Polydopamine-loaded resveratrol sensitization doxorubicin treatment nano particles and preparation and application of polydopamine-loaded resveratrol sensitization doxorubicin treatment nano particles

Abstract

The invention provides a nanoparticle for treatment of polydopamine loaded resveratrol sensibilization adriamycin and a preparation method of the nanoparticle for treatment of polydopamine loaded resveratrol sensibilization adriamycin. The nanoparticle takes polydopamine as an inner core, adriamycin and resveratrol are loaded together, and the targeting property of the nanoparticle is enhanced through folic acid modification. The resveratrol remarkably enhances the cytotoxicity of the adriamycin by down-regulating HSP27, and generates a synergistic effect with the adriamycin, so that the sensitivity of breast cancer cells to the adriamycin is effectively improved, and the treatment effect is enhanced. The nanoparticles provide a new strategy for breast cancer treatment, especially for a treatment path of heat shock protein HSP27 regulation, and show a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomedicine, and in particular to a polydopamine-loaded resveratrol-sensitized doxorubicin-treated nanoparticle, and a preparation method and application thereof. Background Art

[0002] Doxorubicin is an anti-tumor drug that can inhibit the synthesis of RNA and DNA and has significant effects on a variety of tumors, including breast cancer, lung cancer, malignant lymphoma, etc. In the clinical treatment of malignant tumors, especially breast cancer, although the chemotherapy drug doxorubicin (DOX) is widely used, its efficacy is often greatly reduced due to the drug resistance of tumor cells.

[0003] Heat shock protein (HSP) plays an important role in the occurrence and development of tumors. Its family members not only promote the proliferation of tumor cells, but also inhibit their death pathways. As a key stress response protein, HSP27 is highly abundantly expressed in a variety of tumor cells, inhibiting cell apoptosis through complex mechanisms, thereby strengthening the resistance barrier of tumor cells to chemotherapeutic drugs. In the case of breast cancer, high-level expression of HSP27 not only promotes the invasion and metastasis of cancer cells, but also leads to enhanced resistance to a variety of chemotherapeutic drugs including doxorubicin. Therefore, the development of new strategies that can effectively regulate HSP27 expression and thereby enhance the sensitivity of breast cancer cells to chemotherapeutic drugs is of great significance for improving the treatment of breast cancer. Summary of the invention

[0004] Based on the above description, the object of the present invention is to provide a polydopamine-loaded resveratrol nanoparticle for enhancing the sensitization of doxorubicin treatment.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned polydopamine-loaded resveratrol nanoparticles for enhancing the sensitization to adriamycin treatment.

[0006] The present invention is achieved through the following technical solutions:

[0007] (1) Preparation of polydopamine nanoparticles

[0008] 10 mL of anhydrous ethanol and 30 mL of deionized water were mixed by magnetic stirring at room temperature, and then 0.5 mL of ammonia water was added and stirred for 30 minutes. 100 mg of dopamine hydrochloride was dissolved in 2 mL of deionized water to obtain a dopamine hydrochloride solution (50 mg / mL), which was slowly added to the above mixed reaction solution. It can be observed with the naked eye that the reaction solution changes from light brown to brown-black. Magnetic stirring was continued at room temperature for 12 hours. After the chemical reaction was completed, the aqueous solution was centrifuged for 15 minutes (14000 r / min). The resulting precipitate was polydopamine nanoparticles, which were washed with deionized water 2 to 3 times.

[0009] (2) Drug loading

[0010] PDA (5 mg) was placed in 10×Tris buffer (0.5 mL), and then doxorubicin (DOX) aqueous solution (1 mg / mL) and resveratrol (Res) aqueous solution (1 mg / mL) were added. Equal volumes of DOX aqueous solution and Res aqueous solution were added to the above mixture, that is, 1 mL each. The reactants were placed on a shaker and cultured for 24 hours at room temperature to avoid light. After the reaction was completed, centrifuged for 15 minutes (14000 r / min), and the resulting precipitate was DOX@Res@PDA NPs nanoparticles.

[0011] (3) Folic acid modification on the surface of DOX@Res@PDA NPs

[0012] 1 mL of 50 μg / mL folic acid (FA) was added to 1 mL of 1 mg / mL EDC / NHS solution, and the mixture was reacted at room temperature for 2 h. The pH of the reaction was then adjusted to 7.4, and 1 mg of DOX@Res@PDA NPs was added. The mixture was reacted for 8 h to prepare FA@DOX@Res@PDA NPs.

[0013] Beneficial effects of the present invention:

[0014] The nanoparticles of the present invention use PDA as the core of the nanomaterial, then co-encapsulate DOX and Res in the PDA nanoparticles, and use FA to modify the surface of the nanoparticles to enhance the targeting of the nanoparticles. The nanoparticles prepared by the present invention co-load DOX and Res, wherein Res can downregulate HSP27, which not only enhances the cytotoxicity of DOX, but also produces a synergistic effect with DOX, acting together on breast cancer cells, and significantly enhancing the sensitivity of breast cancer cells to DOX. Specifically, the FA@DOX@Res@PDA NPs nanoparticles prepared by the present invention show a stronger ability to kill breast cancer cells and inhibit their growth at the same drug concentration. FA@DOX@Res@PDA NPs can enhance the sensitivity of drug-resistant tumor cells, making drug-resistant tumor cells have a lower survival rate, and provide a new idea for the treatment strategy of breast cancer based on the regulation of heat shock proteins (especially HSP27). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 TEM characterization image of FA@DOX@Res@PDA NPs nanoparticles, scale bar: 200 nm.

[0016] Figure 2 Cell viability experimental results of FA@DOX@Res@PDA NPs nanoparticles.

[0017] Figure 3 This is the expression diagram of apoptosis-related proteins in MCF-7 cells treated with FA@DOX@Res@PDA NPs nanoparticles, where ① is the PBS treatment group; ② is the DOX treatment group; ③ is the Res treatment group; ④ is the DOX@PDA NPs treatment group; ⑤ is the DOX@Res@PDANPs treatment group; ⑥ is the FA@DOX@Res@PDA NPs treatment group.

[0018] Figure 4 The results of FA@DOX@Res@PDA NPs nanoparticles on the expression level of heat shock protein gene in MCF-7 cells.

[0019] Figure 5 This is a graph showing the effect of FA@DOX@Res@PDA NPs nanoparticles on the expression of heat shock proteins in MCF-7 cells, where ① is the PBS treatment group; ② is the DOX treatment group; ③ is the Res treatment group; and ④ is the FA@DOX@Res@PDA NPs treatment group.

[0020] Figure 6 These are the experimental results of drug resistance of FA@DOX@Res@PDA NPs nanoparticles.

[0021] Figure 7 Tumor volume change curves treated with normal saline, DOX, Res, DOX@Res@PDA NPs, and FA@DOX@Res@PDA NPs. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiments.

[0023] Example 1: A method for preparing FA@DOX@Res@PDA NPs nanoparticles, comprising the following steps:

[0024] (1) Preparation of polydopamine nanoparticles

[0025] 10 mL of anhydrous ethanol and 30 mL of deionized water were mixed by magnetic stirring at room temperature, and then 0.5 mL of ammonia water was added and stirred for 30 minutes. 100 mg of dopamine hydrochloride was dissolved in 2 mL of deionized water to obtain a dopamine hydrochloride solution (50 mg / mL), which was slowly added to the above mixed reaction solution. It can be observed with the naked eye that the reaction solution changes from light brown to brown-black. Magnetic stirring was continued at room temperature for 12 hours. After the chemical reaction was completed, the aqueous solution was centrifuged for 15 minutes (14000 r / min). The resulting precipitate was polydopamine nanoparticles, which were washed with deionized water 2 to 3 times.

[0026] (2) Drug loading

[0027] 5 mg of PDA was placed in 0.5 mL of 10×Tris buffer, and then doxorubicin (DOX) aqueous solution (1 mg / mL) and resveratrol (Res) aqueous solution (1 mg / mL) were added, and 1 mL of DOX aqueous solution and Res aqueous solution were added to the above mixture, respectively. The reactants were placed on a shaker and cultured for 24 hours at room temperature, avoiding light. After the reaction was completed, centrifuged for 15 minutes (14000 r / min), and the resulting precipitate was DOX@Res@PDA NPs nanoparticles.

[0028] (3) Folic acid modification on the surface of DOX@Res@PDA NPs

[0029] 1 mL of 50 μg / mL folic acid (FA) was added to 1 mL of 1 mg / mL EDC / NHS solution and reacted at room temperature for 2 h. The reaction pH was then adjusted to 7.4 and 1 mg of DOX@Res@PDA NPs nanoparticles were added. The reaction was continued for 8 h to prepare FA@DOX@Res@PDA NPs nanoparticles.

[0030] Example 2: Characterization of FA@DOX@Res@PDA NPs

[0031] Take an appropriate amount of the prepared sample to be tested and dissolve it in deionized water. After ultradispersion, take 2 μL and drop it on the carbon-coated copper mesh. Naturally adsorb and dry it at room temperature. Observe the morphology of the nanoparticles under a transmission electron microscope at 200 KV.

[0032] The TEM image of FA@DOX@Res@PDA NPs nanoparticles prepared according to Example 1 is as follows: Figure 1 As shown, the particles are well dispersed and have a particle size of about 70-120 nm.

[0033] Example 3: In vitro antitumor effect of FA@DOX@Res@PDA NPs nanoparticles

[0034] Folate receptor-positive breast cancer cells MCF-7 and normal breast cells MCF-10A were resuspended in culture medium and the cell density was adjusted to 1×10 4 / mL, inoculate 100 μL into a 96-well plate, culture at 37 °C for 24 h, discard the original culture medium in the wells, and divide MCF-7 cells and MCF-10A cells into 5 groups: Control, DOX, Res, DOX@Res@PDA NPs and FA@DOX@Res@PDA NPs groups (2 μg / mL), add 100 μL of the culture medium containing the above groups, continue incubation for 24 h, add 10 μL of CCK8 solution to the wells, incubate at 37 °C in the dark for 1.5 h, measure the absorbance at 450 nm, and calculate the cell viability.

[0035] We used the CCK8 method to measure the viability of MCF-7 / MCF-10A cells and found that there were significant differences in the effects of different treatment groups on cell viability. Figure 2 As shown in the figure, the cells in the Control group maintained normal viability levels; the DOX-treated group showed significant cytotoxicity, resulting in a significant decrease in the viability of MCF-7 / MCF-10A cells; the Res-treated group showed a certain killing ability on MCF-7, but had little effect on the viability of normal MCF-10A cells; in the DOX@Res@PDA NPs group, due to the combination of the dual effects of doxorubicin and resveratrol, as well as the carrier effect of PDA nanoparticles, the combination showed a certain inhibitory effect on MCF-7 cells, and had no obvious damage to normal MCF-10A cells. On this basis, the FA@DOX@Res@PDANPs group further introduced folic acid-targeted modification, allowing the nanoparticles to specifically target folate receptor-positive tumor cells. This improvement significantly enhanced the inhibitory effect of the nanoparticles on MCF-7 cells, and the cell viability decreased more significantly; while the effect on normal breast cells was negligible.

[0036] Example 4: Analysis of protein expression in MCF-7 cells sensitive to doxorubicin treatment induced by FA@DOX@Res@PDA NPs

[0037] MCF-7 cells (1×10 5 / well) were inoculated into six-well plates and pre-cultured for 24 hours and then divided into the following 6 groups: ①PBS, ②DOX, ③Res, ④DOX@PDA NPs, ⑤DOX@Res@PDA NPs and ⑥FA@DOX@Res@PDA NPs. The original culture medium was replaced with the culture medium (10nM) in the above group and incubated for 24 hours. After the cells were cultured, they were washed twice with PBS, lysed with RIPA lysis buffer containing PMSF, and total protein was extracted. Equal amounts of protein samples were taken for SDS-PAGE electrophoresis and transferred to PVDF membranes. The PVDF membrane after successful transfer was transferred to 1× protein-free blocking buffer, blocked at room temperature for 30 minutes, washed 3 times with 1×TBST, incubated with primary antibody (1:1000) at 4°C overnight, washed with 1×TBST to remove unbound primary antibody, incubated with secondary antibody for 2 hours (1:5000) at room temperature, washed again and developed with ECL.

[0038] Bax protein is a pro-apoptosis related protein, and Bcl2 protein is an anti-apoptosis related protein. Effects of PBS, DOX, Res, DOX@PDA NPs, DOX@Res@PDA NPs and FA@DOX@Res@PDA NPs on apoptosis related proteins in MCF-7 cells Figure 3 As shown in the figure, compared with the expression of apoptosis-related proteins in the blank control group, DOX increased the expression of Bax protein in MCF-7 cells and inhibited the expression of Bcl2 protein in MCF-7 cells, indicating that DOX effectively induced apoptosis in MCF-7 cells by regulating the expression of Bax and Bcl2 proteins. Similarly, Res increased the expression of Bax protein and decreased the expression of Bcl2 protein. Compared with pure DOX, DOX@PDA NPs showed similar expression regulation of apoptosis-related proteins, with increased expression of Bax protein and decreased expression of Bcl2 protein, indicating that DOX@PDA NPs may more effectively induce apoptosis in MCF-7 cells by improving the intracellular delivery efficiency of DOX. Compared with the use of DOX or Res alone, the combination of DOX@Res@PDA NPs significantly enhanced the expression promotion effect of Bax protein and the expression inhibition effect of Bcl2 protein, further enhancing the expression regulation of apoptosis-related proteins. Finally, after treatment with FA@DOX@Res@PDA NPs, Bax showed the highest degree of promotion and Bcl2 showed the highest degree of inhibition, which may be due to the introduction of FA enhancing the specific uptake of nanoparticles into tumor cells and the synergistic effect of Res increasing the sensitivity of tumors to DOX treatment.

[0039] Example 5: Effect of FA@DOX@Res@PDA NPs on the expression of heat shock proteins in MCF-7 cells

[0040] (1) Effect of FA@DOX@Res@PDA NPs on the expression of heat shock protein genes in MCF-7 cells

[0041] MCF-7 cells (1×10 5 / well) were inoculated into six-well plates and pre-cultured for 24 hours and then divided into the following 4 groups: ①PBS, ②DOX, ③Res, ④FA@DOX@Res@PDA NPs. The original culture medium was replaced with the culture medium (10nM) in the above group and incubated for 24 hours. After the cells were cultured, they were washed twice with PBS, and the intracellular RNA was extracted using the SteadyPure universal RNA extraction kit, and reverse transcribed into cDNA using the Evo M-MLV reverse transcription kit. qRT-PCR experiments were performed in the Fast7500 fluorescent quantitative PCR instrument, and GAPDH was used as the internal reference gene. 2 -△△CT The relative expression levels among samples were analyzed.

[0042] like Figure 4 As shown, in the analysis of gene expression levels, compared with untreated control cells, treatment with PBS or DOX alone did not significantly affect the mRNA expression level of HSP27. However, Res treatment significantly reduced the mRNA expression of HSP27 in breast cancer MCF-7 cells. It is worth noting that when the FA@DOX@Res@PDANPs nanoparticles prepared by the present invention were used for treatment, a significant decrease in the expression level of HSP27 mRNA was also observed. This shows that the nanoparticles can efficiently and targetedly deliver drugs to MCF-7 cells, and by effectively inhibiting the expression of the HSP27 gene, the sensitivity of cells to DOX treatment is significantly enhanced, thereby verifying its enhanced therapeutic effect at the gene level.

[0043] (2) Effect of FA@DOX@Res@PDA NPs on the expression of heat shock protein levels in MCF-7 cells

[0044] MCF-7 cells (1×10 5 / well) were inoculated into six-well plates and pre-cultured for 24 hours and then divided into the following 4 groups: ①PBS, ②DOX, ③Res, ④FA@DOX@Res@PDA NPs. The original culture medium was replaced with the culture medium (10nM) in the above group and incubated for 24 hours. After the cells were cultured, they were washed twice with PBS, lysed with RIPA lysis buffer containing PMSF, and total protein was extracted. Equal amounts of protein samples were taken for SDS-PAGE electrophoresis and transferred to PVDF membranes. The PVDF membrane after successful transfer was transferred to 1× protein-free blocking solution, blocked at room temperature for 30 minutes, washed 3 times with 1×TBST, incubated with primary antibody (1:1000) at 4°C overnight, washed with 1×TBST to remove unbound primary antibody, incubated with secondary antibody for 2 hours (1:5000) at room temperature, washed again and developed with ECL.

[0045] like Figure 5 As shown, compared with the untreated control cells, the use of PBS or DOX alone did not significantly change the expression of HSP27 protein, while Res treatment could effectively downregulate the expression of HSP27 protein in MCF-7 cells. At the same time, after treatment with the FA@DOX@Res@PDA NPs nanoparticles prepared by the present invention, the expression of HSP27 protein also decreased, which is consistent with the results of RT-qPCR, indicating that the nanoparticles not only successfully achieved targeted delivery to MCF-7 cells, but also significantly enhanced the responsiveness of cells to DOX treatment by effectively downregulating the expression of HSP27 protein.

[0046] Example 6: Experimental results of FA@DOX@Res@PDA NPs nanoparticles sensitivity to drug-resistant cells

[0047] The MCF-7 human breast cancer cell line was selected and the cytotoxicity experiment was carried out using the CCK8 method. Unlike Example 3, the same concentration (2 μg / mL) of free DOX, Res, DOX-Res combined (DOX+Res) and FA@DOX@Res@PDA NPs nanoparticles were incubated with MCF-7 cells for 48 hours. After the incubation, 10 μL of CCK8 solution was added, and its absorbance was measured at 450 nm. The half inhibitory concentration (IC50) of the drug incubated for 48 hours was obtained after fitting the cytotoxicity curve.

[0048] The results are as follows Figure 6As shown, the half-inhibitory concentration of the DOX-Res combination group is basically lower than the half-inhibitory concentration of DOX and Res respectively. After incubation for 48 hours, the cytotoxicity curve shows that the cytotoxicity curves of FA@DOX@Res@PDA NPs nanoparticles are lower than those of free DOX, free Res, and FA@DOX@Res@PDA NPs nanoparticles. The half-inhibitory concentration of the combination group can be further reduced. Therefore, the cytotoxicity test shows that FA@DOX@Res@PDA NPs nanoparticles have a stronger ability to kill breast cancer cells and inhibit the growth of breast cancer cells. At the same drug concentration, compared with free doxorubicin or doxorubicin-resveratrol combination, the nanoparticles of the present invention make drug-resistant tumor cells have a lower survival rate, indicating that FA@DOX@Res@PDA NPs can promote doxorubicin to inhibit the proliferation of drug-resistant cells and increase the sensitivity of drug-resistant cells to FA@DOX@Res@PDA NPs.

[0049] Example 7: In vivo antitumor effect of FA@DOX@Res@PDA NPs nanoparticles

[0050] The MCF-7 cells in the logarithmic growth phase were digested with trypsin and centrifuged, the supernatant was discarded, the cells were resuspended in serum-free medium, the cell suspension was mixed with matrix gel at a ratio of 1:1 (v:v), and 0.2 mL of tumor cells were inoculated subcutaneously in the front back of BALB / c nude mice (female, 4 weeks old). 3 At about 14 days, the mice were randomly divided into 5 groups (n=5): saline, DOX, Res, DOX@Res@PDA NPs, and FA@DOX@Res@PDA NPs (30 mg / kg, calculated as the active ingredient of DOX or Res). The drugs were injected into the tail vein every 2 days, and the tumor size was recorded. The tumor volume formula was: length × width 2 / 2, draw the tumor growth curve.

[0051] like Figure 7As shown in the figure, the tumor volume in the saline group continued to grow, and no obvious antitumor effect was observed. Compared with the saline group, DOX inhibited the growth of tumors to a certain extent, and the growth rate of tumor volume slowed down. When Res was used alone, its antitumor effect was poorer than that of DOX. Although the tumor volume showed a trend of reduction, it was not as obvious as that of the DOX group. Compared with the use of DOX or Res alone, the DOX@Res@PDA NPs group showed a stronger antitumor effect, which may be related to the fact that PDA NPs as a carrier improved the cellular uptake efficiency of the drug and the synergistic effect of DOX and Res. Due to the integrated folic acid targeting function, FA@DOX@Res@PDA NPs nanoparticles showed the best tumor inhibition effect. The synergistic effect of Res and DOX, as well as the improved drug uptake efficiency by folic acid targeting, jointly enhanced the therapeutic sensitivity of tumor cells to DOX, greatly improving the antitumor effect.

[0052] The above is an exemplary description of the present invention, which is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A polydopamine-loaded resveratrol nanoparticle for sensitizing adriamycin treatment, characterized in that: The nanoparticles are composed of polydopamine PDA, and are loaded with doxorubicin DOX and resveratrol Res. The surface of the PDA nanoparticles is modified by folic acid FA.

2. A method for preparing polydopamine-loaded resveratrol nanoparticles for enhancing doxorubicin treatment as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Preparation of S1 polydopamine nanoparticles: Anhydrous ethanol and deionized water were mixed in a certain proportion, and magnetically stirred at room temperature until uniform; 0.5 mL of ammonia water was added to the mixture, and stirring was continued for 30 minutes; dopamine hydrochloride was dissolved in deionized water to prepare a 50 mg / mL high concentration solution, and slowly added to the above mixed reaction solution, and the color changed to brown-black; the reaction was continued under magnetic stirring at room temperature for 12 hours, and then centrifuged at 14000 r / min for 15 minutes, and the precipitate was collected and washed to obtain polydopamine nanoparticles PDA NPs; S2 Drug loading to form DOX@Res@PDA NPs nanoparticles: a certain amount of PDA NPs was dispersed in 0.5 mL Tris buffer, and then an equal ratio of doxorubicin DOX and resveratrol Res aqueous solution was added; the mixture was placed on a shaker and cultured for 24 hours under light-proof conditions to allow the drug to be fully loaded on the PDA NPs; after the reaction, the precipitate was collected and washed by centrifugation at 14000 r / min for 15 minutes to obtain DOX@Res@PDA NPs nanoparticles; S3 Folic acid modification on the surface of DOX@Res@PDA NPs nanoparticles: 50 μg / mL folic acid FA was coupled to the surface of nanoparticles using 1 mg / mL LEDC / NHS to form FA@DOX@Res@PDA NPs nanoparticles.

3. The method according to claim 2, characterized in that In step S1 of preparing polydopamine nanoparticles, the mixing volume ratio of anhydrous ethanol and deionized water is 1:

3.

4. The method according to claim 2, characterized in that: In the drug loading step S2, the mass ratio of the PDA NPs, DOX and Res is 5:1:

1.

5. The method according to claim 2, characterized in that: In the folic acid modification step S3, the pH value of the reaction solution is adjusted to 7.

4.

6. The method according to claim 2, characterized in that In the folic acid modification step S3, the reaction time is 8 hours.

7. The method according to claim 2, characterized in that The mass ratio of DOX@Res@PDA NPs, EDC / NHS and folic acid is 20:20:

1.

8. The polydopamine-loaded resveratrol nanoparticles for enhancing doxorubicin treatment according to claim 1, characterized in that: The nanoparticles downregulate HSP27 through Res, thereby enhancing the therapeutic sensitivity of breast cancer cells to DOX.

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