A photo-environment-targeted antitumor nanoparticle IUR, its preparation method and application

Abstract

This invention belongs to the field of biopharmaceuticals, specifically referring to a photo-environment-targeted antitumor nanoparticle IUR, its preparation method, and its application. In this application, HA and RVG29 peptides are chemically linked to generate HA-RVG29. Then, HA-RVG29 is coated onto nanoparticles self-assembled from UA and the photosensitizer IR820 via electrostatic interactions, forming photosensitive nanoparticle IURs with glioma-targeting activity. This IUR exerts a therapeutic effect on gliomas through combination with temozolomide. Cell and animal experiments demonstrate that the combination of these nanoparticles and TMZ has a good antitumor effect. In vivo experiments, using TUNEL cell apoptosis detection, flow cytometry, inverted fluorescence microscopy, and other methods, show that the combination of IUR and TMZ can promote cell apoptosis, inhibit cell proliferation, and has a good inhibitory effect on gliomas.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmaceuticals, and relates to an antitumor drug. BACKGROUND

[0002] Glioma is the most common primary intracranial tumor, accounting for 26.3% of all tumors, with an annual incidence of 3-6.4 / 100,000. According to the World Health Organization, glioma is classified into four grades based on the degree of malignancy of tumor cells in pathology, with grades I and II being low-grade and grades III and IV being high-grade. Compared with other grades, WHO grade IV glioblastoma (GBM) is the most common and has the highest annual incidence, about 4.03 / 100,000 people, 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 is also increasing year by year. As one of the most challenging malignant tumors in tumors, due to its high malignancy and poor prognosis, the current academic community has conducted in-depth research to explore effective treatment strategies for glioblastoma. In addition to traditional surgical treatment, radiotherapy and chemotherapy, advanced treatment methods such as targeted drug therapy, immunotherapy and electric field therapy are also included. However, due to the heterogeneity of glioblastoma and the complexity of the intratumoral immune microenvironment, the current treatment methods still provide limited benefits. Therefore, the scientific community urgently needs to explore new effective treatment options.

[0003] Currently, the standard treatment for glioma is still a combination of surgical resection, radiotherapy and chemotherapy. Chemical drug therapy has always been an indispensable part of glioma treatment. The earliest brain glioma chemotherapy drug was nitrosourea drugs such as carmustine and lomustine, which are lipid-soluble drugs that can penetrate the BBB, but their therapeutic effect is limited and have high cytotoxicity. Foreigners use more combined chemotherapy regimens, such as the PCV regimen, which is a combined chemotherapy method involving the use of three drugs, specifically including procarbazine, lomustine, and vincristine. In recent years, oral chemotherapy drug temozolomide (TMZ) has become the first choice for brain glioma treatment due to its high bioavailability, good BBB permeability, lower side effects than combined chemotherapy regimens, and similar efficacy to combined chemotherapy regimens. The current standard chemotherapy regimen for brain glioma is 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 not satisfactory, and the 5-year survival rate of GBM after standard treatment is less than 10%.

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

[0005] To solve the above problems, the present application provides a light environment targeted anti-tumor nanoparticle IUR and its preparation method and application.

[0006] The technical scheme of the present application is as follows:

[0007] On the one hand, the present application provides a preparation method of light environment targeted anti-tumor nanoparticle IUR, the steps are as follows:

[0008] (1) After the HA is activated with an activating agent, it is mixed with the organic solvent of RVG29 peptide, dialyzed, centrifuged, and the supernatant is freeze-dried to obtain the product HA-RVG29.

[0009] (2) Under ultrasonic conditions, ursolic acid (UA) is added dropwise into the aqueous solution of IR820, the precipitate is collected after washing, and the suspension is obtained. The IU nanoparticles are added to the HA-RVG29 solution, stirred and mixed, and the precipitate is collected after centrifugation, which is the nanoparticle IUR.

[0010] Preferably, the activating agent in the above step (1) is EDCI; the organic solvent is DMSO, and the molar ratio of water and DMSO in the organic solvent is 1:1~2.5; the molar ratio of HA, activating agent and RVG29 peptide is 1~2.5:1:0.01~0.02; the activation temperature is 0~5℃, and the activation time is 30~120 min; the reaction temperature is room temperature, and the reaction time is 12~20 h.

[0011] Preferably, the dialysis bag used in the above step (1) has a model of 3500 kDa, the dialysate is distilled water, and the dialysis time is 20-24 h.

[0012] Preferably, the molar ratio of IR820 to ursolic acid in the above step (2) is 1:1, the molar ratio of IU nanoparticles to HA-RVG29 is 3-5:1, and the ultrasonic treatment is performed at room temperature for 10-15 min.

[0013] In the second aspect, the anti-tumor nanoparticles IUR prepared by the preparation method.

[0014] In the third aspect, the anti-tumor nanoparticles IUR and TMZ are used in combination in the preparation of an anti-glioma drug that can penetrate the blood-brain barrier.

[0015] In the fourth aspect, the anti-tumor nanoparticles IUR and TMZ are used in combination in the preparation of an anti-tumor drug.

[0016] In the fifth aspect, the anti-tumor nanoparticles IUR and TMZ are used in combination in the preparation of a drug for inhibiting the proliferation and growth of human U87 cells.

[0017] Preferably, in the above drug, the concentration of UA in the nanoparticles IUR is 20 μg / mL, and the concentration of TMZ is 20 μg / mL.

[0018] In the sixth aspect, the anti-tumor nanoparticles IUR and TMZ are used in combination in the preparation of a drug for treating brain glioma in a nude mouse orthotopic glioma model and effectively restoring the behavioral ability.

[0019] Preferably, in the above drug, the concentration of UA in the nanoparticles IUR is 20 μg / mL, and the concentration of TMZ is 20 μg / mL.

[0020] The present application has the following advantages:

[0021] 1. In the present application, HA and RVG29 peptides are connected by chemical reaction to generate HA-RVG29, which is then coated on the nanoparticles formed by self-assembly of UA and photosensitizer IR820 through electrostatic interaction to form photosensitive nanoparticles IUR with glioma targeting effect, and the combination of the nanoparticles and TMZ is used to exert a therapeutic effect on glioma. Through cell and animal experiments, it is proved that the combination of the nanoparticles and TMZ has good anti-tumor effect.

[0022] 2、The application is found by TUNEL cell apoptosis detection method, flow cytometry method, inverted fluorescence microscope observation, in vivo experiment evaluation that IUR has better in vitro cytotoxicity effect combined with TMZ, shows excellent tumor cell toxicity, can promote cell apoptosis, inhibit cell proliferation, has better inhibition effect on brain glioma, has better treatment effect on U87-luc orthotopic brain glioma mice in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0024] Figure 1 It is a synthesis route diagram of HA-RVG29.

[0025] Figure 2 It is a synthesis route diagram of HA-RVG29. 1 H-NMR spectrum.

[0026] Figure 3 It is an infrared spectrum analysis spectrum of IU.

[0027] Figure 4 It is the ultraviolet absorption spectrum of the target product IUR and the three kinds of nanoparticles IU, IUH.

[0028] Figure 5 It is a 24-hour cell viability comparison chart of different administration groups; wherein figure a is UA single-drug cell viability, figure b is free IR820 cell viability, figure c is TMZ single-drug cell viability, figure d is UA combined with TMZ cell viability, figure e is the CI value obtained by integrating a, c, d data, and figure f is a 24-hour cell viability comparison chart of different administration groups.

[0029] Figure 6 It is a co-staining of live and dead cells by green fluorescent dye Calcein-AM and red fluorescent dye PI; wherein figure a is a live and dead cell staining chart of U87 cells in different administration groups, and figure b is a quantitative statistical chart of Image J on the data of figure a.

[0030] Figure 7 It is an EdU staining method to monitor cell proliferation; wherein figure a is the influence of different administration groups on U87 cell proliferation, and figure b is a cell proliferation rate statistical chart.

[0031] Figure 8For the detection of the active oxygen generation of IUR nanoparticles; wherein Figure a is the detection of the active oxygen generation of different administration groups under the condition of laser irradiation or not, and Figure b is the quantitative statistical graph of the active oxygen generation of each administration group under laser irradiation.

[0032] Figure 9 For the determination of the apoptosis of IUR nanoparticles and different administration groups by one-step TUNEL apoptosis detection; wherein Figure a is the apoptosis detection of different administration groups on U87 cells, and Figure b is the corresponding apoptosis rate statistical graph.

[0033] Figure 10 For the direct observation and analysis of the apoptosis of early and late apoptotic cells by Annexin V-FITC / PI double staining flow cytometry; wherein Figure a is the flow cytometry detection of the apoptosis rate of U87 cells in each group, and Figure b is the apoptosis rate statistical graph.

[0034] Figure 11 For the inhibition of brain glioma of mice by IUR combined with TMZ when preparing U87-luc orthotopic glioma mouse model experiment; wherein Figure a is the detection result of brain tumor bioluminescence at 0 d, 5 d, 10 d and 15 d after administration, Figure b is the quantitative data of intracranial tumor bioluminescence, and Figure c is the survival curve of mice. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] The experimental methods used in the following experimental examples are all conventional methods unless otherwise specified; and the materials, reagents, etc. used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0037] The main experimental reagents and instrument equipment used in the present application are briefly introduced as follows: all reagents are purchased from Beijing Solarbio Company, Wuhan Servicebio Company or Suzhou Xinsaimei Company.

[0038] Example 1

[0039] The preparation method of the light environment targeted anti-tumor nanoparticles IUR of the present embodiment is as follows:

[0040] (1) HA-RVG29: the synthesis route diagram of HA-RVG29 is as follows: Figure 1As shown, HA (400 mg) was precisely weighed in a flask, and ultrapure water was added. After stirring until the solution was clear, the activated agent EDCI (202 mg) was added at 0°C. The carboxyl group on HA was activated by reacting at room temperature for 30 min. The activated HA was mixed with RVG29 polypeptide (34 mg) dissolved in 0.2 mL of DMSO. After 12 h of reaction at room temperature under argon protection, the solution in the dialysis bag was collected after dialysis for 20 h. After centrifugation, the supernatant was freeze-dried to obtain the dry product HA-RVG29.

[0041] (2) Preparation of IUR: 0.1 mL of ursolic acid (UA) solution (concentration of 10 mg / mL, solvent DMSO) was slowly dropped into 1 mL of IR820 (concentration of 1 mg / mL) aqueous solution in an ultrasonic water bath. Then, the mixed solution was centrifuged after ultrasonic treatment at room temperature. The precipitate was washed with ultrapure water three times 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, and the nanoparticle precipitate was collected by centrifugation and washed twice with sterile deionized water. Finally, the precipitate was collected to obtain IUR.

[0042] An appropriate amount (5 mg) of HA, RVG29 and HA-RVG29 powder was weighed, and deuterated DMSO was used as the solvent to determine the 1 H NMR of the sample to be tested. Figure 2 As shown, the characteristic peak of the nuclear magnetic hydrogen spectrum of HA is mainly at 8.30 ppm, which is the signal peak of the hydrogen proton of the amide group in HA. The characteristic peak signal of RVG29 peptide is at 1.51 ppm (signal peak of sulfhydryl hydrogen on cysteine in the peptide segment) and 6.50-8.75 ppm (signal peak region of aromatic protons in RVG29). In the nuclear magnetic spectrum of the synthesized HA-RVG29, we found that the characteristic peak signals of HA and RVG29 peptide coexist, and the disappearance of the characteristic peak of sulfhydryl hydrogen on RVG29 peptide proves the successful grafting of HA and RVG29 peptide. The grafting rate of RVG29 peptide is calculated to be 40-45%.

[0043] A small amount of samples IR820, UA and IU nanoparticles was weighed, and was mixed with dry KBr. After grinding and tabletting, the obtained samples were analyzed and detected by Fourier transform infrared spectrometer (FT-IR). As shown in Figure 3 , it can be seen from Figure 3 that the characteristic peak of HA is at 1616 cm -1an absorption peak at 3300, 1660 and 1540 cm -1 respectively, which are attributed to the stretching vibration of amide (N-H), the stretching vibration of (NC=O) and the bending vibration of (CN-H) respectively, and the peak at 3070 cm -1 is attributed to the stretching vibration of olefin C-H in the peptide, which is the characteristic of the peptide bond between the amino acids in the RVG 29 peptide, and these bands also exist in the synthesized HA-RVG 29, indicating the successful synthesis of HA-RVG 29.

[0044] Dilute 1 mg / mL of IR820, IU, IUH, IUR, etc. at a certain ratio, and select the appropriate concentration after dilution to determine the absorbance of each group of diluents by ultraviolet spectrophotometry. The results are shown in Figure 4 Figure 4 The ultraviolet absorption peaks of free IR820 and the three kinds of nanoparticles IU, IUH and IUR are shown in Table 1, respectively. Compared with free IR820, the three kinds of nanoparticles show a slight red shift in the ultraviolet-visible light spectrum, which may be due to the change in molecular form after self-assembly.

[0045] Example 2

[0046] The preparation method of the light environment targeted antitumor nanoparticles IUR of the present embodiment is as follows:

[0047] (1) HA-RVG 29: The synthesis route of HA-RVG 29 is shown in Figure 1 Precisely weigh HA (400 mg) in a flask, add ultrapure water, stir until the solution is clear, and then add the activating agent EDCI (81 mg) at 5°C. React for 120 min at room temperature to activate the carboxyl group on HA. Mix the activated HA with RVG 29 polypeptide (28 mg) dissolved in 0.15 mL of DMSO, and react for 20 h at room temperature under argon protection. Then, dialyze for 24 h with distilled water, collect the liquid in the dialysis bag, centrifuge, and freeze-dry the supernatant to obtain the dry product HA-RVG 29.

[0048] ​(2) Preparation of IUR: Ursolic acid (UA) (114 mg) was weighed and slowly added dropwise to an aqueous solution containing IR820 (207 mg) in an ultrasonic water bath. The mixture was then sonicated 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 a pre-prepared HA-RVG29 (26 mg) solution, stirred, centrifuged to collect the nanoprecipitate, and washed twice with sterile deionized water. The final precipitate was the IUR.

[0049] Weigh appropriate amounts (10 mg) of HA, RVG29, and HA-RVG29 powders, and use deuterated DMSO as solvent to determine the properties of the samples. 1 H NMR. Results are as follows: Figure 2 As shown, the characteristic peak of the 1H NMR spectrum of HA is mainly at 8.30 ppm, which is the signal peak of the hydrogen proton on the amide group in HA. The characteristic peak signals of RVG29 peptide are at 1.51 ppm (the signal peak of the mercapto hydrogen on the cysteine ​​in the peptide segment) and 6.50-8.75 ppm (the signal peak region of the aromatic proton in RVG29). In the NMR spectrum of the synthesized HA-RVG29, we found that the characteristic peak signals of HA and RVG29 peptides exist simultaneously, and the characteristic peak of mercapto hydrogen on the corresponding RVG29 peptide disappears, which proves that HA and RVG29 peptide were successfully grafted. The grafting rate of RVG29 peptide was calculated to be 40-45% through scoring.

[0050] A small amount of IR820, UA, and IU nanoparticles were weighed and thoroughly mixed with dry KBr. After grinding and pressing, the resulting sample was analyzed using Fourier transform infrared spectroscopy (FT-IR). The results are as follows: Figure 3 As shown, by Figure 3 It can be seen that HA is at 1616cm -1 An absorption peak appears at this position, attributed to the stretching vibration of the hyaluronic acid carboxyl group (-COOH); the absorption peak intensity at this position decreases in the infrared spectrum of HA-RVG; the RVG29 peptide shows absorption peaks at 3300, 1660, and 1540 cm⁻¹. -1 Peaks are observed at 3070 cm⁻¹, attributed to the stretching vibration of amide (NH₄⁺), the stretching vibration of (NC=O), and the bending vibration of (CN-H), respectively. -1 The peaks at the specified locations are attributed to the stretching vibrations of the olefin CH in the peptide, which are characteristic of peptide bonds between amino acids present in the RVG 29 peptide spectrum. These bands are also present in the synthesized HA-RVG29, indicating the successful synthesis of HA-RVG29.

[0051] Take 1 mg / mL of IR820, IU, IUH, IUR, etc. dilution, select the appropriate concentration after dilution with ultraviolet spectrophotometer to determine the absorbance of each group of diluent. Results as shown in Figure 4 Figure 4 The UV absorption peak of free IR820 and IU, IUH, IUR three kinds of nanoparticles, respectively, compared with free IR820, three kinds of nanoparticles in the ultraviolet visible light spectrum showed a slight red shift of the absorption peak, which may be due to the change of molecular form after self-assembly.

[0052] Example 3

[0053] The preparation method of the light environment targeted anti-tumor nanoparticles IUR of this embodiment is as follows:

[0054] (1) HA-RVG29: The synthesis route of HA-RVG29 is shown in Figure 1 , accurately weigh HA (400 mg) in a flask, add ultrapure water, stir until the solution is clear, then add activated agent EDCI (202 mg) at 3°C, react at room temperature for 90 min to activate the carboxyl group on HA, mix the activated HA with the polypeptide (69 mg) dissolved in 0.4 mL DMSO, under argon protection, react at room temperature for 16 h, then dialyze with distilled water for 22 h, collect the liquid in the dialysis bag, centrifuge, then freeze-dry the supernatant to obtain the dry product HA-RVG29.

[0055] (2) Preparation of IUR: weigh ursolic acid (UA) (114 mg) and slowly drop into the aqueous solution containing IR820 (207 mg) in a sonication water bath. Then, continue to ultrasonic treat this mixed solution at room temperature, then collect the precipitate by centrifugation, then wash the precipitate with ultrapure water for three times to remove DMSO, unreacted IR820 and ursolic acid. Finally, collect the precipitate and resuspend it in ultrapure water to obtain IU nanoparticles. Put the prepared IU nanoparticles (50 mg) into the prepared HA-RVG29 (32 mg) solution, stir, collect the nanoparticle precipitate by centrifugation and wash it with sterile deionized water for 2 times, then collect the precipitate to obtain IUR.

[0056] Weigh an appropriate amount (7 mg) of HA, RVG29 and HA-RVG29 powder, use deuterated DMSO as the solvent, and determine the 1 H NMR of the test sample. Results as shown in Figure 2 ​As shown, the characteristic peak of HA's nuclear magnetic hydrogen spectrum is mainly at 8.30 ppm, which is the signal peak of hydrogen proton on the amide group in HA, and the characteristic peak signal of RVG29 peptide is at 1.51 ppm (signal peak of sulfhydryl hydrogen on cysteine in the peptide segment) and 6.50-8.75 ppm (signal peak region of aromatic proton in RVG29), and we found in the nuclear magnetic spectrum of the synthesized HA-RVG29 that the characteristic peak signals of both HA and RVG29 peptide exist simultaneously, and the disappearance of the characteristic peak of sulfhydryl hydrogen on RVG29 peptide, which proves the successful grafting of HA and RVG29 peptide, and the grafting rate of RVG29 peptide is calculated to be 40-45% by scoring.

[0057] A small amount of samples IR820, UA and IU nanoparticles were weighed, and were mixed with dry KBr, ground and pressed into tablets, and the obtained samples were analyzed and detected by Fourier transform infrared spectrometer (FT-IR). The results are shown in Figure 3 As shown, the characteristic peak of HA's nuclear magnetic hydrogen spectrum is mainly at 8.30 ppm, which is the signal peak of hydrogen proton on the amide group in HA, and the characteristic peak signal of RVG29 peptide is at 1.51 ppm (signal peak of sulfhydryl hydrogen on cysteine in the peptide segment) and 6.50-8.75 ppm (signal peak region of aromatic proton in RVG29), and we found in the nuclear magnetic spectrum of the synthesized HA-RVG29 that the characteristic peak signals of both HA and RVG29 peptide exist simultaneously, and the disappearance of the characteristic peak of sulfhydryl hydrogen on RVG29 peptide, which proves the successful grafting of HA and RVG29 peptide, and the grafting rate of RVG29 peptide is calculated to be 40-45% by scoring. Figure 3 It can be seen that HA appears an absorption peak at 1616 cm -1 , which belongs to the stretching vibration of hyaluronic acid carboxyl (-COOH); the absorption peak intensity at this position in the infrared spectrum of HA-RVG decreases; RVG29 peptide shows peaks at 3300, 1660 and 1540 cm -1 , which respectively belong to the stretching vibration of amide (N-H), the stretching vibration of (NC=O) and the bending vibration of (CN-H), and the peak at 3070 cm -1 belongs to the stretching vibration peak of olefin C-H in the peptide, which is the characteristic of the peptide bond between the amino acids in RVG 29 peptide, and these bands also exist in the synthesized HA-RVG29, indicating the successful synthesis of HA-RVG29.

[0058] IR820, IU, IUH, IUR were diluted at a ratio of 1 mg / mL, and the appropriate concentration after dilution was selected to determine the absorbance of each group of diluents by ultraviolet spectrophotometer. The results are shown in Figure 4 As shown, Figure 4 the ultraviolet absorption peaks of free IR820 and the three kinds of nanoparticles IU, IUH and IUR, respectively, compared with free IR820, the three kinds of nanoparticles all show slight red shift of absorption peak in the ultraviolet visible light spectrum, which may be due to the change of molecular form after self-assembly.

[0059] Application Example 1

[0060] MTT method was used to evaluate the in vitro cytotoxicity of different administration groups on U87 cells

[0061] In this experiment, four groups of UA, IU, IUH and IUR were set up, and the drug concentration gradient of each group was 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.

[0062] MTT method was used to evaluate the in vitro cytotoxicity of different drug groups on U87 cells. Figure 5 (a) is the cell viability graph of different concentrations of UA single drug, and the IC50 value of UA is 36.47 μg / mL, Figure 5 (b) is the cell viability graph of free IR820, and from the data, it can be seen that the cytotoxicity of IR820 can be basically considered as no cytotoxicity within 10 μg / mL. In this paper, the equivalent UA concentration of nanoparticle administration is 20 μg / mL, which is equivalent to containing IR820 3.5 μg / mL. It can be considered that IR820 does not play a cytotoxic role in this paper without laser irradiation. Figure 5 (c) is the cell viability statistical graph of different concentrations of TMZ, and the IC50 value of TMZ is 166.4 μg / mL. Figure 5 (d) is the cell viability statistical graph of the combined application of UA and TMZ. Figure 5 (e) is the evaluation of the drug combination index (CI) of the combined application of UA and TMZ by Chou-Talalay method using software Compusyn. CI<1, CI=1 and CI>1 represent synergistic effect, additive effect and antagonistic effect, respectively. From the graph, it can be seen that the CI value of UA and TMZ is less than 1, indicating that there is a synergistic inhibitory effect of TMZ and UA on U87 cells. Figure 5 (f) is a comparison chart of 24-hour cell viability of different drug groups, which shows that the combination of TMZ and targeted photosensitive nanoparticles has good in vitro cytotoxicity.

[0063] Application Example 2

[0064] Green fluorescent dye Calcein-AM and red fluorescent dye PI were used for co-staining of live and dead cells to observe the killing effect of IUR combined with TMZ on cells in vitro

[0065] In order to directly observe the killing effect of drugs on cells in vitro, green fluorescent dye Calcein-AM and red fluorescent dye PI were used for co-staining of live and dead cells to evaluate the survival and death of cells. As Figure 6As shown, the TMZ+IUR+L treatment group shows the most red fluorescence signal, and shows similar results compared with the cell viability of different drug groups in the cytotoxicity experiment. It is shown that temozolomide combined with targeted photosensitive nanoparticles shows excellent tumor cytotoxicity.

[0066] Application Example 3

[0067] EdU staining method to evaluate the inhibitory effect of IUR combined with TMZ on U87 cell proliferation

[0068] Take the U87 cells in good growth state to 8×10 3 cells per hole in a 96-well plate, and continue to culture in a constant temperature incubator for 24 hours. The experimental groups are: 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, TMZ+IUR group, TMZ+IUR+L group. The concentration of UA used in the experiment is based on the concentration of about 80% cell survival rate within 24 hours when it is used alone, that is, 20 μg / mL. When the cells grow to the appropriate density, give different experimental groups of drug treatment cells for 24 h.

[0069] In order to evaluate the inhibitory effect of different drug treatment groups on U87 cell proliferation, we used EdU staining method to monitor the proliferation of cells. EdU, a nucleoside mimic similar to deoxythymidine, can be embedded in replicating DNA and specifically bind to fluorescent dyes, so that we can directly evaluate the proliferation activity of cells. Among them, the red fluorescence label is the cell in the proliferation state. The results are shown in Figure 7 As can be seen from the experimental results, the nanoparticles show strong proliferation inhibition under laser irradiation, and the combination with temozolomide almost completely inhibits cell proliferation.

[0070] Application Example 4

[0071] Detect the generation of reactive oxygen species of nanoparticles to prove the ability of photodynamic production of ROS

[0072] (1) Cell preparation: take the U87 cells in good growth state to 3×10 5 cells per hole in a 6-well plate, and then continue to culture in a constant temperature incubator for 24 hours.

[0073] (2) Experimental grouping: The grouping is set as: blank control group, IU group, IU+TMZ group, IUR group, IUR+TMZ group. When the cells grow to the appropriate density, add different experimental group drugs to the inoculated cells, inoculate two holes in each drug group, one hole is irradiated by 808 nm laser after two hours, and the other hole is not irradiated as a control. After treatment, incubate the cells in a 37°C cell incubator for 6 hours in the dark.

[0074] (3) Probe loading: Refer to the ROS active oxygen detection kit (product number: R6033) of UElandy for instructions. Dilute DCFH-DA with serum-free medium at a ratio of 1:1000 to achieve a final concentration of 10 μM. After removing the cell culture solution, add the prepared active oxygen probe solution to the cell culture plate, and then incubate in a 37°C incubator for 20 minutes. After incubation, wash with serum-free cell culture medium three times.

[0075] (4) Observe and take pictures with an inverted fluorescence microscope, and then use ImageJ software for quantitative analysis.

[0076] The detection results are shown in Figure 8 The results show that IU and IUR nanoparticles can normally pass through laser irradiation to generate ROS and play a photodynamic role, and the ROS generated by the combination of TMZ and nanoparticles is more than that of the single nanoparticle group, indicating that TMZ promotes the generation of ROS by nanoparticles through photodynamic.

[0077] Application Example 5

[0078] One-step TUNEL cell apoptosis detection is used to determine the apoptosis of different drug groups

[0079] (1) Take U87 cells in good growth state at 8×10 3 cells / well and inoculate in a 96-well plate, and continue to culture in a constant temperature incubator for 24 hours.

[0080] (2) Experimental grouping: 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, TMZ+IUR group, TMZ+IUR+L group. The concentration of UA used in the experiment is based on the concentration at which the cell survival rate is about 80% within 24 hours when used alone, i.e. 20 μg / mL. When the cells grow to the appropriate density, give different experimental group drugs to treat the cells for 24 hours.

[0081] (3) First, fix the cells with 4% paraformaldehyde solution for no more than 30 minutes; then, wash the dye solution in the cells with PBS and treat the cells with 0.5% Triton X-100 PBS solution as a permeabilizing agent for no more than 5 minutes; after completion, wash again with PBS.

[0082] (4) Incubate the TUNEL staining solution with the cells for 1 hour in the dark, and then wash with PBS after incubation.

[0083] (5) Add DAPI staining working solution to the cells for staining for 30 minutes, and then wash the cells with PBS and store in the dark.

[0084] (6) Observe and photograph with an inverted fluorescence microscope, and then perform quantitative analysis using ImageJ software.

[0085] The apoptosis of different drug administration groups was determined. As shown in Table 1, the distribution of cell apoptosis trends was also consistent with the cytotoxicity experiment, indicating that the toxic effect of the drug on the cells mainly occurred through induction of cell apoptosis. Figure 9

[0086] Application Example 6

[0087] Annexin V-FITC / PI double staining flow cytometry directly observed and analyzed the apoptosis of cells

[0088] (1) U87 cells in good growth state were inoculated in a 96-well plate at 8x10 3 cells / well, and continued to be cultured in a constant temperature incubator for 24 hours.

[0089] (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, TMZ+IUR group, TMZ+IUR+L group. The concentration of UA used in the experiment was based on the concentration at which the cell survival rate was about 80% within 24 hours when used alone, i.e., 20 μg / mL. When the cells grew to the appropriate density, the cells were treated with different experimental drugs for 24 hours.

[0090] (3) After drug treatment, the cells were digested and collected, and washed with PBS.

[0091] (4) The cells were operated according to the instructions of the YF®488-Annexin V / PI apoptosis kit. The prepared staining solution was added to the cells and mixed evenly, and then incubated in the dark.

[0092] ​(5) The flow cytometry was used to detect each experimental group, and the results were analyzed by FlowJo-V10 software.

[0093] In order to directly observe and analyze the apoptosis, we used Annexin V-FITC / PI double staining flow cytometry. Annexin V-FITC was used to label early and late apoptotic cells, while PI staining was used to identify necrotic cells. The results of flow cytometry were represented by four quadrant diagrams, in which the upper left quadrant represented necrotic cells, the lower left quadrant showed healthy cells, the upper right quadrant identified late apoptotic cells, and the lower right quadrant represented early apoptotic cells. As shown in Figure 10 The apoptosis rates of the Control group, the IU group, the IUR group, the TMZ+IU group, and the TMZ+IUR group were 6.28%, 26.85%, 34.21%, 32.97%, and 37.71%, respectively. The apoptosis rates of the Control group, the IU group, the IUR group, the TMZ+IU group, and the TMZ+IUR group were 8.68%, 42.86%, 44.28%, 55.63%, and 71.5%, respectively. Through the comparison between the IU and IUR groups, the IUR with strong targeting ability indeed showed stronger apoptosis. The combination of TMZ and IUR showed a significantly stronger apoptosis ability than the single use of TMZ or IUR.

[0094] Application Example 7

[0095] Therapeutic effect of IUR combined with TMZ on U87-luc orthotopic glioma mouse model in vivo

[0096] To prove the therapeutic effect of nanoparticle IUR and its combination with TMZ on glioma mice, we designed the following experiment;

[0097] 1. In vivo distribution investigation

[0098] Since the IR820 contained in the nanoparticles itself has fluorescence, different experimental groups of nanoparticles (nanoparticle dispersion liquid concentration of 500 μg / mL, 10 μL / g of drug) can be directly injected into the tail vein of BALB / c nude mice. The experimental groups are: IR820 group, IUH, IUR group.

[0099] (1) At 1 h, 4 h, 8 h, 12 h, 24 h, and 48 h after injection, the fluorescence distribution images of mice in vivo were taken by small animal live fluorescence imaging.

[0100] (2) At 24 h after injection, the heart, liver, spleen, kidney, and brain of each drug group of nude mice were taken out, washed, and subjected to fluorescence imaging.

[0101] 2. Preliminary study on anti-tumor effect in mice

[0102] The treatment of nude mice in situ glioma, implantation of tumor seven days, at this time the total ROI value of each mouse brain tumor area more than 1 x 10 5 The fluorescence intensity and body weight of the nude mice were consistent, and they were randomly assigned to six different drug groups, and each group received saline (NS), TMZ, IUR, IUR+L, TMZ+IUR and TMZ+IUR+L treatment (L is light), wherein the dose of TMZ is 1 mg / kg, the concentration of IUR nanoparticles is 500 μg / mL, and the dose is 10 μL / g. TMZ is administered by oral gavage, and IUR targeted photosensitive nanoparticles are administered by tail vein injection. Oral administration and tail vein injection are performed every two days. 2 h after tail vein injection of IUR, brain laser irradiation (808 nm, 0.2 W / cm 2 , 5 min) is performed, and the treatment is performed continuously for seven times, for a total of 14 days. During the treatment, the mice are weighed every two days, and the tumor volume is monitored using the bioluminescence channel of the live imaging instrument on the 0th, 5th, 10th and 15th day of treatment (3 mice in each group are recorded). The survival of each group of mice is recorded from the start of treatment to the time of death of the mice (6 mice in each group are recorded).

[0103] As shown in Figure 11 (a), during the treatment period, by monitoring the tumor volume at different time points using a live imaging system, we found that at the beginning of treatment (0th day), the fluorescence intensity of each experimental group was basically flat. However, as the treatment time progresses, the tumor growth rate of each group begins to differ. In particular, our TMZ+IUR+L group has a significantly lower tumor fluorescence area than other groups on the 15th day, and the tumor growth is significantly inhibited compared to other groups. Figure (b) is a quantitative analysis of the fluorescence intensity of the tumor site of each treatment group, which more intuitively presents the differences in tumor growth between each treatment group, and more intuitively shows the inhibitory effect of TMZ+IUR+L on glioma tumor growth, indicating that it has a good therapeutic effect. Figure (c) shows that although TMZ alone and IUR nanoparticles alone combined with laser treatment can prolong the survival time of mice, the survival time of mice in the TMZ+IUR+L group is significantly longer than that of any single drug, indicating that the combination of temozolomide and nanoparticles indeed has a good therapeutic effect on glioma mice.

[0104] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a light environment targeted anti-tumor nanoparticle IUR, characterized in that, The steps are as follows: (1) the hyaluronic acid is activated with an activating agent EDCI and then mixed with the organic solvent of RVG29 peptide, and then the product hyaluronic acid-RVG29 is obtained by dialysis, centrifugation and freeze-drying of the supernatant; (2) under ultrasonic conditions, ursolic acid is dropped into the aqueous solution of IR820, the precipitate is collected after washing, and the IU nanoparticles are obtained by resuspension; the IU nanoparticles are added into the hyaluronic acid-RVG29 solution, stirred and mixed, and then the precipitate is collected after centrifugation, which is the nanoparticles IUR.

2. The method for preparing the antitumor nanoparticle IUR according to claim 1, characterized in that: In the step (1), the organic solvent is DMSO; the molar ratio of hyaluronic acid, EDCI and RVG29 peptide is 1-2.5:1:0.01-0.02; the activation temperature is 0-5℃, 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 anti-tumor nanoparticles IUR according to claim 2, characterized in that: In the step (1), the dialysis bag used in dialysis is 3500 kDa, the dialysis liquid is distilled water, and the dialysis time is 20-24 h.

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

5. The anti-tumor nanoparticles IUR prepared by the preparation method of any one of claims 1-4.

6. The use of the anti-tumor nanoparticles IUR and temozolomide of claim 5 in the preparation of an anti-glioma drug.

7. Use according to claim 6, characterized in that: The anti-glioma drug can penetrate the blood-brain barrier and effectively restore the behavioral ability.

8. Use according to claim 7, characterized in that: The concentration of nanoparticles IUR in the anti-glioma drug is 10 μL / g, and the concentration of temozolomide is 1 mg / kg.

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