Double-targeting nano-material photosensitizer for cervical cancer as well as preparation method and application of double-targeting nano-material photosensitizer

Abstract

The invention provides a dual-targeting nano-material photosensitizer for cervical cancer as well as a preparation method and application of the dual-targeting nano-material photosensitizer. The folic acid modified poly-beta-cyclodextrin (poly-beta-CD) is used as a nano-carrier and is used for loading a photosensitizer PAP (PAP is pyropheophorbide a covalently connected PAAKRVKLD) coupled with a nuclear localization signal (NLSs). The finally prepared FA-CD-coated PAP nano material can specifically recognize cervical cancer cells of overexpressed folate receptors (FR), so that accumulation in tumor cells is remarkably enhanced. In addition, the encapsulated PAP can achieve accurate cell nucleus localization. Under an illumination condition, reactive oxygen species (ROS) generated by PAP accumulated in nuclei can instantly oxidize and damage a DNA chain or DNA repair enzyme, so that cell death is directly induced, and the anti-tumor effect of targeted photodynamic is greatly enhanced. The novel dual-targeting nano drug-loading system prepared by the invention solves the problems of poor targeting and biological solubility and limited treatment effect of a traditional photosensitizer, and provides an innovative intelligent targeting delivery strategy for in-situ cell nucleus photodynamic therapy.

Description

Technical Field

[0001] This invention belongs to the field of novel nanomedicines, specifically relating to the preparation method and application of dual-targeting nanomaterials for the treatment of cervical cancer. Background Technology

[0002] In recent years, despite significant progress in photodynamic therapy (PDT) for the clinical treatment of tumors, traditional photosensitizers still face numerous challenges in their clinical application and development due to issues such as poor tumor targeting and poor biosolubility. Reactive oxygen species (ROS), a key component of PDT, possess strong oxidizing capabilities, but their extremely short lifespan (<20 ns) and limited diffusion range (≈20 nm) restrict their effectiveness to the vicinity of the site of origin, hindering their ability to target distant tumor cells and significantly reducing therapeutic efficacy. Therefore, addressing these issues with traditional photosensitizers is crucial for improving the efficacy and safety of PDT. Currently, the precise delivery of photosensitizers to important subcellular organelles, such as the nucleus, endoplasmic reticulum, and mitochondria, has become a highly sought-after strategy. The nucleus, as the heart of eukaryotic cells, plays a crucial role in cell metabolism, growth, and differentiation. Oxidative damage and irreversible injury to the DNA strands in the nucleus caused by reactive oxygen species (ROS) are considered one of the most direct and effective pathways to cell death. Therefore, the nucleus can serve as a more sensitive and ideal site for phototherapy-targeting (PDT). With the development of nanotechnology, nuclear-targeted nanocomposites can precisely deliver photosensitizers to the nucleus, achieving precise targeting of the nucleus, which undoubtedly provides a broad and highly promising platform for in situ nuclear PDT. However, in practical applications, the non-specific selectivity of nanocomposites not only reduces therapeutic efficacy but may also cause unnecessary damage to normal tissues. Furthermore, the nuclear membrane, as an important barrier to the nucleus, exhibits high selectivity, and its pore size is very limited (9-40 nm), which is undoubtedly a significant hurdle for some larger-sized nanocomposites. These factors severely limit the accumulation of photosensitizers in tumor cells and the nucleus, preventing them from fully exerting their therapeutic effects and becoming a major bottleneck in the application of nuclear-targeted nanocomposites. To overcome the challenges faced by nanocomposites, researchers have focused on dual-targeted drug delivery systems. This strategy, by simultaneously modifying the surface of nanomaterials with tumor-targeting and nuclear-targeting ligands, opens new avenues for addressing nonspecificity and improving the efficacy of nuclear phototherapy (PDT). However, issues such as nuclear-targeting ligand degradation, side effects caused by premature leakage of photosensitizers, and poor biocompatibility of nanomaterials limit the further development of nuclear PDT. Therefore, constructing intelligent targeted nanocomposites that integrate nuclear-targeting ligand protection, controlled release, and good biocompatibility holds significant potential importance for achieving highly efficient and ideal in-situ nuclear PDT.

[0003] PAAKRVKLD, derived from the human C-MyC protein, exhibits exceptional nuclear localization capabilities. At physiological pH, it possesses three cationic residues that interact with specific structures within the cell nucleus, guiding substances precisely into the nucleus. This unique structure endows PAAKRVKLD with superior nuclear localization ability, providing strong support for precise nuclear-targeted therapy. Furthermore, cyclodextrins, composed of multiple glucose units linked by α-1,4 glycosidic bonds, form a unique cyclic structure with a hydrophobic inner lumen and a hydrophilic outer surface. This structure contributes to its excellent biocompatibility, degradability under acidic conditions, and high biosafety. Therefore, cyclodextrins can serve as an ideal carrier for photosensitizer delivery.

[0004] Folic acid plays a crucial role in cellular metabolism, serving as an essential substance for cell growth and proliferation. The folate receptor (FR), a glycoprotein on the cell membrane surface, primarily mediates the efficient uptake of folate by cells. In tumor cells, due to their significantly increased demand for folate, the expression level of the folate receptor is significantly upregulated to meet the needs of their rapid growth and proliferation, making the folate receptor an ideal target for tumor targeting. In recent years, numerous studies have demonstrated the targeting effects mediated by folate and its receptor, providing a highly efficient and low-toxicity strategy for cancer treatment. Summary of the Invention

[0005] The present invention aims to provide a dual-targeting nanomaterial for the treatment of cervical cancer and its preparation method. First, a nuclear-targeting signal peptide (PAAKRVKLD) ligand with nuclear targeting function is covalently coupled with the photosensitizer pyropheophorbide-a (Ppa) to obtain a photosensitizer PAP with nuclear targeting capabilities. Subsequently, a folic acid derivative is modified onto the surface of a polycyclodextrin material through host-guest interactions to obtain a folic acid receptor-targeting nanomaterial FA-CD. Finally, the photosensitizer PAP and FA-CD are mixed and assembled to form a multifunctional dual-targeting nanomaterial FA-CD@PAP with both tumor cell targeting and nuclear targeting functions. The high loading rate and high biocompatibility of polycyclodextrin can reduce biotoxicity. Furthermore, the folic acid 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 cell nucleus-targeting photosensitizer PAP encapsulated in the material can further enrich more photosensitizer in the cell nucleus. The generated ROS can directly act on the cell nucleus, destroying its structure and function, thereby significantly improving the tumor-suppressing ability of targeted photodynamic therapy.

[0006] The multifunctional dual-targeting nanoparticle drug delivery system prepared by this invention exhibits high biocompatibility and significant targeting to tumor cells and nuclei, while demonstrating negligible toxic side effects and significantly enhancing the photodynamic antitumor effect. This provides an innovative strategy for achieving in situ nuclear-targeted photodynamic therapy for cervical cancer. Furthermore, this invention also provides a multifunctional dual-targeting nanodelivery material for cervical cancer treatment obtained by the above method, as well as the applications of this nanodelivery material.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention discloses a method for preparing a dual-targeting nanomaterial photosensitizer for cervical cancer, comprising the following steps: (1) First, prepare a 15wt% NaOH aqueous solution, then weigh out the... β -CD was dissolved in the mixture and stirred at room temperature. Toluene was then slowly added dropwise while stirring. Epichlorohydrin was then slowly added dropwise as a crosslinking agent to continue the reaction. After the reaction was complete, the crude product was pre-precipitated with isopropanol. The precipitate was transferred to an appropriate amount of water and adjusted to neutral with dilute hydrochloric acid. Dialysis was then performed using a dialysis bag to completely remove monomers and oligomers. Finally, the product was freeze-dried under vacuum to obtain a white solid product, Poly- β -CD; (2) Accurately weigh folic acid, add anhydrous DMF solution and stir to mix evenly, then add accurately weighed NHS and EDCI to the mixed reaction solution, react in an ice-water bath for 1 hour, then add accurately weighed adamantane to the mixed solution, stir to react at room temperature, after the reaction is completed, transfer the reaction solution to a mixture of acetone and water to form a precipitate, vacuum filter, and finally vacuum dry at 50°C for 24 hours to obtain orange solid compound FA-Ad; (3) Accurately weighed pyrophyllite a was added to DMF solution, followed by HOBT, EDCI and hexaaminocaproic acid. The mixture was stirred for 24 h under nitrogen protection at 25 °C. After the reaction was completed, the mixture was purified by column chromatography and vacuum dried to obtain brown-black solid compound PAA. (4) Dissolve accurately weighed PAA, HBTU and DIEA in anhydrous DMF solution and react at room temperature in the dark for 30 min. Then, add the N-terminal exposed NLS molecule PAAKRVKLD to the reaction system and continue the reaction in the dark for 24 h under N2 protection. After the reaction is completed, wash away the unreacted substances with DMF and methanol solutions respectively, freeze-dry the sample, and then soak the sample in a mixture of 95% trifluoroacetic acid and 5% water for 4 h. Filter off the solid and collect the filtrate. Add 10 times the volume of anhydrous diethyl ether to precipitate the precipitate. Centrifuge to collect the solid and freeze-dry to obtain the gray-green solid compound PAP. (5) Take the Poly- obtained in step (1) β -CD and PAP obtained in step (4) were dissolved in a mixed solution of DMSO and water and reacted at room temperature in the dark for 2 hours. Then, FA-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water and slowly added to the reaction system. The reaction was continued at room temperature in the dark for 12 hours. After the reaction was completed, the reaction mixture was transferred to a dialysis bag and dialyzed for 24 hours. Finally, the reaction solution was freeze-dried to obtain brown solid powder FA-CD@PAP NPs.

[0008] Furthermore, the dialysis conditions in step (1) are... M W Dialysis was performed for 24 hours in an 8000-well dialysis bag, with the water changed every 3 hours.

[0009] Furthermore, in step (2), the ratio of acetone to water in the mixture is 1:4.

[0010] Furthermore, in step (5), the ratio of the mixed solution of DMSO and water is 1:3; the dialysis conditions are as follows: M W Dialysis was performed for 24 hours in a dialysis bag with a capacity of 3000, and the water was changed every 3 hours.

[0011] The second aspect of this invention is to disclose a dual-targeting nanomaterial for in situ nuclear-targeted photodynamic therapy of cervical cancer, which is prepared by the method described above.

[0012] The third aspect of this invention is to disclose the application of the dual-targeting nanomaterials described above for in situ nuclear-targeted photodynamic therapy of cervical cancer in nano-drug delivery systems.

[0013] The significant advantages of this invention are: (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.

[0014] (2) The folic acid derivative-modified nanodelivery materials exhibit significant targeting recognition ability for cervical cancer cells, providing a promising cancer targeted therapy strategy.

[0015] (3) The nano-drug delivery system has excellent stability and uniformity, which is beneficial for use and long-term storage.

[0016] (4) The preparation method is simple, with few side reactions, readily available raw materials, low cost, and conducive to industrial production. Attached Figure Description

[0017] Figure 1 Transmission electron microscopy and particle size distribution diagrams of different nano-drug delivery systems; Figure 2 A map illustrating the targeting of different nano-drug delivery systems to HeLa tumor cells using laser confocal microscopy and flow cytometry. Figure 3 A diagram illustrating the nuclear targeting capabilities of different drugs characterized by laser confocal microscopy. Figure 4 A graph showing the inhibitory effect of different drug groups on the activity of HeLa tumor cells as determined by the MTT assay. Figure 5 Figure showing the growth inhibition ability of different drugs on a mouse HeLa tumor model; Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0019] This invention provides a method for preparing a multifunctional targeted photodynamic synergistic immunotherapy nanomaterial for various cancers, comprising the following steps: (1) First, accurately weigh 10.0g β-CD was dissolved in 15 mL of NaOH (15 WT%) aqueous solution and stirred at room temperature for 2 h. Then, 2 mL of toluene was added dropwise to the mixture, and stirring continued for 2 h. Next, 3.8 mL of epichlorohydrin as a crosslinking agent was slowly added dropwise, and the reaction continued for 3 h. After the reaction was complete, 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, through... M W Dialyzing the sample with an 8000 dialysis bag for 2 days, changing the water every 3 hours, was performed to completely remove monomers and oligomers. Finally, vacuum freeze-drying was used to obtain a white solid product, Poly-. β -CD.

[0020] (2) Accurately weigh folic acid (0.9 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 complete, the reaction solution is transferred to a 1:4 mixture of acetone and water to form a precipitate, which is then filtered under vacuum. Finally, the precipitate is dried under vacuum at 50 °C for 24 h to obtain 0.65 g of orange solid compound FA-Ad, with a yield of 58%. HRMS (ESI): m / z calculated for C 29 H 35 N8O5[M+H] + :575.2730;found:575.2714.

[0021] (3) Accurately weighed pyrophyllophosphate a (42.8 mg, 0.08 mmol), HOBT (16.0 mg, 0.12 mmol), and EDCI (23.0 mg, 0.12 mmol) were dissolved in 3 mL of anhydrous DMF solution and reacted at room temperature in the dark for 30 min. Then, hexaaminocaproic acid (12.6 mg, 0.10 mmol) was added, and the reaction continued for 24 h under N2 protection. After the reaction was complete, the mixture was purified by column chromatography, finally yielding 14.6 mg of brownish-black solid compound PAA, with a yield of 28%. HRMS (ESI): m / z calculated for C 39 H 46 N5O4[M+H] + :648.3530;found:648.3530.

[0022] (4) Accurately weigh PAA (28.0 mg, 0.04 mmol) and HBTU (48.0 mg, 0.12 mmol). DIEA (93.0 μL) was dissolved in 3 mL of anhydrous DMF solution and reacted at room temperature in the dark for 30 min. Then, the N-terminally exposed NLS molecule PAAKRVKLD (15.0 mg, 0.02 mmol) was added to the reaction system, and the reaction was continued in the dark for 24 h under N2 protection. After the reaction was completed, unreacted substances were washed away with DMF and methanol solutions, respectively, and the sample was lyophilized. Subsequently, the sample was soaked in a mixture of 95% trifluoroacetic acid and 5% water for 4 h, the solid was filtered off and the filtrate was collected. Ten times the volume of anhydrous diethyl ether was added to precipitate the solid, which was collected by centrifugation and lyophilized to obtain 20.5 mg of gray-green PAP molecules, which were stored at -20 °C, with a yield of 29%. HRMS (ESI): m / z calculated for C 83 H 124 N 19 O 15 [M+H] + :1626.9524;found:1626.9496.

[0023] (5) Take the Poly- obtained in step (1) β -CD (21.0 mg) and PAP (2.0 mg) obtained in step (4) were dissolved in a mixed solution of DMSO and water (1:3) and reacted at room temperature in the dark for 2 h. Then, 2.0 mg of FA-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... M W Dialyze the solution in a dialysis bag at 3000 °C for 24 hours, changing the water every 3 hours. Finally, freeze-dry the reaction solution to obtain a brownish-black solid powder FA-CD@PAP NPs.

[0024] Nanomaterial characterization (see appendix) Figure 1 ) The morphology of FA-CD NPs and FA-CD@PAP NPs was characterized using transmission electron microscopy (TEM) at room temperature. Next, the hydrodynamic dimensions of FA-CD NPs and FA-CD@PAP NPs in aqueous solution were determined using a Malvern Nano-ZS ZEN3600. TEM images showed that both nanomaterials exhibited spherical structures with dimensions of approximately 50 nm. Dynamic light scattering (DLS) further confirmed the hydrodynamic diameters of FA-CD NPs and FA-CD@PAP NPs, with average hydrodynamic diameters of approximately 39 nm and 51 nm, respectively, which is almost consistent with the TEM data. The polydispersity indices were 0.32 and 0.21, respectively, indicating that the preparation of FA-CD@PAP NPs did not alter the original morphology of FA-CD NPs, maintaining their smaller particle size, which is more conducive to tumor cell endocytosis.

[0025] Cell targeting characterization (see appendix) Figure 2 ) (1) Cell plating: First, select HeLa cells in good growth condition, remove the old culture medium, wash three times with sterile PBS, digest with 0.25% trypsin, centrifuge, resuspend in fresh culture medium, count the cell 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.

[0026] (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 CD@PAP NPs and FA-CD@PAP NPs at a concentration of 100 μg / mL to the laser confocal dish and incubate in a cell culture incubator at 37℃ and 5% CO2 for 12 h.

[0027] (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 photography. 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 and flow cytometry 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.

[0028] The fluorescence intensity of intracellular FA-CD@PAP NPs was detected using laser confocal microscopy and flow cytometry. Both sets of experimental results showed that in HeLa cells, FA-CD@PAP NPs exhibited a significantly stronger fluorescence signal intensity than CD@PAP NPs, approximately three times stronger, indicating that FA-CD@PAP NPs demonstrated a stronger tumor cell-targeted uptake capability. This suggests that the introduction of folic acid greatly enhanced the specific recognition of the nanomaterials by tumor cells, thereby promoting cellular uptake. Therefore, all cellular uptake experiments indicate that the folic acid-modified nanocarrier can significantly improve the targeting ability of FA-CD@PAP NPs to tumor cells overexpressing folic acid receptors.

[0029] Characterization of nuclear targeting (see appendix) Figure 3 ) (1) Plating: First, select HeLa cells in good growth condition, remove the old culture medium, wash three times with sterile PBS, then digest with 0.25% trypsin, centrifuge, add fresh culture medium to resuspend, count the cell 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.

[0030] (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 100 μg / mL of Ppa, PAP or FA-CD@PAP NPs to the laser confocal dish and incubate in a cell culture incubator at 37℃ and 5% CO2 for 12 h.

[0031] (3) Staining: First, 2 μL of the stock solution of the 1 mM Hoechst 33258 nuclear 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.

[0032] (4) Imaging: After co-incubation, the corresponding probe dyes in the dish were removed by centrifugation, and the cells were washed three times with sterile PBS to remove excess probe dyes to 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 405nm or 488nm multi-argon laser and a 633nm diode laser. The excitation and emission wavelength parameters of the photosensitizer compound were: Ex / Em = 633 / 650-750nm; the excitation and emission wavelength parameters of the nuclear probe dye were: Ex / Em = 405nm / 425-475nm.

[0033] Laser confocal microscopy was used to observe the red fluorescence of Ppa and PAP and the blue fluorescence of the nuclear probe. The red fluorescence of free Ppa showed almost no overlap with the blue fluorescence signal representing the cell nucleus. Further qualitative analysis of the fluorescence intensity line scan spectra showed significant separation signals between the Ppa group and the nuclear dye. Figure 3 A). By introducing a nuclear localization signal peptide, after 12 hours of incubation, the free PAP group showed visible fluorescence overlap between the red fluorescence signal and the nuclear dye, with pink fluorescent spots appearing in the cell nucleus. Qualitative analysis of the fluorescence intensity line scan spectrum also indicated partial overlap between the PAP group and the nuclear dye, suggesting that the free PAP exhibited visible nuclear targeting ability. In contrast, the FA-CD@PAP NPs group showed strong pink fluorescent spots in the cell nucleus, and qualitative analysis of the fluorescence intensity line scan spectrum also showed a high degree of overlap between the PAP and the nuclear dye. Therefore, these data strongly demonstrate that FA-CD@PAP NPs can significantly enhance the enrichment of photosensitizers in the cell nucleus, achieving the goal of targeting the cell nucleus.

[0034] In vitro photodynamic activity characterization (see appendix) Figure 4 ) 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: Mitochondria in 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. Mitochondria in dead cells do not contain this reducing succinate dehydrogenase, and therefore cannot reduce MTT or generate blue-purple crystalline formazan. Related experiments have shown that dimethyl sulfoxide (DMSO) can dissolve the formazan deposited in 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: I%=[(A-A0) / (As-A0)]×100% 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.

[0035] Experimental methods (1) Plating: Select HeLa 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. 4 Cells / 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.

[0036] (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 hours.

[0037] (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=670nm, 4mW / cm²). 2 2.4 J / cm 2 Irradiate for 10 minutes. Finally, incubate in a cell culture incubator at 37°C and 5% CO2 for 24 hours. 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.

[0038] (4) Testing: After culturing for 24 hours, 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 hours. 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 minutes 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.

[0039] The experimental data shows that, in the absence of laser irradiation ( Figure 4 (A) No significant cytotoxicity was observed in HeLa cells across all drug groups, indicating negligible dark toxicity in different drug groups. However, under LED laser plate conditions (λ=670nm, 4mW / cm²), significant cytotoxicity was observed. 2 2.4 J / cm 2 Under the condition of irradiation for 10 minutes ( Figure 4 B) The photocytotoxicity of FA-CD@PAP NPs was significantly higher than that of the free photosensitizer Ppa, FA-CD@PA NPs group and blank control group, indicating that within a smaller concentration range, the drug group with tumor cell and cell nucleus targeting functions showed more significant killing ability on HeLa cells, thereby achieving stronger anti-tumor cell proliferation ability.

[0040] In vivo photodynamic activity characterization (see appendix) Figure 5 ) 200 μL containing HeLa cells (3 × 10⁻⁶) 6 A tumor model was established by subcutaneous inoculation of 100 cells (approximately 1000 cells) into the right hind limb of nude mice, and tumor growth was closely monitored. After 5 days, when the tumor grew to 50-100 mm... 3 Size (using length × width) 2The tumor volume was calculated using the formula / 2. All mice were randomly divided into six groups, including (I) Control (saline), (II) FA-CD NPs, (III) Free PA, (IV) CD@PAP NPs, (V) FA-CD@PA NPs, and (VI) FA-CD@PAP NPs, with five mice in each group (n=5) for the experiment.

[0041] Before intravenous drug injection, mice were weighed and tumor size was measured. Each mouse was then intravenously injected with different drugs (100 μg / mL, 200 μL) and saline solution according to its group. Twelve hours after drug administration, the tumor area was irradiated with a 670 nm laser for 10 minutes, and tumor volume was monitored every other day. The growth status and tumor size of the mice indicated that the single-targeting material groups, including free PA, CD@PAP NPs, and FA-CD@PA NPs, could only partially inhibit tumor growth, while the FA-CD@PAP NPs group almost completely inhibited tumor growth. This clearly demonstrates that the dual-targeting nanomaterial photosensitizer enhances the tumor-inhibiting ability of PDT. Simultaneously, the tumor growth in the FA-CD NPs group was rapid, similar to the control group. The relative tumor growth curves plotted based on the measurement data clearly show that the FA-CD@PAP NPs group almost completely inhibited tumor growth. All experimental results indicate that FA-CD@PAP NPs with dual targeting function can precisely deliver photosensitizers to HeLa cells and further to the cell nucleus, thereby achieving targeted photodynamic therapy for cervical cancer and exhibiting significant in vivo tumor-suppressing efficacy.

Claims

1. A method for preparing a dual-targeting nanomaterial photosensitizer for cervical cancer, comprising the following steps: (1) β -CD dissolved in NaOH aqueous solution reacted with a crosslinking agent to give a white solid product Poly- β -CD; (2) Add folic acid to anhydrous DMF solution, then add NHS, EDCI and adamantane to the mixed reaction solution, react in an ice-water bath, after the reaction is completed, transfer to a mixture of acetone and water to form a precipitate, vacuum filter and dry to obtain orange solid compound FA-Ad; (3) Add pyrophyllite a to DMF solution, then add HOBT, EDCI and hexaaminocaproic acid, react under nitrogen protection at 25℃, and after the reaction is completed, purify the mixture by column chromatography to obtain brown-black solid compound PAA. (4) Dissolve PAA, HBTU and DIEA in anhydrous DMF solution and react in the dark. Then, add PAAKRVKLD to the reaction system and continue the reaction in the dark. After the reaction is completed, wash away the unreacted substances with DMF and methanol solutions respectively. Then soak the sample in a mixture of trifluoroacetic acid and water, collect the filtrate, add 10 times the volume of anhydrous diethyl ether to precipitate the precipitate, centrifuge to collect the solid and freeze dry to obtain the gray-green solid compound PAP. (5) Take the Poly- obtained in step (1) β -CD and PAP obtained in step (4) were dissolved in a mixed solution of DMSO and water and reacted at room temperature in the dark. Then, FA-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water and slowly added to the reaction system at room temperature in the dark to continue the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain brown solid powder FA-CD@PAPNPs.

2. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 1, characterized in that: In step (1), first accurately weigh 10.0g. β -CD was dissolved in 15 mL of 15 wt% NaOH aqueous solution and stirred at room temperature for 2 h. Then, 2 mL of toluene was added dropwise to the mixture and stirring continued for 2 h. Next, 3.8 mL of epichlorohydrin as a crosslinking agent was slowly added dropwise, and the reaction continued for 3 h. After the reaction was complete, 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, it was dialyzed through a dialysis bag to completely remove monomers and oligomers. Finally, it was freeze-dried under vacuum to obtain a white solid product, Poly- β -CD.

3. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 1, characterized in that: In step (2), 0.9 g and 2.0 mmol of folic acid 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 mixed solution. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the reaction solution was transferred to a mixture of 300 mL of acetone and water to form a precipitate. The precipitate was filtered under vacuum and finally dried under vacuum at 50 °C for 24 h to obtain an orange solid compound FA-Ad.

4. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 1, characterized in that: In step (3), 42.8 mg and 0.08 mmol of pyromethesin a, 16.0 mg and 0.12 mmol of HOBT, and 23.0 mg and 0.12 mmol of EDCI were accurately weighed and dissolved in 3 mL of anhydrous DMF solution. The mixture was reacted at room temperature in the dark for 30 min. Then, 12.6 mg and 0.10 mmol of hexaaminocaproic acid were added, and the reaction was continued for 24 h under N2 protection. After the reaction was completed, the mixture was purified by column chromatography to obtain the brown-black solid compound PAA. In column chromatography, the volume ratio of CH2Cl2 / CH3OH is 30:

1.

5. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 1, characterized in that: In step (4), 28.0 mg and 0.04 mmol of PAA, 48.0 mg and 0.12 mmol of HBTU and 93.0 μL of DIEA were accurately weighed and dissolved in 3 mL of anhydrous DMF solution. The reaction was carried out at room temperature in the dark for 30 min. Then, 15.0 mg and 0.02 mmol of N-terminally exposed NLS molecules PAAKRVKLD were added to the reaction system and the reaction was continued in the dark for 24 h under N2 protection. After the reaction was completed, the unreacted substances were washed away with DMF and methanol solutions, respectively. The sample was lyophilized and then soaked in a mixture of 95% trifluoroacetic acid and 5% water for 4 h. The solid was filtered off and the filtrate was collected. Anhydrous diethyl ether was added to 10 times the volume of the filtrate to precipitate the solid. The solid was collected by centrifugation and lyophilized to obtain gray-green PAP molecules, which were stored at -20 °C.

6. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 1, characterized in that: In step (5), 21.0 mg of the Poly- obtained in step (1) is... β -CD and 2.0 mg of PAP obtained in step (4) were dissolved in a mixed solution of DMSO and water and reacted at room temperature in the dark for 2 h. Then, 2.0 mg of FA-Ad obtained in step (2) was dissolved in a mixed solution of DMSO and water 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 for dialysis. Finally, the reaction solution was freeze-dried to obtain brown-black solid powder FA-CD@PAP NPs.

7. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 2, characterized in that: The dialysis conditions in step (1) are as follows: M W Dialysis was performed for 24 hours in an 8000-well dialysis bag, with the water changed every 3 hours.

8. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 3, characterized in that: In step (2), the ratio of acetone to water is 1:

4.

9. The method for preparing a photosensitizer for dual-targeting nanomaterials in cervical cancer according to claim 6, characterized in that: In step (5), the ratio of DMSO to water in the mixed solution is 1:3; the dialysis conditions are as follows: M W Dialysis was performed for 24 hours in a dialysis bag with a capacity of 3000, and the water was changed every 3 hours.

10. A dual-targeting nanomaterial photosensitizer for cervical cancer, characterized in that: The dual-targeting nanomaterial photosensitizer, as a nanomedicine carrier, is prepared by the method described in any one of claims 1-9.

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