CN117482065A - High-load microsphere based on interface effect and preparation method thereof - Google Patents

High-load microsphere based on interface effect and preparation method thereof Download PDF

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CN117482065A
CN117482065A CN202311460838.4A CN202311460838A CN117482065A CN 117482065 A CN117482065 A CN 117482065A CN 202311460838 A CN202311460838 A CN 202311460838A CN 117482065 A CN117482065 A CN 117482065A
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microsphere based
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刘东飞
王宝训
杜春阳
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China Pharmaceutical University
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Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a high-load microsphere based on interface effect and a preparation method thereof. The high-load microsphere based on interface effect provided by the invention comprises an active drug and an amphiphilic polymer carrier, wherein the drug loading rate is 40% -80%, the encapsulation rate of the active drug is 90% -100%, and no burst release phenomenon exists; the high-load microsphere takes the double emulsion as a template, and the amphiphilic polymer formed by grafting small-molecule hydrophilic groups with charge on the polymer in the double emulsion can be rapidly formed in W 1 /O/W 2 The interface forms an interface film to realize the high-efficiency encapsulation of the medicine. The high-load microsphere based on the interface effect can effectively avoid adverse reactions caused by overhigh blood concentration, reduce toxic and side effects and enhance the treatment effect, and has simple preparation process and easy industrial production.

Description

High-load microsphere based on interface effect and preparation method thereof
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a high-load microsphere based on interface effect and a preparation method thereof.
Background
The protein and polypeptide medicines have been widely used for the treatment, diagnosis and prevention of diseases due to the advantages of strong pharmacological activity, strong specificity, low toxicity and the like. The protein polypeptide drugs have wide sources, and along with the continuous and deep research, more and more protein polypeptide drugs become hot spots for drug development, but the application of the drugs is limited by the short half-life in vivo and in vitro, unstable physicochemical properties, low bioavailability and the like. Encapsulation of such drugs into delivery systems such as polymeric microspheres can protect proteins and polypeptides from the external environment, improving their stability. The drug depot formed can provide sustained drug release over a prolonged period of time by subcutaneous or intramuscular administration.
The emulsification method is a conventional method for preparing microspheres, and the simple preparation process is the first choice for industrialization of the microparticle preparation at present. The microsphere can be obtained by taking water-in-oil-in-water as a template and solidifying. However, when the oil phase and the water phase are contacted to form a new oil-water interface, the stability of the oil-water interface determines the stability of the emulsion, and for the water-in-oil-in-water emulsion, the internal water drops can be combined with continuous water phase to destroy the structure of double emulsion, so that the internal phase escapes, and thus, an oil-in-water single emulsion is formed, and the final microsphere encapsulation rate is low. Therefore, the more the escape of the inner water phase is inhibited in the emulsification stage, the higher the encapsulation efficiency and drug loading rate of the drug microsphere are. However, high drug loading often accompanies high burst rate, which severely affects the application of drug loaded microspheres.
Patent technical document cn201810996995.X discloses a slow-release pasireotide microsphere and a preparation method thereof, the microsphere is prepared from pasireotide serving as an active ingredient, a microsphere carrier polymer, a protective agent, a surfactant and a suspending agent, the suspending agent increases the encapsulation efficiency and the drug loading of the slow-release microsphere by increasing the viscosity of an inner aqueous phase so as to further obstruct the migration of the active ingredient, and the stability of the microsphere is improved, the encapsulation efficiency and the drug loading of the microsphere prepared by the method are 86.3% and 8.86%, however, too high colostrum viscosity can cause the increase of transfer loss and sacrifice of yield, which can undoubtedly bring difficulty to the industrialization of the microsphere.
The invention discloses a PLGA drug-loaded microsphere for delaying initial burst release, a preparation method and application thereof, wherein the PLGA drug-loaded microsphere for delaying initial burst release is prepared by adopting a multiple emulsion solvent volatilization method, and the PLGA drug-loaded microsphere for delaying initial burst release is obtained by coating the drug-loaded microsphere with silk fibroin, and the drug-loaded microsphere has lower drug-loading rate and encapsulation rate although the drug-loaded microsphere can be effectively delayed.
Patent technical document CN201010251521.6 discloses recombinant human growth hormone rhGH long-acting slow-release microcapsule and preparation method thereof, which uses amphiphilic copolymer as carrier material, and has encapsulation rate of 99.5% and drug loading rate of 7.37%, but has obvious burst release.
Therefore, there is a need to develop a carrier microsphere with high drug loading rate to solve the problems of low drug loading rate, low encapsulation efficiency and high burst rate of the existing carrier microsphere.
Disclosure of Invention
In view of the above, the present invention aims to provide a high-load microsphere based on interface effect and a preparation method thereof, so as to solve the problems of low drug loading, low encapsulation efficiency and high burst release rate of the existing carrier microsphere.
Based on the above object, the present invention provides a high-load microsphere based on interface action, comprising an active drug and an amphiphilic polymer carrier; the drug loading rate of the high-load microsphere based on the interface effect is 40% -80%, the encapsulation rate of the active drug is 90% -100%, and no burst release phenomenon exists.
Wherein the charged small molecule hydrophilic groups include, but are not limited to: spermine, polylysine, polyaspartic acid, polyglutamic acid, and the molecular weight is 100-1000, preferably 200-700.
Wherein the modified polymer is a hydrophobic long chain containing polymer including, but not limited to: polylactic acid-glycolic acid copolymer, acetalized dextran, polycaprolactone and polylactic acid.
The amphiphilic polymer carrier material is prepared from a polymer modified by a small-molecule hydrophilic group with high interfacial activity, can be quickly adsorbed on an oil-water interface to form an interfacial film, and can obviously reduce the interfacial tension of dimethyl carbonate-water to be below 5mN/m within 100s when the concentration of the polymer is 0.5% (w/v).
Furthermore, the invention also provides a preparation method of the high-load microsphere, which comprises the following specific steps:
(1) Preparing colostrum: dissolving an active drug in a solvent I to prepare an inner water phase, dissolving an amphiphilic polymer carrier material in a solvent II to prepare an oil phase, emulsifying the inner water phase and the oil phase to prepare water-in-oil type colostrum, wherein the volume ratio of the inner water phase to the oil phase is 1:1-10, and the emulsifying method comprises any one or a combination of a plurality of ultrasonic, mechanical stirring, high-pressure homogenization, membrane emulsification and continuous flow.
(2) Preparing compound emulsion: re-emulsifying the primary emulsion with an aqueous solution containing salts and an emulsifier to form a water-in-oil-in-water type compound emulsion, wherein the volume ratio of the primary emulsion to the external aqueous phase is 1:5-10, and the emulsification method comprises any one or a combination of several of ultrasonic, mechanical stirring, high-pressure homogenization, membrane emulsification and continuous flow.
(3) Curing: the prepared water-in-oil-in-water type composite emulsion is solidified by removing the organic solvent to obtain the high-load microsphere based on the interface effect, and the solidifying method comprises any one or a combination of a plurality of solvents including solvent diffusion, rotary evaporation and electrostatic spraying.
Wherein the active pharmaceutical ingredient is water-soluble polypeptide, protein substance, preferably one or more of exenatide, bei Lalu peptide, antegei peptide, liraglutide, so Ma Lutai, abirudin, octreotide, enkephalin, nosiheptide, glutathione, nesiritide, enfuvirtide, romidepsin, goserelin, leuprolide, histrelin, insulin, diniinterleukin somatostatin, salmon calcitonin, glucagon, frizzled, polymyxin, hyaluronidase, lysozyme, catalase, amylase, cellulase, whey protein, ovalbumin, vitellin, bovine serum albumin, histone, ferritin, transferrin, myoalbumin, lactoferrin, and the like
The solvent I in the step (1) is water, an acidic solution, an alkaline solution or a saline solution, preferably, the acidic solution is one or more of hydrochloric acid, sulfuric acid and acetic acid, the alkaline solution is one or two of a sodium hydroxide solution and a potassium hydroxide solution, the saline solution salt comprises one or more of a sodium chloride solution, a potassium nitrate solution, a potassium sulfate solution and a magnesium chloride solution, the salt content range is 0.01% -5% (w/v), and the solvent II is an organic solvent, preferably one or more of dimethyl carbonate, ethyl acetate, dichloromethane, trichloroethane and tetrachloroethane.
Wherein, the emulsifier in the step (2) is one or more of polyvinyl alcohol, poloxamer, sorbitan fatty acid, polysorbate and polyoxyethylene fatty acid ester, the salt is one or more of sodium chloride, potassium nitrate, potassium sulfate, magnesium chloride and zinc chloride, and the content range of the salt is 0.1-60%w/v.
The invention has the beneficial effects that:
(1) The high-load microsphere based on the interface effect has the drug loading rate of 40-80%, the encapsulation rate of active drugs of 90-100%, no burst release phenomenon, unequal in-vitro release period from one week to three months, zero-order release characteristics and particle size of 1-500 mu m.
(2) The invention provides a high-load micro-device based on interface effectThe sphere uses the compound emulsion as a template, and the amphiphilic polymer formed by grafting charged micromolecular hydrophilic groups on the polymer in the compound emulsion can be rapidly coated on W 1 /O/W 2 The interface forms an interface film, and benefits from the solvation effect of the polymer hydrophobic chain in the oil phase, and a large amount of oil phase solvent molecules are adsorbed by the interface film formed on the surface of the inner water phase, so that the inner water phase is more represented as the property of the oil phase, the escape of the inner water phase is avoided, the efficient encapsulation of the medicine is realized, the formation of the interface film can delay the combination of the inner water phase, the internal structure of the microsphere is better controlled, and the curing process of the amphiphilic polymer is regulated by the environment of the inner water phase and the outer water phase, so that a compact polymer matrix is formed, and the abrupt release of the medicine is avoided.
(3) The high-load microsphere based on the interface effect can effectively avoid adverse reactions caused by overhigh blood concentration, reduce toxic and side effects and enhance the treatment effect, and has simple preparation process and easy industrial production.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the following brief description will be given of the drawings used in the description of the embodiments or the prior art:
FIG. 1 is a schematic diagram of an interfacial film formed by adsorption of amphiphilic polymers at an oil-water interface (internal water phase-oil phase);
FIG. 2 is an optical micrograph of exenatide microspheres prepared in example 1;
FIG. 3 is an electron micrograph of the surface of exenatide microspheres prepared in example 1;
FIG. 4 is an optical micrograph of Bilva Lu Dingwei spheres prepared in example 2;
FIG. 5 is an optical micrograph of lysozyme microsphere prepared in example 3;
FIG. 6 is an optical micrograph of salmon calcitonin microsphere prepared in example 7;
FIG. 7 is an optical micrograph of insulin microspheres prepared in example 8;
FIG. 8 is an optical micrograph of exenatide microspheres prepared in example 9;
FIG. 9 is an optical micrograph of insulin microspheres prepared in example 10;
FIG. 10 is an optical micrograph of insulin microspheres prepared in example 13;
FIG. 11 is an optical micrograph of bovine serum albumin microspheres prepared in example 14;
FIG. 12 is an external release profile of insulin microspheres prepared in example 4;
FIG. 13 is an external release profile of insulin microspheres prepared in example 8;
FIG. 14 is an external release profile of insulin microspheres prepared in example 13;
FIG. 15 is an external release profile of bovine serum albumin microspheres prepared in example 14.
Detailed Description
The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 70% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.5mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving exenatide in water to obtain 200uL of 100mg/mL exenatide aqueous solution, and performing ultrasonic treatment with 500uL of 5mg/mL dimethyl carbonate solution of PLGA-KK3 for 1min to obtain W 1 O colostrum, adding 3mL of 2% MgCl saturated with dimethyl carbonate 2 ·6H 2 In O.1%PVA solution, the mixture is stirred for 10min by magnetic stirring at 100rpm to form W 1 /O/W 2 Multiple emulsion, 50mL of 2% MgCl is slowly added into the multiple emulsion 2 ·6H 2 O.1%PVA solution, stirring at 100rpm for 1h to extract dimethyl carbonate, solidifying and washing to obtain the medicine carrying microsphere. The prepared microsphere has an encapsulation rate of 95.99%, a drug loading rate of 79.34% and a cumulative release percentage of 6.42% in 24 hours.
Example 2: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-SP reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving 20mg bivalirudin in 20% acetic acid, preparing 200uL of bivalirudin solution of 100mg/mL, and carrying out ultrasonic treatment on the solution with 500uL of dimethyl carbonate solution of 40mg/mL PLGA-SP for 1min to form W 1 O colostrum, adding 5mL of 1% MgCl saturated with dimethyl carbonate 2 ·6H 2 Stirring the solution of O.1%PVA at 150rpm to form W 1 /O/W 2 Multiple emulsion, 50mL of 4% MgCl is slowly added into the multiple emulsion 2 ·6H 2 O.1%PVA, stirring at 100rpm for 1h to extract dimethyl carbonate, solidifying and washing to obtain the drug-carrying microsphere. The prepared microsphere has the encapsulation rate of 99.67%, the drug loading rate of 49.57% and the cumulative release percentage of 24 hours of 0.28%.
Example 3: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was spermine modified Acetalized Dextran (ADS) with a polymer grafting ratio of 67% and 5mg/mL ADS reduced the interfacial tension of dimethyl carbonate-water to 3.4mN/m. Spermine molecular weight: 202.34; the acetalized dextran has a weight average molecular weight of 9000-11000;
dissolving lysozyme in water to prepare 400uL of 100mg/mL lysozyme aqueous solution, and homogenizing and emulsifying with 1mL of 20mg/mL PLGA-SP ethyl acetate solution for 15s to form W 1 O colostrum, adding the colostrum into 4.5mL of 1% NaCl.1% PVA solution, stirring to form W 1 /O/W 2 And (3) re-emulsifying, stirring with 1L of 0.9% NaCl for 4 hours to extract ethyl acetate, and washing to obtain the drug-loaded microspheres. The prepared microsphere has an encapsulation rate of 95.21%, a drug loading rate of 63.40% and a cumulative release percentage of 9.70% in 24 hours.
Example 4: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
insulin was dissolved in 0.06M HCl to prepare 400uL of 100mg/mL insulin solution, which was emulsified with 1mL of 40mg/mL PLGA-SP in dichloromethane for 15s to form W 1 O colostrum, adding the colostrum into 6mL 1% NaCl.1% PVA solution, stirring magnetically at 400rpm for 5min to form W 1 /O/W 2 Multiple emulsion, 50mL of 2% MgCl is slowly added into the multiple emulsion 2 ·6H 2 O.1%PVA, stirring for 1h by magnetic stirring at 100rpm to slowly solidify the compound emulsion, and obtaining the drug-carrying microsphere. The prepared microsphere has an encapsulation rate of 97.67%, a drug loading rate of 48.84% and a cumulative release percentage of 0.69% in 24 hours.
Example 5: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a pentapolylysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 5) with a polymer grafting rate of 64% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.6mN/m. Pentalysine molecular weight: 658.877; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving exenatide in water to prepare 200uL of 100mg/mL exenatide aqueous solution, and carrying out ultrasonic treatment on the 200uL of 40mg/mL PLGA-KK3 dimethyl carbonate solution for 30s to form W 1 O colostrum, adding 3mL of 2% MgCl saturated with dimethyl carbonate 2 ·6H 2 In O.1%PVA solution, the mixture is stirred for 10min by magnetic stirring at 300rpm to form W 1 /O/W 2 And (3) re-emulsifying, stirring with 1L of 0.9% NaCl for 4 hours to extract dimethyl carbonate, and washing to obtain the drug-loaded microspheres. The prepared microsphere has an encapsulation rate of 98.29%, a drug loading rate of 49.57% and a cumulative release percentage of 0.89% in 24 hours.
Example 6: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a pentapolylysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting rate of 66% and 5mg/mL PLGA-KK5 reduced the interfacial tension of dimethyl carbonate-water to 3.6mN/m. Pentalysine molecular weight: 658.877; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving bovine serum albumin in water to prepare 200uL of 100mg/mL bovine serum albumin solution, and swirling the solution with 500uL of 20mg/mL PLGA-KK3 dimethyl carbonate solution for 30s to form W 1 O colostrum, adding 3mL of 2% MgCl saturated with dimethyl carbonate 2 ·6H 2 In O.1%PVA solution, the mixture is stirred for 5min by magnetic stirring at 500rpm to form W 1 /O/W 2 Multiple emulsion, 50mL of 2% MgCl is slowly added into the multiple emulsion 2 ·6H 2 O.1%PVA is stirred for 2 hours to extract the organic solvent, and the organic solvent is solidified and washed to obtain the drug-loaded microsphere. The prepared microsphere has an encapsulation rate of 95.28%, a drug loading rate of 63.3% and a cumulative release percentage of 9.42% in 24 hours.
Example 7: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 45% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.7mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 24000-38000;
dissolving salmon calcitonin in water to obtain 400uL100mg/mL salmon calcitonin, and ultrasonic treating with 1mL40mg/mL PLGAKK3 solution (dimethyl carbonate) to obtain W 1 and/O colostrum. The colostrum was added to 6mL 1% PVA solution saturated with dimethyl carbonate and stirred to form W 1 /O/W 2 Adding 40ml of 1% PVA into the double emulsion, magnetically stirring at 100rpm for 2 hours to volatilize the organic solvent, solidifying and washing to obtain the drug-loaded microsphere. The prepared microsphere has an encapsulation rate of 97.80%, a drug loading rate of 49.45% and a cumulative release percentage of 2.34% in 24 hours.
Example 8: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 23% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.6mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight of 38000-54000;
insulin is administeredDissolving in 0.06M HCl to prepare 100uL of 100mg/mL insulin solution and 250uL of 40mg/mL PLGA-KK3 chloroform solution, and performing ultrasonic treatment for 30s to form W 1 and/O colostrum. The colostrum was added to 1.5mL of 1% NaCl.1% PVA solution and stirred magnetically at 350rpm for 20min to form W 1 /O/W 2 And (5) re-emulsifying. 40mL of 1% PVA is added into the compound emulsion, the mixture is stirred for 2 hours under magnetic stirring at 100rpm, the volatile organic solvent is volatilized, and the solid is washed to obtain the drug-loaded microsphere. The obtained microsphere has an encapsulation rate of 94.43%, a drug loading rate of 48.57% and a cumulative release percentage of 1.34% in 24 hours.
Example 9: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-SP reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving exenatide solution in water to prepare 400uL of exenatide solution with 100mg/mL of PLGA-SP ethyl acetate solution and homogenizing and emulsifying for 15s to form W 1 and/O colostrum. W was fed at a flow rate of 0.1mL/h 1 introducing/O colostrum into the internal phase of the two-phase microfluidic chip, and introducing 2% MgCl at a flow rate of 7mL/h 2 ·6H 2 Introducing O.1%PVA solution into the external phase of the two-phase microfluidic chip, and shearing at the capillary orifice of the microfluidic chip to form W 1 /O/W 2 And (3) re-emulsifying, stirring for 2 hours by using 1L of 0.9% NaCl to extract ethyl acetate, solidifying and washing to obtain the drug-loaded microsphere. The encapsulation efficiency is 98.87%, the drug loading rate is 65.91%, and the cumulative release percentage is 11.34% in 24 hours.
Example 10: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-SP reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
the insulin solution was dissolved in 0.06M HCl to prepare 400uL of 100mg/mL insulin solution, and 1mL of 20mg/mL PLGA-SP dimethyl carbonate solution, ultrasonic for 30s to form W 1 and/O colostrum. W was fed at a flow rate of 0.5mL/h 1 introducing/O colostrum into the internal phase of the two-phase microfluidic chip, and introducing 2% MgCl at a flow rate of 15mL/h 2 ·6H 2 Introducing O.1%PVA solution into the external phase of the two-phase microfluidic chip, and shearing at the capillary orifice of the microfluidic chip to form W 1 /O/W 2 And (3) re-emulsifying, stirring with 1L of 0.9% NaCl for 2 hours to extract dimethyl carbonate, solidifying and washing to obtain the drug-loaded microsphere. The obtained microsphere has an encapsulation rate of 98.14%, a drug loading rate of 66.12% and a cumulative release percentage of 8.84% in 24 hours.
Example 11: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-SP reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
lysozyme was dissolved in water to prepare 400uL of 100mg/mL lysozyme solution, and 1mL of 20mg/mL PLGA-SP ethyl acetate solution was sonicated for 1min to form colostrum. W was fed at a flow rate of 0.5mL/h 1 introducing/O colostrum into the internal phase of the two-phase microfluidic chip, and introducing 2% MgCl at a flow rate of 15mL/h 2 ·6H 2 Introducing O.1%PVA solution into the external phase of the two-phase microfluidic chip, and shearing at the capillary orifice of the microfluidic chip to form W 1 /O/W 2 And (3) re-emulsifying, stirring for 2 hours by using 1L of 0.9% NaCl to extract ethyl acetate, solidifying and washing to obtain the drug-loaded microsphere. The encapsulation efficiency is 98.34%, the drug loading is 65.91%, the cumulative release percentage is 9.74% in 24 hours, and the cumulative release percentage is 2.14% in 24 hours.
Example 12: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a spermine modified polylactic acid-glycolic acid copolymer (PLGA-SP) with a polymer grafting rate of 68% and 5mg/mL PLGA-SP reduced the interfacial tension of dimethyl carbonate-water to 3.3mN/m. Spermine molecular weight: 202.34; PLGA 50:50, weight average molecular weight 7000-17000;
salmon calcitonin was dissolved in water to prepare 400uL100mg +.The salmon calcitonin solution is homogenized and emulsified with 1mL of a dimethyl carbonate solution of 20mg/mL PLGA-SP for 15s to form W 1 and/O colostrum. Introducing the colostrum into the internal phase of the two-phase microfluidic chip at a flow rate of 0.3mL/h, introducing the 1% NaCl.1wt% PVA solution into the external phase of the two-phase microfluidic chip at a flow rate of 10mL/h, and shearing at the capillary orifice in the microfluidic chip to form W 1 /O/W 2 And (3) re-emulsifying, wherein the receiving phase is a large amount of 1% NaCl.1% PVA solution, extracting the organic solvent, solidifying and washing to obtain the drug-loaded microsphere. The encapsulation efficiency of the obtained microsphere is 96.04%, the drug loading rate is 64.71%, and the cumulative release percentage is 4.32% in 24 hours.
Example 13: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 70% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.5mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 7000-17000;
dissolving insulin in 0.047M HCl, preparing 78mg/mL insulin solution as internal phase, weighing a proper amount of trilysine modified polylactic acid-glycolic acid copolymer, dissolving the copolymer in a certain volume of ethyl acetate to prepare 20mg/mL polymer solution as intermediate phase, 3% NaCl.1% PVA as external phase, and shearing at capillary openings after three-phase microfluidic chip is introduced to obtain W, wherein the flow rates of three phases of the three phases are respectively 0.1mL/h, 0.4mL/h and 15mL/h 1 /O/W 2 Stirring with 1L of 0.9% NaCl for 2h to extract dimethyl carbonate, solidifying and washing to obtain the drug-loaded microsphere. The encapsulation efficiency of the obtained microsphere is 91.2%, the drug loading rate is 58.7%, and the cumulative release percentage is 4.32% in 24 hours.
Example 14: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 70% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.5mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 7000-17000;
weighing a proper amount of bovine serum albumin, dissolving the bovine serum albumin into a certain volume of water to prepare a bovine serum albumin solution with the volume of 78mg/mL as an internal phase, weighing a proper amount of polylactic acid-glycolic acid copolymer modified by trilysine, dissolving the polylactic acid-glycolic acid copolymer into a certain volume of dimethyl carbonate to prepare a polymer solution with the volume of 20mg/mL as an intermediate phase, taking 1% NaCl.2% PVA as an external phase, and respectively cutting at capillary orifices after the three-phase microfluidic chip is introduced into the three-phase microfluidic chip at the flow rates of 0.2mL/h, 0.5mL/h and 12mL/h to obtain W 1 /O/W 2 Stirring with 1L of 0.9% NaCl for 2h to extract dimethyl carbonate, solidifying and washing to obtain the drug-loaded microsphere. The encapsulation efficiency of the obtained microsphere is 95.3%, the drug loading rate is 59.8%, and the cumulative release percentage is 9.42% in 24 hours.
Example 15: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 70% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.5mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 7000-17000;
a hydrophilic SPG membrane with a pore diameter of 5.2 μm was immersed in water to wet the pore membrane sufficiently. 400uL of 100mg/mL bovine serum albumin and 1mL of 40mg/mL ethyl acetate solution of PLGA-KK3 were sonicated for 2min to form W 1 and/O colostrum. Pumping the colostrum through SPG membrane at a speed of 1mL/min by using a quantitative injection pump (nitrogen steel cylinder), and magnetically stirring at 150rpm to make the microspheres drop from the surface of the porous glass membrane to obtain W 1 /O/W 2 And (3) re-emulsifying, stirring for 2 hours with 1L of 0.9% NaCl to extract ethyl acetate, solidifying and washing to obtain the drug-loaded microspheres. The encapsulation efficiency of the obtained microsphere is 93.3%, the drug loading rate is 60.3%, and the cumulative release percentage is 1.42% in 24 hours.
Example 16: the preparation method of the high-load microsphere based on the interface effect comprises the following specific preparation steps:
the polymer used in this example was a trilysine modified polylactic acid-glycolic acid copolymer (PLGA-KK 3) with a polymer grafting ratio of 70% and 5mg/mL PLGA-KK3 reduced the interfacial tension of dimethyl carbonate-water to 3.5mN/m. Molecular weight of trilysine: 402.532; PLGA 50:50, weight average molecular weight 7000-17000;
a hydrophilic SPG membrane with a pore diameter of 5.2 μm was immersed in water to wet the pore membrane sufficiently. Homogenizing 400uL of 100mg/mL insulin solution with 1mL of 40mg/mL ethyl acetate solution of PLGA-SP for 30s to form W 1 and/O colostrum. The colostrum was injected into the reaction vessel by a quantitative injection pump (nitrogen cylinder) at a rate of 1mL/min to obtain a mixture of W and 1 pumping the O-type colostrum through an SPG film, and magnetically stirring at 150rpm to separate the microspheres from the surface of the porous glass film to obtain W 1 /O/W 2 And (3) re-emulsifying, stirring for 2 hours with 1L of 0.9% NaCl to extract ethyl acetate, solidifying and washing to obtain the drug-loaded microsphere, wherein the encapsulation efficiency of the obtained microsphere is 92.2%, the drug loading rate is 46.7%, and the cumulative release percentage is 5.37% in 24 hours.
Examples 17 to 29: high-load microsphere based on interface effect is prepared by the preparation method of examples 1-10, and specific raw material selections are shown in Table 1;
table 1 examples 17-29 raw materials selection table
Examples 30 to 38: high-load microsphere based on interface effect is prepared by the preparation method of examples 11-14, and specific raw material selections are shown in Table 2;
table 2 examples 30-38 raw materials selection table
Examples 39 to 43: high-load microspheres based on interface effect are obtained according to the preparation methods of examples 15-16, and specific raw material selections are shown in Table 3;
TABLE 3 selection of raw materials for examples 39-43
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Examples 44 to 49: high-load microspheres based on interface effect are obtained according to the preparation methods of examples 17-18, and specific raw material selections are shown in Table 4:
TABLE 4 selection of raw materials for examples 44-49
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Comparative example 1: comparative experiments of amphiphilic polymers mPEG550-b-PLGA10000 and PLGA-KK3, the specific results are shown in Table 5 and Table 6:
TABLE 5 interfacial tension of dimethyl carbonate-water at 100s for mPEG550-b-PLGA10000 and PLGA-KK3 at different concentrations
Concentration mg/mL mPEG550-b-PLGA10000 PLGA-KK3
0 7.37 7.37
2 7.27 4.20
5 7.14 3.58
10 6.99 3.17
Table 6 encapsulation efficiency of exenatide microspheres prepared by two polymers respectively using the method of example 1
Ratio of drug to PLGA-KK3 Encapsulation efficiency Drug and mPEG550-b-PLGA10000 Encapsulation efficiency
20：40 99.59％ 20：40 11.03％
20：20 96.43％ 20：20 2.47％
From tables 5 and 6, it can be seen that the amphiphilic polymer containing the small-molecule hydrophilic group has strong interfacial activity, and can realize efficient encapsulation, while the mPEG550-b-PLGA10000 has low interfacial activity, and is difficult to quickly and stably adsorb at an oil-water interface, so that ideal encapsulation is difficult to realize.
Comparative example 2: the drug-loaded microsphere is prepared according to the scheme disclosed in the patent technical document CN 111568877A:
TABLE 7 encapsulation and drug loading after initial drug to polymer ratio in the formulation is improved by the method disclosed in the patent
As can be seen from table 7, when the polymer content is fixed by the method described in patent CN102370630a, when the initial ratio of the drug to the polymer in the formulation is increased, it is difficult to achieve high encapsulation, and for mPEG2000-b-PLGA10000 containing hydrophilic long chains, steric hindrance can hinder the formation of a dense polymer film, and when the ratio of the polymer to the drug is low, it is difficult to achieve an ideal encapsulation effect, and a large amount of polymer is required to achieve high encapsulation, so that it is impossible to simply increase the drug content to achieve both high encapsulation and high drug loading, and the above patent also fails to solve the problem of high burst rate.
Performance test:
encapsulation efficiency and drug loading: the encapsulation efficiency and drug loading of the active drug was indirectly determined by measuring the amount of active drug that was not encapsulated into the microspheres. And immersing the prepared microspheres in the bottom of a container, cleaning the microcapsules for multiple times by using ultrapure water, taking 1mL of supernatant before each cleaning, analyzing the concentration of the supernatant by using HPLC, and calculating the encapsulation efficiency and the drug loading rate.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The present invention is intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.

Claims (10)

1. The high-load microsphere based on the interface effect is characterized by comprising an active drug and an amphiphilic polymer carrier, wherein the drug loading rate is 40% -80%, the encapsulation rate of the active drug is 90% -100%, and no burst release phenomenon exists;
the high-load microsphere based on the interface effect comprises the following preparation steps:
(1) Preparing colostrum: dissolving an active drug in a solvent I to prepare an inner water phase, dissolving an amphiphilic polymer in a solvent II to prepare an oil phase, and emulsifying the inner water phase and the oil phase to prepare water-in-oil type colostrum;
(2) Preparing compound emulsion: re-emulsifying the colostrum with an aqueous solution containing salts and an emulsifier to form a water-in-oil-in-water type compound emulsion;
(3) Curing: removing the solvent II from the water-in-oil-in-water type compound emulsion, and curing to obtain the high-load microsphere based on the interface effect;
the amphiphilic polymer is a polymer modified by a charged micromolecular hydrophilic group;
the molecular weight of the charged micromolecular hydrophilic group is 200-700;
the modified polymer is a polymer containing long hydrophobic chains.
2. The high-load microsphere based on interfacial action according to claim 1, wherein the active drug is one of a water-soluble polypeptide and a protein.
3. The high-load microsphere based on interfacial action according to claim 1, wherein the volume ratio of the internal aqueous phase to the oil phase in step (1) is 1:1-10.
4. The high-load microsphere based on interfacial interactions according to claim 1, wherein the solvent i in step (1) is water, an acidic solution, an alkaline solution or an aqueous salt solution.
5. The high-load microsphere based on interfacial action according to claim 1, wherein the solvent ii in the step (1) is an organic solvent.
6. The high-load microsphere based on interfacial action according to claim 1, wherein the volume ratio of the colostrum to the external water phase in step (2) is 1:5-10.
7. The high-load microsphere based on interfacial action according to claim 1, wherein the emulsifier in the step (2) is one or more of polyvinyl alcohol, poloxamer, sorbitan fatty acid, polysorbate, and polyoxyethylene fatty acid ester.
8. The high-load microsphere based on interfacial action according to claim 1, wherein the salt in the step (2) is one or more of sodium chloride, potassium nitrate, potassium sulfate, magnesium chloride and zinc chloride.
9. The high-load microsphere based on interfacial action according to claim 1, wherein the emulsification method is one or a combination of several of ultrasound, mechanical stirring, high-pressure homogenization, membrane emulsification, continuous flow.
10. The high-load microsphere based on interfacial action according to claim 1, wherein the curing method is one or a combination of several of solvent diffusion, rotary evaporation, electrostatic spraying.
CN202311460838.4A 2023-11-06 2023-11-06 High-load microsphere based on interface effect and preparation method thereof Pending CN117482065A (en)

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