Tumor targeted drug delivery system and drug packaging method thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a tumor targeted drug delivery system and a drug packaging method thereof. The tumor targeted drug delivery system comprises the following components: spermidine, a glutathione inhibitor, polyamine oxidase, mesoporous silica and a target molecule, the drug delivery system intensively exerts the synergistic effect of PAOX, SPD and BSO, tumor cells are effectively killed, and the fatality rate reaches 95% or above. In animal experiments, the compound shows remarkable anti-tumor activity and good lipid peroxidation activity and DNA damage activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a tumor-targeted drug delivery system and a method for packaging drugs thereof. Background Art

[0002] Cancer is currently the second leading cause of death globally. Early diagnosis and finding appropriate treatment options are of great significance for improving the survival rate and prognosis of patients. The treatment methods for malignant tumors mainly include four parts, namely surgical operation, radiotherapy, chemotherapy with chemical drugs, and molecular targeted immunotherapy. Among them, chemotherapy is a systemic treatment. The drug is delivered throughout the body through the vein and concentrated in the tumor tissue for killing. However, due to the lack of selectivity and drug resistance, its efficacy and compliance are reduced. Targeted therapy is currently recognized as the most prospective treatment method. It can inhibit cell growth by directly changing the cell signal pathway, or use targeted biological agents to indirectly deliver anticancer drugs to tumor cells and their microenvironment through the target molecules of the cells to inhibit or kill tumor cells. Since it can specifically target tumor cells and their microenvironment, while greatly improving the treatment effect, it significantly reduces the side effects of broad-spectrum anticancer drugs. Therefore, it is of great significance to find new methods.

[0003] Rationally utilizing substances in tumors and the tumor microenvironment for tumor treatment is an effective solution. The synthesis and expression of polyamine substances in tumors are abnormally expressed and play an important role in the development of cancer [1-5]. Polyamines carry positive charges themselves and interact with negatively charged protein macromolecules and nucleic acids in cells to produce cytotoxicity. Moreover, reactive oxygen species can also cause oxidative stress and cell damage. However, the cytotoxicity of both is much less than that of acrolein. Acrolein is the largest endogenous aldehyde in the body and has strong cytotoxicity. It mainly comes from the oxidative metabolism and decomposition of spermine. Micromolar levels of acrolein can achieve a median lethal effect. Using the polyamine oxidation metabolite acrolein to kill tumor cells may have a more significant effect. However, oxidative metabolism occurs in all cells, but it cannot fully exert the effect of killing tumor cells because acrolein will be depleted by glutathione in the body. Based on this, Patent CN118320106A discloses a scheme of using the combination of spermidine, polyamine oxidase, and glutathione synthase inhibitor to kill tumors. However, whether the three drugs are administered sequentially or simultaneously, they cannot achieve precise killing. Moreover, in actual animal experiments, due to the differences in the onset time and local concentration of different drugs in the body, the treatment effect is limited, lacking precision, and the drug utilization rate is low. Therefore, there is an urgent need to develop a drug delivery system that integrates three effective substances and performs accurate delivery to achieve precise treatment.

[0004] [1] Šebela, M.,&Rašková, M. (2023). Polyamine-derived aminoaldehydesand acrolein: cytotoxicity, reactivity and analysis of theinduced proteinmodifications. Molecules (Basel, Switzerland), 28(21), 7429. [2] Holbert, C. E., Cullen, M. T., Casero, R. A., Jr,&Stewart, T. M.(2022). Polyamines in cancer: integrating organismal metabolism andantitumour immunity. Nature reviews. Cancer, 22(8), 467–480. [3] Casero, R. A., Jr,Murray Stewart, T.,&Pegg, A. E. (2018).Polyamine metabolism and cancer: treatments, challenges and opportunities.Naturereviews. Cancer, 18(11), 681–695. [4] Xuan, M., Gu, X.,Li, J., Huang, D., Xue, C.,&He, Y. (2023).Polyamines: their significance for maintaining health and contributing todiseases.Cell communication and signaling: CCS, 21(1), 348. Pegg A. E. (2013). Toxicity of polyamines and their metabolicproducts. Chemical research in toxicology,26(12), 1782–1800. Summary of the Invention

[0005] To solve the above technical problems and find effective methods for treating prostate cancer and bladder cancer, the present invention provides a packaging method for a combined drug for treating prostate cancer and bladder cancer, and a treatment plan for combining spermidine (SPD), polyamine oxidase (PAOX), and glutathione inhibitor (BSO) to treat tumors. To achieve drug delivery, it is necessary to construct a suitable drug delivery system. Mesoporous silica (MSN) has the advantages of good biocompatibility, degradability, and good tolerance. It can combine multiple molecules on a multifunctional platform for personalized diagnosis and treatment. MSN can be modified in various ways, such as aldehyde group modification and amino group substitution. The present invention uses MSN and MSN-NH2 (MSN+) to package SPD, PAOX, and BSO, and has verified it in prostate cancer and bladder cancer.

[0006] The present invention is based on the idea of using PAOX to metabolize SPD derivatives to produce acrolein, and combining the use of BSO to inhibit the clearance of acrolein to achieve the accumulation of acrolein, thereby killing tumor cells. First, construct MSN and MSN+ drug delivery systems for the combined drugs SPD, PAOX, and BSO. PAOX is negatively charged in solution, and the positively charged modification of MSN can increase the adsorption of PAOX, which helps to exert the therapeutic effect. To achieve the precise delivery of drugs to bladder cancer and prostate cancer cells, based on the above factors, the technical solution of the present invention is as follows: Polyamine oxidase PAOX oxidizes the acetylated product of spermidine to acrolein, which can effectively kill cells. However, due to the detoxification effect of glutathione on acrolein, the effect cannot be fully exerted. When the three act alone, there is drug dispersion and the precise treatment effect cannot be fully exerted. To concentrate the combined effect of the drugs, a method of using MSN and MSN+ to package PAOX, BSO, and SPD to treat bladder cancer and prostate cancer respectively is designed.

[0007] Specifically, the technical solution of the present invention is as follows: A tumor-targeted drug delivery system named BPS@MSN@T, whose components include: SPD, BSO, PAOX, mesoporous silica, and a target molecule (T); The specific structure of BPS@MSN@T is: the mesoporous silica coats BSO and SPD to form BS@MSN, and the surface of BS@MSN is coated in turn from the inside to the outside with: a) A PAOX layer; b) A target molecule layer.

[0008] Furthermore, the present invention also provides a tumor-targeted drug delivery system based on positively charged modified MSN, named BPS@MSN+@T. The specific structure is: MSN+ coats BSO to form B@MSN+, and the surface of B@MSN+ is coated in turn from the inside to the outside with: a) PAOX layer; b) SPD and target molecule mixed layer.

[0009] The PAOX layer, target molecule layer, SPD and target molecule mixed layer respectively refer to the coating hierarchical structures formed by mixing and grafting the corresponding substances (PAOX, target molecule, SPD and target molecule mixture) with intermediate nanoparticles (BS@MSN, B@MSN+).

[0010] Furthermore, the target molecule includes but is not limited to any one of the substances targeting tumor cells, preferably any one of the protein or polypeptide molecules of bladder cancer or adenocarcinoma, including but not limited to one of the PSMA target molecule and Bld target molecule. Preferably, the PSMA target molecule is PSMA-1, and the Bld target molecule is Bld-1.

[0011] The present invention provides a drug packaging method for the above-mentioned tumor-targeted drug delivery system. The steps of BPS@MSN@T include: Using mesoporous silica to electrostatically adsorb and package BSO and SPD to form BS@MSN. The BS@MSN is mixed with PAOX to form a PAOX layer on its surface, obtaining BPS@MSN, and then combining Bld-1 by the grafting method to obtain BPS@MSN@T.

[0012] The steps of BPS@MSN+@T include: Using MSN-NH2 to electrostatically adsorb and package BSO to form B@MSN+. The B@MSN+ is mixed with PAOX to form a PAOX layer on its surface, obtaining BP@MSN+, and then combining the mixture of SPD and PSMA-1 by the grafting method to obtain BPS@MSN+@T.

[0013] Furthermore, the mass ratio of the mesoporous silica, BSO, PAOX, SPD and target molecule is 100 - 300:5 - 20:10 - 40:10 - 20:1. Preferably, the mass ratio is 200:10:30:15:1.

[0014] Furthermore, the mass ratio of the MSN+, BSO, PAOX, SPD and target molecule is 100 - 300:5 - 15:10 - 20:10 - 15:1. Preferably, the mass ratio is 200:5:30:12.8:1.

[0015] Furthermore, the usage concentration ratio of the MSN+ to SPD, PAOX, BSO and PSMA-1 is 20:5:30:12.8:1, and the usage concentration ratio of the MSN to SPD, PAOX, BSO and Bld-1 is 20:10:30:15:1.

[0016] Furthermore, the particle size of the tumor-targeted drug delivery system is 100-200 nm.

[0017] The potential of the MSN+, namely MSN-NH2, is 7-8 mV.

[0018] On the other hand, the present invention also provides the use of the above-mentioned tumor-targeted drug delivery system in the preparation of drugs for treating bladder cancer and adenocarcinoma of the prostate. Specifically, the tumor-targeted drug delivery system is prepared into a pharmaceutical composition for treating bladder cancer and adenocarcinoma of the prostate, and the pharmaceutical composition further includes optional pharmaceutically acceptable excipients.

[0019] The treatment of prostate cancer includes inhibiting prostate cancer cells, and the prostate cancer cells include PC3 and PC3-PSMA; the treatment of bladder cancer includes inhibiting bladder cancer cells, and the bladder cancer cells include SW 780 and T24.

[0020] The present invention also provides a method for administering a tumor-targeted drug delivery system. The steps include: Sequentially administering PAOX encapsulated in mesoporous silica (MSN@PAOX), spermidine, and glutathione synthetase inhibitor; Or, sequentially administering PAOX and SPD (SP@MSN) encapsulated in mesoporous silica and glutathione synthetase inhibitor.

[0021] Furthermore, the administration method further includes administering PAOX encapsulated in mesoporous silica and BSO, and separately administering spermidine. The administration methods include common administration methods for liquid / solid drugs such as injection and oral administration.

[0022] As used herein, "treatment" refers to any manner that can be beneficial to humans or non-human animals. Treatment can be directed at existing conditions, including therapeutic effects, alleviating or preventive effects, and these effects are directed at bladder cancer or prostate cancer-related diseases and include their complications. Specifically, the treatment includes blocking, inhibiting, reducing, or preventing the growth, migration, and metastasis of bladder cancer or prostate cancer cells to any extent.

[0023] The "pharmaceutically acceptable excipients" used in the present invention include one or more of any and all physiologically applicable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents or absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc. and combinations thereof. In many cases, it is preferable to include an isotonic agent in the composition, for example, one or more of sugars, polyols such as mannitol, sorbitol, sorbitan or sodium chloride, etc. Pharmaceutically acceptable excipients may also contain small amounts of auxiliary substances, such as one or more of wetting agents or emulsifying agents, preservatives or buffers, etc., which enhance the shelf life or potency of the pharmaceutical preparation.

[0024] The beneficial effects of the present invention are as follows: It provides a drug delivery system for effectively treating prostate cancer and bladder cancer, which can accurately deliver to bladder cancer and prostate cancer cells and centrally exert the synergistic effects of PAOX, SPD and BSO. Specifically, BPS@MSN+@T targets prostate cancer cells through PSMA-1 and releases the packaged PAOX, SPD and BSO to jointly exert the effect of killing tumor cells; BPS@MSN@T targets prostate cancer cells through Bld-1 and releases the packaged PAOX, SPD and BSO to jointly exert the effect of killing tumor cells. Importantly, under this drug packaging combination scheme, the ratio of drug usage to treatment effect is greatly reduced, significantly reducing the drug cost.

[0025] The drug delivery system constructed by MSN and MSN+ of the present invention containing BSO, SPD and PAOX effectively kills tumor cells, and the lethality rate reaches more than 95%. It shows significant anti-tumor activity and good lipid peroxidation activity and DNA damage activity in animal experiments.

[0026] The drug delivery system constructed by MSN and MSN+ of the present invention containing BSO, SPD and PAOX can induce apoptosis and trigger immunogenic cell death. Description of the Drawings

[0027] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0028] Figure 1 For the nanoparticle size and potential of the drug delivery system constructed by MSN+; Figure 2 For the nanoparticle size and potential of the drug delivery system constructed by MSN; Figure 3 For the release of SPD, BSO and PAOX of the drug delivery system constructed by MSN+; Figure 4 Release profiles of drug delivery systems SPD, BSO, and PAOX constructed for MSN; Figure 5 Effect of the drug delivery system constructed for MSN+ on the viability of prostate cancer cells; Figure 6 Effect of the drug delivery system constructed for MSN on the viability of bladder cancer cells; Figure 7 In vitro targeting of the drug delivery system constructed for MSN+ to prostate cancer cells; Figure 8 In vitro targeting of the drug delivery system constructed for MSN to bladder cancer cells; Figure 9 In vivo targeting of the drug delivery system constructed for MSN+ to prostate cancer; Figure 10 In vivo targeting of the drug delivery system constructed for MSN to bladder cancer; Figure 11 Therapeutic effect of the drug delivery system constructed for MSN+ on prostate cancer; Figure 12 Therapeutic effect of the drug delivery system constructed for MSN on bladder cancer; Figure 13 Drug delivery system-induced lipid peroxidation; Figure 14 Drug delivery system-induced DNA damage; Figure 15 Drug delivery system-induced apoptosis; Figure 16 Drug delivery system-induced immunogenic cell death. Detailed implementation manners

[0029] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0030] Prostate cancers treatable by the methods described in the present invention include, but are not limited to, metastatic prostate cancer, or prostate cancer refractory to standard therapies or recurring after standard therapies. In some embodiments, the prostate cancer is anti-androgen prostate cancer. In some embodiments, the prostate cancer is drug-resistant prostate cancer. In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the treatment is first-line treatment. In some embodiments, the treatment is second-line treatment. Bladder cancers treatable by the methods described in the present invention include, but are not limited to, invasive bladder cancer, non-invasive bladder cancer, or bladder cancer refractory to standard therapies or recurring after standard therapies. In some embodiments, the bladder cancer is drug-resistant bladder cancer. In some embodiments, the bladder cancer is metastatic bladder cancer. In some embodiments, the bladder cancer is invasive bladder cancer. In some embodiments, the treatment is first-line treatment. In some embodiments, the treatment is second-line treatment.

[0031] In some embodiments, the present invention uses MSN packaging in combination with drugs to obtain BPS@MSN@T. SPD, PAOX, and BSO will be uniformly and centrally provided, and multiple doses can be provided over a period of time. The dose will be calculated in the form of the use concentration of PAOX, such that the dose will be 10 nM, 30 nM, 100 nM, 1 μM, 100 μM or higher, depending on the treatment effect. In additional embodiments, the administration will be carried out at a ratio of the total mass of SPD, PAOX, and BSO to the mass of the individual being treated. The proportions of SPD, PAOX, BSO, and PSMA-1 packaged in BPS@MSN+@T can be allocated differently for different individual situations, including but not limited to concentration ratios or mass ratios such as 4 - 10:4 - 10:1 - 5, 3 - 20:4 - 20:1 - 5, etc.

[0032] For the total dose of BPS@MSN@T or BPS@MSN+@T, it can be 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg or higher, depending on the treatment effect. In embodiments of multiple administrations, the administration schedule can be 1 dose / day, 2 doses / day, 3 doses / day or more, and can continue for the necessary time such that the administration can continue for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, or permanently during the lifetime of the individual.

[0033] It should be noted that for the sake of easy understanding, the drug delivery system of the present invention, also referred to as "series of nanoparticles constructed by MSN+", "series of nanoparticles constructed by MSN", corresponding to "BPS@MSN+@T nanoparticles", "BPS@MSN@T nanoparticles", corresponding to BPS@MSN+@T, BPS@MSN@T.

[0034] In some embodiments of the present invention, a number of experiments were carried out to characterize the MSN-packaged drug system, including drug release rate, nanoparticle potential, targeting to bladder cancer and prostate cancer cells, etc., and the effect of the packaged nanoparticles on cell viability at the cellular level. The experiments are as follows: Example 1 Experimental materials: Human prostate cancer cell line PC3, recombinant human overexpressing PSMA plasmid PC3-PSMA; human bladder cancer cell lines SW780 and T24; MTT reagent was purchased from Solarbio, and the mother liquor preparation and reagent addition amount were carried out according to the product instructions. Glutathione synthetase inhibitor BSO, spermidine, cell culture medium, and serum were purchased from Shandong Sikejie Biotechnology Co., Ltd. Polyamine oxidase (PAOX) was purchased from Cloud-Clone Crop. Lentivirus PSMA was purchased from Vigene Biotechnology Co., Ltd., and the reagent operation was carried out according to the product instructions. MSN and MSN+ were customized by Xianfeng Nano Co., Ltd.

[0035] Cell culture: Human prostate cancer cell line PC3 and recombinant human overexpressing PSMA plasmid PC3-PSMA were cultured in 1640 medium containing 10% FBS, and human bladder cancer cell lines SW780 and T24 were cultured in 1640 medium containing 10% FBS.

[0036] Experimental methods: Packaging process of BPS@MSN@T and BPS@MSN+@T nanoparticles Preparation process of BPS@MSN+@T: 1) 2 mg of MSN+ and 128 μg of BSO were reacted in a 200 μl PBS system at room temperature for 4 h, and centrifuged at 3000 r / min to obtain B@MSN+. 2) The B@MSN+ obtained in 1) was reacted with 300 μg of PAOX in a 200 μl PBS system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain BP@MSN+. 3) The BP@MSN+ obtained in 2) was reacted with 10 μg of PSMA-1, 50 μg of SPD, and 2 mg of 8-PEG-CHO in a 200 μl system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain BPS@MSN+@T. And BP@MSN+ was reacted with 50 μg of SPD and 2 mg of 8-PEG-CHO in a 200 μl system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain BPS@MSN+. 4) 2 mg of MSN-NH2 was reacted with 300 μg of PAOX in a 200 μl PBS system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain P@MSN+. Then 2 mg of 8-PEG-CHO and 50 μg of SPD were added and reacted in a 200 μl system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain PS@MSN+.

[0037] Preparation process of BPS@MSN@T: 1) 2 mg of MSN, 150 μg of BSO, and 100 μg of SPD were reacted in a 200 μl PBS system at room temperature for 4 h, and centrifuged at 3000 r / min to obtain BS@MSN. 2) The BS@MSN obtained in 1) was reacted with 300 μg of PAOX in a 200 μl PBS system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain BPS@MSN. 3) The BPS@MSN obtained in 2) was reacted with 10 μg of Bld-1 and 2 mg of 8-PEG-CHO in a 200 μl system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain BPS@MSN@T. 4) 2 mg of MSN was reacted with 300 μg of PAOX in a 200 μl PBS system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain P@MSN. Then 2 mg of 8-PEG-CHO and 50 μg of SPD were added and reacted in a 200 μl system at 4 °C for 4 h, and centrifuged at 3000 r / min to obtain PS@MSN.

[0038] Example 2 Packaging sequence verification experiment: 1. For MSN particles, in the following way: 1) BSO and SPD are first adsorbed onto MSN, and then PAOX is adsorbed. The encapsulation efficiencies of BSO, SPD, and PAOX are 85.67% ± 5.61%, 82.43% ± 3.23%, and 95.71% ± 4.21% respectively.

[0039] 2) PAOX is first adsorbed, and then BSO and SPD are adsorbed. The encapsulation efficiencies of BSO, SPD, and PAOX are 57.21% ± 2.32%, 89.34% ± 1.32%, and 90.43% ± 2.45% respectively.

[0040] Among them, the adsorption rate of BSO in 2) decreases. Therefore, the method in 1) is finally selected for packaging.

[0041] 2. For MSN+ particles, both BSO and SPD can be connected to MSN+ particles by grafting. Due to the different positions of the amino groups in the molecular formulas of SPD and BSO, the grafting rate of SPD is higher, reaching over 80%, and that of BSO is over 70%. Therefore, finally, MSN+ electrostatically adsorbs BSO and PAOX, and the grafting method is used to connect SPD and PSMA-1.

[0042] For the adsorption sequence of BSO and PAOX, in the following way: 1) BSO is first adsorbed, and then PAOX is adsorbed, and SPD is connected by the grafting method. The encapsulation efficiencies of BSO, PAOX, and SPD are 78.32% ± 1.34%, 97% ± 1.23%, and 80% ± 2.22% respectively.

[0043] 2) PAOX is first adsorbed, then BSO is adsorbed, and then SPD is connected by grafting. The encapsulation efficiencies of BSO, PAOX, and SPD are 84.56% ± 1.34%, 97.27% ± 3.21%, and 50.45% ± 1.24% respectively.

[0044] Among them, adsorbing PAOX first in 2) will cause the decrease of the encapsulation rate of SPD. Therefore, the method in 1) is finally selected for packaging.

[0045] Example 3 Particle size and zeta potential of BPS@MSN@T and BPS@MSN+@T nanoparticles The series of drug nanoparticles obtained in Example 1 are diluted with PBS to 1 mg / ml, and the particle size and zeta potential are measured by a nanoparticle size and zeta potential analyzer.

[0046] The results are as Figure 1, as shown in Figure 2: The potentials of the series of nanoparticles constructed by MSN+ are as follows: MSN+ is 7.58 ± 0.33 mv, B@MSN+ is 8.61 ± 0.31 mV, PS@MSN+ is -11.53 ± 0.83 mV, BPS@MSN+ is -9.84 ± 0.39 mV, BPS@MSN+@T is -8.17 ± 0.55 mV. The potentials of the series of nanoparticles constructed by MSN are as follows: MSN is 0.44 ± 0.04 mV, B@MSN is 1.00 ± 0.28 mV, PS@MSN is -9.98 ± 0.79 mV, BPS@MSN is -8.44 ± 0.55 mV, BPS@MSN@T is -6.97 ± 0.46 mV. The particle sizes of the series of nanoparticles constructed by MSN+ are as follows: MSN+ is 114.33 ± 12.66 nm, B@MSN+ is 122.67 ± 10.07 nm, PS@MSN+ is 163.67 ± 6.66 nm, BPS@MSN+ is 163.00 ± 5.00 nm, BPS@MSN+@T is 184.33 ± 5.67 nm. The particle sizes of the series of nanoparticles constructed by MSN are as follows: MSN is 96.00 ± 6.25nm, B@MSN is 103.60 ± 6.08 nm, PS@MSN is 118.67 ± 5.13 nm, BPS@MSN is 155.33 ± 11.06 nm, BPS@MSN@T is 185.67 ± 9.07 nm.

[0047] Example 4 Release of SPD, BSO, and PAOX from BPS@MSN@T and BPS@MSN+@T nanoparticles Under full wavelength scanning, SPD and BSO have a maximum absorption peak at around 200 nm. At a certain concentration, the absorbance value is proportional to the concentration, enabling quantitative analysis.

[0048] The results are as Figure 3 , as shown in Figure 4, for the BPS@MSN+@T nanoparticles containing PAOX, SPD, BSO, and PSMA-1 constructed by MSN+, PAOX can be rapidly released, reaching 80% in 8 hours. BSO is released to 80% within 4 hours. SPD has a faster release rate under acidic conditions and is basically not released under neutral conditions. The BPS@MSN@T nanoparticles constructed by MSN have similar results (±3%).

[0049] Example 5 Effect of BPS@MSN@T and BPS@MSN+@T nanoparticles on the viability of cancer cells The MTT assay was used to verify the effects of BPS@MSN+@T nanoparticles on the viability of prostate cancer cells and BPS@MSN@T nanoparticles on bladder cancer cells. Cells were seeded in 96-well plates at a density of 5000 cells / well and cultured overnight in appropriate cell culture medium. The cells were then changed to 100 μl / well of fresh medium, and different concentrations of BPS@MSN@T or BPS@MSN+@T (0 - 2.0 mM) were added. Calculated based on the PAOX concentration, after continued culture for 24 h, 100 μl of fresh medium containing MTT at a final concentration of 0.5 mg / ml was added to each well, and the culture was continued in an incubator at 37 °C for 4 h. The medium was gently aspirated, 100 μl / well of DMSO was added and reacted for 15 min, and the absorbance at OD490nm was measured using a microplate reader. The group without spermidine treatment was used as a control, and the data were expressed as a percentage relative to the control group. Five parallels were set for each group.

[0050] The results are as Figure 5 and 6 shown. In both groups, when the concentration of PAOX reached 90 μg / ml, the cell killing effect reached more than 95%. When the median lethal dose of 12 μg / ml was taken to verify the viability of cells with different drug delivery systems, at this time, the concentration of BSO in the drug delivery system was 22 - 27 μM, and the concentration of SPD was 10 - 21 μM. The combined effect of the three reached more than 95%, and the single use had only a 20% inhibitory effect, indicating that the drug delivery system could effectively play a role in cells and kill tumor cells. And compared with the previous patent, by using the MSN delivery method, PAOX could be quantified, making the killing method more stable. Secondly, the usage amounts of BSO and SPD were significantly reduced, and it could ensure that the three played a role in the same cell, giving full play to the killing advantage to a greater extent.

[0051] Example 6 In vitro targeting of BPS@MSN@T and BPS@MSN+@T nanoparticles to bladder and prostate cancer cells The in vitro targeting of PSMA-1 and Bld-1 was detected by flow cytometry. For easy fluorescence detection, first, BPS@MSN@T and BPS@MSN+@T and BPS@MSN and BPS@MSN+ without targeting peptides were labeled with FITC. FITC was purchased from Cisco Jie Biotechnology Co., Ltd., and the operation was carried out according to the instructions. Cells were seeded in 6-well plates. When the cells grew to 80%, they were digested with trypsin without EDTA, washed once with PBS, resuspended in 200 μl of PBS, 5 μg of FITC-labeled nanoparticles were added, and incubated for 10 min, 30 min, and 2 h respectively. After washing twice with PBS for 3 min each time, flow cytometry was performed.

[0052] The results are as Figure 7 , shown in Figure 8. The nanoparticles constructed by NSN and MSN+ both showed a significantly enhanced fluorescence intensity containing the targeting peptide. Incubation for only 10 min could promote the rapid binding of the nanoparticles to the cells, and there was still a strong fluorescence intensity at 2 h. It indicates that the addition of the targeting peptide can significantly increase the targeting of the nanodelivery system and promote rapid localization.

[0053] Example 7 In vivo targeting of BPS@MSN@T and BPS@MSN+@T nanoparticles to bladder cancer and prostate cancer cells Animal experiments were used to verify the in vivo targeting of the nanodelivery system. First, BPS@MSN@T, BPS@MSN+@T, BPS@MSN, and BPS@MSN+ without target molecules were labeled with FITC. FITC was purchased from Cisco Jie Biotechnology Co., Ltd., and the operation was carried out according to the instructions. Female C57BL / 6J mice aged 6 - 8 weeks and male C57BL / 6J mice aged 6 - 8 weeks were respectively selected for the bladder cancer and prostate cancer models. Mouse-derived bladder cancer cells MB49 were inoculated into the mouse bladder at 1×105 to construct an orthotopic model, and mouse-derived prostate cancer cells RM-1 were inoculated into the mouse prostate at 1×105 to construct an orthotopic model. Two weeks later, the bladder cancer model was perfused into the bladder in situ, and the drug delivery system was injected into the prostate cancer model via the tail vein. The fluorescence intensity was observed using a small animal in vivo imaging system, and the tumors and important organs were dissected to observe the binding of BPS@MSN@T and BPS@MSN+@T.

[0054] The results are as Figure 9 , shown in Figure 10. In the mouse prostate cancer model, after injecting the drug delivery system containing the target molecule, the fluorescence intensity was concentrated in the abdominal tumor site. After dissection, the fluorescence intensity of the tumor part was strong, and there was also a small amount of binding in the liver, indicating that the target molecule can promote in vivo targeting. In the bladder cancer model, after perfusing the drug delivery system containing the target molecule, fluorescent spots appeared on the abdomen of the mice, indicating that increasing the target molecule can significantly increase the targeting of the cells. After dissecting the tumors and important organs, the fluorescence was only concentrated in the tumor part and did not bind to other internal organs, indicating that this drug delivery system has good biosafety.

[0055] Example 8 Therapeutic effects of BPS@MSN@T and BPS@MSN+@T nanoparticles on bladder cancer and prostate cancer. In this example and the following examples, the target molecule is expressed as "Pep". In the group using MSN+, the target molecule is PSMA-1, and in the group using MSN, the target molecule is Bld-1.

[0056] Mice were used to establish orthotopic models of bladder cancer and prostate cancer respectively according to Example 6. The mice were administered drugs according to the grouping. According to the differences in the treatment methods of prostate cancer and bladder cancer, the prostate cancer groups included (i) negative control group, (ii) P@MSN+@Pep group, (iii) BP@MSN+@Pep group, (iv) PS@MSN+@Pep group, and (v) BPS@MSN+@Pep group. The bladder cancer groups included (i) negative control group, (ii) B@MSN group, (iii) PS@MSN group, (iv) BPS@MSN group, and (v) BPS@MSN@Pep group. Six mice were randomly assigned to each group and the above drugs were injected via the tail vein, twice a week for three weeks. During the drug treatment process, the weight of the mice was measured every other day, and the fluorescence intensity was detected by small animal in vivo imaging every other week until the end of the experiment.

[0057] The results were as Figure 11 , shown in Figure 12, the combined use of BSO, SPD and PAOX had a significant effect, and the tumor volume was significantly reduced. It indicated that the drug delivery system had good in vivo anti-tumor activity.

[0058] Example 9 BPS@MSN@T and BPS@MSN+@T had lipid peroxidation activity.

[0059] Cells were seeded in six-well plates at a density of 50,000 cells / well and cultured overnight in appropriate cell culture medium. The cells were changed to 100 µl / well of fresh medium, and the prostate cancer cells were added with nanoparticles according to the grouping: (i) negative control group, (ii) P@MSN+@Pep group, (iii) BP@MSN+@Pep group, (iv) PS@MSN+@Pep group, (v) BPS@MSN+@Pep group. The bladder cancer groups included (i) negative control group, (ii) B@MSN group, (iii) PS@MSN group, (iv) BPS@MSN group, (v) BPS@MSN@Pep group. After 24 hours, the cells were collected, and DCFH-DA was added at a final concentration of 5 uM, and the fluorescence intensity was detected by flow cytometry. After repeating the above treatment, the cells were collected, lysed with lysis buffer, protein samples were prepared by adding loading buffer, 10% SDS-PAGE was used, PVDF membrane transfer was carried out, and WB experiments were performed. The primary antibodies were incubated with GPX4 and the ferroptosis-related Ferritin-1 antibody, and the secondary antibody was incubated with horseradish peroxidase-labeled goat anti-rabbit antibody, and detected after incubation with chemiluminescence solution.

[0060] The results were as Figure 13As shown, under the combined action of BSO, SPD, and PAOX, the content of DCHF-DA in cells increased, and the expression levels of GPX4 and Ferritin-1 decreased, indicating that the drug delivery system in this combination method can cause lipid peroxidation, thereby triggering ferroptosis.

[0061] Example 10 BPS@MSN@T and BPS@MSN+@T have DNA damage activity.

[0062] Cells were seeded in six-well plates at a density of 50,000 cells / well and cultured overnight in appropriate cell culture medium. The cells were replaced with 100 µl / well of fresh medium, and prostate cancer cells were added with nanoparticles according to the groups: i) negative control group, (ii) P@MSN+@Pep group, (iii) BP@MSN+@Pep group, (iv) PS@MSN+@Pep group, (v) BPS@MSN+@Pep group. The bladder cancer groups included i) negative control group, (ii) B@MSN group, (iii) PS@MSN group, (iv) BPS@MSN group, (v) BPS@MSN@Pep group. After 24 hours, the cells were collected, lysed with lysis buffer, and protein samples were prepared by adding loading buffer. 10% SDS-PAGE was used, followed by PVDF membrane transfer, and WB experiments were performed. The primary antibody was incubated with γ-H2AX antibody, and the secondary antibody was incubated with horseradish peroxidase-labeled goat anti-rabbit antibody. Detection was carried out after incubation with chemiluminescent solution.

[0063] The results are as Figure 14 shown, under the combined action of BSO, SPD, and PAOX, the expression level of γ-H2AX in cells increased, indicating that the drug delivery system in this combination method can cause DNA damage.

[0064] Example 11 BPS@MSN@T and BPS@MSN+@T can induce apoptosis.

[0065] Cells were seeded in six-well plates at a density of 50,000 cells per well and cultured overnight in appropriate cell culture medium. The cells were changed to 100 μl / well of fresh medium, and prostate cancer cells were added with nanoparticles according to the groups: i) negative control group, (ii) P@MSN+@Pep group, (iii) BP@MSN+@Pep group, (iv) PS@MSN+@Pep group, (v) BPS@MSN+@Pep group. The bladder cancer groups included i) negative control group, (ii) B@MSN group, (iii) PS@MSN group, (iv) BPS@MSN group, (v) BPS@MSN@Pep group. After 24 hours, the cells were collected, and the apoptosis of the cells was detected according to the cell apoptosis kit. The cell apoptosis kit was purchased from Novoprotein Biotechnology Co., Ltd., and the operation was carried out according to the product instructions.

[0066] The results are as Figure 15 shown that the drug delivery system with the combined action of BSO, SPD and PAOX significantly increased the proportion of apoptotic cells, up to more than 80%.

[0067] Example 12 BPS@MSN@T and BPS@MSN+@T can induce immunogenic cell death.

[0068] Cells were seeded in 24-well plates containing cell slides at a density of 50,000 cells per well and cultured overnight in appropriate cell culture medium. The cells were changed to 100 μl / well of fresh medium, and prostate cancer cells were added with nanoparticles according to the groups: i) negative control group, (ii) P@MSN+@Pep group, (iii) BP@MSN+@Pep group, (iv) PS@MSN+@Pep group, (v) BPS@MSN+@Pep group. The bladder cancer groups included i) negative control group, (ii) B@MSN group, (iii) PS@MSN group, (iv) BPS@MSN group, (v) BPS@MSN@Pep group. After 24 hours, CRT antibody was added, incubated overnight at 4°C, 488-goat anti-rabbit secondary antibody was added, after washing three times with PBS, a mounting medium containing DAPI was added for mounting, and observed under a laser confocal microscope. The above cells were processed according to Example 10, the cells were collected, lysed with lysis buffer, protein samples were prepared by adding loading buffer, 10% SDS-PAGE was used, PVDF membrane transfer was carried out, and WB experiments were carried out. The primary antibody was incubated with HMGB1 antibody, and the secondary antibody was incubated with horseradish peroxidase-labeled goat anti-rabbit antibody, and detected after incubation with chemiluminescence solution. After the cells were processed according to Example 10, the change in ATP content was detected using an ATP detection kit. The ATP kit was purchased from Solarbio Co., Ltd., and the operation was carried out according to the instructions.

[0069] As Figure 16As shown, after the combined use of BSO, SPD and PAOX, the expression level of CRT increases, the expression level of HMGB1 increases, and the ATP content increases, indicating that this combination method can trigger immunogenic cell death.

Claims

1. A tumor-targeted drug delivery system, characterized in that, The components include: spermidine, glutathione inhibitor, polyamine oxidase, mesoporous silica and target molecules; The mesoporous silica encapsulates the glutathione inhibitor and spermidine to form BS@MSN, and the surface of BS@MSN is sequentially coated with: A polyamine oxidase layer; A target molecule layer.

2. The tumor-targeted drug delivery system according to claim 1, characterized in that, The mesoporous silica is positively charged mesoporous silica, and the positively charged mesoporous silica encapsulates the glutathione inhibitor to form B@MSN+, and the surface of B@MSN+ is sequentially coated with: a) A polyamine oxidase layer; b) A mixed layer of spermidine and target molecules.

3. The tumor-targeted drug delivery system according to claim 1 or 2, characterized in that, The target molecule is a protein or polypeptide molecule targeting bladder cancer or prostate cancer, including one of the PSMA target molecule and the Bld target molecule.

4. A method for packaging a drug of the tumor-targeted drug delivery system according to claim 1 or 2, characterized in that the steps It includes: Using mesoporous silica to electrostatically adsorb and package the glutathione inhibitor and spermidine to form BS@MSN, mixing the BS@MSN with polyamine oxidase to form a polyamine oxidase layer on its surface, and then binding the target molecule by grafting method to obtain BPS@MSN@T; Or, using positively charged mesoporous silica to electrostatically adsorb and package the glutathione inhibitor to form B@MSN+, mixing the B@MSN+ with polyamine oxidase to form a polyamine oxidase layer on its surface, and then binding the mixture of spermidine and target molecule by grafting method simultaneously to obtain BPS@MSN+@T.

5. The tumor-targeted drug delivery system according to claim 1, wherein The mass ratio of the mesoporous silica, glutathione inhibitor, polyamine oxidase, spermidine and target molecule is 200:10:30:15:

1.

6. The tumor-targeted drug delivery system according to claim 2, wherein The mass ratio of the positively charged mesoporous silica, glutathione inhibitor, polyamine oxidase, spermidine and target molecule is 200:5:30:12.8:

1.

7. The tumor-targeted drug delivery system according to claim 3, wherein The PSMA target molecule is PSMA-1, and the Bld target molecule is Bld-1.

8. A tumor-targeted drug delivery system according to claim 1 or 2, characterized in that, The particle size of the tumor-targeted drug delivery system is 100 - 200 nm.

9. The tumor-targeted drug delivery system according to claim 2, wherein The positively charged mesoporous silica is MSN-NH2, and the potential of MSN-NH2 is 7 - 8 mv.

10. Use of the tumor-targeted drug delivery system according to claim 1 or 2 in the preparation of drugs for treating bladder cancer and prostate cancer.

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