CN102614498B - Insulin nanoparticle and preparation method thereof - Google Patents
Insulin nanoparticle and preparation method thereof Download PDFInfo
- Publication number
- CN102614498B CN102614498B CN201110031305.5A CN201110031305A CN102614498B CN 102614498 B CN102614498 B CN 102614498B CN 201110031305 A CN201110031305 A CN 201110031305A CN 102614498 B CN102614498 B CN 102614498B
- Authority
- CN
- China
- Prior art keywords
- insulin
- nanoparticles
- preparation
- insulin nanoparticles
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention relates to the technical field of drugs, and especially relates to an insulin nanoparticle and a preparation method thereof. The preparation method comprises the following steps: preparation of an insulin ion pair compound and preparation of the insulin nanoparticle. According to the insulin nanoparticle of the invention, the entrapment rate and the drug loading are high, the particle size distribution is uniform, and a certain slow releasing effect is possessed, so drugs can be effectively prevented from being degraded by proteases, the stability of the drugs can be increased, and a case that the drugs permeate a biomembrane is promoted, thereby curative effects of the drugs are improved, the toxicity of the drugs is reduced, the biological utilization degree of the drugs is increased, and the insulin nanoparticle is suitable for various administration approaches of the drugs; preparation conditions are mild, no violent conditions of high temperature, and high speed shearing force or the like are needed, so the biological activities of the drugs can be guaranteed; and a surfactant Cremophor EL used in the preparation process can inhibit P-glycoprotein, so the permeability of the drugs going through cell membranes is improved, and the absorption of the drugs going through intestinal tract walls to enter blood is increased.
Description
Technical field
The present invention relates to medical art, particularly a kind of insulin nanoparticles and preparation method thereof.
Background technology
Diabetes are a kind of common chronic metabolic disease, caused by insulin deficit in human body or tolerance.Diabetes have very large harm to health, can cause cardiovascular disease, dyslipidemia, the complication that blind, renal failure and amputation etc. are serious, become the fourth-largest disease causing population in the world death.Estimate according to IDF (IDF), within 2007, China's diabetics number is about 3,980 ten thousand, and wherein the overwhelming majority is type ii diabetes patient, accounts for 90% ~ 95% of diabetes diagnosis case.
Insulin is one of the most effective Remedies for diabetes, and for type i diabetes (accounting for 10% of all diabetes) patient, insulin is unique medicine; For type ii diabetes (accounting for more than 90% of all diabetes) patient, still need to utilize medicine to control blood glucose and raise, prevent and treat the generation of complication, wherein the type ii diabetes patient of 30% ~ 40% finally needs to use insulin.Therefore, insulin be I type and in, indispensable medicine in the daily treatment of severe type ii diabetes patient, be also the most effective unique medicine recognized in the world.Because insulin belongs to protein substance, molecular weight is large, fat-soluble difference, not easily through biomembrane, all the time based on drug administration by injection, but be administered to patient for a long time and frequently and bring very large misery and inconvenience, therefore, the long acting injection of the insulin that abroad begun one's study and non-injection administration preparation.
In recent years, along with the birth of various novel medicament release device and the development of various new formulation technology, develop the broad interest that oral or other insulin preparations easy to use have caused domestic and international researcher, the research of long-acting injection and non-injection insulin preparation has been made significant headway, as protamine zine insulin injection Basulin, inhaled insulin preparation Exubera etc.The Basulin that Bristol-Myers Squibb company and French Flamel Technologies S.A. company develop cooperatively, it is the protamine zine insulin injection carried by nanotechnology Medusa, be designed to drug administration by injection 1 time every day, for the treatment of I type and type ii diabetes, now enter II phase clinical development, demonstrate certain potential applicability in clinical practice (US2009110742 (A1), Long-acting colloidal insulin formulation and its preparation, Alain Constancis, Florence Nicolas, Remi Meyrueix, Olivier Soula, FLAMEL TECHNOLOGIES).The insulin atomized inhalation Exubera through lung inhalation of Sanofi-Aventis (Sanofi-Aventis) company of France and the cooperative development of Pfizer (Pfizer) drugmaker was approved listing by U.S. FDA in January, 2006, use special inhaler direct oral cavity inhalation, the insulin of atomization is sent to patient lungs.Although Exubera likely causes the moderate lesion of abnormal pulmonary function and pulmonary function, and by the unstability of pulmonary administration, in October, 2007, Pfizer announces that Exubera removes city, but this has been except injecting pathway first new insulin administration methods since nineteen twenty insulin invention, be regarded as a large invention in treating diabetes field, certain effect is served to the development of Non-parenteral Delivery Routes.
At present, it is the preparation such as carrier or the nanocapsule adding enzyme inhibitor, protective agent and absorption enhancer, nanoparticle, liposome or emulsion that the insulin novel form developed both at home and abroad also comprises with macromolecular material.The document of domestic and international research insulin nano drug-supplying system is more, especially for the Non injection administration system of insulin, at oral formulations, suck preparation, preparation capable of permeating skin, the aspects such as rectally have made some progress, but still have such as that biological insulin availability is low, dosage is difficult to accurate control, quality stability is poor, use the problems such as compliance difference, therefore, be necessary to research and develop the nano particle preparations had compared with high encapsulation rate and drug loading, reach the compliance that the preparation with certain slow-releasing and multiple route of administration uses to increase patient.
In protein molecular structure, amino or guanidine radicals dissociates and in acid condition with positive charge, and anion surfactant contains electronegative polar group, therefore, protein with positive charge can occur electrically to combine with electronegative anion surfactant, form ion-pair complexes (hydrophobic ion pairs complex, HIP complex).Protein can reduce its water-soluble after formation ion-pair complexes, increases dissolubility in organic solvent, and concerning a variety of protein, ion-pair complexes can also make the structure and activity of protein remain unchanged increasing fat-soluble while.
Insulin contains 6 basic groups, i.e. 1 arginine, 1 lysine, 2 histidine, 2 amino terminals, and 4 acidic-groups.When pH value is 2.5, because the amino acid residues such as the arginine on insulin peptide chain, lysine, histidine are protonated and be with 6 positive charges, anion surfactant is as sodium lauryl sulphate (SDS, sodium laurylsulfate) be with 1 negative charge, the two combines by electrostatic attraction, and the hydrophobic amino acid residue of insulin and the hydrophobic group hydrocarbon chain of SDS also have combination simultaneously.Therefore, under finite concentration condition, the SDS of 6 mol and the insulin of 1 mol form complex, form precipitation in aqueous.Precipitate at most when the ratio of SDS and insulin mole is 6: 1, the distribution simultaneously in water and 1-capryl alcohol also reaches maximum.Above-mentioned formed precipitation, will be more stable than insulin after collection, drying, can be stored in higher temperature and humidity and have no significant effect its biological activity.Simultaneously ion-pair complexes dissolves in some solvents miscible with water, as methanol, ethanol, N-Methyl pyrrolidone, in trimethyl phosphate etc., and at the middle indissoluble such as dioxane, acetonitrile (ACN), ether, monochloro methane and ethane.
In addition, insulin also can form ion-pair complexes with anion surfactants such as NaTDC (DOC), enuatrols (Sodium oleate).
Patent US6613358(Sustained-release composition including amorphous polymer, 2003, Theodore W. Randolph, Mark C. Manning, Richard F. Falk) and WO/1994/008599 (Ion-pairing of drugs for improved efficacy and delivery, SHEFTER, Eli, MANNING, Mark, G.) comparatively detailed research has been carried out to the relevant preparation method of ion-pair complexes and physicochemical property, and proposed ion-pair complexes there is certain slow-releasing.(the Hong Yuan such as Hong Yuan, Sai-Ping Jiang, Yong-Zhong Du, Jing Miao, Xing-Guo Zhang, Fu-Qiang Hu. Strategic approaches for improving entrapment of hydrophilic peptide drugs by lipid nanoparticles. Colloids and surfaces. B, Biointerfaces, 2009, 70 (2): 248-253.) have studied leuprorelin and enuatrol forms ion-pair complexes LR-SA-Na(leuprolide-sodium stearate) after obviously can increase the envelop rate of medicine, but envelop rate is less than 80%, and drug loading is less than 1%.Therefore, can be applicable to clinical pharmaceutical preparation to prepare, entrapment efficiency and the lower problem of drug loading must be solved.
Summary of the invention
The object of the present invention is to provide a kind of insulin nanoparticles and preparation method thereof, the preparation method of a kind of insulin nanoparticles colloid solution and preparation thereof is provided especially.
The present invention seeks to be achieved through the following technical solutions:
The preparation method of insulin nanoparticles of the present invention comprises the steps:
Step one: prepare insulin ion-pair complexes;
Step 2: the insulin ion-pair complexes lyophilization powder obtained with step one, for crude drug, prepares insulin nanoparticles;
Described in above-mentioned steps one, the preparation method of insulin ion-pair complexes comprises the steps:
(1) 100 ~ 200 weight portion insulins, 35 ~ 65 weight portion anion surfactants are dissolved in respectively pH value be less than 5.3 acidic aqueous solution, obtain the anionic surfactant solution that insulin solutions that concentration is 5 ~ 15 mg/ml and concentration are 0.5 ~ 5 mg/ml in buffer or water;
(2) according to insulin in insulin solutions positively charged number and anion surfactant the ratio of electronegative number be the ratio of 4 ~ 8:1, the anionic surfactant solution that step (1) is obtained joins in the solution of insulin, slow dropping is constantly jolting also, namely separates out white or near-white precipitate; White obtained above or near-white precipitation are left standstill, to be combined completely after, solution is carried out centrifugalize, obtain centrifugal after white depositions be insulin ion-pair complexes;
(3) get the insulin ion-pair complexes that step (2) is obtained, after adsorbable free drug and anion surfactant, lyophilization in freezer dryer can be placed in, obtain its lyophilization powder with water or buffer solution removing.
Wherein, described insulin includes but not limited to animal insulin, recombinant human insulin (Recombinant human insulin, rhINS), biosynthetic human insulin, insulin compound analog etc.; Described insulin compound analog is the insulin human, native proinsulin compound etc. with Lys28 and Pro29;
Wherein, the effect of described anion surfactant be make the complex of described insulin compared with the insulin compounds of corresponding non-compound fat-soluble more greatly, more stable, higher temperature and humidity can be stored in and its biological activity is had no significant effect;
Wherein, described ion pair is anion surfactant, includes but not limited to sodium lauryl sulphate (SDS), dodecylbenzene sodium sulfonate, sodium hexadecyl sulfate (spermol sodium sulfate), sodium stearyl sulfate (stearyl alcohol sodium sulfate), NaTDC (DOC), enuatrol etc.;
The ion-pair complexes that above-mentioned steps one obtains dissolves in as dimethyl sulfoxine (DMSO), N, in dinethylformamide (DMF), Aerosol OT (AOT), methanol, ethanol, N-Methyl pyrrolidone, trimethyl phosphate, 2-propanol, and be insoluble in dioxane, ACN, ether, monochloro methane and ethane equal solvent.
The preparation method of the insulin nanoparticles described in above-mentioned steps two comprises solvent diffuse (volatilization) method, polymerization, coprecipitation, multi-emulsion method, self-emulsifying microemulsion etc., and conventional preparation method is solvent diffuse (volatilization) method, and its key step comprises:
(1) 1 parts by weight polymer is dissolved in organic solvent makes the organic facies that concentration is 20 ~ 500 mg/ml; During as formulation properties need be improved, in the above-mentioned organic solvent that appropriate fat-soluble additives can be dissolved in 1 ~ 5 ml or other organic solvent, then join in the organic facies containing polymer;
(2) get 0.05 ~ 1 weight portion insulin ion-pair complexes lyophilization powder join step (1) containing polymer organic facies in make dissolving, or in the organic solvent being dissolved in step (1) or other organic solvent, the concentration of insulin-containing ion-pair complexes in the organic facies obtained is made to be 5 ~ 150 mg/ml, this organic facies joined in the organic facies of step (1), under the condition of room temperature or ice bath, homogenize or ultrasonic homogenize obtain the organic facies containing medicine and polymer again;
(3) organic facies containing medicine and polymer obtained in 1 parts by volume step (2) is got, slowly join 1 ~ 10 volume doubly containing in the aqueous phase solution of certain density surfactant, under the condition of room temperature or ice bath, fully form Emulsion after homogenize or ultrasonic emulsification;
(4) get the Emulsion formed in 1 parts by volume step (3) and join 1 ~ 10 volume doubly containing in the aqueous phase solution of certain density surfactant, rotary evaporation under room temperature or 40 below C water bath condition, or magnetic agitation, organic solvent is spread, volatilizees, namely obtain the obvious nanoparticle colloid solution of opalescence;
(5) nanoparticle colloid solution that will be obtained, according to the requirement of route of administration and dosage form design, can add customary adjuvant or freeze drying protectant, preserve after routine preservation, cryopreservation or lyophilization;
Wherein, in step 2 (1), polymer used is biodegradable high molecular polymer, as polylactic acid (PLA), polyglycolic acid (PGA), Poly(D,L-lactide-co-glycolide (PLGA), the PLA that Polyethylene Glycol (PEG) is modified, the copolymer of PGA or PLGA, poe, polycaprolactone, Polyalkylcyanoacrylanano, polyethyleneglycol modified Polyalkylcyanoacrylanano, PTMC, the copolymer of polylactic acid and trimethylene carbonate, the copolymer of polyglycolic acid and trimethylene carbonate, the copolymer of poly lactic-co-glycolic acid and trimethylene carbonate, one or more in polymethyl methacrylate (PMMA) etc.,
Wherein, the fat-soluble additives described in step 2 (1) comprise phospholipid, medium chain triglycerides, glycerol etc.;
Wherein, in step 2 (1), (2), organic solvent used comprises the mixed liquor of ethyl acetate (EA), dichloromethane (DCM), ACN, acetone (AC), ethanol (EtOH), methanol (MeOH), dimethyl sulfoxine (DMSO), N-Methyl pyrrolidone and multiple organic solvent thereof, if EA and ACN volume ratio is 60 ~ 100: 0 ~ 40(v/v) mixed liquor;
Wherein, in step 2 (3), (4), surfactant used includes but not limited to polyoxyethylene (35) Oleum Ricini (Cremophor EL), polyvinyl alcohol (PVA), phospholipid, sodium cholate, NaTDC, poloxamer class, Tweens, brejs, and its mixed surfactant;
Wherein, in the aqueous phase solution of certain density surfactant described in step 2 (3), (4), the concentration of polyoxyethylene (35) surfactant such as Oleum Ricini, polyvinyl alcohol is 0.01% ~ 10%(g/ml), typical concentrations is 0.05% ~ 3.0%(g/ml), optimal concentration is 0.5% ~ 2.0%(g/ml).
The nanoparticle form rounding that above-mentioned steps two is obtained, without adhesion, particle diameter is mainly distributed within the scope of 50 nm ~ 300 nm, narrower particle size distribution, and envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
The insulin nanoparticles that above-mentioned steps two obtains can be used for preparing the pharmaceutically acceptable various dosage forms such as injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, gel, ointment, patch, membrane, ophthalmic preparation or suppository.
The preparation method of insulin ion-pair complexes of the present invention comprises the steps:
Get 100 ~ 200 weight portion insulins, being dissolved in pH value is in the HCl solution of 2.0 ~ 3.0, and obtained concentration is the insulin solutions of 5 ~ 15 mg/ml; Separately get 35 ~ 65 weight portion SDS, be dissolved in pH value be 2.0 ~ 3.0 HCl solution or water in, obtained concentration is the SDS solution of 0.5 ~ 5 mg/ml; SDS solution joins in insulin solutions by the ratio being 4 ~ 8:1 according to the mol ratio of SDS and insulin, and slowly drip and constantly jolting, namely separating out white precipitate is insulin-sodium lauryl sulphate ion-pair complexes; White precipitate obtained above is left standstill, to be combined completely after, solution is carried out centrifugalize, gets precipitation, can adsorbable free drug and anion surfactant with water or buffer solution removing, namely obtain insulin ion-pair complexes powder through lyophilization;
Wherein, described insulin comprises animal insulin, recombinant human insulin etc.;
Wherein, described SDS can also substitute with DOC.
The preparation method of insulin nanoparticles colloid solution of the present invention comprises the steps:
Get insulin-NaTDC ion-pair complexes powder and be dissolved in DMSO or volume ratio is in DMSO and the DCM mixed solution of 1:1, the concentration obtaining insulin-containing ion-pair complexes is the organic facies of 5 ~ 150 mg/ml, separately get PLGA, wherein lactide/glycolides (LA/GA)=90/10 ~ 50/50, Mw=15000 ~ 50000, be dissolved in DCM or EA, making containing polymer concentration is the organic facies of 20 ~ 500 mg/ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer; The organic facies of above-mentioned drug containing and polymer is joined containing 0.1 ~ 5.0%(w/v under ultrasound condition and room temperature or condition of ice bath) in the aqueous solution of PVA or Cremophor EL, ultrasonicly under room temperature or condition of ice bath make formation O/W type Emulsion, again O/W type Emulsion is joined under stirring 1 ~ 10 times of emulsion volume containing 0.1 ~ 5.0%(w/v) in the aqueous solution of PVA or Cremophor EL, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be insulin nanoparticles colloid solution;
Wherein, described insulin-NaTDC ion-pair complexes can also use recombinant human insulin-sodium lauryl sulphate ion pair (rhINS-SDS) complex to substitute, and obtains recombinant human insulin's nanoparticle colloid solution.
The preparation method of insulin nanoparticles lyophilization powder of the present invention comprises the steps:
Get recombinant human insulin's nanoparticle colloid solution of 90 ~ 110 parts by volume, adding 2 ~ 10 weight portion concentration is 2% ~ 10%(g/ml) mannitol make dissolving as freeze drying protectant, mixing, get 1 ~ 5 ml in 10 ml cillin bottles, in-50 C ~-30 C pre-freeze 3 ~ 5 hours, lyophilization in freezer dryer, obtains recombinant human insulin's nanoparticle lyophilization powder;
Wherein, described mannitol can also substitute with freeze drying protectant acceptable on the pharmaceuticss such as lactose.
The pass of weight portion of the present invention and parts by volume is the relation of g and ml.
The advantages such as insulin nanoparticles of the present invention has envelop rate and drug loading is high, even particle size distribution, also there is certain slow-releasing simultaneously, can medicine be effectively prevented easily to be easily degraded by proteases, increase the stability of medicine, promote agent permeates therethrough biomembrane, thus improve curative effect of medication, reduce drug toxicity, increase drug bioavailability, can be used for the multiple route of administration of medicine; Its preparation condition is gentle, does not need the drastic conditions such as High Temperature And Velocity shearing force, can ensure the biological activity of medicine; Surfactant used can direct injection, and consumption is little, the nanoparticle colloid solution stable in properties of preparation; In insulin nanoparticles preparation process of the present invention, surfactant Cremophor EL used has and suppresses P-glycoprotein (P-gp) to improve medicine and pass through cell permeability of the membrane, increases medicine enters blood absorption by intestinal walls.
Below in conjunction with accompanying drawing, experimental example and embodiment, the invention will be further described.
Accompanying drawing explanation
Fig. 1: the infrared spectrogram being rhINS, DOC in experimental example 2, recombinant human insulin-NaTDC ion pair (rhINS-DOC) complex, rhINS and DOC mixture;
Fig. 2: the infrared spectrogram being rhINS, SDS, rhINS-SDS complex, rhINS and SDS mixture in experimental example 5;
Fig. 3: be the particles size and distribution of recombinant human insulin's nanoparticle in experimental example 8, Zeta potential figure (Zetasizer Nano ZS90; Take DCM as oil phase solvent, 1.0%(w/v) PVA be surfactant; Probe temperature: 30.00 C; Solvent viscosity: 0.89 mPas; Solvent refractive index: 1.33);
Fig. 4: transmission electron microscope figure (the HITACHI S-4800 being recombinant human insulin's nanoparticle in experimental example 10; Take EA as oil phase solvent, 0.5%(w/v) PVA be surfactant);
Fig. 5: the freeze-drying curve figure being recombinant human insulin's nanoparticle in embodiment 10;
Fig. 6: be add equal volume amounts 5.0%(w/v in recombinant human insulin's nanoparticle colloid solution in embodiment 10) mannitol when being freeze drying protectant, recombinant human insulin's nanoparticle colloid solution behind (left side) before lyophilization with lyophilization through jolting (in) and add suitable quantity of water redissolve after the outside drawing on (right side);
Fig. 7: be the particles size and distribution in experimental example 14 after the lyophilizing of recombinant human insulin's nanoparticle, Zeta potential figure (Zetasizer Nano ZS90; Take DCM as oil phase solvent, 1.0%(w/v) PVA be surfactant; Probe temperature: 30.00 C; Solvent viscosity: 0.89 mPas; Solvent refractive index: 1.33).
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
The combination rate of experimental example 1 recombinant human insulin-NaTDC measures
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); TDL-50B low speed desk centrifuge (Max RCF 2500 g, rotor is XTS05-2,15 ml × 8, Anting Scientific Instrument Factory, Shanghai);
Reagent: (tire is 29.7 IUmg to rhINS raw material
-1); RhINS standard substance (26.4 IUmg
-1, 140633-200302, Nat'l Pharmaceutical & Biological Products Control Institute); ICE company of DOC(Italy); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get centrifugal in the embodiment of the present invention 1 after supernatant, utilize high effective liquid chromatography for measuring medicine wherein, be unconjugated free drug, calculated the combination rate of recombinant human insulin and NaTDC by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements.
4, measurement result: the combination rate obtained when DOC and recombinant human insulin's mol ratio are 4:1 is 84.97%(RDS=0.50%).
The infrared spectrum of experimental example 2 recombinant human insulin-NaTDC complex is differentiated
Get each 1 ~ 2 mg of sample of the recombinant human insulin described in recombinant human insulin, NaTDC, the embodiment of the present invention 1-NaTDC complex, recombinant human insulin and NaTDC physical mixed respectively, porphyrize, and the potassium bromide mixing adding 100 ~ 200 mg grinds well rear YP-2 tablet machine and suppresses infrared sample strip, then respectively in the enterprising line scanning of PerkinElmer Spectrum 100 Fourier transform infrared spectrometer, obtain each infrared spectrogram, it the results are shown in Figure 1.
The combination rate of experimental example 3 insulins-sodium lauryl sulphate measures
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); TDL-50B low speed desk centrifuge (Max RCF 2500 g, rotor is XTS05-2,15 ml × 8, Anting Scientific Instrument Factory, Shanghai);
Reagent: (lot number is 0312A02 to insulin raw material, and tire 28 IUmg
-1, Tonghua Dongbao Pharmaceutical Co., Ltd); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73:27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get centrifugal in the embodiment of the present invention 3 after supernatant, utilize high effective liquid chromatography for measuring medicine wherein, be unconjugated free drug, calculated the combination rate of insulin and sodium lauryl sulphate by free drug amount and the ratio of the insulin total amount added of comparative measurements;
4, measurement result: the combination rate obtained when SDS and insulin mol ratio are 6:1 is 98.12%(RDS=0.94%).
The combination rate of experimental example 4 recombinant human insulin-sodium lauryl sulphate measures
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); TDL-50B low speed desk centrifuge (Max RCF 2500 g, rotor is XTS05-2,15 ml × 8, Anting Scientific Instrument Factory, Shanghai);
Reagent: (tire is 29.7 IUmg to rhINS raw material
-1); RhINS standard substance (26.4 IUmg
-1, 140633-200302, Nat'l Pharmaceutical & Biological Products Control Institute); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73:27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm.
3, assay method: get centrifugal in the embodiment of the present invention 4 after supernatant, utilize high effective liquid chromatography for measuring medicine wherein, be unconjugated free drug, calculated the combination rate of recombinant human insulin and sodium lauryl sulphate by free drug amount and the ratio of the insulin total amount added of comparative measurements;
4, measurement result: the combination rate obtained when SDS and recombinant human insulin's mol ratio are 6:1 is 99.82%(RDS=0.08%).
The infrared spectrum of experimental example 5 recombinant human insulin-sodium lauryl sulphate complex is differentiated
Get each 1 ~ 2 mg of sample of the recombinant human insulin described in recombinant human insulin, sodium lauryl sulphate, the embodiment of the present invention 4-sodium lauryl sulphate complex, recombinant human insulin and sodium lauryl sulphate physical mixed respectively, porphyrize, and the potassium bromide mixing adding 100 ~ 200 mg grinds well rear YP-2 tablet machine and suppresses infrared print, then respectively in the enterprising line scanning of PerkinElmer Spectrum 100 Fourier transform infrared spectrometer, obtain each infrared spectrogram, it the results are shown in Figure 2.
The envelop rate of experimental example 6 insulin nanoparticles colloid solution Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: insulin-NaTDC ion-pair complexes (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the insulin nanoparticles colloid solution described in the embodiment of the present invention 5 after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, uses the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in insulin nanoparticles colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of insulin in nanoparticle colloid solution by free drug amount and the ratio of the insulin total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle colloid solution Chinese medicine obtained with insulin-NaTDC ion-pair complexes is for 93.35%(RDS=0.35%), drug loading reaches 15.8%.
The envelop rate of experimental example 7 recombinant human insulin nanoparticle colloid solution Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: rhINS-SDS complex (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the recombinant human insulin's nanoparticle colloid solution described in the embodiment of the present invention 6 after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, use the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in recombinant human insulin's nanoparticle colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of recombinant human insulin in nanoparticle colloid solution by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle colloid solution Chinese medicine obtained with recombinant human insulin-sodium lauryl sulphate ion-pair complexes is for 95.73%(RDS=0.65%), drug loading reaches 16.5%.
The particle diameter of experimental example 8 recombinant human insulin nanoparticle, distribution and Zeta potential measure
Get the recombinant human insulin's nanoparticle colloid solution described in the embodiment of the present invention 6, utilize photon correlation spectroscopy to measure size and the distribution thereof of nanoparticle.At ambient temperature, get nanoparticle colloid solution, after dilute with water, the sample cell being placed in laser particle size analyzer/zeta potential instrument (Malvern Zetasizer Nano ZS90) measures size and the distribution thereof of nanoparticle.Utilize above-mentioned laser particle size analyzer/zeta potential instrument to measure the Zeta potential of nanoparticle simultaneously.As a result, the Z-mean diameter of nanoparticle is 154 nm, and dispersity index (PDI) is 0.16, and Zeta potential is-5.38 mV, records the size of nanoparticle and distribution thereof, Zeta potential figure is shown in Fig. 3.
The envelop rate of experimental example 9 recombinant human insulin nanoparticle colloid solution Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: rhINS-SDS complex (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2. chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the recombinant human insulin's nanoparticle colloid solution described in the embodiment of the present invention 7 after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, use the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in recombinant human insulin's nanoparticle colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of recombinant human insulin in nanoparticle colloid solution by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle colloid solution Chinese medicine obtained with recombinant human insulin-sodium lauryl sulphate ion-pair complexes is for 97.39%(RDS=0.47%), drug loading reaches 17.2%.
The form of experimental example 10 recombinant human insulin nanoparticle
Get the recombinant human insulin's nanoparticle colloid solution described in 1 ~ 2 embodiment of the present invention 7, drip on microscope slide dry, through HITACHI E-1010 ion sputtering instrument metal spraying, in HITACHI S-4800 cold field emission observed under electron microscope nanoparticle form and take pictures, the results are shown in Figure 4.
The envelop rate of experimental example 11 recombinant human insulin nanoparticle colloid solution Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: rhINS-SDS complex (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the recombinant human insulin's nanoparticle colloid solution described in the embodiment of the present invention 8 after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, use the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in recombinant human insulin's nanoparticle colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of recombinant human insulin in nanoparticle colloid solution by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle colloid solution Chinese medicine obtained with recombinant human insulin-sodium lauryl sulphate ion-pair complexes is for 92.14%(RDS=1.22%), drug loading reaches 16.7%.
The envelop rate of experimental example 12 recombinant human insulin nanoparticle colloid solution Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: rhINS-SDS complex (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the recombinant human insulin's nanoparticle colloid solution described in the embodiment of the present invention 9 after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, use the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in recombinant human insulin's nanoparticle colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of recombinant human insulin in nanoparticle colloid solution by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle colloid solution Chinese medicine obtained with recombinant human insulin-sodium lauryl sulphate ion-pair complexes is for 94.82%(RDS=0.97%), drug loading reaches 18.1%.
The envelop rate of experimental example 13 recombinant human insulin nanoparticle lyophilization product Chinese medicine and drug loading measure
1, instrument: high performance liquid chromatograph, comprises Alltech 426 type chromatogram pump, UVIS-200 type UV-vis detector, Alltech Data Processing in Chromatography Workstation (Alltech company of the U.S.); SUPELCO solid-phase extraction device (Visiprep DL SPE vacuum manifold standard, 12-port model, SUPELCO company of the U.S.); Solid-phase extraction column (3 ml, Shenzhen comma Bioisystech Co., Ltd);
Reagent: rhINS-SDS complex (self-control); Polydextran gel (G-75, Superfine, 17-0051-01, Amersham Biosciences company); ACN(chromatographically pure, Chemical Reagent Co., Ltd., Sinopharm Group); Anhydrous sodium sulfate (analytical pure, Tianjin Bo Di Chemical Co., Ltd.); Water is redistilled water; Other reagent such as phosphoric acid, hydrochloric acid is analytical pure;
2, chromatographic condition: chromatographic column: Shim-pack VP-ODS chromatographic column (150 mm × 4.6 mm, 5 μm, Japanese Shimadzu Corporation); Mobile phase: 0.2 moll
-1anhydrous slufuric acid sodium solution (with phosphoric acid adjust pH to 2.3)-ACN(73: 27, v/v); Flow velocity: 0.8 mlmin
-1; Column temperature: 40 C; Determined wavelength: 214 nm;
3, assay method: get the lyophilization of recombinant human insulin's nanoparticle described in the embodiment of the present invention 10 product, with redistilled water redissolve to lyophilization before the solution of same concentrations, after standardize solution, precision measures 0.4 ml, on the Sephadex G-75 of 3 ml solid-phase extraction columns, use the purified water eluting of 1.2 ml, obtain, not containing the nanoparticle of free drug, re-using 0.01 moll
-1hydrochloric acid solution eluting, collect in hydrochloric acid elute soln to 5 ml measuring bottle to scale, shake up, obtain non-encapsulated free drug in recombinant human insulin's nanoparticle colloid solution, utilize the concentration of high effective liquid chromatography for measuring wherein free drug, calculated the envelop rate of recombinant human insulin in nanoparticle colloid solution by free drug amount and the ratio of the recombinant human insulin's total amount added of comparative measurements;
4, measurement result: the envelop rate of the nanoparticle lyophilization product Chinese medicine obtained with recombinant human insulin-sodium lauryl sulphate ion-pair complexes is for 95.42%(RDS=0.90%), drug loading reaches 16.5%.
The particle diameter of experimental example 14 recombinant human insulin nanoparticle, distribution and Zeta potential measure
Get recombinant human insulin's nanoparticle lyophilization product described in the embodiment of the present invention 10, add with lyophilization before wait after water gaging redissolves, the sample cell being placed in laser particle size analyzer/zeta potential instrument (Malvern Zetasizer Nano ZS90) measures size and the distribution thereof of nanoparticle.Utilize above-mentioned laser particle size analyzer/zeta potential instrument to measure the Zeta potential of nanoparticle simultaneously.As a result, the Z-mean diameter of nanoparticle is 251 nm, PDI is 0.13, and Zeta potential is-9.57 mV, records the size of nanoparticle and distribution thereof, Zeta potential figure, sees Fig. 7.
Detailed description of the invention
Embodiment 1: the preparation of recombinant human insulin-NaTDC complex
Getting 100 mg recombinant human insulin, to be dissolved in pH value be in the HCl solution of 2.5, and obtained concentration is recombinant human insulin's solution of 6.8 mg/ml; Separately get 60 mg DOC soluble in water, obtained concentration is the DOC solution of 2.5 mg/ml, DOC solution joins in recombinant human insulin's solution by the ratio being 4:1 according to the mol ratio of DOC and recombinant human insulin, slow dropping is constantly jolting also, and namely separating out white precipitate is recombinant human insulin-NaTDC ion-pair complexes.White precipitate obtained above leaves standstill, to be combined completely after, solution is carried out centrifugalize, gets precipitation, can adsorbable free drug and anion surfactant with suitable quantity of water washing removing, namely obtain recombinant human insulin compound powder through lyophilization.
Embodiment 2: the preparation of insulin-NaTDC complex
Getting 100 mg insulins, to be dissolved in pH value be in the HCl solution of 2.5, obtained concentration is the insulin solutions of 8.4 mg/ml, separately get 60 mg DOC soluble in water, obtained concentration is the DOC solution of 0.7 mg/ml, DOC solution joins in insulin solutions by the ratio being respectively 8:1 according to the mol ratio of DOC and insulin, slow dropping is constantly jolting also, and namely separating out white precipitate is insulin-NaTDC ion-pair complexes; White precipitate obtained above is left standstill, to be combined completely after, solution is carried out centrifugalize, gets precipitation, can adsorbable free drug and anion surfactant with suitable quantity of water washing removing, namely obtain insulin complex substance powder through lyophilization.
Embodiment 3: the preparation of insulin-sodium lauryl sulphate complex
Get 100 mg insulins, to be dissolved in pH value be respectively in the HCl solution of 2.5 to 35 mg SDS, the SDS solution of obtained concentration to be the insulin solutions of 9.1 mg/ml and concentration be 2.1 mg/ml, SDS solution joins in insulin solutions by the ratio being 6:1 according to SDS and insulin mol ratio, slow dropping is constantly jolting also, namely separating out white precipitate is insulin-sodium lauryl sulphate ion-pair complexes, white precipitate obtained above leaves standstill, to be combined completely after, solution is carried out centrifugalize, get precipitation, use 0.01 moll
-1hydrochloric acid solution wash removing in right amount can adsorbable free drug and anion surfactant, namely obtain insulin complex substance powder through lyophilization.
Embodiment 4: the preparation of recombinant human insulin-sodium lauryl sulphate complex
Get 200 mg recombinant human insulin, it is in the HCl solution of 2.5 that 65 mg SDS are dissolved in pH value respectively, the SDS solution of obtained concentration to be recombinant human insulin's solution of 10.5 mg/ml and concentration be 3.4 mg/ml, be that SDS solution joins in recombinant human insulin's solution by the ratio of 6:1 according to SDS and recombinant human insulin's mol ratio, slow dropping is constantly jolting also, namely separating out white precipitate is recombinant human insulin-sodium lauryl sulphate ion-pair complexes, white precipitate obtained above leaves standstill, to be combined completely after, solution is carried out centrifugalize, get precipitation, use 0.01 moll
-1hydrochloric acid solution wash removing in right amount can adsorbable free drug and anion surfactant, namely obtain recombinant human insulin compound powder through lyophilization.
Embodiment 5: the preparation of insulin nanoparticles colloid solution
13 mg insulins-NaTDC ion-pair complexes powder that Example 2 obtains is dissolved in the DMSO of 0.5 ml, separately get the PLGA(LA/GA=75/25 of 50 mg, Mw=25000) be dissolved in the DCM of 0.5 ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer.The organic facies of above-mentioned drug containing and polymer is joined 3 ml under ultrasound condition and condition of ice bath and contains 1.0%(w/v) in the aqueous solution of PVA, ultrasonicly make formation O/W type Emulsion, again O/W type Emulsion is joined the 0.5%(w/v of 20 ml under stirring) in PVA aqueous solution, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be the nanoparticle colloid solution carrying insulin.
Embodiment 6: the preparation of recombinant human insulin's nanoparticle colloid solution
Example 4 obtained recombinant human insulin-sodium lauryl sulphate ion-pair complexes powder 13 mg is dissolved in the DMSO of 1.0 ml, separately get the PLGA(LA/GA=75/25 of 50 mg, Mw=15000) be dissolved in the DCM of 1.5 ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer.The organic facies of above-mentioned drug containing and polymer is joined volume 3 ml under ultrasound condition and condition of ice bath and contains 1.0%(w/v) in the aqueous solution of PVA, ultrasonicly make formation O/W type Emulsion, again O/W type Emulsion is joined the 1.0%(w/v of 50 ml under stirring) in PVA aqueous solution, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be the nanoparticle colloid solution carrying recombinant human insulin.
Embodiment 7: the preparation of recombinant human insulin's nanoparticle colloid solution
Example 4 obtained recombinant human insulin-sodium lauryl sulphate ion-pair complexes powder 14 mg is dissolved in the DMSO of 0.5 ml, separately get the PLGA(LA/GA=75/25 of 50 mg, Mw=15000) be dissolved in the EA of 1.2 ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer.The organic facies of above-mentioned drug containing and polymer is joined volume 5 ml under ultrasound condition and condition of ice bath and contains 0.5%(w/v) in the aqueous solution of PVA, ultrasonicly make formation O/W type Emulsion, again O/W type Emulsion is joined the 0.5%(w/v of 80 ml under stirring) in PVA aqueous solution, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be the nanoparticle colloid solution carrying recombinant human insulin.
Embodiment 8: the preparation of recombinant human insulin's nanoparticle colloid solution
Example 4 obtained recombinant human insulin-sodium lauryl sulphate ion-pair complexes powder 14 mg is dissolved in the DMSO of 2 ml, separately get the PLGA(LA/GA=80/20 of 50 mg, Mw=50000) be dissolved in the DCM of 2 ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer.The organic facies of above-mentioned drug containing and polymer is joined volume 8 ml under ultrasound condition and condition of ice bath and contains 0.5%(w/v) in the aqueous solution of Cremophor EL, ultrasonicly make formation O/W type Emulsion, again O/W type Emulsion is joined the 0.5%(w/v of 100 ml under stirring) in Cremophor EL aqueous solution, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be the nanoparticle colloid solution carrying recombinant human insulin.
Embodiment 9: the preparation of recombinant human insulin's nanoparticle colloid solution
The volume ratio that recombinant human insulin-sodium lauryl sulphate ion-pair complexes powder 15 mg that Example 4 obtains is dissolved in 1 ml is in DMSO and the DCM mixed solution of 1:1, separately get the PLGA(LA/GA=75/25 of 50 mg, Mw=15000) be dissolved in the DCM of 2 ml, above-mentioned two kinds of solution are mixed also supersound process make evenly, to obtain the organic facies of drug containing and polymer.The organic facies of above-mentioned drug containing and polymer is joined volume 7 ml under ultrasound condition and condition of ice bath and contains 1.0%(w/v) in the aqueous solution of PVA, ultrasonicly make formation O/W type Emulsion, again O/W type Emulsion is joined the 0.5%(w/v of 60 ml under stirring) in PVA aqueous solution, volatilization removing organic solvent, obtain the obvious colloid solution of opalescence, be the nanoparticle colloid solution carrying recombinant human insulin.
Embodiment 10: the preparation of recombinant human insulin's nanoparticle lyophilization powder
Recombinant human insulin's nanoparticle colloid solution 100 ml that Example 6 is obtained; the mannitol adding 3.5 g makes dissolving as freeze drying protectant; after mixing; get 2.0 ml in 10 ml cillin bottles; in-45 C pre-freeze 4 h; in LGJ-18S type freezer dryer after-40 C pre-freeze 3 h; lyophilization 8 h under the vacuum condition of 1 Pa ,-45 C; again respectively at lyophilization 2 h under lyophilization 4 h, 5 C conditions under-30 C ,-25 C ,-10 C, 0 C condition, obtain the dried frozen aquatic products of recombinant human insulin's nanoparticle.
Embodiment 11: the preparation of recombinant human insulin-NaTDC complex
Getting 100 mg recombinant human insulin, to be dissolved in pH value be in the HCl solution of 2.5, obtained concentration is recombinant human insulin's solution of 10 mg/ml, separately get 60 mg DOC soluble in water, obtained concentration is the DOC solution of 2 mg/ml, DOC solution joins in recombinant human insulin's solution by the ratio being 8:1 according to the mol ratio of DOC and recombinant human insulin, slow dropping is constantly jolting also, and namely separating out white precipitate is recombinant human insulin-NaTDC ion-pair complexes.White precipitate obtained above leaves standstill, to be combined completely after, solution is carried out centrifugalize, get centrifugal after ion-pair complexes namely obtain recombinant human insulin compound powder through lyophilization.
Embodiment 12: the preparation of recombinant human insulin's nanoparticle lyophilization powder
Recombinant human insulin's nanoparticle colloid solution 100 ml that Example 7 is obtained; the mannitol and the 1.0 g lactose that add 2.0 g make dissolving as freeze drying protectant; after mixing; get 2.0 ml in 10 ml cillin bottles; in-45 C pre-freeze 4 h; in LGJ-18S type freezer dryer after-40 C pre-freeze 3 h; lyophilization 8 h under the vacuum condition of 1 Pa ,-45 C; again respectively at lyophilization 2 h under lyophilization 4 h, 5 C conditions under-30 C ,-25 C ,-10 C, 0 C condition, obtain the dried frozen aquatic products of recombinant human insulin's nanoparticle.
Claims (28)
1. an insulin nanoparticles, is characterized in that the preparation method of this insulin nanoparticles comprises the steps:
Step one: prepare insulin ion-pair complexes;
Step 2: the insulin ion-pair complexes lyophilization powder obtained with step one, for crude drug, prepares insulin nanoparticles;
Wherein, described in step one, the preparation method of insulin ion-pair complexes comprises the steps:
(1) 100 ~ 200 weight portion insulins, 35 ~ 65 weight portion anion surfactants are dissolved in respectively pH value be less than 5.3 acidic aqueous solution, obtain the anionic surfactant solution that insulin solutions that concentration is 5 ~ 15 mg/ml and concentration are 0.5 ~ 5 mg/ml in buffer or water;
(2) according to insulin in insulin solutions positively charged number and anion surfactant the ratio of electronegative number be the ratio of 4 ~ 8:1, the anionic surfactant solution that step (1) is obtained joins in the solution of insulin, slow dropping is constantly jolting also, namely separates out white or near-white precipitate; White obtained above or near-white precipitation are left standstill, to be combined completely after, solution is carried out centrifugalize, obtain centrifugal after white or near-white precipitate be insulin ion-pair complexes;
(3) get the insulin ion-pair complexes that step (2) is obtained, after adsorbable free drug and anion surfactant, lyophilization in freezer dryer can be placed in, obtain its lyophilization powder with water or buffer solution removing;
Wherein, the preparation method of the insulin nanoparticles described in step 2 comprises the steps:
(1) 1 parts by weight polymer is dissolved in organic solvent makes the organic facies that concentration is 20 ~ 500 mg/ml, described polymer includes but not limited to polylactic acid, polyglycolic acid, Poly(D,L-lactide-co-glycolide, polyethyleneglycol modified polylactic acid, the copolymer of polyglycolic acid or poly lactic-co-glycolic acid, poe, polycaprolactone, Polyalkylcyanoacrylanano, polyethyleneglycol modified Polyalkylcyanoacrylanano, PTMC, the copolymer of polylactic acid and trimethylene carbonate, the copolymer of polyglycolic acid and trimethylene carbonate, the copolymer of poly lactic-co-glycolic acid and trimethylene carbonate, one or more in polymethyl methacrylate,
(2) get 0.05 ~ 1 weight portion insulin ion-pair complexes lyophilization powder join step (1) containing polymer organic facies in make dissolving, or in the organic solvent being dissolved in step (1) or other organic solvent, the concentration of insulin-containing ion-pair complexes in the organic facies obtained is made to be 5 ~ 150 mg/ml, this organic facies joined in the organic facies of step (1), under the condition of room temperature or ice bath, homogenize or ultrasonic homogenize obtain the organic facies containing medicine and polymer again;
(3) organic facies containing medicine and polymer obtained in 1 parts by volume step (2) is got, slowly join 1 ~ 10 volume doubly containing in the aqueous phase solution of certain density surfactant, under the condition of room temperature or ice bath, fully form Emulsion after homogenize or ultrasonic emulsification;
(4) get the Emulsion formed in 1 parts by volume step (3) and join 1 ~ 10 volume doubly containing in the aqueous phase solution of certain density surfactant, rotary evaporation under room temperature or less than 40 DEG C water bath condition, or magnetic agitation, organic solvent is spread, volatilizees, namely obtain the obvious nanoparticle colloid solution of opalescence;
(5) by obtained nanoparticle colloid solution, add customary adjuvant or freeze drying protectant, preserve after routine preservation, cryopreservation or lyophilization.
2. a kind of insulin nanoparticles as claimed in claim 1, it is characterized in that the insulin described in preparation method step one (1) of this insulin nanoparticles includes but not limited to animal insulin, recombinant human insulin, biosynthetic human insulin or insulin compound analog, wherein, insulin compound analog is insulin human or the native proinsulin compound with Lys28 and Pro29.
3. a kind of insulin nanoparticles as claimed in claim 1 or 2, is characterized in that the anion surfactant described in preparation method step one (1) of this insulin nanoparticles includes but not limited to sodium lauryl sulphate, dodecylbenzene sodium sulfonate, sodium hexadecyl sulfate, sodium stearyl sulfate, NaTDC or enuatrol.
4. a kind of insulin nanoparticles as claimed in claim 1 or 2, is characterized in that one or more that organic solvent used in the preparation method step 2 (1) of this insulin nanoparticles, (2) includes but not limited in ethyl acetate, dichloromethane, acetonitrile, acetone, ethanol, methanol, dimethyl sulfoxine, N-Methyl pyrrolidone.
5. a kind of insulin nanoparticles as claimed in claim 3, is characterized in that one or more that organic solvent used in the preparation method step 2 (1) of this insulin nanoparticles, (2) includes but not limited in ethyl acetate, dichloromethane, acetonitrile, acetone, ethanol, methanol, dimethyl sulfoxine, N-Methyl pyrrolidone.
6. a kind of insulin nanoparticles as described in claim 1,2 or 5, is characterized in that one or more that surfactant used in the preparation method step 2 (3) of this insulin nanoparticles, (4) includes but not limited in polyoxyethylene (35) Oleum Ricini, polyvinyl alcohol, phospholipid, sodium cholate, NaTDC, poloxamer class, Tweens, brejs.
7. a kind of insulin nanoparticles as claimed in claim 3, is characterized in that one or more that surfactant used in the preparation method step 2 (3) of this insulin nanoparticles, (4) includes but not limited in polyoxyethylene (35) Oleum Ricini, polyvinyl alcohol, phospholipid, sodium cholate, NaTDC, poloxamer class, Tweens, brejs.
8. a kind of insulin nanoparticles as claimed in claim 4, is characterized in that one or more that surfactant used in the preparation method step 2 (3) of this insulin nanoparticles, (4) includes but not limited in polyoxyethylene (35) Oleum Ricini, polyvinyl alcohol, phospholipid, sodium cholate, NaTDC, poloxamer class, Tweens, brejs.
9. a kind of insulin nanoparticles as described in claim 1,2,5,7 or 8, it is characterized in that, in the aqueous phase solution of certain density surfactant described in the preparation method step 2 (3) of this insulin nanoparticles, (4), the concentration of surfactant is 0.01% ~ 10.0%g/ml.
10. a kind of insulin nanoparticles as claimed in claim 3, it is characterized in that, in the aqueous phase solution of certain density surfactant described in the preparation method step 2 (3) of this insulin nanoparticles, (4), the concentration of surfactant is 0.01% ~ 10.0%g/ml.
11. a kind of insulin nanoparticles as claimed in claim 4, it is characterized in that, in the aqueous phase solution of certain density surfactant described in the preparation method step 2 (3) of this insulin nanoparticles, (4), the concentration of surfactant is 0.01% ~ 10.0%g/ml.
12. a kind of insulin nanoparticles as claimed in claim 6, it is characterized in that, in the aqueous phase solution of certain density surfactant described in the preparation method step 2 (3) of this insulin nanoparticles, (4), the concentration of surfactant is 0.01% ~ 10.0%g/ml.
13. a kind of insulin nanoparticles as described in claim 1,2,5,7,8,10,11 or 12, it is characterized in that the particle diameter of this insulin nanoparticles is 50 nm ~ 300 nm, envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
14. a kind of insulin nanoparticles as claimed in claim 3, it is characterized in that the particle diameter of this insulin nanoparticles is 50 nm ~ 300 nm, envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
15. a kind of insulin nanoparticles as claimed in claim 4, it is characterized in that the particle diameter of this insulin nanoparticles is 50 nm ~ 300 nm, envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
16. a kind of insulin nanoparticles as claimed in claim 6, it is characterized in that the particle diameter of this insulin nanoparticles is 50 nm ~ 300 nm, envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
17. a kind of insulin nanoparticles as claimed in claim 9, it is characterized in that the particle diameter of this insulin nanoparticles is 50 nm ~ 300 nm, envelop rate is 50% ~ 100%, and drug loading is 3% ~ 50%.
18. a kind of insulin nanoparticles as described in claim 1,2,5,7,8,10,11,12,14,15,16 or 17, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
19. a kind of insulin nanoparticles as claimed in claim 3, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
20. a kind of insulin nanoparticles as claimed in claim 4, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
21. a kind of insulin nanoparticles as claimed in claim 6, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
22. a kind of insulin nanoparticles as claimed in claim 9, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
23. a kind of insulin nanoparticles as claimed in claim 13, is characterized in that this insulin nanoparticles makes injection, lyophilized injectable powder, tablet, capsule, granule, dry suspension, oral liquid, drop, drop pill, inhalant, patch, ophthalmic preparation or suppository.
24. a kind of insulin nanoparticles as claimed in claim 20, is characterized in that described ophthalmic preparation is unguentum, gel or membrane.
25. a kind of insulin nanoparticles as claimed in claim 19, is characterized in that described ophthalmic preparation is unguentum, gel or membrane.
26. a kind of insulin nanoparticles as claimed in claim 21, is characterized in that described ophthalmic preparation is unguentum, gel or membrane.
27. a kind of insulin nanoparticles as claimed in claim 22, is characterized in that described ophthalmic preparation is unguentum, gel or membrane.
28. a kind of insulin nanoparticles as claimed in claim 23, is characterized in that described ophthalmic preparation is unguentum, gel or membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110031305.5A CN102614498B (en) | 2011-01-28 | 2011-01-28 | Insulin nanoparticle and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110031305.5A CN102614498B (en) | 2011-01-28 | 2011-01-28 | Insulin nanoparticle and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102614498A CN102614498A (en) | 2012-08-01 |
| CN102614498B true CN102614498B (en) | 2014-12-17 |
Family
ID=46554944
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110031305.5A Active CN102614498B (en) | 2011-01-28 | 2011-01-28 | Insulin nanoparticle and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102614498B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104324006B (en) * | 2014-08-06 | 2018-06-12 | 绍兴文理学院 | Degradable poly succinic acid-butanediol ester nano particle and preparation method and application |
| CN105617362B (en) * | 2014-10-27 | 2021-05-11 | 中国医学科学院药物研究所 | Novel insulin-phospholipid-chitosan self-assembled particle carrier and preparation thereof |
| US11491114B2 (en) * | 2016-10-12 | 2022-11-08 | Curioralrx, Llc | Formulations for enteric delivery of therapeutic agents |
| CN107224348B (en) * | 2017-06-01 | 2020-05-05 | 佛山勃林格医疗器械有限公司 | Eye surface sustained-release drug delivery device and manufacturing method thereof |
| CN109260174B (en) * | 2018-09-04 | 2021-10-15 | 中山大学 | High-throughput preparation method of therapeutic protein nanoparticles |
| CN109289041B (en) * | 2018-11-22 | 2021-11-26 | 福州大学 | Vitamin D-insulin nano sustained-release transdermal preparation and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651463A (en) * | 2004-12-02 | 2005-08-10 | 华东师范大学 | Mono methoxy polyethylene glycol-insulin complex substance and its preparation method |
| CN101199482A (en) * | 2007-12-20 | 2008-06-18 | 中国科学院长春应用化学研究所 | Process for preparing degradable polyester microsphere wrapping |
| CN101721375A (en) * | 2009-12-30 | 2010-06-09 | 上海交通大学 | Insulin slow release micron sphere composition and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2910494C (en) * | 2004-07-19 | 2018-10-23 | Biocon Limited | Insulin-oligomer conjugates, formulations and uses thereof |
-
2011
- 2011-01-28 CN CN201110031305.5A patent/CN102614498B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651463A (en) * | 2004-12-02 | 2005-08-10 | 华东师范大学 | Mono methoxy polyethylene glycol-insulin complex substance and its preparation method |
| CN101199482A (en) * | 2007-12-20 | 2008-06-18 | 中国科学院长春应用化学研究所 | Process for preparing degradable polyester microsphere wrapping |
| CN101721375A (en) * | 2009-12-30 | 2010-06-09 | 上海交通大学 | Insulin slow release micron sphere composition and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Metabolic Efficacy of Preprandial Administration of Lvs(B28),Pro(B29) Human Insulin Analog in IDDM Patients;MAARTEN AJ.M. JACOBS等;《DIABETES CARE》;19970831;第20卷(第8期);1279-1286 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102614498A (en) | 2012-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Peng et al. | A rapid-acting, long-acting insulin formulation based on a phospholipid complex loaded PHBHHx nanoparticles | |
| Liu et al. | Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation | |
| CN102614498B (en) | Insulin nanoparticle and preparation method thereof | |
| Suzuki et al. | Long-term oral administration of Exendin-4 to control type 2 diabetes in a rat model | |
| Sarmento et al. | Oral insulin delivery by means of solid lipid nanoparticles | |
| CN102988295B (en) | A kind of nanoparticle and preparation method thereof worn film peptide and modify | |
| Dong et al. | Comparative study of mucoadhesive and mucus-penetrative nanoparticles based on phospholipid complex to overcome the mucus barrier for inhaled delivery of baicalein | |
| US20080145432A1 (en) | Fine Particle and Pharmaceutical Preparation | |
| CN103705469B (en) | A kind of honokiol nanoparticle and preparation method thereof | |
| Yang et al. | Preparation, characterization and in vivo evaluation of pH‐sensitive oral insulin‐loaded poly (lactic‐co‐glycolicacid) nanoparticles | |
| CN105853403B (en) | A kind of paclitaxel palmitate liposome and preparation method thereof | |
| CN100506208C (en) | Chansu-loaded nanoliposome and preparation method thereof | |
| CN101953792B (en) | Irinotecan nano circulating liposome and preparation method thereof | |
| CN101889982B (en) | Novel long-circulating liposome composition and preparation method thereof | |
| Ma et al. | In vivo fate of biomimetic mixed micelles as nanocarriers for bioavailability enhancement of lipid–drug conjugates | |
| Xu et al. | Cell-penetrating peptide enhanced insulin buccal absorption | |
| Meng et al. | The biological fate of the polymer nanocarrier material monomethoxy poly (ethylene glycol)-block-poly (d, l-lactic acid) in rat | |
| CN101780042A (en) | Taxol nano targeting slow-release long-circulating liposome and preparation method thereof | |
| Balducci et al. | Antidiuretic effect of desmopressin chimera agglomerates by nasal administration in rats | |
| CN105726494B (en) | Andrographolide nano suspension composition and its preparation method and application | |
| Li et al. | Construction and anti-tumor activities of disulfide-linked docetaxel-dihydroartemisinin nanoconjugates | |
| Zhang et al. | Preparation, characterization and in vivo distribution of solid lipid nanoparticles loaded with syringopicroside | |
| Hu et al. | Docetaxel-loaded cholesterol-PEG co-modified poly (n-butyl) cyanoacrylate nanoparticles for antitumor drug pulmonary delivery: preparation, characterization, and in vivo evaluation | |
| CN110049759A (en) | The preparation of enteral delivery for therapeutic agent | |
| Kang et al. | Inhaled deep eutectic solvent based-nanoemulsion of pirfenidone in idiopathic pulmonary fibrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SICHUAN KELUN PHARMACEUTICAL RESEARCH INSTITUTE CO Free format text: FORMER NAME: KELUN PHARMACEUTICAL RESEARCH CO., LTD. |
|
| CP03 | Change of name, title or address |
Address after: 611138 Sichuan science and Technology Development Zone, Wenjiang District, Chengdu City, Xinhua Road, the central section of the two paragraph Patentee after: SICHUAN KELUN DRUG RESEARCH INSTITUTE CO., LTD. Address before: 610500, No. 520, industrial road, Xindu District, Xindu, Sichuan, Chengdu Patentee before: Kelun Pharmaceutical Research Co., Ltd. |