CN102293748B - Oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof - Google Patents
Oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof Download PDFInfo
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- CN102293748B CN102293748B CN 201110208468 CN201110208468A CN102293748B CN 102293748 B CN102293748 B CN 102293748B CN 201110208468 CN201110208468 CN 201110208468 CN 201110208468 A CN201110208468 A CN 201110208468A CN 102293748 B CN102293748 B CN 102293748B
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Abstract
The invention discloses an oral PEGylated insulin pH-sensitive naonparticle and a preparation method thereof. The naonparticle is prepared from PEGylated insulin, pH-sensitive polymer, carrier, additives and stabilizer. The preparation method of the naonparticle comprises the following steps: preparing a PEGylated insulin/pH-sensitive polymer/carrier W/O primary emulsion, dispersing the W/O primary emulsion in a stabilizer solution to form a W/O/W multiple emulsion, obtaining a crude product after the solvent volatilizes, purifying the crude product, and carrying out freeze-drying to obtain the naonparticle. The PEGylated insulin pH-sensitive naonparticle disclosed by the invention has the advantages of uniform dispersion and high medicine enveloping rate; the insulin modified by polyethylene glycol can reduce the aggregation of insulin molecules, prolong the half life in vivo, and prolong the time of blood sugar reduction effect; and the release rate of the insulin in the gastric acid environment is reduced, the release rate of the insulin in the intestinal tract environment is enhanced since the naonparticle swells, and thus, the oral PEGylated insulin pH-sensitive naonparticle has an oral application value.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of pH sensitive nanoparticles and preparation method thereof, be specifically related to a kind of oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof.
Background technology
The common route of administration of medicine comprises: oral, intravenous injection, intramuscular injection, subcutaneous injection etc.Compare the advantage such as oral administration has convenience, safety, expense is relatively cheap, patient compliance is good with other administering mode.Yet oral administration route has many restrictions, and several factors all can affect the absorption of oral drugs in the administration process.Most peptide and protein medicines are because molecular weight is large, fat-soluble poor, be difficult to by the biomembrane barrier, and owing to having a large amount of peptidohydrolases and proteolytic enzyme in the gastrointestinal tract, oral very easily first pass effect occurs and eliminated by liver metabolism, in addition, because these medicines itself exist chemistry and conformation unstability, and seldom or hardly absorb through gastrointestinal smoother.Therefore, the oral administration system difficulty of peptide and protein medicine is very large, and the biomembrane permeability and the protease inhibitor degraded that improve peptide class and protein drug are the difficult points of oral formulations research and development.
Insulin is stimulated and a kind of proteohormone of secretion by endogenous or exogenous material such as glucose, lactose, ribose, arginine, glucagon etc. by beta Cell of islet.Exogenous insulin is mainly used to treating diabetes, and therapeutic modality is mainly diabetics is directly carried out injection of insulin, and this mode is larger to the misery that patient brings, and administration inconvenience.Yet insulin exists that the half-life is short, easily degraded by enzymes, insulin easily assembles, is difficult to see through the problems such as intestinal film absorption in intestinal in the stomach sour environment when oral, bioavailability is very low.
Insulin can effectively prolong its half-life after polyethyleneglycol modified, reduces the gathering between the molecule, prolongs the time of staying in vivo.Disclose a kind of PLGA preparation that contains the Pegylation insulin such as Lewis etc. in U.S. Patent No. 09/999,82, said preparation can blood sugar lowering concentration in the animal body through subcutaneous injection, but can not be directly used in orally, and its envelop rate only has 33 ~ 49.2%; And [the Kenneth Hinds such as Hinds, Kathleen Campbell, Kathleen Holland, PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis, J Control Release, 2005,104:447-460] adopt single emulsion process to carry the Pegylation insulin with the PLGA bag, prepared microgranule has higher drug loading, but does not have pH sensitivity equally, can only be used for ejection preparation.Also there are many deficiencies in existing pH responsive type Pegylation insulin, such as [P. Calceti such as Calceti, S. Salmaso, G. Walker, A. Bernkop-Schn ü rch, Development and in vivo evaluation of an oral insulin – PEG delivery system, Eur J Pharm Sci, 2004,22:315] prepare Pegylation insulin tablet for the stability and the oral administration biaavailability that improve the Pegylation insulin, this Pegylation insulin tablet is comprised of the adhering polyacrylic acid-cysteine of film, the tablet top layer is the L100-55 enteric coating, this tablet shows the responsive release characteristics of pH, but the absorbed ability of medicine is relatively poor, is more conducive to the absorption of biomacromolecule not as nanoparticle.
Summary of the invention
The object of the invention is to for above-mentioned the deficiencies in the prior art, the characteristics in conjunction with Pegylation insulin and pH sensitive polymer nanoparticle provide a kind of oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof.The present invention is carried the formation nanoparticle with the Pegylation insulin with pH sensitive polymer bag, stops the release of insulin under the stomach acidity environment, prevents that pepsin is to the Degradation of insulin; And under intestinal environment, pH sensitive polymer swelling, even dissolving promote that insulin discharges fast, and nanoparticle also helps the absorption of insulin simultaneously, improves its relative bioavailability.
In order to achieve the above object, the present invention has adopted following technical scheme:
A kind of oral PEGylated insulin pH-sensitive naonparticle, it consists of: the quality percentage composition is 3 ~ 8% Pegylation insulin, the quality percentage composition is 46 ~ 68% pH sensitive polymer, the quality percentage composition is 22 ~ 47% carrier, and the quality percentage composition is that 0.5 ~ 2% additive and quality percentage composition are 1 ~ 5% stabilizing agent.
The mass ratio of pH sensitive polymer of the present invention and carrier is (1 ~ 3): 1.
Pegylation insulin of the present invention is the insulin that the mono methoxy polyethylene glycol butyraldehyde is modified, and the molecular weight of described mono methoxy polyethylene glycol butyraldehyde is 5000; Described pH sensitive polymer is hydroxypropylmethyl cellulose phthalate; Described carrier is poly (glycolide-lactide) or polylactic acid; Described additive is one or both in glycerol or the starch; Described stabilizing agent is polyvinyl alcohol.
The mean molecule quantity of carrier of the present invention is 5000 ~ 100000, and wherein the mol ratio of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 50:50 in the poly (glycolide-lactide) molecule segment.
The preparation method of oral PEGylated insulin pH-sensitive naonparticle of the present invention comprises the steps:
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing additive by the described prescription of claim 1, be dissolved in the deionized water, the preparation quality mark is 0.1 ~ 0.5% additive solution; Being 10 ~ 40mg/mL by mass volume ratio adds Pegylation insulin and dissolving, water in forming in additive solution; With acetone and dichloromethane by volume 1:4 be mixed to get mixed solvent, in mixed solvent, add pH sensitive polymer and carrier and dissolving by the described prescription of claim 1, form oil phase, the total concentration of pH sensitive polymer and carrier is 20 ~ 40mg/mL in the described oil phase, and the mass ratio of pH sensitive polymer and carrier is (1 ~ 3): 1; Be 1:(5 ~ 20 by volume with interior water and oil phase) mix, 40 ~ 60W ultrasonic emulsification, 0.5 ~ 4min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing stabilizing agent by the described prescription of claim 1, be dissolved in the deionized water, form outer water, described outer aqueous phase stabilizer concentration is 0.1 ~ 3%; Above-mentioned outer water is mixed with the W/O colostrum that step (1) obtains, and 40 ~ 60W ultrasonic emulsification, 0.5 ~ 4min obtains the W/O/W emulsion, and the volume ratio of oil phase is (2 ~ 8) in described outer water and the W/O colostrum: 1;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained volatilization of reducing pressure under 35 ~ 45 ℃ of water-baths, 400 ~ 500mbar obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 5 ~ 15min under 15000 ~ 25000r/min that step (3) is obtained, washing, in-15 ~-25 ℃ of lower precooling 8 ~ 12 h, obtain Pegylation insulin pH sensitive nanoparticles in-40 ~-60 ℃ of vacuum lyophilization 16 ~ 32 h again.
In the step of the present invention (1), described Pegylation insulin is the insulin that the mono methoxy polyethylene glycol butyraldehyde is modified, and the molecular weight of described mono methoxy polyethylene glycol butyraldehyde is 5000; Described pH sensitive polymer is hydroxypropylmethyl cellulose phthalate; Described carrier is poly (glycolide-lactide) or polylactic acid; Described additive is one or both in glycerol or the starch.
In the step of the present invention (2), described stabilizing agent is polyvinyl alcohol.
In the step of the present invention (1), the mean molecule quantity of described carrier is 5000 ~ 100000, and wherein the mol ratio of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 50:50 in the poly (glycolide-lactide) molecule segment.
The present invention also provides the application of a kind of Pegylation insulin pH sensitive nanoparticles in Oral administration treatment diabetes.
The present invention compared with prior art has following beneficial effect:
(1) adopt hydroxypropylmethyl cellulose phthalate (to be called for short: HP55) be the pH sensitive polymer, be used for the release of control Polyethylene Glycol insulin, stop the release of insulin under the stomach acidity environment, prevent that pepsin is to the Degradation of insulin, and under intestinal environment, HP55 swelling, even dissolving promote that insulin discharges fast;
(2) carrier material be poly (glycolide-lactide) (be called for short: PLGA) or polylactic acid (be called for short: PLA), the Polyethylene Glycol insulin is dispersed in the carrier material, prevents from assembling; The pH sensitive polymer is swelling or dissolving under intestinal environment, and when promoting that the Polyethylene Glycol insulin discharges, carrier material can also play control and discharge stable carrying out;
(3) bag of interpolation glycerol or water soluble starch raising Polyethylene Glycol insulin carries rate in interior water A;
(4) adopt polyvinyl alcohol (to be called for short: PVA) as stabilizing agent, stablize the emulsion that forms in the preparation process, and dried nanoparticle, prevent the gathering of emulsion and nanoparticle;
(5) polyethyleneglycol modified dose for the modification insulin is the mono methoxy polyethylene glycol butyraldehyde, and low molecular poly is modified insulin, is conducive to absorb by the cell bypass transhipment;
(6) materials safety of the present invention is nontoxic, and the product safety performance is good, and is harmless;
(7) the Pegylation insulin is dispersed in PLGA/HP55 or the PLA/HP55 mixed polymer nanoparticle, and envelop rate reaches 70 ~ 85%, and the nanoparticle particle diameter is about 300 nm, is evenly distributed;
(8) the responsive response of the pH of Pegylation insulin nanoparticles of the present invention release performance is good, can effectively reduce the release of insulin under the stomach acidity environment, prevents that pepsin is to the Degradation of insulin; And under intestinal environment, pH sensitive polymer swelling, even dissolving promote that insulin discharges fast;
(9) preparation method of the present invention does not affect the biological activity of insulin, and preparation technology is simple, cost is low.
Description of drawings
Fig. 1 is that the scanning electron microscope of the Pegylation insulin pH sensitive nanoparticles of the embodiment of the invention 6 (is called for short: SEM) figure.
Fig. 2 is the particle size distribution figure of the Pegylation insulin pH sensitive nanoparticles of the embodiment of the invention 6.
Fig. 3 is the release in vitro curve of the Pegylation insulin pH sensitive nanoparticles of the embodiment of the invention 6.
The specific embodiment
Below in conjunction with embodiment the present invention is done further and to explain, but the scope of protection of present invention is not limited to this.
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 40mg starch, be dissolved in the 40mL deionized water, obtain mass fraction and be 0.1% starch solution, with 5mg Pegylation insulin be dissolved in form in the above-mentioned starch solution of 0.5mL in water; 1mL acetone and 4mL dichloromethane are mixed, and (be called for short: HP55) (be called for short: PLGA) dissolving forms oil phase with the 50mg poly (glycolide-lactide) to wherein adding the 50mg hydroxypropylmethyl cellulose phthalate; Water in the 0.5mL and 5mL oil phase are mixed, and 40W ultrasonic emulsification 0.5min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: (be called for short: PVA), be dissolved in the 10mL deionized water, formation PVA mass fraction is 1% outer water to take by weighing the 100mg polyvinyl alcohol; The outer water of 10mL is mixed with the W/O colostrum that 5.5mL step (1) obtains, and 60W ultrasonic emulsification 0.5min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 40 ℃ of water-baths, 450 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 10min under 20000r/min that step (3) is obtained, washing,-20 ℃ of lower precooling 10h, a ℃ vacuum lyophilization 24h obtains Pegylation insulin pH sensitive nanoparticles again-50.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 1 obtains is 69.8%, and the residual PVA mass percent of particle surface is 2%.
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 200mg glycerol, add in the 40mL deionized water, obtain mass fraction and be 0.5% glycerite, with the Pegylation insulin of 10mg be dissolved in form in the above-mentioned glycerite of 0.5mL in water; 1mL acetone and 4mL dichloromethane are mixed, and (be called for short: PLA) dissolving forms oil phase to wherein adding 100mg HP55 and 100mg polylactic acid; Water in the 0.5mL and 5mL oil phase are mixed, and 40W ultrasonic emulsification 4min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing 600mg PVA, be dissolved in the 20mL deionized water, formation PVA mass fraction is 3% outer water; The outer water of 20mL is mixed with the W/O colostrum that 5.5mL step (1) obtains, and 60W ultrasonic emulsification 2min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 35 ℃ of water-baths, 400 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 15min under 15000r/min that step (3) is obtained, washing,-15 ℃ of lower precooling 12h, a ℃ vacuum lyophilization 32h obtains Pegylation insulin pH sensitive nanoparticles again-40.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 2 obtains is 65.1%, and the residual PVA mass percent of particle surface is 5%.
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 0.1g starch, be dissolved in the 40mL deionized water, obtain mass fraction and be 0.25% starch solution, with the Pegylation insulin of 10mg be dissolved in form in the above-mentioned starch solution of 0.5mL in water; 1mL acetone and 4mL dichloromethane are mixed, to wherein adding 100mg HP55 and 50mg PLGA dissolving, form oil phase; Water in the 0.5mL and 5mL oil phase are mixed, and 50W ultrasonic emulsification 4min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing 0.8g PVA, be dissolved in the 40mL deionized water, formation PVA mass fraction is 2% outer water; The outer water of 40mL is mixed with the W/O colostrum that 5.5mL step (1) obtains, and 50W ultrasonic emulsification 4min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 45 ℃ of water-baths, 500 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 5min under 25000r/min that step (3) is obtained, washing, repeated centrifugation, washing,-25 ℃ of lower precooling 8h, a ℃ vacuum lyophilization 16h obtains Pegylation insulin pH sensitive nanoparticles again-60.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 3 obtains is 68.3%, and the residual PVA mass percent of particle surface is 4%.
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 0.1g starch, be dissolved in the 40mL deionized water, obtain mass fraction and be 0.25% starch solution, with the Pegylation insulin of 10mg be dissolved in form in the above-mentioned starch solution of 1mL in water; 1mL acetone and 4mL dichloromethane are mixed, to wherein adding 75mg HP55 and 25mg PLA dissolving, form oil phase; Water in the 1mL and 5mL oil phase are mixed, and 60W ultrasonic emulsification 1min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing 0.2g PVA, be dissolved in the 40mL deionized water, formation PVA mass fraction is 0.5% outer water; The outer water of 30mL is mixed with the W/O colostrum that 6mL step (1) obtains, and 40W ultrasonic emulsification 3min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 40 ℃ of water-baths, 450 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 10min under 20000r/min that step (3) is obtained, washing, repeated centrifugation, washing,-20 ℃ of lower precooling 10h, a ℃ vacuum lyophilization 24h obtains Pegylation insulin pH sensitive nanoparticles again-50.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 4 obtains is 78.6%, and the residual PVA mass percent of particle surface is 2%.
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 0.2g starch, be dissolved in the 40mL deionized water, obtain mass fraction and be 0.5% starch solution, with the Pegylation insulin of 10mg be dissolved in form in the above-mentioned starch solution of 0.25mL in water; 1mL acetone and 4mL dichloromethane are mixed, to wherein adding 75mg HP55 and 75mgPLGA dissolving, form oil phase; 0.25mL interior water and 5mL oil phase mix, 60W ultrasonic emulsification 0.5min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing 20mg PVA, be dissolved in the 20mL deionized water, formation PVA mass fraction is 0.1% outer water; The outer water of 20mL is mixed with the W/O colostrum that 5.25mL step (1) obtains, and 40W ultrasonic emulsification 2min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 35 ℃ of water-baths, 500 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 15min under 15000r/min that step (3) is obtained, washing, repeated centrifugation, washing,-15 ℃ of lower precooling 12h, a ℃ vacuum lyophilization 16h obtains Pegylation insulin pH sensitive nanoparticles again-60.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 5 obtains is 80.9%, and the residual PVA mass percent of particle surface is 1%.
Embodiment 6
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing 0.05g starch and 0.05g glycerol, be dissolved in the 40mL deionized water, obtain mass fraction and be 0.25% glycerinum amyli mixed solution, with the Pegylation insulin of 10mg be dissolved in form in the above-mentioned glycerinum amyli mixed solution of 0.25mL in water; 1mL acetone and 4mL dichloromethane are mixed, to wherein adding 75mg HP55 and 25mgPLGA dissolving, form oil phase; Water in the 0.25mL and 5mL oil phase are mixed, and 40W ultrasonic emulsification 2min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing 0.04g PVA, be dissolved in the 40mL deionized water, formation PVA mass fraction is 0.1% outer water; The outer water of 40mL is mixed with the W/O colostrum that 5.25mL step (1) obtains, and 60W ultrasonic emulsification 0.5min obtains the W/O/W emulsion;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained rotary evaporation that reduces pressure under 45 ℃ of water-baths, 400 mbar is removed acetone and dichloromethane, obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 5min under 25000r/min that step (3) is obtained, washing, repeated centrifugation, washing,-25 ℃ of lower precooling 8h, a ℃ vacuum lyophilization 16h obtains Pegylation insulin pH sensitive nanoparticles again-60.
The Pegylation insulin pH sensitive nanoparticles envelop rate that embodiment 6 obtains is 83.5%, and the residual PVA mass percent of particle surface is 1%.
Pegylation insulin of the present invention is dispersed in PLGA/HP55 or the PLA/HP55 mixed polymer nanoparticle, and envelop rate reaches 70 ~ 85%.
As shown in Figure 1, nanoparticle of the present invention is spherical in shape, and particle diameter is at 100 ~ 300nm, distribution uniform.
As shown in Figure 2, nanoparticle mean diameter of the present invention is 251nm, and dispersion index (be called for short: PDI)=0.290, distribution uniform.
As shown in Figure 3, the extracorporeal releasing experiment demonstration, the responsive response of the pH of Pegylation insulin nanoparticles of the present invention release performance is good, under the simulated gastric fluid sour environment of pH=1.2, the insulin release rate effectively reduces, and pepsin significantly reduces the Degradation of insulin; And under the simulation intestinal environment of pH=7.4, pH sensitive polymer swelling, even dissolving promote that insulin discharges fast.
Claims (6)
1. oral PEGylated insulin pH-sensitive naonparticle, it is characterized in that, consisting of of described nanoparticle: the quality percentage composition is 3～8% Pegylation insulin, the quality percentage composition is 46～68% pH sensitive polymer, the quality percentage composition is 22～47% carrier, the quality percentage composition is that 0.5～2% additive and quality percentage composition are 1～5% stabilizing agent, described Pegylation insulin is the insulin that the mono methoxy polyethylene glycol butyraldehyde is modified, and the molecular weight of described mono methoxy polyethylene glycol butyraldehyde is 5000; Described pH sensitive polymer is hydroxypropylmethyl cellulose phthalate; Described carrier is poly (glycolide-lactide) or polylactic acid; Described additive is one or both in glycerol or the starch; Described stabilizing agent is polyvinyl alcohol.
2. oral PEGylated insulin pH-sensitive naonparticle according to claim 1 is characterized in that, the mass ratio of described pH sensitive polymer and carrier is 1:1～3:1.
3. oral PEGylated insulin pH-sensitive naonparticle according to claim 1 is characterized in that, the mean molecule quantity of described carrier is 5000～100000, and wherein the mol ratio of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 50:50 in the poly (glycolide-lactide) molecule segment.
4. the preparation method of oral PEGylated insulin pH-sensitive naonparticle claimed in claim 1 is characterized in that comprising the steps:
(1) ultrasonic emulsification prepares the W/O colostrum of Pegylation insulin and pH sensitive polymer and carrier material: take by weighing additive by the described prescription of claim 1, be dissolved in the deionized water, the preparation quality mark is 0.1～0.5% additive solution; Being 10～40mg/mL by mass volume ratio adds Pegylation insulin and dissolving, water in forming in additive solution; With acetone and dichloromethane by volume 1:4 be mixed to get mixed solvent, in mixed solvent, add pH sensitive polymer and carrier and dissolving by the described prescription of claim 1, form oil phase, the total concentration of pH sensitive polymer and carrier is 20～40mg/mL in the described oil phase, and the mass ratio of pH sensitive polymer and carrier is 1:1～3:1; Be that 1:5～1:20 mixes by volume with interior water and oil phase, 40～60W ultrasonic emulsification, 0.5～4min obtains the W/O colostrum;
(2) W/O colostrum ultra-sonic dispersion forms the W/O/W emulsion in stabiliser solution: take by weighing stabilizing agent by the described prescription of claim 1, be dissolved in the deionized water, form outer water, described outer aqueous phase stabilizer concentration is 0.1～3%; Above-mentioned outer water is mixed with the W/O colostrum that step (1) obtains, and 40～60W ultrasonic emulsification, 0.5～4min obtains the W/O/W emulsion, and the volume ratio of oil phase is 2:1～8:1 in described outer water and the W/O colostrum;
(3) W/O/W emulsion solvent evaporates obtains the nanoparticle crude product: the W/O/W emulsion that step (2) the is obtained volatilization of reducing pressure under 35～45 ℃ of water-baths, 400～500mbar obtains Pegylation insulin pH sensitive nanoparticles crude product;
(4) purification and the lyophilization of Pegylation insulin pH sensitive nanoparticles crude product: Pegylation insulin pH sensitive nanoparticles crude product centrifugal 5～15min under 15000～25000r/min that step (3) is obtained, washing, in-15～-25 ℃ of lower precooling 8～12h, obtain Pegylation insulin pH sensitive nanoparticles in-40～-60 ℃ of vacuum lyophilization 16～32h again.
5. preparation method according to claim 4 is characterized in that, described Pegylation insulin is the insulin that the mono methoxy polyethylene glycol butyraldehyde is modified, and the molecular weight of described mono methoxy polyethylene glycol butyraldehyde is 5000; Described pH sensitive polymer is hydroxypropylmethyl cellulose phthalate; Described carrier is poly (glycolide-lactide) or polylactic acid; Described additive is one or both in glycerol or the starch; Described stabilizing agent is polyvinyl alcohol.
6. preparation method according to claim 5 is characterized in that, the mean molecule quantity of described carrier is 5000～100000, and wherein the mol ratio of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 50:50 in the poly (glycolide-lactide) molecule segment.
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| CN108159403B (en) * | 2018-01-17 | 2019-12-10 | 四川大学 | Preparation method of polypeptide-insulin nanoparticles |
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| CN100391979C (en) * | 2004-12-02 | 2008-06-04 | 华东师范大学 | Mono methoxy polyethylene glycol-insulin complex substance and its preparation method |
| US20090306337A1 (en) * | 2006-07-31 | 2009-12-10 | Novo Nordisk A/S | Pegylated, Extended Insulins |
| CN101743252A (en) * | 2007-07-16 | 2010-06-16 | 诺沃-诺迪斯克有限公司 | protease stabilized, pegylated insulin analogues |
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