Preparation method and application of composite hydrogel based on bioadhesive nanoparticles

Abstract

The present invention belongs to the field of biomedicine technology, and in particular to a preparation method and use of a composite hydrogel based on bioadhesive nanoparticles. The present invention utilizes bioadhesive nanoparticles BNP based on PLA-HPG to react with carboxymethyl chitosan, and a reversible Schiff base bond is formed by the aldehyde group on the surface of BNP and the amino group of carboxymethyl chitosan, so that BNP and the polymer are cross-linked to form a composite hydrogel with a porous network structure and containing bioadhesive nanoparticles. The composite hydrogel has good biocompatibility and injectability, and the BNP and porous structure therein enable the hydrogel to deliver water-insoluble and water-soluble drugs simultaneously, and has a sustained-release effect, can be applied to aspects such as peritoneal drug delivery and skin drug delivery, greatly expanding the application value of BNP, and has huge potential application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a composite hydrogel based on bioadhesive nanoparticles. Background Art

[0002] Nanoparticles with a hyperbranched polyglycerol (HPG) shell and a PLA core offer numerous advantages over nanoparticles with a PEG shell: ① They are more water-soluble, making them better able to disperse poorly water-soluble drugs such as CPT; ② They exhibit enhanced bioinvisibility, resulting in longer blood circulation, less liver accumulation, and higher concentrations in cancerous tissues compared to PEG. Oxidation of the vicinal diol groups on the HPG surface of PLA-HPG nanoparticles to aldehyde groups (NaIO4 oxidation method) allows the nanoparticles to directly interact with proteins, resulting in bioadhesive nanoparticles (BNP) that adsorb to various tissues. BNP can be used in sunscreens to prevent sunscreen ingredients from penetrating into the skin and causing double-strand breaks in the DNA of skin cells (US10272019B2). Furthermore, using the highly lipid-soluble PLA core allows the nanoparticles to load poorly water-soluble drugs, increasing their solubility and providing excellent sustained-release properties. However, BNP cannot load water-soluble drugs, which limits its application as a drug carrier. Therefore, if the drug delivery system of BNP is modified so that it can also load water-soluble drugs, it will be more conducive to expanding its application value. Summary of the Invention

[0003] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a bioadhesive nanoparticle BNP prepared based on PLA-HPG and an amino-containing polymer nanohydrogel, which realizes the simultaneous delivery of water-insoluble and water-soluble drugs, greatly expanding the application value of BNP.

[0004] To achieve the above object, the present invention is implemented through the following technical solutions:

[0005] The present invention provides a composite hydrogel based on bioadhesive nanoparticles. The composite hydrogel comprises bioadhesive nanoparticles BNP based on PLA-HPG and an amino-containing polymer material. The amino-containing polymer material is carboxymethyl chitosan.

[0006] Preferably, BNP is prepared by dissolving PLA-HPG in an organic solvent, then adding the mixture to water under vortexing, ultrafiltration to obtain non-adherent nanoparticles, and oxidation of the vicinal diols in the non-adherent nanoparticles to aldehyde groups. Alternatively, BNP may be prepared according to the method described in CN113975249A.

[0007] Preferably, the final concentration of the carboxymethyl chitosan is not less than 20 mg / mL, and the final concentration of BNP is not less than 5 mg / mL.

[0008] The present invention also provides the use of the above-mentioned composite hydrogel based on bioadhesive nanoparticles in the preparation of a drug delivery system, wherein the drug delivery system uses the above-mentioned composite hydrogel based on bioadhesive nanoparticles as a carrier to load water-insoluble drugs and / or water-soluble drugs.

[0009] The aldehyde structure on the surface of BNP can form a reversible Schiff base with the amino group. By utilizing this feature, BNP is reacted with a polymer material containing multiple amino structures in the molecule and having certain water retention properties, such as carboxymethyl chitosan. BNP is added to a carboxymethyl chitosan aqueous solution and incubated for a period of time to form a new type of nanohydrogel complex. Scanning electron microscopy shows that the BNP nanohydrogel has a typical 3D porous network structure, and the nanoparticles of BNP are clearly visible and evenly dispersed in the nanohydrogel. In addition, the prepared nanohydrogel also has the following important characteristics: First, the nanoparticles formed by PLA-HPG have a fat-soluble core and can deliver water-insoluble drugs, while the nanohydrogel 3D network is rich in water and can deliver water-soluble drugs, achieving the simultaneous delivery of water-insoluble and water-soluble drugs. Based on this feature, the nanohydrogel can be used for the delivery of a variety of drugs, including but not limited to paclitaxel, NO donor C 14 SNO, antibiotics, antibodies, enzymes, etc. Second, the Schiff base formed by the aldehyde and amino groups is reversible, allowing BNP to be slowly released from the nanohydrogel to restore bioadhesion, thereby interacting with proteins to adhere to various tissues, further extending the retention time of the nanoparticles and drugs. Third, because PLA-HPG and carboxymethyl chitosan have low biotoxicity and are biodegradable, the formed nanohydrogel has excellent safety and biocompatibility.

[0010] Preferably, the poorly water-soluble drugs include paclitaxel, NO donor C 14 SNO, the water-soluble drugs include antibiotics, antibodies, and enzymes.

[0011] The present invention also provides a drug delivery system for treating ovarian cancer, which includes PLA-HPG-based bioadhesive nanoparticles BNP, an amino-containing polymer material, paclitaxel, and a PD-1 antibody, wherein the amino-containing polymer material is carboxymethyl chitosan.

[0012] The present invention also provides a method for preparing the above-mentioned drug delivery system for treating ovarian cancer, comprising the following steps:

[0013] S1. PLA-HPG and paclitaxel are dissolved in an organic solvent and then added to ultrapure water under vortexing. Non-adhesive nanoparticles are obtained after ultrafiltration. The vicinal diols in the non-adhesive nanoparticles are then oxidized to aldehyde groups to prepare drug-loaded bioadhesive nanoparticles (BNPs).

[0014] S2. Mix the carboxymethyl chitosan solution with the PD-1 antibody solution, then add the BNP from step S1, mix well, and let stand until a hydrogel is formed, thereby obtaining a drug delivery system.

[0015] The present invention also provides a drug delivery system for treating skin wounds, wherein the drug delivery system comprises bioadhesive nanoparticles BNP based on PLA-HPG, a polymer material containing amino groups, and an NO donor C 14 SNO, alpha-amylase and cefepime, and the amino-containing polymer material is carboxymethyl chitosan.

[0016] The present invention also provides a method for preparing the above-mentioned drug delivery system for treating skin wounds, comprising the following steps:

[0017] S1, PLA-HPG and NO donor C 14 SNO is dissolved in an organic solvent and then added to ultrapure water under vortexing. Non-adhesive nanoparticles are obtained after ultrafiltration. The vicinal diols in the non-adhesive nanoparticles are then oxidized to aldehyde groups to prepare drug-loaded bioadhesive nanoparticles BNP.

[0018] S2. Mix the carboxymethyl chitosan solution with α-amylase and cefepime solution, then add the BNP prepared in step S1, mix well and let stand until a hydrogel is formed, thereby obtaining a drug delivery system.

[0019] In the above two drug delivery systems, the final concentration of the carboxymethyl chitosan is not less than 20 mg / mL, and the final concentration of BNP is not less than 5 mg / mL.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention utilizes PLA-HPG-based bioadhesive nanoparticles (BNP) to react with amino-containing polymer carboxymethyl chitosan. The aldehyde groups on the BNP surface form reversible Schiff base bonds with the amino groups in the polymer, allowing the BNP and the polymer to crosslink, thereby forming a composite hydrogel with a porous network structure and containing the bioadhesive nanoparticles. The composite hydrogel has good biocompatibility and injectability. The BNP and porous structure enable the hydrogel to simultaneously deliver both poorly water-soluble and water-soluble drugs with a sustained-release effect. This hydrogel can be used in intraperitoneal and transdermal drug delivery, greatly expanding the application value of BNP and possessing enormous potential application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The structure of carboxymethyl chitosan (CMCS) and the formation of BNP / CMCS nanohydrogel (a); nanohydrogel formed by different concentrations of BNP and CMCS (b); BNP structure and BNP particle size photographed by transmission electron microscopy, and the structure of BNP / CMCS nanohydrogel at different resolutions photographed by scanning electron microscopy (c).

[0023] Figure 2 Figure 3 shows the stability of BNP / CMCS nanohydrogel at 37°C (a); the degradation curve of BNP / CMCS nanohydrogel under the action of 10KU / mL lysozyme (b); the healing property of BNP / CMCS nanohydrogel loaded with pigments Coomassie Brilliant Blue and curcumin, respectively (c); the periodic changes of storage modulus G' and loss modulus G" of BNP / CMCS nanohydrogel when the strain is 1% and 500%, respectively (d); the change of BNP / CMCS nanohydrogel viscosity with shear rate and the morphology of hydrogel after injection (e).

[0024] Figure 3 Effects of BNP (a), CMCS (b), and BNP / CMCS (c) on the viability of mouse skin fibroblast 3T3 cells; effects of BNP, CMCS, and BNP / CMCS on rat erythrocyte hemolysis (d).

[0025] Figure 4 Effects of different drug administration groups on the body weight of mice in the ovarian cancer ascites metastasis model (a); effects of different drug administration groups on the survival rate of mice in the ovarian cancer ascites metastasis model (b); effects of different drug administration groups on the ratio of Ki67-positive cells in mouse tumors (c).

[0026] Figure 5Effects of different drug administration groups on the proportion of CD4+T cells in ascites cells (a, b); effects of different drug administration groups on the proportion of CD8+T cells in ascites cells (c, d); effects of different drug administration groups on the proportion of CD8+CD44+CD62- effector T cells in ascites cells (e, f); effects of different drug administration groups on the proportion of CD4+T cells in tumor cells (g, h); effects of different drug administration groups on the proportion of CD8+T cells in tumor cells (i, j).

[0027] Figure 6 The residual amount of MSSA biofilm (a); the residual amount of MRSA biofilm (b); the bacterial amount of MSSA biofilm (c); and the bacterial amount of MRSA biofilm (d) after the action of each treatment group in the in vitro experiment.

[0028] Figure 7 The dispersion effect of each treatment group on mouse wound biofilm (a); the live bacteria remaining in the biofilm after the treatment of each treatment group (b). DETAILED DESCRIPTION

[0029] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0031] Example 1 Preparation of Bioadhesive Nanoparticles-Carboxymethyl Chitosan Nanohydrogel (BNP / CMCS)

[0032] 1. Preparation method of bioadhesive nanoparticles

[0033] BNP can be prepared by precipitation method: 100 mg PLA-HPG (which can be prepared according to the method described in patent CN113975249A or purchased) is dissolved in 1 mL DMSO. If drug is loaded, 1-10 mg water-insoluble drug (such as paclitaxel, C 14The method involves dissolving 100mg of PLA-HPG (drug to PLA-HPG ratio of 0.1-1:10) with a 1mL syringe. The solution is then drawn up using a 1mL syringe and slowly added to 40mL of ultrapure water under vortexing. The solution is then ultrafiltered twice using a 100kDa ultrafiltration tube with a molecular cutoff to obtain blank or drug-loaded non-adhesive nanoparticles. The non-adhesive nanoparticles are resuspended to 25mg / mL with ultrapure water, and 1 volume of 0.1M NaIO4 solution is added. The mixture is incubated for 2 minutes to oxidize the vicinal diol to an aldehyde group. The reaction is then terminated by adding 1 volume of 0.2M Na2SO3 solution. The mixture is then ultrafiltered four times using a 100kDa ultrafiltration tube with a molecular cutoff to obtain bioadhesive nanoparticles (BNP). The BNP concentration can be adjusted by resuspending it in ultrapure water. It should be noted that the preparation of BNP can be scaled up. If BNP particles need to have color or fluorescence, 2-5 mg PLA-Cy5 can be prepared together with 100 mg PLA-HPG using the same method as above.

[0034] 2. Preparation of hydrogel

[0035] Dissolve carboxymethyl chitosan (CMCS) in a 50 mM KH2PO4 solution and adjust the solution pH to 7.0 with 1 M HCl. Mix 250 μL of a water-soluble drug solution (the concentration of the solution should be based on the effective concentration of the drug) with 500 μL of the CMCS solution. Add 250 μL of the BNP suspension, immediately vortex mix, and allow to stand until a hydrogel forms. The final concentration of CMCS in the hydrogel should be no less than 20 mg / mL, and the final concentration of BNP should be no less than 2.5 mg / mL.

[0036] For the blank BNP / CMCS hydrogel preparation method, prepare blank BNP nanoparticles using 100 mg of PLA-HPG according to the above method and resuspend in ultrapure water to a BNP concentration of 120 mg / mL. Add 250 μL of ultrapure water to 500 μL of CMCS solution (pH 7.0) and mix thoroughly. Then, add 250 μL of blank BNP at a concentration of 120 mg / mL. Immediately vortex to mix thoroughly and allow to stand until a hydrogel forms.

[0037] For example, in the preparation method of PLA-Cy5 / BNP / CMCS hydrogel, 5 mg PLA-Cy5 and 100 mg PLA-HPG were used to prepare PLA-Cy5 / BNP nanoparticles according to the above method and resuspended in ultrapure water to 120 mg / mL (BNP concentration). 500 μL of CMCS solution (pH 7.0) was added to 250 μL of ultrapure water and mixed well. Then, 250 μL of 120 mg / mL PLA-Cy5 / BNP was added and immediately vortexed to mix well. The mixture was allowed to stand until a hydrogel was formed (the hydrogel was light blue and the color was pale blue). Figure 1 a).

[0038] For example, in the preparation method of paclitaxel / BNP / CMCS@PD-1 hydrogel, 10 mg of paclitaxel and 100 mg of PLA-HPG were used to prepare paclitaxel / BNP nanoparticles according to the above method. The particles were then resuspended in ultrapure water to a BNP concentration of 120 mg / mL. To 500 μL of CMCS solution (pH 7.0), 250 μL of 20 mg / mL PD-1 antibody was added and mixed thoroughly. Then, 250 μL of the 120 mg / mL paclitaxel / BNP suspension was added, the mixture was immediately vortexed, and the mixture was allowed to stand until a hydrogel formed.

[0039] Such as C 14 In the preparation method of SNO / BNP / CMCS@α-amylase@Cefepime hydrogel, 10 mg C 14 SNO and 100 mg PLA-HPG were prepared according to the above method. 14 SNO / BNP nanoparticles. 14 The SNO stock solution contains a portion of DCM. 14 After the SNO / BNP nanoparticles were prepared, a rotary evaporator was used to remove the residual dichloromethane (DCM) (room temperature, 5 min), and the C 14 SNO / BNP nanoparticles were resuspended to 120 mg / mL (BNP concentration). 500 μL of CMCS solution (pH 7.0) was added with 125 μL of 8 mg / mL α-amylase and 125 μL of 0.26 mM cefepime, mixed well, and then 250 μL of 120 mg / mL Cefepime was added. 14 The SNO / BNP suspension was immediately vortexed to mix and allowed to stand until a hydrogel was formed. 14 The synthesis method of SNO is as follows: 540 mg of tetradecanethiol is dissolved in 3 mL of DCM, and 1 mL of 5M HCl and 162 mg of NaNO2 are dissolved in 1 mL of ultrapure water. Then, the mixture is added dropwise to the above solution under an ice-water bath, stirred in the dark for 24 hours, and the resulting solution is washed three times with water. The solvent is dried on a rotary evaporator to obtain a red liquid C. 14 SNO, finally C 14 SNO was dissolved in a mixed solvent (DMSO:DCM=3:1) at a concentration of 50 mg / mL and stored at -80°C.

[0040] For example, in the preparation method of curcumin / BNP / CMCS, 3 mg of curcumin and 100 mg of PLA-HPG were used to prepare curcumin / BNP nanoparticles according to the above method and resuspended in ultrapure water to a BNP concentration of 120 mg / mL. To 500 μL of CMCCS solution (pH 7.0), 250 μL of ultrapure water was added and mixed thoroughly. Then, 250 μL of the 120 mg / mL curcumin / BNP suspension was added, immediately vortexed to mix thoroughly, and allowed to stand until a hydrogel formed. For example, in the preparation method of BNP / CMCS@Coomassie Brilliant Blue, blank BNP nanoparticles were prepared according to the above method using 100 mg of PLA-HPG and resuspended in ultrapure water to a BNP concentration of 120 mg / mL. Take 500 μL of CCMCS solution (pH 7.0), add 250 μL of 0.01 mg / mL Coomassie Brilliant Blue G250 solution, mix evenly, then add 250 μL of 120 mg / mL blank BNP suspension, immediately vortex mix, and let stand until a hydrogel is formed.

[0041] like Figure 1 As shown in a, when the final concentration of CMCS is 20 mg / mL, the hydrogel can be formed when the final concentration of BNP is greater than 5 mg / mL; when the final concentration of CMCS is 30 mg / mL, the final concentration of BNP is 2.5 mg / mL, a softer hydrogel can also be formed, but when the BNP concentration is greater than 5 mg / mL, the hydrogel formed is more stable ( Figure 1 b). The gel formation time is determined by the concentrations of BNP and CMCS. For example, when the BNP concentration is constant, the higher the CMCS concentration, the faster the gelation. When the CMCS concentration is constant, the higher the BNP concentration, the faster the gelation. The gelation time is 10s to 30min. For hydrogels used for injection in animals, NaCl is added to a final concentration of 0.9% during the preparation of the gel (i.e., 3.6% NaCl is added to the water-soluble drug, and the rest of the method is the same). Observations using projection electron microscopy show that the BNP particles are nearly round and have a diameter of approximately 80nm. Observations using scanning electron microscopy show that the BNP nanohydrogel has a typical 3D porous network structure, and the BNP nanoparticles are clearly visible and evenly dispersed in the nanohydrogel ( Figure 1 c).

[0042] Experimental Example 1 Performance Evaluation of Bioadhesive Nanoparticle Composite Hydrogel

[0043] Taking the BNP / CMCS of Example 1 as an example, the relevant properties of the bioadhesive nanoparticle composite hydrogel were tested. When the final concentration of BNP and CMCS was 30 mg / mL, the hydrogel was able to exist stably at 37°C for 21 days, proving that the hydrogel can exist stably in vivo ( Figure 2a). At the same time, multiple enzymes in the body (such as lysozyme) can degrade the CMCS skeleton, thereby degrading the BNP / CMCS hydrogel, proving that the hydrogel has good biocompatibility ( Figure 2 b, Hydrogel formed when the concentration of BNP and CMCS were both 30 mg / mL). Since Schiff base is reversible, the hydrogels were labeled with yellow and brown markers, respectively, to construct curcumin / BNP / CMCS and BNP / CMCS@Coomassie Brilliant Blue hydrogels (where the concentrations of BNP and CMCS were both 30 mg / mL). The two hydrogels of different colors were placed together. After 3 minutes, the two hydrogels were able to connect together, indicating that the BNP / CMCS hydrogel has good healing properties ( Figure 2 c). Rheological parameters were measured using a rotational rheometer (MCR302, Anton Paar, equipped with 25 mm parallel plates). When the strain was set to 1% and 500% alternately, the storage modulus (G') and loss modulus (G") were determined. Figure 2 As shown in d, G' and G" of BNP / CMCS hydrogels with different concentrations also undergo periodic destruction and recovery with the change of strain, indicating that BNP / CMCS hydrogels have good healing properties ( Figure 2 d). Shear viscosity gradually decreases with the increase of shear rate ( Figure 2 e), indicating that BNP / CMCS hydrogel has injectable properties, which is convenient for intraperitoneal administration.

[0044] In addition, the toxicity of the hydrogel was determined using mouse fibroblast 3T3 cells. It was found that after 48 hours of treatment with 0-2 mg / mL BNP, 0-2 mg / mL CMCS, and 0-4 mg / mL BNP / CMCS hydrogels (the mass ratio of BNP to CMCS in the hydrogel was 1:1), the cell viability was detected using CCK-8 reagent and found that the cell viability was greater than 80% ( Figure 3 3a, 3b, 3c), indicating that both BNP and CMCS have extremely low toxicity, giving the hydrogel excellent safety. Hemolysis was measured using a 5% rat erythrocyte suspension in PBS. Addition of 2mg / mL BNP, 2mg / mL CMCS, and 4mg / mL BNP / CMCS hydrogel did not cause erythrocyte lysis (3d), demonstrating the hydrogel's excellent biocompatibility.

[0045] Experimental Example 2 Application of Bioadhesive Nanoparticle Composite Hydrogel in Intraperitoneal Drug Delivery

[0046] Peritoneal metastasis of ovarian cancer refers to the widespread spread and growth of tumor cells within the peritoneal cavity. It is a common type of ovarian cancer metastasis, often seen in the late stages of ovarian cancer, associated with a poor prognosis and a leading cause of death. Current clinical treatments for peritoneal ovarian metastasis primarily include cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. However, these two approaches struggle to completely remove the tumor lesions and are extremely painful for the patient. Many seriously ill or elderly patients cannot tolerate these drastic treatments. While tumor immunotherapy offers new hope for cancer treatment, ovarian cancer and its metastases often harbor minimal immune cell infiltration, representing "cold tumors" and thus being ineffective against immunotherapies, including those targeting PD-1 / PD-L1 antibodies. Therefore, combining tumor immunotherapy antibodies (such as PD-1 antibodies) with chemotherapy drugs (such as paclitaxel) is necessary to enhance their efficacy. By inducing immunogenic cell death through chemotherapy, the chemotherapy increases local immune cell infiltration within the tumor, thereby enhancing the efficacy of immunotherapy.

[0047] Taking the BNP / CMCS hydrogel drug delivery system of Example 1 as an example, a mouse peritoneal metastasis cancer model (5×10 6 cells / mouse, intraperitoneal injection, 28 days), and then drug intervention was performed. The normal group (no modeling) was given PBS intraperitoneally once a week; the model group was given PBS intraperitoneally once a week; the paclitaxel (PTX) group was given paclitaxel 10 mg / kg once a week; the PD-1 antibody group was given PD-1 antibody 10 μg / g intraperitoneally once a week; the paclitaxel + PD-1 antibody combination group was given paclitaxel 10 mg / kg and PD-1 antibody 10 μg / g intraperitoneally once a week. The blank gel group received weekly intraperitoneal injections of 300 μL / 25 g of BNP / CMCS hydrogel, with a BNP and CMCS concentration of 30 mg / mL. The paclitaxel + PD-1 antibody gel group received weekly intraperitoneal injections of 300 μL / 25 g of paclitaxel / BNP / CMCS@PD-1 antibody hydrogel (containing 10 mg / kg of paclitaxel and 10 μg / g of PD-1 antibody), with a BNP and CMCS concentration of 30 mg / mL. Mice were weighed every 2-3 days to assess the progression of peritoneal ovarian cancer. After three doses, some mice were killed under anesthesia, and samples were collected to evaluate the therapeutic effects of the drugs.

[0048] In the ovarian peritoneal metastasis model, ascites caused by the tumor is an important cause of weight changes, so weight is a very accurate indicator for evaluating tumor progression. Figure 4As shown in a, the weight gain rate of the paclitaxel + PD-1 antibody combination group was slower than that of the paclitaxel and PD-1 antibody alone groups, indicating that paclitaxel and PD-1 antibody have a combined effect; the blank gel group could not inhibit the weight gain, indicating that BNP / CMCS gel could not inhibit the progression of peritoneal metastasis of ovarian cancer; the inhibitory effect of the paclitaxel / BNP / CMCS@PD-1 antibody gel group was better than that of the paclitaxel + PD-1 antibody combination group, indicating that the gel can further enhance the combined effect of paclitaxel and PD-1 antibody through its sustained-release effect. Figure 4 The survival curve of tumor-bearing mice in b also reflects the same result. The therapeutic effect of the paclitaxel / BNP / CMCS@PD-1 antibody gel administration group is better than that of the paclitaxel + PD-1 antibody combination group. Figure 4 The results of Ki67 immunohistochemical staining in c showed that the proportion of Ki67-positive cells in the paclitaxel / BNP / CMCS@PD-1 antibody gel-treated group was lower than that in the paclitaxel + PD-1 antibody combination group, further demonstrating that the BNP / CMCS hydrogel delivery system can enhance the efficacy of paclitaxel + PD-1 antibody.

[0049] Multicolor flow cytometry was used to evaluate the effects of different drug groups on immune cells ( Figure 5 ) and found that compared with the paclitaxel and PD-1 antibody alone group, the paclitaxel + PD-1 antibody combination group was able to significantly increase the proportions of CD4+ T cells, CD8+ T cells, and CD8+CD44+CD62- effector T cells in the ascites, and also increased the proportions of CD4+ T cells and CD8+ T cells in the tumor. Compared with the paclitaxel + PD-1 antibody combination group, the paclitaxel / BNP / CMCS@PD-1 antibody gel group was able to further increase the proportions of CD8+ T cells in the ascites and CD4+ T cells and CD8+ T cells in the tumor.

[0050] The above results show that the combined efficacy of paclitaxel and PD-1 antibody can be further enhanced through the BNP / CMCS nanohydrogel delivery system, proving the role of BNP / CMCS in the delivery of water-insoluble drugs and antibody drugs, and proving the application value of BNP / CMCS in the drug treatment of peritoneal tumors.

[0051] Experimental Example 3 Application of Bioadhesive Nanoparticle Composite Hydrogel in Drug Delivery to Skin Wounds

[0052] Biofilms, composed of bacterial communities encapsulated by their own extracellular polymeric substances (EPS), are a major cause of chronic infection. Biofilm-related diseases occur in multiple human systems, including the respiratory, digestive, urinary, reproductive, cardiovascular, and skin systems. Biofilms are commonly found in chronic wounds, such as diabetic foot ulcers, pressure sores, burns, and surgical wounds, potentially leading to systemic infection, amputation, and even life-threatening consequences. Due to their protective properties, bacteria within biofilms are approximately 1,000 times more resistant to antibiotics than planktonic bacteria, making them difficult to completely kill with direct antibiotics. Even with surgical debridement and detergents, biofilm removal and disruption is difficult, and bacteria can invade deeper into tissues, causing recurrence of biofilm infection.

[0053] An effective strategy for treating biofilms is to disperse the biofilms and use sufficient antibiotics to kill the released bacteria. The main components of biofilm EPS include polysaccharides, proteins, DNA, etc., among which polysaccharides are the skeleton structure of EPS. Glycosidase (such as α-amylase) can be used to degrade the EPS structure of biofilms, making the biofilm loose and cracked, promoting the release of bacteria in the biofilm, and also promoting the further penetration of other drugs. In addition to actively degrading biofilms, inducing biofilm diffusion is also an important means. Nitric oxide (NO) is a ubiquitous quorum sensing biological signal molecule. Low doses of NO can induce bacteria to diffuse from biofilms. For this reason, this experiment is based on the above strategy and the BNP / CMCS nanohydrogel of Example 1, using BNP loaded with fat-soluble NO donor C 14 SNO, and loaded α-amylase and antibiotic Cefepime in the hydrogel network structure, to construct a nanocomposite hydrogel system that can simultaneously deliver α-amylase (α-amylase), NO and antibiotics (the hydrogel preparation method is as described above), and then used it to treat biofilms of skin infected wounds to explore whether the three substances have a synergistic effect. 14 The preparation method of SNO / BNP / CMCS@α-amylase gel was the same as that of C 14 SNO / BNP / CMCS@α-amylase@Cefepime gel, except that Cefepime was not added during the preparation process.

[0054] 1 ml LB medium was added to a 12-well plate, and methicillin-sensitive Staphylococcus aureus (MSSA, ATCC No. 6538, purchased from American Type Culture Collection) was inoculated and placed in a 37°C incubator for 48 hours to establish an in vitro biofilm model. PBS (Control group), α-amylase (0.2 mg / mL), C 14 SNO (0.4 mM), Cefepime (32 μM), α-amylase (0.2 mg / mL) + C 14 SNO (0.4 mM), BNP / CMCS gel, C 14 SNO / BNP / CMCS@α-amylase gel (containing 0.2 mg / mL α-amylase and 0.4 mM C 14 SNO), C 14 SNO / BNP / CMCS@α-amylase@Cefepime gel group (containing 0.2 mg / mL α-amylase, 0.4 mM Cefepime 14 The cells were treated with SNO and 32μM Cefepime and stained with crystal violet to determine the amount of residual biofilm. The results are shown in 6a. α-amylase + C 14 Compared with the single-use group, the SNO combined group can reduce the residual amount of biofilm, indicating that α-amylase and NO have a combined effect and can synergistically remove MSSA biofilm, while C 14 SNO / BNP / CMCS@α-amylase gel can further reduce the residual amount of MSSA biofilm. 1 mL of TSB medium (2% glucose and 2% sodium chloride were added to promote biofilm formation) was added to a 12-well plate, inoculated with methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300, purchased from Beijing Biological Collection Center), and placed in a 37°C incubator for 48 hours to establish an in vitro biofilm model. The rest of the treatment was the same as for MSSA. The results are shown in Figure 2. Figure 6 As shown in b, the effects of each treatment group are similar to those on MSSA. α-amylase and NO have a combined effect and can synergistically eliminate MRSA biofilms, while C 14 SNO / BNP / CMCS@α-amylase gel can further reduce the residual amount of MRSA biofilm. By coating LB plates, the amount of residual bacteria in the remaining biofilm was further counted. It was found that in MSSA and MRSA, the antibiotic Cefepime (32μM) could not kill the residual bacteria in the biofilm, while α-amylase + C 14The SNO combination group could significantly reduce the amount of bacteria in the biofilm and supernatant, while α-amylase+C 14 The combination of SNO and Cefepime had the best effect, which could kill the bacteria in the biofilm and the supernatant, indicating that the three substances cooperated with each other and enhanced their synergy ( Figure 6 c-6d).

[0055] Compared with in vitro models, wound biofilms formed in vivo are more difficult to remove. After establishing a wound on the back of mice, MRSA was inoculated and cultured for 2 days to form a clear biofilm. Grouping was set up: the healthy group (Healthy) established a back wound at the same time, but was not inoculated with MRSA and was given PBS (200μL); all other groups were inoculated with MRSA on the wound, of which the model group (Model) was given PBS (200μL), the antibiotic group was given 32μM Cefepime (200μL), and C 14 The SNO+α-amylase combined group was given C 14 SNO and α-amylase (200 μL, containing C 14 SNO 0.4mM and α-amylase 0.2mg / mL); blank gel group was given BNP / CMCS gel (200μL, the concentrations of BNP and CMCS in the gel were both 30mg / mL); C 14 The SNO / BNP / CMCS@α-amylase gel group was given gel (200 μL, containing 0.4 mM C 14 SNO and 0.2 mg / mL α-amylase, BNP and CMCS concentrations in the gel were all 30 mg / mL); C 14 The SNO / BNP / CMCS@α-amylase@Cefepime gel group was given gel (200 μL, containing 0.4 mM C 14 SNO, 0.2 mg / mL α-amylase and 32 μM Cefepime, the concentration of BNP and CMCS in the gel were all 30 mg / mL), and the above-mentioned administration groups were administered once every 2 days, for a total of 5 times.

[0056] The results are as follows Figure 7 As shown in a, C 14 SNO / BNP / CMCS@α-amylase gel group and C 14 The SNO / BNP / CMCS@α-amylase@Cefepime gel group had a significantly better dispersion effect on biofilm than the model group, antibiotic group, and C 14 SNO+α-amylase combined group, BNP / CMCS gel group. Take equal amount of biofilm, further plate and count the bacteria in the biofilm, analyze the residual live bacteria in the biofilm, and find that C 14The SNO / BNP / CMCS@α-amylase@Cefepime gel group was superior to the other groups and had the best bacterial killing effect ( Figure 7 b).

[0057] Through the above experimental examples, the role of BNP / CMCS in delivering enzyme drugs and antibiotics was demonstrated, and the application value of BNP / CMCS in clearing skin biofilms was demonstrated.

[0058] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A drug delivery system for treating skin wounds, characterized in that: Including composite hydrogel and NO donor C 14 SNO, α-amylase and cefepime, the composite hydrogel comprises bioadhesive nanoparticles BNP based on PLA-HPG and a polymer material containing amino groups, wherein the polymer material containing amino groups is carboxymethyl chitosan; the bioadhesive nanoparticles BNP based on PLA-HPG are loaded with fat-soluble NO donor C 14 SNO, and loaded α-amylase and cefepime in the hydrogel network structure; The C 14 The synthesis method of SNO is as follows: 540 mg of tetradecathiols was dissolved in 3 mL of DCM to obtain a solution; 1 mL of 5 M HCl and 162 mg of NaNO2 were dissolved in 1 mL of ultrapure water, and then added dropwise to the above solution under an ice-water bath. The mixture was stirred in the dark for 24 h, and the obtained solution was washed three times with water. The solvent was dried on a rotary evaporator to obtain a red liquid C. 14 SNO.

2. The method for preparing the drug delivery system for treating skin wounds according to claim 1, wherein: The following steps are involved: S1, PLA-HPG and NO donor C 14 SNO is dissolved in an organic solvent and then added to water under vortexing. Non-adhesive nanoparticles are obtained after ultrafiltration. The vicinal diols in the non-adhesive nanoparticles are then oxidized to aldehyde groups to prepare drug-loaded bioadhesive nanoparticles BNP. S2. Mix the carboxymethyl chitosan solution with α-amylase and cefepime solution, then add the drug-loaded bioadhesive nanoparticles BNP prepared in step S1, mix well and let stand until a hydrogel is formed, thereby obtaining a drug delivery system.

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