Preparation method and application of multifunctional targeting nano material for treating various cancers

Abstract

The invention provides a preparation method and application of a multifunctional targeting nano material for treating various cancers. A biotin derivative with a biotin receptor targeting capability is modified on the surface of polycyclodextrin, and is mixed and assembled with a Golgi apparatus targeting photosensitizer and a PD-L1 in-situ inhibitor, so that the constructed novel multifunctional targeting nano material can accurately recognize various tumor cells in a targeting manner, and the enrichment of the photosensitizer at a tumor part is increased; the accumulation in the golf apparatus can be further realized, and the structure and the function of the golf apparatus can be obviously damaged. In addition, the nano drug delivery system can also realize effective PD-L1 down-regulation, and the photodynamic synergistic immune anti-tumor effect is further enhanced. The novel multifunctional targeted nano drug-loading system prepared by the invention solves the problems of poor targeting and biological solubility and limited immune response ability and treatment effect of the body of the traditional photosensitizer, and provides an innovative strategy for realizing targeted photodynamic synergistic immunotherapy of cancers.

Description

Technical Field

[0001] The present invention belongs to the field of novel targeted nanomedicines, and specifically relates to multifunctional targeted nanomaterials for treating various cancers, as well as preparation methods and applications thereof. Background Art

[0002] Cancer is a major disease that poses a serious threat to human health and life worldwide. Characterized by high morbidity and mortality, it brings immense physical and mental suffering and economic burdens to patients and their families, and also has a certain impact on social development and stability. However, cancer treatment still faces several major challenges. With the continuous advancement of science and technology and the increasing awareness of people's health, traditional cancer treatments such as surgery, chemotherapy, and radiotherapy have achieved certain results, but they still have many limitations, such as significant side effects and limited effectiveness in advanced cancer. Therefore, there is an urgent need to develop new treatments and technologies to improve cancer cure rates and patients' quality of life.

[0003] Photodynamic therapy (PDT) has emerged as a novel cancer treatment modality due to its unique advantages, including minimal invasiveness, high efficacy, and lack of significant drug resistance. However, traditional photosensitizers, such as phthalocyanines and porphyrins, suffer from limited tumor tissue targeting and biosolubility, resulting in limited PDT therapeutic efficacy. With the development and application of nanomaterial technologies, the construction of nanodelivery systems as photosensitizer carriers has undoubtedly provided a new platform for the development of PDT, enhancing the selective accumulation of photosensitizers at tumor sites. Unfortunately, this strategy still faces two obstacles: 1) poor biosolubility of nanocarriers and premature leakage of photosensitizers, leading to enhanced toxic side effects; and 2) the short half-life (<0.04 μs) and limited radius of action (<0.02 μm) of ROS, which restrict their effectiveness to the site of generation, further limiting PDT therapeutic efficacy. Therefore, to address these issues, the development of soluble nanodelivery carriers with tumor cell and subcellular organelle targeting capabilities is urgently needed to effectively enhance the anti-tumor efficacy of photodynamic therapy.

[0004] Biotin is a water-soluble B vitamin that plays a vital role in the normal physiological metabolism of fats and proteins in cells, promoting cell proliferation and differentiation. Therefore, biotin is considered essential for cell growth and proliferation, and tumor cells require more biotin than normal cells to maintain their rapid growth, proliferation, and differentiation. Biotin receptors are overexpressed on the surfaces of various tumor cells, making them useful as tumor targets for delivering drugs to tumor sites. Studies have found that covalently linking drugs to biotin can increase drug absorption and uptake by tumor cell tissue. Therefore, biotin can be used as a target for tumor cell lines.

[0005] The Golgi apparatus is the core organelle for protein glycosylation, modification and sorting. In tumor cells, abnormal Golgi apparatus function can lead to changes in protein glycosylation patterns, thereby affecting the signal transduction of cancer cell surface receptors (such as EGFR, HER2), cell adhesion (such as integrin) and invasion ability. For example, abnormally glycosylated proteins may promote the proliferation, metastasis and immune escape of tumor cells. Therefore, targeting the Golgi apparatus may have a more significant inhibitory effect on cancer cells and reduce damage to normal cells. However, how to accurately target the Golgi apparatus of tumor cells and avoid toxicity to normal cells is still a technical difficulty in related fields at this stage. More selective drugs or targeted delivery systems need to be developed to achieve more effective anti-tumor purposes.

[0006] In recent years, therapeutic strategies using anti-PD-L1 monoclonal antibodies to block the PD-1 / PD-L1 signaling pathway have demonstrated enhanced anti-tumor efficacy. However, their widespread use remains limited by the fact that they can only disrupt binding to the tumor surface and cannot modulate the immune regulation of PD-L1 protein in the cytoplasm or nucleus. Furthermore, they are associated with adverse immune responses and high costs. Studies have shown that disrupting mitochondrial oxidative phosphorylation (OXPHOS) can lead to reduced mitochondrial energy content (e.g., increased ADP / ATP ratio), promoting activation of adenosine monophosphate-activated protein kinase (AMPK), and thus leading to the phosphorylation and degradation of PD-L1, thereby inhibiting PD-L1 expression in situ and thereby eliminating the immune checkpoint inhibitory pathway at its source. However, while several commonly used anticancer drugs (e.g., lonidamine (LND) and papaverine) can act as OXPHOS disruptors, they still face challenges such as insufficient localization within tumor cells and mitochondria, and high dosage requirements. Based on this, increasing the mitochondrial targeting of OXPHOS disruptors and seeking a safe and efficient tumor-targeted delivery strategy will hopefully achieve in situ inhibition of PD-L1, thereby further enhancing the immune anti-tumor effect. Summary of the Invention

[0007] The present invention aims to provide a method for preparing multifunctional targeted nanomaterials for the treatment of various cancers. First, the indomethacin (IMD) ligand with Golgi targeting function is covalently coupled with the photosensitizer pyropheophorbide-a (Ppa) to obtain the photosensitizer IPA with Golgi targeting ability. Subsequently, a biotin derivative is modified on the surface of a polycyclodextrin material through host-guest interaction to obtain a nanodelivery material Bi-CD with biotin receptor targeting ability. Simultaneously, triphenylphosphine with mitochondrial precise targeting ability is covalently linked with the anti-tumor drug lonidamine (LND) to obtain a lonidamine derivative PND with mitochondrial specific targeting ability. Finally, the photosensitizers IPA and PND are mixed with Bi-CD to assemble into a multifunctional nanomaterial Bi-CD@IPA@PND with tumor cell targeting, Golgi targeting, and mitochondrial targeting functions. The high loading rate and high biocompatibility of polycyclodextrin can reduce biological toxicity. In addition, the biotin derivatives loaded on its surface can accurately target and identify tumor cells, thereby promoting the enrichment of photosensitizers and anticancer drugs in the tumor site. The Golgi-targeted photosensitizer IPA wrapped in the material can further achieve the accumulation of more photosensitizers in the Golgi apparatus, and the generated ROS can directly act on the Golgi apparatus, destroying its structure and function, thereby significantly improving the tumor inhibition ability of targeted photodynamic therapy. In addition, the anticancer drug PND with precise mitochondrial targeting ability can significantly destroy mitochondrial function, enhance the inhibition of mitochondrial oxidative phosphorylation (OXPHOS), achieve effective PD-L1 downregulation, and further enhance the photodynamic synergistic immune anti-tumor effect. The multifunctional targeted nano-drug delivery system prepared by the present invention has high biocompatibility and significant tumor cell, Golgi apparatus and mitochondrial targeting, and exhibits negligible toxic side effects and significantly improves the photodynamic anti-tumor effect, providing an innovative strategy for achieving targeted photodynamic synergistic immunotherapy of tumors. At the same time, the present invention also provides a multifunctional targeted nano-delivery material obtained by the above method for treating various cancers, as well as applications of the nano-delivery material.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention discloses a method for preparing a multifunctional targeted nanomaterial for treating various cancers, comprising the following steps:

[0010] (1) First, a 15 wt% NaOH aqueous solution is prepared, and then the weighed β-CD is dissolved therein and stirred at room temperature. Subsequently, toluene is slowly added dropwise to the mixture while continuing to stir, and then epichlorohydrin is slowly added dropwise as a cross-linking agent to continue the reaction. After the reaction is completed, the crude product is pre-precipitated with isopropanol, and the precipitate is transferred to an appropriate amount of water and adjusted to neutrality with dilute hydrochloric acid. It is dialyzed with a dialysis bag to completely remove monomers and oligomers, and finally vacuum freeze-dried to obtain a white solid product, Poly-β-CD;

[0011] (2) Accurately weigh biotin, add anhydrous DMF solution and stir to mix evenly, then accurately weigh NHS and EDCI are added to the mixed reaction solution, react in an ice-water bath, then accurately weigh adamantane amine is added to the mixed solution, and react with stirring at room temperature. After the reaction is completed, the mixture is purified by column chromatography and vacuum dried to obtain a white solid compound Bi-Ad;

[0012] (3) Accurately weighed indomethacin was added to the DMF solution, followed by the addition of tert-butyl 6-aminohexylcarbamate, HATU, and DIEA, and stirred at 25°C under nitrogen for 2 h. After the reaction, the mixture was extracted, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography to obtain a solid product, IMDH-Boc.

[0013] (4) HCl was added to the ethyl acetate solution and stirred at 0°C for 2 h to obtain tert-butyl 6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)hexylcarbamate. After the reaction was completed, the mixture was extracted and the organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a solid product, IMDH.

[0014] (5) Accurately weigh pyropheophorbide a and add it to the DMF solution, then add the IMDH obtained in step (4), and add TCFH and NMI to the mixed solution, and stir at 25°C under nitrogen protection for 2 hours. After the reaction is completed, the reaction mixture is extracted, and the organic phase is dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The mixture is purified by column chromatography to obtain a solid product IPA;

[0015] (6) Accurately weigh 6-bromo-1-hexanol and dissolve it in anhydrous acetonitrile solution, and add triphenylphosphine. Under nitrogen protection, heat under reflux at 85°C for 48 hours. After the reaction is completed, remove the solvent under reduced pressure, and purify the mixture by column chromatography to obtain white solid TPH;

[0016] (7) Accurately weighed lonidamine was added to DCM, and TPH obtained in step (6) was added, and then DCC and DMAP were added to the reaction mixture, and stirred at 25°C under nitrogen protection for 2 hours. After the reaction was completed, the reaction mixture was extracted, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography to obtain white solid PND;

[0017] (8) The Poly-β-CD obtained in step (1), the IPA obtained in step (5), and the PND obtained in step (7) were dissolved in a mixed solution of DMSO and water, and reacted at room temperature in the dark for 2 h. Subsequently, the Bi-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water, and then slowly added dropwise to the reaction system. The reaction was continued at room temperature in the dark for 12 h. After the reaction was completed, the reaction mixture was transferred to a dialysis bag for dialyzation. Finally, the reaction solution was freeze-dried to obtain brown-black solid powder Bi-CD@IPA@PND NPs.

[0018] Furthermore, the dialysis condition in step (1) is W =8000 dialysis bag for 24 h, changing the water every 3 h.

[0019] Furthermore, the extraction solution in step (3) is 10 mL of H2O and 20 mL of ethyl acetate.

[0020] Furthermore, the extraction solution in step (4) is 20 mL of ethyl acetate.

[0021] Furthermore, the extraction solution in step (5) is 15 mL of H2O() and 20×2 mL of dichloromethane.

[0022] Furthermore, the extraction solution in step (7) is 15 mL of H2O and 20 mL of dichloromethane.

[0023] Furthermore, the ratio of the mixed solution of DMSO and water in step (8) is 1:3. W =3000 dialysis bag for 24 h, changing the water every 3 h.

[0024] The second aspect of the present invention is to disclose a multifunctional targeted nanomaterial for treating various cancers, which is prepared by the above method.

[0025] The third aspect of the present invention is to disclose the application of the multifunctional targeted nanomaterial for treating various cancers in a nano-drug delivery system.

[0026] The significant advantages of the present invention are:

[0027] (1) Polycyclodextrin-based nanodelivery carriers have good biocompatibility and stability under physiological conditions, and have the ability to release drugs in response to the disintegration of the tumor microenvironment. They can significantly reduce the toxic side effects and adverse reactions caused by premature leakage and poor solubility of nanodrugs during transportation, and can serve as ideal nanodrug carriers.

[0028] (2) Nano-delivery materials modified with biotin derivatives exhibit significant targeting recognition capabilities for a variety of cancer cells, providing a promising strategy for targeted cancer therapy.

[0029] (3) The nano-drug delivery system has excellent stability and uniformity, which is conducive to use and long-term storage.

[0030] (4) The preparation method is simple, with few side reactions, readily available raw materials, and low cost, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Transmission electron microscopy and particle size distribution diagrams of different nano-drug delivery systems;

[0032] Figure 2 This is a laser confocal microscopy image characterizing the targeting of different nano-drug delivery systems to 4T1 tumor cells and KYSE150 tumor cells;

[0033] Figure 3 :Confocal laser scanning to characterize the Golgi targeting ability of different drugs;

[0034] Figure 4 : The MTT assay was used to detect the inhibitory effects of different drug groups on the activity of 4T1 and KYSE150 tumor cells; DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] The present invention provides a method for preparing a multifunctional targeted nanomaterial for treating various cancers, comprising the following steps:

[0037] (1) First, accurately weigh 10.0 g of β-CD and dissolve it in 15 mL of a 15 wt% aqueous solution of NaOH. Stir at room temperature for 2 h. Subsequently, 2 mL of toluene was added dropwise to the mixture and stirring continued for 2 h. 3.8 mL of epichlorohydrin as a cross-linking agent was then slowly added dropwise and the reaction continued for 3 h. After the reaction, the crude product was pre-precipitated with 200 mL of isopropanol. The precipitate was transferred to an appropriate amount of water and adjusted to neutrality with dilute hydrochloric acid. Finally, the product was dialyzed for 2 days using a MW = 8000 dialysis bag, with the water changed every 3 h to completely remove monomers and oligomers. Finally, the product was freeze-dried in vacuo to obtain a white solid product, Poly-β-CD.

[0038] (2) Accurately weighed biotin (0.5 g, 2.0 mmol) was placed in a 50 mL round-bottom flask, and 30 mL of anhydrous DMF solution was added and stirred to mix evenly. Accurately weighed NHS (0.3 g, 2.4 mmol) and EDCI (0.5 g, 2.4 mmol) were then added to the mixed reaction solution, and the mixture was reacted in an ice-water bath for 1 h. Subsequently, accurately weighed adamantane (0.3 g, 2.1 mmol) was added to the mixture, and the mixture was stirred at room temperature for 12 h. After the reaction, the mixture was purified by column chromatography (CH2Cl2 / CH3OH 30:1 v / v) to obtain 0.48 g of a white solid compound, Bi-Ad, with a yield of 62%. 1 H NMR(400MHz, DMSO-d6)δ=8.07(s,1H),7.22(s,1H),4.66(s,1H),3.51(s,2H),1.99(s,4 H), 1.91 (d, J = 2.8Hz, 4H), 1.60 (d, J = 2.2Hz, 4H), 1.47 (s, 4H), 1.36 (s, 4H), 0.86 (s, 4H). 13 C NMR (101MHz, DMSO-d6) δ=171.50, 163.80, 62.87, 61.96, 54.79, 44.23, 41.05, 36.69, 35.93, 28.73, 27.93, 25.36, 24.15. HRMS(ESI):m / z calculated for C 20 H 32 N3O2S[M+H] + :378.2215;found:378.2204.

[0039] (3) Accurately weighed indomethacin (0.33 g, 0.93 mmol) was added to DMF (8 mL), followed by the addition of tert-butyl 6-aminohexylcarbamate (0.20 g, 0.93 mmol), HATU (0.70 g, 1.85 mmol) and DIEA (0.24 g, 1.85 mmol), and the mixture was stirred at 25 °C under nitrogen for 2 h. After the reaction, H2O (10 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography (petroleum ether / ethyl acetate 1:1 v / v) to obtain 0.31 g of the product IMDH-Boc with a yield of 61%. HRMS (ESI): m / z calculated for C 30 H 39 ClN3O5[M+H] +:557.2656;found:557.2579.

[0040] (4) HCl (2 mL) was added to an ethyl acetate (0.5 mL) solution and stirred at 0°C for 2 h to obtain tert-butyl 6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)hexylcarbamate (0.3 g). After the reaction was completed, saturated NaHCO3 was added to neutralize the reaction solution, and the mixture was extracted with ethyl acetate (20 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain the product IMDH (0.2 g) with a yield of 85%. 1 H NMR (400MHz, DMSO-d6) δ = 8.16 (s, 1H), 7.94 (br s,2H),7.72-7.62(m,4H),7.15(d,J=2.5Hz,1H),6.93(d,J=9.0Hz,1H),6.71(dd,J=2.6,8.9Hz ,1H),3.77(s,3H),3.51(s,2H),3.11-3.01(m,2H),2.73(t,J=7.5Hz,2H),2.24(s,3H),1.52(br t,J=7.3Hz,2H),1.41(br t,J=6.6Hz,2H),1.33-1.22(m,4H). HRMS(ESI):m / z calculated for C 25 H 32 ClN3O3[M+H] + :457.2132;found:457.2156.

[0041] (5) Accurately weigh pyropheophorbide a (0.1 g, 0.187 mmol) and add it to a DMF (2 mL) solution. Then add IMDH (0.1 g, 0.224 mmol) obtained in step (5), and add TCFH (105 mg, 0.374 mmol) and NMI (31 mg, 0.374 mmol) to the mixed solution. Stir under nitrogen at 25 ° C for 2 h. After the reaction is completed, H2O (15 mL) is added to the mixture and extracted with dichloromethane (20×2 mL). The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The mixture is purified by column chromatography (CH2Cl2 / CH3OH 20:1 v / v) to obtain the product IPA (0.09 g) with a yield of 43%. 1H NMR(400MHz,DMSO-d6)δ=9.70-9.55(m,1H),9.42-9.31(m,1H),8.92-8.81(m,1H),8.28-8.08(m,1H),7.94-7.84(m,1H),7.73-7.51(m,5H),7.09-6.99(m,1H),6.92-6.82(m,1H),6.68-6.56(m,1H),6.43-6.31(m,1H),6.25-6.14(m,1H),5.36-5.01(m,2H),4.66-4.50(m,1H),4.35-4.23(m,1H),4.11-3.99(m,1H),3.73-3.56(m,8H),3.42(brs,3H),3.18(br s,2H),3.02-2.88(m,4H),2.65-2.58(m,1H),2.39-2.29(m,1H),2.13(br s,4H),1.85-1.74(m,3H),1.68-1.56(m,3H),1.38-1.07(m,10H),0.26-0.13(m,1H),1.91-2.08(m,1H)。 13 CNMR(101MHz,DMSO-d6)δ=195.71,172.21,169.44,168.18,162.18,155.99,154.44,150.50,145.26,139.91,137.80,136.49,135.76,135.39,134.69,131.29,130.61,129.42,128.28,115.02,111.29,106.48,102.30,101.88,96.84,94.25,60.19,55.92,51.74,49.85,47.98,32.67,31.50,30.66,29.36,26.49,23.56,18.96,17.87,14.56,13.74,12.53,12.19,11.36。HRMS(ESI):m / z calculated for C 58 H 64 ClN7O5[M+H] + :973.4653;found:973.4618。

[0042] (6) Accurately weigh 6-bromo-1-hexanol (0.72 g, 4.0 mmol) and dissolve it in anhydrous acetonitrile (25 mL). Add triphenylphosphine (1.05 g, 4.0 mmol). Heat under reflux at 85°C for 48 h under nitrogen. After the reaction is complete, remove the solvent under reduced pressure. Purify the mixture by column chromatography (CH2Cl2 / CH3OH 20:1 v / v) to obtain TPH (0.6 g) as a white solid in a 35% yield. 1 H NMR(400MHz, DMSO-d6)δ=7.94-7.88(m,3H),7.85-7.76(m,12H),4.38-4.31(m,1 H),3.65-3.53(m,2H),3.38-3.34(m,2H),1.57-1.42(m,4H),1.39-1.27(m,4H).

[0043] (7) Accurately weighed lonidamine (0.1 g, 0.31 mmol) was added to DCM (10 mL), and TPH (0.14 g, 0.31 mmol) obtained in step (7) was added. Subsequently, DCC (0.096 g, 0.465 mmol) and DMAP (0.057 g, 0.465 mmol) were added to the reaction mixture, and stirred under nitrogen protection at 25°C for 2 h. After the reaction was completed, H2O (15 mL) was added to the mixture, and the mixture was extracted with dichloromethane (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography (CH2Cl2 / CH3OH 30:1 v / v) to obtain white solid PND (0.1 g) with a yield of 43%. 1 H NMR (400MHz, DMSO-d6)δ=8.11-8.06(m,1H),7.92-7.73(m,16H),7.72-7.68(m,1H),7.56-7.50(m,1H),7.40-7.34(m,2H),6.99- 6.91(m,1H),5.90-5.83(m,2H),4.36-4.29(m,2H),3.64-3.52(m,2H),1.77-1.65(m,2H),1.63-1.51(m,4H),1.48-1.39(m,2H). 13C NMR (101MHz, DMSO-d6)δ=162.22,141.36,135.35,134.06,133.66,131.44,130.70,129.58,128.26,127 .75,123.98,123.23,121.81,119.46,118.61,111.35,64.75,50.38,29.95,28.34,25.08,22.11,20.85. HRMS(ESI):m / z calculated for C 39 H 37 Cl2N2O2P + [M-Br] + :667.6022;found:667.5907.

[0044] (8) Poly-β-CD (21.0 mg) obtained in step (1), IPA (2.0 mg) obtained in step (5), and PND (2.0 mg) obtained in step (7) were dissolved in a mixed solution of DMSO and water (1:3) and reacted at room temperature in the dark for 2 h. Subsequently, 0.5 mg of Bi-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water (1:3) and then slowly added dropwise to the reaction system. The reaction was continued at room temperature in the dark for 12 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with MW = 3000 and dialyzed for 24 h, with the water changed every 3 h. Finally, the reaction solution was freeze-dried to obtain brown-black solid powder Bi-CD@IPA@PND NPs.

[0045] Nanomaterial characterization (see Appendix Figure 1 )

[0046] The morphology of Bi-CD NPs and Bi-CD@IPA@PND NPs was characterized using a transmission electron microscope (TEM) at room temperature. Secondly, the hydrodynamic size of Bi-CD NPs and Bi-CD@IPA@PND NPs in aqueous solution was measured using a Malvern Nano-ZSZEN3600. TEM images showed that both nanomaterials exhibited a spherical structure with a size of approximately 100 nm. Then, dynamic light scattering (DLS) further confirmed the hydrodynamic diameter and stability of Bi-CD NPs and Bi-CD@IPA@PND NPs. Their average hydrodynamic diameters were approximately 110 nm and 105 nm, respectively, which was almost consistent with the TEM data, and the polydispersity indices were 0.16 and 0.13, respectively. This indicates that the preparation of Bi-CD@IPA@PND NPs did not change the original morphology of Bi-CD NPs, and they also exhibited a smaller particle size, which was more conducive to tumor cell endocytosis.

[0047] Cell targeting characterization (see Appendix Figure 2 )

[0048] (1) Cell plating: First, select 4T1 cells and KYSE150 cells in good growth condition, remove the old culture medium, wash them three times with sterile PBS, and then digest them with 0.25% trypsin. After centrifugation, add fresh culture medium and resuspend them. Count the cell suspension using a cell counter, and then dilute the cell suspension to 2.0×10 5 cells / mL, add 1 mL to the confocal dish. Finally, all confocal dishes were placed in a cell culture incubator at 37°C and 5% CO2 and incubated overnight.

[0049] (2) Drug addition: After the cells are completely attached, the culture medium in the laser confocal dish is removed, and then washed three times with sterile PBS. Finally, 1 mL of fresh culture medium containing Bi-CD@IPA@PND NPs and CD@IPA@PND NPs at a concentration of 20 μg / mL is added to the laser confocal dish and placed in a cell culture incubator at 37°C and 5% CO2 for 12 h.

[0050] (3) Test: After the incubation is completed, remove the drug-containing culture medium from all laser confocal dishes, wash them three times with sterile PBS, and then add 1 mL of sterile PBS and wait for photography. At the same time, add 0.5 mL of 0.25% trypsin to other cleaned confocal dishes for digestion, centrifuge and resuspend in 1 mL of sterile PBS. Finally, use a laser confocal electron microscope to perform intracellular drug fluorescence testing (excitation wavelength: 633 nm, emission wavelength: 650-750 nm). In the experiment, all cell blank control groups were incubated with drug-free culture medium, and other operations were consistent with the experimental group.

[0051] The fluorescence intensity of Bi-CD@IPA@PND NPs within cells was detected by laser confocal microscopy. In both 4T1 cells and KYSE150 cells, Bi-CD@IPA@PND NPs exhibited a stronger fluorescence signal intensity than CD@IPA@PND NPs, approximately 5 times that of CD@IPA@PND NPs, indicating that Bi-CD@IPA@PND NPs exhibited stronger tumor cell targeting and uptake ability. This indicates that the introduction of biotin greatly enhances the specific recognition of nanomaterials for tumor cells, thereby promoting cellular uptake. Therefore, all cell uptake experimental results indicate that biotin-modified nanocarriers can significantly improve the targeting ability of Bi-CD@IPA@PND NPs to tumor cells overexpressing biotin receptors.

[0052] Characterization of Golgi targeting (see Appendix Figure 3 )

[0053] (1) Plating: First, select 4T1 cells in good growth condition, remove the old culture medium, wash three times with sterile PBS, and then digest with 0.25% trypsin. After centrifugation, add fresh culture medium and resuspend. Count the cell suspension using a cell counter, and then dilute the cell suspension to 1.0×10 4 cells / mL, add 1 mL to the confocal dish. Finally, all confocal dishes were placed in a cell culture incubator at 37°C and 5% CO2 and incubated overnight.

[0054] (2) Drug addition: After the cells are completely attached, the culture medium in the laser confocal dish is removed, and then washed three times with sterile PBS. Finally, 1 mL of fresh culture medium containing Ppa, IPA, or Bi-CD@IPA@PND NPs at a concentration of 20 μg / mL is added to the laser confocal dish and placed in a cell culture incubator at 37°C and 5% CO2 for 12 h.

[0055] (3) Staining: First, 2 μL of the 1 mM Golgi Green probe stock solution was taken out and mixed with fresh culture medium to achieve a probe solution concentration of 2 μM. Then, the drug-containing culture medium in the laser confocal microscopy dish was removed, and the cells were washed three times by centrifugation with sterile PBS. The cells were then resuspended in 1 mL of culture medium containing the probe dye and transferred to the corresponding laser confocal microscopy dish. Finally, the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 20 min.

[0056] (4) Photography: After the co-incubation is completed, the corresponding probe dye in the dish is removed by centrifugation, and sterile PBS is used for centrifugation and washing three times to remove the excess probe dye to prevent interference with the photography background. Then, a small amount of PBS is added to resuspend the cells, and the cells are transferred to a new confocal dish before taking pictures. Finally, the cells are photographed using a laser confocal microscope equipped with a 405nm or 488nm multi-argon laser and a 633nm diode laser. Among them, the excitation and emission wavelength parameters of the photosensitizer compound are: Ex / Em=633 / 650-750nm; the excitation and emission wavelength parameters of the Golgi probe dye are: Ex / Em=488 / 510-570nm.

[0057] Laser confocal microscopy was used to observe the red fluorescence of Ppa and IPA and the green fluorescence of the Golgi probe. The red fluorescence of free Ppa had almost no overlap with the green fluorescence signal representing the Golgi apparatus. Further qualitative analysis of the fluorescence intensity line scan spectrum showed that the Ppa group and the Golgi dye had significantly separated signals. By introducing indomethacin, the red fluorescence signal of the free Ppa group showed significant fluorescence overlap with the Golgi dye after 12 hours of incubation, with yellow fluorescent spots appearing within the Golgi apparatus. Qualitative analysis of the fluorescence intensity line scan spectrum also showed that the IPA group and the Golgi dye had partially overlapping signals, indicating that free IPA has demonstrated visible Golgi targeting ability. Compared with free IPA, the Bi-CD@IPA@PND NPs group showed strong yellow fluorescent spots within the Golgi apparatus. At the same time, qualitative analysis of the fluorescence intensity line scan spectrum also showed a high degree of overlap between IPA and the Golgi dye. Therefore, these data strongly demonstrate that Bi-CD@IPA@PNDNPs can significantly enhance the enrichment of photosensitizers in the Golgi apparatus and achieve the purpose of targeting the Golgi apparatus.

[0058] In vitro photodynamic activity characterization (see Appendix Figure 4 )

[0059] During photodynamic therapy, the cytotoxicity exhibited by photosensitizers is a key component in evaluating their properties. The MTT assay is a widely used method for evaluating a drug's ability to induce cell apoptosis. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide) is a yellow dye, also known as thiazolyl blue. The principle of the MTT assay can be summarized as follows: the mitochondria of living cells contain a reducing enzyme, succinate dehydrogenase, which reduces the yellow MTT to an insoluble, blue-purple crystalline substance, formazan, which then deposits within the cell. However, the mitochondria of dead cells lack this reducing enzyme, making it unable to reduce MTT and produce blue-purple formazan crystals. Related experiments have shown that the organic solvent dimethyl sulfoxide (DMSO) can dissolve the deposited formazan in cells. The absorbance (OD) value at 490 nm or 570 nm is measured using a microplate reader, indirectly providing a corresponding measure of viable cell count. The formula for calculating cell viability (1%) is as follows:

[0060] I%=[(A-A0) / (As-A0)]×100%

[0061] Where A, A0, and As represent the OD values ​​of the drug group, blank solvent control group, and cell solvent control group, respectively. During the experiment, the logarithm of the corresponding drug concentration was plotted on the horizontal axis, and the cell viability (%) was plotted on the vertical axis. Experimental data are presented as the mean ± SD of three independent experiments. Finally, the experimental data were plotted using GraphPad Prism 6.0 software to generate a curve showing the relationship between cytotoxicity and drug dosage.

[0062] Experimental methods

[0063] (1) Plating: Select 4T1 cells and KYSE150 cells in good growth condition, remove the old culture medium, and wash three times with sterile PBS. Then, add 1 mL of trypsin (containing 0.25% EDTA) for digestion and centrifugation. Resuspend the cells in fresh cell culture medium, count them using a cell counter, and dilute them to a density of 8.0 × 10 4 cells / mL. Then, 100 μL of the diluted cell suspension was added to each well of a 96-well plate. Six replicates were set up for each drug concentration group and all control groups to reduce experimental error. Finally, all 96-well plates were placed in a cell culture incubator at 37°C and 5% CO2 for adherent culture.

[0064] (2) Drug addition: First, remove the old culture medium from the 96-well plate and wash each well three times with sterile PBS. Then, add the drug prepared in fresh cell culture medium to different 96-well plates in sequence and incubate in a cell culture incubator at 37°C and 5% CO2 for 12 hours.

[0065] (3) Illumination: After incubation, remove the old medium containing drugs from the 96-well plate and wash it three times with sterile PBS to completely remove the unabsorbed drugs. Then, add 100 μL of new cell culture medium to each well, and place the 96-well plate for phototoxicity experiment under the LED laser plate (λ = 670 nm, 4 mW / cm 2 , 1.2J / cm 2 ) for 10 minutes. Finally, incubate in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The dark toxicity experiment was performed without light exposure. Other steps were the same as those for the phototoxicity experiment and were performed in a dark environment.

[0066] (4) Test: After 24 hours of culture, the 96-well plates containing both cells were removed from the incubator and 10 μL of the pre-prepared MTT solution was added to each well. The plates were then placed in a cell culture incubator and cultured for another 4 hours. The supernatant in the 96-well plates was then removed and 100 μL of DMSO was added to each well. The plates were then shaken at 37°C for 30 minutes to fully dissolve any formazan that might have formed. Finally, the OD value of the 96-well plates was measured at 570 nm using a microplate reader.

[0067] The experimental data show that in the absence of laser irradiation, no significant cytotoxicity was observed in all drug groups on 4T1 cells or KYSE150 cells, indicating that the dark toxicity of different drug groups was negligible. However, under the LED laser panel (λ = 670nm, 4mW / cm 2 , 1.2J / cm 2 ) for 10 min, the photocytotoxicity of the Bi-CD@IPA@PND NPs group and Bi-CD@IPANPs was significantly higher than that of the single-targeted IPA, CD@IPA@PND NPs group and the blank control group, indicating that within a smaller concentration range, the drug group with tumor cell and Golgi apparatus targeting function showed more significant killing ability against 4T1 cells and KYSE150 cells, thereby achieving stronger anti-tumor cell proliferation ability.

[0068] Based on the above embodiments, the present invention provides a method for preparing a multifunctional targeted nanomaterial for the treatment of various cancers. The resulting multifunctional targeted nanomaterial has been verified to accurately target and recognize various tumor cells, increase the accumulation of photosensitizers at the tumor site, and further achieve accumulation in the Golgi apparatus, significantly disrupting its structure and function. This overcomes the problems of traditional photosensitizers, such as poor targeting and biosolubility, limited immune response, and limited therapeutic efficacy, and provides an innovative strategy for achieving targeted photodynamic synergistic immunotherapy for cancer. This has achieved outstanding technical results.

[0069] It should be further noted that the above embodiments are intended only to facilitate understanding of the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. Any obvious adjustments and modifications to the technical solution of the present invention that fall within the technical concept of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional targeted nanomaterial for treating multiple cancers, comprising the following steps: (1) β-CD is dissolved in a NaOH aqueous solution and reacted with a cross-linking agent to obtain poly-β-CD; (2) adding biotin to an anhydrous DMF solution, then adding NHS, EDCI, and adamantane to the mixed reaction solution, and reacting in an ice-water bath to obtain Bi-Ad; (3) dissolving indomethacin, tert-butyl 6-aminohexylcarbamate, HATU, and DIEA in anhydrous DMF solution to react and obtain IMDH-Boc; (4) adding HCl to an ethyl acetate solution, stirring at 0°C to obtain tert-butyl 6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)hexylcarbamate, and extracting and drying to obtain IMDH; (5) adding pyropheophorbide a to a DMF solution, then adding IMDH obtained in step (4), and adding TCFH and NMI to the mixed solution. After the reaction is completed, the mixture is extracted and dried, and the mixture is purified by column chromatography to obtain a solid product IPA; (6) Accurately weigh 6-bromo-1-hexanol and dissolve it in anhydrous acetonitrile solution, and add triphenylphosphine. Heat under reflux under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure and purify the mixture by column chromatography to obtain white solid TPH; (7) Add accurately weighed lonidamine to DCM, and add TPH obtained in step (6), then add DCC and DMAP to the reaction mixture, and stir under nitrogen protection. After the reaction is completed, extract the reaction mixture, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate in vacuo to obtain a residue, and purify the mixture by column chromatography to obtain white solid PND; (8) The Poly-β-CD obtained in step (1), the IPA obtained in step (5), and the PND obtained in step (7) are dissolved in a mixed solution of DMSO and water, and the Bi-Ad obtained in step (2) is dissolved in a mixed solution of DMSO and water, and then slowly added dropwise to the reaction system to react to obtain the final product Bi-CD@IPA@PND NPs.

2. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: In step (1), first, 10.0 g of β-CD was accurately weighed and dissolved in 15 mL of a 15 WT% NaOH aqueous solution, and stirred at room temperature for 2 h. Subsequently, 2 mL of toluene was added dropwise to the mixture and stirring was continued for 2 h. Then, 3.8 mL of epichlorohydrin as a cross-linking agent was slowly added dropwise and the reaction was continued for 3 h. After the reaction was completed, the crude product was pre-precipitated with 200 mL of isopropanol, and the precipitate was transferred to an appropriate amount of water and adjusted to neutrality with dilute hydrochloric acid. Finally, it was dialyzed with a MW = 8000 dialysis bag for 2 days, and the water was changed every 3 h to completely remove monomers and oligomers. Finally, the white solid product Poly-β-CD was obtained by vacuum freeze drying.

3. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: In step (2), 0.5 g, 2.0 mmol of biotin was accurately weighed and placed in a 50 mL round-bottom flask, 30 mL of anhydrous DMF solution was added and stirred to mix evenly, then 0.3 g, 2.4 mmol of NHS and 0.5 g, 2.4 mmol of EDCI were accurately weighed and added to the mixed reaction solution, and the mixture was reacted in an ice-water bath for 1 h. Subsequently, 0.3 g, 2.1 mmol of adamantane amine was accurately weighed and added to the mixed solution, and the mixture was stirred and reacted at room temperature for 12 h. After the reaction, the mixture was purified by column chromatography to obtain a white solid compound Bi-Ad; In column chromatography, the volume ratio of CH2Cl2 / CH3OH was 30:

1.

4. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: Accurately weighed 0.33 g and 0.93 mmol of indomethacin were added to 8 mL of DMF, followed by the addition of 0.20 g and 0.93 mmol of tert-butyl 6-aminohexylcarbamate, 0.70 g and 1.85 mmol of HATU, and 0.24 g and 1.85 mmol of DIEA. The mixture was stirred at 25°C under nitrogen for 2 h. After the reaction, 10 mL of H2O was added to the mixture, and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography to obtain the product IMDH-Boc. In column chromatography, the volume ratio of petroleum ether / ethyl acetate was 1:

1.

5. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: 2 mL of HCl was added to 0.5 mL of ethyl acetate solution, and the mixture was stirred at 0°C for 2 h to obtain tert-butyl 6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)hexylcarbamate. After the reaction, saturated NaHCO3 was added to neutralize the reaction solution, and the mixture was extracted with 20 mL of ethyl acetate. The organic layer was washed with 10 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain the product IMDH.

6. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: Accurately weigh 0.1 g, 0.187 mmol of pyropheophorbide a and add it to 2 mL of DMF solution, then add 0.1 g, 0.224 mmol of IMDH obtained in step (4), and add 105 mg, 0.374 mmol of TCFH and 31 mg, 0.374 mmol of NMI to the mixed solution, and stir at 25 ° C under nitrogen protection for 2 h. After the reaction is completed, 15 mL of H2O is added to the mixture, and the mixture is extracted with 20×2 mL of dichloromethane. The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The mixture is purified by column chromatography to obtain the product IPA; The volume ratio of CH2 Cl2 / CH3 OH in column chromatography was 20:

1.

7. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: Accurately weigh 0.72 g, 4.0 mmol of 6-bromo-1-hexanol and dissolve it in 25 mL of anhydrous acetonitrile solution. Then add 1.05 g, 4.0 mmol of triphenylphosphine. Under nitrogen protection, heat under reflux at 85°C for 48 h. After the reaction is complete, remove the solvent under reduced pressure and purify the mixture by column chromatography to obtain TPH as a white solid. The volume ratio of CH2 Cl2 / CH3 OH in column chromatography was 20:

1.

8. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: Accurately weighed 0.1 g, 0.31 mmol of lonidamine was added to 10 mL of DCM, and 0.14 g, 0.31 mmol of TPH obtained in step (6) was added. Subsequently, 0.096 g, 0.465 mmol of DCC and 0.057 g, 0.465 mmol of DMAP were added to the reaction mixture, and stirred at 25°C for 2 h under nitrogen protection. After the reaction was completed, 15 mL of H2O was added to the mixture, and the mixture was extracted with 20 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a residue. The mixture was purified by column chromatography to obtain white solid PND; The volume ratio of CH2 Cl2 / CH3 OH in column chromatography was 30:

1.

9. The method for preparing a multifunctional targeted nanomaterial for treating multiple cancers according to claim 1, characterized in that: 21.0 mg of Poly-β-CD obtained in step (1), 2.0 mg of IPA obtained in step (5), and 2.0 mg of PND obtained in step (7) were dissolved in a mixed solution of DMSO and water in a volume ratio of 1:3, and reacted at room temperature in the dark for 2 h. Subsequently, 0.5 mg of Bi-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water in a volume ratio of 1:3, and then slowly added dropwise to the reaction system. The reaction was continued at room temperature in the dark for 12 h. After the reaction was completed, the reaction mixture was transferred to a dialysis bag with MW = 3000 and dialyzed for 24 h, with water changed every 3 h. Finally, the reaction solution was freeze-dried to obtain brown-black solid powder Bi-CD@IPA@PND NPs.

10. A multifunctional targeted nanomaterial for treating multiple cancers, characterized by: It is prepared by the method according to any one of claims 1 to 9.

Images