An in-situ polymerization drug perfusion delivery system for PDA and its application

Abstract

This study proposed a PDA in-situ polymerization drug perfusion delivery system. By investigating the interaction relationship between the perfusion drug system and the body's signaling pathways and immune responses, the inhibitory mechanisms of manganese metal immune regulation, copper death-based synergistic therapy, novel protein drug immunotherapy, and enhanced traditional chemotherapy on bladder cancer cells were elucidated. Dopamine-mediated in-situ polymerization in the bladder cavity was used to load drugs, and the characteristics of polydopamine were combined to organically combine various drugs to form a new multi-functional bladder perfusion drug system with multiple effects in one. This simplifies the complexity of drug design, improves drug bioavailability, and ensures the realization of multi-modal synergistic therapy in the bladder.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to an anti-tumor drug perfusion delivery system, a preparation method and uses thereof. Background Art

[0002] Bladder cancer is the fourth most common malignant tumor, ranking first in the incidence of urogenital tumors in China. In recent years, its incidence and mortality have been showing an increasing trend year by year. The conventional treatment for bladder cancer is transurethral resection of bladder tumor followed by intravesical chemotherapy or Bacillus Calmette-Guérin (BCG) perfusion therapy, but the postoperative recurrence rate is still as high as 40%-70%. Many patients experience multiple relapses, repeated surgeries and long-term perfusion, which cause great physical and mental pain to the patients and increase the economic burden. Therefore, preventing and reducing the recurrence of bladder cancer has always been one of the key points and difficulties in the treatment of bladder cancer. Although adjuvant perfusion therapy can reduce the recurrence and progression rate of bladder cancer, due to the lack of more effective treatment methods and the short residence time and low permeability of drugs in the bladder, the overall recurrence and progression rate of bladder cancer remains high. Therefore, to solve this key problem, developing a new type of drug perfusion delivery system that is simple and safe, can effectively increase the drug concentration of the perfused drug in the bladder lesion tissue, and can effectively prevent the further development and recurrence of bladder cancer cells has become the key point and difficulty in the research field of bladder cancer.

[0003] Drug Delivery System (DDS) plays an important role in the intravesical perfusion therapy of bladder cancer. DDS can enhance the perfusion therapy effect, reduce side effects, and provide precise drug delivery. For example, paclitaxel (PTX) and docetaxel (DTX) are commonly used in the systemic chemotherapy and neoadjuvant chemotherapy for treating bladder cancer. However, they have poor solubility in water, significant systemic adverse reactions, and cause pain to patients after intravenous infusion. Therefore, in order to improve the solubility and dispersibility of PTX, administer drugs accurately, and reduce adverse reactions, it is very promising to improve PTX using nanotechnology. The preparation of nanoparticle formulation of nab-paclitaxel by binding PTX with human serum albumin has been found to significantly improve the therapeutic index of paclitaxel. This is because the binding of paclitaxel and albumin in the nanoparticles changes the drug transport mode in cells, improves the drug uptake efficiency in cells, and achieves better therapeutic effects. In addition, a nanosystem with specific targeting can be developed using the DDS system. Pan et al. developed disulfide-crosslinked PLZ4 nanobundles (DC-PNM) to deliver PTX. The loading efficiency of PTX was greater than 99%, and PTX could be released at physiological glutathione concentration. Most importantly, after modification with PLZ4, the nanoparticles could specifically target bladder tumors without accumulating in lung cancer xenografts, thus greatly reducing the systemic toxicity caused by PTX chemotherapy. Currently, this nanodrug has entered the first-phase clinical trial in the United States (NCT05519241).

[0004] Traditional perfusion therapy involves directly dissolving drugs in normal saline or glucose solution, which has the defects of poor drug absorption and low bioavailability. To solve the above problems, researchers have currently developed a method of delivering drugs in the human body through in-situ polymerization to improve drug absorption and retention time. For example, in the US20210177938A1 and US20230101687A1 patent documents, researchers at the Massachusetts Institute of Technology injected the polymer monomer dopamine, drugs, and an oxygen source (hydrogen peroxide) into the human body to achieve the effect of drug delivery. However, this process requires adding an oxygen source to the composition and contacting the endogenous catalyst of the subject to polymerize the monomer by the catalyst. The introduction of these oxygen sources will limit the use of organs sensitive to these oxidants, and at the same time, these oxidant residues will accelerate the decomposition of the polymer, reducing the effect of the perfusion carrier to achieve long-term retention and slow release on the bladder mucosa. Therefore, a new perfusion system needs to be developed. How to design and prepare a suitable perfusion carrier to achieve long-term retention and slow release on the bladder mucosa and transport drugs across the bladder mucosal barrier to the lesion site is the key scientific problem to be solved first in this project. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present application creatively uses dopamine-mediated in-situ polymerization in the bladder cavity to load drugs, combines various drugs by virtue of the characteristics of polydopamine, makes full use of the oxygen in the bladder, and forms a new multi-functional bladder perfusion drug system that combines multiple effects without adding other oxidants, simplifies the complexity of drug design, improves drug bioavailability, and ensures the realization of multi-modal synergistic treatment for bladder cancer.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An in-situ polymerization drug perfusion delivery system, characterized in that the drug perfusion delivery system is composed of a polymer monomer capable of in-situ polymerization, a therapeutic drug, and a Tris buffer solution. The drug perfusion delivery system is injected into the bladder cavity, and the polymer monomer undergoes auto-oxidative polymerization by using the oxygen inside the bladder to form an adhesive polymer coating on the bladder mucosa surface. The polymer coating wraps the therapeutic drug to enable the slow release of the therapeutic drug.

[0008] In some embodiments, the polymer monomer capable of in-situ polymerization is one or more of dopamine (DA), ethylene glycol (EG), methyl acrylate, or ethyl acrylate.

[0009] In some embodiments, preferably, the polymer monomer capable of in-situ polymerization is dopamine (DA), the polymer is polydopamine (PDA), and the pH value of the Tris buffer solution is 8-9. PDA is a synthetic material inspired by the adhesive protein in mussels, which contains rich catechol and quinone groups and has strong universal adhesiveness. The bladder mucosa structure contains mercapto amino acids (such as serine, methionine, etc.), which are rich in nucleophilic groups such as amino and thiol groups. They can form covalent bonds or hydrogen bond interactions through polydopamine to extend the residence time of the drug in the bladder cavity and improve drug bioavailability. Some viral pathogens have the ability to significantly reduce the interaction with mucosal mucus and effectively diffuse through the mucus barrier due to their near-neutral or slightly negatively charged and hydrophilic surface characteristics. PDA has similar surface characteristics (hydrophilicity and slightly negative charge) to these pathogens, enabling it to enhance the penetration of mucus by reducing the interaction with negatively charged and hydrophobic vesicles in mucosal mucus, and at the same time, it can also promote the interaction with positively charged choline groups on the lipid membrane, thereby enhancing the absorption of nanoparticles by cells.

[0010] Preferably, the Tris buffer solution also contains urease. The addition of urease can enhance the movement of the drug delivery system by providing power through the decomposition of urea in urine. Using this autonomous movement can increase the bladder mucosa penetrability of the drug delivery system and its residence time in the bladder, providing a new scheme for bladder perfusion therapy.

[0011] The bladder mucosa layer is an endogenous viscoelastic biopolymer barrier that restricts the entry of foreign pathogens and therapeutic vectors into mucosal cells. This porous and dense viscoelastic biopolymer mucosal layer is mainly composed of mucin glycoproteins wrapped with short glycans, most of which are negatively charged (carboxyl or sulfate groups) and are separated by cysteine-rich hydrophobic domains. The functional groups on the surface of PDA make it zwitterionic, and its isoelectric point is around 4. When the pH is lower than 4, the amino groups on the PDA structure are protonated, making it positively charged; when the pH is higher than 4, the phenolic hydroxyl groups on the PDA structure are deprotonated, making polydopamine negatively charged. The carrier surface of PDA with hydrophilic and zwitterionic properties can minimize the interaction with mucin glycoprotein fibers to overcome the mucosal barrier. At the same time, when the pH value in the system is set to 8.5, the phenolic hydroxyl groups on the DA structure are deprotonated, making polydopamine negatively charged, which can promote the interaction with the positively charged choline groups on the lipid membrane, thereby enhancing the absorption of nanoparticles by cells, affecting the structure and function of tight junction proteins, changing the epithelial cell resistance, opening cell tight junctions, and increasing mucosal permeability.

[0012] In certain embodiments, the therapeutic drug is Mn 2+ Combined with one or more of cGMAP mixture, novel albumin-coated flubendazole (FBZ) drug, or other first-line clinical chemotherapy drugs.

[0013] Preferably Mn 2+ Combined with cGMAP mixture, Mn 2+ As an excellent activator of the cGAS-STING signaling pathway, it can activate cGAS, catalyze the synthesis of cGAMP from ATP and GTP. cGAMP binds to the STING protein on the endoplasmic reticulum membrane and activates it. The activated STING transfers from the endoplasmic reticulum to the Golgi apparatus, where it recruits TBK-binding enzyme 1 and IKK kinase. These kinases phosphorylate IRF3 and the NF-κB inhibitor IκBα, respectively. Phosphorylated IRF3 dimerizes and transfers to the nucleus, activating the gene transcription encoding type I IFN. Phosphorylation of IκBα leads to the transfer of NF-κB to the nucleus, where it activates the gene transcription encoding pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor (TNF), and type I IFN, exerting an immunomodulatory effect and thus effectively anti-tumor. At the same time, since Mn 2+ is an essential inorganic trace element required by the immune system and is used in drugs approved by the US Food and Drug Administration, so combining Mn 2+ with the STING agonist cGAMP, this combination can exert a stronger and more effective activation of the STING pathway for immunity and a more effective anti-tumor effect.

[0014] On the other hand, the present invention also provides a method for in-situ forming a polymer in the bladder of a subject. The method includes injecting into the bladder of the subject a composition comprising a polymer monomer capable of in-situ polymerization, a therapeutic drug, and a Tris buffer solution, and using the oxygen inside the bladder to cause the self-oxidative polymerization reaction of the polymer monomer to form an adhesive polymer coating on the surface of the bladder mucosa.

[0015] Preferably, the Tris buffer solution further contains urease. The addition of urease can enhance the movement of the drug delivery system by providing power through the decomposition of urea in urine. Using this autonomous movement can increase the bladder mucosa penetrability of the drug delivery system and its residence time in the bladder, providing a new scheme for intravesical perfusion therapy.

[0016] In some embodiments, the polymer monomer capable of in-situ polymerization is one or more of dopamine (DA), ethylene glycol (EG), methyl acrylate, or ethyl acrylate.

[0017] In some embodiments, preferably, the polymer monomer capable of in-situ polymerization is dopamine (DA), and the polymer is polydopamine (PDA).

[0018] In some embodiments, the therapeutic drug is Mn 2+ in combination with a cGMAP mixture, a novel albumin-coated flubendazole (FBZ) drug, or one or several of other first-line clinical chemotherapy drugs.

[0019] Use of the above in-situ polymerization drug perfusion delivery system or the above method for in-situ forming a polymer in the bladder of a subject in the preparation of an anti-tumor drug; the tumor is bladder cancer.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) In terms of the drug system, the raw materials are widely sourced and inexpensive. The preparation process of the drug system is simple, and no oxidant needs to be added. While prolonging the residence time of the drug in the bladder, it can also be commercially prepared on a large scale. In addition to metal elements (such as Mn), albumin drugs and first-line clinical chemotherapy drugs can also be efficiently loaded to play the role of drug controlled release and dose reduction.

[0022] (2) The carrier surface of PDA with hydrophilic and zwitterionic properties can minimize the interaction with mucin glycoprotein fibers to overcome the mucosal barrier. At the same time, when the pH value in the system is set in the alkaline range of 8-9, the phenolic hydroxyl groups on the DA structure are deprotonated, making polydopamine negatively charged, which can promote the interaction with the positively charged choline groups on the lipid membrane, thereby enhancing the absorption of nanoparticles by cells, affecting the structure and function of tight junction proteins, changing the epithelial cell resistance, opening the cell tight junctions, and increasing the mucosal permeability.

[0023] (3) In terms of the anti-cancer mechanism, fully utilize the cGAS-STING pathway of Mn ion-activated cells and metal immunology, and combine Mn 2+ with the STING agonist cGAMP. This combination can activate the immune system through the STING pathway more strongly and effectively, and achieve a more effective anti-tumor effect.

[0024] (4) The addition of urease can enhance the movement of the drug delivery system by decomposing urea in urine to provide power. Using this autonomous movement can increase the bladder mucosa penetrability of the drug delivery system and its residence time in the bladder, providing a new scheme for intravesical perfusion therapy. Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the PDA-mediated in-situ polymerization drug system of the present invention.

[0026] Figure 2 It is the mouse bladder drug retention experiment of the drug system in the examples and comparative examples of the present invention after perfusion.

[0027] Figure 3 It is a schematic diagram of the tumor volume of the treatment endpoint mice in the examples and comparative examples of the present invention.

[0028] Figure 4 It is a schematic diagram of the killing ability of the DMC drug system in the examples and comparative examples of the present invention against tumors shown in the cell colony formation experiment. Detailed Description of the Invention

[0029] To make the technical solutions and advantages of the present invention clearer, the present invention and its beneficial effects will be further described in detail below in combination with the specific embodiments and the drawings of the specification. However, the embodiments of the present invention are not limited thereto.

[0030] In the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one (item)" or similar expressions thereof refer to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can all represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0031] The present invention and its beneficial effects will be further described in detail. However, the embodiments of the present invention are not limited thereto.

[0032] In the following examples, unless otherwise specified, the raw materials and instruments used are all commercially available.

[0033] Example 1

[0034] DA + Mn + cGAMP (abbreviated as DMC) system:

[0035] At room temperature, an appropriate amount of DA monomer solution, MnCl2 solution and cGAMP solution were added to Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0036] Example 2

[0037] DA + Mn + cGAMP + urease (abbreviated as DMCU) system:

[0038] At room temperature, a certain amount of urease was added to Tris buffer solution (pH = 8.5, 50 mM) to obtain a mixed solution, and then an appropriate amount of DA monomer solution, MnCl2 solution and cGAMP solution were added to the mixed solution, and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0039] Example 3

[0040] DA + albumin-coated flubendazole system:

[0041] At room temperature, an appropriate amount of DA solution and a novel albumin-coated flubendazole (FBZ) solution were added to Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0042] Example 4

[0043] DA + new gemcitabine system:

[0044] At room temperature, an appropriate amount of DA hydrochloride solution and the clinical first-line chemotherapy drug gemcitabine solution were added to a Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a drug-loaded PDA coating was formed on the mucosal surface in the bladder cavity to exert a therapeutic effect.

[0045] Example 5:

[0046] At room temperature, an appropriate amount of DA hydrochloride solution, MnCl2 solution, cGAMP solution and hydrogen peroxide oxidant were added to a Tris buffer solution (pH = 7, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity to enable the oxidation polymerization of DA, and a drug-loaded PDA coating was formed on the mucosal surface in the bladder cavity to exert a therapeutic effect.

[0047] Example 6:

[0048] At room temperature, an appropriate amount of DA hydrochloride solution, MnCl2 solution and cGAMP solution were added to a Tris buffer solution (pH = 8, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a drug-loaded PDA coating was formed on the mucosal surface in the bladder cavity to exert a therapeutic effect.

[0049] Example 7:

[0050] At room temperature, an appropriate amount of DA hydrochloride solution, MnCl2 solution and cGAMP solution were added to a Tris buffer solution (pH = 9, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a drug-loaded PDA coating was formed on the mucosal surface in the bladder cavity to exert a therapeutic effect.

[0051] Example 8:

[0052] At room temperature, an appropriate amount of DA hydrochloride solution, MnCl2 solution and cGAMP solution were added to Tris buffer solution (pH = 10, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the bladder cavity of mice, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0053] Control Example 1:

[0054] DA + Mn (abbreviated as DM) system:

[0055] At room temperature, an appropriate amount of DA hydrochloride solution and MnCl2 solution were added to Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the bladder cavity of mice, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0056] Control Example 2:

[0057] DA + cGAMP (abbreviated as DC) system:

[0058] At room temperature, an appropriate amount of DA hydrochloride solution and cGAMP solution were added to Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the bladder cavity of mice, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity to enable the auto-oxidation polymerization of DA, a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0059] Control Example 3:

[0060] At room temperature, an appropriate amount of DA hydrochloride solution, MnCl2 solution, cGAMP solution and hydrogen peroxide oxidant were added to Tris buffer solution (pH = 8.5, 50 mM), and mixed for about 1 hour to obtain a dopamine in-situ polymerization drug system. When polymerizing in the bladder cavity of mice, the above dopamine in-situ polymerization drug system was immediately perfused into the bladder cavity to enable the oxidation polymerization of DA, and a PDA coating loaded with drugs was formed on the mucosal surface in the bladder cavity, exerting a therapeutic effect.

[0061] (1) Establishment and evaluation of the mucosal barrier model of bladder cancer cells

[0062] An in vitro model of the bladder mucosal barrier was established using a mixed cell line of SV-HUC-1 (immortalized human bladder epithelial cells), MB49 (mouse bladder cancer cells), and T24 (human bladder cancer cells): The Transwell permeability support produced by Corning was used as the Transwell insert and multi-well plate for the experiment. The culture medium was first added to the multi-well plate and then to the Transwell insert. The culture plate was placed in the incubator for at least one hour or overnight for this pre-equilibration to improve the cell attachment efficiency. Subsequently, the cell concentration was diluted to 250,000 cells / mL with freshly prepared medium, with over 90% of the cells being single cells. The cell suspension was inoculated into the Transwell insert and returned to the incubator for culture, recorded as day 0. After 24 hours of inoculation, the culture medium inside and outside the inserts was changed. In the first week, the medium was changed every other day, and in the following two weeks, the medium was changed daily. After 21 days of cell culture, it was ready for use.

[0063] (2) Evaluation of the monolayer cell model of bladder cancer cells

[0064] ① Cell morphological examination: Observe the growth status of bladder cancer cells, the formation of the monolayer, and check the color of the culture medium inside and outside the inserts under an inverted optical microscope every day; when changing the medium each time, an appropriate amount of the culture medium can be aspirated and observed under the microscope for contamination and large-scale cell detachment.

[0065] ② Measurement of the trans-epithelial electrical resistance (TEER) of monolayer cells: In this project, a transmembrane resistance meter was used to measure the TEER of the cells. Before measurement, the HBSS balanced salt solution was placed in a 37°C water bath and preheated to 37°C. The electrodes were placed in the preheated HBSS at 37°C and equilibrated for 20 minutes. The culture medium in the culture plate was removed, and 0.5 mL and 1.5 mL of preheated HBSS at 37°C were added to the AP side (apical side or mucosal side) and the BL side (basolateral side or serosal side) respectively, and equilibrated at 37°C for 20 minutes while washing away the impurities on the cell surface. The HBSS was removed, and preheated HBSS was added again. The resistance meter mode was set to Ohms, and the switch was turned to ON. The short end of the electrode was immersed in the AP side, and the long end was immersed in the BL side, taking care that the short end did not touch the cells growing on the membrane and the long end just touched the bottom of the BL side, with the electrode at a 90° angle to the culture plate. There are three holes around the Transwell chamber. Each time, measurements should be taken in the three holes respectively. Each hole was repeatedly measured until the same result appeared three times in a row before recording, and then the average value of the measurements in the three holes was taken as the measured resistance value. In addition, a blank chamber without inoculated cells was set up for each experiment, and the blank resistance value was measured according to the above steps. TEER = (measured resistance value - blank resistance value) × Transwell chamber membrane area, in units of Ω·cm2.

[0066] (3) Immunofluorescence staining analysis of tight junction proteins

[0067] After co - culturing SV - HUC - 1 cells with the drug system for 1 h, the cells were washed with PBS. Fixed with 4% paraformaldehyde at 4 °C for 20 min, permeabilized with 0.5% Triton X - 100 at room temperature for 20 min, blocked with 2% BSA (in PBS) at room temperature for 1 h, and incubated with the following primary antibodies: ZO - 1 (1:100), E - cadherin (1:500), occluding (1:100), claudin (1:1000), MLC (1:100), pMLC (1:100) overnight at 4 °C, incubated with FITC - conjugated secondary antibody (1:1000) at room temperature for 1 h, and incubated with DAPI at room temperature for 15 min. Finally, the cells were observed and imaged using a confocal microscope.

[0068] (4) Observation of cell tight junctions under transmission electron microscope

[0069] ① Pre - fixation of experimental samples: Remove the culture medium from the bladder cancer cells inoculated on Transwell and cultured for about 21 days after the action of experimental drugs, and wash with HBSS. Gently scrape the cells from the microporous membrane, place them in 2.5% glutaraldehyde, gently shake to disperse the cells, fix for 3 min, then centrifuge and form a cell mass (with a volume between 1 - 2 mm3), blot the excess fixative with filter paper, add a few drops of serum, and gently break the cell mass with a fine needle to suspend it in the serum. Let it stand for 10 - 20 min, then centrifuge (at a speed of 5000 r / min), suck off the upper serum with a pipette, leave a little serum, and finally add 2.5% glutaraldehyde fixative to continue fixation, and store it in a 4 °C refrigerator for standby.

[0070] ②Embedding block preparation, sectioning and observation under transmission electron microscope: Rinse with HBSS for 5 min each time, and change the solution 3 times in total to thoroughly remove the residual glutaraldehyde as much as possible. After rinsing, transfer the cells into a centrifuge tube containing 1% osmium tetroxide fixative, shake well to allow the osmium tetroxide to fully penetrate, and fix for about 1 h. Rinse with HBSS for 5 min each time, change the solution 3 times in total, and finally rinse with double-distilled water 2 times, shake well to completely remove the residual HBSS. Immerse the cells in 1% uranyl acetate staining solution for about 2 h. Immerse the specimens in 50%, 70%, 80%, and 90% acetone for gradient dehydration for 15 min each, and dehydrate with acetone 2 times, 10 min each time. Use EPON-812 epoxy resin as the embedding agent. Through the impregnation steps: pure acetone + embedding solution (1:1) in an oven at 37 °C for 2 h, pure acetone + embedding solution (1:2) in an oven at 37 °C overnight, pure embedding solution in an oven at 45 °C for 2 h, and then carry out embedding polymerization: the polymerization time is: 6 h in an oven at 45 °C; 48 h in an oven at 65 °C. After obtaining the embedding block, use a microtome to section and stain the embedding block, and locate the area that needs to be ultrathin sectioned under an optical microscope. The ultrathin section is 60 - 80 nm, double-stained with uranyl acetate-lead citrate, and finally observe the cell ultrastructure and tight junctions of the section under a transmission electron microscope and take pictures.

[0071] (5) Evaluation of mucosal retention and transmembrane penetration of the in-situ polymerized drug delivery system in the bladder cavity:

[0072] Multiple methods can be used to investigate the drug retention and penetration in the bladder mucosa. The fluorescence method uses a near-infrared Cy5.5 fluorescent dye co-loaded with the drug system for labeling and tracing. The experimental procedure is as follows: Anesthetize the mice with sodium pentobarbital (50 mg / kg), and then perfuse each mouse's bladder with a mixed solution of Cy5.5-SO3H and DA + MnCl2 + cGAMP, clamp the urethral orifice to keep the drug acting for 2 h. Use a small animal in vivo imaging system to perform fluorescence imaging on the mouse bladder to monitor the retention of Cy5.5 at different times. Or after the mice are perfused with the drug containing Cy5.5, collect the bladder tissues at different time points to prepare frozen sections. After the sections are stained with DAPI for the cell nucleus, use a confocal microscope to detect the fluorescence distribution of Cy5.5 in the bladder sections, and perform semi-quantitative analysis of Cy5.5 fluorescence using Image J software. For quantitative analysis, ICP-MS quantitative analysis can be used. Randomly divide 6 mice into 2 groups, perfuse the bladder with the same volume of normal saline or drug system solution and keep it for 2 h, take out the bladder, clean, weigh, digest, and then use ICP-MS to determine the content of manganese or copper in the bladder. The copper salt staining method uses rhodanine to stain the bladder tissue sections, which can confirm the presence of copper elements in the bladder tissue and analyze its transmembrane penetration.

[0073] (6) Establishment of an orthotopic model of bladder cancer:

[0074] The bladder cancer cell line MB49 was labeled with luciferase by lentiviral transfection. Healthy female C57BL / 6 mice were anesthetized, and the bladder was exposed by dissection. Referring to the literature reports, the cell solution was injected into the bladder wall of the mice with an insulin needle, the wound was sutured and the mice were continued to be raised. An animal in vivo imaging system and a photoacoustic imaging system were used to monitor the growth of the tumor. The administration method was transurethral bladder perfusion for 1 h once a week for a total of 4 times.

[0075] (7) Study on the long-term synergistic treatment effect of orthotopic tumors in bladder cancer:

[0076] The in-situ polymerization drug delivery system in the bladder cavity was injected into the bladder of the mice, and the urethra was clamped for 2 h to enable the drugs in the system to fully enter the lesion tissue. The growth of orthotopic tumors was evaluated by detecting the fluorescence intensity with a mouse in vivo imager. The ablation ability of the primary tumor mass by the co-treatment with the perfused drugs was investigated by means of Western blot of proteins in tissues and sera and other immune indices determination, tissue dissection, pathological observation, immunohistochemical analysis, tumor quantitative measurement, etc.

[0077] (8) Study on the tumor immunotherapy effect:

[0078] ① Determination of DC maturation: DC cells were isolated from the bone marrow of 8-week-old C57BL / 6 mice by the method in the reference. For the in vitro stimulation of DC cells experiment, bone marrow-derived dendritic cells were incubated with different materials and lipopolysaccharide (LPS, used as a positive control) for 20 h. Then they were stained with anti-CD11c FITC, anti-CD86 PE and anti-CD80 APC, detected by a C6 plus flow cytometer, and analyzed with FlowJo software. The DC maturation was studied by analyzing the surface expression of the co-stimulatory molecules CD80 and CD86. The levels of IL-6 and TNF in the culture medium were detected with an ELISA kit.

[0079] ② ELISA and RNA analysis: The mouse bladder was homogenized in a lysis solution and transferred to a microtube, and centrifuged at 12,000 g for 10 min. The supernatant was transferred to a new microtube, and the concentrations of IL-6, IL-12, TNF-α and IFN-γ were detected with an ELISA kit. To evaluate the transcription of IFN-β and CXCL-10, RNA was collected and transcribed into complementary DNA (cDNA) with a RevertAid First Strand cDNA Synthesis Kit. Quantitative RT-PCR was performed using a FastStart Universal SYBR Green Master (Rox) kit with an ABI StepOnePlus real-time fluorescence quantitative PCR instrument in the United States according to the method attached by the manufacturer.

[0080] ③ In vivo experiments: Establish orthotopic / subcutaneous dual-tumor models and systemic metastatic tumor models. Infuse the drug solution into the orthotopic bladder cancer tumor (the first tumor). After treatment, observe and measure the tumor sizes of the first tumor and the second tumor (simulating metastatic tumors) to evaluate the inhibitory effect of combination therapy on tumor metastasis. After treatment, further remove the tumor tissues and blood from the mice, and extract the corresponding cells and serum. Then, use flow cytometry and ELISA reagent detection methods to measure the proportions of cytokines such as TNF-α and IFN-γ, cytotoxic T lymphocytes (CTL), and regulatory T cells in the system, and deeply analyze and evaluate the effect of radiotherapy-generated antigens and immune activators acting synergistically as a "vaccine" to stimulate the body to produce an anti-tumor immune response.

[0081] Appendix Figure 1 Figure Figure 1 is a schematic diagram of the PDA-mediated in-situ polymerization drug system in this application. By delivering the drug and dopamine monomers into the bladder of mice, polymerization is carried out using the oxygen in the bladder.

[0082] Figure 2 Figure shows the drug retention experiment in the bladder of mice after perfusion with the drug systems of Example 1 and Example 2 of the present invention. Imaging was performed at 0day, 1day, 2day, 3day, 4day, 6day, and 10day respectively. It can be seen that the retention time of the drug in DMCU is significantly better than that in DMC. It can be seen that the addition of urease can enhance the movement of the drug delivery system by decomposing urea in urine to provide power. Using this autonomous movement can increase the bladder mucosa penetrability of the drug delivery system and its retention time in the bladder.

[0083] Figure 3 Figure

[0082] shows the schematic diagram of the tumor volumes of mice at the end point of tumor treatment in Example 1, Example 2, and Comparative Examples 1-2 of the present invention. It can be seen from the pictures that the size of the tumors in the mice using the DMCU system is the smallest, and the size of the tumors in the mice using the DMC system is significantly smaller than those in the DM system and the DC system. Figure 4 Figure Figure 2 shows the schematic diagram of the killing ability of the DMC drug system against tumors in the cell colony formation experiment. It can be seen that the killing ability of the DMC system against tumor cells is significantly better than that of the DM system and the DC system. It can be seen that by fully utilizing the cGAS-STING pathway of cells activated by Mn ions and metal immunology, combining Mn 2+ and the STING agonist cGAMP, this combination can activate the immune system through the STING pathway more effectively and achieve a more effective anti-tumor effect.

[0084] Table 1: Drug retention degree of DMC in different pH systems

[0085] Number System pH value D10 retention situation Example 1 DMC 8.5 Retention of 10% Example 5 DMC 7 Emptying Example 6 DMC 8 Retention of 8% Example 7 DMC 9 Retention of 6% Example 8 DMC 10 Emptying Comparative Example 3 <![CDATA[DMC+H2O2]]> 8.5 Emptying

[0086] The drug systems in Examples 1, 5 - 8 and Comparative Example 3 were respectively injected into the bladders of mice, and the residence time of the drugs was observed. The test results are shown in Table 1. It can be seen that the drugs in the bladders of mice in Example 5, Example 8 and Comparative Example 3 were almost completely emptied after 10 days. About 10% of the drugs remained in the bladders of mice in Example 1 after 10 days. About 8% of the drugs remained in the bladders of mice in Example 6 after 10 days. About 6% of the drugs remained in the bladders of mice in Example 7 after 10 days. From Example 1 and Examples 5 - 8, it can be seen that when the pH value in the system is set in the alkaline range of 8 - 10, the phenolic hydroxyl groups on the DA structure are deprotonated, making polydopamine negatively charged, which can promote the interaction with the positively charged choline groups on the lipid membrane, thereby enhancing the absorption of nanoparticles by cells, affecting the structure and function of tight junction proteins, changing the epithelial cell resistance, opening the cell tight junctions, and increasing the mucosal permeability. However, if the pH is too high, it may affect the physicochemical environment in the bladder, resulting in a relatively short drug residence time. From Comparative Example 3 and Example 1, it can be seen that when an oxidant is added to the DMC system, the drug residence time is reduced instead. It can be seen that these oxidant residues will accelerate the decomposition of the polymer, reducing the effect of the perfusion carrier to achieve long-term retention and slow release in the bladder mucosa.

[0087] According to the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the above specific embodiments. Any obvious improvements, substitutions or variations made by those skilled in the art based on the present invention fall within the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. An in-situ polymerization drug perfusion delivery system, characterized in that, The drug perfusion delivery system is composed of polymer monomers capable of in-situ polymerization. The polymer monomer is dopamine (DA), a therapeutic drug, and Tris buffer solution. The pH value of the Tris buffer solution is 8.5, and the Tris buffer solution also contains urease. The drug perfusion delivery system is injected into the bladder cavity, and the self-oxidative polymerization reaction of the polymer monomer is carried out using the oxygen inside the bladder to form an adhesive polymer coating on the surface of the bladder mucosa. The polymer coating is a polydopamine coating, and the polymer coating wraps the therapeutic drug to enable the slow release of the therapeutic drug; the therapeutic drug is Mn 2+ -binding cGMAP mixture.

2. Use of the in-situ polymerized drug perfusion delivery system according to claim 1 in the preparation of anti-tumor drugs; the tumor is bladder cancer.

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