Preparation method and application of visual supramolecular drug carrier

Abstract

The invention belongs to the technical field of biological medicine, and relates to a preparation method and application of a visual supramolecular drug carrier. The preparation method comprises the following steps: respectively dissolving oxaliplatin, 3, 6-bis (benzothiophene-2-yl) pyrrolo [3, 4-d] pyrimidine and cucurbituril [8] in water according to a molar ratio of (0.8-1.5): (0.8-1.5): 1, adding the oxaliplatin and the 3, 6-bis (benzothiophene-2-yl) pyrrolo [3, 4-d] pyrimidine in equal proportion, carrying out ultrasonic dissolution for 20-40 minutes after each substance is added, and carrying out self-assembly to prepare the supramolecular diagnosis and treatment system host-guest compound. The preparation method is simple and easy to implement, the tumor killing capacity of the prepared OTC host-guest compound is higher than that of a single chemotherapy drug, the position of the chemotherapy drug in tumor cells and the form of the cancer cells can be traced and positioned, and supermolecule diagnosis and treatment integration is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, relates to supramolecular chemotherapy combined with photodynamic therapy technology, and specifically relates to a preparation method and application of a visualized supramolecular drug carrier. Background Art

[0002] Cancer is a heterogeneous disease characterized by the uncontrolled growth and reproduction of tumor cells in normal tissues. The first-line treatment for most cancers is chemotherapy, but chemotherapy has problems such as nonspecificity and high toxicity, while phototherapy can selectively destroy tumor cells. The combined effect of the two can optimize the treatment effect and achieve the purpose of inhibiting tumors and improving survival rates. Supramolecular materials help to realize this treatment system and provide new ideas for tumor treatment. Based on its precise drug loading, targeted drug release ability and the ability to maintain high concentrations of drug accumulation in tumors, the supramolecular combined treatment system can improve the shortcomings of traditional therapies such as poor solubility, strong drug resistance, and poor accumulation in tumor tissues, obtain higher anticancer activity, and reduce the toxicity of treatment to normal tissues. In addition to facilitating high-concentration drug treatment, targeted controlled release and prolonged accumulation time are also one of the advantages of supramolecular combined photoimmunotherapy. These show that the use of supramolecular materials for photoimmunotherapy is reasonable and effective, and has clinical application prospects. Chinese patent CN110934830A discloses a supramolecular drug-loaded vesicle and its preparation method and use, which utilizes the host-guest recognition interaction between water-soluble column [5] aromatic hydrocarbons and aniline tetramers to construct a supramolecular vesicle. Chinese patent CN113499312A discloses a supramolecular core-shell complex, a supramolecular drug-loaded core-shell complex, and the preparation method and use of the two, using water-soluble column [5] aromatic hydrocarbons as the main body and amphiphilic aniline tetramers modified with polyethylene glycol segments as guest molecules to self-assemble to form a supramolecular core-shell complex with photothermal properties. The above-mentioned disclosed vesicles and core-shell complexes both belong to supramolecular chemotherapy in the prior art, but their preparation process is complicated, and it is impossible to determine the location of chemotherapy drugs in cells and the size of cancer cells. Summary of the invention

[0003] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a preparation method and application of a visualized supramolecular drug carrier. The preparation method is simple and easy, and the prepared supramolecular diagnostic and therapeutic system (OTC) can trace and locate the position of chemotherapy drugs in tumor cells and the morphology of cancer cells, and its tumor killing ability is stronger than that of chemotherapy drugs alone.

[0004] The technical solution of the present invention is:

[0005] The present invention provides a method for preparing a visualized supramolecular drug carrier, comprising the following steps:

[0006] Oxaliplatin, 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine and cucurbituril[8] were dissolved in water at a molar ratio of (0.8-1.5):(0.8-1.5):1, wherein oxaliplatin and 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine were added in equal proportions. After each substance was added, it was dissolved by ultrasonic for 20-40 minutes. Cucurbituril[8] was used as a drug carrier molecule to coat oxaliplatin and then coat 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine molecules, and a supramolecular diagnostic and therapeutic system host-guest complex was prepared by self-assembly.

[0007] CB[8] material was used as the drug carrier molecule. It has a large internal cavity and can encapsulate both OxPt molecules and TTVP molecules at the same time. By using supramolecular chemotherapy in conjunction with photodynamic therapy, the prepared assembly OTC (OxPt-TTVP@CB[8]) showed a stronger anti-tumor effect than OxPt.

[0008] In the above preparation steps, there is no specific requirement for the order of adding CB[8], TTVP, and OxPt, and the three can form an OTC host-guest complex through self-assembly.

[0009] Furthermore, the molar ratio of oxaliplatin, 3,6-bis(benzothiophene-2-yl)pyrrolo[3,4-d]pyrimidine and cucurbituril[8] is 1:1:1.

[0010] Furthermore, the ultrasonic time is 30 minutes.

[0011] Furthermore, the preparation method of cucurbituril [8] comprises the following steps:

[0012] Weigh a certain amount of ethylene glycol and add it to a reaction container, add hydrochloric acid, and then slowly add polymethanol. The mass ratio of the three is 1:(1.6-2.0):(0.4-0.5). After mixing, stir at room temperature for 25-35 minutes, heat to 95-105°C and keep it for 0.5-1.5 hours, then heat to 110°C and keep it for 15-19 hours. Centrifuge at 4000 rpm for 5-10 minutes, collect the precipitate, wash it several times with 60% formic acid solution, and then wash it with water until the supernatant is neutral. Collect the precipitate, freeze-dry it, and obtain cucurbituril[8] powder.

[0013] Furthermore, in the step of preparing cucurbituril [8], after stirring, the mixture is heated to 100° C. and maintained for 1 hour, and then heated to 110° C. and maintained for 17 hours.

[0014] The present invention also provides the use of the host-guest complex of the supramolecular diagnosis and treatment system prepared by the above preparation method in the preparation of anti-tumor drugs.

[0015] The present invention further provides the use of the host-guest complex of the supramolecular diagnosis and treatment system prepared by the above preparation method in tracing and positioning chemotherapy drugs.

[0016] Furthermore, the host-guest complex of the supramolecular diagnostic and therapeutic system can track and locate the position of chemotherapy drugs in tumor cells and the morphology of cancer cells.

[0017] Furthermore, the host-guest complex of the supramolecular diagnostic and therapeutic system is used to observe the localization of chemotherapy drugs in tumor cells and visualize tumor cells through laser confocal microscopy.

[0018] Beneficial effects of the present invention:

[0019] (1) The preparation method provided by the present invention is simple and easy. Through host-guest interaction, oxaliplatin, 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine and cucurbituril[8] are dissolved in water at a certain molar ratio to form a supramolecular diagnostic and therapeutic system (OTC) by self-assembly.

[0020] (2) The present invention is visualized and verified by an intestinal tumor cell model. The research results show that OTC can track and locate the location of chemotherapy drugs in tumor cells and the morphology of cancer cells;

[0021] The results of the study, verified by intestinal tumor cell models and nude mouse tumor-bearing models, showed that OTC achieved supramolecular chemotherapy combined with photodynamic therapy, and its tumor-killing ability was stronger than that of chemotherapy drugs alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the H-NMR spectrum of OxPt, TTVP and OTC;

[0023] Figure 2 is the H NMR spectrum of CB[8];

[0024] Figure 3 Fluorescence spectra of CB[8], OxPt, TTVP, TTVP+CB[8] and OTC under 488 nm excitation light;

[0025] Figure 4 The fluorescence spectra of OTC drugs after adding different concentrations of SPD;

[0026] Figure 5 are the UV absorption spectra of CB[8], OxPt, TTVP, TTVP+CB[8], and OTC;

[0027] Figure 6 This is the UV absorption spectrum of the sustained release process of OTC drugs in spermidine solution;

[0028] Figure 7Laser confocal microscopy images of HCT-116 cells uptake of TTVP and OTC;

[0029] Figure 8 The fluorescence quantitative results of TTVP and OTC uptake by HCT-116 cells;

[0030] Fig. 9 The cell viability changes of HCT-116 cells with drug concentration;

[0031] Fig.10 The cell viability changes of NCM460 cells with drug concentration;

[0032] Fig.11 is the tumor growth curve of mice in the control group, OxPt, and OTC groups;

[0033] Fig.12 The weight of mouse tumor in vitro on day 26;

[0034] Fig.13 Figure 2 is the body weight change of tumor-bearing mice during the drug administration period. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in 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.

[0036] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0037] The experimental methods in the following embodiments or test examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following embodiments or test examples, unless otherwise specified, can be obtained from commercial channels.

[0038] Example 1

[0039] Preparation of CB[8]:

[0040] 100 g of ethylene glycol was placed in a flask, 160 mL of hydrochloric acid was added, and 42.2 g of polymethanol was slowly added. The mixture was stirred at room temperature for 30 minutes, heated to 100 ° C for 1 hour, and then heated to 110 ° C for 17 hours. Centrifuged at 4000 rpm for 7 minutes, and the precipitate was collected. The precipitate was washed three times with 60% formic acid solution to remove incompletely reacted by-products and raw materials, and then washed with pure water until the supernatant was neutral. The precipitate was collected and freeze-dried to obtain about 6.4 g of CB[8] powder.

[0041] Preparation of supramolecular theranostic system (OTC):

[0042] OxPt, TTVP and CB[8] were dissolved in water at a molar ratio of 1:1:1. There was no specific requirement for the order of adding the three substances. After each substance was added, ultrasound was applied for 30 min to allow the three substances to self-assemble into an OTC host-guest complex.

[0043] The above-mentioned assembly process was characterized by nuclear magnetic resonance spectroscopy and spectroscopy, and the anti-tumor properties of the OTC host-guest complex were verified at the cellular level. The localization of chemotherapy drugs in tumor cells and tumor cell visualization were observed by laser confocal microscopy to achieve integrated supramolecular diagnosis and treatment.

[0044] Example 2

[0045] Preparation of CB[8]:

[0046] 100 g of ethylene glycol was placed in a flask, 136.8 mL of hydrochloric acid was added, and 40 g of polymethanol was slowly added. The mixture was stirred at room temperature for 25 minutes, heated to 95 ° C for 1.5 hours, and then heated to 110 ° C for 19 hours. Centrifuged at 4000 rpm for 5 minutes, and the precipitate was collected. The precipitate was washed three times with 60% formic acid solution to remove incompletely reacted by-products and raw materials, and then washed with pure water until the supernatant was neutral. The precipitate was collected and freeze-dried to obtain about 6.4 g of CB[8] powder.

[0047] Preparation of supramolecular theranostic system (OTC):

[0048] OxPt, TTVP and CB[8] were dissolved in water at a molar ratio of 0.8:0.8:1. There was no specific requirement for the order of adding the three substances. After each substance was added, ultrasound was applied for 20 minutes, and the three substances self-assembled to form an OTC host-guest complex.

[0049] Example 3

[0050] Preparation of CB[8]:

[0051] 100 g of ethylene glycol was placed in a flask, 170.9 mL of hydrochloric acid was added, and 50 g of polymethanol was slowly added. The mixture was stirred at room temperature for 35 minutes, heated to 105 ° C for 0.5 hours, and then heated to 110 ° C for 15 hours. The mixture was centrifuged at 4000 rpm for 10 minutes and the precipitate was collected. The precipitate was washed three times with 60% formic acid solution to remove incompletely reacted by-products and raw materials, and then washed with pure water until the supernatant was neutral. The precipitate was collected and freeze-dried to obtain about 6.4 g of CB[8] powder.

[0052] Preparation of supramolecular theranostic system (OTC):

[0053] OxPt, TTVP and CB[8] were dissolved in water at a molar ratio of 1.5:1.5:1. There was no specific requirement for the order of adding the three substances. After each substance was added, ultrasound was applied for 40 min, and the three substances self-assembled to form an OTC host-guest complex.

[0054] Test Example 1

[0055] The assembly process of Example 1 was subjected to nuclear magnetic resonance measurement, and the results were Figure 1 and Figure 2 The hydrogen spectrum.

[0056] like Figure 1 As shown in the figure, the hydrogen atoms of the methylene groups on the six-membered ring of OxPt can be seen with five clear peaks between 1.0 and 2.75 ppm. After adding CB[8], the five peaks originally located between 1.0 and 2.75 ppm all disappeared, indicating that OxPt was almost completely loaded into CB[8]. The peaks corresponding to the chemical shifts of the hydrogen atoms on the aromatic ring of TTVP between 6.5 and 7.5 ppm were reduced to almost disappearance, and the peak intensity corresponding to the chemical shift of the three methyl groups at the end was about 3.25 ppm, which was reduced from 500 to 100, which means that except for the methyl groups at the end, almost all of TTVP entered the interior of CB[8]. Figure 2 As shown, the three positions of hydrogen atoms in CB[8] are consistent with the NMR results.

[0057] Fluorescence spectrometry

[0058] CB[8], OxPt, TTVP, TTVP@CB[8] and OxPt-TTVP@CB[8] were dissolved in ultrapure water at a concentration of 10 μM, placed in a cuvette, and scanned from 900 nm to 500 nm using a fluorescence spectrophotometer. The wavelength was used as the horizontal axis and the fluorescence intensity was used as the vertical axis to plot the fluorescence spectrum. After adding 2 μL of SPD (1 mM) to 1 mL of OTC (10 μM), the fluorescence spectrum was measured and repeated 10 times.

[0059] like Figure 3As shown in the figure, the fluorescence spectrum shows that CB[8] can enhance the fluorescence intensity of TTVP. The fluorescence intensity of TTVP at 730nm is increased from 13942 to 25924 of OTC, nearly twice as much. Figure 4 As shown, with the addition of SPD, the fluorescence intensity of OTC decreased significantly and gradually approached the fluorescence intensity of TTVP alone, proving that SPD helps the disassembly of OTC.

[0060] UV spectrometry

[0061] CB[8], OxPt, TTVP, TTVP@CB[8] and OxPt-TTVP@CB[8] were dissolved in ultrapure water at a concentration of 50 μM, placed in a cuvette, and scanned from 900 nm to 200 nm using a UV spectrophotometer. The wavelength was used as the horizontal axis and the absorbance was used as the vertical axis to plot the graph. In 1 mL of OTC (50 μM), 2 μL of SPD (5 mM) was added each time and the UV spectrum was measured. This was repeated 10 times.

[0062] like Figure 5 As shown in the figure, the UV spectrum changed significantly after OTC binding. Compared with the single TTVP control, the UV absorption peak at 488nm was significantly reduced and slightly red-shifted, and the peak red-shifted from 478nm to 499nm; compared with the single OxPt control, the absorption value of OxPt at 250nm increased from 0.13 to 0.33, and the peak valley here was significantly smaller, which indicates that TTVP and OxPt entered the hydrophobic cavity of CB[8] together. Figure 6 As shown, after adding SPD to the OTC solution, it can be found that the UV absorption peak of OTC at 488nm increased again, and gradually returned to the UV absorption peak intensity of free TTVP. The UV absorption spectrum can also prove that SPD helps OTC disassembly.

[0063] Test Example 2

[0064] Cellular uptake assay:

[0065] After HCT116 cells were placed in a laser confocal culture dish for 4 hours, they were treated with serum-free medium containing OxPt, TTVP, CB[8], and OTC (at a concentration of 25 μM, respectively), and the control group was treated with serum-free medium. Then, the cells were incubated with Dio and Hoechst in turn, and cell fluorescence images were taken under a laser confocal microscope, and fluorescence quantification was performed using Image J.

[0066] Cell culture and viability assays:

[0067] Normal human colorectal mucosal cells (NCM460) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2. Cells were passaged when the cell density reached 80-90% or every other day. Human colorectal cancer cells (HCT116) were also cultured in RPMI-1640 medium in a similar manner. The cytotoxicity of OxPt, TTVP, CB[8] and OTC on NCM460 and HCT116 cells was evaluated using the CCK-8 assay according to the manufacturer's instructions (Bio-Optic, China). 2×10 3 The cells were seeded into 96-well plates at a density of 10 cells and cultured for 12 hours to allow them to adhere to the bottom. Then, they were cultured with different concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM) of OxPt, TTVP, CB[8], and OTC for 24 hours. After washing the cells with PBS buffer, 10 μL of CCK-8 solution was injected into each well. After incubation at 37°C for 30-40 minutes, the CCK-8 solution was removed. The reagent was developed, and the absorbance of each well was detected at a wavelength of 450 nm using a microplate reader (Bio-Tek, USA).

[0068] like Figure 7 As shown in the figure, the distribution of OTC drugs in cells and the location of cancer cells can be observed by laser confocal microscopy. In the Merge diagram of the OTC group, OTC appears in granular and vesicular form. Arrow 1 indicates that OTC gathers around the nuclear membrane to play a role, and arrow 2 indicates that OTC has the function of locating cancer cells. The Hoechst group is the cell nucleus stained with Hoechst, and the DiO group is the cell membrane stained with DiO.

[0069] like Figure 8 As shown, the uptake of TTVP and OTC by HCT-116 cells was quantitatively analyzed by fluorescence. The results showed that the average fluorescence intensity of OTC was 40.36, which was significantly stronger than the average fluorescence intensity of TTVP, which was 34.56. This was consistent with the results of fluorescence spectroscopy characterization (see Figure 3 ).

[0070] like Fig. 9 As shown in the figure, under the conditions of visible light irradiation for 12h and OTC action for 24h, the cytotoxicity experiment showed that OTC had stronger anti-tumor ability than OxPt alone. The survival rate of HCT-116 cells decreased with the increase of the concentration of each poisoning solution. When the concentration of OTC reached 50μM, the survival rate of HCT-116 cells was only 34.28%, and at each poisoning concentration, the cell survival rate of the OTC group was the lowest, indicating that it caused the greatest cell damage, and the drug system can effectively kill tumor cells.

[0071] like Fig.10As shown in the figure, under the condition of 24h of OTC action, the results of cytotoxicity experiments showed that OTC drugs had no obvious killing effect on normal cells at low concentrations. When the OTC poisoning concentration was 12.5μM, the survival rate of NCM460 cells was still as high as 98.49%, with no significant difference from the control group. Only when the OTC poisoning concentration reached 25μM and above, it had a significant toxic effect on NCM460 cells, indicating that under normal circumstances, this drug system had no obvious damage to normal human intestinal epithelial cells.

[0072] Test Example 3

[0073] Detection of anti-tumor therapeutic effects in vivo:

[0074] HCT-116 cells (1×10 7 cells, 0.1 mL saline), and the tumor male mice were randomly divided into 4 groups (5 mice in each group). The tumor volume reached about 500 mm 3 Afterwards, CB[8], OxPt or OTC (3.5 mg / kg) were injected into the tail vein three times a week for two weeks. Body weight and tumor volume were measured every other day, and tumor volume (V) was calculated according to the following formula: V = 0.52 × L × W 2 , where L and W represent the length and width of the tumor, respectively. On day 26, the tumors were removed for analysis.

[0075] like Fig.11 As shown, the tumors of mice treated with normal saline in the control group (Ctrl) grew rapidly during the treatment period, while OxPt and OTC could inhibit tumor growth, and the anti-tumor effect of OTC was more obvious than that of the OxPt group.

[0076] like Fig.12 As shown, the tumors obtained at the end of treatment were weighed, and the results showed that the tumor weights of mice in the OxPt and OTC groups were much lower than those in the control group (Ctrl), and the tumor weight in the OTC group was lower than that in the OxPt group, indicating that the anti-tumor effect of OTC was significantly better than that of the OxPt group.

[0077] like Fig.13 As shown in Figure 2, during the treatment process, the weight of mice in the OTC group remained stable, which indicates that during the anti-tumor treatment process, the OTC system constructed by the present invention has a protective effect on normal tissues and organs, which is consistent with the results of cell viability determination (see Fig. 9 and Fig.10 ).

[0078] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a visual supramolecular drug carrier, characterized in that: The following steps are involved: Oxaliplatin, 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine and cucurbituril[8] were dissolved in water at a molar ratio of (0.8-1.5):(0.8-1.5):1, wherein oxaliplatin and 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine were added in equal proportions. After each substance was added, it was dissolved by ultrasonic for 20-40 minutes. Cucurbituril[8] was used as a drug carrier molecule to coat oxaliplatin and then coat 3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine molecules, and a supramolecular diagnostic and therapeutic system host-guest complex was prepared by self-assembly.

2. The preparation method according to claim 1, characterized in that: The molar ratio of oxaliplatin, 3,6-bis(benzothiophene-2-yl)pyrrolo[3,4-d]pyrimidine and cucurbituril[8] is 1:1:

1.

3. The preparation method according to claim 1, characterized in that: The ultrasonic time is 30 min.

4. The preparation method according to claim 1, characterized in that: The preparation method of cucurbituril [8] comprises the following steps: Weigh a certain amount of ethylene glycol and add it to a reaction container, add hydrochloric acid, and then slowly add polymethanol, the mass ratio of the three being 1:(1.6-2.0):(0.4-0.5). After mixing, stir at room temperature for 25-35 minutes, heat to 95-105°C and keep it for 0.5-1.5 hours, then heat to 110°C and keep it for 15-19 hours, centrifuge at 4000 rpm for 5-10 minutes, collect the precipitate, wash it several times with 60% formic acid solution, and then wash it with water until the supernatant is neutral, collect the precipitate, and freeze-dry it to obtain cucurbituril[8] powder.

5. The preparation method according to claim 4, characterized in that: In the preparation step of cucurbituril [8], after stirring, the mixture is heated to 100° C. and maintained for 1 hour, and then heated to 110° C. and maintained for 17 hours.

6. Use of a host-guest complex of a supramolecular diagnosis and treatment system prepared by the preparation method according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

7. Use of a host-guest complex of a supramolecular diagnosis and treatment system prepared by the preparation method according to any one of claims 1 to 5 in tracing and localizing chemotherapy drugs.

8. The use according to claim 7, characterized in that: The host-guest complex of the supramolecular diagnosis and treatment system can track and locate the position of chemotherapy drugs in tumor cells and the morphology of cancer cells.

9. The use according to claim 7, characterized in that: The host-guest complex of the supramolecular diagnostic and therapeutic system is used to observe the localization of chemotherapy drugs in tumor cells and visualize tumor cells through laser confocal microscopy.

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