Anti-tumor intelligent double-crosslinking hydrogel drug delivery system based on purple phosphorus nanosheet as well as preparation method and application of anti-tumor intelligent double-crosslinking hydrogel drug delivery system

Abstract

The invention discloses an anti-tumor intelligent double-crosslinking hydrogel drug delivery system based on a purple phosphorus nanosheet and a preparation method and application thereof, the system comprises a hydrogel carrier, a photodynamic carrier material and a drug, and the hydrogel carrier is a drug delivery carrier formed by crosslinking polydimethyldiguanide with Fe < 3 + > and a crosslinking agent; the photodynamic carrier material is a purple phosphorus nanosheet; the medicine is a medicine with two effects of inhibiting hypoxia-inducible factor-1 alpha and lowering PD-L1 down. According to the invention, the purple phosphorus nanosheet and the polydimethyldiguanide / Fe < 3 + > bi-crosslinking hydrogel are taken as carriers to prepare the tumor microenvironment response type hydrogel drug delivery system with good pH responsiveness, injectability and self-healing property, so that an effective solution is provided for tumor precise drug delivery mediated by a new material, namely the purple phosphorus nanosheet, as a nano-drug carrier; the invention also provides an effective photodynamic therapy strategy for anti-tumor application of the intelligent double-crosslinking hydrogel and precise treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on violet phosphorus nanosheets, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer is a common type of cancer in women and has become the number one killer seriously threatening women's health. At present, breast-conserving surgery, mastectomy, radiotherapy, and chemotherapy are the main clinical strategies for breast cancer treatment, but there are still certain deficiencies in these treatment methods. For example, surgery cannot completely remove tumors, which is likely to cause recurrence and metastasis; while treatment methods such as chemotherapy and radiotherapy are prone to cause many adverse reactions such as myelosuppression, cardiotoxicity, and drug resistance, bringing great troubles to patients. Therefore, there is an urgent need to find a breast cancer treatment strategy with better efficacy, lower toxicity and side effects, and higher patient compliance.

[0003] Photodynamic therapy (PDT) is a new minimally invasive anti-tumor method with many excellent properties such as spatiotemporal responsiveness and non-invasiveness. In the presence of O2, the photosensitizer distributed in tumor cells undergoes a photochemical reaction with a specific wavelength laser in a certain absorption band, generating reactive oxygen species (ROS) with cell-killing effects, which can kill tumor cells and cause immunogenic cell death (ICD) of tumor cells, thereby activating the body's anti-tumor immune response. However, after PDT, the expression of PD-L1 in tumor cells will be up-regulated, thereby promoting tumor immune escape and greatly reducing the anti-tumor treatment effect of PDT. Therefore, combining PDT with inhibiting the PD-1 / PD-L1 signaling pathway is an important means to improve the anti-tumor treatment effect.

[0004] Violet phosphorus (VP) was first discovered by German scientists in molten lead in 1865. It wasn't until 2019 that the research group of Zhang Jinying at Xi'an Jiaotong University first synthesized macroscopic single-crystal violet phosphorus, which was also proven to have a higher thermal decomposition temperature compared to black phosphorus (BP) and is the most stable allotrope of phosphorus discovered so far. VP has unique optoelectronic properties and good biocompatibility, and has great application potential in the field of biomedicine and is considered a new "dream material" expected to surpass BP. Violet phosphorus nanosheets (VPN) can be prepared by liquid-phase exfoliation of layered VP crystals. Due to its high drug loading efficiency, excellent photodynamic effect, and good biocompatibility, it shows unique advantages different from other inorganic materials in the research of tumor-targeted therapy mediated by nanodrug delivery systems.

[0005] Ferroptosis is a new type of programmed cell death characterized by the accumulation of lipid peroxides (LPO), which is different from apoptosis, necrosis and autophagy. It has great application potential as a new cancer treatment strategy. Ferroptosis is an iron-dependent form of tumor cell killing, which is usually achieved by the input of exogenous iron. 3+ Reduced to Fe by various reducing agents in the tumor (especially the high concentration of glutathione in tumor cells) 2 + , which avoids the consumption of ROS produced by these reducing agents during PDT, thereby fully ensuring the anti-tumor effect of PDT; at the same time, Fe 3 + It can also catalyze the excessive H2O2 in cells to generate O2. The continuous generation of O2 further alleviates the hypoxic microenvironment of the tumor, provides more sufficient O2 for PDT, and thus improves the anti-tumor therapeutic effect of PDT. 2+ It can consume excess H2O2 in the tumor and generate ·OH through the Fenton reaction. Highly toxic ·OH oxidizes unsaturated fatty acids on the tumor cell membrane, leading to a large accumulation of LPO, further disrupting the redox homeostasis of tumor cells, inducing cell death, and producing a certain tumor immune activation effect, reflecting the killing of tumor cells based on iron death. Studies have shown that HIF-1α is the main driving factor for inhibiting iron death in solid tumors under hypoxic conditions, which inhibits iron death in tumor cells by regulating glycolysis and glutamate metabolism. Therefore, Fe 3+ Combined with HIF-1α inhibitors, it will ultimately further enhance the effect of ferroptosis in tumor cells. 3+ It also has a strong coordination ability and can form coordination compounds with many groups containing oxygen and nitrogen atoms such as hydroxyl, primary amino, secondary amino, etc., reflecting the Fe 3+ It has good prospects in the field of drug delivery.

[0006] As a multifunctional drug, acridine yellow hydrochloride (ACF) was initially synthesized by German medical researcher Paul Ehrlich in 1912. It can inhibit the expression of HIF-1α, protein kinases, topoisomerase I and II, and also has a certain immune activation effect. It has been proven to have good therapeutic effects on various cancers (such as breast cancer, lung cancer, liver cancer, etc.). Research has shown that under hypoxic conditions, HIF-1α is upregulated in a variety of cancer cells and is closely related to disease progression and increased patient mortality. During the treatment of cancer, ACF, as an efficient HIF-1α inhibitor, can effectively bind to HIF-1α and inhibit the expression of HIF-1α, relieve the tumor hypoxic microenvironment, and at the same time downregulate downstream signaling molecules (PD-L1, VEGF, MMP-2), thereby effectively inhibiting the proliferation of tumor cells. In the process of combined use with PDT for anti-tumor treatment, on the one hand, ACF can relieve the hypoxic microenvironment of the tumor by inhibiting HIF-1α, providing more sufficient O2 for the implementation of PDT. On the other hand, it can reverse the upregulation of PD-L1 expression in tumor cells after PDT, trigger ICD to activate tumor immunity, reflecting the high efficiency and synergy of ACF and PDT in the anti-tumor treatment process.

[0007] Hydrogel is a three-dimensional network structure material with high hydrophilicity, strong bioadhesion and good biocompatibility, and has been widely used in the fields of drug delivery, tissue engineering and biosensing. The porous structure of hydrogel endows it with a high drug loading capacity, which can act as a drug reservoir to achieve sustained release after local administration, improving the in vivo retention ability of drugs. According to the responsiveness to external stimuli, hydrogels can be divided into traditional hydrogels and intelligent hydrogels. As an emerging material, intelligent hydrogels can not only possess the basic properties of traditional hydrogels, but also effectively respond to external physical, chemical or biological stimuli, causing conformational changes in the hydrogels and realizing the controlled release of drugs, showing good development prospects in the field of drug delivery.

[0008] Metformin hydrochloride (Met) is a model of "repurposing old drugs" for small molecule drugs. As the first-choice drug for the treatment of type 2 diabetes, it has been widely used in clinical practice. In recent years, studies have found that Met has definite anti-tumor efficacy, which has attracted much attention. Met has a broad anti-cancer spectrum and has good efficacy against breast cancer, lung cancer, prostate cancer, pancreatic cancer, etc. Moreover, Met has no significant effect on the growth and proliferation of normal tissue cells. The anti-tumor mechanism of Met is relatively complex, mainly by activating adenosine monophosphate-activated protein kinase (AMPK), blocking the mammalian target of rapamycin (mTOR) signaling pathway, inducing apoptosis and autophagy of tumor cells, and inhibiting the growth of tumor cells. Poly metformin (PM) is a high molecular polymer containing a large number of biguanide groups prepared by the addition reaction of chitosan and dicyandiamide. It has good water solubility, biodegradability and biocompatibility. Due to the large number of biguanide groups in its structure, PM excellently inherits the selective killing effect of Met on tumor cells and is a new type of anti-tumor polymer drug with great application prospects. In addition, the oxygen- and nitrogen-containing groups such as hydroxyl groups, primary amino groups and secondary amino groups in the PM structure can form coordination bonds directly with Fe 3+ to directly generate coordination bonds. At the same time, PM can also form Schiff base bonds with diphenylaldehyde polyethylene glycol (DF-PEG) under physiological conditions. Under the coexistence of coordination bonds and Schiff base bonds, an intelligent double-crosslinked hydrogel with both direct killing of tumor tissues and induction of ferroptosis is prepared.

[0009] To sum up, the following problems exist in the prior art: 1. Conventional hydrogel carriers do not have anti-tumor activity themselves and cannot produce a synergistic anti-tumor effect with other active ingredients; 2. The application of the new photodynamic material VPN in the field of drug delivery is very limited, and further research on its drug-loading performance is still needed; 3. Limited by the hypoxic microenvironment of tumor tissues, the photochemical reaction mediated by PDT lacks sufficient O2 participation, which weakens the anti-tumor effect of PDT; 4. The immune activation effect induced by PDT is usually weak, and it can also lead to up-regulation of PD-L1 expression after treatment, which is likely to ultimately cause immunosuppression and reduce the curative effect; 5. High expression of HIF-1α in the tumor microenvironment promotes ferroptosis resistance of tumor cells by regulating glycolysis and glutamate metabolism, reducing the occurrence of ferroptosis of tumor cells mediated by ferroptosis inducers; 6. VPN, Fe 3+ and ACF have weak tumor targeting after intravenous administration and are easily cleared by the body, which is not conducive to long-term effective treatment and limits their application in the anti-tumor field. Summary of the Invention

[0010] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides an anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on violet phosphorus nanosheets. The present invention uses violet phosphorus nanosheets (VPN) and poly metformin / Fe 3+ double-crosslinked hydrogel (PM / Fe3+ Using a gel as the carrier, based on the pharmaceutical concept of "multiple uses of one drug - multi - mode combined therapy", a tumor microenvironment - responsive hydrogel drug delivery system with good pH responsiveness, injectability and self - healing ability is prepared. By means of in - situ intratumoral injection, high selectivity for tumor tissues is achieved, providing an effective photodynamic therapy strategy for the application of an anti - tumor intelligent double - crosslinked hydrogel drug delivery system and the precise treatment of breast cancer.

[0011] For the first time, the present invention is based on Fe 3+ A hydrogel carrier with both direct killing of tumor tissues and induction of ferroptosis is prepared for drug delivery; the hydrogel carrier of the present invention can simultaneously exert the effects of direct killing of tumor tissues and induction of ferroptosis, and the anti - tumor ability of the carrier itself is stronger; compared with the existing PM hydrogel carrier, the dosage of the active ingredient PM is increased, the dosage of the non - active ingredient cross - linker diphenylaldehyde polyethylene glycol is reduced, and at the same time, the active ingredient Fe 3+ is introduced, further increasing the content of the anti - tumor active ingredient, and having a stronger anti - tumor effect at the same dosage.

[0012] The present invention also provides a preparation method and application of the anti - tumor intelligent double - crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets.

[0013] Technical solution: To achieve the above object, the present invention provides an anti - tumor intelligent double - crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets, including a hydrogel carrier, a photodynamic carrier material and a drug that can produce a direct killing effect on tumor tissues. The hydrogel carrier is a drug delivery carrier formed by the cross - linking of poly - metformin (PM) with Fe 3+ and diphenylaldehyde polyethylene glycol (DF - PEG); the photodynamic carrier material is VPN; the drug is a drug with both hypoxia - inducible factor - 1α (HIF - 1α) inhibition and PD - L1 down - regulation effects. The drug is loaded on the surface of VPN to form a nanocomplex and encapsulated in the hydrogel to form a drug delivery system, realizing the synergistic anti - tumor effect of phototherapy, ferroptosis therapy, immunotherapy and direct killing of tumor tissues.

[0014] Among them, the hydrogel carrier is a double - crosslinked hydrogel network formed by PM forming coordination bonds and Schiff base bonds with Fe 3+ and DF - PEG respectively. Among them, PM has a direct killing effect on tumor tissues and can react with Fe 3+ to be reduced by glutathione in the tumor to obtain Fe 2+ mediating ferroptosis of tumor cells for combined anti - tumor treatment.

[0015] Among them, the photodynamic carrier material is VPN dispersed in acetone, which can serve as a nanodrug carrier to efficiently load drugs that can kill tumor tissues, and at the same time has excellent photodynamic effects, good biocompatibility, and the potential for anti-tumor photodynamic therapy.

[0016] Among them, the drug is acridine yellow hydrochloride (ACF) which has both the effects of inhibiting HIF-1α and downregulating PD-L1. It can reverse the tumor hypoxic microenvironment by inhibiting the expression of HIF-1α, enhance the photodynamic therapy effect, and synergistically enhance Fe 3+ Fe obtained by glutathione reduction in the tumor 2+ mediated ferroptosis of tumor cells.

[0017] The preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets of the present invention includes the following steps:

[0018] (1) Mix purple phosphorus (VP) particles with an organic solvent, obtain a crude dispersion after ultrasonic treatment, collect the upper-layer nanosheet dispersion by low-speed centrifugation, then collect the precipitate at the bottom of the centrifuge tube by high-speed centrifugation treatment, and add ultrapure water for ultrasonic dispersion to obtain material A;

[0019] (2) Dissolve the drug that can directly kill tumor tissues in ultrapure water to obtain material B. Under stirring conditions, add material B to material A dispersed in step (1), continuously stir and react, then ultrafilter and centrifuge to remove the excess drug, and add an appropriate amount of ultrapure water for ultrasonic treatment to obtain the drug / purple phosphorus nanocomposite material C;

[0020] (3) Dissolve PM in ultrapure water to obtain material D, dissolve anhydrous iron salt in ultrapure water to obtain material E, dissolve DF-PEG in ultrapure water to obtain material F. Add material E and material C to material D in sequence, fully stir, adjust the pH of the system to physiological pH, then add material F, fully mix and let stand for a while to obtain the anti-tumor intelligent double-crosslinked hydrogel drug delivery system.

[0021] Among them, in step (1), every 20-100 mg of VP particles are dispersed in 10-50 mL of acetone; the probe ultrasonic power for preparing the VPN crude dispersion is 100-300 W, working for 1-4 s, intermittent for 1-4 s, and the total ultrasonic time is 18-30 h; the low-speed centrifugation speed of the crude dispersion is 2000-5000 rpm, and the centrifugation time is 0.5-5 min; the centrifugation speed for removing the supernatant by high-speed centrifugation is 12000-15000 rpm, and the centrifugation time is 10-30 min; the probe ultrasonic power for redispersing small-sized VPN after resuspension is 100-200 W, working for 1-4 s, intermittent for 1-4 s, and the total ultrasonic time is 5-35 min.

[0022] Preferably, in step (1), every 40 mg of VP particles are dispersed in 30 mL of acetone; when preparing the VPN coarse dispersion, the probe ultrasonic power is 200 W, working for 2 s and intermittent for 2 s, and the total ultrasonic time is 24 h; the low-speed centrifugation speed of the coarse dispersion is 4000 rpm and the centrifugation time is 1 min; when centrifuging to remove the supernatant at high speed, the centrifugation speed is 12,000 rpm and the centrifugation time is 15 min; when redispersing small-sized VPN after resuspension, the probe ultrasonic power is 150 W, working for 2 s and intermittent for 2 s, and the total ultrasonic time is 20 min.

[0023] Among them, in step (2), the feeding mass ratio of VPN to drug ACF is 1:0 - 1:8, and the stirring time is 4 - 10 h.

[0024] Preferably, in step (2), the feeding mass ratio of VPN to ACF is one of 1:0, 1:1, 1:2, 1:4, 1:6, 1:8; the stirring reaction time is 6 h.

[0025] In step (3), the final concentration of VPN in the hydrogel system is 0.02 - 5 mg / mL, the final concentration of ingredient D is 1.5 - 3% (g / mL), the final concentration of ingredient E is 0 - 100 mmol / L, the final concentration of ingredient F is 0.04 - 8.5% (g / mL), and the volume ratio of ingredient D to ingredient F is 2:1 - 100:1.

[0026] Preferably, in step (3), the final concentration of VPN in the hydrogel system is 0.03 mg / mL, the final concentration of PM is 2.81% (g / mL); 3+ the final concentration of Fe is 25 mmol / L; the final concentration of DF-PEG is 1.24% (g / mL); the feeding volume ratio of PM to DF-PEG is 15:1.

[0027] Preferably, the preparation method is as follows:

[0028] (1) Mix VP with acetone, after ultrasonic treatment, centrifuge at a low speed of 4000 rpm for 1 min to collect the upper-layer dispersion, then centrifuge at a high speed of 12,000 rpm for 15 min to collect the precipitate, add ultrapure water for ultrasonic dispersion to obtain ingredient A, pass N2 into ingredient A, and store it sealed in a 4°C refrigerator;

[0029] (2) Dissolve drug ACF in ultrapure water to obtain ingredient B. Under stirring conditions, add ingredient B to ingredient A dispersed in step (1) in different mass ratios in sequence. After continuous stirring reaction, place the mixed solution in an ultrafiltration centrifugal tube with a molecular weight cut-off of 3000 Da, centrifuge at 2000 rpm for 45 min to remove the excess drug, and add an appropriate amount of ultrapure water for ultrasonic treatment to obtain the ACF / VPN nanocomposite ingredient C;

[0030] (3) PM was dissolved in ultrapure water to obtain material D, anhydrous FeCl3 was dissolved in ultrapure water to obtain material E, and DF-PEG was dissolved in ultrapure water to obtain material F. Material E and material C were added to material D in sequence, and the system was adjusted to physiological pH after thorough stirring. Material F was then added to make the final concentration of VPN in the hydrogel system 0.03 mg / mL, the final concentration of material D 2.81% (g / mL), the final concentration of material E 25 mmol / L, and the final concentration of material F 1.24% (g / mL). After thorough mixing and standing for a while, the anti-tumor intelligent double-crosslinked hydrogel drug delivery system ACF / VPN@PM / Fe was obtained. 3+ gel.

[0031] The invention relates to the application of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets in the preparation of phototherapy anti-tumor drugs.

[0032] The anti-tumor intelligent double-cross-linked hydrogel drug delivery system based on purple phosphorus nanosheets constructed by the present invention is different from the common PDT treatment methods. First of all, unlike the prior art that uses MnO2 to load ACF through electrostatic action, the encapsulation of the present invention is based on the electrostatic interaction between ACF and VPN, and is further encapsulated in the hydrogel to form a drug delivery system, which prolongs the intratumoral retention time of the drug and reduces the number of drug administrations. The present invention is based on the ACF / VPN nanocomposite and is encapsulated in the hydrogel drug delivery system. Through the combined application of multiple treatment methods, it effectively solves the problems of insufficient O2 supply during the PDT process and immunosuppression caused by upregulation of PD-L1 after treatment, and synergistically enhances the ability of PDT to induce immunogenic cell death (ICD). At the same time, precise treatment is achieved through in situ intratumoral injection, which effectively prolongs the in vivo retention time of the drug, which is conducive to long-term, effective and controllable anti-tumor treatment.

[0033] The present invention uses ultraviolet full wavelength scanning and compares the appearance to find PM / Fe 3+ The coordination compound solution has a characteristic absorption peak at 486nm that can be attributed to the coordination bond. 3+ Under the condition of equal concentration, the corresponding PM / Fe 3+ The color of the coordination compound solution is different from that of Fe 3+ The solution darkened significantly, which proved that PM and Fe 3+ Based on this property, the present invention forms a coordination bond between PM and Fe 3+Coordination bonds and Schiff base bonds are respectively formed with DF-PEG to prepare a double-crosslinked hydrogel drug delivery carrier, which encapsulates the ACF / VPN nanocomposite formed by electrostatic interaction, and constructs a tumor microenvironment-responsive hydrogel drug delivery system with good pH responsiveness, injectability and self-healing properties for in-situ precise treatment of breast cancer and effectively inhibits lung metastasis of tumors. The design of this drug delivery system effectively solves the following problems: (1) Based on Fe 3+ For the first time, a "one-gel multi-use" hydrogel carrier with direct killing of tumor tissue and induction of ferroptosis is prepared, which can efficiently encapsulate the ACF / VPN nanocomposite and synergistically enhance the anti-tumor performance to further improve the anti-tumor treatment effect. PM can form coordination bonds and Schiff base bonds with Fe 3+ and DF-PEG respectively, and then obtain a double-crosslinked hydrogel drug delivery carrier with both direct killing of tumor tissue and induction of ferroptosis; due to the large number of biguanide groups in its structure, PM excellently inherits the anti-tumor effect of biguanide drugs and can be combined with Fe 3+ Reduced by glutathione in the tumor to obtain Fe 2+ Mediated ferroptosis of tumor cells is combined for anti-tumor treatment; (2) The combination of VPN, ACF and Fe 3+ On the one hand, it effectively solves the problem of insufficient O2 content during the PDT process mediated by VPN, and on the other hand, ACF can down-regulate the up-regulated PD-L1 of tumor cells after PDT, avoiding the immunosuppression caused by the up-regulation of PD-L1 expression and further ensuring the treatment effect; (3) Fe 3+ And the HIF-1α inhibitor ACF are used as hydrogel gelling materials and drugs respectively, which can effectively reverse the hypoxic microenvironment of tumors. While ACF exerts a direct killing effect on tumor tissue, it can also synergistically enhance Fe 3+ Reduced to Fe by glutathione in the tumor 2+ Mediated ferroptosis, achieving the effect of "one drug multi-use"; (4) By encapsulating the ACF / VPN nanocomposite in a hydrogel carrier and administering it by in-situ intratumoral injection, the problems of poor aggregation of drug components at the target site and easy clearance by the body are effectively solved, which is beneficial to the local sustained release of drugs, effectively ensures the efficacy of each drug component, and is beneficial to long-term effective treatment; (5) The prepared hydrogel drug delivery system is sensitive to the tumor microenvironment and can release drugs in a responsive manner at the tumor site. Due to the sensitivity of coordination bonds and Schiff base bonds to pH conditions, reversible bond cleavage occurs in the weakly acidic tumor microenvironment, which is beneficial to the targeted release of drug components in the tumor; (6) The drug delivery system constructed in the present invention involves multi-modal combined treatment of photodynamic therapy-ferroptosis therapy-immunotherapy-direct killing of tumor tissue, overcomes the limitations and deficiencies of single treatment methods, and provides an effective treatment strategy for the application of anti-tumor intelligent double-crosslinked hydrogel drug delivery systems and the precise treatment of breast cancer.

[0034] Principle of the invention: The present invention constructs an ACF / VPN nanocomposite by utilizing the electrostatic interaction between ACF and VPN, and co - encapsulates it in a hydrogel carrier based on PM and Fe 3+ constructed with the functions of directly killing tumor tissues and inducing ferroptosis. After in - situ intratumoral injection, the coordination bonds and Schiff base bonds of the hydrogel gradually break in the weakly acidic tumor microenvironment, ultimately causing the disassembly of the hydrogel skeleton. The encapsulated ACF / VPN is released in - situ within the tumor, and synergistically exerts an anti - tumor effect with the free PM and Fe 3+ produced by the degradation of the hydrogel. Among them, VPN has excellent photodynamic effects under laser irradiation, can generate reactive oxygen species (ROS) with cell - killing effects, kill tumor cells and cause ICD, thereby activating the body's anti - tumor immune response; meanwhile, Fe 3+ can not only reverse the hypoxic microenvironment of tumors in combination with ACF, provide sufficient O2 for the PDT process, effectively improve the anti - tumor effect of PDT, but also be reduced to Fe 2+ by glutathione in the tumor, and then mediate the ferroptosis of tumor cells, producing a certain immune activation effect; both PM and ACF can act as drugs to produce good tumor cell killing effects. In addition, in addition to having a certain immune activation effect itself, ACF can also synergistically enhance Fe 3+ reduced to Fe 2+ by glutathione in the tumor. It mediates the ferroptosis of tumor cells, and can down - regulate the up - regulated PD - L1 of tumor cells after PDT, effectively alleviating the resulting immunosuppression. By enhancing the tumor immune activation effect induced by PDT, the anti - tumor treatment effect is ensured, and the synergistic anti - tumor treatment effect of photodynamic therapy - ferroptosis therapy - immunotherapy - direct killing of tumor tissues is overall achieved.

[0035] The VPN adopted in the present invention is a new type of inorganic nanomaterial with high drug - loading efficiency and potential for photodynamic therapy of tumors. By loading positively charged ACF on VPN, the anti - tumor treatment effect of PDT mediated by VPN can be effectively synergistically enhanced; chitosan, as a carrier material widely used in the field of hydrogels, has a large number of amino groups in its molecular structure and can directly react with aldehyde groups to form Schiff base bonds. In previous studies, a pharmacologically active PM polymer synthesized from chitosan can form Schiff base bonds and coordination bonds with DF - PEG and Fe 3+ respectively, to construct a new type of intelligent double - crosslinked hydrogel carrier with the functions of directly killing tumor tissues and inducing ferroptosis, for encapsulating the above - mentioned nanocomposite. After in - situ intratumoral injection, the sustained and controllable release and long - term retention of drugs in the nanocomposite can be achieved, reducing the toxic and side effects on normal tissues of traditional drug - delivery methods such as intravenous injection, etc.; in addition, the Fe 3+On the one hand, it can act in combination with ACF to reverse the hypoxic microenvironment of tumors, providing sufficient O2 for PDT. On the other hand, it can be reduced to Fe by glutathione in the tumor 2+ Induce the occurrence of tumor ferroptosis; at the same time, ACF can also down-regulate the up-regulated PD-L1 expressed by tumor cells after PDT, effectively relieve the immunosuppression caused by the up-regulation of PD-L1 expression, avoid the limitations of PDT treatment, and further prevent the occurrence of tumor metastasis by enhancing the tumor immune activation effect while ensuring the treatment effect.

[0036] The present invention adopts a brand-new composition and preparation method to construct an anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets, and synthesizes PM / Fe with both direct killing of tumor tissues and induction of ferroptosis 3+ gel and the drug-loaded VPN nanocomposite. The present invention uses VPN as a nano-drug carrier, utilizes the electrostatic interaction between ACF and VPN, loads ACF on the surface of VPN to form an ACF / VPN nanocomposite, and then encapsulates it into a hydrogel carrier, solving the problems of short in vivo half-life and easy clearance of ACF; in addition, the present invention solves the problems of insufficient O2 supply during PDT and immunosuppression caused by the up-regulation of PD-L1 expression after treatment by combining Fe 3+ and ACF with VPN. Finally, the anti-tumor intelligent double-crosslinked hydrogel drug delivery system of the present invention is prepared, which has the ability to be highly synergistic for anti-tumor treatment.

[0037] Compared with the existing VPN hydrogel carrier, the present invention is based on Fe 3+ For the first time, a "multi-functional" hydrogel carrier with direct killing of tumor tissues and induction of ferroptosis is prepared, and an anti-tumor intelligent double-crosslinked hydrogel drug delivery system is further prepared based on Schiff base bonds and coordination bonds, which has good injectability and self-healing properties, reduces the dosage of the inactive ingredient diphenylaldehyde polyethylene glycol, increases the dosage of the anti-tumor active ingredient PM, and at the same time introduces the active ingredient Fe 3+ and has stronger drug efficacy at the same dosage, which is beneficial to improving the treatment effect. The coordination bond formed between Fe 3+ and PM and the Schiff base bond formed between DF-PEG and PM together constitute the crosslinked network of the hydrogel. The PM / Fe 3+ hydrogel carrier can, on the one hand, act as a drug carrier to deliver drugs, and on the other hand, it can down-regulate the highly expressed GSH in the tumor before Fe 3+ is reduced to Fe 2+ to exert the ferroptosis effect, which is beneficial to realizing the synergy of direct killing of tumor tissues and induction of ferroptosis, and plays the effect of "multi-functional".

[0038] Existing PM hydrogel carriers only have a direct killing effect on tumor tissues, but the PM / Fe 3+ hydrogel carrier of the present invention has dual effects of directly killing tumor tissues and inducing ferroptosis. The present invention first combines the synthesized PM and Fe to construct a hydrogel carrier, and the combination of the two has an obvious synergistic effect. On the one hand, PM and Fe 3+ can play their respective anti-tumor effects, and on the other hand, a hydrogel carrier is formed that can be used to deliver anti-tumor active components and has excellent intratumoral retention ability. The present invention is based on Fe 3+ and first prepares a hydrogel carrier with direct killing effect on tumor tissues and ferroptosis induction effect. Fe 3+ is reduced to Fe 2+ by overexpressed glutathione in the tumor site. The Fe 2+ -mediated Fenton reaction can, on the one hand, relieve tumor hypoxia and synergistically enhance PDT, and on the other hand, play a role in ferroptosis to kill tumors. In summary, the anti-tumor effect of the carrier of the present invention is stronger.

[0039] Different from the existing anti-tumor active components encapsulated by VPN hydrogel being M&D / VPN&aPD-L1, the anti-tumor active components encapsulated by the present invention are ACF / VPN. The two active components can play the roles played by the original four active components, and the drug effect is stronger, and the preparation is simpler. It only requires a simple mixture of the two active components, which is convenient for quality control.

[0040] The present invention effectively increases the content of PM when preparing the hydrogel. The purpose is to use PM-Fe coordination bonds to replace part of the Schiff base bonds formed by PM-DF-PEG to participate in the formation of the hydrogel cross-linking network. The content of the active component PM is significantly increased, and the anti-tumor effect is better, while the dosage of the cross-linking agent DF-PEG is significantly reduced. The number of Schiff base bonds formed by directly mixing PM and DF-PEG is limited and cannot form a gel directly. By introducing the active component Fe 3+ to form coordination bonds with PM to synergistically form a gel, and no gel can be formed without adding Fe 3+ .

[0041] The hydrogel drug delivery system prepared by the present invention has four active components PM, Fe 3+ , ACF, and VPN that cooperate with each other. Among them, (1) PM: on the one hand, it forms coordination bonds and Schiff base bonds with Fe 3+ and diphenylaldehyde polyethylene glycol respectively to prepare a hydrogel carrier for delivering ACF / VPN nanocomplexes, and on the other hand, it can play a direct killing effect on tumor tissues and cooperate with the other three active components to play an anti-tumor role. (2) Fe 3+ : First, it forms coordination bonds with PM to form a gel skeleton for drug delivery; second, it is reduced to Fe 2+Furthermore, it induces ferroptosis in tumor cells; third, Fe 3+ can react with excessive hydrogen peroxide in the tumor to generate oxygen, relieve the hypoxic environment of the tumor, provide sufficient oxygen for PDT, and synergize with the other three active ingredients in terms of efficacy. (3) ACF: First, by inhibiting the expression of hypoxia-inducible factor-1α, and Fe 3+ synergistically relieve the hypoxic microenvironment of the tumor, solve the limitation of the hypoxic tumor microenvironment in the process of VPN-induced PDT, and synergistically enhance PDT; second, inhibit the upregulation of PD-L1 expression after PDT, producing an effect similar to that of PD-L1 antibodies; third, ACF also has a certain immune activation effect, and synergistically exerts an anti-tumor effect with the other three active ingredients. (4) VPN: First, it delivers the drug ACF as a drug carrier; second, it is a novel photosensitizer that can exert PDT effects, and synergistically exerts an anti-tumor effect with the other three active ingredients.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0043] (1) The drug carrier VPN used in the present invention is a novel inorganic material with high drug loading capacity, excellent photodynamic effect and good biocompatibility. By combining VPN with ACF and Fe 3+ in combination has a significant synergistic effect compared with Fe 3+ alone, and the combined Fe 3+ +VPN, ACF+Fe 3+ (ACF / VPN@PM / Fe 3+ -L group) has a significant synergistic effect. On the one hand, it effectively solves the problem of insufficient O2 content during PDT. On the other hand, ACF can down-regulate the up-regulated PD-L1 after PDT, avoiding the immunosuppression caused by the up-regulation of PD-L1 expression, and further ensuring the treatment effect.

[0044] (2) The ACF used in the present invention is an effective HIF-1α inhibitor and also has a certain immune activation effect. In various tumors, ACF can bind to HIF-1α, effectively inhibit the expression of HIF-1α, relieve the tumor hypoxic environment and down-regulate the expression of downstream signaling molecules (PD-L1, VEGF, MMP-2) at the same time, effectively inhibiting the proliferation of tumor cells. In addition, ACF can also regulate glycolysis and glutamate metabolism by inhibiting HIF-1α, and finally enhance the ferroptosis of tumor cells, synergistically improving the anti-tumor efficacy of ferroptosis, and achieving the therapeutic effect of "one drug with multiple uses".

[0045] (3) The present invention uses electrostatic interaction to load ACF on VPN to prepare the ACF / VPN nanocomposite. It has simple components and a simple and easy-to-control preparation process, achieving a minimalist design of "one drug with multiple functions". At the same time, it can also play an anti-tumor role synergistically and has good clinical transformation potential.

[0046] (4) The polybiguanide / Fe 3+ double-crosslinked hydrogel carrier (PM / Fe 3+ gel) has both direct killing effect on tumor tissues and ferroptosis induction effect. The anti-tumor intelligent double-crosslinked hydrogel drug delivery system prepared by encapsulating the ACF / VPN nanocomposite not only has excellent anti-tumor effects, but also solves the problems of weak targeting and easy clearance of each component drug in the body. It can prolong the drug retention time in the tumor and achieve controllable and sustained release of the drug, effectively ensuring the anti-tumor effects of each component drug.

[0047] (5) After the anti-tumor intelligent double-crosslinked hydrogel drug delivery system constructed by the present invention is injected into the in-situ tumor, it can effectively respond to the weakly acidic microenvironment of the tumor. The coordination bonds and Schiff base bonds inside the hydrogel gradually break, and finally cause the disassembly of the hydrogel skeleton. The encapsulated ACF / VPN nanocomposite is precisely released in the in-situ tumor. After that, ACF can gradually fall off from the surface of VPN, and the two can cooperate with PM and Fe 3+ produced by the degradation of the hydrogel to play an anti-tumor role synergistically. Among them, VPN has excellent photodynamic effect under laser irradiation, can generate ROS with cell killing effect, kill tumor cells and cause ICD at the same time, and then activate the anti-tumor immune response of the body; at the same time, Fe 3+ is reduced to Fe 2+ by glutathione in the tumor, mediating the ferroptosis of tumor cells and producing a certain immune activation effect. On the other hand, it can also combine with ACF falling off from the surface of VPN to reverse the hypoxic microenvironment of the tumor, providing sufficient O2 for the PDT process and effectively improving the anti-tumor effect of VPN. Both PM and ACF can be used as drugs to produce good direct killing effects on tumor cells. In addition, in addition to having a certain immune activation effect itself, ACF can also synergistically enhance the ferroptosis of tumor cells mediated by Fe 3+ reduced by glutathione in the tumor to obtain Fe 2+ , and can down-regulate the up-regulated PD-L1 of tumor cells after PDT, effectively alleviating the immune suppression caused by the up-regulation of PD-L1 expression. By enhancing the tumor immune activation effect induced by PDT, the treatment effect is ensured, and the synergistic anti-tumor treatment effect of photodynamic therapy-ferroptosis therapy-immunotherapy-direct killing of tumor tissues is achieved as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1PM / Fe in the present invention 3+ Characterization data graph of coordination bonds in the coordination compound solution. Among them, Figure A is the PM / Fe prepared in Examples 1-10 3+ Ultraviolet spectrum graph of the coordination compound solution, and Figure B is EDTA, PM, Fe 3+ , PM / Fe 3+ And PM / Fe 3+ + Ultraviolet spectrum graph of the EDTA solution, and Figure C is the PM / Fe of different concentrations 3+ Coordination compound solution and Fe 3+ Solution color appearance comparison graph.

[0049] Figure 2 Characterization data graph of the ACF / VPN nanocomposite in the present invention. Among them, Figure A is the hydrated particle size data graph of the ACF / VPN nanocomposite dispersion liquid prepared in Examples 48-53, Figure B is the Zeta potential data graph of the nanocomposite dispersion liquid under the foregoing various embodiments, Figure C is the drug loading data graph of the nanocomposite under the foregoing various embodiments, and Figure D is the transmission electron microscope (TEM) micrograph of VPN and ACF / VPN nanocomposite obtained under the optimal preparation conditions.

[0050] Figure 3 ACF / VPN@PM / Fe in the present invention 3+ Characterization data graph of the gel drug delivery system. Among them, Figure A is the scanning electron microscope-energy dispersive spectroscopy analysis (SEM-EDS mapping) data graph of VPN obtained under the optimal preparation conditions, Figure B is the SEM-EDS mapping data graph of ACF / VPN obtained under the optimal preparation conditions, Figure C is the SEM-EDS mapping data graph of ACF / VPN@PM / Fe 3+ Gel, Figure D is the comparison graph of the processes before and after gel formation of PM + DF-PEG solution, PM / Fe 3+ Gel, ACF / VPN, ACF / VPN@PM / Fe 3+ Gel of the four, Figure E is the injectability characterization graph of ACF / VPN@PM / Fe 3+ Gel, and Figures F and G are both the self-healing characterization graphs of ACF / VPN@PM / Fe 3+ Gel.

[0051] Figure 4 ACF / VPN@PM / Fe in the present invention 3+ Characterization data graph of the rheological properties of the gel drug delivery system.

[0052] Among them, Figure A is the time scan diagram of ACF / VPN@PM / Fe 3+ gel obtained under the optimal preparation conditions, Figure B is the step strain scan diagram of ACF / VPN@PM / Fe 3+ gel obtained under the optimal preparation conditions, Figure C is the step strain scan diagram of ACF / VPN@PM / Fe 3+ gel obtained under the optimal preparation conditions, Figure D is the frequency scan diagram of ACF / VPN@PM / Fe 3+ gel obtained under the optimal preparation conditions, Figure E is the shear flow scan diagram of ACF / VPN@PM / Fe 3+ gel.

[0053] Figure 5 It is the data diagram for investigating the in vitro (based on mouse breast cancer cell 4T1) anti-tumor performance of the ACF / VPN@PM / Fe 3+ gel drug delivery system in the present invention. Among them, Figure A is the data diagram of the cell uptake experiment of ACF / VPN@PM / Fe 3+ gel obtained under the optimal preparation conditions, Figure B is the data diagram of the cytotoxicity experiment under different drug administration treatment conditions, Figure C is the data diagram of the down-regulation effect of cell PD-L1 under different drug administration treatment conditions, Figure D is the data diagram of the changes in cell mitochondrial membrane potential and the generation effect of lipid peroxide (LPO) under different drug administration treatment conditions, and Figure E is the data diagram of the cell apoptosis induction effect under different drug administration treatment conditions.

[0054] Figure 6 It is the data diagram of the in vivo retention of PM / Fe 3+ gel in the present invention. Among them, Figure A is the in vivo imaging diagram of the retention of PM / Fe 3 + gel at the site of the in-situ tumor at different time points, and Figure B is the fluorescence distribution diagram in the excised tumor and other normal tissues of the tumor-bearing mice at the last investigated time point.

[0055] Figure 7 It is the ACF / VPN@PM / Fe in the present invention 3+Data graphs of the in vivo anti-tumor efficacy, in situ and systemic tumor immune activation, and anti-tumor metastasis performance of the gel drug delivery system. Among them, Figure A is a fresh ex vivo in situ tumor tissue image of tumor-bearing mice after treatment under different drug administration and treatment conditions, Figure B is a statistical graph of the volume data of fresh ex vivo in situ tumor tissues of tumor-bearing mice after treatment under different drug administration and treatment conditions, Figure C is a data graph of the immune activation in the in situ tumors of tumor-bearing mice under different drug administration and treatment conditions, Figure D is a data graph of the systemic immune activation of tumor-bearing mice under different drug administration and treatment conditions, Figure E is an effect graph of the inhibition of HIF-1α expression in the in situ tumor tissue, Figure F is an effect graph of the inhibition of lung metastasis in tumor-bearing mice under different drug administration and treatment conditions, and Figure G is an H&E stained section image of the lung tissue of tumor-bearing mice under different drug administration and treatment conditions.

[0056] Figure 8 This is ACF / VPN@PM / Fe in the present invention 3+ Data graphs for the in vivo safety investigation of the gel drug delivery system.

[0057] Among them, Figure A is a data graph of the body weight changes of tumor-bearing mice during treatment under different drug administration and treatment conditions, and Figure B is an H&E stained section image of various tissues and organs of tumor-bearing mice under different drug administration and treatment conditions. Detailed implementation manners

[0058] To make the present invention easier to understand, the following further illustrates the present invention in conjunction with specific embodiments, which do not limit the present invention in any way. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the technical solution of the present invention, any modification or change that is easily achievable by those of ordinary skill in the art to the present invention will fall within the scope of the claims of the present invention.

[0059] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions. The experimental methods without specific conditions in the embodiments usually follow conventional conditions or the conditions recommended by the manufacturer.

[0060] Among them, anhydrous FeCl3 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: I141414; disodium ethylenediaminetetraacetate (EDTA) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., product number: E909976; Cy5-N-hydroxysuccinimide ester (Cy5-NHS) was purchased from Dalian Meilun Biotechnology Co., Ltd., product number: MB12193-2; deferoxamine mesylate (DFO) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., product number: D873692; acridine yellow hydrochloride (ACF) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: A113289.

[0061] Purple phosphorus (VP) particles were synthesized and provided by Professor Zhang Jinying's team at Xi'an Jiaotong University. The synthesis method was based on the literature: Structure and Properties of Violet Phosphorus and Its Phosphorene Exfoliation, Angew. Chem. Int. Ed., 2020; the synthesis methods of dibenzaldehyde polyethylene glycol (DF-PEG) and polymethformin (PM) were based on Examples 1-2 in Chinese Patent No. 2023117713397.

[0062] Examples 1-10

[0063] A. Polymethacin / Fe 3+ (PM / Fe 3+ ) Solution preparation:

[0064] Weigh a certain amount of PM solid and prepare it with ultrapure water to make a PM solution. Weigh a certain amount of anhydrous FeCl3 powder and prepare FeCl3 aqueous solutions of varying concentrations with ultrapure water. Take a certain volume of PM solution and add a certain volume of FeCl3 solution to make the final PM concentration in the system 2.81% (g / mL). After thorough mixing, PM / Fe 3+ coordination compound.

[0065] The PM / Fe prepared in Examples 1-10 3+ The concentration of FeCl3 in the coordination compound solution and the volume ratio of PM to FeCl3 are shown in Table 1:

[0066] The initial concentration of FeCl3 in Table 1 is millimolar volume ratio (mmol / L).

[0067] Table 1 is the PM / Fe prepared in Examples 1-10. 3+ Raw material concentration and volume ratio of coordination compound solution

[0068] Process conditions Example 1 Example 2 Example 3 Example 4 Example 5 <![CDATA[FeCl3 feeding concentration]]> 0 25 50 100 250 <![CDATA[Volume ratio of PM to FeCl3 solution]]> 84.4:5 84.4:5 84.4:5 84.4:5 84.4:5 Process conditions Example 6 Example 7 Example 8 Example 9 Example 10 <![CDATA[FeCl3 feeding concentration]]> 500 750 1000 1500 2000 <![CDATA[Volume ratio of PM to FeCl3 solution]]> 84.4:5 84.4:5 84.4:5 84.4:5 84.4:5

[0069] Embodiment 11

[0070] B.PM / Fe 3+ Preparation of EDTA solution:

[0071] Weigh a certain amount of EDTA solid and prepare EDTA solution with ultrapure water. Prepare a certain volume of PM / Fe 3+ The coordination compound solution (Example 6) was then added with EDTA solution of the same molar mass as FeCl3 to make the final concentration of PM in the system 2.81% (g / mL). 3+The final concentration of EDTA is 25mmol / L, and the final concentration of PM / Fe is 25mmol / L. After mixing evenly, PM / Fe 3+ +EDTA solution.

[0072] Examples 12-46

[0073] C. Hydrogel carrier polymetformin / Fe 3+ Double cross-linked hydrogel (PM / Fe 3+ Preparation of gel:

[0074] Weigh a certain amount of DF-PEG solid and prepare a series of DF-PEG solutions with ultrapure water. Take a certain volume of PM solution and add a certain volume of FeCl3 solution (Example 6) to prepare PM / Fe 3+ The coordination compound was then adjusted to physiological pH (pH 7.4), and a certain volume of DF-PEG solution was added to make the final concentration of PM in the hydrogel system 2.81% (g / mL). 3+ The final concentration is 25mmol / L. After fully mixing and letting it stand for a while, PM / Fe 3+ gel.

[0075] The final concentration of DF-PEG is: mass-to-volume ratio (g / mL).

[0076] Table 2 shows the PM / Fe prepared in Examples 12-46. 3+ Final concentration and volume ratio of gel raw materials

[0077]

[0078]

[0079] Embodiment 47

[0080] D. Preparation of Purple Phosphorus Nanosheets (VPN) Aqueous Dispersion:

[0081] Accurately weigh 40 mg of VP particles and place them in a brown narrow-necked bottle. Add 30 mL of acetone and mix. Use an ultrasonic cell disruptor and continue ultrasonic treatment for 24 hours at a power of 200 W, a working time of 2 seconds, and an interval of 2 seconds. Then, the crude VPN dispersion is divided into 2 mL centrifuge tubes and centrifuged at 4000 rpm for 1 minute to remove large VPN particles that are not fully stripped. The upper dispersion is then centrifuged at 12000 rpm for 15 minutes. After discarding the supernatant, add an appropriate amount of ultrapure water to resuspend the VPN solid at the bottom of the centrifuge tube to an appropriate concentration. Use an ultrasonic cell disruptor at a power of 150 W, a working time of 2 seconds, an interval of 2 seconds, and continue ultrasonic treatment for 20 minutes to obtain a uniformly dispersed VPN aqueous dispersion, which is stored at 4°C under N2 protection.

[0082] Examples 48-53

[0083] E. Preparation of Acridinium Yellow Hydrochloride / Purple Phosphorus Nanocomposite (ACF / VPN):

[0084] A certain amount of acriflavine hydrochloride (ACF) was weighed and dissolved in an appropriate amount of ultrapure water to prepare an ACF solution of a certain concentration. The ACF solution was added dropwise to the VPN aqueous dispersion prepared in Example 47. The mixture was stirred at room temperature for 6 hours. After continuous stirring, the mixture was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000 Da and centrifuged at 2000 rpm for 45 minutes to remove excess drug. An appropriate amount of ultrapure water was added and ultrasonic treatment was performed to obtain the ACF / VPN nanocomposite.

[0085] The mass ratios of the materials used in preparing the ACF / VPN nanocomposites in Examples 48-53 are shown in Table 3:

[0086] The feed mass ratio is the absolute mass ratio of VPN to ACF in the VPN aqueous dispersion.

[0087] Table 3 shows the mass ratios of the materials used in preparing the ACF / VPN nanocomposites in Examples 48-53.

[0088]

[0089] Embodiment 54

[0090] F. Construction of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system ACF / VPN@PM / Fe3+gel:

[0091] Take a certain volume of PM solution and add a certain volume of FeCl3 solution (Example 6) to prepare PM / Fe 3+ The coordination compound was then added with the prepared ACF / VPN nanocomposite (Example 52, the mass ratio of VPN to ACF was 1:6, wherein the VPN aqueous dispersion was prepared according to Example 47), and after sufficient stirring, the pH was adjusted to physiological pH (pH 7.4). Finally, DF-PEG solution was added at a volume ratio of PM to DF-PEG of 15:1, so that the final concentration of PM in the hydrogel system was 2.81% (g / mL). 3+ The final concentration of 25mmol / L, the final concentration of DF-PEG was 1.24% (g / mL), and the final concentration of VPN was 0.03mg / mL. After mixing evenly and standing for a while, the anti-tumor intelligent double cross-linked hydrogel drug delivery system ACF / VPN@PM / Fe was obtained. 3+ gel.

[0092] Embodiment 55

[0093] PM / Fe 3+ Characterization of coordination bonds in coordination compound solutions:

[0094] (1) Ultraviolet spectrum scanning: Use an ultraviolet-visible spectrophotometer to scan the wavelengths in the range of 400 - 700 nm for coordination compound solutions of different concentrations of PM / Fe 3+ (Examples 1 - 10), PM solution (Example 6, replacing the FeCl3 solution with an equal volume of ultrapure water), Fe 3+ solution (Example 6, replacing the PM solution with an equal volume of ultrapure water), EDTA solution (Example 11, replacing the PM and FeCl3 solutions with an equal volume of ultrapure water), PM / Fe 3+ +EDTA solution (Example 11). As shown in Attachment Figure 1 A and Attachment Figure 1 B, the coordination compound solution of PM / Fe 3+ has a characteristic absorption peak attributable to coordination bonds at 486 nm, while neither the PM solution nor the Fe 3+ solution has an absorption peak at 486 nm, proving the formation of a coordination bond between PM and Fe 3+ . At the same time, the content of the coordination bond formed between PM and Fe 3+ increases with the increase in the feeding amount of Fe 3+ , and when the feeding concentration of Fe 3+ is 500 mmol / L, the amount of the coordination bond formed with PM has basically reached saturation, and when the concentration of Fe 3+ is further increased, the content of the coordination bond in the system no longer increases significantly. In addition, the absorption peak intensity of the PM / Fe 3+ +EDTA solution at 486 nm is lower than that of the PM / Fe 3+ coordination compound solution, and the EDTA solution has no absorption peak at 486 nm. At the same time, the color of the PM / Fe 3+ +EDTA solution changes significantly compared with that of the PM / Fe 3+ coordination compound solution, proving that EDTA destroys the coordination bond formed between PM and Fe 3+ , and once again proving the formation of the coordination bond between PM and Fe 3+ .

[0095] (2) Solution color comparison: Use a camera to take pictures of the appearances of coordination compound solutions of different concentrations of PM / Fe 3+ (Examples 1 - 10) and Fe 3+ solution (Examples 1 - 10, replacing the PM solution with an equal volume of ultrapure water) for investigation. As shown in Attachment Figure 1 C, when the Fe 3+ concentration in the system is equal, corresponding to PM / Fe3+ The color of the coordination compound solution is significantly darker than that of the Fe 3+ solution, indicating the formation of a coordination bond between PM and Fe 3+ .

[0096] Example 56

[0097] Characterization of the properties of the ACF / VPN nanocomposite:

[0098] (1) Determination of particle size, Zeta potential and drug loading: The hydrodynamic particle size and Zeta potential of the ACF / VPN nanocomposite dispersion (the VPN dispersion was prepared according to Example 47) in Examples 48 - 53 were measured using a laser particle size analyzer, and then the drug loading of ACF was measured using a UV-visible spectrophotometer. As shown in Figure 2 Appendix A, with the addition of ACF, the particle size of the ACF / VPN nanocomposite first increased and then decreased, and finally stabilized. As shown in Figure 2 Appendix B, with the addition of ACF, the Zeta potential of different groups of ACF / VPN nanocomposites gradually increased, indicating that the electropositive ACF was loaded on the surface of the electronegative VPN through electrostatic binding. From the drug loading results in Figure 2 Appendix C, with the addition of ACF, the drug loading on the surface of VPN gradually increased. As shown in Figure 2 Appendix A - 2C, when the feeding mass ratio of VPN to ACF reached 1:8, there were no significant differences in the particle size, potential and drug loading of the ACF / VPN nanocomposite compared with those when the feeding mass ratio was 1:6. This indicates that when the feeding ratio of VPN to ACF was controlled at 1:6, the maximum drug loading of ACF on the surface of VPN had been reached. Further increasing the feeding amount of ACF could not increase its loading on the surface of VPN, and the excessive ACF would be free in the solution and removed during the purification process of ACF / VPN. Therefore, from the perspective of saving raw materials, the feeding mass ratio of VPN to ACF at 1:6 was used as the optimal preparation prescription for the ACF / VPN nanocomposite. It can be seen that when the feeding mass ratio of VPN to ACF was 1:6, the hydrodynamic particle size of the ACF / VPN nanocomposite was (264.34 ± 5.48) nm, the potential was (13.70 ± 2.47) mV, and the drug loading of ACF was (84.91 ± 3.82)%.

[0099] (2) Microscopic morphology characterization: The VPN dispersion (Example 47) and the ACF / VPN nanocomposite dispersion (Example 52, the VPN dispersion was prepared according to Example 47) were respectively prepared, and the microscopic morphology characteristics of the two groups of samples were observed by transmission electron microscopy (TEM). As shown in Figure 2As shown in D, the VPN has a flaky structure. After drug loading, the thickness of the ACF / VPN nanocomposite lamella increases and the lamellar structure tends to be blurred, indicating the successful loading of ACF on the surface of VPN.

[0100] Example 57

[0101] Antitumor intelligent double-crosslinked hydrogel drug delivery system ACF / VPN@PM / Fe 3+ Characterization of the properties of gel:

[0102] (1) Microscopic morphology characterization: Prepare a VPN dispersion (Example 47), an ACF / VPN nanocomposite dispersion (Example 52, the VPN dispersion is prepared according to Example 47), and an ACF / VPN@PM / Fe 3+ gel (Example 54, the VPN dispersion is prepared according to Example 47) respectively. Use a freeze dryer for vacuum freeze-drying treatment, and observe the microscopic morphology characteristics and element distribution of the three groups of samples through SEM-EDS mapping, as shown in Appendix Figure 3 A and Appendix Figure 3 B. The VPN has a flaky structure. After drug loading, the thickness of the ACF / VPN nanocomposite lamella increases, and some protruding VPN lamellae can be seen on the surface, indicating the successful loading of ACF on the surface of VPN. Appendix Figure 3 C shows the internal three-dimensional porous structure unique to the hydrogel in the ACF / VPN@PM / Fe 3+ gel. At the same time, compared with the VPN and ACF / VPN nanocomposite, the Fe element content in the ACF / VPN@PM / Fe 3+ gel increases, thus proving the successful construction of the antitumor intelligent double-crosslinked hydrogel drug delivery system.

[0103] (2) Sol-gel transition: The PM+DF-PEG solution (Example 30, replacing the FeCl3 solution with an equal volume of ultrapure water, with a total volume of 500 μL), the precursor solution of the polybiguanide / Fe 3+ hydrogel (PM / Fe 3+ gel, prepared according to Example 30) (the solution before the components are mixed and not yet gelled, with a total volume of 500 μL), the ACF / VPN nanocomposite dispersion (Example 52, the VPN dispersion is prepared according to Example 47, with a volume of 500 μL), the ACF / VPN@PM / Fe 3+The precursor solution of the gel (Example 54, the VPN dispersion was prepared according to Example 47) (the solution in which each component was mixed but not yet gelled, where the total volume of the drug delivery system was 500 μL, and the volume of each component was obtained according to the volume ratio and concentration of the corresponding components in Example 54) was placed in a 3 mL glass vial and mixed evenly. After tilting the vial and taking a photo, it was immediately placed vertically and allowed to stand until the gel formed. After 10 min, it was tilted again and photographed. As shown in Figure 3 Appendix D, it can be seen by comparison that PM / Fe 3+ gel and ACF / VPN@PM / Fe 3+ gel underwent a transition from sol to gel. By tilting the vial, observing whether the liquid level in the vial was tilted before and after gel formation indicated the successful formation of the hydrogel, while the ACF / VPN nanocomposite dispersion did not have this effect; it was observed that the mixed solution of PM and DF-PEG did not form a gel, and after introducing Fe 3+ gelation occurred smoothly, indicating that the number of Schiff base bonds formed by directly mixing PM and DF-PEG was limited and could not form a gel directly. By introducing the active ingredient Fe 3+ to form coordination bonds with PM to synergistically form a gel, and without adding Fe 3+ gelation could not occur.

[0104] (3) Injectability investigation: The precursor solution (500 μL) of ACF / VPN@PM / Fe 3+ gel (Example 54, the VPN dispersion was prepared according to Example 47) was placed in a 1 mL syringe. After the hydrogel was formed at room temperature, it was slowly pushed out of the syringe through the needle, and the text "CPU WW" was written and photographed. As shown in Figure 3 Appendix E, the hydrogel could be smoothly and relatively easily pushed out of the syringe needle at room temperature to form the text "CPU WW" with a smooth appearance and clear outline, indicating that the ACF / VPN@PM / Fe 3+ gel drug delivery system had good injectability.

[0105] (4) Self-healing investigation: ACF / VPN@PM / Fe 3+ gel (Example 54, orange, where the VPN dispersion was prepared according to Example 47) and ACF / VPN@PM-Cy5 / Fe 3+gel (Example 54, green, the PM solution was replaced with an equal amount of PM-Cy5 solution, keeping the concentration of PM-Cy5 and PM consistent, the final concentration of PM-Cy5 in the hydrogel system was 2.81% (g / mL), 1g PM and 1mg Cy5-NHS were weighed and dissolved in 25mL and 5mL phosphate buffer (0.01M, pH 7.4), respectively. The Cy5-NHS solution was added dropwise to the PM solution under stirring, and then stirred for 8h in the dark. After the reaction was completed, the mixture was transferred to a dialysis bag (molecular weight cutoff: 3500Da) and dialyzed in ultrapure water for 48h to remove excess Cy5-NHS. Finally, the purified solution was lyophilized to obtain PM-Cy5 solid). The above hydrogel was divided into four pieces, and two pieces were randomly selected and placed diagonally close to each other to form a hydrogel with alternating colors and placed on a glass slide. The self-healing condition was observed and photographed at 0, 2, 6, 12, 24, 36, and 48h, respectively. As shown in the attached figure Figure 3 As shown in F, as the static time increases, the hydrogels of different colors penetrate each other, the gaps at the joints gradually fade and disappear, and they are completely fused at 48h, which shows that the anti-tumor intelligent double-crosslinked hydrogel drug delivery system has good self-healing properties. 3+ A small hole with a diameter of 4 mm was carefully dug in the center of the hydrogel (Example 54, orange, wherein the VPN dispersion was prepared according to Example 47). The size of the hole was observed and photographed at 0, 2, 6, 12, 24, 36, and 48 h. Figure 3 As shown in Figure G, as the static time increases, the small holes in the center of the hydrogel gradually become smaller, and the small holes basically disappear after 48 hours, which indicates that the anti-tumor intelligent double-cross-linked hydrogel drug delivery system has good self-healing properties.

[0106] (5) Rheological properties investigation: ACF / VPN@PM / Fe was tested using a rotational rheometer. 3+ The gel (Example 54, VPN dispersion prepared according to Example 47) was subjected to time scan, step strain scan, step strain scan, frequency scan and shear flow scan. Figure 4 As shown in Figure A, with the increase of gelation time, the storage modulus (G') and loss modulus (G") of the hydrogel gradually increased, and the increase in G' decreased significantly around 10 minutes, indicating the gradual formation of the hydrogel network. Figure 4 The step strain scan results of Figure B show that when the stress applied to the hydrogel is small, its G' and G" remain basically unchanged, and G' is always greater than G", at this stage the hydrogel structure is still intact; when the stress increases to a certain value, the G' and G" of the hydrogel decrease rapidly, and when it reaches around 185%, G' begins to be less than G", indicating that the internal structure of the hydrogel has been destroyed. Figure 4As shown in C, with the applied stress instantaneously increasing from 1% to 200%, G’ instantaneously drops from about 1200 Pa on average to about 400 Pa on average and is less than G”, indicating that the hydrogel structure has been damaged under stress; when the large strain is removed, both G’ and G” rapidly return to their original states within a few seconds, and G’ is greater than G”, indicating that the hydrogel rapidly restores its original structure after the stress is removed, thereby reflecting the reversibility of Schiff base bonds and coordination bonds. As shown in the appendix Figure 4 D, it can be seen that within a certain frequency range (1 - 100 rad / s), the G’ of ACF / VPN@PM / Fe 3+ gel is greater than G” at the same frequency. As the frequency changes, G’ remains unchanged, and the value of G” changes dynamically with the change of frequency, further indicating that this hydrogel is composed of a dynamically cross-linked hydrogel network rather than a covalently cross-linked network with constant G’ and G”. As shown in the appendix Figure 4 E, it can be seen that the viscosity of the hydrogel decreases with the increase of the shear rate, indicating that the prepared hydrogel has good injectability, further proving the injectability of the hydrogel from another perspective and detection method.

[0107] Example 58

[0108] Investigation on the in vitro antitumor effect of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system ACF / VPN@PM / Fe 3+ gel:

[0109] In this example, ACF / VPN@PM / Fe 3+ gel prepared by the method of Example 54 was used, in which the VPN dispersion was prepared according to Example 47, and the final concentration of VPN was 0.03 mg / mL;

[0110] Among them: PM / Fe 3+ gel was obtained by the preparation method of Example 30;

[0111] Among them: VPN@PM / Fe 3+ gel was obtained by the preparation method of Example 54. The ACF / VPN nanocomposite was replaced with an equal volume of VPN aqueous dispersion, and the final concentration of VPN was maintained at 0.03 mg / mL;

[0112] Among them: ACF / VPN@PM / Fe 3+ +DFO gel was obtained by the preparation method of Example 54. The final concentration of DFO in the hydrogel was controlled to be 100 μmol / L. DFO is a commonly used ferroptosis inhibitor at present. In the present invention, the ACF / VPN@PM / Fe 3+ +DFO gel group was designed to verify Fe 3+ reduced by glutathione in the tumor to obtain Fe 2+The occurrence of ferroptosis in tumor cells mediated;

[0113] Among them: ACF / VPN@PM-Cy5 / Fe 3+ gel was obtained by the preparation method of Example 54. The PM solution was replaced with an equal amount of PM-Cy5 solution, and the concentrations of PM-Cy5 and PM were kept the same. The final concentration of PM-Cy5 in the hydrogel system was 2.81% (g / mL), and the PM-Cy5 solution was prepared according to Example 57.

[0114] In this example, except for the apoptosis experiment, mouse breast cancer cells 4T1 were seeded into 24-well plates at a density of 4×10 4 cells / well. When the confluence reached about 80%, each hydrogel preparation was prepared on the upper layer of the Transwell chamber and drug administration was completed for each well to start subsequent experimental operations.

[0115] In the apoptosis experiment, mouse breast cancer cells 4T1 were seeded into 12-well plates at a density of 1×10 5 cells / well. When the confluence reached about 80%, each hydrogel preparation was prepared on the upper layer of the Transwell chamber and drug administration was completed for each well to start subsequent experimental operations.

[0116] (1) Cell uptake experiment: Using flow cytometry, the drug uptake of ACF / VPN@PM-Cy5 / Fe 3+ gel (where the final concentration of VPN was 0.03 mg / mL, and the other drug concentrations were converted according to Example 54) co-cultured with mouse breast cancer cells 4T1 for 2, 4, 6, and 12 h was investigated. As shown in Appendix Figure 5 A, when the uptake time was 6 h, ACF and PM-Cy5 had a relatively high double-uptake percentage (56.4%). When the uptake time was further extended to 12 h, the double-uptake percentage of ACF and PM-Cy5 decreased compared with that at 6 h (46.2%). Therefore, 6 h was determined as the optimal cell uptake time of ACF / VPN@PM-Cy5 / Fe 3+ gel.

[0117] (2) Cytotoxicity experiment: Based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, mouse breast cancer cells 4T1 were used to investigate PM / Fe 2 gel (Example 30), VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+gel (Example 54, where the final concentration of VPN is 0.03 mg / mL), and Control without laser irradiation, ACF / VPN@PM / Fe 3+ The cytotoxicity of gel (Example 54, where the final concentration of VPN is 0.03 mg / mL). As shown in the appendix Figure 5 B, PM / Fe 3+ gel+L and VPN@PM / Fe 3+ gel+L both have a certain degree of tumor cell cytotoxicity. At the same time, VPN@PM / Fe 3+ gel+L group has significantly lower cell viability than PM / Fe 3+ gel+L group. This is because ROS with cell-killing effects are generated during the PDT process mediated by VPN as a photosensitizer, which can have a strong killing effect on cells. At the same time, ACF / VPN@PM / Fe 3+ gel+L group has significantly lower cell viability than ACF / VPN@PM / Fe 3+ gel group, further verifying the excellent anti-tumor effect of PDT; by comparing the cell viability of VPN@PM / Fe 3+ gel+L and ACF / VPN@PM / Fe 3+ gel+L groups, it can be confirmed that ACF has an excellent anti-tumor effect; compared with other control groups, ACF / VPN@PM / Fe 3+ gel under laser irradiation has the strongest tumor cell killing effect. The viability of 4T1 cells is only 20.10% after administration and light irradiation, and the tumor cell cytotoxicity generated by this group is significantly higher than that of other control groups; at the same time, the cell viability of ACF / VPN@PM / Fe 3+ +DFO gel+L group is 32.65%, which is significantly higher than the cell viability of ACF / VPN@PM / Fe 3+ gel+L group (20.10%), indicating that ACF / VPN@PM / Fe 3+ gel can effectively induce the occurrence of ferroptosis in tumor cells, and the ferroptosis-inducing effect of ACF / VPN@PM / Fe 3+ gel on tumor cells can be alleviated by the ferroptosis inhibitor DFO.

[0118] (3) Cell PD-L1 downregulation experiment: Using a flow cytometer, based on mouse breast cancer cells 4T1, investigate PM / Fe 2 under 660 nm, 0.25 W / cm 3+ 、10 min laser irradiation (+L) of gel (Example 30), VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3++DFO gel, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL), and Control, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL) without laser irradiation. As shown in Figure 5 Figure C, compared with other control groups, ACF / VPN@PM / Fe 3+ gel has the most prominent PD-L1 downregulation effect, indicating that it can produce the strongest PD-L1 downregulation effect on 4T1 cells under this condition.

[0119] (4) Cell mitochondrial membrane potential change experiment: Based on the mitochondrial membrane potential detection kit (JC-1), mouse breast cancer cells 4T1 were used to investigate the intracellular mitochondrial membrane potential changes of PM / Fe 2 gel (Example 30), VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL), and Control, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL) without laser irradiation. As shown in 3+ Figure D, 4T1 cells treated with ACF / VPN@PM / Fe Figure 5 gel and laser irradiation have the fewest JC-1 aggregates (red fluorescence) compared to other groups, indicating that it can produce the strongest effect of reducing mitochondrial membrane potential on 4T1 cells under this condition. At the same time, the JC-1 aggregates produced by the ACF / VPN@PM / Fe 3+ +DFO gel + L group are more than those of the ACF / VPN@PM / Fe 3+ gel + L group, indicating that ACF / VPN@PM / Fe 3+ gel under laser irradiation can effectively induce ferroptosis in tumor cells, and the ferroptosis of tumor cells induced by ACF / VPN@PM / Fe 3+ gel can be alleviated by the ferroptosis inhibitor DFO. 3+

[0120] (5) Intracellular lipid peroxide (LPO) production experiment in cells: Based on the fluorescent probe BODIPY 581 / 591 ​-C11, using mouse breast cancer cells 4T1 to investigate the generation of intracellular LPO in PM / Fe 2 gel (Example 30), VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL), as well as Control without laser irradiation, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL). As shown in 3+ Figure D, the 4T1 cells treated with ACF / VPN@PM / Fe Figure 5 gel and laser irradiation generated the highest level of LPO compared to other groups, indicating that the strongest ferroptosis-inducing effect on 4T1 cells could be produced under this condition. At the same time, the LPO level generated in the ACF / VPN@PM / Fe 3+ +DFO gel + L group was lower than that in the ACF / VPN@PM / Fe 3+ gel + L group, indicating that ACF / VPN@PM / Fe 3+ gel under laser irradiation could effectively induce the occurrence of ferroptosis in tumor cells, and the ferroptosis of tumor cells induced by ACF / VPN@PM / Fe 3+ gel could be alleviated by the ferroptosis inhibitor DFO. 3+ (6) Cell apoptosis experiment: Based on the Annexin V-PE / Cy7 / DAPI cell apoptosis detection kit, using mouse breast cancer cells 4T1 to investigate the apoptosis-inducing effects of PM / Fe

[0121] gel (Example 30), VPN@PM / Fe 2 gel, ACF / VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL), as well as Control without laser irradiation, ACF / VPN@PM / Fe 3+ gel (Example 54, where the final concentration of VPN is 0.03 mg / mL). As shown in 3+ Figure E, the apoptosis-inducing effects of ACF / VPN@PM / Fe Figure 5 gel were investigated. As shown in 3+4T1 cells treated with gel and laser irradiation had the largest number of apoptotic cells compared to other groups, indicating that the strongest apoptosis-inducing effect on 4T1 cells could be produced under this condition.

[0122] Example 59

[0123] Hydrogel carrier PM / Fe 3+ Investigation of the in vivo retention of gel:

[0124] Among them: PM-Cy5 / Fe 3+ gel was obtained by the preparation method of Example 54. The ACF / VPN nanocomposite was replaced with an equal volume of ultrapure water, and the PM solution was replaced with an equal amount of PM-Cy5 solution, keeping the concentrations of PM-Cy5 and PM the same. The PM-Cy5 solution was prepared according to Example 57.

[0125] A 4T1 breast cancer orthotopic model was established using 6-8-week-old female BALB / c mice. When the volume of the orthotopic tumor in the tumor-bearing mice grew to 100-150 mm 3 , the tumor-bearing mice were randomly grouped for intratumoral injection.

[0126] (1) In vivo imaging investigation: Using a small animal in vivo imager, the retention of 50 μL of PM-Cy5 / Fe 3+ gel (the final concentration of PM-Cy5 in the hydrogel system was 2.81% (g / mL), and the final concentration of Fe 3+ was 25 mmol / L) in the tumor-bearing mice was investigated on days 0-11 after injection. As shown in Appendix 3+ A, as time extended, PM-Cy5 / Fe Figure 6 gel still had strong fluorescence at the tumor site on day 11 after administration, indicating its excellent intratumoral retention ability. 3+

[0127] (2) Ex vivo tissue imaging investigation: On day 11, the mice in step (1) were sacrificed, and the normal tissues (heart, liver, spleen, lung, kidney) and tumor tissues of each group of mice were taken out. Using a small animal in vivo imager, the fluorescence distribution in the tumor tissues and normal tissues was investigated. As shown in Appendix Figure 6 B, the tumor imaging results of PM-Cy5 / Fe 3+ gel also showed excellent intratumoral retention effect of PM-Cy5 / Fe 3+ gel, and there was no fluorescence distribution in the normal tissues. Therefore, the PM-Cy5 / Fe 3+ gel hydrogel carrier could significantly prolong the retention time of the drug at the orthotopic site in mice and enhance the anti-tumor efficacy.

[0128] Example 60

[0129] Antitumor intelligent double-crosslinked hydrogel drug delivery system ACF / VPN@PM / Fe 3+ Investigation of the in vivo antitumor, in situ and systemic tumor immune activation, and antitumor metastasis properties of gel:

[0130] (1) Investigation of in situ tumor pharmacodynamics: A 4T1 breast cancer in situ model was established using 6-8-week-old female BALB / c mice. When the in situ tumor volume of the tumor-bearing mice grew to 100-150 mm 3 ³, the tumor-bearing mice were randomly divided into 6 groups. The injection dose of VPN in each treatment group was controlled at 5 mg / kg of mouse body weight, and the injection dose of DFO was 20 mg / kg of mouse body weight. After intratumoral injection, the in vivo in situ tumor effects of PM / Fe 2 gel (Example 30), VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+ gel (Example 54, where the injection dose of VPN is 5 mg / kg and the injection dose of DFO is 20 mg / kg), and PBS and ACF / VPN@PM / Fe 3+ gel (Example 54, where the injection dose of VPN is 5 mg / kg) without laser irradiation were investigated under 660 nm, 0.25 W / cm Figure 7 A and 7B show that compared with other control groups, the ACF / VPN@PM / Fe 3+ gel group under laser irradiation had the smallest in situ tumor volume, indicating the strongest in situ tumor efficacy under this treatment condition. In addition, the in vitro tumor volume under the treatment of VPN@PM / Fe 3+ gel+L was significantly smaller than that of the PM / Fe 3+ gel+L group. This is because ROS with cell-killing effects are generated during the PDT process mediated by VPN as a photosensitizer, which can have a strong killing effect on tumor tissues. At the same time, the in vitro tumor volume under the treatment of ACF / VPN@PM / Fe 3+ gel+L was significantly smaller than that of the ACF / VPN@PM / Fe 3+ gel group, further verifying the excellent antitumor effect of PDT; Through ACF / VPN@PM / Fe 3++DFO gel+L group and ACF / VPN@PM / Fe 3+ Comparison between gel+L group shows that ACF / VPN@PM / Fe 3+ gel+L can effectively induce ferroptosis of tumor cells, and ACF / VPN@PM / Fe 3+ The gel+L-induced ferroptosis of tumor cells can be attenuated by the ferroptosis inhibitor DFO; VPN@PM / Fe 3+ gel+L group and ACF / VPN@PM / Fe 3+ The comparison between the gel+L group shows that ACF has an excellent anti-tumor effect. The synergistic effect between the components shows that the anti-tumor intelligent double cross-linked hydrogel drug delivery system ACF / VPN@PM / Fe 3+ gel+L has the strongest anti-tumor effect.

[0131] (2) Investigation of immune activation of in situ tumor tissue: Tumor-bearing mice were grouped and treated with medication according to the method in step (1) of Example 60. Mice were killed 48 hours after the end of treatment and in situ tumors were removed. Each tumor tissue was minced into 1-2 mm 3 Small pieces of tissue were placed in 15 mL centrifuge tubes with 5 mL of DMEM high-glucose complete medium containing 3.33 mg collagenase I, 0.45 mg dispase II, 0.013 mg DNA enzyme I and 0.08 mg hyaluronidase, mixed evenly and placed in a 37 ° C shaker at 300 rpm for 10 minutes. The digested mixture was fully ground and sieved with a cell sieve with a 70 μm pore size to prepare a single cell suspension of each in situ tumor sample. The single cell suspension was then centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and 4 mL of 1× red blood cell lysis buffer (0.01 M) was added respectively. After thorough mixing, each group of samples was treated for 5 minutes to completely lyse the red blood cells in the suspension. After lysis, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 1 mL of ice PBS to resuspend the cells at the bottom of the tube, and after accurate counting, take 1×10 6 Each cell was placed in 100 μL of staining buffer and fully suspended. PE / Cy7-labeled anti-CD45, APC-labeled anti-CD3, and PE-labeled anti-CD8 flow cytometry antibodies were added to each cell suspension under light-proof conditions. After fully mixing, staining was performed for 15 minutes. Finally, flow cytometry was used to detect the cytotoxic T cells (CD8 + T cell) infiltration. Figure 7 As shown in C, compared with other control groups, the ACF / VPN@PM / Fe 3+ In situ tumor CD8 +The T cell infiltration reached the maximum (29.1%), producing the strongest in situ tumor immune activation effect.

[0132] (3) Investigation of systemic tumor immune activation: Tumor-bearing mice were grouped and treated with drugs according to the method in step (1) of Example 60. Mice were sacrificed 48 hours after the end of treatment and lymph nodes were obtained. Pre-cooled PBS was used and each lymph node sample was placed on a cell sieve with a pore size of 70 μm and ground into a single cell suspension. Each sample was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. The cells were resuspended in 200 μL PBS and accurately counted. 1×10 6 Each cell was placed in 100 μL of staining buffer and fully suspended. APC-labeled anti-CD11c, PE-labeled anti-MHC-Ⅱ, FITC-labeled anti-CD80, and PE / Cy7-labeled anti-CD86 flow cytometry antibodies were added to each cell suspension under light-proof conditions. After thorough mixing, staining was performed for 15 minutes. Finally, flow cytometry was used to detect the infiltration of mature dendritic cells (DCs) in the lymph nodes of tumor-bearing mice under different treatment conditions. Figure 7 As shown in D, compared with other control groups, the ACF / VPN@PM / Fe 3+ Mature DCs (CD11c + MHC-Ⅱ + CD80 + CD86 + The intracellular filtration of DCs reached a maximum of 29.7%, producing the most prominent systemic tumor immune activation effect.

[0133] (4) Investigation of HIF-1α inhibition in in situ tumor tissue: Tumor-bearing mice were grouped and treated with medication according to the method in step (1) of Example 60. Mice were killed 48 hours after the end of treatment and the in situ tumors were removed and placed in a 10-fold volume of paraformaldehyde solution for fixation at room temperature for one week. After fixation, each tumor sample was prepared into paraffin sections and stained with HIF-1α and DAPI. The sections of each group were observed under an upright fluorescence microscope. Figure 7 As shown in E, red fluorescence indicates the expression of HIF-1α, and blue fluorescence indicates the distribution of cell nuclei. Among them, the red fluorescence of the PBS group was the strongest, indicating that the tumor tissue was in a hypoxic environment; 3+ +DFO gel+L and ACF / VPN@PM / Fe 3+ The comparison with gel+L group showed that Fe 3+ It can alleviate tumor hypoxia; at the same time, by combining VPN@PM / Fe 3+ gel+L and ACF / VPN@PM / Fe 3+When compared with the gel+L group, it can be demonstrated that ACF can effectively inhibit the expression of HIF-1α, thereby alleviating tumor hypoxia; in addition, by using PM / Fe 3+ gel+L and VPN@PM / Fe 3+ the gel+L group and ACF / VPN@PM / Fe 3+ gel and ACF / VPN@PM / Fe 3+ When compared with the gel+L group, it can be demonstrated that the PDT mediated by VPN consumes oxygen during the treatment process, thereby upregulating the expression of HIF-1α in tumor tissues.

[0134] (5) Investigation of anti-tumor lung metastasis performance: The tumor-bearing mice were grouped and treated with drugs according to the method in step (1) of Example 60. After the treatment, they were continuously raised for 15 days. Subsequently, all the mice were sacrificed, and the lung tissues of each mouse were dissected, rinsed with PBS, and then placed in 10 times the volume of Bouin's fixative, and fixed in the dark at room temperature for one week. After the fixation, each lung tissue sample was photographed. At the same time, each lung tissue sample was separately processed to prepare paraffin sections, stained with hematoxylin and eosin (H&E) dyes, and observed under an upright optical microscope for each group of sections to investigate the inhibitory effect of in-situ tumor lung metastasis in mice of different treatment groups. As shown in Figure 7 Figure F, the black arrows indicate the nodules of tumor lung metastasis. Compared with other control groups, the number of tumor lung metastasis nodules in the ACF / VPN@PM / Fe 3+ gel+L group was the least; at the same time, the ACF / VPN@PM / Fe 3+ gel+L group had the most complete lung structure, and tumor metastasis was almost completely inhibited, which was the result of the synergistic anti-tumor effect of PM, Fe 3+ , VPN, and ACF, indicating that the treatment of this group had the strongest inhibitory effect on tumor lung metastasis. Combining with the H&E sections of the lung tissues of the tumor-bearing mice shown in Figure 7 Figure G, it can be seen that the ACF / VPN@PM / Fe 3+ gel+L group had the most complete lung tissue structure, and the number of nodules was significantly less than that of other control groups, also indicating that the treatment of this group had the strongest inhibitory effect on tumor lung metastasis.

[0135] (6) In vivo safety evaluation of the preparation: A 4T1 breast cancer in-situ model was established using 6-8-week-old female BALB / c mice. When the volume of the in-situ tumor of the tumor-bearing mice grew to 100-150 mm 3 , the tumor-bearing mice were randomly divided into 5 groups. The injection dose of VPN in each treatment group was controlled at 5 mg / kg of the mouse body weight. After intratumoral injection, PBS, PM / Fe 3+ gel (Example 30), VPN@PM / Fe 3+gel, ACF / VPN@PM / Fe 3+ +DFO gel, ACF / VPN@PM / Fe 3+ The in vivo safety of gel (Example 54, where the injection dose of VPN is 5 mg / kg and the injection dose of DFO is 20 mg / kg). During this period, the body weight of the mice was measured every two days. Ten days after the administration ended, the mice in each group were sacrificed, and the heart, liver, spleen, lungs, and kidneys were collected and placed in 10 times the volume of 4% paraformaldehyde solution for fixation at room temperature for one week. Each fixed tissue was processed to prepare paraffin sections and stained with H&E. The sections of each group were observed under an upright optical microscope to complete the in vivo safety evaluation of the mice in different administration groups. As shown in Figure 8 Figure A, there were no obvious fluctuations in the body weight of the mice in each group during the treatment period. At the same time, as shown in Figure 8 Figure B, no obvious pathological damage was found in the heart, liver, spleen, lungs, and kidneys of the mice in each group after administration, indicating that all the preparations had good in vivo safety.

Claims

1. An anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets, characterized in that, It includes a hydrogel carrier, a photodynamic carrier material and a drug. The hydrogel carrier is a drug delivery carrier formed by crosslinking polybiguanide with Fe 3+ and a crosslinking agent; the photodynamic carrier material is violet phosphorus nanosheets; the drug is a drug with both hypoxia-inducible factor-1α inhibition and PD-L1 downregulation effects. The drug is loaded on the surface of the violet phosphorus nanosheets to form a nanocomplex and encapsulated in the hydrogel to form a drug delivery system.

2. The anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 1, wherein The hydrogel carrier is formed by metformin poly(dimethylbiguanide) forming coordination bonds and Schiff base bonds with Fe 3+ and crosslinker diphenylaldehyde polyethylene glycol respectively to crosslink and form a hydrogel network. Among them, the direct killing effect of metformin poly(dimethylbiguanide)-mediated tumor tissue and Fe 3+ reduced by glutathione in the tumor to obtain Fe 2+ mediated ferroptosis of tumor cells are combined for anti-tumor treatment.

3. The anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 1, wherein The photosensitizer carrier material is purple phosphorus nanosheets, which are used as a nano-drug carrier to efficiently load drugs that can produce a tumor tissue killing effect. At the same time, it has excellent photosensitizing effect, good biocompatibility and the potential for photodynamic anti-tumor therapy.

4. The anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 1, wherein The drug is acriflavine hydrochloride, which has two functions: inhibiting hypoxia-inducible factor-1α and downregulating PD-L1. By inhibiting the expression of hypoxia-inducible factor-1α, it reverses the tumor hypoxic microenvironment, enhances the effect of photodynamic therapy, and synergistically enhances the ferroptosis of tumor cells mediated by Fe 3+ reduced by glutathione in the tumor to obtain Fe 2+ ​ 5. A preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 1, characterized in that, It includes the following steps: (1) Mix purple phosphorus particles with an organic solvent, and obtain a crude dispersion after ultrasonic treatment. Centrifuge at a low speed to collect the upper-layer purple phosphorus nanosheet dispersion, and then centrifuge at a high speed to collect the precipitate at the bottom of the centrifuge tube. Add ultrapure water and perform ultrasonic dispersion to obtain Material A. (2) Dissolve the drug that can directly kill tumor tissue in ultrapure water to obtain Material B. Under stirring conditions, add Material B to Material A dispersed in step (1). After continuous stirring reaction, ultrafiltration and centrifugation are used to remove the excess drug, and then add ultrapure water and perform ultrasonic treatment to obtain the drug / purple phosphorus nanocomposite Material C. (3) Dissolve polybiguanide in ultrapure water to obtain Material D, dissolve anhydrous iron salt in ultrapure water to obtain Material E, and dissolve dibenzaldehyde polyethylene glycol in ultrapure water to obtain Material F. Add Material E and Material C to Material D in sequence. After sufficient stirring, adjust the system to physiological pH, and then add Material F. After sufficient mixing, let it stand for a while to obtain the anti-tumor intelligent double-crosslinked hydrogel drug delivery system.

6. The preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 5, characterized in that, In step (1), 20-100 mg of purple phosphorus particles are dispersed in 10-50 mL of acetone.

7. The preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 5, characterized in that, In step (1), when preparing the crude dispersion of purple phosphorus nanosheets, the probe ultrasonic power is 100-300 W, working for 1-4 s, intermittent for 1-4 s, and the total ultrasonic time is 18-30 h; the low-speed centrifugation speed of the crude dispersion is 2000-5000 rpm, and the centrifugation time is 0.5-5 min; the high-speed centrifugation speed is 12000-15000 rpm, and the centrifugation time is 10-30 min; when adding ultrapure water to resuspend and then disperse the small-sized purple phosphorus nanosheets, the probe ultrasonic power is 100-200 W, working for 1-4 s, intermittent for 1-4 s, and the total ultrasonic time is 5-35 min.

8. The preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 5, characterized in that, In step (2), the feeding mass ratio of purple phosphorus nanosheets to the drug is 1:0-1:8, and the stirring time is 4-10 h.

9. The preparation method of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets according to claim 5, characterized in that, In step (3), the final concentration of purple phosphorus nanosheets in the hydrogel system is 0.02-5 mg / mL, the final concentration of Material D is 1.5-3% (g / mL), the final concentration of Material E is 0-100 mmol / L, and the final concentration of Material F is 0.04-8.5% (g / mL).

10. An application of the anti-tumor intelligent double-crosslinked hydrogel drug delivery system based on purple phosphorus nanosheets as claimed in claim 1 in the preparation of anti-tumor drugs for photodynamic therapy.

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