A nano drug crystal and its preparation method and application

Abstract

The present invention belongs to the field of biomedical nanomaterials, and discloses a nano drug crystal and its preparation method and application. The nano drug crystal components include Au / HA, Ag / HA, CPT / HANC, and can be used to inhibit conventional breast cancer cells and BCSCs. Based on the diversity and complementarity of the mechanism of action of AuNPs and AgNPs in inhibiting CSCs, the present application prepares Au‑Ag@CPT / HANC, which can integrate the characteristics of two metal particles and show unique optical and biological properties, supplemented by the anti-cancer properties of chemotherapeutic drugs, to achieve the purpose of multi-target inhibition of CSCs remnants.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical nanomaterials, and specifically relates to a bimetallic nanoparticle-modified nanomedicine crystal (Au-Ag@CPT / HANC) for resisting breast cancer stem cells (BCSCs), and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common cancers in women. Despite the high survival rate after treatment, breast cancer patients still have a high risk of tumor recurrence and metastasis. The reason is that although chemotherapy drugs can kill the vast majority of conventional tumor cells in the rapid proliferation phase in tumor tissues and reduce the volume of tumor tissue masses, it is difficult to eliminate a relatively low-content cell subpopulation called cancer stem cells (CSCs). CSCs are located at the top of the cell hierarchy and are present in very low levels in tumors, but they may be key cells in tumor evolution. They have similar characteristics to normal stem cells, such as self-renewal, multidirectional differentiation, and high expression of certain stemness-related genes. They are closely related to tumor progression, drug resistance, recurrence, and metastasis, and are the root cause of cancer recurrence and metastasis. The development of a multi-component combined treatment strategy for CSCs can inhibit the residual phenomenon of CSCs caused by multiple mechanisms at multiple targets.

[0003] In recent years, nanoparticles have been found to be an important option for the treatment of CSCs resistance. There is also evidence that photothermal therapy mediated by nanoparticles can stimulate thermal physiological responses to kill cancer cells, and as a non-invasive treatment, it causes DNA damage and protein denaturation in tumors, thereby inducing apoptosis. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a nano drug crystal and a preparation method and application thereof. The nano drug crystal has high biocompatibility and a certain photothermal effect, and can effectively inhibit the formation of tumor spheres.

[0005] The above object of the present invention is achieved by the following technical solution: A method for preparing nano drug crystals, comprising the following steps:

[0006] 1. HA solution and HAuCl 4 AgNO 3 The solutions were mixed, and the pH of the solutions was adjusted after being made up with ultrapure water. The solutions were then heated and stirred in the dark to obtain single gold nanoparticles (Au / HA) coated with hyaluronic acid (HA) and single silver nanoparticles (Ag / HA) coated with hyaluronic acid, respectively. Au / HA and Ag / HA were then collected by centrifugation and resuspended for storage.

[0007] 2. Add 400 μL of 0.01-10 mg / mL CPT solution to 0.001-1 mg / mL HA solution, stir magnetically in the dark after ultrasonic disruption, and filter through a carbonate membrane (pore size range of 10-4000 nm) to obtain CPT / HANC.

[0008] 3. Take the Au / HA and Ag / HA solutions obtained in step 1 and mix them evenly in a certain molar ratio, then make up to volume with ultrapure water, add the CPT / HANC obtained in step 2, and stir magnetically in the dark to obtain nano drug crystals modified with bimetallic nanoparticles (Au-Ag@CPT / HANC).

[0009] 4. The nano drug crystals Au-Ag@CPT / HANC obtained in step 3 were irradiated with 808 nm near-infrared laser at a power of 2 to 4 W for 0 to 2 min.

[0010] Furthermore, in step 1, the HA solution and HAuCl 4 The solution mixing molar ratio is 1:1 to 1:4, the pH is adjusted to 7.5 to 9.5, and the light-proof heating temperature is 65 to 75°C.

[0011] Furthermore, in step 1, the HA solution and AgNO 3 The solution mixing molar ratio is 1:10-1:3, the pH is adjusted to 8.5-11.0, and the light-proof heating temperature is 65-75°C.

[0012] Furthermore, in step 3, the molar ratio of Au / HA:Ag / HA:CPT / HANC is 10:10:1 to 1:4:3.

[0013] Another object of the present invention is to protect the nano drug crystals prepared by the above-mentioned nano drug crystal preparation method, wherein the particle size of Au / HA is 20±20nm~500±100nm, the particle size of Ag / HA is 20±20nm~500±100nm, the particle size of CPT / HANC is 50±20nm~3000±100nm, and the particle size of Au-Ag@CPT / HANC is 100±50nm~4000±100nm, and the Au / HA and Ag / HA are respectively dotted around the rod-shaped CPT / HANC in a spherical shape in an aggregated or dispersed manner.

[0014] Another object of the present invention is to protect the application of the above-mentioned nano drug crystals, including the application in the inhibition of conventional breast cancer cells and BCSCs.

[0015] Under 808nm near-infrared laser, the temperature increment of Au-Ag@CPT / HANC suspension can continue to increase with the increase of laser power and irradiation time. When the CPT concentration is 1-10μg / mL, it can reach 40-50℃ within 0-2min under 2-4W power conditions.

[0016] Compared with the prior art, the present invention has the following beneficial effects: Based on the diversity and complementarity of the mechanism of AuNPs and AgNPs in inhibiting CSCs, the Au-Ag@CPT / HANC prepared by the present invention can integrate the characteristics of two metal particles and exhibit unique optical and biological properties, and with the anti-cancer properties of chemotherapeutic drugs, achieve the purpose of multi-target inhibition of CSCs. At the same time, by effectively adjusting the ratio of gold and silver in the nano drug crystals, the balance point between high anti-cancer activity and low physiological toxicity can be determined; the present invention uses natural linear polysaccharide HA as a bioreductant to prepare bimetallic nanoparticles in a simple and green manner, that is, the reducing groups of HA are used to induce metal ions (Au 3+ 、Ag + ) and fix the newly formed metal nanoparticles (Au 0 、Ag 0 ). In addition, HA can be used as a targeting molecule to modify the surface of the nano drug delivery system, thereby specifically targeting breast cancer cells that express CD44 at high levels, improving the compatibility of nanomaterials, and prolonging the blood circulation cycle of drugs. The Au-Ag@CPT / HANC and Au / HA prepared in this application have high photothermal conversion efficiency and significant inhibitory effect on tumor spheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0018] Figure 1 The scanning electron microscope image (SEM) (a) and elemental analysis image (EDX) (b) of Au-Ag@CPT / HANC prepared by the present invention;

[0019] Figure 2 This is a graph showing the results of the MDA-MB-231 cytotoxicity experiment of Au-Ag@CPT / HANC prepared by the present invention;

[0020] Figure 3 This is a bar graph showing the inhibitory effect of Au-Ag@CPT / HANC prepared in the present invention on BCSCs;

[0021] Figure 4 Schematic diagram of the inhibitory effect of Au-Ag@CPT / HANC prepared in the present invention on BCSCs. DETAILED DESCRIPTION

[0022] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0023] Example 1

[0024] The preparation method of bimetallic nanoparticle-modified nano drug crystals for anti-breast cancer stem cells comprises the following specific steps:

[0025] S1. Use a microsampler to pipette 100 μL of HAuCl 4 solution and 1mL HA solution to make HA:Au in the reaction solution 3 + The ratio of the reaction mixture was 1:2, and the pH of the reaction solution was adjusted to 7.5-9.5 after the ultrapure water was used to make the volume. The Au / HA was obtained by heating and stirring in the dark. 3+ With the increase of the content, the color of the solution gradually deepened from light purple to ruby ​​red. Then, Au / HA was obtained by centrifugation, and then resuspended in ultrapure water and stored in the dark.

[0026] S2. Use a microsampler to pipette 50 μL AgNO 3 solution and 1mL HA solution to make the HA:Ag + The ratio is 1:1, and the pH of the reaction solution is adjusted to 8.5-11.0 after ultrapure water is used to make the volume. Ag / HA is obtained by heating and stirring in the dark. + With the increase of the content, the color of the solution gradually deepened from light brown to dark brown. Then, the Ag / HA was obtained by centrifugation, and then resuspended in ultrapure water and stored in the dark.

[0027] S3. Use a microsampler to transfer ultrapure water with a pH of 4, add 100 μL of HA solution, mix well, then drop 400 μL of CPT solution, ultrasonically disrupt, and stir magnetically at room temperature in the dark for 3 hours, then filter through a carbonate membrane to obtain CPT / HANC.

[0028] S4. Use a microsampler to transfer 1 mL Au / HA and 4 mL Ag / HA solutions obtained in steps S1 and S2 respectively; make the Au:Ag ratio in the reaction solution 1:2, mix well and make up to volume with ultrapure water, then add CPT / HANC obtained in step S3. Magnetic stirring at room temperature in the dark to obtain Au-Ag@CPT / HANC, filter with carbonate membrane, resuspend in ultrapure water and store in the dark. Then, the physicochemical properties of the nanoparticles provided by the present invention were analyzed. Figure 1The SEM and EDX images of Au-Ag@CPT / HANC at the optimal 1:2 volume ratio provided by the present invention are consistent with the results of dynamic light scattering (DLS) analysis, and the particle size is 100±50nm~4000±100nm and is evenly distributed.

[0029] S5. In vitro anticancer activity analysis (MTT method): The present invention selected the MDA-MB-231 breast cancer cell line to explore the in vitro anticancer activity of Au-Ag@CPT / HANC. Cells in the logarithmic growth phase were taken at 1×10 4 Cells were inoculated into 96-well cell culture plates, and after overnight adherence culture, different nanoparticle formulations (negative control, CPT / HANC, CPT / HANC+L, Au-Ag@CPT / HANC, Au-Ag@CPT / HANC+L) were added for 24 hours. The negative control was cells without any treatment. CPT / HANC+L and Au-Ag@CPT / HANC+L were treated with drugs for 24 hours and then irradiated with 808 nm laser (3.5 W / cm 2 After drug treatment, 20 μL of 5 mg / mL thiazolyl blue solution was added to each well, and the cells were incubated at 37°C for 4 h, then the supernatant was discarded. Then, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the OD value of the sample was measured at 570 nm and 650 nm using an enzyme marker to calculate the cell survival rate.

[0030] Figure 2 The cytotoxicity of different drug dosage forms to MDA-MB-231 cells. From the analysis of the figure, it can be seen that CPT / HANC has a small degree of cell damage. Even when the CPT concentration reaches 25μg / mL, nearly 70% of the cells still survive. The cell survival rate of the Au-Ag@CPT / HANC treatment group dropped to 40% compared with the control group. The most significant was the Au-Ag@CPT / HANC+L treatment group. The drug had the greatest cell killing effect on cells, with only 18% of the cells surviving. In addition, it was calculated that the nanodrug crystals modified with bimetallic nanoparticles combined with near-infrared laser irradiation had the strongest cytotoxicity, IC 50 The results showed that the combined use of the bimetallic nanoparticle-modified nano drug crystals and photothermal therapy provided by the present invention significantly improved the anticancer activity of CPT.

[0031] S6. Tumor sphere inhibition: The formation of tumor spheres is related to the proportion of CSCs in cancer cells. Therefore, the inhibitory effect of Au-Ag@CPT / HANC on CSCs can be evaluated by the number and volume of tumor spheres formed. MDA-MB-231 cells in the logarithmic growth phase were passaged according to the conventional method and cultured at 1×10 6Each well was inoculated in a 6-well cell culture plate, and the culture medium was discarded after overnight adherence culture. Different nanoparticle formulations (negative control, CPT / HANC, CPT / HANC+L, Au-Ag@CPT / HANC, Au-Ag@CPT / HA NC+L, each group containing 10μg / mL CPT) were added and incubated for 24h. CPT / HANC+L and Au-Ag@CPT / HANC+L were treated with drugs for 24h and then 808nm laser treatment (3.5W / cm 2 , 1 min) and incubated again for 3 h. The cells were then digested with 25% trypsin and counted. The treated cells were counted at 1×10 4 Each well was inoculated into an ultra-low adhesion 24-well plate, and cultured in suspension in stem cell culture medium for 10 days. The number of tumor spheres (>50 μm was defined as stem cells) was counted under an inverted microscope.

[0032] Figure 3 and Figure 4 The following are schematic diagrams showing the number of tumor spheres and tumor sphere inhibition results after cells were treated with different nanoparticles. Compared with the CPT / HANC and CPT / HANC+L treatment groups, the Au-Ag@CPT / HANC group significantly reduced the ability to form tumor spheres (~75%). After treatment, the Au-Ag@CPT / HANC+L group was even difficult to aggregate into spheres, and most of them were dispersed single cells. Therefore, nanomedicine crystals modified with bimetallic nanoparticles can treat drug-resistant tumors based on the remnants of tumor stem cells, and the effect is significant.

[0033] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing nano drug crystals, characterized in that: The following steps are involved: A. HA solution is mixed with HAuCl4 and AgNO3 solution respectively, and the pH of each solution is adjusted after the volume is fixed with ultrapure water, and then heated and stirred in the dark to obtain hyaluronic acid-coated single gold nanoparticles, namely Au / HA, and hyaluronic acid-coated single silver nanoparticles, namely Ag / HA, respectively. Then, Au / HA and Ag / HA are collected by centrifugation and resuspended for storage. The molar ratio of the HA solution to the HAuCl4 solution is 1:1-1:4, the pH is adjusted to 7.5-9.5, and the light-proof heating temperature is 65-75°C. The molar ratio of the HA solution to the AgNO3 solution is 1:10-1:3, the pH is adjusted to 8.5-11.0, and the light-proof heating temperature is 65-75°C. B. Add 400 μL of 0.01-10 mg / mL CPT solution to 0.001-1 mg / mL HA solution, stir magnetically in the dark after ultrasonic disruption, and filter through a carbonate membrane with a pore size range of 10-4000 nm to obtain CPT / HANC; C. The Au / HA and Ag / HA solutions obtained in step A were mixed evenly and then fixed to volume with ultrapure water, and the CPT / HANC obtained in step B was added, and the mixture was stirred under magnetic force in a dark environment to obtain bimetallic nanoparticle-modified nano drug crystals, namely Au-Ag@CPT / HANC, wherein the molar ratio of Au / HA:Ag / HA:CPT / HANC was 10:10:1 to 1:4:3; D. The nano drug crystal Au-Ag@CPT / HANC obtained in step C is irradiated with 808 nm near-infrared laser at a power of 2 to 4 W for 0 to 2 min.

2. The nano drug crystal prepared by the preparation method according to claim 1, characterized in that: The particle size of Au / HA is 20±20nm~500±100nm, the particle size of Ag / HA is 20±20nm~500±100nm, the particle size of CPT / HANC is 50±20nm~3000±100nm, and the particle size of Au-Ag@CPT / HANC is 100±50nm~4000±100nm. The Au / HA and Ag / HA are dotted around the rod-shaped CPT / HANC in spherical aggregates or dispersed forms.

3. Use of the nano drug crystals as claimed in claim 2 in the preparation of drugs.

4. The use of the nano drug crystals according to claim 3 in the preparation of drugs, characterized in that: Applied to the inhibition of conventional breast cancer cells and BCSCs.

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