Biodegradable enteric polymer microsphere and preparation method thereof

Abstract

The invention relates to the technical field of biodegradable enteric polymer microspheres, in particular to the technical field of preparation of biodegradable enteric polymer microspheres. The invention relates to a biodegradable enteric polymer microsphere and a preparation method thereof, the enteric polymer microsphere comprises a carrier and a drug, the carrier is poly (N-isopropylacrylamide)-b-poly (gamma-benzyl-L-glutamate-co-L-glutamic acid), the drug is insulin / absorption enhancer composite particles, and the carrier is a carrier-b-poly (gamma-benzyl-L-glutamate-co-L-glutamic acid) or a carrier-b-poly (gamma-benzyl-L-glutamate-co-L-glutamic acid). The absorption enhancer is a polyethyleneimine modified bile acid substance, the mass ratio of the absorption enhancer to the insulin is (1-50): 1, and the drug accounts for 1%-15% of the mass of the polymer drug-loaded microspheres. The enteric polymer microsphere provided by the invention can be used for an insulin oral absorption enhancing carrier, during oral administration, insulin can be released in the intestinal tract without being released or with little release in the stomach, the retention time of the medicine in the intestinal tract is prolonged, the medicine absorption is enhanced, and the blood sugar reducing effect of the medicine carrying microsphere can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biodegradable enteric polymer microspheres, in particular to the technical field of preparation of biodegradable enteric polymer microspheres. Technical Background

[0002] Diabetes has become one of the three most serious health threats to humanity, following cancer and cardiovascular and cerebrovascular diseases, and is becoming increasingly prevalent among younger patients. While regular insulin injections can quickly lower blood sugar, frequent injections cause significant physical and psychological distress, leading to poor patient tolerance.

[0003] While oral insulin delivery offers numerous advantages, including high patient compliance, convenient administration, and improved glucose homeostasis, insulin, a protein drug, has very low oral bioavailability due to its inherent physicochemical properties (large molecular weight, poor lipid solubility), coupled with multiple physical and chemical barriers to oral administration (gastric acid denaturation, digestive enzyme barriers, and intestinal absorption barriers). Improving insulin's oral bioavailability has long been a significant challenge in drug delivery research and a key focus and challenge in the development of novel oral insulin formulations.

[0004] Faced with the challenge of low bioavailability of oral insulin delivery, various strategies can be used to improve oral bioavailability of insulin. The pH-triggered release mechanism can safely deliver drugs to the intestinal tract and enhance intestinal drug absorption. Although existing technologies have explored a variety of carriers (such as microspheres and composite gels), there are still significant defects: although traditional single-layer microspheres are biodegradable, they lack enteric targeting capabilities, resulting in premature leakage of insulin in the stomach; although pH-responsive systems (poly-γ-glutamic acid / chitosan) can achieve intestinal release, they have insufficient adhesion and low transmembrane efficiency (bioavailability <10%); the use of absorption enhancers (bile acids, polyamines, etc.) alone is difficult to overcome the dual limitations of mucus layer clearance and cell paracellular barriers.

[0005] The use of mucosal adhesive polymer materials such as poly (N-isopropylacrylamide) (PNIPAM) to construct a delivery system can prolong the retention time of drugs in the intestinal absorption site, providing another potential way to improve drug absorption.

[0006] The present invention proposes to prepare drug-loaded microspheres using enteric adhesive polymers. The pH sensitivity of the polymer microspheres can achieve safe intestinal release of drugs through the stomach. The adhesion of PNIPAM can prolong the retention time of drugs in the intestinal absorption site, and the absorption enhancer promotes intestinal absorption of drugs, opening up a new way for oral insulin. Summary of the Invention

[0007] In view of this, the purpose of the present application is to provide a biodegradable enteric polymer microsphere for insulin oral absorption enhancement carrier and a preparation method thereof, the drug-loaded microsphere provided by the present application can realize drug protection in the stomach, target release in the intestinal tract, prolong intestinal retention time and promote intestinal absorption of the drug, and can improve the bioavailability of the drug.

[0008] The present application provides a biodegradable enteric polymer microsphere and a preparation method thereof, comprising a carrier material and a drug, wherein the carrier material is poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid);

[0009] The drug is an insulin / absorption enhancer composite particle, and the absorption enhancer is oligomeric ethyleneimine modified deoxycholic acid.

[0010] The mass ratio of the absorption enhancer to the insulin is 0.01-10:1.

[0011] The mass percentage of the drug in the carrier is 1%-15%.

[0012] The present application provides a biodegradable enteric polymer microsphere and a preparation method thereof, comprising a carrier material and a drug, wherein the carrier material is poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid), the drug is an insulin / absorption enhancer composite particle, the absorption enhancer is oligomeric ethyleneimine modified deoxycholic acid, the mass ratio of the absorption enhancer to the insulin is 0.01-10:1, and the mass percentage of the drug in the carrier is 1%-15%.

[0013] A preparation method of a biodegradable enteric polymer microsphere, specifically comprising the following steps:

[0014] The polymer microsphere is prepared by using an oil-in-oil solid emulsion method, the drug and the carrier material are mixed in an organic solvent to obtain an oil-in-solid emulsion, and then mixed and emulsified with vegetable oil containing a surfactant to obtain an oil-in-oil solid emulsion, and the emulsion is extracted with diethyl ether to obtain the drug-loaded microsphere. The mass percentage of the drug in the carrier is 1%-15%.

[0015] The poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid) provided by the present application has the following structural formula, wherein m is the polymerization degree of poly(N-isopropyl acrylamide), 10≤m≤150; n is the polymerization degree of poly L-glutamic acid, 20≤n≤300; and i is the relative molar number, 0.01≤i / n≤0.7.

[0016] The structural formula is:

[0017]

[0018] The content of benzyl in the poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid) is 1% to 70%.

[0019] The absorption promoter is oligomeric ethylenimine modified deoxycholic acid, characterized in that the molecular weight of the oligomeric ethylenimine is 300 to 10000.

[0020] The insulin / absorption promoter composite particle is characterized in that the mass ratio of the absorption promoter to the insulin is 0.01 to 10:1.

[0021] The polymer drug-loaded microsphere is characterized in that the size of the microsphere is 0.5 to 100 microns.

[0022] The biodegradable enteric polymer microsphere can be used as an oral absorption enhancement carrier for insulin.

[0023] Compared with the prior art, the present application first complexes the insulin with an absorption promoter to form a drug, and then prepares a drug-loaded microsphere including a carrier and the drug by an oil-in-oil solid anhydrous emulsion method together with the carrier material. The carrier is poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid), wherein poly(gamma-benzyl-L-glutamate-co-L-glutamic acid) is a pH-sensitive polymer that can protect the drug activity in gastric juice when orally administered; after reaching the intestine, the carrier material swells, dissolves or degrades due to the change in pH value, achieving intestinal targeted release of the drug; poly(N-isopropyl acrylamide) is an adhesive polymer that can prolong the residence time of the drug in the absorption site of the intestine; the absorption promoter can promote the absorption of the drug, ultimately improving the therapeutic effect of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Preparation route of poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid) for Example 2;

[0025] Figure 2 Nuclear magnetic resonance hydrogen spectrum of poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate) provided for Example 2;

[0026] Figure 3 Nuclear magnetic resonance hydrogen spectrum of poly(N-isopropyl acrylamide)-b-poly(gamma-benzyl-L-glutamate-co-L-glutamic acid) provided for Example 3;

[0027] Figure 4This is a transmission electron micrograph of the insulin / absorption enhancer composite particles prepared in Example 5;

[0028] Figure 5 This is a scanning electron microscope photograph of the drug-loaded microspheres prepared in Example 6. DETAILED DESCRIPTION

[0029] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other examples improved or modified by ordinary technicians in this field are within the scope of protection of the present invention.

[0030] Example 1

[0031] 33.948g N-isopropylacrylamide (NIPAM), 0.7715g mercaptoethylamine (AET) and a small amount of AIBN were placed in an ampoule with a stirrer, and the argon atmosphere was replaced three times. Methanol was then injected at low temperature to dissolve the mixture. The gas in the ampoule was then removed by freeze-melt method. The ampoule was sealed and placed in an oil bath at 60°C and stirred for 24 hours. After the reaction was completed, the precipitate was filtered off, and the filtrate was precipitated with ether, filtered, washed, and dried in vacuum at 25°C for 24 hours to obtain amino-terminated poly (N-isopropylacrylamide) (PNIPAM) 30 -NH2).

[0032] PNIPAM 20 -NH2 and PNIPAM 70 The preparation of -NH2 refers to PNIPAM 30 -NH2 preparation method.

[0033] Example 2

[0034] Under anhydrous conditions, 6.9440 g (2 mmol) of amino-terminated poly (N-isopropylacrylamide) (PNIPAM) prepared in Example 1 was added to three reaction bottles respectively. 30 -NH2), dissolved in dimethyl sulfoxide to obtain PNIPAM 30 -NH2 solution; respectively, 36.848g (140mmol), 52.640g (200mmol) and 78.960g (30mmol) γ-benzyl-L-glutamate-N-inner carboxylic anhydride (BLG-NCA) were dissolved in dimethyl sulfoxide and added to the PNIPAM 30 -NH2 solution, reacted at 25 ° C with stirring for 72 hours. After the reaction, the solution was poured into ether with a volume 10 times that of the solvent for precipitation, filtered, washed, and dried in vacuo at 25 ° C for 24 hours to obtain poly (N-isopropylacrylamide) -b-poly (γ-benzyl-L-glutamate) (PNIPAM30 -b-PBLG), the results are shown in Table 1.

[0035] Table 1 Performance parameters of poly (N-isopropylacrylamide)-b-poly (γ-benzyl-L-glutamate) prepared in Example 2

[0036]

[0037] In Table 1, A / I is the ratio of γ-benzyl-L-glutamate-N-internal carboxylic anhydride (BLG-NCA) to PNIPAM. 30 -NH2 molar feed ratio; Mn is the number average molecular weight of poly (N-isopropylacrylamide) -b--poly (γ-benzyl-L-glutamate), 1 The reaction yield is the ratio of the mass of the actually obtained poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate) to the theoretical mass of the poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate).

[0038] For experimental number PNIPAM 30 -b-PBLG 56 The samples were analyzed by nuclear magnetic resonance, and the results are shown in Figure 2 .

[0039] PNIPAM 20 -b-PBLG and PNIPAM 70 -b-PBLG was prepared by referring to PNIPAM 30 -b-PBLG preparation method.

[0040] Example 3

[0041] 3.4 g of PNIPAM prepared in Example 2 were respectively 30 -b-PBLG 56 The product was dissolved in 34 mL of dichloroacetic acid, and then 10.2 mL of a 33% mass concentration of hydrogen bromide in glacial acetic acid solution was added at room temperature. After stirring at 30°C for 8, 25 and 120 minutes, the obtained product was precipitated with ether and washed with ether. The product was then dissolved in N,N-dimethylformamide and dialyzed with a 3500Da dialysis bag for 3 days. After lyophilization, poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) (PNIPAM 30 -bP(BLG-co-GA)), the obtained products are shown in Table 2.

[0042] Table 2 Performance parameters of poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) obtained in Example 3

[0043]

[0044] In Table 2, A / I is the ratio of γ-benzyl-L-glutamate-N-internal carboxylic anhydride (BLG-NCA) to PNIPAM. 30 -NH2 molar feed ratio; Mn is poly (N-isopropylacrylamide) -b-poly (γ-benzyl-L-glutamate-co-L-glutamic acid) (PNIPAM 30 -bP(BLG-co-GA)) number average molecular weight, given by 1 The reaction was carried out under the following conditions: H NMR was used to determine the reaction mass; DP(BLG) was the average degree of polymerization of poly(γ-benzyl-L-glutamate) in poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) obtained from the number average molecular weight; and the reaction yield was the ratio of the actual mass of poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) obtained to the theoretical mass of poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid).

[0045] For experimental number PNIPAM 30 -bP(BLG 19 -co-GA) samples were analyzed by nuclear magnetic resonance, and the results are shown in Figure 3 .

[0046] PNIPAM 20 -bP(BLG-co-GA) and PNIPAM 70 -bP(BLG-co-GA) was prepared by referring to PNIPAM 30 -b-PBLG preparation method.

[0047] Example 4

[0048] 392.5mg deoxycholic acid (DC), 230.7mg EDC . HCl and 231.0mg NHS are dissolved in DMSO / ethanol solution (1:3, v:v) and stirred overnight, then 600.0mg oligoethyleneimine OEI600 is added.Reaction is carried out at room temperature 3 days.Use the dialysis tubing that molecular weight cut-off is 600Da by reaction solution deionized water dialysing 3d.By freeze-drying, obtain the deoxycholic acid (DCO) of the final product oligoethyleneimine modification of white solid.

[0049] The preparation of other oligoethyleneimine-modified deoxycholic acids refers to the preparation method of DCO.

[0050] Example 5

[0051] Dissolving insulin in an aqueous hydrochloric acid solution having a pH value of 2.6 is performed at a concentration of 1 mg / mL. After insulin is completely dissolved, the pH value is slowly adjusted to the isoelectric point of insulin, 5.3, with 0.1 M NaOH solution under agitation. The solution produces a blue turbidity, generating insulin nanoparticles. Leaving the mixture for 2 hours, the insulin nanoparticles are deposited in the lower floor, and the supernatant is sucked off. The concentrated insulin nanoparticle suspension of the lower floor is placed in liquid nitrogen for quick freezing, and freeze-dried to obtain the insulin nanoparticle lyophilized powder. 50 mg of DCO prepared in Example 4 and 50 mg of insulin are dissolved in water having a pH of 2.5. Then 0.1 M NaOH is added in the above-mentioned solution until the pH is 9.0. The milky white suspension is left standstill for 2 hours, and then centrifuged and freeze-dried to obtain white insulin / absorption enhancer composite particles.

[0052] The insulin / absorption enhancer composite particles of Example 5 were analyzed by transmission electron microscopy. Figure 4 .

[0053] Example 6

[0054] Using the PNIPAM in Example 2 30 -bP(BLG2-co-GA) to prepare polymer drug-loaded microspheres. 30 mg PNIPAM 30 -bP(BLG2-co-GA) was dissolved in 30 mL of dimethylformamide. After the solution was clarified, 3 mg of the insulin / absorption enhancer composite particles prepared in Example 4 were added thereto to obtain a polymer solution; the polymer solution was added to 30 mL of corn oil containing 0.25 g of Span80 under high-speed shearing at 3000 rpm / min. After emulsification for 4 minutes, the obtained emulsion was transferred to a beaker and extracted with ether three times under stirring. After standing and removing the ether, drug-loaded microspheres were obtained.

[0055] The electron microscope scanning photos of the drug-loaded microspheres are shown in Figure 5 The results showed that the particle size distribution of the drug-loaded microspheres provided by the present invention was relatively uniform, with a particle size below 10 μm. The drug loading of the drug-loaded microspheres was 8.84%, and the encapsulation efficiency was 90.2%.

[0056] The preparation of drug-loaded microspheres of other materials refers to PNIPAM 30 -bP (BLG2-co-GA) drug-loaded microspheres preparation method.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biodegradable enteric polymer microsphere, characterized in that: The invention comprises a carrier material and a drug, wherein the carrier material is poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid), the drug is insulin / absorption enhancer composite particles, the absorption enhancer is oligopolyethylenimine-modified deoxycholic acid, the mass ratio of the absorption enhancer to the insulin is 0.01-10:1, and the mass percentage of the drug to the carrier is 1% to 15%.

2. The biodegradable enteric polymer microspheres according to claim 1, wherein the preparation method comprises: preparing the polymer microspheres by a solid-in-oil-in-oil emulsion method, mixing the drug and the carrier material in an organic solvent to obtain a solid-in-oil emulsion, then emulsifying the mixture with a vegetable oil containing a surfactant to obtain a solid-in-oil-in-oil emulsion, extracting the emulsion with ether as an extractant to obtain the biodegradable enteric polymer microspheres, wherein the mass percentage of the drug in the carrier is 1% to 15%.

3. The carrier material poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) according to claim 1, wherein: m is the degree of polymerization of poly(N-isopropylacrylamide), 10≤m≤150; n is the degree of polymerization of poly-L-glutamic acid, 20≤n≤300; i is the relative molar number, 0.01≤i / n≤0.

7.

4. The carrier material poly(N-isopropylacrylamide)-b-poly(γ-benzyl-L-glutamate-co-L-glutamic acid) according to claim 1, characterized in that The benzyl content is 1%-70%.

5. The absorption enhancer oligopolyethylenimine-modified deoxycholic acid according to claim 1, characterized in that: The molecular weight of oligoethyleneimine is 300-10,000.

6. The biodegradable enteric polymer microspheres according to claim 1, characterized in that: The size of the microspheres is 0.5-100 μm.

7. The insulin / absorption enhancer composite particles according to claim 1, characterized in that The mass ratio of the absorption enhancer to the insulin is 0.01-10:1.

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