Self-assembled hybrid photosensitizer nanoparticles and preparation method and application thereof

Abstract

The present invention provides self-assembled hybrid photosensitizer nanoparticles, and a preparation method and application thereof. The photosensitizer protoporphyrin PpIX and the HIF-1α inhibitor KC7F2 drug powder are dissolved in dimethyl sulfoxide to obtain a mixed solution, and the mixed solution is dissolved in a FeCI3 solution for self-assembly, centrifuged, and freeze-dried to obtain nanoparticles. For the first time, the small molecule inhibitor KC7F2 is used as a prodrug for a nanophotosensitizer, and its chemical structure is used to achieve the results of GSH response and quenching, thereby reducing the consumption of ROS and achieving the purpose of oxygenation. The above-mentioned simple preparation method is used to construct nanohybrid particles formed by the self-assembly of iron ions, protoporphyrin PpIX and KC7F2, aiming to realize a new oxygenation therapy strategy of PDT synergizing with CDT to reverse the hypoxic microenvironment of tumors.

Description

Technical Field

[0001] This patent belongs to the field of biochemistry, and in particular relates to self-assembled hybrid photosensitizer nanoparticles and their preparation methods and applications. Background Art

[0002] Due to the limitations of traditional tumor treatment methods such as surgical resection, radiotherapy or chemotherapy, such as high damage to the body, easy to produce drug resistance and side effects, photodynamic therapy (PDT), as a photobiological reaction involving oxygen molecules, has become an emerging treatment for superficial oral cancer (OSCC) due to its advantages such as high targeting ability, low invasiveness, low side effects, and maximum preservation of tissue function. Its principle is to stimulate the photosensitizer (PS) to react with the surrounding medium (such as O2, H + or e - ) reacts to generate singlet oxygen (1O2) or superoxide anion (O 2- ), hydroxyl radicals (OH·), etc. can cause selective photodamage to tumor tissues to achieve the purpose of treatment.

[0003] However, due to the rapid proliferation and abnormal microvascular structure and distribution of solid tumors, a hypoxic tumor microenvironment with high concentrations of GSH and H2O2 forms. Further oxygen consumption during type II PDT treatment severely reduces the production of ROS. Furthermore, the presence of GSH and H2O2 enhances the reduction of ROS, thereby reducing PDT efficacy. Furthermore, limitations such as limited tissue penetration depth and non-selective accumulation of photosensitizers (PS) further impair PDT efficacy, leading to PDT resistance.

[0004] Xiang Zhou et al. developed a self-releasing nanodrug CYC-1 (Xiang Zhou, Jia-Qi Huang, Ling-Shan Liu, Fu-An Deng, Yi-Bin Liu, Yan-Mei Li, A-Li Chen, Xi-Yong Yu, Shi-Ying Li, and Hong Cheng. Self-Remedied Nanomedicine for Surmounting the Achilles'Heel of Photodynamic Tumor Therapy. ACS Appl. Bio Mater. 2021, 4, 8023-8032.). This drug downregulates the expression of HIF-1α in tumor cells through YC-1, inhibits tumor growth, and alleviates the hypoxic microenvironment, thereby sensitizing cells to PDT and achieving a therapeutic effect. Junnan He et al. developed a nanomicelle composed of polyporphyrin and HIF-1α inhibitor YC-1 (Junnan He, Kangkai Xia, Binggong Zhao, Wangze Song, Yubin Zheng, Guishan Xiao, Huijian Wu and Nan Zheng. Codelivery of High-Molecular-Weight Poly-porphyrins and HIF-1αInhibitors for In Vivo Synergistic Anticancer Therapy. Biomacromolecules 2021, 22, 4783-4793.). The Cu-catalyzed multicomponent polymerization (MCP) method was used to prepare biodegradable porphyrin polymers using dialkyl-containing porphyrins and cleavable diols with flexible thioketone linkers as monomers. Then, micelles were prepared by self-assembly and nanoprecipitation of mPEG-PCL, polyporphyrin and YC-1 in aqueous solution. The purpose was to increase the Mw value of porphyrin by designing a new polymerization method to solve the aggregation-induced quenching (ACQ) problem of traditional porphyrin, improve its ability to produce 1O2, and at the same time synergize with YC-1 to inhibit tumor hypoxia-related pathways, thereby alleviating tumor hypoxia in disguise and improving the PDT efficacy.

[0005] The preparation process of the above-mentioned drugs is complicated, and the HIF-1α inhibitor YC-1 only alleviates tumor hypoxia and inhibits the proliferation and metastasis of tumor cells by inhibiting HIF-related survival pathways. However, because its structure does not contain disulfide bonds, it cannot quench GSH and increase the content of ROS in disguise. In addition, the drug does not contain iron ions and cannot achieve self-oxygen supply and ROS generation through the Fenton reaction while saving oxygen in disguise, thereby realizing synergistic chemodynamic therapy (CDT) treatment. Therefore, it does not have the dual-modal performance of fluorescence and nuclear magnetic resonance, and cannot well realize integrated diagnosis and treatment.

[0006] Mans B. et al. developed a nanodrug consisting of liposomes (purchased from Aventi Polar Lipids) encapsulating the photosensitizer ZnPC (purchased from Sigma-Aldrich) and the HIF-1 inhibitor acriflavine (ACF) (purchased from Sigma-Aldrich) (Mans Broekgaarden1, Ruud Weijer1, Massis Krekorian1, Bas van den IJssel1, Milan Kos1, Lindy K. Alles1, Albert C. van Wijk1, Zsolt Bikadi2, Eszter Hazai2, Thomas M. van Gulik1, and Michal Heger1. Inhibition of hypoxia-inducible factor1 with acriflavine sensitizes hypoxic tumor cells to photodynamic therapy with zinc phthalocyanine-encapsulating cationic liposomes. Nano Research 2016,9(6):1639–1662.), the preparation formed by combining the dimerization domain between ZnPC and ACF molecules and then encapsulating them in a single liposome significantly improved the efficacy of PDT. The high molar extinction coefficient of ZnPC and the retention effect (EPR) produced by liposome encapsulation allowed a large amount of photosensitizer to reach the interior of the tumor. At the same time, ACF could inhibit the polymerization of HIF-1α and HIF-1β, thereby increasing the degree of cell death induced by PDT under hypoxic conditions and reducing the expression of HIF-1 target genes VEGF, PTGS2 and EDN1.

[0007] The liposome encapsulation process is relatively complicated, and the HIF-1 inhibitor ACF only alleviates tumor hypoxia and inhibits tumor cell proliferation and metastasis by inhibiting HIF-related survival pathways. However, because its structure does not contain disulfide bonds, it cannot quench GSH and increase the ROS content in disguise. In addition, the drug does not contain iron ions and cannot achieve self-oxygen supply and ROS generation through the Fenton reaction while saving oxygen in disguise, and cooperate with CDT treatment. Its photosensitizer ZnPC does not have the dual-modal performance of fluorescence and nuclear magnetic resonance, and cannot better realize integrated diagnosis and treatment. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing self-assembled hybrid photosensitizer nanoparticles. For the first time, the small molecule inhibitor KC7F2 is used as a prodrug for nanophotosensitizers, and its chemical structure is utilized to achieve GSH response and quenching results. Nanohybrid particles are formed by self-assembly of iron ions, protoporphyrin PpIX and the HIF-1α inhibitor KC7F2. The preparation method is simple and realizes a new oxygen enhancement therapy strategy for PDT combined with CDT.

[0009] The second object of the present invention is to provide self-assembled hybrid photosensitizer nanoparticles.

[0010] The third object of the present invention is to provide an application of self-assembled hybrid photosensitizer nanoparticles.

[0011] In order to achieve the above-mentioned first objective, the present invention provides a method for preparing self-assembled hybrid photosensitizer nanoparticles, comprising dissolving the photosensitizer protoporphyrin PpIX and the HIF-1α inhibitor KC7F2 drug powder in dimethyl sulfoxide (DMSO) to obtain a mixed solution, dissolving the mixed solution in an FeCl3 solution for self-assembly, centrifuging, and freeze-drying to obtain nanoparticles.

[0012] As a preferred solution, the molar ratio of protoporphyrin PpIX and HIF-1α inhibitor KC7F2 is 2:1. Initial preliminary experiments have confirmed that the optimal nanoparticle drug loading ratio of 2:1 maximizes drug delivery and maximizes drug efficiency.

[0013] As a preferred embodiment, the pH of the FeCI3 solution is 6.5.

[0014] In order to achieve the above second object, the present invention provides self-assembled hybrid photosensitizer nanoparticles prepared by the above method, comprising iron ions, protoporphyrin PpIX and HIF-1α inhibitor KC7F2.

[0015] In order to achieve the third objective mentioned above, the present invention provides the use of the self-assembled hybrid photosensitizer nanoparticles in the preparation of oral cancer diagnosis or treatment drugs.

[0016] The HIF-1α inhibitor KC7F2 is a cell-permeable, potent hypoxia-inducible factor that inhibits the activation of HIF target genes. Its molecular formula is: C 16 H 16 C l 4N2O4S4.

[0017] Oral cancer clinically includes tongue cancer, cheek cancer, gum cancer, palate cancer, lip cancer, maxillary and mandibular cancer, floor of mouth cancer, etc., and tissue types include mucoepidermoid carcinoma and squamous cell carcinoma.

[0018] The role of the photosensitizer protoporphyrin PpIX: It can not only use O2 to produce ROS to kill tumors under irradiation, but it also has its own fluorescence effect, which can realize the integration of diagnosis and treatment.

[0019] Fe 3+ Function: Inducing Fenton reaction to consume high concentrations of H2O2 inside the tumor, increasing the oxygen content in the cell, which is equivalent to providing a self-oxygen supply system to alleviate TME hypoxia, while also increasing ROS content to achieve synergistic CDT treatment; In addition, Fe 3+ The presence of enables KPF to have the function of magnetic resonance imaging and also realize the integration of diagnosis and treatment.

[0020] The function of KC7F2: Inhibits the expression of HIF-1α and its downstream genes, thereby blocking the HIF-1α / VEGF hypoxia-related survival pathway of the tumor and inhibiting tumor proliferation and metastasis; at the same time, the disulfide bonds contained in it can quench the high concentration of GSH inside the tumor, inhibiting tumor cells from clearing ROS to achieve the purpose of saving oxygen.

[0021] The advantages of the present invention are that it directly self-supplies oxygen and generates ROS by inducing the Fenton reaction under low pH response through internal iron ions to achieve combined chemodynamic therapy. For the first time, the small molecule inhibitor KC7F2 is used as a prodrug in a nanophotosensitizer, and its chemical structure is utilized to achieve GSH response and quenching results. HIF-1α inhibitors can not only reverse the hypoxia-related survival pathway of tumors, but also use internal disulfide bonds to quench excess GSH to further increase ROS production and reduce the clearance rate of ROS in tumor cells, thereby improving the tumor hypoxic microenvironment, inhibiting tumor proliferation and metastasis, enhancing the efficacy of PDT in selectively killing cancer cells, and reducing toxic side effects on normal cells. In addition, the presence of iron ions gives the system the performance of nuclear magnetic resonance imaging. In addition, the photosensitizer PpIX itself has a fluorescence effect, making the system a supramolecular polymer with both fluorescence and nuclear magnetic resonance dual modalities, achieving dual functions of diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM image of KPF nanoparticles;

[0023] Figure 2 DLS results of KPF nanoparticles;

[0024] Figure 3 Variable temperature infrared results of KPF nanoparticles;

[0025] Figure 4 Field emission electron microscopy results of KPF nanoparticles;

[0026] Figure 5 MRI T1 angiography results of KPF nanoparticles;

[0027] Figure 6 MTT verifies the low toxicity results of KPF;

[0028] Figure 7 MTT verifies the results of KPF photodynamic killing effect;

[0029] Figure 8 Intracellular ROS detection results under laser confocal microscopy;

[0030] Figure 9 Flow cytometry was used to detect the results of intracellular ROS production;

[0031] Figure 10 Flow cytometry was used to detect apoptosis in the non-illumination group;

[0032] Figure 11 Flow cytometry was used to detect the results of KPF illumination group;

[0033] Figure 12 These are the cellular uptake results of the PpIX group and the KPF group under laser confocal microscopy;

[0034] Figure 13 The results of cellular uptake were detected by flow cytometry in the PpIX and KPF groups respectively;

[0035] Figure 14 Results of tumor volume changes in each group after PDT treatment in PDX models;

[0036] Figure 15 Verify the changes in GSH content in tumor cells after KPF treatment;

[0037] Figure 16 Characteristic UV absorption peak results of photosensitizer PpIX;

[0038] Figure 17 Characteristic UV absorption peak results of KPF nanoparticles;

[0039] Figure 18Results of high performance liquid chromatography-mass spectrometry (LC-MS) analysis of the photophysical properties of KPF nanoparticles;

[0040] Figure 19 Western blot experiments were used to verify the effects on the HIF-1α / VEGF hypoxia-related pathway under hypoxic and aerobic conditions;

[0041] Figure 20 PCA analysis of metabolomics data of intracellular metabolites in the treatment and control groups;

[0042] Figure 21 Fluorescence emission peak analysis diagram of KPF nanoparticles and PpIX photosensitizer. DETAILED DESCRIPTION

[0043] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only intended to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0044] Example 1. Preparation and physicochemical characterization of self-assembled hybrid photosensitizer nanoparticles (KPF)

[0045] PpIX and inhibitor KC7F2 drug powders were dissolved in DMSO (the molar ratio of the two was 2:1, and the unit was μM) to obtain a mixed solution. According to the principle of trivalent iron ion coordination drive, the DMSO solution was dissolved in a PBS solution of FeCl3 with a pH of 6.5 (Fe 3+ The sample was then self-assembled in a 0.1 μM solution of KPF nanoparticles (PpIX, KC7F2, and DMSO). Unassembled PpIX, KC7F2, and DMSO were removed by centrifugation to obtain a red aqueous solution of KPF nanoparticles. Some samples were freeze-dried to obtain a powder, which was stored at room temperature in the dark.

[0046] KPF nanoparticle morphology and physicochemical characterization analysis:

[0047] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to detect the particle size, dispersion and morphology of KPF. TEM showed that the particle size was spherical, uniform in size and dispersion was acceptable (see Appendix). Figure 1 ); DLS detected the average particle size to be around 90-100 nm (see Appendix Figure 2 ), indicating that the KPF system has good stability, which is consistent with the characteristics of nanoparticles.

[0048] The photophysical properties and drug content of KPF nanoparticles were analyzed by UV-visible photometer, fluorescence spectrophotometer and high performance liquid chromatography-mass spectrometry (LC-MS). The results showed that the nanoparticles contained PpIX, Fe 3+ and KC7F2 characteristic ultraviolet absorption peaks (see attached Figure 16 、17 ); contains the characteristic fluorescence emission peak of PpIX (see Appendix Figure 21 ); LC-MS proved that it has good photophysical properties and the drug loading capacity can be (see Appendix Figure 18 ).

[0049] X-ray photoelectron spectroscopy (XPS) was used to analyze the iron content of the nanoparticles, and field emission electron microscopy was used to characterize the elemental composition of the nanoparticles (see Appendix). Figure 4 ), proving the successful preparation of KPF.

[0050] MRI studies have analyzed the relaxation rate and imaging effects of nanoparticles: it can be seen that the relaxation rate is almost proportional to the concentration, and the effect is good. (See Appendix Figure 5 )

[0051] Coarse-grained molecular dynamics (CGMD) simulations of PpIX, KC7F2 and FeCl 3 The assembly process of the α-Hydrogen bond was detected by infrared spectroscopy at variable temperature. (See Appendix Figure 3 )

[0052] Example 2. KPF-mediated in vitro cell experimental study

[0053] MTT cytotoxicity test showed that KPF has low cytotoxicity (see Appendix Figure 6 )

[0054] (1) Experimental design:

[0055] In order to verify the effect of KPF nanoparticles on the proliferation of oral squamous cell carcinoma cells (CAL27, HN4, SCC4), oral epidermoid carcinoma cells (KB) and normal cells (L929), each cell type was divided into 6 groups according to the experimental requirements, namely

[0056] Control group (no drug stimulation, cultured with regular complete medium)

[0057] Iron ion group (concentration gradient: 0.1μM, 0.2μM, 0.4μM, 0.6μM, 0.8μM, 1μM)

[0058] KC7F2 group (concentration gradient: 0.5μM, 1μM, 2μM, 3μM, 4μM, 5μM)

[0059] PpIX group (concentration gradient: 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM)

[0060] KC7F2 and PpIX mixed group (PpIX:KC7F2 ratio was 2:1)

[0061] KPF group (PpIX concentration gradient: 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM)

[0062] (2) Experimental methods:

[0063] Tumor cells were seeded into 96-well plates (10,000 cells per well) according to the group. After the cells adhered, they were stimulated by adding drugs according to a concentration gradient and incubated in a 37°C, 5% CO2 incubator for 24 hours. 10 μL of MTT reagent was added to each well and the cells were incubated in a 37°C, 5% CO2 incubator for 4 hours. The culture medium was discarded, and 100 μL of DMSO was added to each well to dissolve the blue-purple precipitate. The OD value at 490 nm was measured, and cell growth curves were plotted at different concentrations. By comparing the cell proliferation activity of each group, the low toxicity of KPF nanoparticles was confirmed. The results showed that all groups of cells had a high survival rate, indicating that KPF has good biosafety.

[0064] Example 3. Laser confocal microscopy and flow cytometry were used to detect the intracellular uptake of nanophotosensitizers (see Appendix Figure 12 、 13 )

[0065] (1) Experimental design:

[0066] In order to verify the effect of KPF nanoparticles on the uptake of oral squamous cell carcinoma cells (CAL27), the cells were divided into two groups, namely PpIX group and KPF group (the fixed concentration of PpIX was 10 μM), and confocal microscopy and flow cytometry were used to verify the drug uptake of tumor cells at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration.

[0067] (2) Experimental methods:

[0068] Conventional cells were seeded into six-well plates. After the cells adhered to the wall, drugs were added according to the above time gradient for stimulation and the cells were cultured in a 37° C., 5% CO 2 incubator in the dark.

[0069] Confocal laser scanning microscopy analysis:

[0070] After the cell density reached approximately 50%-60%, 10 μM PpIX and KPF solutions were added to six-well plates and incubated for 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours, respectively. The drug-containing medium was then aspirated and the cells were washed 2-3 times with PBS. 1 ml of paraformaldehyde solution was added to each well to fix the cells and incubated in a 37°C incubator in the dark for 20-30 minutes. After aspirating the paraformaldehyde, the cells were washed 2-3 times with PBS for 3-5 minutes each. A small amount of DAPI staining solution was added to each well to cover the cells and incubated in the dark at room temperature for 3-5 minutes. The DAPI staining solution was aspirated and the cells were washed 2-3 times with PBS for 3-5 minutes each. The distribution and intensity of red fluorescence (PpIX) and blue fluorescence (nuclei) were observed under a confocal microscope to confirm cellular uptake. The results showed that as the time PpIX and KPF entered the cells increased, the intracellular fluorescence signal gradually increased, reaching its peak after 4 hours.

[0071] Flow cytometric analysis:

[0072] After the cell density grew to approximately 80%-90%, 10 μM PpIX and KPF solutions were added to six-well plates and incubated for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h, respectively. The medium containing the drugs was then aspirated and washed 2-3 times with PBS. The cells were then digested with EDTA and transferred to a centrifuge tube. The tubes were centrifuged at low speed (1000 r, 3 min), washed 2-3 times with PBS, and centrifuged again. The supernatant was aspirated, 1 ml of PBS was added to each well, and the mixture was pipetted and transferred to a flow cytometer. The tubes were stored on ice and the red fluorescence intensity of PpIX and KPF was measured by flow cytometry as the drug treatment time increased. The results showed that the fluorescence intensity of PI-A-labeled cells gradually increased with the extension of drug treatment time, and the number also increased accordingly, reaching the highest level after 4 h, demonstrating that the cells had a good uptake of PpIX and KPF.

[0073] Example 4. GSH assay kit was used to detect changes in GSH content in tumor cells (see Appendix Figure 15 )

[0074] (1) Experimental design:

[0075] Since the KC7F2 inhibitor contains disulfide bonds, it can quench GSH inside tumor cells, thereby reducing the consumption of ROS. Therefore, a GSH detection kit was used to detect the changes in GSH content in tumor cells after drug treatment. The cells were divided into 9 groups: PBS control group, KC7F2 group, PpIX group, KC7F2 and PpIX mixed group, KPF group, PpIX + light group, mixed + light group, KPF light group, Fe 3+ group (in which the fixed effective concentration of PpIX was 4 μM).

[0076] (2) Experimental methods:

[0077] The relevant reagents required for the experiment were prepared according to the requirements of the kit, and standard solutions with a certain concentration gradient (15, 10, 5, 2, 1, 0.5 μM GSSG solution) and standard solutions after GSH removal were prepared. The GSH content was detected at 412 nm and a standard curve was drawn.

[0078] Tumor cells were routinely inoculated into six-well plates and added after culture until 80%-90% cells were grown. The light-exposed group was irradiated for 5 minutes 4 hours after drug addition. All cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. After aspirating the culture medium, the cells were washed once with PBS, and the cells of each group were collected by centrifugation, and the supernatant was aspirated. Protein removal reagent M solution was added in an amount 3 times the volume of the cell pellet and fully vortexed. The sample was quickly frozen and thawed twice using liquid nitrogen and a 37°C water bath, and then placed at 4°C or on ice for 5 minutes. After centrifugation at 4°C and 10,000g for 10 minutes, the supernatant was collected and used for the determination of total glutathione and GSSG content after GSH removal (OD value at 412nm) according to the requirements of the kit.

[0079] The final GSH content of each group = total glutathione content - GSSG content after GSH removal. The results showed that the GSH content of each group containing KC7F2 was significantly reduced, and the KPF + light group had the best effect.

[0080] Example 5. MTT cytotoxicity test proves the killing effect of KPF-mediated PDT on tumor cells (see Appendix Figure 7 )

[0081] (1) Experimental design:

[0082] In order to verify the effect of KPF nanoparticles on the proliferation of oral squamous cell carcinoma cells (CAL27, HN4, SCC4), oral epidermoid carcinoma cells (KB) and normal cells (L929), each cell type was divided into 9 groups according to the experimental requirements, namely

[0083] Control group (no drug stimulation, cultured with regular complete medium)

[0084] Iron ion group (concentration gradient: 0.1μM, 0.2μM, 0.4μM, 0.6μM, 0.8μM, 1μM)

[0085] KC7F2 group (concentration gradient: 0.5μM, 1μM, 2μM, 3μM, 4μM, 5μM)

[0086] PpIX group (concentration gradient: 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM)

[0087] KC7F2 and PpIX mixed group (PpIX:KC7F2 ratio was 2:1)

[0088] KPF group (PpIX concentration gradient: 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM)

[0089] PpIX+light group (concentration gradient: 1μM, 2μM, 4μM, 6μM, 8μM, 10μM)

[0090] KC7F2 and PpIX mixed + light group (PpIX:KC7F2 ratio is 2:1)

[0091] KPF+light group (PpIX concentration gradient: 1μM, 2μM, 4μM, 6μM, 8μM, 10μM)

[0092] (2) Experimental methods:

[0093] Cells were seeded into 96-well plates according to the group, with 10,000 cells per well. After the cells attached to the wall, the drug was added. Four hours after the drug exposure, the cells were illuminated with a 650nm semiconductor laser (100mW / cm2, 12J / cm2) for 600s. After exposure, the cells were incubated at 37°C and 5% CO2 for another 24 hours. Then, 10μl of MTT reagent was added to each well and incubated for 4 hours before being dissolved in DMSO. The OD value was measured at 490nm to detect the cell-killing effect of KPF-mediated PDT. The cell survival rate of each group was calculated, and a cell killing curve was plotted. The results showed that the cell survival rate of the PpIX, KC7F2, and KPF groups decreased with increasing concentration, but the decrease was more significant in the KPF group. Furthermore, at 6μM PpIX, the cell survival rate in the KPF group was almost zero, fully demonstrating the strong phototoxicity and cell-killing ability of KPF.

[0094] Example 6. Using a ROS kit to detect the effect of KPF-mediated PDT on ROS production in tumor cells (see Appendix Figure 8 、 9 )

[0095] (1) Experimental design:

[0096] The cells were divided into 9 groups: PBS control group, KC7F2 group, PpIX group, KC7F2 and PpIX mixed group, KPF group, PpIX + light group, mixed + light group, KPF light group, Fe 3+The cells were treated with the aforementioned groups and drug concentrations (where PpIX was fixed at a 4 μM effective concentration). After incubation with the drug for 4 hours, the cells were illuminated for 5 minutes and then cultured for an additional 24 hours. The cells in each group were treated with a ROS probe, and the intracellular fluorescence intensity was qualitatively and quantitatively measured using laser confocal microscopy and flow cytometry.

[0097] (2) Experimental methods:

[0098] Cal27 cells were seeded in a conventional six-well plate and stimulated with drugs after reaching 80%-90% cell growth. For the light-exposed group, cells were exposed to light for 5 minutes after 4 hours of drug stimulation. After 24 hours of incubation, the cell culture medium was removed and an appropriate volume of diluted DCFH-DA probe was added. Incubation was performed in a 37°C cell culture incubator in the dark for 20 minutes. The cells were washed three times with PBS (5 minutes each time) to fully remove the DCFH-DA that had not entered the cells.

[0099] If using a confocal microscope to maintain cell morphology, after aspirating PBS, add 1 ml of 4% paraformaldehyde to each well for fixation. Place in the dark at room temperature for 20-30 minutes, then aspirate the paraformaldehyde and wash with PBS three times (5 minutes each time). Observe the production of green ROS fluorescence inside the cells. If used for flow cytometry, it is necessary to collect the cells by conventional EDTA digestion and centrifugation, wash with 1 ml of PBS, and transfer to a flow tube for processing. The results showed that the ROS content in the KPF group was significantly higher than that in the control group; the ROS content in the KC7F2 group was also significantly higher than that in the blank group, indicating that the disulfide bonds in KC7F2 consumed the GSH inside the cells, thereby reducing the quenching of ROS.

[0100] Example 7. Detection of KPF-mediated PDT-induced tumor cell apoptosis (see Appendix Figure 10 、 11 )

[0101] (1) Experimental design:

[0102] The tumor cells were divided into three blank groups (Blank group, FITC group, PI group) and six non-illumination groups (Control group, Fe 3+ The cells were divided into three groups (group A, group B, KC7F2, group C, PpIX, a mixture of KC7F2 and PpIX, and group D), and three light-exposed groups (group B, group C, PpIX, a mixture of KC7F2 and PpIX, and group D). After drug treatment, the percentage of apoptosis in each group was determined by flow cytometry, demonstrating the cell-killing effect of KPF.

[0103] (2) Experimental methods:

[0104] Cells were seeded into a conventional six-well plate to 80%-90% cytotoxicity. After drug addition and illumination treatment, the cells were cultured for 24 hours. The culture medium was aspirated, digested with EDTA, and placed in a centrifuge tube. The cells were centrifuged (1000 rpm for 3 minutes). The supernatant was discarded and the cells were washed 2-3 times with PBS, centrifuged each time. After discarding the supernatant, the cells were gently resuspended in 400 μl of Annexin V-FITC conjugate solution. 195 μl of the suspension was placed in a flow cytometer tube, 5 μl of Annexin V-FITC was added, and the cells were gently mixed. 10 μl of PI was added and the cells were gently mixed. The cells were incubated at room temperature in the dark for 10-20 minutes, then stored on ice in the dark and processed. The results showed that the KPF + illumination group had the strongest killing effect and the highest percentage of apoptosis.

[0105] Example 8. Effect of KPF-mediated PDT on tumor cell apoptosis-related pathways

[0106] Total protein was extracted from cells in each group and then assayed for Caspase-3 expression. Western blot was also performed to examine the expression of apoptosis-related proteins: Caspase-3, Caspase-6, Caspase-7, and Cleaved Caspase-3; pro-apoptotic proteins: Fas, Bax, Bak, and Bad; and anti-apoptotic proteins: Bcl-2 and Survivin. This study aimed to investigate whether KPF could inhibit tumor cell proliferation and metastasis by blocking tumor apoptosis pathways. The results demonstrated that KPF significantly inhibited apoptosis-related pathways, as evidenced by the upregulation of both pro- and anti-apoptotic proteins.

[0107] Example 9. Effect of KPF-mediated PDT on tumor HIF-1α / VEGF hypoxia-related pathway (see Appendix Figure 19 )

[0108] Each cell line was placed in a normoxic environment (21% O2) and a hypoxic microenvironment (1% O2), respectively. After laser treatment, Western blot was used to detect the expression of key proteins related to the hypoxia-inducible factor HIF-1α / VEGF pathway, such as HIF-1α, VEGF, EPO, GLUT-1, GLUT-3, LDHA, and CAN.

[0109] A HIF-1α-overexpressing lentiviral vector was constructed and recHIF-1α was transfected into L929 cells, oral squamous cell carcinoma cells CAL27, HN4, and SCC4 cells, and oral epidermoid carcinoma KB cells, respectively. Stable HIF-1α-overexpressing strains were screened. After laser treatment in normoxic (21% O2) and hypoxic microenvironment (1% O2), the expressions of key proteins involved in the hypoxia-inducible factor (HIF-1α) pathway, including HIF-1α, VEGF, EPO, GLUT-1, GLUT-3, LDHA, and CNA, were detected by Western blot.

[0110] The results showed that KPF significantly inhibited the HIF-1α / VEGF hypoxia-related pathway, as manifested by the downregulation of hypoxia-related proteins.

[0111] Example 10. Study on the mechanism of cell death induced by KPF-mediated PDT (see Appendix Figure 20 )

[0112] That is, metabolomics methods are used to study the effects of KPF nanoparticle-mediated PDT on the redox process, glycolysis, and mitochondrial metabolism in tumor cells at the cellular and molecular level. Pathway analysis is performed through statistical analysis of metabolites to further clarify its effects on various pathways at the cellular and molecular level, thereby verifying whether it affects the HIF-1α / VEGF hypoxia pathway and apoptosis pathway.

[0113] The experiment was divided into PBS control group, PpIX group, KC7F2 group and KPF group: CAL27 cells were cultured at 5×10 7 Cells were plated at a density of 100 cells / well in 100 mm culture dishes. Each group of drugs (PpIX concentration was 4 μM and KC7F2 concentration was 2 μM) was added to different culture dishes, with 4 replicates per group. After 4 hours, the cells were illuminated for 5 minutes and cultured for another 48 hours. The supernatant was removed by washing and centrifugation, and the samples were sent for LC-MS (AB 5500) detection. The data were subjected to unidimensional statistical analysis and multivariate statistical analysis by T-test and SIMCA-P, respectively.

[0114] The results showed that KPF had a significant inhibitory effect on the HIF-1α / VEGF hypoxia pathway and apoptosis pathway.

[0115] Example 11. KPF-mediated anti-tumor experimental study in vivo (see Appendix Figure 14 )

[0116] Construction of human oral squamous cell carcinoma PDX model:

[0117] The human oral squamous cell carcinoma specimens were removed during surgery and trimmed into 1 mm 3 The tumor was implanted subcutaneously in the right axilla of severely immunodeficient nude mice (type BALB / c) until the tumor grew to 150-200 mm. 3 When the transplanted tumor is removed and trimmed into small tissue blocks, it is then transplanted into the subcutaneous tissue of other nude mice through subculture. After three generations of reproduction, KPF-related experiments are performed on the PDX model of the third generation of nude mice.

[0118] In vivo drug distribution studies and pharmacokinetic experiments based on PDX models:

[0119] Nude PDX mice were randomly divided into three groups of three. PpIX and KPF nanoparticles were injected via the tail vein at a 1 mg / kg dose of PpIX. A control group received an equal volume of saline. After anesthesia took effect, in vivo fluorescence imaging was used to measure the fluorescence intensity of major organs and tumor sites at different time points. This analysis allowed for analysis of drug distribution and tumor accumulation, as well as the pharmacokinetic and temporal characteristics of the nanophotosensitizer.

[0120] In vivo PDT efficacy experiments based on PDX models:

[0121] (1) Grouping:

[0122] Non-illumination groups: normal saline group, KC7F2 group, PpIX group, KPF nanoparticle group

[0123] Light exposure groups: normal saline group, KC7F2 group, PpIX group, KPF nanoparticle group

[0124] (2) Experimental method: PpIX: 1 mg / kg, KC7F2: 0.64 mg / kg, both administered by tail vein injection. Six hours after administration, the tumor area was irradiated with a semiconductor laser (100 mW / cm 2 , 12J / cm 2 , 600s). The control group was injected with 200μL of normal saline. One day later, the nude mice were sacrificed and tumor tissues were harvested.

[0125] a. Section staining analysis: After PDT treatment, tumor tissues of mice in each group were obtained and HE staining was performed to observe the degree of tumor tissue necrosis;

[0126] b. Immunofluorescence staining was followed by observation of the tumor-targeting distribution of nanoparticles and the induction of tumor cell apoptosis under a laser confocal microscope;

[0127] c. PDT treatment was performed once every two weeks for a total of three treatments. Tumor volume was measured and recorded each time. At the end of the treatment course, a tumor growth curve was drawn to compare the effects of photosensitizer photodynamic therapy in each group.

[0128] d. Tumor samples were collected and IHC and Western blot were performed to detect apoptosis-related proteins and key proteins related to the HIF-1α / VEGF pathway to demonstrate its anti-tumor mechanism.

[0129] In vivo fluorescence-NMR dual-modality analysis of KPF nanoparticles:

[0130] (1) Establishment of tumor-bearing mouse model: BALB / c nude mice were subcutaneously inoculated with 100 μL of 1×10 7CAL27 oral squamous cell carcinoma cells were cultured and, when the tumor tissue grew to a size of 0.5 cm × 0.5 cm, the tumor tissue was removed and divided into 0.1 cm × 0.1 cm pieces and transplanted subcutaneously into nude mice to establish a transplanted tumor mouse model. The tumor was allowed to grow for 2 weeks before use.

[0131] (2) MRI Scanning: Nude mice were randomly divided into three groups, each with three mice. PpIX and KPF nanoparticles were injected via the tail vein at a dose of 1 mg / kg PpIX. The control group received an equal volume of saline. 100 μL of 1% sodium pentobarbital was injected intraperitoneally. After anesthesia took effect, the mice were placed in a prone position in the magnetic resonance imaging small animal coil and anesthesia was maintained with isoflurane. The tumor was positioned in the center of the coil. After successful localization, the mice were moved to the scanning position of a Bruker biospec 7.0TMR and scans were started. Axial T1WI dynamic monitoring was performed before injection and at 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after injection.

[0132] (3) Data analysis: Measure the signal intensity (SI) and relative enhancement of signal intensity (RESI) of the region of interest.

[0133] The following formula was used for calculation: RESI (%) = SI enhancement / SI before injection × 100%. The measurement was repeated three times to obtain the average value.

[0134] The present invention selects iron ions and the novel HIF-1α inhibitor KC7F2 as a new PDT combined treatment strategy. On the one hand, the iron ion-mediated Fenton reaction directly increases the intracellular oxygen content, reversing the hypoxic microenvironment of the tumor; it has an indirect inhibitory effect on tumor metastasis and proliferation. In addition, the increased oxygen can increase the singlet oxygen yield of the photosensitizer, greatly increasing the efficacy of photodynamic tumor killing. On the other hand, KC7F2, as a small molecule HIF-1α inhibitor containing a disulfide bond structure, inhibits the expression of HIF-1α and its downstream genes. At the same time, its internal disulfide bond can also be activated by intracellular GSH and quench the GSH continuously produced in the tumor, blocking the intracellular reduction pathway, inhibiting the ROS scavenging ability of tumor cells, and controlling the level of ROS through redox balance. This is the first time that the small molecule inhibitor KC7F2 has been selectively used as a prodrug for nanophotosensitizers, and its chemical structure is used to achieve the results of GSH response and quenching.

[0135] The present invention clarifies the regulatory mechanism of oxygenation on the hypoxia pathway HIF-1α / VEGF and the cell death mechanism of photodynamic therapy, that is, blocking the hypoxia-related pathway and apoptosis pathway for tumor survival, inhibiting tumor angiogenesis and invasion and metastasis, thereby effectively expanding its application in the field of biomedicine, and providing an ideal drug delivery system for photodynamic therapy of tumors clinically. In addition, unlike the previous simple photosensitizer system that only has fluorescence imaging capabilities, due to the presence of iron ions, the system has the performance of nuclear magnetic resonance imaging. Based on the GSH response and the passive targeting of nanomedicines, the system has both fluorescence and nuclear magnetic dual modalities, and can have both diagnostic and therapeutic functions. This will be a direction with great academic value and clinical application prospects. At present, there are few reports on carrier-free self-assembled hybrid nanophotosensitizer diagnostic and therapeutic systems, which will enrich the construction of intelligent oxygenation materials and provide new means for photodynamic therapy of anaerobic diseases.

[0136] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing self-assembled hybrid photosensitizer nanoparticles, characterized in that: The photosensitizer protoporphyrin PpIX and the HIF-1α inhibitor KC7F2 drug powders were dissolved in dimethyl sulfoxide to obtain a mixed solution, which was then dissolved in a FeCI3 solution for self-assembly, centrifuged, and freeze-dried to obtain nanoparticles. The molar ratio of the protoporphyrin PpIX and the HIF-1α inhibitor KC7F2 was 2:1, and the pH of the FeCI3 solution was 6.

5.

2. The self-assembled hybrid photosensitizer nanoparticles prepared by the method of claim 1, characterized in that: The nanoparticles contain iron ions, protoporphyrin PpIX and HIF-1α inhibitor KC7F2. 3+ The coordination with protoporphyrin PpIX and the disulfide bond-mediated GSH response of KC7F2 achieve synergistic treatment of chemodynamic therapy and photodynamic therapy.

3. Use of the self-assembled hybrid photosensitizer nanoparticles according to claim 2 in the preparation of drugs for diagnosing or treating oral cancer.

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