Preparation method and application of drug delivery system containing drug-loaded liposome and BCG

Abstract

Bladder cancer perfusion treatment is limited by the bladder mucosal barrier, and the drug treatment efficiency is low. The present invention provides a drug delivery system containing a drug-loaded liposome and BCG, the drug delivery system includes drug-loaded liposomes, BCG, and chitosan solution, the drug-loaded liposomes are drug-loaded liposomes modified with cell-penetrating peptides, the drug delivery system can increase the retention time of liposomes and BCG in the bladder, enhance the penetration and uptake capacity of drug-loaded liposomes modified with cell-penetrating peptides in the bladder, and combine with BCG to exert immune effects, which can significantly improve the anti-tumor effect of drugs on bladder cancer, realize chemical and immunotherapy, reduce the use of chemical drugs and BCG, and reduce systemic side effects.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a drug-loaded liposome and BCG composite delivery system for treating bladder cancer, and a preparation method and application thereof. Background Art

[0002] Bladder cancer is one of the most common and expensive malignancies of urinary tract tumors worldwide, and patients need lifelong monitoring and follow-up of their condition. About 75% of patients are initially diagnosed with non-muscle invasive bladder cancer (NMIBC). Transurethral resection of bladder tumors (TURBT) is the standard surgery for NMIBC. However, the 5-year recurrence rate after TURBT is still as high as 78%. Low-risk NMIBC intravesical chemotherapy drugs mainly include mitomycin C, doxorubicin, epirubicin and gemcitabine, etc. The clinical treatment method for medium / high-risk NMIBC is mainly Bacillus Calmette-Guérin (i.e., attenuated live Mycobacterium bovis, BCG) maintenance treatment for 1-3 years, but some patients do not respond to BCG treatment. Despite great progress in bladder cancer treatment, about 60% of patients will relapse after 1 year, and 20% of high-risk NMIBC patients may progress to muscle invasive bladder cancer (MIBC) within 2 years after BCG treatment. For MIBC, radical cystectomy is the gold standard treatment. However, cystectomy has a high rate of postoperative complications and many patients are very reluctant to undergo or cannot undergo this surgery. Regardless of whether the bladder is preserved, cisplatin combined with other chemotherapy drugs (such as nicotinamide or gemcitabine) is the first-line chemotherapy regimen for patients with MIBC.

[0003] Bacillus Calmette-Guérin (BCG) is the main immunotherapy drug for NMIBC, and its mechanism of action is still unclear. Some studies have shown that BCG can directly interact with immune cells and urothelial cells (including tumor cells) in the body, induce antigen presentation, and activate innate and adaptive immune responses. Although intravesical instillation of BCG is the standard treatment for medium- and high-risk NIMBC, as mentioned above, its efficacy still cannot meet the needs. At the same time, adverse reactions after BCG treatment are also common, such as bacterial cystitis, rash, high fever, and other systemic discomfort, and even induce life-threatening sepsis. BCG combined with mitomycin for the treatment of bladder cancer can significantly increase the proportion of 5-year disease-free intervals, which has inspired more exploration of the direction of intravesical chemoimmunotherapy. In addition, in the past decade, BCG has been in short supply worldwide due to a decrease in the number of companies approved for production and an increase in demand, which has also promoted the exploration of low-dose BCG instillations.

[0004] The bladder is a multi-layered hollow organ that stores urine and urinates, and is composed of mucosa (urothelium), submucosa, muscularis propria, and serosa. The surface cells of the urothelium are tightly connected and rich in glycosaminoglycans, which control and limit the absorption of intravesical drugs from urine to the deep bladder, and are called the bladder permeability barrier. Due to the special physiological structure of the bladder, factors such as frequent drug dilution, short retention time, and poor drug absorption greatly limit the therapeutic effect of drugs in the bladder. Patients need to endure frequent and large-dose bladder instillations of drugs, which leads to serious systemic side effects, reduced quality of life, and a heavy burden on family finances. Effective, safe, and feasible intravesical drug therapy that avoids radical cystectomy and reduces side effects during treatment is an urgent need for clinical treatment. Therefore, improving the absorption, targeting, and retention time of drugs in the bladder, thereby improving the therapeutic effect, is a problem to be solved. Summary of the invention

[0005] In view of the defects of the above-mentioned prior art, the present invention proposes a drug delivery system containing a drug-loaded liposome drug and BCG, and its preparation method and application, wherein the drug-loaded system comprises the following components: drug-loaded liposome, BCG, chitosan solution, and the drug-loaded liposome is a drug-loaded liposome modified with a cell-penetrating peptide. The present invention is based on the good biocompatibility and adhesion properties of chitosan, and uses chitosan as a carrier to increase the retention time of the drug-loaded liposome modified with a cell-penetrating peptide and BCG in the bladder, enhance the penetration and uptake capacity of the drug-loaded liposome modified with a cell-penetrating peptide in the bladder, and combine with BCG to exert an immune effect, which can significantly improve the anti-tumor effect of the drug, realize chemical and immune combined therapy, reduce the use of chemical drugs and BCG, and reduce systemic side effects.

[0006] The term "liposome" refers to the situation that when amphiphilic molecules are dispersed in the water phase, the hydrophobic tails of the molecules tend to gather together and avoid the water phase, while the hydrophilic heads are exposed in the water phase, forming a closed vesicle with a bilayer structure. The water phase and bilayer membrane in the vesicle can encapsulate a variety of drugs including water-soluble and fat-soluble drugs, similar to the structure of ultramicrocapsules. This ultramicrosphere carrier preparation made by encapsulating drugs in the middle of lipid molecular layer films is called liposome. Liposomes are generally composed of phospholipids, cholesterol, and modified materials.

[0007] The term "membrane-penetrating peptide" refers to a class of short peptides that have the ability to penetrate the cell membrane, and the length of the short peptide is between 5 and 30. The membrane-penetrating peptides include cationic membrane-penetrating peptides, amphipathic membrane-penetrating peptides and hydrophobic membrane-penetrating peptides.

[0008] One of the purposes of the present invention is to provide a complex drug delivery system containing drug-loaded liposomes and BCG, characterized in that the drug delivery system comprises drug-loaded liposomes, BCG, and chitosan solution; and the drug-loaded liposomes are drug-loaded liposomes modified with a membrane-penetrating peptide.

[0009] One of the purposes of the present invention is to provide a drug delivery system containing a complex of drug-loaded liposomes and BCG, characterized in that the drug-loaded liposomes modified with a cell-penetrating peptide comprise phospholipids, cholesterol, a cell-penetrating peptide and an anti-tumor drug.

[0010] One of the purposes of the present invention is to provide a composite drug delivery system containing drug-loaded liposomes and BCG, characterized in that the liposome phospholipid component includes but is not limited to neutral phospholipids, namely phosphatidylcholine and its derivatives, such as dipalmitoylcholine, distearoylcholine, dimyristoylphosphatidylcholine, etc., sphingomyelin; negatively charged phospholipids, namely phospholipids, such as phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, etc.; positively charged phospholipids, such as one or more of stearamide, oleyl fatty amine derivatives, cholesterol derivatives, etc.

[0011] One of the purposes of the present invention is to provide a drug delivery system containing a drug-loaded liposome and a BCG complex, characterized in that the cell-penetrating peptide is a stearic acid-containing cationic cell-penetrating peptide, preferably a cell-penetrating peptide containing 5-10 arginines and 3 histidines modified by a fatty acid carbon chain containing 8-18 carbon atoms, wherein the carbon atoms are preferably stearic acid containing 18 carbon atoms, and the cell-penetrating peptide preferably has 8-10 arginines. More preferably, the cell-penetrating peptide is RRRRRRRRHHH, and further preferably, the cell-penetrating peptide is a stearic acid-containing cell-penetrating peptide CH3(CH2) 16 -CONH-RRRRRRRRHHH.

[0012] One of the purposes of the present invention is to provide a drug delivery system comprising a drug-loaded liposome and a BCG complex, characterized by a drug-loaded liposome modified with a membrane-penetrating peptide, wherein the drug is an anti-tumor drug, preferably an anti-bladder cancer drug, preferably an anti-bladder cancer drug including paclitaxel, docetaxel, oxaliplatin, gemcitabine, cisplatin, mitomycin, 5-fluorouracil and one or more of the prodrug forms of the above drugs, more preferably an anti-bladder cancer drug is an oxaliplatin prodrug, and further preferably an anti-bladder cancer drug is octadecyl oxaliplatin (IV) carboxylic acid, as shown in formula (I):

[0013]

[0014] One of the purposes of the present invention is to provide a drug delivery system comprising a drug-loaded liposome and a BCG complex, wherein the particle size of the liposome is in the range of 50 to 200 nm, the zeta potential is +8 to +30 mv, and the zeta potential is preferably +10 to +20 mv.

[0015] One of the purposes of the present invention is to provide a drug delivery system containing a drug-loaded liposome and a BCG complex, characterized in that the components of the liposome are, by mass ratio, 50-88% phospholipids, 10-30% cholesterol, 1%-10% cell-penetrating peptide, and 0.5%-20% anti-tumor drug, and the sum of the components is 100%.

[0016] One of the purposes of the present invention is to provide a drug delivery system comprising a complex of drug-loaded liposomes and BCG, wherein the drug delivery system comprises any type of aqueous phase, including but not limited to distilled water, purified water, water for injection, deionized water and solutions (such as HEPES solutions), and the aqueous phase can be further sterile and pyrogen-free.

[0017] One of the purposes of the present invention is to provide a drug delivery system containing a drug-loaded liposome and BCG, characterized in that the concentration of BCG is 10 5 -10 8 CFU / mg, preferably 10 6 CFU / mg.

[0018] One of the purposes of the present invention is to provide a drug delivery system comprising a drug-loaded liposome and a BCG complex, characterized in that the chitosan deacetylation degree of the chitosan solution is ≥95%, the viscosity is 100-200 mPa.s, the concentration is 1-10 mg / mL, preferably 1-5 mg / mL, and further preferably 3-5 mg / mL.

[0019] One of the purposes of the present invention is to provide a drug delivery system comprising a drug-loaded liposome and a BCG complex, wherein the pH value of the drug delivery system is between 4.0 and 10.0, preferably between 5.0 and 8.0.

[0020] One of the purposes of the present invention is to provide a drug delivery system of a drug-loaded liposome drug and BCG, characterized in that the preparation method of the drug delivery system comprises the following steps:

[0021] (1) preparing drug-loaded liposomes by one or more of a thin film dispersion method, a reverse evaporation method, a chemical gradient method, and a calcium fusion method, wherein the particle size of the liposomes is 50 to 200 nm and the zeta potential is +8 to +30 mv;

[0022] (2) slowly drop the BCG solution into the chitosan solution and mix well, then slowly drop the drug-loaded liposome solution modified with the cell-penetrating peptide into the BCG chitosan solution and vortex mix well to obtain the product.

[0023] The present invention has the following beneficial effects:

[0024] One of the purposes of the present invention is to combine anti-tumor drugs and BCG chitosan solution in the same drug delivery system, so as to achieve the combination of chemotherapy and immunotherapy, enhance the effect of anti-tumor treatment, and thus reduce the number and dosage of bladder instillation.

[0025] Compared with the prior art, the present invention has the following characteristics

[0026] (1) The drug-loaded liposomes modified with membrane-penetrating peptides can encapsulate different anti-tumor drugs and improve the uptake of liposomes in mouse bladder cancer cells and their penetration into tumor tissues;

[0027] (2) Anti-tumor chemotherapy drugs and immune drug BCG are delivered simultaneously to further enhance the anti-tumor effect;

[0028] (3) Chitosan solution can enhance the retention time of cell-penetrating peptide-modified drug-loaded liposomes and BCG in bladder tissue, thereby enhancing the therapeutic effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings:

[0030] Figure 1 NMR and mass spectra of oxaliplatin prodrug;

[0031] Figure 2 Particle size and potential diagram of drug-loaded liposomes modified with membrane-penetrating peptides;

[0032] Figure 3 Transmission electron microscopy image of drug-loaded liposomes modified with cell-penetrating peptides;

[0033] Figure 4 Uptake of cell-penetrating peptide-modified drug-loaded liposomes in mouse bladder cancer cells;

[0034] Figure 5 The lysosomal escape ability of drug-loaded liposomes modified with membrane-penetrating peptides;

[0035] Figure 6 Investigation of the permeability of drug-loaded liposomes modified with cell-penetrating peptides in tumor cell spheroids;

[0036] Figure 7 Investigation of the penetration ability of transmembrane modified drug-loaded liposomes in tumor tissues in a drug delivery system containing drug-loaded lipids and BCG complexes;

[0037] Figure 8 Study on the retention time of drug-loaded liposomes and BCG in bladder tissue in the drug delivery system containing drug-loaded liposomes and BCG;

[0038] Fig. 9 Scanning electron micrograph of the drug delivery system containing drug-loaded liposomes and BCG;

[0039] Fig.10 The therapeutic effect of the drug delivery system containing drug-loaded liposomes and BCG in a mouse bladder cancer model; DETAILED DESCRIPTION

[0040] The following examples are further explanations of the present invention, but are by no means limitations of the scope of the present invention. The present invention will be further described in detail below with reference to the examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art may carry out equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0041] Example 1 Preparation and characterization of cell-penetrating peptide-modified drug-loaded liposomes using oxaliplatin prodrug as an example

[0042] In the present invention, in order to reflect the drug loading performance of the cell-penetrating peptide modified liposome (LR), octadecyl-oxaliplatin (OXA) prodrug octadecyl-oxaliplatin carboxylic acid, i.e., octadecyl-OXA (IV) carboxylic acid (OOC) is taken as an example. In the subsequent examples, unless otherwise specified, in the cell-penetrating peptide modified drug-loaded liposome and BCG complex, the liposomes are all cell-penetrating peptide (stearoyl cell-penetrating peptide, CH3(CH2) 16 -CONH-RRRRRRRRHHH, abbreviated as C18-R8H3) modified liposomes (LRO).

[0043] Specific operation: (1) Synthesize oxaliplatin prodrug, the synthesis route is as follows

[0044]

[0045] OXA (100 mg, 0.25 mmol) was dissolved in 10 ml of deionized water, and 3 ml of 30% H2O2 was added to react at room temperature for 24 h. The mixture was concentrated by rotary evaporation to obtain OXA(IV)-OH as a white powder (105 mg, yield 98%). Then, succinic anhydride (30 mg, 0.29 mmol) dissolved in 3 mL of dimethyl sulfoxide (DMSO) was added to OXA(IV)-OH and reacted in the dark for 12 h at room temperature to obtain OXA(IV)-COOH. The DMSO solution was then precipitated twice with cold ether. The precipitate was collected by centrifugation and rotary evaporation. According to calculations, the yield of this process can reach 90%. The precipitate was dissolved with 2 mL of N,N-dimethylformamide (DMF), octadecyl isocyanate (97.5 mg, 0.33 mmol) was added, stirred in the dark for 24 h, and DMF was removed by rotary evaporation at 60 ° C. Cold ether was added and centrifuged to obtain a precipitate, and the precipitation was repeated twice. Finally, the precipitate was collected to obtain octadecyl oxaliplatin (IV) carboxylic acid (OOC, 145.7 mg, yield 80%). The hydrogen spectrum and mass spectrum were as shown in the attached Figure 1 As shown in a and 1b.

[0046] (2) Preparation of drug-loaded liposomes by thin film hydration method: 3 mg of neutral phospholipid dioleoylphosphatidylcholine (DOPC), 1 mg of cholesterol, 0.2 mg of membrane-penetrating peptide (C18-R8H3), and 0.2 mg of OOC were dissolved in 5 mL of methanol:chloroform = 1:4 solution, and the mixture was shaken and mixed. After removing the organic solvent by rotary evaporation for 30 min, a uniform film was formed on the wall of the bottle. After the end, vacuum was continued overnight to remove the residual organic solvent. After completion, the aqueous phase (HEPES buffer at pH 7.4) was added, and the mixture was gently shaken at room temperature until the film fell off. After treatment with probe ultrasound (200 W, 10 min), the membrane-penetrating peptide-modified oxaliplatin prodrug OOC liposomes LRO were obtained. The control sample liposomes (LO) without membrane-penetrating peptide modification were prepared without adding membrane-penetrating peptide, and the rest of the preparation method was the same. Take 50 μL of LRO and dilute it with 1 mL of ultrapure water. Use Zetasizer Nano ZS90 laser particle size analyzer to measure the particle size and potential. Figure 2 In the preparation method, the particle size of the liposomes is between 50 and 200 nm, the LRO potential of the liposomes containing the cell-penetrating peptide is +14.23±1.10 mV (SD), and the LO potential of the liposomes without the cell-penetrating peptide is -21.37±1.59 mV.

[0047] Example 2 Transmission electron microscopy of liposomes

[0048] The liposomes were dropped onto the copper mesh and allowed to stand for 3 min. They were then stained with 2% phosphotungstic acid for 2 min. The excess dye on the copper mesh was removed with filter paper. The samples were dried at room temperature and observed under 200 kV. The morphologies of the prepared LO and LRO were observed using a transmission electron microscope. Figure 3 As shown, the results show that the prepared liposomes have uniform morphology, are spherical, and have a particle size between 50 and 120 nm.

[0049] Example 3 Investigation of liposome uptake in bladder cancer cells

[0050] Specific operation: In this experiment, C57 BL / 6J mouse bladder cancer MB49 cells were used to investigate the cellular uptake of drug-loaded liposomes;

[0051] Preparation of liposomes labeled with fluorescent dye DiD: 3 mg of neutral phospholipid dioleoylphosphatidylcholine (DOPC), 1 mg of cholesterol, 0.2 mg of cell-penetrating peptide (C18-R8H3), and 1.5 ug of DiD were dissolved in 5 mL of methanol: chloroform = 1:4 solution, and the mixture was shaken and mixed. After removing the organic solvent by rotary evaporation for 30 min, a uniform film was formed on the wall of the bottle. After the completion, the vacuum was continued overnight to remove the residual organic solvent. After completion, the aqueous phase (HEPES buffer at pH 7.4) was added, and the film was gently shaken at room temperature until the film fell off. After treatment with probe ultrasound (200 W, 10 min), cell-penetrating peptide-modified liposomes loaded with fluorescent dye DiD (Lip-R8H3-DiD, LRD) were prepared. The control sample liposomes without cell-penetrating peptide modification (Lip-DiD, LD) did not add cell-penetrating peptide, and the rest of the preparation method was the same. All the above operations were performed under strict light-proof conditions.

[0052] Cell uptake experiment: MB49 cells were plated in 12-well plates, with 1.0×10 cells per well. 5 When the cells grew to about 80%, free DiD, LD and LRD were added to the well plate at a rate of 50 ng DiD per well in serum-free medium, incubated in a 37°C incubator for 4 h, the supernatant was removed, washed twice with PBS, and the cells were collected and the positive cell ratio was detected by flow cytometry. The results are shown in the attached figure. Figure 4 As shown, it can be seen that compared with the liposome group not modified with the cell-penetrating peptide, the liposome group modified with the cell-penetrating peptide has a stronger uptake rate and has a significant difference. It can be seen that the cell-penetrating peptide modification can significantly improve the penetration of liposomes on cancer cells.

[0053] Example 4 Investigation of the ability of drug-loaded liposomes to escape from lysosomes in cells

[0054] Specific operation: MB49 cells were cultured at 2×10 5Each well is inoculated in 12 wells and placed in a carbon dioxide incubator to wait for the cells to adhere. According to Example 3, liposomes LD and LRD labeled with the fluorescent dye DiD are prepared, the culture medium in the well plate is discarded, and 0.5 mL of DMEM complete culture medium containing LD and LRD is added to each well, including 50 ng DiD in each well. The culture medium is aspirated at 1h and 5h time points, respectively, and washed once with PBS. 1 mL of lysosomal fluorescent dye LysoTracker-Green is added to each well at a concentration of 50 nM, and the well is placed in a carbon dioxide incubator for incubation and staining for 1 hour, then the culture medium is discarded, and 1 mL of paraformaldehyde fixative is added and incubated at room temperature for 15 minutes to fix the cells after washing once with PBS. After washing once with PBS, DAPI solution is added and incubated at 37°C for 10 minutes, centrifuged and washed twice with PBS, and finally resuspended in 500 μL PBS solution, transferred to a confocal dish, and observed and photographed with a laser confocal microscope. The results are shown in the attached Figure 5 It can be seen that the liposomes modified with the membrane-penetrating peptide can obviously achieve lysosomal escape because of the proton sponge effect with positive charge, while the liposomes without the membrane-penetrating peptide modification cannot achieve lysosomal escape. It can be seen that the liposomes modified with the membrane-penetrating peptide are conducive to the escape of drugs from the lysosomes and exert better efficacy.

[0055] Example 5 Investigation of the permeability of drug-loaded liposomes modified with cell-penetrating peptides in bladder cancer tumor cell spheroids

[0056] Specific operation: First, add a pre-sterilized low-melting point agarose solution (2%, w / v) to a 96-well culture plate to prevent cell attachment, and place it in a UV clean bench for irradiation sterilization for at least 30 minutes. MB49 cells were inoculated into a 96-well plate at a density of 2000 cells / well and cultured in a 37°C constant temperature incubator containing 5% CO2. The 96-well plate was placed under a microscope for observation every 2 days until it grew into a sphere with a diameter of about 200 μm. Select tumor spheres with uniform and rounded morphology and dense cell growth, carefully discard the original culture medium and wash 2-3 times with sterile PBS solution, replace the serum-free culture medium, prepare fluorescent dye DiD samples, non-penetrating peptide-modified DiD-loaded liposomes (LD) and penetrating peptide-modified DiD-loaded liposomes (LRD) according to Example 3, add free DiD, LD and LRD under light-proof conditions, and the administration concentration is 400 ng / mL DiD. After 4 hours of incubation, the tumor spheres were washed twice with pre-cooled PBS buffer, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and the tumor spheres were tomographically scanned using a laser scanning confocal microscope to observe the penetration of different preparations into the tumor spheres. The results are shown in the attached Figure 6As shown in the figure, both LD and LRD can penetrate into the tumor sphere, but the fluorescence intensity of LD is significantly weaker than that of LRD at the depth of 60μm in the tumor sphere, and LRD can still show obvious fluorescence at the depth of 140μm in the tumor sphere. This shows that LRD has stronger penetration ability than LD, which may be because LRD contains membrane-penetrating peptides and has a positive charge, which has stronger tumor cell gap penetration ability and cell surface adsorption, which is more conducive to the efficacy of anticancer drugs.

[0057] Example 6 Investigation of the penetration ability of liposomes in tumor tissues in the drug delivery system of cell-penetrating peptide-modified drug-loaded liposomes and BCG complex

[0058] Simulated free drug control group: an equal amount of DiD was added dropwise to the BCG chitosan solution CS, labeled as DiD-BCG / CS;

[0059] Cell-penetrating peptide modified drug-loaded liposome group without CS solution: Since the cell-penetrating peptide modified drug-loaded liposome has a positive charge and BCG has a negative charge, it is very easy to form precipitation when the two are directly mixed in physiological saline or buffered saline. In this experiment, cell-penetrating peptide modified DiD-loaded liposome LRD was selected as the sample group;

[0060] Preparation of the complex delivery system of DiD liposomes loaded with fluorescent dye modified by cell-penetrating peptide, BCG and chitosan solution: 0.1 mL of 20 mg / mL BCG physiological saline solution was slowly added dropwise to 0.4 mL of 20 mg / mL chitosan solution and vortexed evenly to form a BCG chitosan solution, and 1 mL of DiD liposomes LRD modified by cell-penetrating peptide prepared according to the method in Example 3 was added dropwise to the above BCG chitosan solution and vortexed evenly to form a DiD fluorescently labeled cell-penetrating peptide modified liposome and BCG complex drug delivery system (LRD-BCG / CS), wherein the DiD concentration was 40 ug / mL and the chitosan concentration was 5 mg / mL.

[0061] MB49 cells were divided into 1*10 5 The cells were inoculated into the bladder tissue of C57 BL / 6J female mice to form an orthotopic bladder cancer model in mice. Seven days after tumor implantation, three groups of samples, DiD-BCG / CS, LRD, and LRD-BCG / CS, were instilled into the bladder. Each mouse received 3 μg DiD and the instillation volume was 75 μL. The mice were killed 4 hours later to remove the bladder tissue. Each group of tissue samples was embedded with an embedding agent and pre-frozen, and then cut into 8 μm thick tissue sections using a freezing microtome. The sections were fixed with 4% paraformaldehyde at room temperature, and the cell nuclei were stained with DAPI for 15 minutes. After being carefully washed three times with PBS, the sections were sealed after adding an anti-fluorescence quencher. The tumor sections were observed using a confocal microscope to observe the penetration of each group of preparations in the tumor tissue. The results are shown in the attached figure. Figure 7As shown in the data, the permeation intensity of LRD-BCG / CS was significantly stronger than that of LRD and DiD-BCG / CS groups, proving that the permeation ability of the drug-loaded liposomes modified with the membrane-penetrating peptide in the drug-loaded liposome and BCG complex drug delivery system was further enhanced under the adhesion and retention effect of the CS solution, and the membrane-penetrating peptide and CS made the composite delivery system have stronger permeability and adhesion.

[0062] Example 7 Investigation of the retention capacity of drug-loaded liposomes and BCG in the drug delivery system containing drug-loaded liposomes and BCG complex

[0063] The specific operation of the composite delivery system retention experiment was as follows: MB49 cells were divided into 1*10 5 The cells were inoculated into the bladder tissue of C57 BL / 6J female mice to form an orthotopic bladder cancer model in mice. The cells were instilled into the bladder 7 days after tumor inoculation. Three groups of samples, DiD-BCG / CS, LRD, and LRD-BCG / CS, were prepared according to the preparation method of Example 6. The fluorescence intensity was recorded using a mouse in vivo imaging device to observe the retention of the cell-penetrating peptide-modified liposomes in the composite drug delivery system in the bladder tissue at different time points of 4, 8, 12, 24, and 48 hours. The results are shown in the attached figure. Figure 8 As shown in a mouse in vivo fluorescence imaging and 8b the fluorescence intensity graph of the in vivo imaging picture, fluorescence can still be detected in the fluorescently labeled transmembrane peptide-modified liposomes in the composite delivery system 48 hours after bladder instillation, and the retention time is significantly stronger than that of the control group DiD-BCG / CS and LRD. At different time points, the fluorescence intensity of the transmembrane peptide-modified liposomes in the composite delivery system is also significantly stronger than that of the control group and has significant differences.

[0064] Preparation of a cell-penetrating peptide-modified liposome drug and BCG complex delivery system: Take 0.1 mL of a 20 mg / mL BCG saline solution and slowly add it dropwise to 0.4 mL, 20 mg / mL chitosan solution, and vortex evenly to form a BCG chitosan solution. Take 1 mL of the cell-penetrating peptide-modified liposome LRO loaded with the oxaliplatin prodrug OCC in Example 1 and add it dropwise to the above BCG chitosan solution, and vortex evenly to form a cell-penetrating peptide-modified liposome drug loaded with the oxaliplatin prodrug OOC and BCG complex delivery system (LRO-BCG / CS). The bladder of tumor-bearing mice was instilled with a BCG saline mixture and LRO-BCG / CS, respectively, with an instillation volume of 75 μL and BCG 0.1 mg / mouse. The retention of BCG in the complex delivery system was observed by acid-fast staining at different time points of 4, 8, 12, 24 and 48 hours after bladder instillation. The results of acid-fast staining are shown in the attached figure. Figure 8As shown in Figure c, BCG can still be detected in the composite drug delivery system 48 hours after BCG bladder instillation, while BCG saline group can no longer detect BCG at 12 hours. It can be seen that the composite drug delivery system of the present invention can significantly enhance the retention time of the cell-penetrating peptide-modified drug-loaded liposomes and BCG in the mouse bladder, further confirming the adhesion effect of chitosan and the penetration ability of the cell-penetrating peptide-modified drug-loaded liposomes.

[0065] Example 8 Scanning electron microscopy of drug-loaded liposome and BCG complex delivery system

[0066] The drug delivery system containing drug-loaded liposomes and BCG complex (LRO-BCG / CS) was prepared according to the method of Example 7. The composite delivery system and chitosan solution of the same concentration were freeze-dried and then examined by scanning electron microscopy. The results are shown in the attached figure. Fig. 9 As shown in the attached figure, it can be seen that the chitosan scanning electron microscopy Fig. 9 a is relatively smooth, and the scanning electron microscopy of the composite delivery system is shown in the attached Fig. 9 As shown in Figures b and 9c, the liposomes and BCG are evenly distributed in chitosan, and the size of the liposomes is consistent with the results of transmission electron microscopy. It can be seen that the composite delivery system of the present invention has the performance of maintaining the even distribution of liposomes and BCG, which is beneficial to the exertion of drug efficacy.

[0067] Example 9 Preliminary evaluation of the efficacy of drug delivery system containing drug-loaded liposomes and BCG complex

[0068] Specific operation: MB49 cells were divided into 1*10 5 The cells were inoculated into the bladder tissue of C57 BL / 6J female mice to form an orthotopic bladder cancer model in mice. The cells were instilled into the bladder 7 days after tumor inoculation. The groups of mice for the efficacy experiment are as follows:

[0069] 1. Saline group;

[0070] 2. Oxaliplatin chitosan solution group, labeled as OXA / CS;

[0071] 3. Simulated clinical perfusion method group: BCG saline group, marked as BCG;

[0072] 4. The CS solution group of the cell-penetrating peptide-modified oxaliplatin prodrug OOC liposomes, labeled as LRO / CS; 5. The alternating perfusion group of chemical drugs and immune preparations simulating the clinical treatment plan, that is, the drug-loaded liposomes were first perfused and then BCG was perfused after 24 hours, labeled as LRO+BCG (24h);

[0073] 6. A drug delivery system group containing drug-loaded liposomes and BCG complex, labeled as LRO-BCG / CS.

[0074] The cell-penetrating peptide-modified oxaliplatin-loaded OOC liposomes LRO were prepared according to the method in Example 1. The cell-penetrating peptide-modified drug-loaded liposomes and BCG complex drug delivery system (LRO-BCG / CS) were prepared according to the method in Example 8. The LRO / CS group was not added with BCG during the preparation process. The dosage was OXA 0.6 mg / kg, BCG 0.1 mg / head (10 6 CFU / mg), and the treatment was performed by intravesical instillation once every 7 days. After 3 consecutive instillations, the mice were killed on the 7th day, and the bladder tumor tissue was dissected. The anti-tumor treatment effect was evaluated by weighing and calculating the size of the tumor tissue. The results are shown in the attached Fig.10 As shown, 10a is a visual diagram of bladder tumor tissues in different drug administration groups, and 10b and 10c are statistical diagrams of the volume and weight of bladder tumor tissues in different drug administration groups. Fig.10 It can be seen that the drug-loaded liposome drug and BCG complex drug delivery system has the most obvious therapeutic effect, and the tumor volume and weight are significantly different from those of other groups. It can be seen that the complex drug delivery system of the present invention has a significant therapeutic effect on the treatment of bladder cancer.

[0075] In summary, the drug-loaded liposomes in the composite delivery system containing drug-loaded liposomes and BCG of the present invention have good anti-tumor drug loading capacity, uniform size, positive charge, high tumor cell uptake capacity, lysosomal escape capacity and strong penetration capacity in tumor spheres and tumor tissues. The composite delivery system of the present invention can significantly increase the penetration capacity of drug-loaded liposomes and the retention time of BCG in the bladder and has good uniformity, and has excellent therapeutic effects in the efficacy experiment of bladder cancer-bearing mice.

[0076] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A drug delivery system containing a drug-loaded liposome and BCG, characterized in that: The drug delivery system comprises the following components: drug-loaded liposomes, BCG, and chitosan solution. The drug-loaded liposomes are drug-loaded liposomes modified with a cell-penetrating peptide, the cell-penetrating peptide is C18-RRRRRRRRHHH, and the anti-tumor drug in the drug-loaded liposomes is octadecyl oxaliplatin (IV) carboxylic acid represented by formula (I):

2. The drug delivery system according to claim 1, characterized in that: The components of the drug-carrying liposome modified by the cell-penetrating peptide include phospholipids, cholesterol, cell-penetrating peptide and anti-tumor drugs.

3. The drug delivery system according to claim 1 or 2, characterized in that: The phospholipid component in the drug-carrying liposome modified with the membrane-penetrating peptide is selected from one or more of neutral phospholipids, negatively charged phospholipids or positively charged phospholipids.

4. The drug delivery system according to claim 2, characterized in that: The components of the drug-loaded liposome modified with the cell-penetrating peptide are 50-88% phospholipid, 10-30% cholesterol, 1%-10% cell-penetrating peptide, and 0.5%-20% anti-tumor drug in terms of mass ratio, and the sum of the components is 100%.

5. The drug delivery system according to claim 1, wherein the particle size of the drug-loaded liposome modified with the cell-penetrating peptide is in the range of 50 to 200 nm, and the zeta potential is in the range of +8 to +30 mv. 6 . The drug delivery system according to claim 5 , wherein the zeta potential of the drug-loaded liposome modified with the cell-penetrating peptide is +10 to +20 mv.

7. The drug delivery system according to claim 1, characterized in that: The drug delivery system includes any type of aqueous phase, including purified water and buffered solutions.

8. The drug delivery system according to claim 7, characterized in that: The purified water includes distilled water and deionized water.

9. The drug delivery system according to claim 1, characterized in that: The concentration of BCG is 10 5 ~10 8 CFU / mg.

10. The drug delivery system according to claim 1, characterized in that: The chitosan solution has a chitosan deacetylation degree of ≥95%, a viscosity of 100-200 mpa.s, and a concentration of 1-10 mg / mL.

11. The drug delivery system according to claim 10, characterized in that: The concentration of the chitosan solution is 1-5 mg / mL.

12. The drug delivery system according to claim 11, characterized in that: The concentration of the chitosan solution is 3-5 mg / mL.

13. The drug delivery system according to claim 1, characterized in that: The pH value of the drug delivery system is between 4.0 and 10.

0.

14. The drug delivery system according to claim 13, characterized in that: The pH value of the drug delivery system is 5.0-8.

0.

15. The method for preparing the drug delivery system according to any one of claims 1 to 14, characterized in that The following steps are involved: (1) preparing the drug-loaded liposomes modified with the cell-penetrating peptide by one or more of the following methods: thin film dispersion method, reverse evaporation method, chemical gradient method, and calcium fusion method; (2) After slowly dropping the BCG solution into the chitosan solution and mixing, slowly drop the drug-loaded liposome solution modified with the cell-penetrating peptide into the BCG chitosan solution and vortex mix to obtain the product.

16. Use of the drug delivery system according to any one of claims 1 to 14 or the drug delivery system prepared by the preparation method according to claim 15 in preparing drugs for treating bladder cancer.

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