CN116813714A

CN116813714A - Injection type medicine slow-release peptide hydrogel and preparation method thereof

Info

Publication number: CN116813714A
Application number: CN202310592565.2A
Authority: CN
Inventors: 洪莉; 王颖; 郝卓文
Original assignee: Renmin Hospital of Wuhan University
Current assignee: Renmin Hospital of Wuhan University
Priority date: 2023-05-24
Filing date: 2023-05-24
Publication date: 2023-09-29

Abstract

The invention provides an injection type medicine slow-release peptide hydrogel and a preparation method thereof, wherein the injection type medicine slow-release peptide hydrogel comprises ion complementary polypeptides and a polypeptide for reversing cell drug resistance; the mixture of the ion complementary polypeptide and the polypeptide reversing cell drug resistance is self-assembled into peptide hydrogel in salt solution; the polypeptide for reversing cell drug resistance consists of a self-assembled peptide segment, a flexible connecting peptide segment and a cell penetrating peptide segment. The peptide hydrogel can be locally injected, has good tumor cell selective toxicity and biocompatibility, has a slow release effect on the active ingredients of the medicine, and can prevent bleeding after tumor operation. In addition, the peptide hydrogel can enhance the uptake of the tumor to the drug, and simultaneously reduce the expression level of the cell drug efflux protein, thereby maintaining higher drug concentration in cells, improving the killing capacity of the drug with the same dosage and resisting the drug resistance of tumor cells.

Description

Injection type medicine slow-release peptide hydrogel and preparation method thereof

Technical Field

The invention belongs to the technical field of nano biological medicines, and particularly relates to an injection type medicine slow-release peptide hydrogel and a preparation method thereof.

Background

Malignant tumors are the main cause of life and health risk for humans. Surgery combined postoperative chemotherapy is the main treatment mode of the current malignant tumor, however, the recurrence rate and death rate of tumor patients are high due to the occurrence of unresectable tumor residual foci and multi-drug resistance, systemic toxicity of systemic application of chemotherapy drugs also severely reduces the life quality and medication compliance of patients, which is a serious challenge facing current tumor treatment. In addition, postoperative bleeding from imperfect intraoperative hemostasis increases the risk of re-surgery, spreading iatrogenic tumors, and wound infection, even endangering patient lives.

Compared with systemic administration, local administration can maximize the drug concentration at the focus and reduce the systemic toxicity of the drug, so local therapeutic means such as intratumoral injection, intervention and perfusion have been successfully developed for tumor treatment. With the development of biological nano material technology, functional hydrogel as a chemotherapeutic drug delivery carrier becomes an ideal strategy for local treatment of tumors. The peptide nanofiber hydrogel is a gel material formed by self-assembling small molecular polypeptides into nanofibers and then cross-linking the nanofibers, and has wide application in tumor treatment, tissue repair regeneration and cell three-dimensional culture due to good in-vivo degradability, biocompatibility and drug slow release function. RADA16 is a classical ion-complementary polypeptide that spontaneously assembles into lamellar nanofibers based on beta sheet structures in saline ion solution, thereby forming a high water content gel. The degradation products in the body are amino acids, and the degradation products are not long-term in the body, so that immune rejection reaction and inflammatory reaction are caused. At present, RADA16 has been proved to be effective in delaying the release of hydrophilic and hydrophobic drugs and to have excellent hemostatic and anti-adhesion capabilities, but has no anticancer activity per se and has a limited synergistic effect on antitumor drugs.

The functionalized peptide fragments can be linked to the basic ion-complementary peptide flanking ends by solid phase synthesis to construct functional ion-complementary peptides. Therefore, the polypeptide with anticancer function is connected to RADA16 to form new peptide, and the new peptide is mixed with RADA16 according to a certain proportion to form an ideal hydrogel carrier for tumor postoperative drug treatment, and the hydrogel has multiple functions of controlling drug slow release, enhancing tumor killing and preventing postoperative bleeding and adhesion. However, this method still has a limitation that the sequence of the linked functionalized fragment should not be too long (e.g., melittin: GIGAVLKVLTTGLPALISWIKRKRQQ), otherwise a stable beta-sheet structure cannot be formed and self-assembled into gel. And the production cost of the overlong peptide chain is relatively high, and the peptide chain is put into clinic or the economic burden of a patient is increased.

Cell penetrating peptides (cell penetratingpeptides, CPPs) are a series of short peptides that can directly cross cell membranes, typically no more than 30 amino acids in length, and are classified into cationic peptides, amphiphilic peptides, and hydrophobic peptides. The sequences typically contain a relatively high relative abundance of positively charged amino acids, or an alternating pattern of polar charged amino acids and nonpolar hydrophobic amino acids. This pattern of polycation or amphiphilic structure of CPPs has prompted it to assist drugs or nanocarriers to more easily pass through cell membranes by endocytic pathways, formation of lipid rafts, or formation of pore structures, etc., which has been demonstrated to enhance tumor uptake of chemotherapeutics, proteins, sirnas, etc., thereby increasing intracellular drug concentrations to enhance drug efficacy. On the other hand, it has been reported that CPPs can reduce the level of a drug efflux protein such as P-glycoprotein (P-gp), thereby inhibiting the efflux effect of tumor cells on a drug to resist tumor resistance. Octapoly-arginine (RRRRRRRRRR, R8 for short) is a cationic cell penetrating peptide with short peptide chain, easy availability and relatively low cost, and has been widely used in recent years for the construction of liposomes and peptide-drug covalent conjugates for tumor treatment. However, most of the nano-materials modified by R8 and CPPs are in systemic administration mode, and systemic toxic reaction of the drugs cannot be improved and avoided.

Disclosure of Invention

In order to improve and avoid systemic toxic reaction of the drug, the invention provides an injection type drug slow-release peptide hydrogel capable of being locally administrated, and provides a polypeptide for preparing the injection type drug slow-release peptide hydrogel and reversing cell drug resistance, a kit and a preparation method thereof.

The technical scheme provided by the invention is as follows:

in a first aspect, the invention provides a polypeptide for reversing cellular resistance, the polypeptide consisting of a self-assembling peptide, a flexible linking peptide, and a cell penetrating peptide. The self-assembled peptide is at the C-terminus, the cell penetrating peptide is at the N-terminus, and the flexible linking peptide is between the two.

Preferably, the self-assembled peptide segment is RADA16, the cell penetrating peptide comprises 5 to 15 amino acids, and the flexible connecting peptide segment comprises 2 to 5 amino acids.

Preferably, in the above technical scheme, the cell penetrating peptide is octapolyarginine; the flexible connecting peptide segment is GG.

In a second aspect, the present invention provides an injectable drug-eluting peptide hydrogel comprising a pharmaceutically active ingredient, an ion-complementary polypeptide and a polypeptide that reverses cellular resistance as described above; the mixture of the ion complementary polypeptide and the polypeptide reversing the cell drug resistance is self-assembled into peptide hydrogel in salt solution, and the drug active ingredient is loaded in the peptide hydrogel.

The injection type medicine slow-release peptide hydrogel has tumor cell selective toxicity and biocompatibility, can enhance the medicine killing capacity, can reverse medicine resistance and can prevent postoperative bleeding. The peptide hydrogel loaded with the anti-tumor drug is locally injected into the postoperative tumor residual focus and residual cavity of a tumor patient, especially a drug-resistant tumor patient, so that the killing capacity of the anti-tumor drug can be enhanced and the tumor drug resistance can be reversed to a certain extent. Meanwhile, the injection type medicine slow-release peptide hydrogel has certain controlled release capability on medicines, is beneficial to maintaining stable medicine concentration around tumor tissues, prolonging the action time of the medicines, and avoiding the use of high-dose medicines, thereby reducing the occurrence rate of toxic reaction and acquired drug resistance of the medicines. In addition, the injection type medicine slow-release peptide hydrogel is delivered to a target area through an injection mode, can be regularly administered according to a curative effect, does not need other invasive operations, is beneficial to improving the dependency of a patient, can avoid medicine loss and serious toxic reaction caused by system transportation through a local administration mode, and can be absorbed locally to reach a potential transfer focus position. The peptide hydrogel degradation product is amino acid, does not induce inflammatory reaction and immune rejection reaction, has no toxic reaction to important organs of the whole body, and has excellent biocompatibility.

As a preferable mode of the above technical scheme, the pharmaceutically active ingredient of the injectable drug-eluting peptide hydrogel is a drug that acts in cells.

Preferably, the pharmaceutically active ingredient is an anticancer drug, such as doxorubicin.

In a third aspect, the present invention provides a kit for preparing an injectable drug sustained release peptide hydrogel comprising an independently packaged peptide stock solution and a salt solution; the peptide storage solution comprises the ion complementary polypeptide and the polypeptide for reversing cell drug resistance, and the ion complementary polypeptide and the polypeptide are mixed into a whole or independently packaged.

In a fourth aspect, the present invention provides a method for preparing an injectable drug sustained release peptide hydrogel comprising:

providing a peptide hydrogel precursor solution; the peptide hydrogel precursor solution comprises ion complementary polypeptides, the polypeptides for reversing cell drug resistance, salt ions and medicinal active ingredients;

and standing the peptide hydrogel precursor solution, and self-assembling to obtain the peptide hydrogel.

As the optimization of the technical scheme, the peptide hydrogel precursor solution is placed in an environment of not higher than 4 ℃ to self-assemble into the peptide hydrogel, so that the stability and the pharmaceutical activity of the peptide hydrogel are better kept.

As a preferred embodiment of the above-described method, the total concentration of the ion-complementary polypeptide and the polypeptide for reversing cell resistance in the peptide hydrogel precursor solution is 1 to 5w/v%, preferably 2w/v%.

Preferably, the self-assembled peptide and the ion complementary polypeptide are the same substance.

Preferably, the mass ratio of the ion complementary polypeptide to the polypeptide reversing cell resistance is 1:0.1 to 1.5, preferably 1:1.

as a preferred aspect of the above-described technical solution, the method for providing a peptide hydrogel precursor solution comprises:

respectively dissolving ion complementary polypeptide and the polypeptide for reversing cell drug resistance by using ultrapure water to obtain two polypeptide stock solutions;

the mass ratio of the two polypeptide stock solutions is 1:0.1 to 1.5 to obtain a mixed polypeptide storage solution;

and mixing the mixed polypeptide stock solution with a salt solution of the pharmaceutical active ingredient to obtain a peptide hydrogel precursor solution.

Compared with the prior art, the peptide hydrogel has the following advantages and beneficial effects:

the injection type medicine slow-release peptide hydrogel provided by the invention can be locally injected into tumor focus or postoperative tumor residual focus, can greatly increase the tumor treatment effect of the medicine, and provides more treatment opportunities for patients with drug resistance; meanwhile, the administration mode of local injection can avoid serious toxic reaction of systemic administration, can relieve pain of patients and improve compliance of the patients. The polypeptide amino acid sequence for reversing cell drug resistance is shorter, the synthesis cost is low, the polypeptide has tumor drug resistance reversing activity, and the polypeptide can be self-assembled with ion complementary polypeptide in a salt particle solution to form the peptide hydrogel which has good biocompatibility, is degradable in vivo, has tumor cell selective toxicity, can reverse tumor drug resistance, enhances the sensitivity of tumors to drugs, realizes the controlled release of the drugs and can prevent bleeding after tumor operation.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the structure of the RADA16-GG-R8 peptide;

FIG. 2 shows chemical structural formulas of RADA16 peptide (R16 in short) and RADA16-GG-R8 peptide (R16 GGR8 in short);

FIG. 3 shows a mass spectrum detection chart (A) and a high performance liquid chromatography HPLC detection chart (B) of RADA16-GG-R8 peptide;

FIG. 4 is a macroscopic view (A) of a RADA16 peptide hydrogel, a RADA16-R8 solution, a RADA16-GG-R8/RADA16 nanofiber peptide hydrogel (RR), and doxorubicin-loaded RADA16-GG-R8/RADA16 nanofiber peptide hydrogel (RRD) and an atomic force microscope view (B) of peptide nanofibers;

FIG. 5 is a circular dichroism spectrum of RADA16 peptide hydrogel, RADA16-R8 solution, RR peptide hydrogel, and RRD peptide hydrogel;

FIG. 6 shows in vitro degradation (A) and drug release profile (B) of RRD peptide hydrogels;

FIG. 7 is a graph showing the cytotoxicity profile of RR peptide hydrogels against normal cells (IOSE 80 ovarian cells and L929 fibroblasts) and ovarian cancer cells (A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells);

FIG. 8 is a graph of CCK8 assay (A) of the effect of doxorubicin solution (DOX), doxorubicin-loaded RADA16 peptide hydrogel (RD) and RRD peptide hydrogel on proliferation activity of A2780 ovarian carcinoma cells and cytotoxicity (B) of different concentrations of DOX and RRD peptide hydrogels on SKOV3 ovarian carcinoma parent cell line and SKOV3/DDP ovarian carcinoma resistant cell line;

FIG. 9 is a photomicrograph of the effect of DOX solution, RD peptide hydrogel, and RRD peptide hydrogel on migration and invasiveness of A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells;

FIG. 10 is a live-dead staining chart of the killing effect of DOX solution, RD peptide hydrogel and RRD peptide hydrogel on A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells;

FIG. 11 is a flow cytometry detection of DOX uptake in A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells following treatment with DOX solution and RRD peptide hydrogel;

FIG. 12 is a confocal micrograph of DOX uptake and nuclear localization in A2780 cells and SKOV3/DDP ovarian cancer resistant cells following treatment with DOX solution and RRD peptide hydrogel;

FIG. 13 is a real-time fluorescent quantitative PCR detection chart of the expression levels of the drug-resistance related drug efflux proteins P-gp (A) and BRCP (B) mRNA in SKOV3 ovarian cancer cells, SKOV3/DDP ovarian cancer resistant cells and RR peptide hydrogel treated SKOV3/DDP ovarian cancer resistant cells;

FIG. 14 is a statistical plot of tumor volume (A) and mass (B) of subcutaneous xenograft tumor nude mice after DOX solution, RD peptide hydrogel, and RRD peptide hydrogel treatment;

FIG. 15 is a chart of visceral HE staining of subcutaneous tumor-bearing nude mice treated with DOX solution, RD peptide hydrogel and RRD peptide hydrogel;

FIG. 16 is a graph of in vivo fluorescence imaging of small animals released in vivo from DOX solution, RD peptide hydrogel, and RRD peptide hydrogel;

FIG. 17 is a statistical plot of in vivo hemostasis time (A) and blood volume (B) for RADA16 peptide hydrogels and RR peptide hydrogels;

fig. 7 to 17: CON represents the group treated with 0.9% nacl solution, DOX represents the group treated with DOX solution, RADA16 represents the group treated with RADA16 peptide hydrogel, RR represents the group treated with RR peptide hydrogel, RD represents the group treated with RD peptide hydrogel, and RRD represents the group treated with RRD peptide hydrogel.

Detailed Description

The technical scheme of the invention is further described below with reference to specific examples. However, the examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way so that the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

The polypeptides provided in the examples below that reverse cell resistance consist of self-assembling peptides, flexible connecting peptides, and cell penetrating peptides; wherein the self-assembled peptide segment is RADA16 peptide (SEQ ID NO. 1) and is positioned at the C end; the flexible linker peptide comprises 2 amino acids, i.e., GG, located between the self-assembling peptide and the cell penetrating peptide; the cell penetrating peptide is R8 and is located at the N-terminus.

The inventor covalently bonds the cationic peptide R8 to the RADA16 peptide through the flexible connecting peptide segment GG in the experimental process to form the RADA16-GG-R8 peptide (SEQ ID NO. 2), and discovers that the introduction of the cationic peptide R8 at the side end does not influence the spontaneous assembly of the self-assembled peptide segment RADA into the beta-sheet-like nanofiber structure, however, the beta-sheet-like nanofiber structure assembled by the RADA16-GG-R8 peptide cannot be further crosslinked with each other, so that peptide hydrogel cannot be formed. The inventors have surprisingly found that when the RADA16-GG-R8 peptide is mixed with the RADA16 peptide, the two can be co-assembled to form a peptide hydrogel, namely RADA16-GG-R8/RADA16 nanofiber peptide hydrogel (RR), which has the effects of slowly releasing drugs and reversing cell resistance.

The structure of the RADA16-GG-R8 peptide provided by the invention is shown in the figures 1 and 2, and the RADA16-GG-R8 peptide comprises an RADA16 as an assembly domain, GG as a flexible connection domain and R8 as a reverse drug resistance activity domain. The cell penetrating peptide R8 is anchored at the side end of the RADA16 peptide and can still self-assemble into nano-fibers with the length of micron, and the width of the nano-fibers is wider than that of the nano-fibers self-assembled by the RADA16 peptide, which shows that the side end of the nano-fibers self-assembled by the RADA16-GG-R8 has rich cell penetrating peptide R8 epitopes, can enhance the uptake of drugs by tumors and reduce drug-resistance related drug efflux proteins, and realizes reversion of tumor drug resistance.

The technical scheme of the invention is described in detail through specific embodiments.

Example 1: synthesis of RADA16-GG-R8 peptide

The polypeptide molecules required in the experiment are synthesized by an automatic polypeptide synthesizer, purified by high performance liquid chromatography, and detected by a mass spectrometer, amino acid and polypeptide analyzer for purity and sequence. The mass spectrum MS detection chart and the high performance liquid chromatography HPLC detection chart of the RADA16-GG-R8 peptide are shown in FIG. 3 (A) and FIG. 3 (B).

The amino acid sequence of RADA16-GG-R8 peptide is Ac-RADARADAADARADA-GG-RRRRRRRRRR-CONH 2, the molecular weight of RADA16-GG-R8 is 3076.50Da as shown in FIG. 3 (A), and the purity of RADA16-GG-R8 is more than 95% as shown in FIG. 3 (B).

Example 2: construction and characterization of RADA16-GG-R8/RADA16 peptide hydrogels (RR)

10mg of RADA16-GG-R8 peptide and 10mg of RADA16 peptide powder were dissolved in 500. Mu.l of sterile ultra-pure water, respectively, to obtain a RADA16-GG-R8 stock solution and a RADA16 stock solution having a mass/volume ratio of 2%. Ultrasonic dispersion was carried out at 4℃for 30 minutes. Mixing the RADA16-GG-R8 stock solution and the RADA16 stock solution in a vortex mode according to the volume ratio of 50:50, and performing low-temperature ultrasonic dispersion for 30 minutes to obtain the RADA16-GG-R8/RADA16 stock solution.

The resulting 100. Mu.l of RADA16 stock solution, RADA16-GG-R8 stock solution and RADA16-GG-R8/RADA16 stock solution in a mass to volume ratio of 2% were mixed with an equal volume of 100. Mu.l of 1.8% NaCl solution and left at 4℃for 6 hours. In addition, 100. Mu.l of RADA16-GG-R8/RADA16 stock solution was mixed with 100. Mu.l of 1.8% NaCl solution (containing doxorubicin 2 mg/mL) in equal volume to obtain a RADA16-GG-R8/RADA16/DOX mixed solution, which was left at 4℃for 6 hours.

The RADA16-GG-R8 stock solution is mixed with a salt solution, so that peptide hydrogel cannot be effectively formed, hereinafter referred to as RADA16-R8 solution, and the RADA16 stock solution and the RADA16-GG-R8/RADA16 stock solution can be mixed with the salt solution to form peptide hydrogel, hereinafter referred to as RADA16 peptide hydrogel and RR peptide hydrogel, respectively. In addition, the mixture of RADA16-GG-R8/RADA16/DOX can also form peptide hydrogels, abbreviated as RRD peptide hydrogels, after mixing with saline solution, indicating that the loading of doxorubicin does not prevent the formation of peptide hydrogels from the reservoir of RADA16-GG-R8/RADA16, and FIG. 4 (A) shows a macroscopic view of each peptide hydrogel or solution. As shown in FIG. 4 (B), RADA16-GG-R8, RR and RRD all self-assemble to form peptide nanofibers. The width of the peptide nanofibers self-assembled by RADA16-GG-R8 was significantly wider than the peptide nanofibers self-assembled by RADA16, while the width of the peptide nanofibers self-assembled by RR and RRD was between the peptide nanofibers self-assembled by RADA16-GG-R8 and RADA16, and there were many thickened portions in the peptide nanofibers, indicating that R8 was exposed to the peptide nanofiber side ends. In addition, since the peptide nanofibers formed by self-assembly of RADA16, RR or RRD are crosslinked with each other, peptide hydrogel can be further formed, while the peptide nanofibers formed by self-assembly of RADA16-GG-R8 are often arranged in parallel, and the crosslinking is less, so that peptide hydrogel with a three-dimensional structure cannot be further formed.

The secondary structural characterization of the peptide hydrogels was analyzed by scanning circular dichroism in the 190-260 nm range. As shown in FIG. 5, the RADA16 peptide hydrogel, the RR peptide hydrogel and the RRD peptide hydrogel all formed peaks and valleys around 195nm and 216nm, respectively, indicating that they formed a beta sheet-like secondary structure, whereas RADA16-GG-R8 alone failed to form a beta sheet structure. This result suggests that R8 affects to some extent the formation of β -sheet secondary structure, whereas loading DOX does not affect the formation of secondary structure of the polypeptide.

Example 3: in vitro degradation and drug release assays for RRD peptide hydrogels

500. Mu.l of RRD peptide hydrogel (containing 30. Mu.g DOX) was added to the bottom of a 1.5 mL EP tube, and after standing at 4℃for 6 hours, 500. Mu.l of 0.9% NaCl solution containing or not containing 5unit/mL of protein kinase K was added, respectively, and the mixture was left standing at 37 ℃. The top liquid was removed at a specific time point, the remaining gel was weighed and 500. Mu.l of 0.9% NaCl solution with or without 5 units/mL protein kinase K was added again and the solution was kept at 37 ℃. The supernatant was measured for fluorescence absorbance at excitation and emission wavelengths of 478nm and 596nm, respectively, using a PerlinElmer multifunctional microplate reader to determine the DOX concentration.

As shown in fig. 6 (a), the RRD peptide hydrogel degraded more rapidly than the pure NaCl solution in the NaCl solution to which protein kinase K (which mimics the in vivo enzyme environment) was added, and was substantially completely degraded around day 12, indicating that the RRD peptide hydrogel had good biodegradability and did not remain in the body for a long period of time to generate immune and inflammatory reactions. As shown in fig. 6 (B), in the presence of proteinase K, the overall drug release rate was faster. The first 12 hours show burst release, the accumulated release amount reaches about 50%, and the release rate gradually and continuously releases for 8 days after 12 hours, which indicates that the RRD peptide hydrogel has good drug release function.

Example 4: selective toxicity of RR peptide hydrogels to tumor cells

Normal IOSE80 ovarian cells, L929 fibroblasts, a2780 ovarian cancer cells and cisplatin-induced SKOV3 multidrug resistant cells (SKOV 3/DDP ovarian cancer resistant cells for short) are seeded in 96-well plates at a density of 5000 cells/well, cultured in an incubator, fresh culture medium is replaced after the cells are in logarithmic growth phase, RR peptide hydrogels with different concentrations are respectively added, and cytotoxicity of the RR peptide hydrogels on tumor cells a2780, SKOV3/DDP and normal cells IOSE80 and L929 is quantitatively detected by adopting a CCK8 experiment after the culture for 24 hours.

As shown in FIG. 7, RR peptide hydrogels were more cytotoxic to tumor cells than normal cells, and had half inhibitory concentrations of 23.19, 39.76, 100.40 and 51.77. Mu.L/mL for A2780, SKOV3/DDP, IOSE80 and L929 cells, respectively. When the concentration of the RR peptide hydrogel is less than 20 mu L/mL, the RR peptide hydrogel has small cytotoxicity to normal cells, can inhibit 40-50% of tumor cell growth, and shows excellent tumor cell selectivity.

Example 5: RR peptide hydrogels enhance tumor sensitivity to drugs and reverse tumor resistance

(1) CCK8 cell proliferation Activity assay

A2780 ovarian cancer cells, SKOV3 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells were seeded at a density of 5000 cells/well in 96 well plates and cultured in an incubator, and intervention was performed when they were in the logarithmic phase. For A2780 ovarian cancer cells, DOX solution containing equivalent DOX, RADA16 peptide hydrogel loaded with DOX (abbreviated as RD peptide hydrogel) and RRD peptide hydrogel are respectively added, a blank control group (shown by CON in the figure) is added with 0.9% NaCl solution with equivalent volume, and after 24 hours, 48 hours and 72 hours of culture, the cell activity is detected by CCK 8. And (3) respectively adding DOX solutions and RRD peptide hydrogels with different DOX concentration gradients in equal volumes into SKOV3 ovarian cancer cells and SKOV3/DDP ovarian cancer drug-resistant cells, culturing for 24 hours, and quantitatively detecting the cell activity by adopting a CCK8 experiment.

As shown in fig. 8 (a), RRD peptide hydrogel had stronger cellular activity on a2780 ovarian cancer cells than DOX solution, and the inhibition rate of cellular activity was continuously enhanced with time, whereas RD peptide hydrogel had no statistical difference although having slightly stronger inhibition effect than DOX solution. Therefore, the RR peptide hydrogel can improve the sensitivity of A2780 ovarian cancer cells to DOX, and can reduce the administration concentration of DOX on the premise of achieving the same cell activity inhibition effect. As shown in FIG. 8 (B), half-inhibitory concentrations of DOX solution on SKOV3 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells were 0.36 and 3.34. Mu.M, respectively, and the drug resistance coefficient of SKOV3/DDP ovarian cancer resistant cells to DOX was as high as 9.28, indicating an effective drug-resistant strain. Compared with DOX solution, the half inhibition concentration of the RRD peptide hydrogel on SKOV3/DDP ovarian cancer resistant cells is 1.02 mu M, which is about 1/3 of the half inhibition concentration of DOX solution, so that the RRD peptide hydrogel effectively reverses the drug resistance of SKOV3/DDP ovarian cancer resistant cells to DOX and can effectively inhibit the growth and proliferation of SKOV3/DDP ovarian cancer resistant cells.

(2) Transwell invasion and migration experiments

Invasive migration of tumor cells is one of the key factors for cancer metastasis, and thus inhibition of tumor cell action is also an important measure for tumor therapy. 600 microliters of 10% fetal bovine serum medium was added to the lower chamber of a Transwell 24-well plate (8 micron pore size), and the equivalent amount of DOX-containing DOX solution, RD peptide hydrogel, and RRD peptide hydrogel were added separately, and the blank was added with an equivalent volume of 0.9% NaCl solution. A2780 ovarian cancer cells or SKOV3/DDP ovarian cancer resistant cells were resuspended in medium without fetal bovine serum, 200. Mu.l of the cell suspension was added to the matrigel-free and pre-matrigel-laid upper chamber (A2780 ovarian cancer cells: 6X 10) ⁴ A/hole; SKOV3/DDP ovarian cancer resistant cells: 3X 10 ⁴ And/or holes). The upper chamber is lightly placed in a lower chamber culture medium, after the culture in an incubator for 24 hours, the culture medium in the upper chamber is discarded, PBS is used for washing 3 times, a cotton swab is used for erasing cells on the upper layer of a polyester fiber film, PBS is used for washing 3 times, 4% paraformaldehyde is fixed for 30min, crystal violet is dyed for 30min, PBS is used for washing 3 times again, and the cells are photographed under an inverted microscope after being dried.

As shown in fig. 9, RRD peptide hydrogel inhibited proliferation and migration ability of a2780 ovarian cancer cells more significantly than DOX solution, while RD peptide hydrogel acts similarly to DOX solution. Compared with a blank control group, the DOX solution and the RD peptide hydrogel have poor inhibition effect on proliferation and migration of SKOV3/DDP ovarian cancer resistant cells, but the RRD peptide hydrogel has obvious inhibition effect, which shows that the RR peptide hydrogel effectively reverses the drug resistance of the SKOV3/DDP ovarian cancer resistant cells to DOX and can inhibit invasion and migration of the SKOV3/DDP ovarian cancer resistant cells, thereby inhibiting tumor metastasis.

(3) Cell live-dead staining experiment

At 1X 10 ⁵ Density of individuals/wells A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells were seeded in 12 well plates and culturedAfter 24h fresh medium was changed and the same amount of DOX containing DOX solution, RD peptide hydrogel and RRD peptide hydrogel were added separately, the blank was added with an equal volume of 0.9% NaCl solution, after 72h the medium was removed and washed clean with PBS, 1mL of Calcein-PI working solution was added to each well, incubated at 37℃in the absence of light for 30min, medium was aspirated, washed 2 times with PBS, serum-free medium was added, and then photographed under a fluorescent inverted microscope.

As shown in fig. 10, RRD peptide hydrogel had a stronger killing effect on a2780 ovarian cancer cells than DOX solution, whereas RD peptide hydrogel had an effect similar to that of DOX solution. Compared with a blank control group, the DOX solution and the RD peptide hydrogel have poor killing effect on the SKOV3/DDP ovarian cancer drug-resistant cells, but the RRD peptide hydrogel has obvious killing effect, which indicates that the RR peptide hydrogel effectively reverses the drug resistance of the SKOV3/DDP ovarian cancer drug-resistant cells to DOX and can effectively kill the drug-resistant cells.

Example 6: RR peptide hydrogels enhance uptake of drugs by tumor cells to enhance drug sensitivity and reverse drug resistance

1. Flow cytometry

At 3X 10 ⁵ Density of individuals/wells A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer drug-resistant cells were seeded in 6-well plates, fresh medium was changed after 24h of incubation, DOX solution containing equivalent amount of DOX and RRD peptide hydrogel were added, respectively, incubated at 37℃for 2h, cells were digested with pancreatin without EDTA, washed with PBS after centrifugation, centrifuged again, washed 3 times repeatedly, 100. Mu.l PBS cell suspension (containing 1X 10) ⁵ Cells) were transferred to a flow tube and 1h flow-through-the-air was used to detect the fluorescence intensity of intracellular DOX autofluorescence.

As shown in FIG. 11, on the premise of consistent DOX concentration, the uptake capacity of SKOV3/DDP ovarian cancer drug-resistant cells to DOX is obviously lower than that of A2780 ovarian cancer cells, and the drug resistance of the SKOV3/DDP ovarian cancer drug-resistant cells is consistent with that of the A2780 ovarian cancer cells. At DOX concentrations of 2.5. Mu.M, 5. Mu.M and 10. Mu.M, the fluorescence intensity of DOX of both the RRD peptide hydrogel-treated A2780 ovarian cancer cells and the SKOV3/DDP ovarian cancer drug-resistant cells is stronger than that of the DOX solution-treated group, which shows that the RR peptide hydrogel can effectively enhance the uptake of DOX by tumor cells, thereby increasing the intracellular DOX concentration, enhancing the drug sensitivity of tumor cells and reversing the drug resistance. Therefore, the use of RR peptide hydrogels for loading antitumor drugs can reduce the concentration of drug administered to achieve an effective therapeutic dose, and to some extent, the risk of developing acquired drug resistance.

2. Confocal laser microscope

At 5X 10 ⁴ Density of individuals/wells A2780 ovarian cancer cells and SKOV3/DDP ovarian cancer resistant cells were seeded in 12 well plates pre-plated with coverslips and incubated for 24 hours, fresh medium was changed and DOX solution and RRD peptide hydrogel containing equivalent amount of DOX were added separately, incubated for 2 hours at 37℃and washed 3 times with PBS, 4% paraformaldehyde was fixed for 30min, washed 3 times with PBS again, stained with DAPI working solution for 5min, washed 3 times with PBS, capped with anti-fluorescence quenching capper, and photographed under confocal microscope to see intracellular DOX fluorescence intensity and distribution. DOX is an anthracycline that exerts an antitumor effect by inhibiting synthesis of DNA and RNA in the nucleus.

As shown in FIG. 12, in A2780 and SKOV3/DDP cells, the fluorescence intensity of DOX in the nuclei of the RRD peptide hydrogel group was significantly enhanced over that of the DOX solution group, indicating that RR peptide hydrogel increased nuclear uptake of DOX by tumor cells. Notably, in SKOV3/DDP ovarian cancer resistant cells, there is a significant portion of DOX in the DOX solution group that accumulates in the vicinity of the cell membrane and cannot enter the nucleus to act, which may be one of the reasons for its resistance. Whereas in the RRD peptide hydrogel group, DOX fluorescence near the cell membrane was substantially lost, while fluorescence intensity in the nucleus was significantly enhanced, suggesting that RR peptide hydrogels could be resistant to drug resistance by increasing nuclear uptake of DOX by tumor cells.

Example 7: RR peptide hydrogel for reducing tumor drug-resistant cell drug efflux protein level and reversing drug resistance

ATP-binding cassette transporters P-glycoprotein (P-gp), breast Cancer Resistance Protein (BCRP), are transmembrane proteins that are highly expressed in tumors and involved in the development of tumor resistance, and they confer tumor resistance by actively pumping antitumor drugs out of the cells. At 3X 10 ⁵ Density of individuals/holes SKOV3 ovarian cancer cells and SKOV3/DDP ovarian cancer drug-resistant cells were seeded in 6-well plates and cultured for 24h, fresh medium was changed, and one group of SKOV3/DDP ovarian cancer drug-resistant cell medium was added theretoAdding RR peptide hydrogel, continuously culturing for 24h, respectively extracting SKOV3 ovarian cancer cells, SKOV3/DDP ovarian cancer drug-resistant cells and total RNA of the SKOV3/DDP ovarian cancer drug-resistant cells treated by the RR peptide hydrogel, removing residual DNA, performing reverse transcription to obtain cDNA, and detecting the mRNA expression levels of P-gp and BCRP in the cells by real-time fluorescence quantitative PCR.

As shown in FIG. 13, compared with the SKOV3 ovarian cancer cells as a parent, the content of P-gp and BRCP in the SKOV3/DDP ovarian cancer drug-resistant cells is obviously increased, and the RR peptide hydrogel treatment effectively reduces the expression level of P-gp and BRCP in the SKOV3/DDP ovarian cancer drug-resistant cells, so that the RR peptide hydrogel can not only increase the drug intake, but also reduce the drug discharge by reducing the level of the drug efflux proteins P-gp and BRCP, thereby reversing the drug resistance of tumor cells.

Example 8: in vivo anticancer effects and biocompatibility of RRD peptide hydrogels

Establishing a nude mice xenograft subcutaneous tumor model: 1X 10 per mouse ⁶ The inoculation amount of the ovarian cancer cells of the A2780 is inoculated under the skin of a nude mouse, and the tumor volume reaches about 100mm ³ 100 microliters of 0.9% NaCl solution (blank), DOX solution (DOX treated), RD peptide hydrogel (RD treated) and RRD peptide hydrogel (RRD treated) were injected (about 10 days), respectively, tumor size was measured every 3 days, mice were euthanized after 2 weeks, tumor tissue was peeled off, weighed and photographed, and the viscera of the mice were HE stained to observe drug toxicity.

As shown in fig. 14, the tumor volume of the nude mice in the RRD treatment group is always kept at a small level, the tumor mass of the nude mice after the nude mice are stripped on the 14 th day of the group is the lowest, the treatment effect is obviously stronger than that of the DOX treatment group and the RD treatment group, and it is fully proved that the RR peptide hydrogel can effectively enhance the anti-tumor effect of the DOX, and the RR peptide hydrogel can reduce the drug dosage as a carrier on the premise of achieving the same treatment effect. As shown in fig. 15, heart, liver, spleen, lung and kidney tissues of each group of nude mice are all in normal form, no obvious toxic or side reaction is observed, and the RR peptide hydrogel has good safety and biocompatibility as an antitumor drug carrier.

Example 9: in vivo drug sustained release performance of RRD peptide hydrogels

C57BL/6 mice were subcutaneously injected with 100. Mu.l DOX solution and RRD peptide hydrogel (DOX concentration: 1 mg/mL) after back shaving, and IVIS was used at specific time points ^@ The luminexrms III small animal in vivo imaging system performs fluorescence imaging of mice with excitation light and emission light at 478 and 596nm, respectively, to detect the fluorescence intensity and distribution of DOX in the mice. As shown in fig. 16, the fluorescence signal of the DOX solution is basically disappeared at day 1 after administration, and the RRD peptide hydrogel and the RD peptide hydrogel group still have stronger fluorescence signal until day 5 after administration, which indicates that the RR peptide hydrogel with the R8 active domain introduced can still effectively realize the controlled release of DOX, is beneficial to maintaining the stable drug concentration around tumor tissues and prolonging the drug action time, and avoids the use of high-dose drugs, thereby reducing the incidence rate of drug toxic reaction and acquired drug resistance.

Example 10: in vivo hemostatic Properties of RR peptide hydrogels

The RADA16 peptide hydrogel has been used as a commercial hemostatic material, and the invention uses a mouse liver incision model to explore whether RR peptide hydrogel still has good in vivo hemostatic function. Mouse liver incision model construction: c57BL/6 mice were anesthetized, then were cut in the abdomen, peripheral body fluid was sucked with gauze after exposing the liver, the liver was placed on pre-weighed filter paper, a scalpel made a cut 3mm deep and 10mm long in the liver, and the cut was rapidly injected with an equal amount of 0.9% nacl solution (negative control), RADA16 peptide hydrogel (positive control) and RR peptide hydrogel, clotting time was observed and recorded, and filter paper weight was weighed again after hemostasis to calculate bleeding amount. As shown in fig. 17, both the RADA16 peptide hydrogel and the RR peptide hydrogel showed excellent in vivo hemostatic effect compared to the 0.9% nacl solution, and there was no statistical difference between the two, indicating that the RR peptide hydrogel after R8 introduction still maintained good hemostatic performance. After operation, RR peptide hydrogel carrying medicine is injected to the periphery of tumor residual focus, and the functions of resisting tumor and preventing postoperative bleeding are simultaneously exerted.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A polypeptide that reverses cellular resistance, characterized by: the polypeptide consists of self-assembled peptide segments, flexible connecting peptide segments and cell penetrating peptide segments.

2. The polypeptide for reversing cellular resistance according to claim 1, wherein: the self-assembled peptide segment is RADA16, the cell penetrating peptide comprises 5-15 amino acids, and the flexible connecting peptide segment comprises 2-5 amino acids.

3. The polypeptide for reversing cellular resistance according to claim 1, wherein: the cell penetrating peptide is octapolyarginine; the flexible connecting peptide segment is GG.

4. An injectable drug sustained-release peptide hydrogel, comprising a pharmaceutically active ingredient, an ion-complementary polypeptide and a polypeptide for reversing cell resistance according to any one of claims 1 to 3; the mixture of the ion complementary polypeptide and the polypeptide reversing the cell drug resistance is self-assembled into peptide hydrogel in salt solution, and the drug active ingredient is loaded in the peptide hydrogel.

5. The injectable drug eluting peptide hydrogel of claim 4, wherein: the medicine active component of the injection medicine slow-release peptide hydrogel is a medicine which plays a role in cells.

6. A kit for preparing an injectable drug sustained release peptide hydrogel, comprising an independently packaged peptide stock solution and a saline solution; the peptide stock solution comprises an ion complementary polypeptide and the polypeptide for reversing cell resistance according to any one of claims 1 to 3, which are mixed together or independently packaged.

7. A method for preparing an injectable drug delivery peptide hydrogel comprising:

providing a peptide hydrogel precursor solution; the peptide hydrogel precursor solution comprises an ion complementary polypeptide, the polypeptide for reversing cell drug resistance according to any one of claims 1 to 3, salt ions and a drug active ingredient;

8. The method for preparing an injectable drug eluting peptide hydrogel according to claim 7, wherein: the total concentration of the ion-complementary polypeptide and the polypeptide for reversing cell resistance according to any one of claims 1 to 3 in the peptide hydrogel precursor solution is 1 to 5w/v%.

9. The injectable drug eluting peptide hydrogel according to claim 4, the kit for preparing injectable drug eluting peptide hydrogel according to claim 6, the method for preparing injectable drug eluting peptide hydrogel according to claim 7, characterized in that: the self-assembled peptide segment and the ion complementary polypeptide are the same substance.

10. The injectable drug eluting peptide hydrogel according to claim 4, the kit for preparing injectable drug eluting peptide hydrogel according to claim 6, the method for preparing injectable drug eluting peptide hydrogel according to claim 7, characterized in that: the mass ratio of the ion complementary polypeptide to the polypeptide for reversing cell resistance according to any one of claims 1 to 3 is 1:0.1 to 1.5.