A tumor-targeted drug-loaded nanoformulation based on polyglutamic acid conjugated photosensitizer

Abstract

The present invention belongs to the field of tumor-targeted nanoformulations, and provides a tumor-targeted drug-loaded nanoformulation based on a polyglutamic acid conjugated photosensitizer. The polyglutamic acid conjugated photosensitizer adopts PGA and a common second-generation photosensitizer Ce6 through chemical bond conjugation coupling, and the tumor-targeted nanoformulation adopts a polyglutamic acid conjugated photosensitizer to encapsulate a hydrophobic chemotherapy drug, and the surface is coated with a tumor cell membrane. The use of chemotherapy drugs with different mechanisms for joint treatment can effectively improve the tumor killing effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor-targeted nano-preparations, and specifically relates to a preparation method of a polyglutamic acid conjugated photosensitizer and a related tumor-targeted nano-preparation, a pharmaceutical composition comprising the tumor nano-preparation, and an application of the same in the preparation of anti-tumor drugs. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Photodynamic therapy (PDT) is a relatively new non-invasive treatment for tumors. Photosensitizers can produce reactive oxygen species (ROS) to damage tumors after being irradiated with a light source of a specific wavelength. The first generation of photosensitizers are mainly hematoporphyrins, and the second generation of photosensitizers are dihydrochlorins. The second generation of photosensitizers that are currently used more frequently include dihydrochlorin e6 (Ce6). The third generation of photosensitizers are second-generation photosensitizers coupled with different targeting molecules such as monoclonal antibodies to improve their targeting and reduce adverse reactions during PDT treatment. Compared with traditional tumor therapies, PDT can play a local therapeutic advantage without systemic toxic side effects. More importantly, PDT can activate the body's anti-tumor immune response, such as achieving immunogenic death of tumor cells and producing cytokines. Although this treatment method has many advantages, the characteristics of the photosensitizer itself greatly limit its full effect. For example: (1) Photosensitizers have poor dispersibility in water and tend to aggregate in solution to produce a fluorescence quenching effect; (2) Photosensitizers have poor tumor specificity and are easily captured and cleared by the reticuloendothelial system after intravenous injection, and are unable to reach the tumor site; (3) Third-generation photosensitizers have improved their targeting through structural modifications such as monoclonal antibodies, but at the same time they come with high costs, making the application of PDT increasingly distant from the general clinical market.

[0004] Polyglutamic acid (PGA) is a green and environmentally friendly polymer material with good biocompatibility. It is composed of α-amine and γ-carboxyl groups of glutamic acid cross-linked by amide bonds. The main chain molecule of PGA contains a large number of carboxyl groups, which can be conjugated to small molecule drugs, gene drugs, monoclonal antibodies, etc. with poor water solubility, and has great application prospects as a drug delivery platform. At the same time, studies have shown that PGA can induce a highly efficient immune response and is a safe and effective vaccine adjuvant for tumor immunotherapy. For example: (1) The prodrug CT2103 formed by direct connection of PGA and paclitaxel has entered Phase III clinical research for non-small cell lung cancer. (2) The hydrogen on the side chain carboxyl group of the PGA molecule replaces the chlorine atom in the cisplatin (CDDP) molecule to form an active and relatively stable CDDP-PGA complex, which has high kinetic stability and low toxicity to normal cells. (3) After PGA is coupled with poorly soluble 10-hydroxycamptothecin (CPT) or 9-aminocamptothecin to form a PGA-CPT complex, its water solubility is greatly increased and its anti-tumor activity is maintained at a high level. The existing PGA is mainly used in anti-tumor applications by directly or through linkers to chemotherapy drugs, but rarely conjugated with photosensitizers. Although Zhejiang University CN104491863A discloses an anti-tumor drug based on a polyglutamic acid and tetrachlorotetraiodine fluorescein bond, its preparation method and application, the drug combination method still needs to be improved.

[0005] Although photodynamic therapy can damage tumors, it often results in incomplete tumor elimination. The main reason is that the tumor has its own repair mechanism. Poly ADP ribose polymerase (PARP) is a DNA repair enzyme that can bind to the single-strand damage site of damaged DNA to repair DNA damage. Normal cells have both homologous recombination repair and PARP repair mechanisms, while triple-negative breast cancer cells have a higher probability of homologous recombination repair defects.

[0006] Disguising nanoparticles with cell membranes is a mature biomimetic approach. Various cell membrane coating strategies have been developed for different application scenarios. Red blood cell membranes are used to evade immune clearance to increase long circulation, platelet membranes are used to target injury sites, etc. Cancer cell membranes have attracted much attention due to their unique tumor homing effect and the ability to carry tumor antigens. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a tumor-targeted drug-loaded nanoformulation based on polyglutamic acid conjugated photosensitizer. The use of chemotherapy drugs with different mechanisms for co-treatment can effectively improve the tumor killing effect. Among them, the polyglutamic acid conjugated photosensitizer adopts PGA and the common second-generation photosensitizer Ce6 through chemical bond conjugation coupling, and the tumor-targeted nanoformulation adopts polyglutamic acid conjugated photosensitizer to encapsulate hydrophobic chemotherapy drugs, and the surface is coated with tumor cell membrane.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer, comprising:

[0010] Polyglutamic acid conjugated photosensitizer;

[0011] The polyglutamic acid conjugated photosensitizer is loaded with a hydrophobic chemotherapeutic drug;

[0012] The polyglutamic acid conjugated photosensitizer is coated with a tumor cell membrane.

[0013] The present invention is the first to administer a polyglutamic acid conjugated photosensitizer in combination with a chemotherapeutic drug, introduce a PARP inhibitor to prevent tumor DNA repair, and achieve synergistic treatment to completely eliminate the tumor. At the same time, by coating the tumor cell membrane, the specific targeting of the same type of primary tumor is improved.

[0014] The second aspect of the present invention provides a method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer, comprising:

[0015] The photosensitizer is connected to the PGA molecule by covalent bonding and electrostatic adsorption to obtain a polyglutamic acid conjugated photosensitizer;

[0016] The hydrophobic chemotherapeutic drug is encapsulated in the polyglutamic acid conjugated photosensitizer by means of hydrophobic interaction, electrostatic adsorption, and π-π conjugation to obtain a nano preparation;

[0017] The nano preparation is coated with the cell membrane of tumor cells to obtain the nano preparation.

[0018] Beneficial Effects of the Invention

[0019] (1) The present invention conjugates the photosensitizer with PGA through a chemical reaction, retaining its property of generating reactive oxygen species and enhancing its dispersibility, biocompatibility and permeability in water. At the same time, due to the presence of linker cystamine, it can achieve responsive release in the high GSH environment inside the tumor;

[0020] (2) The invented polyglutamic acid photosensitizer that can damage tumor cell DNA is used as a drug carrier and combined with a PARP inhibitor that has the function of preventing DNA repair to form a nanoformulation to achieve synergistic anti-tumor treatment.

[0021] (3) The above-mentioned nanoformulation is further coated with the cell membrane of tumor cells to achieve specific targeting of homologous tumors, so as to construct a biomimetic targeted nanoformulation.

[0022] (4) The preparation method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention, and do not constitute improper limitations on the present invention.

[0024] Figure 1 , the synthesis process of polyglutamic acid conjugated photosensitizer PC.

[0025] Figure 2 , 1HNMR spectra of PGA, PGA-Cys, and PC.

[0026] Figure 3 , FT-IR spectra of PGA, PGA-Cys and PC.

[0027] Figure 4 , UV-Vis spectra of Ce6, PGA-Cys, PC and the Tyndall phenomenon of PC in water.

[0028] Figure 5 , DPBF was used to detect the singlet oxygen production ability of Ce6 and PC.

[0029] Figure 6 , TEM images of PC polymer in water.

[0030] Figure 7 , TEM and particle size images of PCO, MPCO and MPCO+GSH.

[0031] Figure 8 , SDS-PAGE gel electrophoresis images of PCO, 4T1 cell membrane, MPCO and 4T1 cell whole protein.

[0032] Fig. 9 , Changes in particle size and potential of PCO and MPCO nanoparticles in PBS within one week.

[0033] Fig.10 , Drug release curves of Ola from MPCO nanoparticles under different release media conditions.

[0034] Fig.11 , Flow cytometry results of 4T-1 cells uptake of free Ce6 and MPCO nanoparticles.

[0035] Fig.12 , The cytotoxicity results of free Ola, free Ce6, PC polymer, PCO and MPCO nanoparticles under no light and light conditions.

[0036] Fig.13, Fluorescence images of the biodistribution of Ce6, PCO, and MPCO in tumor-bearing mice at 2 h, 4 h, 8 h, and 12 h, and ex vivo fluorescence imaging of tumors and major organs 24 h after intravenous injection. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] Explanation of terms

[0039] In this application, "Olaparib" is the full name of Olaparib, abbreviated as Ola.

[0040] A tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer, comprising:

[0041] Polyglutamic acid conjugated photosensitizer;

[0042] The polyglutamic acid conjugated photosensitizer is loaded with a hydrophobic chemotherapeutic drug;

[0043] The polyglutamic acid conjugated photosensitizer is coated with a tumor cell membrane.

[0044] In some embodiments, the preparation method of the polyglutamic acid conjugated photosensitizer is: connecting the photosensitizer to the PGA molecule by covalent bond coupling or electrostatic adsorption.

[0045] In some embodiments, the photosensitizer is at least one of dihydrochlorin e6, indocyanine green, chlorophyll-a, pheophorbide a, pyropheophorbide-a, and pyropheophorbide a hexyl ether.

[0046] In some embodiments, the hydrophobic chemotherapeutic drug is at least one of a PARP inhibitor, paclitaxel, docetaxel, doxorubicin, oxaliplatin, quercetin, and curcumin.

[0047] In some embodiments, the PARP inhibitor is at least one of olaparib, niraparib, fluzoparib, and pamiparib.

[0048] In some embodiments, the tumor is at least one of melanoma, colon cancer, lung cancer, gastric cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, liver cancer, head and neck cancer, lymphoma, sarcoma, chronic lymphocytic leukemia, thyroid cancer, testicular cancer, and anal cancer.

[0049] A method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer, comprising:

[0050] The photosensitizer is connected to the PGA molecule by covalent bonding and electrostatic adsorption to obtain a polyglutamic acid conjugated photosensitizer;

[0051] The hydrophobic chemotherapeutic drug is encapsulated in the polyglutamic acid conjugated photosensitizer by means of hydrophobic interaction, electrostatic adsorption, and π-π conjugation to obtain a nano preparation;

[0052] The nano preparation is coated with the cell membrane of tumor cells to obtain the nano preparation.

[0053] In some embodiments, the preparation method of the polyglutamic acid conjugated photosensitizer comprises:

[0054] PGA with a molecular weight of 5000-200000 is dissolved in a solution, EDC·HCl and NHS are added for activation, and then cystamine dihydrochloride is added to react at room temperature. After the reaction is completed, the reaction is dialyzed and freeze-dried to obtain a polyglutamic acid-cystamine polymer;

[0055] The photosensitizer is dissolved in a solution, EDC·HCl and NHS are added for activation, and then the polyglutamic acid-cystamine polymer is added for reaction. After the reaction is completed, the polymer is dialyzed and freeze-dried to obtain the product.

[0056] In some embodiments, the preparation method of the nanoformulation comprises: dissolving PC and Ola in a solution, mixing evenly, rotary evaporating to obtain a thin film, then injecting PBS for hydration, ultrasonically dispersing, and removing unencapsulated Ola with a water filter membrane to obtain the nanoformulation.

[0057] In some embodiments, the specific steps of cell membrane coating include: adding tumor cells to a lysis solution and repeatedly freezing and thawing for 5-10 times, and completely breaking the cells by probe ultrasound for 20-40 minutes; after low-temperature and low-speed centrifugation, taking the supernatant, and then centrifuging at a high speed to collect the precipitate to obtain tumor cell membrane fragments;

[0058] The tumor cell membrane and the nanoparticle preparation are mixed and sonicated, and then squeezed out through a polycarbonate membrane filter for 10-30 times to obtain the mixture.

[0059] In some embodiments, in order to synthesize polyglutamic acid conjugated photosensitizer PC (PGA-Cys-Ce6, PC), 200 mg of PGA with a molecular weight of 75,000 is dissolved in 10-30 mL (preferably 20 ml) of distilled water, 1.8 g of EDC·HCl and 630 mg of NHS are added and stirred for 10-30 min (preferably 20 min) to fully activate the carboxyl group of PGA. 100-500 mg (preferably 300 mg) of cystamine dihydrochloride is added and reacted at room temperature for 6-24 h (preferably 12 h). The unreacted EDC, NHS and cystamine are removed by dialyzing with a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h. The polymer solution is freeze-dried to obtain a polyglutamic acid-cystamine (PGA-Cys) polymer. Weigh 20 mg Ce6 and dissolve it in 3-8 mL (preferably 5 mL) of DMF, add 37 mg EDC·HCl and 26 mg NHS and stir for 10-30 min (preferably 20 min) to fully activate the carboxyl group of Ce6. Add 3-8 mL (preferably 5 mL) of PGA-cys aqueous solution containing 30-80 mg (preferably 50 mg) and react at room temperature for 6-48 h (preferably 12 h). Use a dialysis bag with a molecular weight cutoff of 3500 Da to dialyze in DMF:H2O (1:1) for two days and in ultrapure water for two days. Freeze-dry the polymer solution to obtain PC and store it at 4°C away from light. The final product is characterized by nuclear magnetic resonance hydrogen spectrum, infrared spectrum and ultraviolet spectrum.

[0060] In some embodiments, in order to obtain PARP inhibitor-loaded nanoparticles, a thin film dispersion method is used for preparation. PC and Ola containing a molar ratio of (1-20): (1-5) (preferably 10:3) are dissolved in methanol, and mixed and stirred for 0.5-4h (preferably 1h), and the mixed solution is injected into an eggplant-shaped bottle and rotary evaporated to remove methanol to obtain a thin film. 2-10mL (preferably 4mL) of PBS is injected for hydration, and after ultrasonication for 1-30min (preferably 5min), unloaded Ola is removed through a 0.22μm water filter membrane.

[0061] In some embodiments, in order to obtain a drug-loaded nanoformulation coated with a tumor cell membrane, 4T-1 tumor cells are collected, lysate is added, and the cells are repeatedly frozen and thawed in a liquid nitrogen tank for 5-10 times (preferably 7 times), and the cells are completely broken by probe ultrasound for 20-40 minutes (preferably 30 minutes). The supernatant is taken by low-speed centrifugation at 500-1000g (preferably 700g) for 10 minutes at 4°C, and then the precipitate is collected by high-speed centrifugation at 12000-16000 (preferably 14000g) for 30 minutes to obtain 4T-1 tumor cell membrane fragments. The 4T-1 cell membrane is mixed with PCO and ultrasound is performed, and 10-30 (preferably 20) are extruded back and forth through a 200-800nm ​​(preferably 400nm) polycarbonate membrane filter to obtain PC / Ola polymer nanoparticles (MPCO) wrapped with 4T-1 cell membranes.

[0062] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0063] Example 1

[0064] 1. Synthesis and characterization of polyglutamic acid conjugated photosensitizer PC

[0065] In order to synthesize polyglutamic acid conjugated photosensitizer PC (PGA-Cys-Ce6, PC), 200 mg of PGA with a molecular weight of 75,000 was dissolved in 20 ml of distilled water, and 1.8 g of EDC·HCl and 630 mg of NHS were added and stirred for 20 min to fully activate the carboxyl group of PGA. 300 mg of cystamine dihydrochloride was added and reacted at room temperature for 12 h. Dialysis was performed for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da to remove unreacted EDC, NHS and cystamine. The polymer solution was freeze-dried to obtain polyglutamic acid-cystamine (PGA-Cys) polymer. 20 mg of Ce6 was weighed and dissolved in 5 ml of DMF, and 37 mg of EDC·HCl and 26 mg of NHS were added and stirred for 20 min to fully activate the carboxyl group of Ce6. 5 ml of an aqueous solution containing 50 mg of PGA-cys was added and reacted at room temperature for 12 h. The polymer solution was lyophilized to obtain PC, which was stored at 4°C in the dark. The final product was characterized by H NMR, IR, and UV spectra. 1 The production efficiency of O2 was measured using 1,3-diphenylisobenzofuran (DPBF) as an indicator. In short, 10 μL DPBF (3 mg / mL in DMSO) and 2 ml free Ce6 or PC solution (equivalent to 5 μg / mL Ce6) were added. Then, after irradiation with a 660 nm laser (200 mW / cm) for different times, the absorbance of DPBF at 417 nm was recorded using a UV-visible spectrophotometer. DPBF aqueous solution was used as a control. The ROS production efficiency was evaluated by the residual DPBF rate, and the calculation formula was RemainDPBF=A t / A0×100%, where A t and A0 are the absorbance of residual DPBF at 417 nm after t seconds and 0 seconds of irradiation, respectively.

[0066] 2. Synthesis and characterization of drug-loaded nanoparticles PCO

[0067] In order to obtain PARP inhibitor-loaded nanoparticles, a thin film dispersion method was used for preparation. PC and Ola with a molar ratio of 10:3 were dissolved in methanol and mixed and stirred for 1 hour. The mixed solution was injected into an eggplant-shaped bottle and rotary evaporated to remove methanol to obtain a thin film. 4 mL of PBS was injected for hydration, and after ultrasonication for 5 minutes, unloaded Ola was removed through a 0.22 μm water filter membrane. The particle size distribution, potential and appearance morphology of the nanoformulation were characterized by a Malvern laser particle size analyzer and a transmission electron microscope.

[0068] 3. Synthesis and characterization of tumor cell membrane-coated nanoparticles MPCO

[0069] In order to obtain drug-loaded nanoparticles coated with tumor cell membranes, 4T-1 tumor cells were collected, lysate was added, and the cells were repeatedly frozen and thawed in a liquid nitrogen tank for 7 times, and the cells were completely broken by probe ultrasound for 30 minutes. The supernatant was taken by low-speed centrifugation at 700g for 10 minutes at 4°C, and the precipitate was collected by high-speed centrifugation at 14000g for 30 minutes to obtain 4T-1 tumor cell membrane fragments. The 4T-1 cell membrane was mixed with PCO and ultrasound, and extruded 20 times through a 400nm polycarbonate membrane filter to obtain PC / Ola polymer nanoparticles (MPCO) wrapped with 4T-1 cell membranes. The particle size distribution, potential and appearance morphology of the nanoparticles were characterized by Malvern laser particle size analyzer and transmission electron microscopy.

[0070] SDS-PAGE analysis was performed to identify the protein components of PCO, 4T1 cell membranes, coated preparations MPCO, and 4T1 whole cells. Briefly, equal amounts of protein from different samples (quantified using a BCA protein assay kit) were added to a 10% SDS polyacrylamide gel to separate proteins of different molecular weights. The gel was then treated with Coomassie Brilliant Blue and imaged.

[0071] 4. In vitro release experiment of tumor cell membrane-coated nanoformulation MPCO

[0072] The release behavior of Ola from MPCO under different conditions was evaluated by dialysis. In short, 1 mL of MPCO was sealed in a dialysis bag (MWCO = 3500 Da) and immersed in 20 mL of release medium (10 mM) containing or not containing GSH, and placed in a water bath constant temperature oscillator (37 ° C, 100 rpm). At the predetermined time point, 1 mL of release medium was collected and 1 mL of fresh release medium was added. The concentration of released Ola was determined by HPLC. In addition, the release behavior of free Ola was also studied for comparison.

[0073] 5. Cellular Uptake Experiments of Different Treatment Groups

[0074] The effects of different preparations on cell uptake were detected by flow cytometry. 4T1 cells were cultured at 1*105 / well of a 12-well plate was inoculated and incubated for 12 hours, and free Ce6 and MPCO with a Ce6 concentration of 5 μg / mL were added and incubated for 1 hour, 2 hours, and 4 hours, respectively. The cells were washed three times with cold PBS and then digested with trypsin, resuspended in PBS, and detected by flow cytometry.

[0075] 6. Cytotoxicity Experiments of Different Treatment Groups

[0076] In order to detect the in vitro cytotoxicity experiment, the CCK8 method was used to detect cell viability. After digestion of 4T-1 cells in the exponential growth phase, the cells were seeded into 96-well plates at a density of 8000 cells per well. After incubation in a cell incubator for 24 hours, 100 μL of Ola, Ce6, PC, PCO, and MPCO drug-containing culture medium were added, where the Ola concentrations were 0.06, 0.13, 0.32, 0.64, and 1.29 μg / mL, respectively, and the Ce6 concentrations were 0.05, 0.1, 0.25, 0.5, and 1.0 μg / mL. After incubation for 12 hours, for the light-treated group, 100 μL of fresh culture medium was used, and each well was irradiated with a 660 nm laser (100 mW, 1 min); for the non-light-treated group, 100 μL of fresh culture medium was used, and no NIR irradiation was performed. After incubation for 12 hours, 10 μL of CCK8 reagent was added and the cells were incubated in the incubator for about 1 hour. The cells were moved to a microplate reader to measure the absorbance of each well at 450 nm and the cell survival rate was calculated. All experiments were repeated 3 times independently.

[0077] 7. Fluorescence Imaging of Biodistribution in Mice with Orthotopic Breast Cancer in Different Treatment Groups

[0078] To establish a tumor-bearing mouse model, female Balb / c mice (6-8 weeks old) were injected subcutaneously in the fourth mammary fat pad on the right side with 1×10 6 About 7 days after implantation, the tumor size was 100 mm 3 Mice were randomly divided into groups. After intravenous injection of Ce6, PCO and MPCO nanoparticles at a concentration of 4 mg / kg, imaging was performed using an in vivo imaging system instrument. 24 hours after injection, mice were killed and tumors and major organs (heart, liver, spleen, lungs and kidneys) were collected for ex vivo imaging.

[0079] Example 2

[0080] Example 1 A polyglutamic acid conjugated photosensitizer PC was prepared by a two-step amide reaction, wherein the linker was a disulfide-bonded cystamine ( Figure 1 ). The proton peak around 3.0 in the H NMR spectrum proves that cystamine is successfully connected; the proton peak around 8.0 proves that Ce6 is successfully connected ( Figure 2 ). The infrared spectrum at 1734 is the carbonyl stretching vibration peak in the Ce6 structure, indicating that Ce6 was successfully grafted ( Figure 3The synthesized PC is easily dispersed in aqueous media such as water, PBS, saline and cell culture medium. It is brown-green and evenly dispersed in water. The UV-Vis spectrum shows that the grafted polymer has an absorption peak close to that of Ce6 in water and exhibits an obvious Tyndall effect. At the same time, the methodology of Ce6 was established by UV and the content of Ce6 in PC was calculated to be 18.79±0.06% ( Figure 4 ). The generation of singlet oxygen under laser irradiation was detected by DPBF. The results showed that the grafted polyglutamic acid conjugated photosensitizer PC has a similar singlet oxygen generation ability as Ce6 ( Figure 5 ). PC is an amphiphilic polymer, and TEM images show that it can self-assemble into nanovesicles in water ( Figure 6 ), the outer layer is a hydrophilic PGA polymer, the inner layer is a grafted photosensitizer, and each PC nanoparticle has a large hydrophobic cavity. This structural feature lays the foundation for the development of drug-loaded nanoformulations.

[0081] Ola-loaded nanoparticles PCO were prepared by thin film dispersion method, and the optimal drug-to-mass ratio was 10:3, with drug loading and encapsulation efficiency up to 19.52% and 80.86%, respectively. 4T1 tumor cell membrane was coated by ultrasonic extrusion. Particle size potential and morphology ( Figure 7 ). PCO nanoparticles are uniformly round, and the hydrophobic drug Ola is encapsulated in its cavity. The particle size is 191.30±1.50nm, and the zeta potential is -9.54±0.96mV. MPCO forms a coating shadow, indicating the successful coating of 4T-1 cell membrane, with a particle size of 202.43±1.50nm and a zeta potential of -17.5±0.52mV. The membrane protein of the coating preparation was detected by SDS-PAGE gel electrophoresis, and MPCO retained the integrity characteristics of 4T1 cell membrane protein ( Figure 8 The stability of the particle size and potential of PCO and MPCO in PBS was investigated. The results showed that both PCO and MPCO could remain stable within 7 days ( Fig. 9 ).

[0082] The release behavior of MPCO was studied by simulating different physiological environments through dialysis. Fig.10). PBS at pH 7.4 was used to simulate the normal body fluid environment, while PBS at pH 5.0 containing 10 mM GSH was used to simulate the acidic and high GSH conditions in tumor cells. As shown in the release curve, free Ola was almost completely released in the first 6 hours. However, at pH 7.4, MPCO nanoparticles released less than 20% of Ola within 2 days, indicating that it can maintain its integrity during body fluid transport and the drug will not leak before reaching the tumor site. In sharp contrast, when MPCO was incubated under acidic and high GSH conditions, the release of Ola was greater than 50%. The reason may be that the disulfide bond in cysteine ​​of MPCO nanoparticles is broken under high glutathione conditions, achieving smart response to tumor microenvironment release.

[0083] Flow cytometry was used to observe the cellular uptake of free Ce6 and MPCO nanoparticles ( Fig.11 ). The results showed that at 1 h, the mean fluorescence intensity of the MPCO group was nearly an order of magnitude higher than that of the Ce6 group. At 4 h, the MPCO nanoparticles were taken up the most. The study showed that 4T1 cell membrane-modified nanoparticles can be homologously targeted by cell surface interactions mediated by multiple molecules, including tumor-specific binding proteins such as TF antigen and E-cadherin. Possibly due to the homing effect mediated by the tumor cell membrane, MPCO nanoparticles significantly enhanced the drug uptake ability of cells.

[0084] CCK-8 assay was used to evaluate cytotoxicity ( Fig.12 ). At the same Ola concentration, free Ola alone had little cytotoxicity due to the absence of the initial event of ROS damaging DNA. The four preparations in the non-laser treated group had high biocompatibility, and their cytotoxicity increased significantly after laser irradiation. PC(+) achieved a greater tumor killing effect than the Ce6(+) group, which may be due to the improved dispersibility and cellular uptake of Ce6 using the PGA conjugate. IC 50 They are 0.4898 μg / mL and 0.4736 μg / mL respectively.

[0085] An orthotopic breast cancer tumor model was established by injecting 4T1 tumor cells into the fat pad of BALB / c mice, and the biodistribution of free drug and nanoformulation was evaluated by IVIS imaging ( Fig.13). After intravenous injection of free Ce6, PCO or MPCO nanoparticles, free Ce6 was rapidly metabolized and there was almost no fluorescence signal at 12 hours. However, the MPCO nanoparticle group still had higher tumor accumulation at 12 hours, which was attributed to the long blood circulation and tumor homing mechanism mediated by the tumor cell membrane. Based on the above results, in subsequent animal experiments, laser irradiation was selected 12 hours after administration to obtain the best PDT effect. After 24 hours, the mice were killed and the main organs and tumors were collected to check the fluorescence intensity. The results of in vitro organ fluorescence imaging were consistent with the results of in vivo imaging, and MPCO nanoparticles achieved the strongest tumor area enrichment.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer, characterized in that: include: Polyglutamic acid conjugated photosensitizer; The polyglutamic acid conjugated photosensitizer is loaded with a hydrophobic chemotherapeutic drug; The polyglutamic acid conjugated photosensitizer is coated with a tumor cell membrane; The preparation method of the polyglutamic acid conjugated photosensitizer is as follows: connecting the photosensitizer to polyglutamic acid via covalent bond coupling; The photosensitizer is dihydrochlorin e6; The hydrophobic chemotherapy drug is Olaparib; The preparation method of the polyglutamic acid conjugated photosensitizer comprises: Dissolve polyglutamic acid with a molecular weight of 5000-200000 in a solution, add EDC∙HCl and NHS for activation, then add cystamine dihydrochloride, react at room temperature, and after the reaction is completed, dialyze and freeze-dry to obtain a polyglutamic acid-cystamine polymer; The photosensitizer is dissolved in a solution, EDC∙HCl and NHS are added for activation, and then the polyglutamic acid-cystamine polymer is added for reaction. After the reaction is completed, the polymer is dialyzed and freeze-dried to obtain the product.

2. The tumor-targeting nanoformulation based on polyglutamic acid conjugated photosensitizer according to claim 1, characterized in that: The tumor is at least one of melanoma, colon cancer, lung cancer, stomach cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, liver cancer, head and neck cancer, lymphoma, sarcoma, chronic lymphocytic leukemia, thyroid cancer, testicular cancer, and anal cancer.

3. A method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer as claimed in claim 1, characterized in that: include: The photosensitizer is coupled to polyglutamic acid via a covalent bond to obtain a polyglutamic acid conjugated photosensitizer; The hydrophobic chemotherapeutic drug is encapsulated in the polyglutamic acid conjugated photosensitizer by means of hydrophobic interaction, electrostatic adsorption, and π-π conjugation to obtain a nano preparation; The nano preparation is coated with the cell membrane of tumor cells to obtain the nano preparation.

4. The method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer according to claim 3, characterized in that: The preparation method of the polyglutamic acid conjugated photosensitizer comprises: Dissolve polyglutamic acid with a molecular weight of 5000-200000 in a solution, add EDC∙HCl and NHS for activation, then add cystamine dihydrochloride, react at room temperature, and after the reaction is completed, dialyze and freeze-dry to obtain a polyglutamic acid-cystamine polymer; The photosensitizer is dissolved in a solution, EDC∙HCl and NHS are added for activation, and then the polyglutamic acid-cystamine polymer is added for reaction. After the reaction is completed, the polymer is dialyzed and freeze-dried to obtain the product.

5. The method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer according to claim 3, characterized in that: The preparation method of the nano preparation comprises: dissolving polyglutamic acid conjugated photosensitizer and olaparib in a solution, mixing evenly, rotary evaporating to obtain a film, then injecting PBS for hydration, ultrasonically dispersing, and removing unencapsulated olaparib with a water filter membrane to obtain the nano preparation.

6. The method for preparing a tumor-targeting nanoformulation based on a polyglutamic acid conjugated photosensitizer according to claim 3, characterized in that: The specific steps of cell membrane coating include: adding tumor cells to a lysis solution and repeatedly freezing and thawing for 5-10 times, and completely breaking the cells by probe ultrasound for 20-40 minutes; after low-temperature and low-speed centrifugation, taking the supernatant, and then centrifuging at a high speed to collect the precipitate, thus obtaining tumor cell membrane fragments; The tumor cell membrane and the nanoparticle preparation are mixed and sonicated, and then squeezed out through a polycarbonate membrane filter for 10-30 times to obtain the mixture.

Images