Luminous environment targeted anti-tumor nanoparticle IUR as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a light environment targeted anti-tumor nanoparticle IUR as well as a preparation method and application thereof. HA and RVG29 peptides are connected through chemical action to generate HA-RVG29, then nanoparticles formed by self-assembly of UA and a photosensitizer IR820 are coated with the HA-RVG29 through electrostatic interaction to form the photosensitive nanoparticles IUR with a glioma targeting effect, the photosensitive nanoparticles IUR and temozolomide are combined for use to play a role in treating glioma, and through cell and animal experiments, the photosensitive nanoparticles IUR and temozolomide can be used for treating glioma. The combination of the nanoparticles and the TMZ is proved to have a good anti-tumor effect; through a TUNEL cell apoptosis detection method, a flow cytometry method and inverted fluorescence microscope observation, in-vivo experimental evaluation finds that the IUR and the TMZ are combined for use, so that a relatively good inhibition effect on brain glioma can be achieved by promoting cell apoptosis and inhibiting cell proliferation.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals and relates to an anti-tumor drug. Background Art

[0002] Glioblastoma is the most common primary intracranial tumor, accounting for 26.3% of all tumors, with an annual incidence of 3-6.4 / 100,000. The World Health Organization classifies glioma into four levels according to the pathological malignancy of tumor cells, with grades I and II as low-grade and grades III and IV as high-grade. Compared with the other three levels, WHO grade IV glioblastoma (GBM) is the most common and has the highest annual incidence, about 4.03 / 100,000, accounting for 14.2% of all tumors and 50.9% of all malignant tumors. The five-year relative survival rate of glioblastoma is extremely low, only 5%. In recent years, the incidence of glioma has been rising year by year. As one of the most challenging malignant tumors, brain glioma has a high degree of malignancy and poor prognosis. The current academic community has conducted in-depth research on it to explore effective treatment strategies for glioblastoma. In addition to traditional surgery, radiotherapy and chemotherapy, the treatment of glioblastoma also includes advanced treatments such as targeted drug therapy, immunotherapy and electric field therapy. However, due to the heterogeneity of glioblastoma and the complexity of the immune microenvironment within the tumor, the benefits provided by current treatments are still relatively limited. Therefore, the scientific community urgently needs to explore new and effective treatment options.

[0003] At present, the standard treatment for glioma is still a combination of surgical resection, radiotherapy and chemotherapy. Chemotherapy has always been an indispensable part of glioma treatment. The earliest chemotherapy drugs for brain glioma were nitrosourea drugs, such as carmustine and lomustine, which are fat-soluble drugs that can pass through the BBB, but their therapeutic effect is limited and their cytotoxicity is large. Combination chemotherapy regimens are more commonly used abroad, such as the PCV regimen. The PCV regimen is a combination chemotherapy method involving the combined use of three drugs, including methylbenzylhydrazine, lomustine, and vincristine. In recent years, the oral chemotherapy drug temozolomide (TMZ) has the advantages of high bioavailability, good BBB permeability, lower side effects than the side effects of the combination chemotherapy regimen, and the efficacy is no less than the combination chemotherapy regimen. Therefore, it quickly became the first choice for the treatment of brain glioma. At present, the standard chemotherapy regimen for brain glioma mainly uses temozolomide, which can sometimes be combined with genetic testing. Although the clinical application of TMZ plays an important role in glioma chemotherapy, its therapeutic effect is still unsatisfactory, and the 5-year survival rate of GBM after standardized treatment is less than 10%.

[0004] The application of chemotherapy drugs after surgery for glioma patients plays an important role in preventing postoperative recurrence. Considering the existence of the blood-brain barrier in the central nervous system, the ideal chemotherapy drug should be small in molecular weight, lipophilic, non-protein, and able to diffuse through the blood-brain barrier to the brain matrix. At present, the main obstacles to effective chemotherapy of gliomas are the development of drug resistance and toxic side effects. Therefore, it is still of practical significance to explore more effective chemotherapy drugs. TMZ is an oral anti-tumor chemotherapy drug, which is characterized by its ability to enter tumor tissue through blood circulation. Due to its high lipid solubility, TMZ can easily cross the blood-brain barrier (BBB), making it the first choice for the treatment of brain tumors such as gliomas. Although TMZ has better efficacy in the treatment of gliomas than other chemotherapy drugs, the high dose requirement of temozolomide alone and the possibility of tumor resistance limit its therapeutic effect. Patent CN202411381730.0 discloses an anti-glioblastoma pharmaceutical composition and its application, wherein the pharmaceutical composition comprises a SETD8 inhibitor and temozolomide or a pharmaceutically acceptable salt thereof. This application first discovered that the combination of the SETD8 inhibitor UNC0379 and temozolomide has a synergistic effect in the treatment of glioblastoma, providing an effective drug combination strategy for the treatment of glioblastoma. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a light environment targeted anti-tumor nanoparticle IUR and a preparation method and application thereof.

[0006] The technical solution of the present invention is achieved in this way: On the one hand, the present application proposes a method for preparing a light environment targeted anti-tumor nanoparticle IUR, the steps of which are as follows: (1) HA is activated with an activator and then mixed with an organic solvent of RVG29 peptide for reaction, dialyzed, centrifuged, and the supernatant is freeze-dried to obtain the product HA-RVG29; (2) Under ultrasonic conditions, ursolic acid (UA) was dropped into the aqueous solution of IR820, and the precipitate was collected after washing and resuspended to obtain IU nanoparticles; IU nanoparticles were added to the HA-RVG29 solution, stirred and mixed, and the precipitate was collected after centrifugation to obtain nanoparticles IUR.

[0007] Preferably, in the above step (1), the activator is EDCI; the organic solvent is DMSO, and the molar ratio of water to DMSO in the organic solvent is 1:1~2.5; the molar ratio of HA, activator and RVG29 peptide is 1~2.5:1:0.01~0.02; the activation temperature is 0~5°C, and the activation time is 30~120 min; the reaction temperature is room temperature, and the reaction time is 12~20 h.

[0008] Preferably, in the above step (1), the dialysis bag model used for dialysis is 3500k Da, the dialysis fluid is distilled water, and the dialysis time is 20-24 h.

[0009] Preferably, in the above step (2), the molar ratio of IR820 to ursolic acid is 1:1; the molar ratio of IU nanoparticles to HA-RVG29 is 3-5:1; the temperature of ultrasonic treatment is room temperature and the time is 10-15 min.

[0010] In the second aspect, the anti-tumor nanoparticles IUR prepared by the above preparation method.

[0011] Thirdly, the application of the above-mentioned anti-tumor nanoparticles IUR and TMZ in combination in the preparation of anti-glioma drugs that can pass through the blood-brain barrier.

[0012] Fourthly, the application of the above-mentioned anti-tumor nanoparticles IUR and TMZ in combination in the preparation of anti-tumor drugs.

[0013] In a fifth aspect, the above-mentioned anti-tumor nanoparticles IUR and TMZ are used in combination to prepare drugs for inhibiting the proliferation and growth of human U87 cells.

[0014] Preferably, the effective concentration of UA in the nanoparticle IUR in the above-mentioned drug is 20 μg / mL, and the effective concentration of TMZ is 20 μg / mL.

[0015] In a sixth aspect, the above-mentioned anti-tumor nanoparticles IUR and TMZ are used in combination to prepare a drug for treating brain glioma in a nude mouse orthotopic glioma model and effectively restoring behavioral ability.

[0016] Preferably, the administration concentration of nanoparticle IUR in the above-mentioned drug is 10 μL / g, and the administration concentration of TMZ is 1 mg / kg.

[0017] The present invention has the following beneficial effects: 1. In the present application, HA and RVG29 peptides are connected by chemical reaction to generate HA-RVG29, and then HA-RVG29 is coated on nanoparticles self-assembled by UA and photosensitizer IR820 through electrostatic interaction to form photosensitive nanoparticles IUR with glioma targeting effect, and the combination with temozolomide exerts a therapeutic effect on glioma. Cell and animal experiments have proved that the combination of the nanoparticles and TMZ has a good anti-tumor effect.

[0018] 2. The present application found through TUNEL cell apoptosis detection method, flow cytometry, inverted fluorescence microscopy observation, and in vivo experimental evaluation that the combination of IUR and TMZ has a good in vitro cytotoxic effect and exhibits excellent tumor cell toxicity. It can promote cell apoptosis, inhibit cell proliferation, and have a good inhibitory effect on brain gliomas. It has a good therapeutic effect on U87-luc orthotopic brain glioma mice in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The figure shows the schematic diagram of the synthetic route of HA-RVG29.

[0021] Figure 2 For HA-RVG29 1 H-NMR spectrum.

[0022] Figure 3 This is the infrared spectrum analysis chart of IU.

[0023] Figure 4 The UV absorption spectra of the target products IUR, IU and IUH nanoparticles.

[0024] Figure 5 Figure 2 is a comparison chart of cell viability in different dosing groups at 24 hours; Figure a is the cell viability of UA alone, Figure b is the cell viability of free IR820, Figure c is the cell viability of TMZ alone, Figure d is the cell viability of UA combined with TMZ, Figure e is the CI value obtained by combining the data of a, c, and d, and Figure f is a comparison chart of cell viability in different dosing groups at 24 hours.

[0025] Figure 6 The green fluorescent dye Calcein-AM and the red fluorescent dye PI were used to co-stain live and dead cells; Figure a is the live and dead cell staining of U87 cells in different drug groups, and Figure b is the quantitative statistical graph of the data in Figure a using Image J.

[0026] Figure 7 The EdU staining method is used to monitor cell proliferation; Figure a shows the effect of different drug administration groups on U87 cell proliferation, and Figure b is a statistical chart of cell proliferation rate.

[0027] Figure 8To detect the generation of reactive oxygen species by IUR nanoparticles; Figure a is the detection of the generation of reactive oxygen species in different dosing groups with and without laser irradiation, and Figure b is a quantitative statistical chart of the generation of reactive oxygen species in each dosing group under laser irradiation.

[0028] Fig. 9 One-step TUNEL apoptosis detection was used to measure apoptosis in IUR nanoparticles and different drug administration groups; Figure a is the apoptosis detection of U87 cells in different drug administration groups, and Figure b is the corresponding apoptosis rate statistics.

[0029] Fig.10 Annexin V-FITC / PI double staining flow cytometry was used to mark early and late apoptotic cells to directly observe and analyze cell apoptosis; Figure a is the apoptosis rate of U87 cells in each group detected by flow cytometry, and Figure b is the statistical chart of apoptosis rate.

[0030] Fig.11 In the experiment of preparing U87-luc orthotopic brain glioma mouse model for tumor inoculation, the combined use of IUR and TMZ inhibited mouse brain glioma; Figure a shows the detection results of brain tumor bioluminescence at 0 d, 5 d, 10 d, and 15 d after administration, Figure b shows the quantitative data of brain tumor bioluminescence, and Figure c shows the survival curve of mice. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] The main experimental reagents and instruments used in the present invention are briefly introduced as follows: All reagents were purchased from Beijing Solarbio Company, Wuhan Servicebio Company or Suzhou Xinsaimei Company.

[0034] Example 1 The preparation method of the light environment targeted anti-tumor nanoparticles IUR of this embodiment comprises the following specific steps: (1) HA-RVG29: The synthetic route of HA-RVG29 is shown in the figure below: Figure 1As shown, HA (400 mg) was accurately weighed into a flask, ultrapure water was added, and the solution was stirred until it was clear. Then, the activator EDCI (202 mg) was added at 0°C and reacted at room temperature for 30 min to activate the carboxyl groups on HA. The activated HA was mixed with RVG29 polypeptide (34 mg) dissolved in 0.2 mL DMSO, and reacted at room temperature for 12 h under argon protection. Then, the mixture was dialyzed with distilled water for 20 h, the liquid in the dialysis bag was collected, centrifuged, and the supernatant was lyophilized to obtain the dry product HA-RVG29.

[0035] (2) Preparation of IUR: 0.1 mL of ursolic acid (UA) solution (concentration of 10 mg / mL, solvent is DMSO) was measured and slowly dripped into 1 mL of aqueous solution containing IR820 (concentration of 1 mg / mL) in an ultrasonic water bath. Then, the mixed solution was subjected to ultrasonic treatment at room temperature and centrifuged to collect the precipitate. The precipitate was then washed three times with ultrapure water to remove DMSO, unreacted IR820 and ursolic acid. Finally, the precipitate was collected and resuspended in ultrapure water to obtain IU nanoparticles. The prepared IU nanoparticles (50 mg) were added to the HA-RVG29 (42 mg) solution prepared in advance, stirred, centrifuged to collect the nanoprecipitate and washed twice with sterile deionized water. Finally, the precipitate was collected as IUR.

[0036] Weigh an appropriate amount (5 mg) of HA, RVG29 and HA-RVG29 powder, use deuterated DMSO as solvent, and measure the 1 H NMR. The results are as Figure 2 As shown, the characteristic peak of the H-NMR spectrum of HA is mainly at 8.30 ppm, which is the signal peak of the hydrogen protons on the amide group in HA. The characteristic peak signals of the RVG29 peptide are at 1.51 ppm (the signal peak of the thiol hydrogen on cysteine ​​in the peptide) and 6.50-8.75 ppm (the signal peak area of ​​the aromatic protons in RVG29). In the H-NMR spectrum of the synthesized HA-RVG29, we found the simultaneous presence of the characteristic peak signals of HA and RVG29 peptide, as well as the disappearance of the corresponding characteristic peak of the thiol hydrogen on the RVG29 peptide, which proved the successful grafting of HA and RVG29 peptide, and the grafting rate of RVG29 peptide was calculated to be 40~45% through scoring.

[0037] A small amount of sample IR820, UA and IU nanoparticles were weighed and mixed with dry KBr, ground and pressed, and the obtained samples were analyzed and tested using Fourier transform infrared spectrometer (FT-IR). Figure 3 As shown by Figure 3 It can be seen that HA is at 1616 cm -1There is an absorption peak at 3300, 1660 and 1540 cm-1, which is attributed to the stretching vibration of the carboxyl group (-COOH) of hyaluronic acid. The intensity of the absorption peak at this position in the infrared spectrum of HA-RVG decreases. -1 There are peaks at 3070 cm-1, which are attributed to the stretching vibration of amide (NH), the stretching vibration of (NC=O) and the bending vibration of (CN-H). -1 The peaks at are attributed to the stretching vibration peaks of olefin CH in the peptide, which are characteristic of the peptide bonds between amino acids present in the spectrum of the RVG 29 peptide. These bands also exist on the synthesized HA-RVG29, indicating the successful synthesis of HA-RVG29.

[0038] Take 1 mg / mL of IR820, IU, IUH, and IUR and dilute them in equal proportions. Select the appropriate concentration after dilution and use a UV spectrophotometer to measure the absorbance of each dilution. Figure 4 As shown, Figure 4 These are the ultraviolet absorption peaks of free IR820 and three kinds of nanoparticles, IU, IUH and IUR. Compared with free IR820, the three kinds of nanoparticles show a slight red shift in the absorption peak in the UV-visible light spectrum, which may be attributed to the change of molecular form after self-assembly.

[0039] Example 2 The preparation method of the light environment targeted anti-tumor nanoparticles IUR of this embodiment comprises the following specific steps: (1) HA-RVG29: The synthetic route of HA-RVG29 is shown in the figure below: Figure 1 As shown, HA (400 mg) was accurately weighed into a flask, ultrapure water was added, and the solution was stirred until it was clear. Then, the activator EDCI (81 mg) was added at 5°C and reacted at room temperature for 120 min to activate the carboxyl groups on HA. The activated HA was mixed with RVG29 polypeptide (28 mg) dissolved in 0.15 mL DMSO, and reacted at room temperature for 20 h under argon protection. Then, the mixture was dialyzed with distilled water for 24 h, the liquid in the dialysis bag was collected, centrifuged, and the supernatant was lyophilized to obtain the dry product HA-RVG29.

[0040] (2) Preparation of IUR: Weigh ursolic acid (UA) (114 mg) and slowly drip it into an aqueous solution containing IR820 (207 mg) in an ultrasonic water bath. Then, the mixed solution was subjected to ultrasonic treatment at room temperature and centrifuged to collect the precipitate. The precipitate was then washed three times with ultrapure water to remove DMSO, unreacted IR820 and ursolic acid. Finally, the precipitate was collected and resuspended in ultrapure water to obtain IU nanoparticles. The prepared IU nanoparticles (50 mg) were added to the HA-RVG29 (26 mg) solution prepared in advance, stirred, centrifuged to collect the nanoprecipitate and washed twice with sterile deionized water. Finally, the precipitate was collected as IUR.

[0041] Weigh an appropriate amount (10 mg) of HA, RVG29 and HA-RVG29 powder, use deuterated DMSO as solvent, and measure the 1 H NMR. The results are as Figure 2 As shown, the characteristic peak of the H-NMR spectrum of HA is mainly at 8.30 ppm, which is the signal peak of the hydrogen protons on the amide group in HA. The characteristic peak signals of the RVG29 peptide are at 1.51 ppm (the signal peak of the thiol hydrogen on cysteine ​​in the peptide) and 6.50-8.75 ppm (the signal peak area of ​​the aromatic protons in RVG29). In the H-NMR spectrum of the synthesized HA-RVG29, we found the simultaneous presence of the characteristic peak signals of HA and RVG29 peptide, as well as the disappearance of the corresponding characteristic peak of the thiol hydrogen on the RVG29 peptide, which proved the successful grafting of HA and RVG29 peptide, and the grafting rate of RVG29 peptide was calculated to be 40~45% through scoring.

[0042] A small amount of sample IR820, UA and IU nanoparticles were weighed and mixed with dry KBr, ground and pressed, and the obtained samples were analyzed and tested using Fourier transform infrared spectrometer (FT-IR). Figure 3 As shown by Figure 3 It can be seen that HA is at 1616cm -1 There is an absorption peak at 3300, 1660 and 1540 cm-1, which is attributed to the stretching vibration of the carboxyl group (-COOH) of hyaluronic acid. The intensity of the absorption peak at this position in the infrared spectrum of HA-RVG decreases. -1 There are peaks at 3070 cm-1, which are attributed to the stretching vibration of amide (NH), the stretching vibration of (NC=O) and the bending vibration of (CN-H). -1 The peaks at are attributed to the stretching vibration peaks of olefin CH in the peptide, which are characteristic of the peptide bonds between amino acids present in the spectrum of the RVG 29 peptide. These bands also exist on the synthesized HA-RVG29, indicating the successful synthesis of HA-RVG29.

[0043] Take 1 mg / mL of IR820, IU, IUH, and IUR and dilute them in equal proportions. Select the appropriate concentration after dilution and use a UV spectrophotometer to measure the absorbance of each dilution. Figure 4 As shown, Figure 4 These are the ultraviolet absorption peaks of free IR820 and three kinds of nanoparticles, IU, IUH and IUR. Compared with free IR820, the three kinds of nanoparticles show a slight red shift in the absorption peak in the UV-visible light spectrum, which may be attributed to the change of molecular form after self-assembly.

[0044] Example 3 The preparation method of the light environment targeted anti-tumor nanoparticles IUR of this embodiment comprises the following specific steps: (1) HA-RVG29: The synthetic route of HA-RVG29 is shown in the figure below: Figure 1 As shown, HA (400 mg) was accurately weighed into a flask, ultrapure water was added, and the solution was stirred until it was clear. Then, the activator EDCI (202 mg) was added at 3°C ​​and reacted at room temperature for 90 min to activate the carboxyl groups on HA. The activated HA was mixed with a polypeptide (69 mg) dissolved in 0.4 mL DMSO, and the mixture was reacted at room temperature for 16 h under argon protection. The mixture was dialyzed with distilled water for 22 h, the liquid in the dialysis bag was collected, centrifuged, and the supernatant was lyophilized to obtain the dry product HA-RVG29.

[0045] (2) Preparation of IUR: Weigh ursolic acid (UA) (114 mg) and slowly drip it into an aqueous solution containing IR820 (207 mg) in a sonic water bath. Then, the mixed solution was subjected to ultrasonic treatment at room temperature and centrifuged to collect the precipitate. The precipitate was then washed three times with ultrapure water to remove DMSO, unreacted IR820 and ursolic acid. Finally, the precipitate was collected and resuspended in ultrapure water to obtain IU nanoparticles. The prepared IU nanoparticles (50 mg) were added to the HA-RVG29 (32 mg) solution prepared in advance, stirred, centrifuged to collect the nanoprecipitate and washed twice with sterile deionized water. Finally, the precipitate was collected as IUR.

[0046] Weigh an appropriate amount (7 mg) of HA, RVG29 and HA-RVG29 powder, use deuterated DMSO as solvent, and measure the 1 H NMR. The results are as Figure 2As shown, the characteristic peak of the H-NMR spectrum of HA is mainly at 8.30 ppm, which is the signal peak of the hydrogen protons on the amide group in HA. The characteristic peak signals of the RVG29 peptide are at 1.51 ppm (the signal peak of the thiol hydrogen on cysteine ​​in the peptide) and 6.50-8.75 ppm (the signal peak area of ​​the aromatic protons in RVG29). In the H-NMR spectrum of the synthesized HA-RVG29, we found the simultaneous presence of the characteristic peak signals of HA and RVG29 peptide, as well as the disappearance of the corresponding characteristic peak of the thiol hydrogen on the RVG29 peptide, which proved the successful grafting of HA and RVG29 peptide, and the grafting rate of RVG29 peptide was calculated to be 40~45% through scoring.

[0047] A small amount of sample IR820, UA and IU nanoparticles were weighed and mixed with dry KBr, ground and pressed, and the obtained samples were analyzed and tested using Fourier transform infrared spectrometer (FT-IR). Figure 3 As shown by Figure 3 It can be seen that HA is at 1616 cm -1 There is an absorption peak at 3300, 1660 and 1540 cm-1, which is attributed to the stretching vibration of the carboxyl group (-COOH) of hyaluronic acid. The intensity of the absorption peak at this position in the infrared spectrum of HA-RVG decreases. -1 There are peaks at 3070 cm-1, which are attributed to the stretching vibration of amide (NH), the stretching vibration of (NC=O) and the bending vibration of (CN-H). -1 The peaks at are attributed to the stretching vibration peaks of olefin CH in the peptide, which are characteristic of the peptide bonds between amino acids present in the spectrum of the RVG 29 peptide. These bands also exist on the synthesized HA-RVG29, indicating the successful synthesis of HA-RVG29.

[0048] Take 1 mg / mL of IR820, IU, IUH, and IUR and dilute them in equal proportions. Select the appropriate concentration after dilution and use a UV spectrophotometer to measure the absorbance of each dilution. Figure 4 As shown, Figure 4 These are the ultraviolet absorption peaks of free IR820 and three kinds of nanoparticles, IU, IUH and IUR. Compared with free IR820, the three kinds of nanoparticles show a slight red shift in the absorption peak in the UV-visible light spectrum, which may be attributed to the change of molecular form after self-assembly.

[0049] Application Example 1 MTT assay was used to evaluate the in vitro cytotoxicity of different drug groups on U87 cells In this experiment, four dosing groups, UA, IU, IUH and IUR, were set up, and the drug concentration gradients in each group were 0.625 μg / mL, 1.250 μg / mL, 2.500 μg / mL, 5.000 μg / mL, 10.000 μg / mL, 20.000 μg / mL and 40.000 μg / mL, respectively.

[0050] The MTT method was used to evaluate the in vitro cytotoxicity of different drug groups on U87 cells. Figure 5 (a) is a statistical chart of cell viability of different concentrations of UA alone. The IC50 value of UA was 36.47 μg / mL. Figure 5 (b) is the cell viability diagram of free IR820. It can be seen from the data that IR820 concentration within 10 μg / mL can be basically considered to have no cytotoxicity. The equivalent UA concentration of nanoparticle administration in subsequent experiments in this article is 20 μg / mL, which is equivalent to containing 3.5 μg / mL of IR820. It can be considered that IR820 in this article does not exert cytotoxic effects in the absence of laser irradiation. Figure 5 (c) is a statistical chart of cell viability at different concentrations of TMZ, and the IC50 value of TMZ was measured to be 166.4 μg / mL. Figure 5 (d) is the statistical graph of cell viability after combined use of UA and TMZ. Figure 5 (e) The drug synergy index (CI) of the combined use of UA and TMZ was evaluated by the Chou-Talalay method using the software Compusyn. The combined drug index (CI) <1, CI = 1, and CI > 1 represent synergy, additive effect, and antagonism, respectively. As can be seen from the figure, the CI values ​​of the combined use of UA and TMZ are all less than 1, indicating that there is a synergistic inhibitory effect between TMZ and UA on U87 cells. Figure 5 (f) is a comparison chart of cell viability in different dosing groups at 24 hours, indicating that the combination of TMZ and targeted photosensitive nanoparticles does have a good in vitro cytotoxic effect.

[0051] Application Example 2 Green fluorescent dye Calcein-AM and red fluorescent dye PI were used to co-stain live cells and dead cells to observe the killing effect of IUR combined with TMZ on cells in vitro In order to directly observe the killing effect of drugs on cells in vitro, the green fluorescent dye Calcein-AM and the red fluorescent dye PI will be used to co-stain live cells and dead cells to evaluate the survival and death of cells. Figure 6As shown, the TMZ+IUR+L treatment group showed the most red fluorescence signals and showed similar results to the cell viability comparison of different drug groups in the cytotoxicity experiment, indicating that temozolomide combined with targeted photosensitive nanoparticles exhibited excellent tumor cell toxicity.

[0052] Application Example 3 EdU staining method to evaluate the inhibitory effect of IUR combined with TMZ on the proliferation of U87 cells U87 cells in good growth state were taken and 8×10 3 The cells were inoculated into 96-well plates and cultured in a constant temperature incubator for 24 hours. The experimental groups were: free IR820 group, single-drug UA group, single-drug TMZ group, IU group, IU+L group, TMZ+IU group, TMZ+IU+L group, IUR group, IUR+L group, TMZ+IUR group, and TMZ+IUR+L group. The concentration of UA used in the experiment was based on the concentration at which the cell survival rate was approximately 80% within 24 hours when it was used alone, that is, 20 μg / mL. When the cells grew to an appropriate density, the cells were treated with drugs in different experimental groups for 24 hours.

[0053] In order to evaluate the inhibitory effects of different drug treatment groups on U87 cell proliferation, we used EdU staining to monitor cell proliferation. EdU, a nucleoside analog with a similar structure to deoxythymine, can be embedded in replicating DNA and specifically bind to fluorescent dyes, allowing us to visually evaluate the proliferation activity of cells. Among them, red fluorescence marks cells in a proliferative state. The results are shown in Figure 2. Figure 7 As shown in the figure, it can be seen from the experimental results that the nanoparticles exhibit a strong proliferation inhibition effect under laser irradiation, and the combination with temozolomide almost completely inhibits cell proliferation.

[0054] Application Example 4 The generation of reactive oxygen species by nanoparticles was tested to prove their ability to generate ROS by photodynamics (1) Cell preparation: U87 cells in good growth condition were cultured at 3×10 5 Each well of the plate was evenly inoculated and then cultured in a constant temperature incubator for 24 hours.

[0055] (2) Experimental grouping: The groups were set as blank control group, IU group, IU+TMZ group, IUR group, and IUR+TMZ group. When the cells grew to an appropriate density, the drugs in different experimental groups were added to the inoculated cells. Two wells were inoculated in each drug-treated group. One well was irradiated with 808 nm laser two hours after drug administration, and the other well was not irradiated with laser as a control. After treatment, the cells were incubated in a 37°C constant temperature incubator in the dark for 6 h.

[0056] (3) Probe loading: Refer to the instructions for use of UElandy's ROS reactive oxygen species detection kit (product number: R6033) and dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to achieve a final concentration of 10 μM. After removing the cell culture medium, add the pre-prepared reactive oxygen species probe solution to the cell culture plate and incubate in a constant temperature incubator at 37°C for 20 minutes. After incubation, wash three times with serum-free cell culture medium.

[0057] (4) Observe and photograph using an inverted fluorescence microscope, and then perform quantitative analysis using ImageJ software.

[0058] Test results such as Figure 8 As shown, the results showed that both IU and IUR nanoparticles could normally produce ROS through laser irradiation to exert photodynamic effects, and the ROS generation in the combined TMZ group was more than that in the nanoparticles group alone, indicating that TMZ promoted the generation of ROS by nanoparticles through photodynamics.

[0059] Application Example 5 One-step TUNEL apoptosis assay was used to determine the apoptosis of different drug groups. (1) Take U87 cells in good growth state and culture them at 8×10 3 Each well was inoculated into a 96-well plate and cultured in a constant temperature incubator for 24 hours.

[0060] (2) The experimental groups were: free IR820 group, single-drug UA group, single-drug TMZ group, IU group, IU+L group, TMZ+IU group, TMZ+IU+L group, IUR group, IUR+L group, TMZ+IUR group, and TMZ+IUR+L group. The concentration of UA used in the experiment was based on the concentration at which the cell survival rate was approximately 80% within 24 hours when used alone, that is, 20 μg / mL. When the cells grew to an appropriate density, the cells were treated with drugs in different experimental groups for 24 hours.

[0061] (3) First, fix the cells with 4% paraformaldehyde solution for no more than 30 minutes. Then, use PBS to wash the staining solution in the cells, and treat the cells with 0.5% TritonX-100 in PBS as a permeabilizing agent for no more than 5 minutes. After completion, wash again with PBS.

[0062] (4) Incubate the cells with TUNEL staining solution for 1 hour in a dark environment. After incubation, wash the cells with PBS.

[0063] (5) Add DAPI staining working solution to the cells for staining for 30 minutes. After staining, wash the cells with PBS and store them in a dark place.

[0064] (6) Observe and photograph using an inverted fluorescence microscope, and then perform quantitative analysis using ImageJ software.

[0065] The apoptosis of different drug-administered groups was determined. Fig. 9 The trend distribution of cell apoptosis is also consistent with the cytotoxicity experiment, indicating that the toxic effect of drugs on cells mainly occurs by inducing cell apoptosis.

[0066] Application Example 6 Annexin V-FITC / PI double staining flow cytometry to directly observe and analyze cell apoptosis (1) Take U87 cells in good growth state and culture them at 8×10 3 Each well was inoculated into a 96-well plate and cultured in a constant temperature incubator for 24 hours.

[0067] (2) The experimental groups were: free IR820 group, single-drug UA group, single-drug TMZ group, IU group, IU+L group, TMZ+IU group, TMZ+IU+L group, IUR group, IUR+L group, TMZ+IUR group, and TMZ+IUR+L group. The concentration of UA used in the experiment was based on the concentration at which the cell survival rate was approximately 80% within 24 hours when used alone, that is, 20 μg / mL. When the cells grew to an appropriate density, the cells were treated with drugs in different experimental groups for 24 hours.

[0068] (3) After drug treatment, cells were digested and collected and washed with PBS.

[0069] (4) Operate the cells according to the instructions of the YF®488-Annexin V / PI Cell Apoptosis Kit. Add the prepared staining solution to the cells, mix evenly, and then incubate in the dark.

[0070] (5) Each experimental group was tested using flow cytometry, and the results were analyzed using FlowJo-V10 software.

[0071] In order to directly observe and analyze cell apoptosis, we used Annexin V-FITC / PI double staining flow cytometry. Annexin V-FITC is used to mark early and late apoptotic cells, while PI staining is used to identify necrotic cells. The results of flow cytometry are represented by a four-quadrant graph, in which the upper left quadrant represents necrotic cells, the lower left quadrant shows healthy cells, the upper right quadrant identifies late apoptotic cells, and the lower right quadrant represents early apoptotic cells. The results are shown in Fig.10As shown, the apoptosis rates of the control group, IU group, IUR group, TMZ+IU group, and TMZ+IUR group without laser irradiation were 6.28%, 26.85%, 34.21%, 32.97%, and 37.71%, respectively; the apoptosis rates of the control group, IU group, IUR group, TMZ+IU group, and TMZ+IUR group after laser irradiation were 8.68%, 42.86%, 44.28%, 55.63%, and 71.5%, respectively. By comparing the IU and IUR groups, the highly targeted IUR does show stronger apoptosis; the combined TMZ group showed significantly stronger apoptosis ability than the single-use group.

[0072] Application Example 7 The therapeutic effect of IUR combined with TMZ on the U87-luc orthotopic brain glioma mouse model in vivo In order to demonstrate the therapeutic effects of nanoparticle IUR and its combination with TMZ on glioma mice, we designed the following experiment; 1. Investigation of distribution in vivo Since the IR820 contained in the nanoparticles is fluorescent, different experimental groups of nanoparticles can be directly injected into BALB / c nude mice through the tail vein (the concentration of the prepared nanoparticle dispersion is 500 μg / mL, and the dosage is 10 μL / g). The experimental groups are: IR820 group, IUH group, and IUR group.

[0073] (1) In vivo fluorescence imaging was used to capture fluorescence distribution images of mice at 1 h, 4 h, 8 h, 12 h, 24 h, and 48 h after injection.

[0074] (2) 24 hours after injection, the hearts, livers, spleens, kidneys, and brains of nude mice in each treatment group were removed, cleaned, and subjected to fluorescence imaging.

[0075] 2. Preliminary study on anti-tumor effect in mice Treatment plan for nude mice with orthotopic glioma. Seven days after tumor implantation, the total ROI value of the brain tumor area of ​​each mouse exceeded 1×10 5. Nude mice with consistent fluorescence intensity and body weight were selected as experimental subjects and randomly assigned to six different dosing groups. Each group received treatment with normal saline (NS), TMZ, IUR, IUR+L, TMZ+IUR, and TMZ+IUR+L (L is light), respectively. The dose of TMZ was 1 mg / kg, and the concentration of IUR nanoparticles was 500 μg / mL, administered at a dose of 10 μL / g. Temozolomide was administered by oral gavage, while IUR targeted photosensitive nanoparticles were administered by tail vein injection, with one oral administration and one tail vein injection every two days. 2 h after tail vein injection of IUR, brain laser irradiation (808 nm, 0.2 W / cm 2 , 5 min), and seven treatments were performed continuously for a total of 14 days. During the treatment period, the mice were weighed every two days, and the tumor volume was tracked and monitored using the bioluminescence channel of the in vivo imager on days 0, 5, 10, and 15 of treatment (data of 3 mice were recorded in each group). The survival of mice in each group was recorded from the start of treatment to the time of mouse death (data of 6 mice were recorded in each group).

[0076] like Fig.11 As shown in (a), during the treatment cycle, by using the in vivo imaging system to monitor the changes in tumor volume at different time points, we found that at the beginning of treatment (day 0), the fluorescence intensity between the experimental groups was basically the same. However, as the treatment time progressed, the tumor growth rate of each group began to differ. In particular, our TMZ+IUR+L group had a significantly lower tumor fluorescence area than other groups on day 15, and the tumor growth showed significant inhibition compared with other groups. Figure (b) statistically analyzed the fluorescence intensity of the tumor site of each treatment group, and more intuitively presented the difference in tumor growth between the treatment groups through quantitative analysis, and more intuitively showed the inhibitory effect of the TMZ+IUR+L group on glioma tumor growth, indicating that it has a good therapeutic effect. Figure (c) shows that although TMZ alone and nanoparticle IUR combined with laser treatment can also prolong the survival time of mice, the survival time of mice in the TMZ+IUR+L group is significantly longer than any single drug, indicating that the combination of temozolomide and nanoparticles does have a good therapeutic effect on glioma mice.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing light environment targeted anti-tumor nanoparticles IUR, characterized in that: Here are the steps: (1) HA is activated with an activator and then mixed with an organic solvent of RVG29 peptide for reaction, dialyzed, centrifuged, and the supernatant is freeze-dried to obtain the product HA-RVG29; (2) Under ultrasonic conditions, UA was dropped into the aqueous solution of IR820, the precipitate was collected after washing, and resuspended to obtain IU nanoparticles; IU nanoparticles were added to the HA-RVG29 solution, stirred and mixed, and the precipitate was collected after centrifugation to obtain nanoparticles IUR.

2. The method for preparing the antitumor nanoparticle IUR according to claim 1, characterized in that: In the step (1), the activator is EDCI, HATU or HBTU; the organic solvent is DMSO; the molar ratio of HA, activator and RVG29 peptide is 1-2.5:1:0.01-0.02; the activation temperature is 0-5°C, and the activation time is 30-120 min; the reaction temperature is room temperature, and the reaction time is 12-20 h.

3. The method for preparing the antitumor nanoparticle IUR according to claim 2, characterized in that: In the step (1), the dialysis bag model used for dialysis is 3500k Da, the dialysis fluid is distilled water, and the dialysis time is 20-24 h.

4. The method for preparing the antitumor nanoparticle IUR according to claim 3, characterized in that: In the step (2), the molar ratio of IR820 to ursolic acid is 1:1; the molar ratio of IU nanoparticles to HA-RVG29 is 3-5:1; the temperature of ultrasonic treatment is room temperature and the time is 10-15 min.

5. The antitumor nanoparticles IUR prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the anti-tumor nanoparticles IUR according to claim 5 in combination with TMZ in the preparation of an anti-glioma drug that can penetrate the blood-brain barrier.

7. Use of the anti-tumor nanoparticles IUR according to claim 5 in combination with TMZ in the preparation of anti-tumor drugs.

8. The use of the anti-tumor nanoparticles IUR and TMZ in combination in the preparation of a drug for inhibiting the proliferation and growth of human U87 cells according to claim 5, characterized in that: The effective concentration of UA in the nanoparticle IUR in the drug is 20 μg / mL, and the effective concentration of TMZ is 20 μg / mL.

9. Use of the anti-tumor nanoparticles IUR and TMZ according to claim 5 in combination for preparing a drug for treating brain glioma in a nude mouse orthotopic glioma model and effectively restoring behavioral ability.

10. The use according to claim 9, characterized in that: The drug has an administration concentration of 10 μg / mL for nanoparticle IUR and 1 mg / kg for TMZ.

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