Multifunctional targeted nanomaterials for treating various cancers and preparation method and application thereof

Abstract

This invention provides a method for preparing and applying a multifunctional targeted nanomaterial for the treatment of various cancers. By modifying the surface of polycyclodextrin with a biotin receptor-targeting derivative and mixing it with a Golgi-targeting photosensitizer and a PD-L1 in situ inhibitor, the constructed novel multifunctional targeted nanomaterial can precisely target and recognize various tumor cells, increase the accumulation of photosensitizers at tumor sites, and further achieve accumulation in the Golgi apparatus, significantly disrupting its structure and function. Furthermore, this nano-drug delivery system can effectively downregulate PD-L1, further enhancing the photodynamic synergistic immunotherapy effect against tumors. The novel multifunctional targeted nano-drug delivery system prepared by this invention solves the problems of poor targeting and biosolubility of traditional photosensitizers, limited immune response capacity, and limited therapeutic effects, providing an innovative strategy for achieving targeted photodynamic synergistic immunotherapy for cancer.

Description

[0001] Technical Field: This invention belongs to the field of novel targeted nanomedicines, specifically relating to multifunctional targeted nanomaterials for the treatment of various cancers, their preparation methods, and applications. Background Technology

[0002] Cancer is a major disease that seriously threatens human health and life worldwide. It is characterized by high incidence and mortality rates, bringing immense physical and mental suffering and economic burden to patients and their families, and also impacting social development and stability. However, cancer treatment still faces several significant challenges. While traditional cancer treatments such as surgery, chemotherapy, and radiotherapy have achieved some success with continuous technological advancements and increased public awareness of health, 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 treatment methods and technologies to improve cancer cure rates and patients' quality of life.

[0003] Photodynamic therapy (PDT) has become a novel cancer treatment method due to its unique advantages such as being minimally invasive, highly effective, and having no significant drug resistance. However, the therapeutic effect of PDT is limited due to problems such as insufficient tumor tissue targeting and poor biosolubility of traditional photosensitizers such as phthalocyanine and porphyrin. With the development and application of nanomaterials technology, the strategy of constructing nanodelivery systems as photosensitizer carriers has undoubtedly provided a new platform for the development of PDT, increasing the selective enrichment of photosensitizers at tumor sites. Unfortunately, this strategy still faces two obstacles: 1) the poor biosolubility of nanocarriers and premature leakage of photosensitizers leading to stronger toxic side effects; 2) due to the short half-life (< 0.04 μs) and limited radius of action (< 0.02 μm) of ROS, its effectiveness is undoubtedly limited to the vicinity of the site of origin, thus limiting the therapeutic effect of PDT. Therefore, to address these problems, it is urgent to develop a soluble nanodelivery carrier with tumor cell and subcellular organelle targeting functions to effectively improve the anti-tumor effect of photodynamic therapy.

[0004] Biotin is a water-soluble B vitamin that plays a crucial 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. Overexpression of biotin receptors exists on the surface of various types of tumor cells, making it a potential target for targeted drug delivery to tumor sites. Studies have shown that covalently linking drugs to biotin can increase drug absorption and uptake by tumor cell tissues. Therefore, biotin can serve as a target for tumor cell lines.

[0005] The Golgi apparatus is a core organelle for protein glycosylation, modification, and sorting. In tumor cells, Golgi dysfunction leads to altered protein glycosylation patterns, which in turn affects signaling of cancer cell surface receptors (such as EGFR and HER2), cell adhesion (such as integrins), and invasiveness. For example, abnormally glycosylated proteins may promote tumor cell proliferation, metastasis, and immune evasion. Therefore, targeting the Golgi apparatus may produce a more significant inhibitory effect on cancer cells and reduce damage to normal cells. However, precisely targeting the Golgi apparatus of tumor cells while avoiding toxicity to normal cells remains a technical challenge in this field. More selective drugs or targeted delivery systems need to be developed to achieve more effective anti-tumor effects.

[0006] In recent years, while the therapeutic strategy of blocking the PD-1 / PD-L1 signaling pathway with anti-PD-L1 monoclonal antibodies has shown enhanced anti-tumor effects, its widespread use remains limited due to its inability to regulate the immune response of PD-L1 proteins in the cytoplasm or nucleus, as it can only disrupt the binding on the tumor surface. Furthermore, it suffers from adverse immune responses and high costs. Studies have shown that disrupting mitochondrial oxidative phosphorylation (OXPHOS) can lead to a decrease in mitochondrial energy content (e.g., an increase in the ADP / ATP ratio), promoting the activation of adenosine monophosphate-activated protein kinase (AMPK), thereby leading to the phosphorylation and degradation of PD-L1 and achieving in situ inhibition of PD-L1 expression, thus eliminating the presence of immune checkpoint inhibitory channels at their source. However, while several commonly used anticancer drugs (such as londamine (LND) and papaverine) can act as OXPHOS disruptors, they still suffer from insufficient localization to tumor cells and mitochondria, and require large doses. Based on this, increasing the mitochondrial targeting of OXPHOS disruptors and seeking a safe and efficient tumor-targeted delivery strategy may enable in situ inhibition of PD-L1, thereby further enhancing the immunomodulatory antitumor effect. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing multifunctional targeted nanomaterials for the treatment of various cancers. First, an indomethacin (IMD) ligand with Golgi targeting function is covalently coupled with the photosensitizer pyropheophorbide-a (Ppa) to obtain the photosensitizer IPA with Golgi targeting capability. Subsequently, a biotin derivative is modified onto the surface of a polycyclodextrin material through host-guest interactions to obtain a biotin receptor-targeting nanomaterial Bi-CD. Simultaneously, triphenylphosphine with precise mitochondrial targeting capability is covalently linked with the antitumor drug lonidamine (LND) to obtain the lonidamine derivative PND with mitochondrial-specific targeting capability. Finally, the photosensitizers IPA and PND are mixed with Bi-CD and assembled 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 biotoxicity. Furthermore, the biotin derivative loaded on its surface can precisely target and recognize tumor cells, thereby promoting the accumulation of photosensitizers and anticancer drugs at the tumor site. The Golgi-targeting photosensitizer IPA encapsulated in the material can further accumulate more photosensitizers in the Golgi apparatus. The generated ROS can directly act on the Golgi apparatus, disrupting its structure and function, thus significantly improving the tumor-suppressive ability of targeted photodynamic therapy. In addition, the anticancer drug PND with precise mitochondrial targeting capabilities can significantly disrupt mitochondrial function, enhance the inhibition of mitochondrial oxidative phosphorylation (OXPHOS), achieve effective PD-L1 downregulation, and further enhance the photodynamic synergistic immunotherapy antitumor effect. The multifunctional targeted nanocarrier system prepared in this invention has high biocompatibility and significant targeting to tumor cells, the Golgi apparatus, and mitochondria, exhibiting negligible toxic side effects and significantly improving the photodynamic antitumor effect, providing an innovative strategy for achieving targeted photodynamic synergistic immunotherapy for tumors. In addition, the present invention also provides a multifunctional targeted nanodelivery material for the treatment of various cancers obtained by the above method, and the application of the nanodelivery material.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of this invention discloses a method for preparing multifunctional targeted nanomaterials for the treatment of various cancers, comprising the following steps:

[0010] (1) First, prepare a 15WT% NaOH aqueous solution, then dissolve the weighed β-CD in it and stir at room temperature. Then, slowly add toluene to the mixture and continue stirring. Then slowly add epichlorohydrin as a crosslinking agent to continue the reaction. After the reaction is completed, pre-precipitate the crude product with isopropanol, transfer the precipitate to an appropriate amount of water and adjust it to neutral with dilute hydrochloric acid. Dialyze it through a dialysis bag to completely remove the monomer and oligomer. Finally, freeze dry under vacuum to obtain the white solid product Poly-β-CD.

[0011] (2) Accurately weigh biotin, add anhydrous DMF solution and stir to mix evenly, then add accurately weighed NHS and EDCI to the mixed reaction solution and react in an ice-water bath. Subsequently, accurately weighed adamantane is added to the mixture and stirred 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. The mixture was stirred for 2 h under nitrogen protection at 25 °C. After the reaction was completed, the mixture was extracted, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under vacuum to obtain the residue. The mixture was purified by column chromatography to obtain the solid product IMDH-Boc.

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

[0014] (5) Accurately weigh pyrophyllite 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. Stir for 2 h under nitrogen protection at 25°C. After the reaction is complete, extract the reaction mixture, dry the organic phase with anhydrous Na2SO4, filter and concentrate under vacuum to obtain the residue, and purify the mixture by column chromatography to obtain the solid product IPA.

[0015] (6) Accurately weigh 6-bromo-1-hexanol and dissolve it in anhydrous acetonitrile solution, and add triphenylphosphine. Under nitrogen protection, 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 white solid TPH.

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

[0017] (8) Dissolve the Poly-β-CD obtained in step (1), the IPA obtained in step (5), and the PND obtained in step (7) in a mixed solution of DMSO and water, and react at room temperature in the dark for 2 h. Then dissolve the Bi-Ad obtained in step (2) in a mixed solution of DMSO and water, and slowly add it dropwise to the reaction system. Continue the reaction at room temperature in the dark for 12 h. After the reaction is completed, transfer the reaction mixture to a dialysis bag for dialysis. Finally, freeze-dry the reaction solution to obtain brown-black solid powder Bi-CD@IPA@PND NPs.

[0018] Furthermore, the dialysis conditions in step (1) are in M W Dialysis was performed for 24 hours in an 8000-fill dialysis bag, with the water changed every 3 hours.

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

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

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

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

[0023] Furthermore, in step (8), the ratio of the mixed solution of DMSO and water is 1:3. The dialysis conditions are as follows: W Dialysis was performed for 24 hours in a 3000-fill dialysis bag, with the water changed every 3 hours.

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

[0025] The third aspect of this invention is to disclose the application of the multifunctional targeted nanomaterials described above for the treatment of various cancers in nano-drug delivery systems.

[0026] The significant advantages of this invention are:

[0027] (1) Polycyclodextrin-type nanodelivery carriers have good biocompatibility and stability under physiological conditions, and have the characteristic of releasing drugs in response to the tumor microenvironment. They can significantly reduce the toxic side effects and adverse reactions caused by premature leakage and poor solubility of nanomedicines during transportation, and can be used as ideal nanomedicine carriers.

[0028] (2) Biology white The derivative-modified nanodelivery materials 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 beneficial for use and long-term storage.

[0030] (4) The preparation method is simple, with few side reactions, readily available raw materials, low cost, and conducive to industrial production. Attached Figure Description

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

[0032] Figure 2 The target images for different nano-drug delivery systems on 4T1 and KYSE150 tumor cells were characterized by laser confocal microscopy.

[0033] Figure 3 : A graph representing the Golgi apparatus targeting capability of different drugs characterized by laser confocal microscopy;

[0034] Figure 4 : This is a graph showing the inhibitory effect of different drug groups on 4T1 tumor cells and KYSE150 tumor cells as detected by the MTT assay. Detailed Implementation

[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 multifunctional targeted nanomaterials for the treatment of various cancers, comprising the following steps:

[0037] (1) First, accurately weigh 10.0 g of β-CD and dissolve it in 15 mL of NaOH (15 WT%) aqueous solution, and stir at room temperature for 2 h. Then, add 2 mL of toluene dropwise to the mixture and continue stirring for 2 h, and then slowly add 3.8 mL of epichlorohydrin as a crosslinking agent and continue the reaction for 3 h. After the reaction is completed, pre-precipitate the crude product with 200 mL of isopropanol, transfer the precipitate to an appropriate amount of water and adjust it to neutral with dilute hydrochloric acid. Finally, dialyze it through a dialysis bag with MW = 8000 for 2 days, changing the water every 3 h to completely remove the monomer and oligomers. Finally, freeze-dry under vacuum to obtain the white solid product Poly-β-CD.

[0038] (2) Accurately weigh biotin (0.5 g, 2.0 mmol) and place it in a 50 mL round-bottom flask. Add 30 mL of anhydrous DMF solution and stir until homogeneous. Then, accurately weigh NHS (0.3 g, 2.4 mmol) and EDCI (0.5 g, 2.4 mmol) are added to the mixture, and the reaction is carried out in an ice-water bath for 1 h. Subsequently, accurately weigh adamantane (0.3 g, 2.1 mmol) is added to the mixture, and the reaction is carried out at room temperature with stirring for 12 h. After the reaction is completed, the mixture is purified by column chromatography (CH2Cl2 / CH3OH 30:1v / v) to obtain 0.48 g of 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, 4H), 1.91 (d, J = 2.8 Hz, 4H), 1.60 (d, J = 2.2 Hz, 4H), 1.47 (s, 4H), 1.36 (s, 4H), 0.86 (s, 4H). 13 C NMR (101 MHz, 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 / zcalculated 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 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). The mixture was stirred for 2 h under nitrogen protection at 25 °C. After the reaction was complete, H2O (10 mL) was added to the mixture, and it was extracted with ethyl acetate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The mixture was purified by column chromatography (petroleum ether / ethyl acetate 1:1 v / v) to obtain product IMDH-Boc 0.31 g, 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 ethyl acetate (0.5 mL) 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 (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 bound organic layer was washed with brine (10 mL), dried on anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain product IMDH (0.2 g), with a yield of 85%. 1 HNMR (400 MHz, DMSO-d6) δ = 8.16 (s, 1H), 7.94 (br s, 2H), 7.72 - 7.62 (m,4H), 7.15 (d, J = 2.5 Hz, 1H), 6.93 (d, J =9.0 Hz, 1H), 6.71 (dd, J = 2.6,8.9 Hz, 1H), 3.77 (s, 3H), 3.51 (s, 2H), 3.11 - 3.01 (m, 2H), 2.73 (t, J =7.5 Hz, 2H), 2.24 (s,3H), 1.52 (br t, J = 7.3 Hz, 2H), 1.41 (br t, J = 6.6Hz, 2H), 1.33 - 1.22 (m, 4H). HRMS (ESI): m / z calculated for C 25 H32 ClN3O3 [M + H] + :457.2132; found:457.2156.

[0041] (5) Accurately weigh pyrophyllin a (0.1 g, 0.187 mmol) and add it to a DMF (2 mL) solution. Then add the 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 for 2 h under nitrogen protection at 25 °C. After the reaction is complete, add H2O (15 mL) to the mixture and extract with dichloromethane (20 × 2 mL). Dry the organic phase with anhydrous Na2SO4, filter, and concentrate under vacuum to obtain the residue. Purify the mixture by column chromatography (CH2Cl2 / CH3OH 20:1 v / v) to obtain product IPA (0.09 g), with a yield of 43%. 1 H NMR (400 MHz, 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). 13C NMR(101 MHz, 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 / zcalculated for C 58 H 64 ClN7O5 [M + H] + :973.4653; found:973.4618.

[0042] (6) Accurately weigh 0.72 g (4.0 mmol) of 6-bromo-1-hexanol and dissolve it in 25 mL of anhydrous acetonitrile solution, and add triphenylphosphine (1.05 g, 4.0 mmol). Under nitrogen protection, reflux at 85 °C for 48 h. 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 a white solid TPH (0.6 g), with a yield of 35%. 1 H NMR (400 MHz, DMSO-d6) δ = 7.94 - 7.88 (m, 3H), 7.85 -7.76 (m, 12H), 4.38 - 4.31 (m, 1H), 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 chlordamine (0.1 g, 0.31 mmol) was added to DCM (10 mL), and TPH (0.14 g, 0.31 mmol) obtained in step (6) 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 the mixture was stirred for 2 h under nitrogen protection at 25 °C. After the reaction was completed, H2O (15 mL) was added to the mixture, and it was extracted with dichloromethane (20 mL). The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The mixture was purified by column chromatography (CH2Cl2 / CH3OH 30:1 v / v) to obtain a white solid PND (0.1 g), with a yield of 43%. 1 H NMR (400 MHz, 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). 13 C NMR (101 MHz, 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 / zcalculated 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. Then, 0.5 mg of Bi-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water (1:3) and 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 transmission electron microscopy (TEM) at room temperature. Next, the hydrodynamic dimensions of Bi-CD NPs and Bi-CD@IPA@PND NPs in aqueous solution were determined using a Malvern Nano-ZSZEN3600. TEM images showed that both nanomaterials exhibited spherical structures with dimensions of approximately 100 nm. Dynamic light scattering (DLS) further confirmed the hydrodynamic diameters and stability of Bi-CD NPs and Bi-CD@IPA@PND NPs, with average hydrodynamic diameters of approximately 110 nm and 105 nm, respectively, which is almost consistent with the TEM data. The polydispersity indices were 0.16 and 0.13, respectively, indicating that the preparation of Bi-CD@IPA@PND NPs did not alter the original morphology of Bi-CD NPs, and they also exhibited smaller particle sizes, which are 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 three times with sterile PBS, and then digest with 0.25% trypsin. After centrifugation, resuspend the cells in fresh culture medium. Count the cells in the suspension using a cell counter, and then dilute the cell suspension to 2.0 × 10⁻⁶. 5 Add 1 mL of cells / mL to each confocal dish. Finally, incubate all confocal dishes overnight in a cell culture incubator at 37°C and 5% CO2.

[0049] (2) Adding drugs: After the cells have completely adhered to the wall, remove the culture medium in the laser confocal dish, then wash three times with sterile PBS, and finally add 1 mL of fresh culture medium of Bi-CD@IPA@PND NPs and CD@IPA@PND NPs at a concentration of 20 μg / mL to the laser confocal dish and incubate in a cell culture incubator at 37℃ and 5% CO2 for 12 h.

[0050] (3) Testing: After incubation, remove the drug-containing culture medium from all laser confocal dishes and wash three times with sterile PBS. Add 1 mL of sterile PBS and wait for imaging. Simultaneously, add 0.5 mL of 0.25% trypsin to the other washed confocal dishes for digestion, centrifuge, and resuspend in 1 mL of sterile PBS. Finally, use laser confocal electron microscopy to test intracellular drug fluorescence (excitation wavelength: 633 nm, emission wavelength: 650-750 nm). All cell blank control groups were incubated with drug-free culture medium, and other procedures were the same as the experimental group.

[0051] The fluorescence intensity of Bi-CD@IPA@PND NPs within cells was detected using laser confocal microscopy. In both 4T1 and KYSE150 cells, Bi-CD@IPA@PND NPs exhibited a significantly stronger fluorescence signal intensity than CD@IPA@PND NPs, approximately five times stronger, indicating that Bi-CD@IPA@PND NPs possess a stronger tumor cell-targeted uptake capability. This suggests that the introduction of biotin significantly enhances the specific recognition of the nanomaterials by tumor cells, thereby promoting cellular uptake. Therefore, all cellular uptake experiments demonstrate that biotin... white Modified nanocarriers can significantly enhance the targeting ability of Bi-CD@IPA@PND NPs to tumor cells overexpressing biotin receptors.

[0052] Golgi body targeting characterization (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, resuspend the cells in fresh culture medium. Count the cells in the suspension using a cell counter, and then dilute the cell suspension to 1.0 × 10⁻⁶. 4 Add 1 mL of cells / mL to each confocal dish. Finally, incubate all confocal dishes overnight in a cell culture incubator at 37°C and 5% CO2.

[0054] (2) Adding drugs: After the cells have completely adhered to the wall, remove the culture medium in the laser confocal dish, then wash three times with sterile PBS, and finally add 1 mL of fresh culture medium with a concentration of 20 μg / mL of Ppa, IPA or Bi-CD@IPA@PND NPs to the laser confocal dish and incubate in a cell culture incubator at 37℃ and 5% CO2 for 12 h.

[0055] (3) Staining: First, 2 μL of the stock solution of the 1 mM Golgi green probe was taken and mixed with fresh culture medium to bring the probe solution concentration to 2 μM. Then, the drug-containing culture medium in the laser confocal 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 dishes. Finally, the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 20 min.

[0056] (4) Imaging: After co-incubation, the corresponding probe dye in the dish was removed by centrifugation, and the cells were washed three times with sterile PBS to remove excess probe dye and prevent interference with the imaging background. A small amount of PBS was then added to resuspend the cells, and the cells were transferred to a new confocal dish before imaging. Finally, the cells were imaged using a laser confocal microscope equipped with a 405 nm or 488 nm multi-argon laser and a 633 nm diode laser. The excitation and emission wavelength parameters of the photosensitizer compound were: Ex / Em = 633 / 650 - 750 nm; the excitation and emission wavelength parameters of the Golgi probe dye were: Ex / Em = 488 / 510 - 570 nm.

[0057] Laser confocal microscopy was used to observe the red fluorescence of Ppa and IPA and the green fluorescence of the Golgi apparatus probe. The red fluorescence of free Ppa showed almost no overlap with the green fluorescence signal representing the Golgi apparatus. Further qualitative analysis of the fluorescence intensity line scan spectrum revealed significant separation signals between the Ppa group and the Golgi apparatus dye. By introducing indomethacin, after 12 h of incubation, the free Ppa group showed significant fluorescence overlap between the red fluorescence signal and the Golgi apparatus dye, with yellow fluorescent spots appearing within the Golgi apparatus. Qualitative analysis of the fluorescence intensity line scan spectrum also indicated partial overlap between the IPA group and the Golgi apparatus dye, suggesting that free IPA exhibits visible Golgi apparatus targeting capability. In contrast to free IPA, the Bi-CD@IPA@PND NPs group showed strong yellow fluorescent spots within the Golgi apparatus, and qualitative analysis of the fluorescence intensity line scan spectrum also revealed a high degree of overlap between the IPA and the Golgi apparatus dye. Therefore, these data strongly demonstrate that Bi-CD@IPA@PNDNPs can significantly enhance the enrichment of photosensitizers in the Golgi apparatus, thereby achieving the goal of targeting the Golgi apparatus.

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

[0059] In photodynamic therapy, the cytotoxicity of photosensitizers is a key research focus in evaluating their properties. The MTT assay is a widely used experimental method for assessing the ability of drugs to induce apoptosis. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide), a yellow dye, is commercially known as thiazolium blue. The detection principle of MTT can be summarized as follows: The mitochondria of living cells contain a reducing succinate dehydrogenase that can reduce yellow MTT to insoluble blue-purple crystalline formazan, which is then deposited in the cell. The mitochondria of dead cells do not contain this reducing succinate dehydrogenase, and therefore cannot reduce MTT, nor can they generate blue-purple crystalline formazan. Related experiments have shown that dimethyl sulfoxide (DMSO) organic solvent can dissolve the formazan deposited in the cells, and the absorbance (OD value) at wavelengths of 490 nm or 570 nm can be measured using a microplate reader, thus indirectly obtaining the corresponding number of living cells. The formula for calculating cell viability (I%) is as follows:

[0060] I%=[(A-A0) / (As-A0)] × 100%

[0061] Where A, A0, and As represent the OD values ​​of the cell drug group, the blank solvent control group, and the cell solvent control group, respectively. During the experiment, the logarithm of the corresponding drug concentration was plotted on the x-axis, and cell viability (%) was plotted on the y-axis. Experimental data are expressed as the mean ± SD of the three independent experiments. Finally, the experimental data were plotted using GraphPad Prism 6.0 software to obtain the relationship curve 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 the density to 8.0 × 10⁻⁶. 4Cells / mL. Then, 100 μL of diluted cell suspensions of different concentrations were added to each well of a 96-well plate. Different drug concentration groups and all control groups were set to 6 replicates 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 with 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 h.

[0065] (3) Irradiation: After incubation, remove the old drug-containing culture medium from the 96-well plate and wash three times with sterile PBS to completely remove any unabsorbed drug. Then, add 100 μL of fresh cell culture medium to each well and place the 96-well plate for phototoxicity experiments under an LED laser plate (λ = 670 nm, 4 mW / cm²). 2 1.2 J / cm 2 Irradiate for 10 min. Finally, incubate in a cell culture incubator at 37℃ and 5% CO2 for 24 h. The dark toxicity experiment did not involve light treatment; other procedures were the same as the phototoxicity experiment and were performed in a dark environment.

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

[0067] Experimental data show that, without laser irradiation, no significant cytotoxicity was observed in any of the drug groups against 4T1 or KYSE150 cells, indicating negligible dark toxicity in different drug groups. However, under LED laser irradiation (λ = 670 nm, 4 mW / cm²), significant cytotoxicity was observed. 2 1.2 J / cm 2 Under 10 min irradiation conditions, the photocytotoxicity of the Bi-CD@IPA@PND NPs group and Bi-CD@IPA NPs was significantly higher than that of the single-target IPA, CD@IPA@PND NPs group and the blank control group. This indicates that within a smaller concentration range, the drug group with tumor cell and Golgi targeting functions exhibits more significant killing ability against 4T1 cells and KYSE150 cells, thereby achieving stronger anti-tumor cell proliferation ability.

[0068] Based on the above embodiments, this invention provides a method for preparing multifunctional targeted nanomaterials for the treatment of various cancers. Verification has shown that the obtained multifunctional targeted nanomaterials can accurately target and identify 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 solves the problems of poor targeting and biosolubility of traditional photosensitizers, limited immune response capacity, and limited therapeutic effects, providing an innovative strategy for targeted photodynamic and synergistic immunotherapy of cancer. Significant technical results have been achieved.

[0069] It should be further noted that the above embodiments are merely for understanding 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 made to the technical solution of the present invention that fall within the inventive concept should also fall within the scope of protection of the present invention.

Claims

1. A method for preparing multifunctional targeted nanomaterials for the treatment of various cancers, comprising the following steps: (1) The step of dissolving β-CD in NaOH aqueous solution and reacting it under the action of a crosslinking agent to obtain Poly-β-CD; (2) Add biotin to anhydrous DMF solution, then add NHS, EDCI and adamantane to the mixed reaction solution, and react in an ice-water bath to obtain Bi-Ad; (3) The step of dissolving indomethacin, tert-butyl 6-aminohexylcarbamate, HATU and DIEA in anhydrous DMF solution and reacting to obtain IMDH-Boc; (4) Add HCl to ethyl acetate solution and stir at 0°C to obtain tert-butyl 6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)hexylcarbamate, extract and dry to obtain IMDH. (5) Add pyrophyllophospholipid a to the DMF solution, then add the IMDH obtained in step (4), and add TCFH and NMI to the mixed solution. After the reaction is complete, extract and dry the mixture, and purify the mixture by column chromatography to obtain the solid product IPA. (6) Accurately weigh 6-bromo-1-hexanol and dissolve it in anhydrous acetonitrile solution, add triphenylphosphine, reflux and heat under nitrogen protection. After the reaction is complete, remove the solvent under reduced pressure and purify the mixture by column chromatography to obtain white solid TPH. (7) Accurately weighed chlordamine was added to DCM, and TPH obtained in step (6) was added. Then DCC and DMAP were added to the reaction mixture. The mixture was stirred under nitrogen protection. After the reaction was completed, the reaction mixture was extracted. The organic phase was dried with anhydrous sodium sulfate, filtered, and vacuum concentrated to obtain the residue. The mixture was purified by column chromatography to obtain white solid PND. (8) Dissolve the Poly-β-CD obtained in step (1), the IPA obtained in step (5), and the PND obtained in step (7) in a mixed solution of DMSO and water. Dissolve the Bi-Ad obtained in step (2) in a mixed solution of DMSO and water. Then slowly add the solutions to the reaction system to obtain the final product Bi-CD@IPA@PND NPs. Step (7) involves accurately weighing 0.1 g and 0.31 mmol of chlordamine and adding it to 10 mL of DCM, along with 0.14 g and 0.31 mmol of TPH obtained in step (6). Subsequently, 0.096 g and 0.465 mmol of DCC and 0.057 g and 0.465 mmol of DMAP are added to the reaction mixture. The mixture is stirred for 2 h under nitrogen protection at 25 °C. After the reaction is complete, 15 mL of H2O is added to the mixture, and it is extracted with 20 mL of dichloromethane. The organic phase is dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The mixture is then purified by column chromatography to obtain a white solid, PND. In column chromatography, the volume ratio of CH2Cl2 / CH3OH is 30:

1.

2. The method for preparing multifunctional targeted nanomaterials for the treatment of various cancers according to claim 1, characterized in that: In step (1), 10.0 g of β-CD was first accurately weighed and dissolved in 15 mL of 15 WT% NaOH aqueous solution. The mixture was stirred at room temperature for 2 h. Then, 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 crosslinking 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. The precipitate was transferred to an appropriate amount of water and adjusted to neutral with dilute hydrochloric acid. Finally, the product was dialyzed for 2 days using a dialysis bag with MW = 8000, and the water was changed every 3 h to completely remove the monomers and oligomers. Finally, the product was freeze-dried under vacuum to obtain a white solid product, Poly-β-CD.

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

1.

4. The method for preparing multifunctional targeted nanomaterials for the treatment of various 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 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 for 2 h under nitrogen protection at 25 °C. After the reaction was completed, 10 mL of H2O was added to the mixture, and it was extracted with 20 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the residue. The mixture was purified by column chromatography to obtain the product IMDH-Boc. In column chromatography, the volume ratio of petroleum ether to ethyl acetate is 1:

1.

5. The method for preparing multifunctional targeted nanomaterials for the treatment of various 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 was completed, 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 on anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the product IMDH.

6. The method for preparing multifunctional targeted nanomaterials for the treatment of various cancers according to claim 1, characterized in that: Accurately weigh 0.1 g and 0.187 mmol of pyromethes chlorophyll a and add it to 2 mL of DMF solution. Then add 0.1 g and 0.224 mmol of IMDH obtained in step (4), and add 105 mg and 0.374 mmol of TCFH and 31 mg and 0.374 mmol of NMI to the mixed solution. Stir for 2 h under nitrogen protection at 25 °C. After the reaction is complete, add 15 mL of H2O to the mixture and extract with 20 × 2 mL of dichloromethane. Dry the organic phase with anhydrous Na2SO4, filter and concentrate under vacuum to obtain the residue. Purify the mixture by column chromatography to obtain the product IPA. In column chromatography, the volume ratio of CH2Cl2 / CH3OH is 20:

1.

7. A multifunctional targeted nanomaterial for the treatment of various cancers, characterized in that: It is prepared by the method described in any one of claims 1-6.

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