Preparation method and application of a triple combined tumor active targeting nano drug delivery system

Abstract

The present invention discloses a preparation method and application of a three-combination tumor active targeting nano drug delivery system, and relates to the construction and preparation of a novel tumor active targeting nano drug delivery system in which a folic acid-polydopamine carrier material is co-loaded with camptothecin and iron, which can be used for active targeted treatment of tumors and synergistic killing of drug-resistant tumor cells in multiple ways. The present invention selects folic acid-modified dopamine and dopamine polymerization to obtain a carrier material, and co-loads camptothecin and iron to obtain a three-combination nano therapy system of chemotherapy / ferroptosis / photothermal co-loaded with camptothecin / iron having tumor active targeting effect and photothermal conversion performance. Compared with the traditional chemotherapy drug camptothecin, the present invention can realize tumor active targeting drug delivery, and utilize the photothermal conversion characteristics of nanoparticles to realize apoptosis, ferroptosis and photothermal combined therapeutic effects, significantly improve the therapeutic effect of drug-resistant tumors, and has a good clinical application prospect.

Description

Technical Field

[0001] The invention relates to a method for preparing a tumor active targeting nano drug delivery system of a folic acid modified polydopamine carrier material co-loaded with camptothecin and iron. The nano drug delivery system can be used for anti-tumor, especially for the treatment of drug-resistant breast cancer. Background Art

[0002] Camptothecin is a natural alkaloid extracted and separated from Camptotheca acuminata. It has strong cytotoxicity and exhibits significant anti-tumor activity [Natural Product Research and Development, 2016, 28(11): 1830-1837]. Studies have found that camptothecin mainly acts on topoisomerase I by entering tumor cells, thereby inhibiting DNA synthesis to achieve anti-tumor effects. It has certain therapeutic effects on various malignant tumors such as breast cancer, gastric cancer, rectal cancer and leukemia [Chinese Pharmacist, 2003, 6(7): 402-405]. Under different pH environments, camptothecin exists in two forms: lactone and carboxylate [Journal of Biological Chemistry, 1985, 260(27): 14873-14878], and its complete lactone ring is an essential group for inhibiting topoisomerase I. When intravenously administered at a physiological pH of 7.4, camptothecin mainly exists in the form of low-activity carboxylate, and its ion-type cell entry efficiency is extremely low, resulting in poor clinical therapeutic effects. At the same time, since camptothecin kills tumor cells mainly by inducing apoptosis through topoisomerase I, it is very easy to develop multidrug resistance and lead to chemotherapy failure. Therefore, how to efficiently deliver it to tumor cells, exert effective anti-tumor effects with the lactone ring, and reverse tumor resistance to camptothecin is of great research value.

[0003] Ferroptosis is a cell death mode that depends on iron accumulation. It is essentially caused by the Fenton reaction due to iron overload, which produces a large amount of ROS and causes cell death caused by lipid oxidation metabolism disorders in cells. Ferroptosis is a cell death mode different from apoptosis. Therefore, constructing a carrier system for co-delivering iron and camptothecin is expected to synergistically kill tumor cells through the ferroptosis pathway, thereby reversing tumor resistance [Cell, 2012, 149(5): 1060-1072].

[0004] Studies have confirmed that dopamine can undergo oxidative self-polymerization to form polydopamine (PDA) nanoparticles with photothermal therapeutic properties, which can play a role in photothermal treatment of tumors. However, independent polydopamine nanoparticles are not selective for tumor cells and will also produce photothermal killing effects on normal tissues around the tumor. Studies have confirmed that the folate receptors highly expressed on the surface of tumor cells can specifically bind to folic acid (FA) and have an active targeting and recognition effect on tumor cells. Therefore, the present invention selects folic acid-modified dopamine (DA-FA) and dopamine as common carrier materials, and utilizes the coordination effect of phenolic hydroxyl groups in the dopamine structure with iron ions and the π-π stacking of the polydopamine grid structure and the camptothecin planar structure to achieve the co-loading of iron and camptothecin, and obtains tumor active targeting nanoparticles CPT / Fe@PDA-FA10 that co-load iron (Fe) and camptothecin (CPT). The nanoparticles can achieve active uptake and efficient intracellular entry into tumor cells through the targeted effect of folic acid, and utilize the photothermal conversion characteristics of PDA to achieve three combined treatment methods of tumor cell apoptosis, ferroptosis and photothermal effect, synergistically killing drug-resistant tumor cells in multiple ways. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a triple combined tumor active targeted nano drug delivery system, and obtain a tumor active targeted nano drug delivery system that can simultaneously load camptothecin and iron and has three combined therapeutic effects of chemotherapy / photothermal therapy / ferroptosis. This type of drug delivery system uses folic acid-modified polydopamine as a carrier material, which can achieve co-loading of camptothecin and iron and exert photothermal therapy. The folic acid targeting group in the carrier material can achieve active targeted delivery of tumors, obtain an efficient and low-toxic drug delivery system, and kill drug-resistant tumors in a multi-pathway synergistic manner, providing new ideas for the research of anti-tumor drugs.

[0006] One of the purposes of the present invention is to provide a method for preparing a triple combined tumor active targeted nano drug delivery system, which specifically comprises the following steps:

[0007] Step (1) Under light-proof conditions, weigh a certain amount of folic acid and dissolve it in 20 mL of DMSO, add DCC and NHS in the corresponding molar ratio and stir in a 50°C oil bath for 6 hours. Weigh dopamine hydrochloride in the corresponding molar ratio and add it to the above reaction solution. Stir at room temperature for 5 hours and then stop the reaction. After standing, filter and remove the precipitate, pour the filtrate into 80 mL of acetone / ether (1 / 3, V / V) mixed solution, filter the precipitate, wash the filter cake with 40 mL of DCM, 40 mL of acetone, and 40 mL of ether, respectively, and vacuum dry to obtain folic acid-modified dopamine (DA-FA).

[0008] Step (2) Weigh a certain amount of dopamine hydrochloride and add it to a 250mL flask with a magnetic stirrer. Weigh the corresponding molar ratio of folic acid-modified dopamine (DA-FA) and dissolve it in 400μL of DMSO. After vortexing and ultrasonication, it is completely dissolved and then added to the above 250mL flask. Weigh the corresponding ratio of FeCl3·6H2O, add 20mL of deionized water, and pour it into the 250mL flask after ultrasonication. Continue to add deionized water to 130mL and stir for 1h. Quickly pour an appropriate amount of tris aqueous solution into the above solution and continue stirring for 2h. Centrifuge (15000g, 10min), wash with deionized water (100mL×3 times), and then dissolve the precipitate in 50mL of deionized water.

[0009] Step (3) Weigh an appropriate amount of camptothecin and dissolve it in 500 μL of DMSO. After it is completely dissolved by vortexing and ultrasound, it is slowly dripped into the 50 mL sediment solution obtained in step (2) and stirred for 2 h. Centrifuge (15000 g, 10 min), wash once with 50 mL of deionized water to obtain a sediment, and dilute it to 25 mL with deionized water to obtain CPT / Fe@PDA-FA10 nanoparticles.

[0010] Furthermore, in step (1), the molar ratio of folic acid to dopamine is 1:2 to 2:1, and the molar ratio of folic acid, DCC and NHS is 1:0.5:0.5 to 1:5:5.

[0011] Furthermore, in step (2), the molar ratio of folic acid-modified dopamine to dopamine is 1:50-2:1, the mass ratio of dopamine to ferric chloride is 200:1-1:5, and the final concentration of the tris aqueous solution is 0.01%-15%.

[0012] Furthermore, in step (3), the mass of camptothecin fed is 1.0 mg to 15 mg.

[0013] The second purpose of the present invention is to provide a triple combined tumor active targeting nano drug delivery system for use in the treatment of drug-resistant breast cancer.

[0014] The present invention has the following outstanding beneficial effects:

[0015] 1. The present invention uses folic acid and dopamine as raw materials to synthesize folic acid-modified dopamine tumor active targeting materials, and obtains tumor active targeting nanoparticles CPT / Fe@PDA-FA10 by loading camptothecin and iron. The obtained nanoparticles have a small particle size (276nm) and are evenly distributed (PDI=0.086). The drug loading of camptothecin is 12.17%, the drug loading of iron is 10.05%, and the storage stability is good. Cell uptake experiments show that CPT / Fe@PDA-FA10 nanoparticles have a good tumor cell targeting effect. In vitro activity evaluation shows that CPT / Fe@PDA-FA10 can kill drug-resistant breast cancer cells.

[0016] 2. The results of the in vivo activity evaluation experiment of the above-mentioned camptothecin tumor-targeted nanoparticles show that compared with the traditional chemotherapy drug camptothecin used in clinical practice, this type of drug delivery system can achieve targeted drug delivery to tumor cells, exert an effective anti-tumor effect, and has good clinical application prospects for the treatment of drug-resistant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the characterization of the CPT / Fe@PDA-FA10 nano drug delivery system. (A) Particle size distribution of CPT / Fe@PDA-FA10. (B) Transmission electron microscopy of CPT / Fe@PDA-FA10. (C) Scanning electron microscopy of CPT / Fe@PDA-FA10. (D) Field emission transmission electron microscopy of CPT / Fe@PDA-FA10. (E) Stability of CPT / Fe@PDA-FA10.

[0018] Figure 2 The in vitro antitumor test and photothermal conversion performance of CPT / Fe@PDA-FA10. (A) Cytotoxicity test of CPT / Fe@PDA-FA10 on 4T1-Luc / CPT resistant cells (n=6, ). * P<0.05, ** P < 0.01, compared with the Control group; ζ P<0.05, ζζ P < 0.01, compared with the CPT group; & P<0.05, && P < 0.01, compared with the CPT@PDA-FA10 group; # P<0.05, ## P < 0.01, CPT / Fe@PDA-FA10+Laser group compared with CPT / Fe@PDA-FA10 group; ＄ P<0.05, ＄＄P<0.01, CPT / Fe@PDA-FA10 group compared with CPT / Fe@PDA group. (B) Cytotoxicity of CPT@PDA-FA10 and CPT / Fe@PDA-FA10 on 4T1-Luc / CPT resistant cells in the presence of ferroptosis inhibitors. n=6, ** P<0.01, compared with the group without inhibitor. (C) Infrared thermal imaging of CPT / Fe@PDA-FA10 with different concentrations after laser irradiation. (D) Temperature change curve of CPT / Fe@PDA-FA10 with different concentrations after laser irradiation.

[0019] Figure 3 This is a study on the targeting effect of CPT / Fe@PDA-FA10. Competitive cell growth inhibition experiment of CPT / Fe@PDA (A) and CPT / Fe@PDA-FA10 (B) after folic acid pretreatment. n=6, *P<0.05, **P<0.01, compared with the group without folic acid. (C) CLSM images of competitive uptake of CPT / Fe@PDA and CPT / Fe@PDA-FA10 by cells after folic acid pretreatment (60×).

[0020] Figure 4 This is the in vivo combined anti-resistant breast cancer experiment and toxicity experiment of CPT / Fe@PDA-FA10. (A) Tumor growth inhibition curve of each group of nude mice during treatment, n=6, * P<0.05, ** P < 0.01, compared with the Normal saline group; ζ P<0.05, ζζ P < 0.01, compared with the CPT group; && P < 0.01, compared with the CPT@PDA-FA10 group; ## P < 0.01, CPT / Fe@PDA-FA10+Laser group compared with CPT / Fe@PDA-FA10 group; ＄＄ P<0.01, CPT / Fe@PDA-FA10 group compared with CPT / Fe@PDA group. (B) Body weight changes of nude mice in each group during treatment. (C) H&E staining of various organs and tissues of nude mice with in situ camptothecin-resistant breast cancer (15×); ALT (D) and AST (E) activities in the serum of nude mice in each group on the 14th day of administration, as well as BUNCREA (F) and CREA (G) contents. n=6, DETAILED DESCRIPTION

[0021] The specific implementation methods of the present invention, the technical problems solved by the present invention, and the technical solutions are further described in detail below through examples. The following description is only a specific 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 principles of the present invention should be included in the protection scope of the present invention.

[0022] Example 1

[0023] Preparation and characterization of tumor active targeting nanoparticles CPT / Fe@PDA-FA10

[0024] Step (1): Weigh 0.9 g of folic acid (FA, 2 mmol) and dissolve it in 20 mL of dimethyl sulfoxide (DMSO) under light-proof conditions. Add 0.5 g of N,N'-dicyclohexylcarbodiimide (DCC, 2.4 mmol) and 0.5 g of N-hydroxysuccinimide (NHS, 4 mmol) and stir in a 50°C oil bath for 6 h. Weigh 3.8 g of dopamine hydrochloride (DA, 20 mmol) and add it to the above reaction solution. Stir at room temperature for 5 h and stop the reaction. After standing, filter to obtain a filtrate, add 80 mL of acetone / ether (1 / 3, V / V) mixed solution to the filtrate, filter the precipitate, wash the filter cake with 40 mL of DCM, 40 mL of acetone, and 40 mL of ether, respectively, and vacuum dry to obtain 1.1 g of folic acid-modified dopamine (DA-FA).

[0025] Step (2): Weigh 40.5 mg of dopamine hydrochloride and add it to a 250 mL flask with a magnetic stirrer. Weigh 3.6 mg of dopamine-folic acid (DA-FA) and dissolve it in 400 μL of DMSO. Vortex and sonicate until it is completely dissolved and then add it to the above 250 mL flask. Weigh 20.4 mg of FeCl3·6H2O, add 20 mL of deionized water, and pour it into the 250 mL flask after it is dissolved. Continue to add deionized water to 130 mL and stir for 1 hour. Quickly pour 20 mL of 12.5% ​​tris aqueous solution into the above solution and continue stirring for 2 hours. Centrifuge (15000g, 10 min), wash with deionized water (100 mL×3 times), and then dissolve the precipitate in 50 mL of deionized water.

[0026] Step (3): Weigh 6.0 mg of camptothecin and add it to 500 μL of DMSO. After dissolving, slowly drip it into the 50 mL sediment solution obtained in step (2), stir for 2 h, centrifuge (15000 g, 10 min), wash once with 50 mL of deionized water to obtain a sediment, and dilute it to 25 mL with deionized water to obtain CPT / Fe@PDA-FA10.

[0027] The particle size, PDI and zeta potential of the prepared CPT / Fe@PDA-FA10 nanoparticles were determined by laser particle size analyzer. The drug loading of iron was determined by o-phenanthroline UV spectrophotometry, and the drug loading of camptothecin was determined by high performance liquid chromatography. The prepared nanoparticles were morphologically examined by transmission electron microscopy and scanning electron microscopy. The elements of the nanoparticles were analyzed by field emission transmission electron microscopy. The CPT / Fe@PDA-FA10 nanoparticles were stored at 4°C, and their particle size and PDI were determined by laser particle size analyzer. The changes in particle size of the nanoparticles after different storage times were observed to study their stability.

[0028] Experimental results: The particle size, PDI and zeta potential of CPT / Fe@PDA-FA10 nanoparticles were determined. Figure 1A As shown in the figure, the particle size of the nano drug delivery system is 298±19nm, the dispersion coefficient PDI is 0.082±0.015, and the particle size distribution range is narrow (PDI<0.2), that is, a uniformly dispersed nano drug delivery system is obtained, which can meet the requirements of tumor targeted delivery. The zeta potential of the nanoparticles is -37.9±3.6mV, and it is negatively charged. It is calculated that the drug loading of camptothecin in the nano drug delivery system is 12.17%, and the drug loading of iron is 10.05%. Figure 1B Transmission electron microscopy images and Figure 1C The results of scanning electron microscopy showed that the nanoparticles were spherical with regular shape and good uniformity. Figure 1D The results of field emission transmission electron microscopy showed that the nanoparticles contained C, N, O and Fe elements. The particle size and dispersion coefficient of the nanoparticles were measured on the first, 14th and 28th days, and the changes were small, indicating that the nanoparticles had good storage stability.

[0029] Example 2

[0030] In vitro combined anti-drug-resistant tumor effect of CPT / Fe@PDA-FA10

[0031] 4T1-Luc / CPT resistant cells were constructed by low-dose continuous induction. The in vitro antitumor activity of CPT, CPT@PDA-FA10, CPT / Fe@PDA, CPT / Fe@PDA-FA10 and CPT / Fe@PDA-FA10+Laser was evaluated on 4T1-Luc / CPT resistant cells using MTT assay. 4T1-Luc / CPT resistant cells in logarithmic growth phase were digested with 0.25% EDTA-trypsin solution and then incubated at 20×10 4The cells were inoculated in a 96-well plate (200 μL per well) at a density of 100 μg / mL. After incubation for 24 h in an incubator at 37°C with 5.0% carbon dioxide, the old culture medium was discarded and nanoparticles with different CPT concentrations of 0, 320, 640, 1280, 2560, and 5120 nmol / L were added. Free drug camptothecin (CPT) was used as a positive control. The drug solution was diluted stepwise with RPMI 1640, and the volume of each well was 200 μL. Half an hour after administration, the laser irradiation group was given 808 nm laser (1.2 W / cm 2 , 5min) irradiation, and then continue incubation in the incubator for 48h. At the end of incubation, 20μL of MTT solution (5mg / mL, freshly prepared in PBS) was added to each well. After incubation for 4h, the old culture medium was discarded, 150μL of DMSO was added to each well, and the absorbance value of each well was measured at a wavelength of 490nm using an enzyme reader, and the cell survival rate was calculated.

[0032] Experimental results: The study showed that ( Figure 2 A), CPT has little effect on drug-resistant cells, IC 50 As high as 21483nmol / L. CPT / Fe@PDA-FA10 nanoparticles have good killing effect on drug-resistant cells. Its IC 50 The CPT / Fe@PDA-FA10 nanoparticles combined with laser showed stronger anti-drug resistant cell activity, and its IC 50 It is 603nmol / L, which confirms that the combination of nanoparticles and lasers has excellent tumor treatment effect and can treat drug-resistant cells through chemotherapy / photothermal combination therapy.

[0033] Example 3

[0034] Verification of the ferroptosis effect of CPT / Fe@PDA-FA10 in vitro

[0035] The essence of ferroptosis is the Fenton reaction, which is caused by Fe 2+ The reaction with hydrogen peroxide generates toxic hydroxyl radicals. The MTT experiment was used to observe the changes in the anti-tumor effects of CPT@PDA-FA10 and CPT / Fe@PDA-FA10 after the addition of ferroptosis inhibitors Fer-1 and DFO, confirming the ferroptosis anti-tumor effect of CPT / Fe@PDA-FA10. The 4T1-Luc / CPT drug-resistant cells in the logarithmic growth phase were digested with 0.25% EDTA-trypsin solution and then 20×10 4The cells were inoculated in a 96-well plate (200 μL per well) at a cell density of 100 μg / mL. After incubation for 24 h at 37 °C in an incubator containing 5.0% carbon dioxide, the old culture medium was discarded, and CPT@PDA-FA10 and CPT / Fe@PDA-FA10 containing 5120 nmol / L of CPT were added. The concentrations of Fer-1 and DFO were 10 μmol / L and 100 μmol / L, respectively. The dosing volume per well was 200 μL. Incubate in an incubator for 48 h. At the end of the incubation, 20 μL of MTT solution (5 mg / mL, freshly prepared in PBS) was added to each well. After incubation for 4 h, the old culture medium was discarded, 150 μL of DMSO was added to each well, and the absorbance value of each well was measured at a wavelength of 490 nm using an enzyme reader, and the cell survival rate was calculated.

[0036] Experimental results: After adding ferroptosis inhibitors Fer-1 and DFO, the anti-tumor effect of CPT@PDA-FA10 did not decrease significantly, while the anti-tumor effect of CPT / Fe@PDA-FA10 decreased significantly ( Figure 2 B), indicating that CPT / Fe@PDA-FA10 exhibits ferroptosis anti-tumor effects.

[0037] Example 4

[0038] In vitro photothermal effect investigation of CPT / Fe@PDA-FA10

[0039] Using 808nm laser (1.2W / cm 2 , 300s) irradiated CPT / Fe@PDA-FA10 nanoparticle aqueous solutions with different concentrations (250, 500, 1000, 2000, 4000 μg / mL, 1.0 mL). A near-infrared camera (FLIR E40) recorded the temperature and infrared images of the solution every 30 seconds.

[0040] Experimental results: CPT / Fe@PDA-FA10 nanoparticles of different concentrations were exposed to 808nm laser at 1.2W / cm 2 Irradiation, temperature rise curve and infrared thermal imaging Figure 2 C and Figure 2 As shown in D, the surface temperature of the nanoparticles increases with the irradiation time. The experimental results show that the nanoparticles have good concentration and irradiation time-dependent photothermal conversion performance.

[0041] Example 5

[0042] Study on the active tumor targeting effect of CPT / Fe@PDA-FA10

[0043] The competitive growth inhibition of CPT / Fe@PDA and CPT / Fe@PDA-FA10 nanoparticles after the addition of folic acid was studied by MTT assay. 4T1-Luc / CPT drug-resistant cells in the logarithmic growth phase were digested with 0.25% EDTA-trypsin solution and then cultured at 20×10 4 The cells were inoculated in a 96-well plate (200 μL per well) at a cell density of 100 μg / mL. After incubation in an incubator at 37°C with 5.0% carbon dioxide for 24 hours, the old culture medium was discarded, and a serum-free culture medium with a concentration of 400 μmol / L FA was added. After incubation in the incubator for 1 hour, the old culture medium was discarded, and CPT / Fe@PDA and CPT / Fe@PDA-FA10 nanoparticles with different CPT concentrations of 0, 320, 640, 1280, 2560, and 5120 nmol / L were added. The drug solution was diluted stepwise with RPMI 1640, and the volume of each well was 200 μL. Then the incubation continued in the incubator for 48 hours. At the end of incubation, 20 μL of MTT solution (5 mg / mL, freshly prepared in PBS) was added to each well. After incubation for 4 h, the old culture medium was discarded, 150 μL of DMSO was added to each well, the absorbance value of each well was measured at a wavelength of 490 nm using an enzyme reader, and the cell survival rate was calculated.

[0044] CLSM was used to investigate the competitive uptake of CPT / Fe@PDA and CPT / Fe@PDA-FA10 by cells after folic acid pretreatment. A sterilized 12 mm cell slide was added to each well of a 24-well cell culture plate. 4T1-Luc / CPT resistant cells were plated at a rate of 1×10 5 The cells were inoculated in 24-well plates at a density of 1 mL / well and incubated in a 37°C incubator for 24 h. The culture medium was removed, and 1 mL of RPMI 1640 solution containing 400 μmol / L FA was added to the folic acid group and 1 mL of RPMI 1640 solution was added to the non-folic acid group, and the cells were incubated for 1 h. The culture medium was removed, and 1 mL of CPT / Fe@PDA or CPT / Fe@PDA-FA10 solution (prepared in RPMI 1640) containing 100 μmol / L CPT was added, respectively, and the cells were incubated for 4 h. After the incubation, the culture medium was removed, 1 mL of PBS was added to each well, and the cells were washed 3 times. 1 mL of 70% ice ethanol was added to each well, and the cells were fixed at -20°C for 20 min. After the fixation, 1 mL of PBS was added to each well, and the cells were washed 3 times. 1 mL of 10 μg / mL PI / PBS staining solution was added, and the cells were stained for 30 min at room temperature away from light. 1 mL of PBS was added to each well, and the cells were washed three times. The cell slides were mounted on glass slides with glycerol, and the samples were observed using a laser confocal microscope. The excitation wavelength of CPT was 405 nm, and the excitation wavelength of PI was 543 nm.

[0045] Experimental results: After adding free FA to saturate the folate receptors highly expressed on the surface of 4T1-Luc / CPT resistant tumor cells, FA could not significantly inhibit the killing effect of CPT / Fe@PDA nanoparticles on 4T1-Luc / CPT resistant cells without folate target head ( Figure 3 A). However, FA can significantly inhibit the killing effect of CPT / Fe@PDA-FA10 nanoparticles on 4T1-Luc / CPT resistant cells ( Figure 3 B). After adding free FA to competitively inhibit the folate receptors on the surface of tumor cells, FA can significantly inhibit the entry of CPT / Fe@PDA-FA10 nanoparticles into cells, but has no effect on the entry of CPT / Fe@PDA nanoparticles without folate target heads ( Figure 3 C). It can be seen that folic acid competitively inhibits the uptake of CPT / Fe@PDA-FA10 nanoparticles, indicating that CPT / Fe@PDA-FA10 nanoparticles have a targeting effect mediated by folate receptors on the surface of tumor cells and can present good tumor active targeting properties.

[0046] Example 6

[0047] In vivo antitumor activity and toxicity evaluation of CPT / Fe@PDA-FA10

[0048] BALB / c female nude mice aged 6 to 8 weeks were selected as the experimental model. 100 μL 4T1-Luc / CPT breast cancer resistant cells (density 2×10 6 The tumor volume and mouse weight were measured every other day. When the tumor volume reached 75 mm 3 At about 14:00 p.m., the mice were divided into 6 groups after taking in vivo images, namely, saline group, free drug CPT group, CPT@PDA-FA10 group without iron, CPT / Fe@PDA group without target head, CPT / Fe@PDA-FA10 group and PT / Fe@PDA-FA10+Laser group. Camptothecin was calculated at 3 mg / kg and injected into the tail vein three times on days 0, 3 and 6. After 24 hours, laser irradiation (1.2 W / cm 2 , 5min). The tumor volume and body weight of nude mice were measured every 2 days. The length and width of mouse tumors were measured with a digital vernier caliper, and the tumor volume was calculated as V = (a × b 2) / 2, a is the longest diameter of the tumor, and b is the shortest diameter of the tumor. On the 14th day after administration, the experiment was terminated, and the serum of each group of mice was collected to detect the activity of ALT and AST in the serum, as well as the content of BUN and CREA, to evaluate the hepato-renal toxicity to mice with in situ drug-resistant breast cancer. Then, the nude mice in each group were killed, and the hearts, livers, spleens, lungs, and kidneys of the mice were obtained for H&E staining.

[0049] Experimental results: By comparison, the CPT / Fe@PDA-FA10+Laser group showed good apoptosis, ferroptosis and photothermal combined therapeutic effects, and showed excellent anti-tumor activity against CPT-resistant in situ breast cancer ( Figure 4 A). The changes in body weight of nude mice in each group during treatment showed that except for the free drug CPT group, the changes in body weight of other groups were small and there was no significant difference. The body weight of the free drug CPT group decreased by 80% ( Figure 4 B). The results of H&E staining of various organ tissues of mice bearing in situ drug-resistant breast cancer showed that no obvious pathological changes were observed in the heart, liver, spleen, lung, and kidney tissues of mice in all treatment groups ( Figure 4 C). ALT in the serum of nude mice in each group on the 14th day after administration ( Figure 4 D) and AST( Figure 4 E) activity, and BUN ( Figure 4 F) and CREA content ( Figure 4 G) are all within the normal range, indicating that CPT / Fe@PDA-FA10 treatment will not cause damage to the liver and kidney functions of model mice.

[0050] The nano drug delivery system CPT / Fe@PDA-FA10 constructed by the present invention has a small particle size, uniform distribution and good stability. CPT / Fe@PDA-FA10 has good photothermal conversion performance and exhibits significant ferroptosis activity. It can show a significant killing effect on drug-resistant breast cancer cells 4T1-Luc / CPT through apoptosis / ferroptosis / photothermal triple therapy. Through the targeting effect mediated by folic acid, CPT / Fe@PDA-FA10 can accumulate at the tumor site and be actively taken up by 4T1-Luc / CPT drug-resistant cells, exerting apoptosis / ferroptosis / photothermal triple combined therapeutic effects. This topic provides new ideas for the clinical treatment and preparation research of drug-resistant breast cancer, and provides experimental support for the establishment of a chemotherapy / ferroptosis / photothermal combined treatment system based on nanotechnology.

Claims

1. A method for preparing a triple combined tumor active targeting nano drug delivery system, characterized in that: Using folic acid-modified dopamine and dopamine as materials, through oxidative self-polymerization reaction, camptothecin and iron were loaded at the same time to prepare a polydopamine tumor active targeted nanodrug delivery system for chemotherapy / ferroptosis / photothermal triple synergistic treatment.

2. The method for preparing the triple combined tumor active targeting nano drug delivery system according to claim 1, characterized in that: The specific steps include: Step (1) Under light-proof conditions, weigh a certain amount of folic acid and dissolve it in 20 mL of DMSO, add DCC and NHS in a corresponding molar ratio, and stir in a 50°C oil bath for 6 h; weigh dopamine hydrochloride in a corresponding molar ratio and add it to the above reaction solution, stir at room temperature for 5 h, and then stop the reaction; after standing, filter out the precipitate, pour the filtrate into 80 mL of acetone / ether mixture, filter the precipitate, wash the filter cake with 40 mL of DCM, 40 mL of acetone, and 40 mL of ether, respectively, and vacuum dry to obtain folic acid-modified dopamine (DA-FA); Step (2) Weigh a certain amount of dopamine hydrochloride and add it to a 250 mL flask with a magnetic stirrer; weigh a corresponding molar ratio of folic acid-modified dopamine (DA-FA) and dissolve it in 400 μL of DMSO, vortex and sonicate it to completely dissolve it, and then add it to the above 250 mL flask; weigh a corresponding ratio of FeCl3·6H2O, add 20 mL of deionized water, sonicate it to completely dissolve it, pour it into the above 250 mL flask, continue to add deionized water to 130 mL, and stir for 1 h; quickly pour an appropriate amount of tris aqueous solution into the above solution, continue to stir for 2 h, centrifuge at 15000 g for 10 min, wash it with 100 mL of deionized water 3 times, and then dissolve the precipitate in 50 mL of deionized water; Step (3) Weigh an appropriate amount of camptothecin and dissolve it in 500 μL of DMSO. After completely dissolving it by vortexing and ultrasound, slowly drip it into 50 mL of the precipitate solution obtained in step (2), stir for 2 h, centrifuge at 15000 g for 10 min, wash once with 50 mL of deionized water to obtain a precipitate, and then dilute it to 25 mL with deionized water to obtain CPT / Fe@PDA-FA10 nanoparticles.

3. The method for preparing the triple combined tumor active targeting nano drug delivery system according to claim 2, characterized in that: In step (1), the volume ratio of acetone to ether is 1:

3.

4. The method for preparing the triple combined tumor active targeting nano drug delivery system according to claim 2, characterized in that: In step (1), the molar ratio of folic acid to dopamine is 1:2 to 2:1, and the molar ratio of folic acid, DCC and NHS is 1:0.5:0.5 to 1:5:

5.

5. The method for preparing the triple combined tumor active targeting nano drug delivery system according to claim 2, characterized in that: In step (2), the molar ratio of folic acid-modified dopamine to dopamine is 1:50 to 2:1, the mass ratio of dopamine to ferric chloride is 200:1 to 1:5, and the final concentration of the tris aqueous solution is 0.01% to 15%.

6. The method for preparing the triple combined tumor active targeting nano drug delivery system according to claim 2, characterized in that: In step (3), the feed mass of camptothecin is 1.0 mg to 15 mg.

7. Use of the triple combined tumor active targeting nano drug delivery system prepared according to any one of claims 1 to 6 in the preparation of drugs for treating drug-resistant breast cancer.

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