CN114224827A - Hydrogel and application thereof in preparation of postoperative treatment reagent for tumors - Google Patents

Abstract

The invention discloses a hydrogel and application thereof in preparation of a treatment reagent for tumor postoperation, and belongs to the technical field of biological medicines. The hydrogel is prepared by crosslinking chitosan, pullulan and MMPs sensitive polypeptide, wherein sulfydryl is modified on the molecules of the chitosan and the pullulan. The hydrogel has MMP enzyme responsiveness, can regulate and control the development of a tumor microenvironment after a tumor operation by consuming the MMP enzyme, and prevents tumor metastasis promoted by up-regulation of the MMP enzyme; but also can realize the release of drugs sensitive to MMP enzyme; a slow-release drug storage is also provided for in-situ tumor drug delivery, and multiple drugs can be carried in the drug storage; meanwhile, the carrier material also has the function of absorbing tissue fluid, provides a multifunctional carrier for local hemostasis, fixing and killing residual tumor cells and preventing tumor metastasis after tumor resection, and can be applied to combined immunotherapy after tumor resection.

Description

Hydrogel and application thereof in preparation of postoperative treatment reagent for tumors

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a matrix metalloproteinase responsive hydrogel and application thereof in preparation of a postoperative treatment reagent for tumors.

Background

Surgical resection is the first treatment of choice for the local treatment of malignancies, but surgically induced tissue damage, particularly the subsequent local pro-inflammatory and wound healing responses, has been shown to increase growth factor levels, support local and distant tumor recurrence, and improve the invasiveness and motility of residual tumor cells by enhancing the expression of tumor cell adhesion molecules and inducing the release of Matrix Metalloproteinases (MMPs), particularly a massive increase in local MMP-9 following tumor resection.

In recent years, with the great success of immunotherapy based on "Immune Checkpoint Blockade (ICB)" in clinical cancer treatment, ICB is gradually and widely applied in different tumor treatments, and a treatment mode for improving the anti-tumor curative effect through tumor immunotherapy has received wide attention. However, due to the complex tumor microenvironment, the response rate of cancer treatment with ICB immunotherapy alone is only about 20%. Therefore, by regulating and controlling the tumor microenvironment and designing an immune combination treatment means, the method has important significance for improving the responsiveness of the tumor immunotherapy, and the effective tumor immunotherapy can stimulate the systemic immune effect to generate immune memory and has good inhibition effect on the recurrence and metastasis of the tumor.

At present, chitosan/pullulan sugar water gel is mainly used as wound dressing, drug carrier and tissue engineering scaffold in the biomedical field. The preparation of the hydrogel is mainly carried out by physical crosslinking and chemical crosslinking. Compared with the hydrogel which is physically crosslinked, the hydrogel which is chemically crosslinked has higher stability and mechanical property. However, gels are currently prepared by chemically cross-linked hydrogels, such as CN113512206A, using acrolein as the cross-linking agent; CN112940291A gel was prepared using glyoxylic acid as a small molecule crosslinker. The biocompatibility of the introduced small-molecule cross-linking agent is unknown, and the release of the drug can be influenced by the residue of the excessive small-molecule cross-linking agent in the gel carrier.

Therefore, the gel slow-release system which is suitable for postoperative treatment of tumors, has the functions of stopping bleeding, promoting wound healing, regulating and controlling tumor matrix change, stimulating responsive drug release, and has good biocompatibility and biodegradability is developed, and the gel slow-release system can be used for combined immunotherapy after the tumor operation and has very important significance.

Disclosure of Invention

The invention aims to provide a matrix metalloproteinase responsive hydrogel and application thereof in preparing a postoperative treatment reagent for tumors. Through modifying the gel material and utilizing the amino acid characteristic of MMPs sensitive polypeptide, the sulfhydryl group of the gel material and cysteine in a polypeptide sequence are subjected to spontaneous oxidation so as to crosslink chitosan and pullulan, thereby avoiding the disadvantage of a small molecular crosslinking agent and endowing the carrier with sensitivity of response to the MMPs in a tumor microenvironment.

In order to achieve the purpose, the invention adopts the following technical scheme:

a hydrogel is prepared by cross-linking chitosan, pullulan and MMPs sensitive polypeptide;

the molecules of the chitosan are modified with sulfydryl, and the molecules of the pullulan are modified with sulfydryl;

the sequence of the MMPs sensitive polypeptide is selected from CPVGLIGC, CEGPLGVRGKC, CPGLAGGC, CGGALGLPC or CRDPLGLAGDRC, preferably CRDPLGLAGDRC.

Further, the mass ratio of the chitosan to the pullulan is 1:4-4:1, preferably 1: 2-2: 1, more preferably 1: 1. 1:2 or 2: 1.

further, the amount of the MMPs sensitive polypeptide is 10-80%, preferably 20% of the total molar amount of the thiol-modified chitosan and pullulan.

Furthermore, the hydrogel also contains active drugs, and the active drugs are chemotherapeutic drugs and/or antibody drugs.

In the invention, the chemotherapeutic drug can be selected from alkylating agents, antibiotics, plant alkaloids, platinum compounds and the like, and specific drugs include paclitaxel, adriamycin, deoxypodophyllotoxin, oxaliplatin and the like. The antibody drug can be selected from molecular targeted drugs, immune checkpoint inhibitors and the like, and specific drugs comprise anti-HER2 antibody, anti-CTLA4 antibody, anti-PDL1 antibody, anti-CD47 antibody and the like.

The preparation method of the hydrogel comprises the following steps:

step 1, preparing sulfhydryl-modified chitosan;

step 2, preparing sulfhydryl-modified pullulan;

step 3, dissolving the sulfhydryl-modified chitosan and the sulfhydryl-modified pullulan in water, then adding MMPs sensitive polypeptide, and reacting for 12-16h at 35-40 ℃ to obtain a cross-linked product;

step 4, repeatedly freezing and thawing the crosslinked product to obtain gel;

and 5, freezing and drying the gel obtained in the step 4 to obtain the hydrogel.

The application of the hydrogel in preparing a therapeutic agent for tumor postoperation.

A therapeutic agent for tumor postoperation, which comprises the hydrogel.

The hydrogel can effectively absorb liquid, protein drugs and nano or micro carriers, and can even effectively adsorb and capture cells. The freeze-dried hydrogel blank carrier can sufficiently and quickly absorb the substances and can be used as a drug storage to slowly release the substances along with the degradation of the gel. Therefore, the hydrogel is used for drug delivery, and on one hand, the hydrogel can maintain the structural stability and the biological activity of an entrapped protein drug, such as an anti-PDL1 antibody, because drug carrying means such as chemical bond connection or freeze-drying is not needed; on the other hand, the self-induced tumor cell implantation material is used for implantation after a tumor operation, can efficiently absorb blood, tissue fluid and fallen tumor cells at an operation part, captures the tumor cells while stopping bleeding after the operation, induces an autoimmune system to kill the tumor cells at the operation part, and prevents relapse and metastasis after the operation.

The hydrogel has MMP enzyme responsiveness, and can regulate and control the development of a tumor microenvironment after a tumor operation by consuming the MMP enzyme, so that tumor metastasis promoted by up-regulation of the MMP enzyme is prevented; but also can realize the release of drugs sensitive to MMP enzyme; a slow-release drug storage is also provided for in-situ tumor drug delivery, and multiple drugs can be carried in the drug storage; meanwhile, the carrier material also has the function of absorbing tissue fluid, provides a multifunctional carrier for local hemostasis, fixing and killing residual tumor cells and preventing tumor metastasis after tumor resection, and can be applied to combined immunotherapy after tumor resection.

Drawings

FIG. 1 is an infrared spectrum of the preparation of thiol-modified chitosan and the product.

FIG. 2 is an infrared spectrum of the preparation of NAC modified pullulan and the product.

FIG. 3 is an infrared spectrum of the preparation of thiol-modified pullulan and the product.

FIG. 4 is a scanning electron microscope image of the preparation of hydrogel and the gel.

FIG. 5 shows the viscoelasticity measurements of hydrogels with different formulations.

FIG. 6 shows the measurement results of the swelling ratio of the hydrogel.

FIG. 7 is an enzyme sensitive release profile of a hydrogel.

FIG. 8 is a photomicrograph of a hydrogel after drug loading.

FIG. 9 shows the results of the adsorption of the hydrogel to blood cells.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

In the following examples, chitosan was used from Sigma-Aldrich with a molecular weight distribution range of 50-375 kDa; the pullulan used was purchased from Aladdin and had a molecular weight distribution ranging from 20 to 2000 kDa.

Example 1

1. Preparation of sulfhydryl modified chitosan

Weighing about 0.5 g of chitosan, dispersing in 50ml of water under magnetic stirring, adding HoBt (0.3485 g, 2.58 mmol), stirring the solution until the solution is clear, adding N-Acetyl-L-Cysteine (N-Acetyl-L-Cysteine, NAC) (0.8420 g, 5.16 mmol) and EDC & HCl (3.9568 g, 20.64 mmol) for dissolving, adjusting the pH to 5.0 with diluted hydrochloric acid (about 1 mol/L), and reacting for 24h in the dark under the protection of nitrogen. After completion of the reaction, the pH was adjusted to a range of 7.5 to 8.0 with 1 mol/L NaOH, dithiothreitol (1.5918 g, 10.32 mmol) was weighed out, dissolved in about 2 ml of water, and added to the above reaction system. The mixture is stirred for reaction for 30 hours in the dark under the protection of nitrogen. Dialyzing the reaction solution in 5 mmol/L HCl + 2 mu mol/L EDTA at 10 ℃ in the dark for 3 days (3500 Da dialysis bag), then dialyzing in 5 mmol/L HCl + 1% NaCl system for one day, finally dialyzing in water for one day, and freeze-drying to obtain the final product, namely the sulfhydryl-modified chitosan (CS-SH).

The reaction formula of the reaction is shown in figure 1a, and the infrared spectra of the chitosan and the sulfhydryl modified chitosan are shown in figures 1b and 1 c.

2. Preparation of sulfhydryl modified pullulan

(1) Preparation of thiol-modified pullulan from NAC-modified pullulan:

NAC (0.6260 g, 3.84 mmol) was weighed out accurately, placed in a 50mL three-necked flask, dissolved by adding DMSO 4 mL under nitrogen protection, EDCI (0.2610 g, 13.62 mmol) and DMAP (0.1350 mg, 1.11 mmol) were weighed, dissolved by adding DMSO 2 mL, added dropwise slowly to the three-necked flask, stirred magnetically under nitrogen protection, protected from light and activated for the carboxyl group for 1 h.

About 600 mg of pullulan (Pul) was weighed, 50mL of DMSO was added to the above system to dissolve the pullulan, and the reaction was stirred at room temperature for 24 hours under nitrogen protection. After the reaction is finished, 65 mL of glacial ethanol is added, precipitation and centrifugation (3000 rpm, 5 min) are carried out to obtain a product, the product is washed three times by the glacial ethanol, rotary evaporation is carried out at 40 ℃ for about 7 min, drying is carried out to obtain an intermediate product, the intermediate product is dissolved in 30 mL of water, 188 mg of dithiothreitol is added, and stirring is carried out for 24h under the protection of nitrogen to reduce disulfide bonds. And (3) dialyzing the reaction solution in 5 mmol/L HCl + 2 mu mol/L EDTA at 10 ℃ in the dark for 3 days (3500 Da dialysis bag), dialyzing in 5 mmol/L HCl + 1% NaCl for one day, finally dialyzing in water for one day, and cold drying to obtain the final product, namely the sulfhydryl modified pullulan.

The reaction formula of the reaction is shown in fig. 2a, the infrared spectroscopy is adopted for structure verification, the infrared spectrums of the pullulan and the thiol-modified pullulan are shown in fig. 2b and fig. 2c, and no functional group is introduced or disappears, which indicates that the thiol cannot be successfully modified to the pullulan through the reaction.

(2) Preparation of thiol-modified pullulan from lipoic acid-modified pullulan:

lipoic Acid (LA) was weighed to approximately 46 mg and placed in a 50mL three-necked flask, and dissolved by adding 4 mL of DMSO under nitrogen. 261 mg of EDC & HCl and 135 mg of DMAP were weighed out, dissolved in 2 mL of DMSO and added dropwise slowly to the three-necked flask, stirred magnetically under nitrogen, protected from light and activated for 1 h.

About 600 mg of pullulan (Pul) is weighed, 50mL of DMSO is added into the system to be dissolved, and the reaction is stirred for 24 hours at room temperature in the dark under the protection of nitrogen. After the reaction is finished, 65 mL of glacial ethanol is added, precipitation and centrifugation (3000 rpm, 5 min) are carried out to obtain a product, the product is washed three times by the glacial ethanol, rotary evaporation is carried out at 40 ℃ for about 7 min, drying is carried out to obtain an intermediate product, the intermediate product is dissolved in 30 mL of water, 188 mg of dithiothreitol is added, and stirring is carried out for 24h under the protection of nitrogen to reduce disulfide bonds. And (3) dialyzing the reaction solution in 5 mmol/L HCl + 2 mu mol/L EDTA at 10 ℃ in the dark for 3 days (3500 Da dialysis bag), dialyzing in 5 mmol/L HCl + 1% NaCl for one day, finally dialyzing in water for one day, and carrying out cold drying to obtain the final product, namely the sulfhydryl modified pullulan (Pul-SH).

The reaction formula of the reaction is shown in figure 3a, the infrared spectroscopy is adopted for structure verification, the infrared spectroscopy of the pullulan and the sulfhydryl modified pullulan is shown in figure 3b, and the infrared spectroscopy is 1727.57 cm^-1C = O characteristic absorption peak for ester bond of Pul with LA, indicating that carboxyl group of LA forms ester bond with hydroxyl group of Pul. Therefore, the sulfhydryl modified pullulan can be successfully synthesized through the reaction.

3. Preparation of hydrogels

100 mg of the prepared sulfhydryl modified chitosan (CS-SH), 50 mg of pullulan (Pul-SH) and 3.82 mg of polypeptide (CRDPLGLAGDRC) are respectively weighed, added into 1.5 mL of water, stirred and dissolved, placed at 37 ℃ for 12 h to enable sulfhydryl to fully react and crosslink, the equation of crosslinking reaction is shown in figure 4a, and then repeatedly frozen and thawed at-20 ℃ and room temperature for three times to form gel. Freeze drying the gel to obtain dry gel product, and storing.

The gel was freeze dried and stored dry at-20 ℃ and the scanning electron microscopy image of the gel is shown in FIG. 4 b.

Example 2

Viscoelastic determination of hydrogels of different formulations

According to Pul-SH: CS-SH mass ratio is 2:1, 1:1 and 1:2, the molar weight of sulfydryl of two materials is determined by Ellman reaction, then polypeptides (CRDPLGLAGDRC) with the molar ratio of 80%, 40%, 20% and 10% of sulfydryl are added to prepare gel, the change trend of storage modulus (G ') and loss modulus (G ' ') of hydrogel along with the change of oscillation frequency is determined by a rheometer under the condition of 25 ℃ and 1% deformation, and the influence of different polypeptide modifications on the gel strength is examined. The viscoelastic properties of the hydrogels are shown in figures 5 a-b. As can be seen, the storage modulus, loss modulus and viscosity of the gel tend to decrease with increasing amounts of polypeptide added in FIG. 5 a. Under the condition of ensuring a certain mechanical strength, the proportion (20%) of the large amount of the polypeptide is selected to achieve a better enzyme response effect. Respectively preparing Pul-SH according to the polypeptide content with the mercapto molar ratio of 20 percent: CS-SH is gel with the mass ratio of 2:1, 1:1 and 1:2, the rheological property is measured, and the result in figure 5b shows that the storage modulus, the loss modulus and the viscosity of the gel tend to increase along with the increase of the chitosan ratio.

Example 3

Measurement of swelling ratio of hydrogel

Taking CS-SH: three dry gels with Pul-SH mass ratios of 2:1, 1:1 and 1:2 were weighed to W0 in triplicate, added to water, removed from the water at 0.5, 1, 2, 4, 6, 24, 8, 72, 96, 120, 144, 168 h, carefully blotted with filter paper and weighed, and the weights at different time points were recorded as Wh (h is time). Swelling Ratio (SR) calculation formula: SR = (Wh-W0)/W0. SR changes over time were plotted to characterize the swelling kinetics of the gel. The maximum SR is the saturation swelling of the gel. FIG. 6 is a graph showing the measurement of the swelling ratio of the hydrogel. The results show that the gels with the three proportions can quickly absorb water and swell to reach the saturation swelling degree within 6 hours, and the higher the chitosan proportion is, the higher the saturation swelling degree is.

According to the experimental results of viscoelasticity and swelling degree, the CS-SH: Pul-SH is 2:1, and a prescription with 20 percent of polypeptide is used as an optimal prescription of the gel preparation.

Example 4

Enzyme sensitive release of hydrogels

30 mg of a dry gel product (CS-SH: Pul-SH is 2: 1), 100 mu L of IgG concentrated solution (containing 2 mg lgG) and 100 mu L of DOX liposome (containing 0.6 mg DOX) are dripped on the dry gel product in advance, the dry gel product is kept stand for 10 min to be completely absorbed, then the drug-loaded gel is placed in a centrifuge tube to carry out in-vitro drug release experiments, Hepes buffer solution and Hepes buffer solution containing MMP-9 enzyme with the concentration of 7.5 mu g/ml MMP-9 are respectively used as release media, and each group is divided into three parts in parallel. Placing the centrifuge tube at 37 ℃, shaking and incubating at a constant temperature of 50 rpm on a shaking table, sampling and detecting supernatant at 1 h, 2 h, 4h, 6h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d and 7 d, taking 200 mu L of sample for DOX detection and 2 mu L of sample for IgG detection at each time point, wherein the drug release curve of the gel is shown in figures 7 a-b. FIG. 7a is the release profile of IgG and FIG. 7b is the release profile of DOX, which shows that the gel has sustained release effect within 14 days, and simultaneously the release of the drug is accelerated by the action of MMP-9, thus demonstrating the MMP enzyme sensitivity of the gel.

Example 5

Drug entrapment investigation of gel materials

And (3) placing 5 mg of a gel dry product (CS-SH: Pul-SH is 2: 1) on a glass slide, respectively dripping 100 mu L DOX liposome and 100 mu L IgG-FITC concentrated solution upwards, covering a cover glass after the drug-containing concentrated solution is fully absorbed into the gel, placing under a fluorescence microscope for photographing, and observing the drug entrapment condition of the carrier. FIG. 8a is the distribution of IgG-FITC in gel, FIG. 8b is the distribution of DOX liposome in gel, and the experimental results show that both drugs can be uniformly distributed in the drug carrier, thus proving that the gel can effectively load nano-drugs and protein drugs.

Example 6

Examination of blood cell absorption Capacity of gel Material

10 mg of gel dry product (CS-SH: Pul-SH is 2: 1) is put into a penicillin bottle, and 50 mu L of anticoagulated whole blood is dripped on the surface of the gel. After the blood is completely absorbed into the gel after standing, 4 mL of pure water is added into a Xilin bottle, and the absorption capacity of the gel material on blood cells is inspected by photographing at different time points. As shown in FIG. 9, the gel can adsorb blood cells in a three-dimensional network structure, and after water is added, the gel can capture blood cells within a certain time even if the gel swells after absorbing water, thus proving that the gel can effectively adsorb the cells.

Sequence listing

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Claims (7)

1. A hydrogel, characterized by: prepared by cross-linking chitosan, pullulan and MMPs sensitive polypeptide;

the molecules of the chitosan are modified with sulfydryl, and the molecules of the pullulan are modified with sulfydryl;

the sequence of the MMPs sensitive polypeptide is selected from CPVGLIGC, CEGPLGVRGKC, CPGLAGGC, CGGALGLPC or CRDPLGLAGDRC.

2. The hydrogel of claim 2, wherein: the mass ratio of the chitosan to the pullulan is 1:4-4: 1.

3. The hydrogel of claim 1, wherein: the dosage of the MMPs sensitive polypeptide is 10-80% of the total molar weight of the sulfhydryl modification of the chitosan and the pullulan.

4. The hydrogel of claim 1, wherein: the hydrogel contains active drugs, and the active drugs are chemotherapeutic drugs and/or antibody drugs.

5. The method for producing the hydrogel according to claim 1, wherein: the method comprises the following steps:

step 1, preparing sulfhydryl-modified chitosan;

step 2, preparing sulfhydryl-modified pullulan;

step 3, dissolving the sulfhydryl-modified chitosan and the sulfhydryl-modified pullulan in water, then adding MMPs sensitive polypeptide, and reacting for 12-16h at 35-40 ℃ to obtain a cross-linked product;

step 4, repeatedly freezing and thawing the crosslinked product to obtain gel;

and 5, freezing and drying the gel obtained in the step 4 to obtain the hydrogel.

6. Use of the hydrogel of any one of claims 1 to 5 for the preparation of a therapeutic agent for post-operative use on tumors.

7. A therapeutic agent for post-operation of tumor comprising the hydrogel according to any one of claims 1 to 5.

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