumption accounts for 1～5% of liposome membrane material gross weight, described complex is after treating that liposome preparation is good, adopts the back imbedding method that this complex is modified surface of liposome; Polyacrylic molecular weight is between 5 in described cysteine-polyacrylic acid-PHOSPHATIDYL ETHANOLAMINE complex, 000～5,000,000, connect the amount of cysteine on the polyacrylic acid molecule between 50～5000 μ mol/g, carry the medicine for the treatment of ocular disease with bag in the described liposome, its dosage accounts for 0.1～10% of liposome membrane material gross weight.
2. eye according to claim 1 is used the bio-adhesive Liposomal formulation, and the phospholipid that it is characterized in that constituting liposome membrane is soybean phospholipid and lecithin.
3. eye according to claim 1 is used the bio-adhesive Liposomal formulation, it is characterized in that in cysteine-polyacrylic acid-PHOSPHATIDYL ETHANOLAMINE complex polyacrylic molecular weight between 100,000～5,000,000.
4. eye according to claim 1 is used the bio-adhesive Liposomal formulation, it is characterized in that connecting on the polyacrylic acid molecule in cysteine-polyacrylic acid-PHOSPHATIDYL ETHANOLAMINE complex the amount of cysteine between 500～2000 μ mol/g.
5. eye according to claim 1 is used the bio-adhesive Liposomal formulation, it is characterized in that the dosage that wraps the medicine carrying thing accounts for 0.1～5% of liposome membrane material gross weight.
6. eye according to claim 5 is used the bio-adhesive Liposomal formulation, it is characterized in that described medicine is a kind of in the anti-glaucoma medicine or their compound recipe.
7. use the bio-adhesive Liposomal formulation for a kind of according to claim 5, it is characterized in that described medicine is a kind of in the antibiotics or their compound recipe.
8. eye according to claim 5 is used the bio-adhesive Liposomal formulation, it is characterized in that described medicine is a kind of in the antibacterials or their compound recipe.
9. eye according to claim 5 is used the bio-adhesive Liposomal formulation, it is characterized in that described medicine is a kind of in the antiviral drugs or their compound recipe.
10. eye according to claim 5 is used the bio-adhesive Liposomal formulation, it is characterized in that described medicine is a kind of in the steroidal anti-inflammatory class medicine or their compound recipe.
11. eye according to claim 5 is used the bio-adhesive Liposomal formulation, it is characterized in that described medicine is a kind of in protein and the polypeptide drug or their compound recipe.
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| CN102793672A (en) * | 2012-08-21 | 2012-11-28 | 南京医科大学 | Chitosan-modified methazolamide solid lipid nanoparticles and preparation method thereof |
| CN103614799A (en) * | 2013-11-06 | 2014-03-05 | 东华大学 | Preparation method of natural material-ethosome composite nanofiber |
| CN104492396B (en) * | 2014-12-10 | 2016-08-17 | 西南大学 | Preparation method of target liposomes based on protein molecular trace and products thereof |
| CN104922069B (en) * | 2015-05-28 | 2019-04-09 | 燕山大学 | A kind of nano gold spherical shell photosensitive liposomes and preparation method thereof |
| CN116759581B (en) * | 2023-08-17 | 2024-04-19 | 广东工业大学 | Thiolated polyacrylic acid aqueous polymer binder for silicon-based negative electrode of lithium ion battery, and preparation method and application thereof |
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| CN1528271A (en) * | 2003-09-25 | 2004-09-15 | 复旦大学 | Method for producing bio-adhered micro-ball medicine preparation for gastrointestinal tract |
| CN101199505A (en) * | 2007-12-20 | 2008-06-18 | 沈阳药科大学 | Verapamil liposome and preparing method thereof |
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| CN1528271A (en) * | 2003-09-25 | 2004-09-15 | 复旦大学 | Method for producing bio-adhered micro-ball medicine preparation for gastrointestinal tract |
| CN101199505A (en) * | 2007-12-20 | 2008-06-18 | 沈阳药科大学 | Verapamil liposome and preparing method thereof |
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| 刘哲鹏等.生物粘附微球研究及前景.《国外医药－合成药 生化药 制剂分册》.2001,第22卷(第2期),第111－114页. * |
| 徐雯等.受体介导脂质体的研究进展.《国外医药-- 合成药、生化药、制剂分册》.2002,第23卷(第5期),第293-297页. * |
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