CN112516290A

CN112516290A - Injectable protein hydrogel stent and application thereof in prolonging in-vivo stability of drug

Info

Publication number: CN112516290A
Application number: CN202011442647.1A
Authority: CN
Inventors: 夏栋林; 鲍鸿一; 陈静茹; 马乾; 蒋肖含; 王馨蓉
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-19

Abstract

The invention provides an injectable protein hydrogel bracket and application thereof in prolonging in vivo stability of a medicament, wherein the hydrogel is prepared by Fmoc-Phe and Phe2, and is mixed with a Pseudomonas Fluorescens Lipase (PFL) solution to prepare and obtain an amino acid hydrogel precursor. The invention has the beneficial effects that: the injectable protein hydrogel bracket has a good volume bracket effect, can provide a supporting effect for 5-7 days, is completely metabolized by an organism, and can prolong the clearance of the organism to medicines after subcutaneous injection and inhibit the infiltration of macrophages, so that the inflammatory reaction of an injection area is reduced.

Description

Injectable protein hydrogel stent and application thereof in prolonging in-vivo stability of drug

Technical Field

The invention relates to the technical field of injectable protein hydrogel stents, which can be used as a drug carrier to prolong the action time of drugs by subcutaneous injection, in particular to an injectable protein hydrogel stent and application thereof to the prolongation of the in vivo stability of drugs.

Background

Hydrogels are hydrophilic water-swellable polymer networks formed from a variety of natural and synthetic polymeric synthetic building blocks. These synthetic blocks are engineered to crosslink through chemical reactions or physical interactions of cells and proteins, during which injection or in situ hydrogel formation is rapidly carried out. However, most of the existing injectable hydrogel has poor biocompatibility and biodegradability, and is not beneficial to the fixed-point forming of the gel after injection and the tolerance problem caused by long-term movement.

How to solve the above technical problems is the subject of the present invention.

Disclosure of Invention

The invention aims to provide an injectable protein hydrogel stent and application thereof in prolonging in-vivo stability of a medicament.

The invention is realized by the following measures: the erythrocyte-carried insulin medicament is characterized in that erythrocytes are used as carriers, insulin and protoporphyrin (PPIX) are carried, and hydrogel is coated.

As a further optimization scheme of the erythrocyte insulin-loaded medicine provided by the invention, the insulin loading amount in the carrier is 30mg/mL, the carrier is dissolved by a DMSO solution, and the content of protoporphyrin IX (PPIX) is 0.3%.

As a further optimization scheme of the erythrocyte insulin-loaded medicine provided by the invention, a hypotonic dialysis method is adopted to load insulin, and the dialysis time is 12 h.

As one provided by the inventionThe further optimized scheme of the erythrocyte insulin-loaded medicine adopts ultrasonic wave to stimulate protoporphyrin IX, the ultrasonic frequency is 28KHz, and the ultrasonic intensity is 0.5w/cm²The sonication time was 30 s.

In order to better achieve the above object, the present invention further provides a method for preparing an insulin-loaded erythrocyte medicine, which comprises the following steps:

(1) preparing an erythrocyte carrier: collecting 2mL of fresh SD rat whole blood by adopting a cardiac apex blood collection mode, performing anticoagulant treatment on the fresh SD rat whole blood in a 10mL EP tube by using heparin, centrifuging the whole blood for 5min at 2000r/min, discarding the upper plasma layer, repeating the process, suspending the whole blood in PBS with the same volume and the pH value of 7.4 to obtain a red blood cell suspension with the specific volume of 50 percent, and storing the red blood cell suspension in a dark place at 4 ℃;

(2) construction of erythrocyte vector: dialyzing by hypotonic dialysis for 12h, loading insulin and protoporphyrin IX into erythrocytes, wherein the concentration of insulin is 30mg/mL, and the concentration of protoporphyrin IX is 0.3%.

As a further optimization scheme of the preparation method of the erythrocyte insulin-loaded medicine provided by the invention, in the step (1), the erythrocytes are centrifuged at 2000r/min for 5min during centrifugation, the supernatant is discarded, and then the erythrocytes are resuspended in PBS with the same volume and the pH value of 7.4 to obtain erythrocyte suspension with 50% specific volume, and the erythrocyte suspension is stored at 4 ℃ in a dark place.

The application of the erythrocyte insulin-loaded medicine provided by the invention in preparing the medicine for treating diabetes mellitus.

In order to better achieve the above objects, the present invention further provides an injectable protein hydrogel scaffold, which is prepared from Fmoc-Phe, Phe2 and PFL.

Fmoc-Phe 16mg and Phe 212 mg were weighed accurately into the above starting materials and slowly added to a mixed solution of 1mL of PBS (pH 7.4) and 420. mu.L of 0.5mol/L NaOH and stirred slowly until completely dissolved, at which time the final concentrations of Fmoc-Phe 11.3mg/mL and Phe 28.5 mg/mL were obtained. The pH was adjusted to 7.0 with 0.1mol/L HCl solution, at which time the volume of the solution was about 3.5 mL. And finally, adding the drug to be encapsulated, fully stirring and dissolving, slowly adding the PFL solution into the amino acid hydrogel precursor solution, uniformly mixing, then placing the solution in a constant-temperature water bath at 37-80 ℃ for 2-10 minutes, and after complete solidification, sealing at room temperature and keeping away from light.

Preferably, the mass ratio of Fmoc-Phe to Phe2 in the raw material is 4: 3, the solvent was a mixture of 1mL of PBS (pH 7.4) and 420. mu.L of 0.5mol/L NaOH, and the pH of the reaction solution was 7.4.

The invention also provides a preparation method of the hydrogel bracket, which comprises the following specific steps:

(1) preparation of hydrogel: Fmoc-Phe 16mg and Phe 212 mg were weighed accurately and added slowly to a mixed solution of 1mL of PBS (pH 7.4) and 420. mu.L of 0.5mol/L NaOH and stirred slowly until complete dissolution, at which time the final concentration of the solution was Fmoc-Phe 11.3mg/mL and Phe 28.5 mg/mL, and the pH was adjusted to 7.0 with 0.1mol/L HCl solution;

(2) completely dissolving Pseudomonas Fluorescens Lipase (PFL) in PBS to prepare 100 mu L of 50mg/mL PFL solution, slowly adding the prepared PFL solution into the amino acid hydrogel precursor solution, uniformly mixing, and then putting the solution in a thermostatic water bath at 37-80 ℃ for 2-10 minutes to obtain the compound.

Further, in the step (2), the prepared injectable protein hydrogel scaffold needs to be sealed at room temperature and stored away from light after being completely cured.

Further, in the step (2), the injectable protein hydrogel scaffold obtained was prepared, and PFL was added before subcutaneous injection and injection was performed within 6 to 10 minutes (in a fluid state) after the addition.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention can effectively achieve the effect of prolonging the in vivo stability of the medicament by combining Fmoc-Phe and Phe 2;

(2) the invention can effectively inhibit the infiltration of macrophages, thereby reducing the inflammatory reaction of a hydrogel injection area;

(3) the invention has good volume support function, can be completely metabolized in a short time, and has no body damage function;

(4) the early-stage animal simulation experiment research result of the invention provides a foundation for the clinical application of the injectable protein hydrogel (the hydrogel of the invention) for prolonging the in vivo stability of the medicine;

(5) the composition does not damage human bodies and has good biocompatibility;

(6) the injectable protein hydrogel bracket has high coagulation speed, good volume bracket effect and can be completely metabolized by an organism, and metabolites are nontoxic.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a FTIR spectra of solution phase and gel phase amino acid hydrogels of example 1 of the present invention;

FIG. 2 is a graph showing the transmittance at different wavelengths and the absorbance at different wavelengths of ICG of a 1cm thick hydrogel according to example 2 of the present invention;

FIG. 3 is an SEM image of INS @ ER-ICG in a hydrogel of example 3 of the present invention;

FIG. 4 is an injectable hydrogel of example 4 of the present invention, (A)2 minute image; (B)10 minute images; (C) it has plasticity and can be molded into a required shape;

FIG. 5 is a graph showing the swelling curve of the hydrogel in example 4 of the present invention;

FIG. 6 is a graph showing the inhibition of macrophage infiltration by amino acid hydrogel on

days

3, 5, 7 and 10, respectively, in the example of the present invention;

FIG. 7 is a photograph of subcutaneous gel scaffolds for 0, 1, 2, and 10 days in an example of the present invention;

FIG. 8 is a graph of the pathological results of skin after injection of a subcutaneous gel scaffold in an embodiment of the present invention;

FIG. 9 is a graph of subcutaneous gel scaffold volume as a function of time in an example of the invention;

FIG. 10 is a pathological section of the skin at the injection site and the main organs of the animal model on

days

3, 5, 7 and 10 in the example of the present invention;

FIG. 11 is a blood biochemical index map of animal models at

days

0, 1, 3, 5, and 7 in the example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Of course, the specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Example 1

The influence of the decomposition of the hydrogel on the body:

preparation process of injectable protein hydrogel:

Fmoc-Phe 16mg and Phe 212 mg were weighed accurately and added slowly to a mixed solution of 1mL of PBS (pH 7.4) and 420. mu.L of 0.5mol/L NaOH and stirred slowly until complete dissolution, at which time the final concentrations of Fmoc-Phe 11.3mg/mL and Phe 28.5 mg/mL were obtained. The pH was adjusted to 7.0 with 0.1mol/L HCl solution, at which time the volume of the solution was about 3.5 mL. Pseudomonas Fluorescens Lipase (PFL) was completely dissolved in PBS (pH 7.4) to prepare 100. mu.L of a 50mg/mL PFL solution. And slowly adding the prepared PFL solution into the amino acid hydrogel precursor solution and uniformly mixing.

And transferring the prepared hydrogel sample onto a quartz glass slide, fully dehydrating and drying the sample by using a vacuum dryer, grinding, and measuring and recording the Fourier Transform Infrared (FTIR) spectrum of the hydrogel dry powder.

FIG. 1 shows a Fourier transform infrared (FT-IR) spectrum. As can be seen from the figure, the characteristic C ═ O stretching band peaks for the Fmoc-Phe & Phe2 solution phase and the Fmoc-Phe & Phe2 gel phase shifted from 1714cm-1 to 1665cm-1 compared to the FTIR spectra for the solution phase, indicating the presence of intermolecular hydrogen bonding motifs in the gel phase compared to the solution phase. Fmoc-Phe & Phe2 therefore constitutes a gel that relies on the formation of intermolecular hydrogen bonds. No new chemical bond is generated, no new metabolite is generated after the gel is decomposed, and the toxic and side reaction after the subcutaneous hydrogel is decomposed is avoided.

Example 2

Light transmittance at different wavelengths of 1cm thick hydrogel:

preparation process of injectable protein hydrogel:

placing the amino acid hydrogel sample in a detection cell, and recording a UV spectrum by using an ultraviolet-visible spectrophotometer, wherein the wavelength range of the recorded spectrum is from 200nm to 1000 nm. And calculating the light transmittance of the current wavelength according to the absorbance value of the corresponding wavelength.

As shown in FIG. 2, the transmittance at different wavelengths of the hydrogel with a thickness of 1cm is shown, and it can be seen from the graph that the transmittance of the amino acid hydrogel with a thickness of 1cm (cell optical path) is about 84.5% at 808nm at the peak of the ICG absorption wavelength, and the amino acid hydrogel has higher light transmittance.

Example 3

The injectable hydrogel scaffold entraps red blood cells-insulin:

the preparation process of the erythrocyte-insulin hydrogel comprises the following steps:

Fmoc-Phe 16mg and Phe 212 mg were weighed accurately and added slowly to a mixed solution of 1mL of PBS (pH 7.4) and 420. mu.L of 0.5mol/L NaOH and stirred slowly until complete dissolution, at which time the final concentrations of Fmoc-Phe 11.3mg/mL and Phe 28.5 mg/mL were obtained. The pH was adjusted to 7.0 with 0.1mol/L HCl solution, at which time the volume of the solution was about 3.5 mL.

Pseudomonas Fluorescens Lipase (PFL) was completely dissolved in PBS (pH 7.4) to prepare 100. mu.L of a 50mg/mL PFL solution. And slowly adding the prepared PFL solution into the amino acid hydrogel precursor solution and uniformly mixing.

1mL of insulin-coated red blood cells are taken and added with the amino acid hydrogel precursor solution according to the proper volume ratio, then 100 mul of 50mg/mL PFL solution is added, the mixture is slowly and uniformly mixed and then is put in a thermostatic water bath at 37 ℃ for 10 minutes, the red blood cell-insulin hydrogel can be solidified, and subcutaneous injection is finished by adopting a No. 20 syringe within 10 minutes.

Preparing an SEM sample:

in order to study morphological changes of erythrocytes during the preparation of erythrocyte-insulin hydrogel, erythrocytes were fixed with glutaraldehyde, dehydrated with ethanol at a concentration gradient of 35% to 100%, and finally dried using a vacuum drier. SEM sample preparation of erythrocyte-insulin hydrogel was as follows: the samples were treated with a PBS (0.1mol/L, pH 7.4) solution containing 2.5% (v/v) glutaraldehyde at 4 ℃ for 24 hours. After repeated centrifugal washing with PBS solution, the samples were treated with ethanol solutions of different concentrations for 15 minutes at room temperature, respectively, dried using a vacuum drier, and then all samples were coated with a 10nm thick gold film using a sputter coater. The coating samples were examined using a scanning electron microscope at an electron acceleration voltage of 10 keV.

As can be seen in fig. 3, the red blood cells mixed with the injectable protein hydrogel still showed the typical biconcave disc-like structure, morphologically approaching that of normal red blood cells. Thus, red blood cells within injectable protein hydrogels may be able to retain their basic biological properties, being able to withstand certain mechanical stretching or compression forces.

As can be seen from FIG. 4, the constructed erythrocyte-insulin hydrogel was able to remain fluid (A)2 minutes after the PFL addition, set to a gel (B), and was plastic (C).

Example 4

Swelling ratio of hydrogel:

the swelling ratio (%) of the hydrogel was measured using a gravimetric method. The sample was slowly immersed in PBS (pH 7.4) and in a thermostatic water bath at 37 ℃, and after removing the sample every hour and allowing it to stand for 5 minutes to clock dry surface moisture, the remaining weight of the sample was recorded.

As shown in FIG. 5, the hydrogel prepared, erythrocyte-insulin hydrogel (ER @ hydrogel), has a nearly similar swelling curve, and thus both behave similarly in swelling kinetics. After 6 hours of absorption, the weight increased from 4.7mg to 6.0mg and the curve gradually leveled off and did not increase. The lower swelling capacity prevents subcutaneous hydrogel from absorbing a large amount of surrounding tissue fluid, inhibits hydrogel expansion, compresses the epidermis and damages the hydrogel porous structure.

Example 5

Hydrogel biocompatibility and safety:

the preparation process of the INS @ ER-ICG hydrogel comprises the following steps:

1. Animal experiments

Protein hydrogel was injected subcutaneously into the back of the mouse model as a scaffold.

As shown in FIG. 6, skin tissues of the hydrogel injection sites of the mice were taken from 3, 5, 7 and 10 days, respectively. Green fluorescence is macrophages specifically labeled by the F4/80 antibody. From the control group, macrophages slightly aggregated on day 5, significantly increased on day 7, and gradually aggregated on day 10. Meanwhile, in the hydrogel-treated group, it was found that the fluorescence was low and the intensity was weak, and the fluorescence intensity and number were still at a low level at day 10, which is in sharp contrast to the control group, thus demonstrating that the amino acid hydrogel in this test can effectively inhibit the infiltration of macrophages, thereby reducing the inflammatory response in the hydrogel injection region.

As shown in fig. 7, the shape of the subcutaneous gel is similar to a half ellipsoid, and it was calculated that the hydrogel scaffold could maintain a low collapse rate for a long time, and still maintain 50% of the volume on the sixth day after injection, and completely disappear after 10 days (fig. 8). This demonstrates that the hydrogel has a good volume scaffold effect and can be completely metabolized in a short time.

As shown in FIG. 9, pathological analysis after subcutaneous injection revealed that the hydrogel (M) could be found subcutaneously for 3 to 7 days, and that the hydrogel disappeared by 10 days. The hydrogel can be naturally decomposed and absorbed by the body after subcutaneous injection.

2. Evaluation of safety

To investigate the potential risk of injectable protein hydrogels on healthy tissue, diabetic mice were sacrificed on

days

3, 5, 7 and 10 after injection of injectable protein hydrogels and histological analysis of skin tissue of each major organ and injection site was performed. Organs including heart, liver, kidney, lung and spleen were examined by hematoxylin and eosin (H & E) method.

As shown in fig. 10, H & E staining showed no detectable damage in the tissue sections, indicating that injectable protein hydrogels did not cause damage to these organs.

As shown in FIG. 11, liver function markers including alkaline phosphatase (ALP), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), renal function markers urea nitrogen (BUN), creatinine (Cr) and Globulin (GLB) were also normal in the blood biochemical analysis. Thus, during the test, it was shown that the rats had no significant hepatorenal toxicity after 1, 3, 5 and 7 days of injectable protein hydrogel treatment.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. An injectable protein hydrogel stent extension drug, wherein the injectable protein hydrogel is prepared by Fmoc-Phe and Phe 2.

2. The injectable protein hydrogel stent extension drug of claim 1, wherein the mass ratio of Fmoc-Phe to Phe2 is 4: 3, the solvent is a mixed solution of 1mL of PBS and 420 mu L of 0.5mol/L NaOH, and the pH value of the reaction solution is 7.4-8.0.

3. The injectable protein hydrogel stent extension drug of claim 2, wherein an HCl solution is added to the solution, the HCl solution having a concentration of 0.1mol/L and adjusted to a PH of 7.0.

4. The injectable protein hydrogel stent extension drug of claim 3, wherein 100 μ L of 50mg/mL PFL solution is added to the solution.

5. A process for the preparation of a medicament according to any one of claims 1 to 4, comprising the steps of:

(1) preparation of hydrogel: Fmoc-Phe 16mg and Phe 212 mg were weighed into a mixture of 1mL of PBS and 420. mu.L of 0.5mol/L NaOH and stirred to dissolve completely, to give Fmoc-Phe 11.3mg/mL and Phe-28.5 mg/mL, the pH was adjusted to 7.0 with 0.1mol/L HCl solution, and the volume of the solution was about 3.5 mL;

(2) completely dissolving Pseudomonas Fluorescens Lipase (PFL) in PBS to prepare 100 mu L of 50mg/mL PFL solution, adding the prepared PFL solution into the amino acid hydrogel precursor solution, uniformly mixing, and then putting the solution in a thermostatic water bath at 37-80 ℃ for 2-10 minutes to obtain the injectable protein hydrogel stent prolonging drug.

6. The preparation method according to claim 5, wherein the injectable protein hydrogel scaffold obtained in step (2) is stored in a sealed and light-proof manner at room temperature after being completely cured.

7. The method for preparing the protein hydrogel scaffold according to claim 5, wherein the injectable protein hydrogel scaffold obtained in the step (2) is injected by adding PFL before subcutaneous injection and within 6 to 10 minutes after the addition.

8. Use of a medicament according to any one of claims 1 to 4 for the preparation of injectable protein hydrogel scaffolds and their prolonged drug in vivo stability.