PDA in-situ polymerization drug perfusion delivery system and application thereof

Abstract

According to the PDA in-situ polymerization drug perfusion delivery system provided by the invention, the interaction relationship of a perfusion drug system on an organism signal channel and immune response is studied; through manganese metal immunoregulation, based on copper death synergistic therapy, novel protein drug immunotherapy and enhancement of the inhibition action mechanism of traditional chemotherapy on bladder cancer cells, various drugs are organically combined by utilizing dopamine-mediated in-situ bladder intracavity polymerization loaded drugs and combining with the characteristics of polydopamine, so that the bladder cancer cell inhibition effect is enhanced, and the bladder cancer cell inhibition effect is enhanced. A novel multi-effect-in-one multifunctional bladder perfusion drug system is formed, the complexity of drug design is simplified, the bioavailability of drugs is improved, and it is ensured that multi-mode collaborative treatment in the bladder is achieved.

Description

Technical Field

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

[0002] Bladder cancer is the fourth most common malignant tumor, and ranks first in the incidence of urogenital tumors in my country. In recent years, its incidence and mortality have been increasing year by year. The conventional treatment for bladder cancer is intravesical chemotherapy or BCG intravesical therapy after transurethral bladder tumor resection, but the postoperative recurrence rate is still as high as 40%-70%. Many patients have multiple recurrences, repeated surgeries and long-term infusions, which cause great physical and mental trauma to 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 infusion therapy can reduce the recurrence and progression rate of bladder cancer, due to the lack of more effective treatment methods, the short residence time of drugs in the bladder, and low permeability, the overall recurrence and progression rate of bladder cancer is still very high. Therefore, in order to solve this key problem, the development of a simple and safe new drug infusion delivery system that can effectively increase the drug concentration of infusion drugs in bladder lesion tissues and effectively prevent the further development and recurrence of bladder cancer cells has become a key point and difficulty in the field of bladder cancer research.

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

[0004] Traditional perfusion therapy is to dissolve the drug directly in saline or glucose solution, which has the defects of poor drug absorption and low bioavailability. To solve the above problems, researchers have now delivered drugs in the human body through in situ polymerization to improve the absorbability and retention time of drugs. For example, in the US patent documents US20210177938A1 and US20230101687A1, researchers from 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 the addition of an oxygen source to the composition, and the subject's endogenous catalyst must also be contacted to allow the catalyst to polymerize the monomer. The introduction of these oxygen sources will result in the use of organs that are sensitive to these oxidants. At the same time, these oxidant residues will accelerate the decomposition of the polymer and reduce the effect of the perfusion carrier on achieving long-term retention and slow release in 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 in the bladder mucosa and deliver the drug to the lesion across the bladder mucosal barrier is the key scientific problem that this subject must first solve. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present application creatively utilizes dopamine-mediated in situ polymerization in the bladder cavity to load drugs, combines the characteristics of polydopamine to organically combine various drugs, fully utilizes the oxygen in the bladder, does not add other oxidants, and forms a new multifunctional bladder instillation drug system with multiple effects in one, simplifies the complexity of drug design, improves drug bioavailability, and ensures the realization of multimodal synergistic treatment of bladder cancer.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] An in situ polymerized drug infusion delivery system, characterized in that the drug infusion delivery system is composed of polymer monomers that can be polymerized in situ, therapeutic drugs and Tris buffer solution. The drug infusion delivery system is injected into the bladder cavity to use the oxygen inside the bladder to cause the polymer monomers to undergo self-oxidative polymerization reaction, thereby forming an adhesive polymer coating on the surface of the bladder mucosa. The polymer coating wraps the therapeutic drug, so that the therapeutic drug is slowly released.

[0008] In certain embodiments, the in situ polymerizable monomer is one or more of dopamine (DA), ethylene glycol (EG), baculoyl ester or baculoyl ester.

[0009] In certain embodiments, the preferred in situ polymerizable monomer 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 abundant catechol and quinone groups and has strong universal adhesion. The bladder mucosal structure contains thiol amino acids (serine, methionine, etc.), which contain abundant amino and thiol nucleophilic groups, which can form covalent bonds or hydrogen bonds through polydopamine to prolong the residence time of the drug in the bladder cavity and improve the bioavailability of the drug. Certain 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 properties. PDA has surface properties similar to those of these pathogens (hydrophilicity and slightly negative charge), which enables it to enhance mucus penetration by reducing interactions with negatively charged and hydrophobic pockets in mucous membranes, while also promoting interactions with positively charged choline groups on lipid membranes, thereby enhancing cellular uptake of nanoparticles.

[0010] Preferably, the Tris buffer solution also contains urease. The addition of urease can provide power to enhance the movement of the drug delivery system by decomposing urea in urine. This autonomous movement can increase the bladder mucosal penetration of the drug delivery system and the retention time in the bladder, providing a new solution for bladder perfusion therapy.

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

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

[0013] Mn is preferred 2+ Combined with cGMAP mixture, Mn 2+ As an excellent cGAS-STING signaling pathway activator, it can activate cGAS and catalyze ATP and GTP to synthesize cGAMP. 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, which phosphorylate IRF3 and nuclear factor-κB inhibitor IκBα, respectively. Phosphorylated IRF3 dimerizes and transfers to the nucleus, activating the transcription of genes encoding type I IFN. IκBα phosphorylation causes NF-κB to transfer to the nucleus, where it activates the transcription of genes encoding proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor (TNF), and type I IFN, exerting an immunomodulatory effect and thus effectively fighting tumors. At the same time, due to the Mn 2+ It is an essential inorganic trace element required by the immune system and is used in drugs approved by the U.S. Food and Drug Administration. 2+ Combined with the STING agonist cGAMP, this combination can exert a more effective STING pathway immune activation and a more effective anti-tumor effect.

[0014] On the other hand, the present invention also provides a method for forming a polymer in situ in the bladder of a subject, the method comprising injecting a composition of an in situ polymerizable polymer monomer, a therapeutic drug and a Tris buffer solution into the bladder of the subject, utilizing oxygen inside the bladder to cause a 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 also contains urease. The addition of urease can provide power to enhance the movement of the drug delivery system by decomposing urea in urine. This autonomous movement can increase the bladder mucosal penetration of the drug delivery system and the retention time in the bladder, providing a new solution for bladder perfusion therapy.

[0016] In certain embodiments, the in situ polymerizable monomer is one or more of dopamine (DA), ethylene glycol (EG), baculoyl ester or baculoyl ester.

[0017] In certain embodiments, the in situ polymerizable monomer is preferably dopamine (DA), and the polymer is polydopamine (PDA).

[0018] In certain embodiments, the therapeutic agent is Mn 2+ Combined with cGMAP mixture, novel albumin-coated flubendazole (FBZ) drug or one or more of other clinical first-line chemotherapy drugs.

[0019] Use of the above-mentioned in situ polymerization drug infusion delivery system or the above-mentioned method for forming a polymer in situ 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:

[0021] (1) In terms of drug systems, the raw materials are widely available and inexpensive, the drug system preparation process is simple, and no oxidant needs to be added. This not only prolongs the retention time of the drug in the bladder, but also enables commercial large-scale preparation and application. In addition to metal elements (such as Mn), albumin drugs and first-line clinical chemotherapy drugs can also be efficiently loaded, thereby playing a role in drug controlled release and dose reduction.

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

[0023] (3) In terms of anti-cancer mechanism, we make full use of the cGAS-STING pathway and metal immunology of cells activated by Mn ions. 2+ Combined with the STING agonist cGAMP, this combination can exert a more effective STING pathway immune activation and a more effective anti-tumor effect.

[0024] (4) The addition of urease can enhance the movement of the drug delivery system by providing power to decompose urea in urine. This autonomous movement can increase the bladder mucosal penetration of the drug delivery system and the retention time in the bladder, providing a new solution for bladder instillation therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the PDA-mediated in situ polymerization drug system of the present invention.

[0026] Figure 2 This is a drug retention experiment in the mouse bladder after the drug system in the examples and comparative examples of the present invention was instilled.

[0027] Figure 3 Schematic diagram of the tumor volume of mice at the treatment endpoint in the examples of the present invention and the comparative examples.

[0028] Figure 4 It is a schematic diagram of the tumor-killing ability of the DMC drug system in the examples and comparative examples of the present invention in the cell clone formation experiment. DETAILED DESCRIPTION

[0029] To make the technical solutions and advantages of the present invention more clear, the present invention and its beneficial effects will be described in further detail below in conjunction with specific implementation methods and accompanying drawings, but the implementation methods of the present invention are not limited thereto.

[0030] In the present invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple.

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

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

[0033] Example 1

[0034] DA+Mn+cGAMP (DMC for short) system:

[0035] At room temperature, add appropriate amounts of DA monomer solution, MnCl2 solution and cGAMP solution to Tris buffer solution (pH = 8.5, 50mM) and mix 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 is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0036] Example 2

[0037] DA+Mn+cGAMP+urease (DMCU for short) system:

[0038] A certain amount of urease is added to a Tris buffer solution (pH=8.5, 50mM) at room temperature to obtain a mixed solution, and then appropriate amounts of DA monomer solution, MnCl2 solution and cGAMP solution are 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-mentioned dopamine in situ polymerization drug system is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized, forming a drug-loaded PDA coating on the mucosal surface of the bladder cavity to exert a therapeutic effect.

[0039] Example 3

[0040] DA+albumin coated flubendazole system:

[0041] At room temperature, add an appropriate amount of DA solution and a novel albumin-coated flubendazole (FBZ) solution to a Tris buffer solution (pH=8.5, 50 mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the dopamine in situ polymerization drug system is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0042] Example 4

[0043] DA+New Gemcitabine System:

[0044] At room temperature, add an appropriate amount of DA hydrochloride solution and a clinical first-line chemotherapy drug, gemcitabine solution, to a Tris buffer solution (pH=8.5, 50mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the dopamine in situ polymerization drug system is immediately infused into the bladder cavity. Since there is sufficient oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0045] Embodiment 5:

[0046] At room temperature, add appropriate amounts of DA hydrochloride solution, MnCl2 solution, cGAMP solution and hydrogen peroxide oxidant to Tris buffer solution (pH = 7, 50mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the above-mentioned dopamine in situ polymerization drug system is immediately infused into the bladder cavity to cause DA to oxidatively polymerize and form a drug-loaded PDA coating on the mucosal surface of the bladder cavity to exert a therapeutic effect.

[0047] Embodiment 6:

[0048] At room temperature, add appropriate amounts of DA hydrochloride solution, MnCl2 solution and cGAMP solution to Tris buffer solution (pH=8, 50mM) and mix 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 is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0049] Embodiment 7:

[0050] At room temperature, add appropriate amounts of DA hydrochloride solution, MnCl2 solution and cGAMP solution to Tris buffer solution (pH = 9, 50mM) and mix 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 is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0051] Embodiment 8:

[0052] At room temperature, add appropriate amounts of DA hydrochloride solution, MnCl2 solution and cGAMP solution to Tris buffer solution (pH = 10, 50mM) and mix 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 is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0053] Comparative Example 1:

[0054] DA+Mn (DM for short) system:

[0055] At room temperature, add appropriate amounts of DA hydrochloride solution and MnCl2 solution to a Tris buffer solution (pH=8.5, 50mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the dopamine in situ polymerization drug system is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0056] Comparative Example 2:

[0057] DA+cGAMP (DC for short) system:

[0058] At room temperature, add appropriate amounts of DA hydrochloride solution and cGAMP solution to a Tris buffer solution (pH = 8.5, 50 mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the dopamine in situ polymerization drug system is immediately infused into the bladder cavity. Since there is enough oxygen in the bladder cavity, DA can be self-oxidized and polymerized to form a drug-loaded PDA coating on the mucosal surface of the bladder cavity, thereby exerting a therapeutic effect.

[0059] Comparative Example 3:

[0060] At room temperature, add appropriate amounts of DA hydrochloride solution, MnCl2 solution, cGAMP solution and hydrogen peroxide oxidant to Tris buffer solution (pH=8.5, 50mM) and mix for about 1 hour to obtain a dopamine in situ polymerization drug system. When polymerizing in the mouse bladder cavity, the dopamine in situ polymerization drug system is immediately infused into the bladder cavity to allow DA to oxidatively polymerize and form a drug-loaded PDA coating on the mucosal surface of the bladder cavity to exert a therapeutic effect.

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

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

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

[0064] ① Cell morphology examination: Observe the growth status and monolayer formation of bladder cancer cells under an inverted optical microscope every day, and check the color of the culture medium inside and outside the inserts; each time the medium is changed, take an appropriate amount of culture medium and observe under a microscope to see if it is contaminated or if there are large pieces of cells falling off.

[0065] ② Determination of transepithelial electrical resistance (TEER) of monolayer cells: This project uses a transepithelial resistance meter to measure the TEER of cells. Before measurement, place the HBSS balanced salt solution in a 37℃ water bath and preheat it to 37℃. Place the electrode in the HBSS preheated to 37℃ and balance for 20 minutes. Remove the culture medium in the culture plate, add 0.5 and 1.5mL of HBSS preheated to 37℃ to the AP side (apical side or mucosal side) and BL side (basal side or serosal side), respectively, and balance at 37℃ for 20 minutes. At the same time, wash away impurities on the cell surface. Remove the HBSS, add the preheated HBSS again, turn the resistance meter mode to Ohms, turn the switch to ON, immerse the short end of the electrode in the AP side, and the long end in the BL side. Be careful not to touch the cells growing on the membrane, and the long end just touches the bottom of the BL side. The electrode is 90° to the culture plate. There are three holes around the Transwell chamber. Each time, the measurement should be made in the three holes respectively. Each hole should be measured repeatedly until the same result appears three times in a row, and then the average of the three hole measurements is taken as the measured resistance value. In addition, a blank chamber without cells inoculated should be set up for each experiment, and the blank resistance value should be measured according to the above steps. TEER = (measured resistance value - blank resistance value) × Transwell chamber membrane area, unit Ω·cm2.

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

[0067] After SV-HUC-1 cells were co-cultured with the drug system for 1 hour, the cells were washed with PBS, fixed with 4% paraformaldehyde at 4°C for 20 minutes, permeabilized with 0.5% Triton X-100 at room temperature for 20 minutes, blocked with 2% BSA (in PBS) at room temperature for 1 hour, 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) at 4°C overnight, incubated with FITC-conjugated secondary antibodies (1:1000) at room temperature for 1 hour, and incubated with DAPI at room temperature for 15 minutes. Finally, the cells were observed and imaged using a confocal microscope.

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

[0069] ① Fixation of experimental samples before experiment: Remove the culture medium from the bladder cancer cells inoculated on Transwell for about 21 days and treated with experimental drugs, and wash with HBSS. Gently scrape the cells from the microporous membrane, place them in 2.5% glutaraldehyde, shake them gently to disperse the cells, fix them for 3 minutes, centrifuge them, form a mass (volume between 1-2mm3), absorb the excess fixative with filter paper, add a few drops of serum, and use a fine needle to gently break up the cell mass, suspend it in the serum, let it stand for 10-20 minutes, centrifuge it again (speed 5000r / min), use a pipette to absorb the upper serum, leaving a little serum, and finally add 2.5% glutaraldehyde fixative to continue fixation, and store it in a 4℃ refrigerator for future use.

[0070] ② Preparation of embedded blocks, sectioning and observation under transmission electron microscope: Rinse with HBSS for 5 minutes each time, change the solution 3 times in total, and remove the residual glutaraldehyde as thoroughly as possible. After rinsing, transfer the cells to a centrifuge tube containing 1% osmium acid fixative, shake thoroughly to allow the osmium acid to fully penetrate, and fix for about 1 hour. Rinse with HBSS for 5 minutes each time, change the solution 3 times in total, and finally rinse with double distilled water twice, shake thoroughly, and completely remove the HBSS residue. Transfer the cells to 1% uranyl acetate staining solution for about 2 hours. Immerse the specimens in 50%, 70%, 80%, and 90% acetone for gradient dehydration for 15 minutes each, and dehydrate with acetone twice, each time for 10 minutes. EPON-812 epoxy resin was used as embedding agent, and the steps of impregnation were as follows: pure acetone + embedding solution (1:1) in a 37℃ oven for 2h, pure acetone + embedding solution (1:2) in a 37℃ oven overnight, pure embedding solution in a 45℃ oven for 2h, and then embedding polymerization was performed: polymerization time was: 45℃ oven for 6h; 65℃ oven for 48h. After the embedding block was obtained, the embedded block was sliced ​​and stained with a microtome, and the part that needed to be ultrathinly sliced ​​was located under an optical microscope. The ultrathin section was 60-80nm, and double-stained with uranyl acetate-lead citrate. Finally, the cell ultrastructure and tight junctions of the section were observed under a transmission electron microscope and filmed.

[0071] (5) Evaluation of mucosal retention and permeation of in situ polymerized drug delivery system in the cystic cavity:

[0072] A variety of methods can be used to investigate the retention and penetration of drugs in the bladder mucosa. The fluorescence method uses near-infrared Cy5.5 fluorescent dye to co-load with the drug system to mark the tracer. The experimental steps are: anesthetize the mice with sodium pentobarbital (50 mg / kg), then infuse the bladder of each mouse with a mixed solution of Cy5.5-SO3H and DA+MnCl2+cGAMP, and clamp the urethra to maintain the drug effect for 2 hours. Use a small animal in vivo imager to perform fluorescence imaging of the mouse bladder to monitor the retention of Cy5.5 at different times. Or after the mice are infused with drugs containing Cy5.5, bladder tissues are collected at different time points to prepare frozen sections. After the sections are stained with DAPI for nuclei, the fluorescence distribution of Cy5.5 in the bladder sections is detected by confocal microscopy, and the Cy5.5 fluorescence semi-quantitative analysis is performed using Image J software. For quantitative analysis, ICP-MS quantitative analysis can be used. Six mice are randomly divided into two groups, and the same volume of saline or drug system solution is instilled into the bladder for 2 hours. The bladder is removed and washed, weighed, and digested, and then the manganese or copper content in the bladder is determined by ICP-MS. The copper salt staining method uses the rhodamine method to stain bladder tissue sections, which can confirm the presence of copper in bladder tissue and analyze its transmembrane permeability.

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

[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. The cell solution was injected into the mouse bladder wall with an insulin needle according to the literature report. The wound was sutured and the mice were continued to be raised. The growth of the tumor was monitored by animal in vivo imaging system and photoacoustic imaging system. The drug was administered by transurethral bladder instillation for 1 hour, once a week, for a total of 4 times.

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

[0076] The in situ polymerized drug delivery system was injected into the mouse bladder, and the urethra was clamped for 2 hours to allow the drug in the system to fully enter the lesion tissue. The fluorescence intensity was detected by a mouse in vivo imaging device to evaluate the growth of the in situ tumor. The protein immunoblotting and other immune index determinations of tissue and serum, tissue dissection, pathological observation, immunohistochemical analysis, and tumor quantitative measurement were used to investigate the ablation ability of the primary tumor by the synergistic treatment of the infusion drug.

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

[0078] ① DC maturation assay: DC cells were isolated from the bone marrow of 8-week-old C57BL / 6 mice according to the method in the reference. For in vitro DC cell stimulation experiments, bone marrow-derived dendritic cells were incubated with different materials and lipopolysaccharide (LPS, used as a positive control) for 20 hours. Then, anti-CD11c FITC, anti-CD86 PE and anti-CD80 APC were used for staining, C6 plus flow cytometer detection, and FlowJo software analysis. The surface expression of co-stimulatory molecules CD80 and CD86 was analyzed to study DC maturation. The levels of IL-6 and TNF in the culture medium were detected using ELISA kits.

[0079] ②ELISA and RNA analysis: Take the mouse bladder and homogenize it in the lysis buffer, transfer it into a microtube, and centrifuge it at 12000g for 10 minutes. Transfer the supernatant to a new microtube and detect the concentrations of IL-6, IL-12, TNF-α and IFN-γ using an ELISA kit. To evaluate the transcription of IFN-β and CXCL-10, collect RNA and transcribe it into complementary DNA (cDNA) using the RevertAid first-chain cDNA synthesis kit. According to the manufacturer's attached method, quantitative RT-PCR was performed using the American ABI StepOnePlus real-time fluorescence quantitative PCR instrument using the FastStart Universal SYBR Green Master (Rox) kit.

[0080] ③ In vivo experiments: Establish an orthotopic / subcutaneous dual tumor model and a systemic metastasis model, and infuse the drug solution into the orthotopic tumor of bladder cancer (the first tumor). After treatment, observe and test the size of the first tumor and the second tumor (simulated metastatic tumor), so as to evaluate the inhibitory effect of combined treatment on tumor metastasis. After treatment, the mouse tumor and blood were further removed, and the corresponding cells and serum were extracted to test the proportion of cytokines such as TNF-α and IFN-γ and cytotoxic T lymphocytes (CTL) and regulatory T cells in the system by flow cytometry and ELASA reagent detection, and in-depth analysis and evaluation of the antigens produced by radiotherapy and immune activators synergistically as "vaccines" to stimulate the body to produce anti-tumor immune responses.

[0081] Attached Figure 1 This is a schematic diagram of the PDA-mediated in situ polymerization drug system in the present application, which polymerizes the drug and dopamine monomer by delivering them into the bladder of mice and utilizing the oxygen in the bladder.

[0082] Figure 2 This is a drug retention experiment in the mouse bladder after infusion of the drug systems of Example 1 and Example 2 of the present invention. Imaging was performed at 0 day, 1 day, 2 day, 3 day, 4 day, 6 day, and 10 day, 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 providing power to decompose urea in urine. This autonomous movement can increase the bladder mucosal penetration of the drug delivery system and the retention time in the bladder.

[0083] Figure 3 The figure is a schematic diagram of the mouse tumor volume at the end point of mouse tumor treatment in Example 1, Example 2, and Comparative Examples 1-2 of the present invention. It can be seen from the figure that the size of the mouse tumor using the DMCU system is the smallest, while the size of the mouse tumor using the DMC system is significantly smaller than that of the DM system and the DC system. Figure 4 The schematic diagram of the tumor killing ability of the DMC drug system in the cell clone formation experiment shows that the killing ability of the DMC system on tumor cells is significantly better than that of the DM system and the DC system. It can be seen that the cGAS-STING pathway and metal immunology of cells activated by Mn ions are fully utilized. 2+ Combined with the STING agonist cGAMP, this combination can exert a more effective STING pathway immune activation and a more effective anti-tumor effect.

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

[0085] serial number system pH D10 detention situation Example 1 DMC 8.5 10% retention Example 5 DMC 7 Empty Example 6 DMC 8 8% retention Example 7 DMC 9 6% retention Example 8 DMC 10 Empty Comparative Example 3 <![CDATA[DMC+H2O2]]> 8.5 Empty

[0086] The drug systems in Examples 1, 5-8 and Comparative Example 3 were respectively injected into the bladder of mice to observe the retention time of the drugs. The test results are shown in Table 1. It can be seen that the drugs in the mouse bladders in Examples 5, 8 and Comparative Example 3 were almost completely emptied after 10 days, and 10% of the drugs in the mouse bladders in Example 1 were still retained after 10 days. The drugs in the mouse bladders in Example 6 were retained after 10 days. The drugs in the mouse bladders in Example 7 were retained after 10 days. It can be seen from Examples 1 and 5-8 that when the pH value in the system is set to the alkaline range of 8-10, the phenol on the DA structure The deprotonation of the hydroxyl group makes polydopamine negatively charged, which can promote the interaction with the positively charged choline group on the lipid membrane, thereby enhancing the cell absorption of nanoparticles, affecting the structure and function of tight junction proteins, changing the electrical resistance of epithelial cells, opening cell tight junctions, and increasing the permeability of the mucosa. If the pH is too high, it may affect the physical and chemical environment in the bladder, resulting in a short drug retention time. It can be seen from Comparative Example 3 and Example 1 that the addition of an oxidant to the DMC system reduces the drug retention time. It can be seen that these oxidant residues will accelerate the decomposition of the polymer and reduce the effect of the perfusion carrier to achieve long-term retention and slow release in the bladder mucosa.

[0087] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs can also change and modify the above embodiment. Therefore, the present invention is not limited to the above specific embodiment, and any obvious improvement, replacement or modification made by those skilled in the art on the basis of the present invention belongs to the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the 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 infusion delivery system is composed of polymer monomers that can be polymerized in situ, therapeutic drugs and Tris buffer solution. The drug infusion delivery system is injected into the bladder cavity to use the oxygen inside the bladder to cause the polymer monomers to undergo self-oxidative polymerization reaction, thereby forming an adhesive polymer coating on the surface of the bladder mucosa. The polymer coating wraps the therapeutic drug, allowing the therapeutic drug to be slowly released.

2. The in situ polymerization drug infusion delivery system according to claim 1, characterized in that: The in-situ polymerizable polymer monomer is one or more of dopamine (DA), ethylene glycol (EG), methyl bamate or ethyl bamate.

3. According to the in situ polymerization drug perfusion delivery system as claimed in claim 2, the in situ polymerizable polymer monomer is dopamine (DA), the polymer is polydopamine (PDA), and the pH value of the Tris buffer solution is 8-9, preferably 8.

5.

4. According to the in situ polymerized drug infusion delivery system as described in claims 1-3, the therapeutic drug is Mn 2+ Combined with cGMAP mixture, novel albumin-coated flubendazole (FBZ) drug or one or more of other first-line clinical chemotherapy drugs, preferably Mn 2+ Combine cGMAP mixture.

5. According to the in situ polymerization drug perfusion delivery system as described in claims 1-3, the Tris buffer solution further contains urease.

6. A method for forming a polymer in situ in the bladder of a subject, the method comprising injecting a composition consisting of an in situ polymerizable polymer monomer, a therapeutic drug and a Tris buffer solution into the bladder of the subject, using oxygen inside the bladder to cause the polymer monomer to undergo a self-oxidative polymerization reaction to form an adhesive polymer coating on the surface of the bladder mucosa.

7. The preparation method according to claim 6, characterized in that: The in-situ polymerizable polymer monomer is one or more of dopamine (DA), ethylene glycol (EG), methyl bamate or ethyl bamate.

8. The preparation method according to claim 7, characterized in that The in-situ polymerizable monomer is dopamine (DA), the polymer is polydopamine (PDA), the pH value of the Tris buffer solution is 8-10, preferably the pH value is 8.5, and preferably, the Tris buffer solution also contains urease.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The therapeutic drug is Mn 2+ Combined with cGMAP mixture, novel albumin-coated flubendazole (FBZ) drug or one or more of other clinical first-line chemotherapy drugs.

10. Use of the in situ polymerized drug infusion delivery system according to any one of claims 1 to 5 or the method for forming a polymer in situ in the bladder of a subject according to claims 6 to 9 in the preparation of an anti-tumor drug; the tumor is bladder cancer.

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