CN107233570B

CN107233570B - VEGF antibody-loaded ophthalmic temperature-sensitive hydrogel implant and preparation method thereof

Info

Publication number: CN107233570B
Application number: CN201710303559.5A
Authority: CN
Inventors: 李磊; 孟艳秋; 胡丹丹; 宁偎锋; 马蕾
Original assignee: Shenyang University of Chemical Technology
Current assignee: Shenyang University of Chemical Technology
Priority date: 2017-07-04
Filing date: 2017-07-04
Publication date: 2021-11-23
Anticipated expiration: 2037-07-04
Also published as: CN107233570A

Abstract

A VEGF antibody-loaded eye temperature-sensitive hydrogel implant and a preparation method thereof relate to a pharmaceutical preparation and a preparation method thereof, the implant is used for temperature-sensitive hydrogel for eye injection, and the phase transition temperature of the temperature-sensitive hydrogel is 34-35 ℃; the temperature-sensitive hydrogel is selected from temperature-sensitive NIPAAm hydrogel and carries VEGF antibody; the mass concentration of the temperature-sensitive hydrogel solution is 10-20%. The invention makes the anti-VEGF monoclonal antibody drug release slowly, and the method is relatively non-invasive, the anti-VEGF monoclonal antibody drug and the gel are mixed evenly to prepare a gel system which is liquid in vitro and is quickly converted into semi-solid after being injected into the eyeball, so that the drug reaches the back end of the eye in an injection way, and the effect of local slow release is achieved.

Description

VEGF antibody-loaded ophthalmic temperature-sensitive hydrogel implant and preparation method thereof

Technical Field

The invention relates to a pharmaceutical preparation and a preparation method thereof, in particular to a VEGF antibody-loaded ophthalmic temperature-sensitive hydrogel implant and a preparation method thereof.

Background

Diabetic Retinopathy (DR) is one of the common and serious microvascular complications of diabetes, and is the leading cause of acquired blindness in adults. DR is characterized by neovascularization, the proliferation of retinal capillary endothelial cells being a prerequisite for neovascularization. At present, the methods for treating the diseases mainly comprise laser photocoagulation, photodynamic therapy, vitrectomy and the like, but the treatment methods cannot fundamentally solve the problem of angiogenesis.

In recent years, with the emergence and verification of the theory that Vascular Endothelial Growth Factor (VEGF) is an important promoter of neovascularization, the use of anti-VEGF monoclonal antibody drugs for treating neovascular eye diseases has achieved surprising effects. However, the main defects of the method are that the retention time of the monoclonal antibody drug in the eyes is uncontrollable, the half-life period of the monoclonal antibody drug in the vitreous body is short (about 3-5 days), the drug metabolism is fast, 1 repeated injection is needed every 4-6 weeks, and the occurrence probability of complications caused by the invasive operation of intraocular injection is increased.

The temperature-sensitive hydrogel can change its volume or state drastically with the change of temperature, for example, the volume changes by more than ten times, or the system changes from liquid to solid, from solid to liquid, etc., so as to release the loaded drug at the target site.

Disclosure of Invention

The invention aims to provide an eye temperature-sensitive hydrogel implant carrying VEGF antibody and a preparation method thereof, the invention enables the anti-VEGF monoclonal antibody medicament to be slowly released, and the invention is a relatively noninvasive method, the anti-VEGF monoclonal antibody medicament and gel are uniformly mixed to prepare a gel system which is liquid in vitro and quickly converted into a semi-solid state after being injected into an eyeball, so that the medicament reaches the back end of the eye in an injection manner to achieve the effect of local slow release.

The purpose of the invention is realized by the following technical scheme:

an ocular temperature-sensitive hydrogel implant carrying VEGF antibody is used for temperature-sensitive hydrogel for ocular injection, and the phase transition temperature of the temperature-sensitive hydrogel is 34-35 ℃; the temperature-sensitive hydrogel is selected from temperature-sensitive NIPAAm hydrogel and carries VEGF antibody; the mass concentration of the temperature-sensitive hydrogel solution is 10-20%.

A preparation method of an ophthalmic temperature-sensitive hydrogel implant carrying VEGF antibody comprises the preparation of a poly N-isopropylacrylamide (NIPAAm) hydrogel slow-release system of VEGF antibody, and comprises the following steps:

1) synthesis of poly (N-isopropylacrylamide) hydrogel

Dissolving a monomer NIPAAm and a cross-linking agent N, N' -methylene bisacrylamide (the mass ratio is 99: 1) in 4m L deionized water, uniformly mixing, introducing nitrogen for 30 min, adding 12 mg of initiator ammonium persulfate, adding 20 mu L of addition polymerization agent tetramethylethylenediamine, and polymerizing for 6 h at 25 ℃ to obtain hydrogel; soaking the obtained gel in deionized water at room temperature for 3d, and replacing the deionized water at intervals to remove unreacted monomers and the like; cutting the obtained gel into uniform wafers, and drying the wafers in a vacuum drying oven at 60 ℃ to constant weight;

2) preparation of VEGF monoclonal antibody poly N-isopropyl acrylamide hydrogel slow-release system

Weighing 200mg of NIPAAm hydrogel powder, placing the NIPAAm hydrogel powder into a 1.5ml balanced salt solution measuring cylinder, quickly stirring for 2min to fully dissolve the powder to form a mixed solution, placing the mixed solution into a water tank at 60 ℃ for water bath for 5min, then cooling, adding 33mg of VEGF monoclonal antibody in the gelatinized state, then placing the mixture into ice water for ice bath for 5min to obtain an injectable VEGF monoclonal antibody hydrogel slow-release system, namely the ophthalmic temperature-sensitive hydrogel implant.

The invention has the advantages and effects that:

the preparation of the invention not only can be accurately positioned at the diseased part to reduce side effects, but also can reduce the harm of diseases to patients, and the hydrogel can be used for maintaining the blood concentration in vivo and has a long-term treatment effect on symptoms.

The phase transition temperature of the temperature-sensitive hydrogel is 34-35 ℃. The preparation can reduce the injection frequency, reduce the dependence of patients on the medicine, reduce the fluctuation of the blood concentration, stabilize the blood concentration at the treatment concentration for a long time, and play the roles of slow release and local effect, thereby improving the curative effect of the medicine and increasing the compliance of the patients.

Drawings

FIG. 1 is a graph showing the change of equilibrium swelling ratio of PNIPAAm gel with temperature.

Detailed Description

The present invention will be described in detail with reference to examples.

The method comprises the following steps of 1, synthesizing poly N-isopropyl acrylamide hydrogel; 2. preparation of poly-N-isopropylacrylamide (NIPAAm) hydrogel sustained-release system for VEGF antibody: weighing 200mg of NIPAAm hydrogel powder, placing the NIPAAm hydrogel powder into a 1.5ml Balanced Salt Solution (BSS) graduated cylinder, rapidly stirring for 2min to fully dissolve the powder into a mixed solution, placing the mixed solution into a water tank at 60 ℃ for water bath for 5min, cooling, adding 25mg of VEGF antibody in a peptized state, and then placing into ice water for ice bath for 5min to obtain the VEGF antibody hydrogel slow-release system capable of being injected into gel.

Preparation of VEGF monoclonal antibody poly-N-isopropyl acrylamide (NIPAAm) hydrogel sustained-release system

1. Synthesis of poly (N-isopropylacrylamide) hydrogel

Dissolving NIPAAm monomer and crosslinking agent BIS (mass ratio of 99: 1) in 4m L deionized water, mixing, introducing N230 min, adding 12 mg initiator APS, adding 20 μ L addition polymerization agent TEMED, and polymerizing at 25 deg.C for 6 hr to obtain hydrogel. The gel was soaked in deionized water at room temperature for 3 days, and the deionized water was changed at regular intervals to remove unreacted monomers, etc. The resulting gel was cut into uniform discs and dried in a vacuum oven at 60 ℃ to constant weight.

2. Preparation of VEGF monoclonal antibody poly N-isopropyl acrylamide (NIPAAm) hydrogel sustained-release system

Weighing 200mg of NIPAAm hydrogel powder, placing the NIPAAm hydrogel powder into a 1.5ml Balanced Salt Solution (BSS) graduated cylinder, rapidly stirring for 2min to fully dissolve the powder into a mixed solution, placing the mixed solution into a water tank at 60 ℃ for water bath for 5min, cooling, adding 33mg of VEGF antibody in a peptized state, and then placing the mixture into ice water for ice bath for 5min to obtain the hydrogel slow-release system capable of injecting the gelatinous VEGF antibody.

Second, measurement of equilibrium swelling ratio

Accurately weighing the xerogel, placing the xerogel in deionized water at a certain specific temperature to fully swell the xerogel, absorbing water on the surface of the gel by using filter paper, and weighing.

The equilibrium Swelling Ratio (SR) at a certain temperature was SR = ws/wd.

Wherein ws is the mass of water in the gel (swollen gel mass-xerogel mass) and wd is xerogel mass.

The relation of the change of the equilibrium swelling ratio with the temperature is an important parameter for evaluating the temperature-sensitive performance of the gel. FIG. 1 is a graph of equilibrium swelling ratio of PNIPAAm hydrogel in deionized water as a function of temperature. Therefore, the equilibrium swelling ratio of the gel is in a descending trend along with the increase of the temperature, and the gel has obvious thermal shrinkage temperature-sensitive characteristics. When the temperature is increased to about 33 ℃, the equilibrium swelling ratio of the gel is sharply reduced, indicating that the gel undergoes a significant volume phase transition, which is considered to be the LCST of the PNIPAAm gel. This is because at temperatures below the LCST, the amide chains in the gel (-CONH-) can form intermolecular hydrogen bonds with water molecules, causing the chains of the gel to spread out and swell, and at temperatures above the LCST, the amide chains in the gel break up with the intermolecular hydrogen bonds between water molecules and with the intramolecular hydrogen bonds between adjacent amide chains, thereby draining the water out of the polymer network. See figure 1 for a graph of equilibrium swelling ratio of PNIPAAm gel as a function of temperature.

Third, detection of VEGF antibody Activity

The activity of the antibody was detected using an ELISA kit. The method is carried out according to the instructions in the kit. And (3) calculating the preservation rate of the activity of the antibody after loading the hydrogel sustained-release system according to a formula:

retention rate of antibody activity (%) = OD of VEGF antibody in poly N-isopropylacrylamide hydrogel/OD × 100 of VEGF antibody aqueous solution at the same concentration.

The results are shown in Table 1, in which the retention rate of the antibody activity of the hydrogel was 85% or more, as compared with the activity of the monoclonal antibody at the same concentration. It was shown that the hydrogel retained the activity of the VEGF antibody well.

Table 1: activity retention after loading of VEGF antibodies into hydrogel implants

。

Claims

1. A preparation method of an ophthalmic temperature-sensitive hydrogel implant carrying a VEGF antibody is characterized in that the ophthalmic temperature-sensitive hydrogel implant comprises a poly N-isopropyl acrylamide hydrogel slow release system of the VEGF antibody, and the preparation process of the hydrogel slow release system is as follows:

1) synthesis of poly (N-isopropylacrylamide) hydrogel

Dissolving a monomer NIPAAm and a crosslinking agent N, N '-methylene bisacrylamide in 4mL of deionized water, uniformly mixing, introducing nitrogen for 30 min, adding 12 mg of initiator ammonium persulfate, adding 20 mu L of addition polymerization agent tetramethyl ethylenediamine, and polymerizing at 25 ℃ for 6 h to obtain hydrogel, wherein the mass ratio of the monomer NIPAAm to the crosslinking agent N, N' -methylene bisacrylamide is 99: 1; soaking the obtained gel in deionized water at room temperature for 3d, and replacing the deionized water at intervals to remove unreacted monomers; cutting the obtained gel into uniform wafers, and drying the wafers in a vacuum drying oven at 60 ℃ to constant weight;

Weighing 200mg of NIPAAm hydrogel prepared in the step 1) and placing the NIPAAm hydrogel into a 1.5ml balanced salt solution measuring cylinder, quickly stirring for 2min to fully dissolve the NIPAAm hydrogel into a mixed solution, placing the mixed solution into a water tank at 60 ℃ for water bath for 5min and then cooling, adding 33mg of VEGF monoclonal antibody in the gel-dissolved state, then placing the mixture into ice water for ice bath for 5min to obtain an injectable VEGF monoclonal antibody hydrogel slow-release system, namely the ophthalmic temperature-sensitive hydrogel implant;

the implant is used for temperature-sensitive hydrogel for eye injection, and the phase transition temperature of the temperature-sensitive hydrogel is 34-35 ℃; the temperature-sensitive hydrogel is selected from temperature-sensitive NIPAAm hydrogel and carries VEGF antibody; the mass concentration of the temperature-sensitive hydrogel solution is 10-20%.