CN111603564A

CN111603564A - High-load Y-type molecular sieve-adriamycin nano-drug and preparation method and application thereof

Info

Publication number: CN111603564A
Application number: CN202010496401.6A
Authority: CN
Inventors: 李林; 任晶; 付玉静
Original assignee: Taiyuan Normal University
Current assignee: Taiyuan Normal University
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2020-09-01
Anticipated expiration: 2040-06-03
Also published as: CN111603564B

Abstract

The invention provides a high-load Y-type molecular sieve-adriamycin nano-drug as well as a preparation method and application thereof. The preparation method of the nano-drug is to use a Y-type molecular sieve as a carrier and adsorb the adriamycin by adopting hydrogen bonds and Van der Waals force under the condition of pH9.0 to prepare the nano-drug YMS-DOX. The nano-drug interacts with human breast cancer cells (MM-231), and the result shows that YMS-DOX has the characteristic of slow-release drug and can induce tumor cell apoptosis through EPR passive targeting; through interaction with dendritic normal cells (DC), the result shows that YMS-DOX has the characteristic of reducing toxic and side effects of the medicine. The result shows that YMS-DOX can reduce the toxic and side effect of chemotherapeutic medicine on normal cells, can improve the killing power on tumor cells, and can be applied to the preparation of antitumor medicines.

Description

High-load Y-type molecular sieve-adriamycin nano-drug and preparation method and application thereof

Technical Field

The invention relates to a nano-drug, in particular to a high-load Y-type molecular sieve-adriamycin nano-drug, a preparation method thereof and application of the drug in preparing anti-breast cancer and anti-tumor drugs.

Background

Chemotherapy is currently the most common method of treating tumors. However, although chemotherapy can kill tumor cells, during the treatment process, due to the lack of specific selectivity and large toxic and side effects of the chemotherapy drugs, patients often suffer from physical fatigue, listlessness, sweating caused by deficiency, and the like, which seriously affects the life quality of the patients.

The emergence of nanotechnology provides a new field of view for overcoming cancer at an early date, and the design of a drug delivery system by utilizing the advantages of nanomaterials to load anticancer drug Doxorubicin (DOX) has become a hot trend of research, such as: a nano-drug delivery system (COPY-DOX) with amphiphilic block polymer loaded with DOX is utilized, and fullerene with good water dispersibility is used as a nano-carrier to prepare an anti-tumor prodrug system with three characteristics of active targeting, photodynamic therapy and pH response chemotherapy. Although nano-drug delivery systems have been extensively developed, their drug release profile in the tumor microenvironment is less than satisfactory and the anti-tumor effect is weaker than free DOX.

Vallet-Regi, equal to 2001, first proposed that silicon-based mesoporous materials can be used for drug sustained release, and researchers have conducted a great deal of research, such as: the influences of the surface property, the aperture, the morphology and the like of the mesoporous carrier on the drug-loading and drug-releasing performance of the mesoporous carrier are explored. In view of the characteristics of uniform pore paths, rich surface groups, high stability, no toxicity and the like of the molecular sieve, the application of the molecular sieve in crude drugs is concerned. The research results are combined: the mesoporous molecular sieve not only can be used as a drug carrier to become a preferred nano material in clinic, but also provides safety guarantee, which is important for clinical medicine. However, the research on molecular sieves is limited to the slow release of drugs so far, and the regulation factors of the load and release of the anti-tumor drugs are not deeply researched.

Disclosure of Invention

The invention aims to provide a high-load Y-type molecular sieve-adriamycin nano-drug, a preparation method and application thereof.

The invention provides a high-load Y-type molecular sieve-adriamycin nano drug which is characterized in that a Y-type molecular sieve is taken as a carrier, and the high-load Y-type molecular sieve-adriamycin nano drug is prepared by adsorbing adriamycin by hydrogen bonds and Van der Waals force in an environment with pH9.0.

The preparation method of the high-load Y-type molecular sieve-adriamycin nano-drug comprises the following steps:

(1) adding 0.5-1.0mL of ethanol solution and ultrapure water into each mg of Y-type molecular sieve in turn for ultrasonic treatment, centrifuging at 12000rpm for 2 times, adding 0.5-1.0mL of buffer solution (prepared from citric acid and disodium hydrogen phosphate) with pH of 3.0-12.0 into each mg of Y-type molecular sieve, and ultrasonically dispersing for 0.5h to form suspension;

(2) adding an adriamycin solution into the Y-type molecular sieve suspension according to the weight ratio of the Y-type molecular sieve to the adriamycin of 1-10:1, uniformly mixing, placing the mixture on an infrared heating electromagnetic stirrer for reacting at room temperature for 20 hours, after the reaction is finished, centrifuging at a high speed, washing with ultrapure water for 2-3 times until the supernatant is nearly colorless, after the washing is finished, placing the YMS-DOX nano-drug in a freeze dryer for drying in the dark place for later use, collecting all washing liquid by using a brown reagent bottle, and storing in the dark place.

Preferably, the weight ratio of the Y-type molecular sieve to the adriamycin is 5: 1.

Preferably, the pH value of the buffer solution is 9.0.

Preferably, the high-speed centrifugal rotating speed is 12000 rpm.

The high-load Y-type molecular sieve-adriamycin nano-drug can be applied to preparation of anti-breast cancer and anti-tumor drugs.

Compared with the prior art, the invention has the beneficial effects that:

1. the Y-type molecular sieve selected by the invention has the advantages of good biocompatibility, no toxicity, stable chemical property, rich functional groups on the surface and the like;

2. the invention takes the Y-type molecular sieve as a carrier, and the drug loading rate is up to YMS-DOX of 99.61 percent;

3. MTT toxicity test tests show that the nano-drug has low toxic and side effects on normal cells and can improve the killing power of the chemotherapeutic drug on breast tumor cells.

Drawings

FIG. 1 is a photograph of a nano-drug YMS-DOX under visible light and 365nm ultraviolet light

FIG. 2A is a graph of UV-VIS absorption spectra of different pH vs. loading of the nano-drug YMS-DOX

FIG. 2B Effect of different pH on NanoTaharmaceutical YMS-DOX loading

FIG. 3A UV-VISIBLE absorption Spectroscopy of action time vs. NanoTaharmaceutical YMS-DOX

FIG. 3B Effect of action time on NanoTaharmaceutical YMS-DOX Loading

FIG. 4A is a graph of UV-VIS absorption spectra of different amounts of doxorubicin added versus YMS-DOX loading

FIG. 4B Effect of different Doxorubicin addition on YMS-DOX Loading

FIG. 5 UV-VIS absorption spectra of various nanoparticles

FIG. 6 fluorescence spectra of various nanoparticles

FIG. 7 Infrared Spectroscopy of various nanoparticles

FIG. 8A Effect of nanocarrier YMS on the Activity of MM-231 cells cultured in vitro

FIG. 8B Effect of nanocarrier YMS on the Activity of DC cells cultured in vitro

FIG. 9A Effect of Nanopaharmaceutical YMS-DOX on the Activity of MM-231 cells cultured in vitro

FIG. 9B Effect of Nanoparticulate YMS-DOX on the Activity of DC cells cultured in vitro

Detailed Description

The materials used in the examples are as follows:

type Y molecular sieves (YMS, about 10nm in diameter).

Adriamycin (DOX, C)₂₇H₂₉NO₁₁HCl, molecular weight 579.99) was produced by mixebome biotechnology ltd.

Disodium hydrogen phosphate (Na)₂HPO₄·12H₂O, molecular weight 358.14) and citric acid (C)₆H₈O₇·H₂O, molecular weight 210.14) was manufactured by aladin (Aladdin) reagent limited.

Example 1

(1) Accurately weighing 1.0mgY type molecular sieve (YMS), washing with anhydrous ethanol and ultrapure water by ultrasonic high-speed centrifugation for 2 times,placing in a buffer solution (C) with pH of 9.0 at 1.0 mLl₆H₈O₇·H₂O and Na₂HPO₄·12H₂O blending), ultrasonic dispersion for 0.5 hour to form suspension,

(2) and then adding 0.2mg of adriamycin (DOX) into the suspension, uniformly mixing, placing on an infrared heating electromagnetic stirrer, reacting for 20 hours in a dark place at room temperature, after the reaction is finished, centrifuging at 12000rpm for 5 minutes at a high speed, washing for three times by using ultrapure water until the supernatant is nearly colorless, and after the washing is finished, placing the Y-type molecular sieve-adriamycin (YMS-DOX) nano medicine in a freeze dryer for drying for later use. Collecting all the washing liquid by using a brown reagent bottle, storing in a dark place, determining the mass of DOX adsorbed on the YMS nanoparticles, detecting the absorbance at 480nm of the adriamycin by using an ultraviolet-visible light spectroscopy, and calculating the adsorption amount of the DOX, wherein the microgram of the mass (199.22 +/-1.98 microgram/mg) of the adriamycin adsorbed per milligram of the YMS can be calculated.

Example 2

10 parts of 1.0mgYMS were weighed, washed first by the method (1) in example 1, and then 1.0ml of a buffer solution (C) having a pH of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 was added thereto₆H₈O₇·H₂O and Na₂HPO₄·12H₂O blending), ultrasonic dispersing for 0.5 hour, then adding 0.2mg of adriamycin, uniformly mixing, placing on an infrared heating electromagnetic stirrer for reaction for 20 hours in a dark place at room temperature, after the reaction is finished, centrifuging at 12000rpm for 5 minutes at high speed, washing with ultrapure water for three times until the supernatant is nearly colorless, and after the washing is finished, placing the Y-type molecular sieve-adriamycin (YMS-DOX) nano-drug in a freeze dryer for drying and standby. All washes were collected with a brown reagent bottle, stored away from light, and used to determine the mass of DOX adsorbed on YMS nanoparticles. And detecting the absorbance of the adriamycin at 480nm by using an ultraviolet-visible light spectrophotometric method, and calculating the load of the DOX under different pH conditions.

The results are shown in fig. 2A and 2B, the pH is in the range of 3.0-12.0, the loading amount of DOX shows a trend of increasing first and then decreasing with increasing pH, and the insets are the nano-drug supernatant images. The DOX loading was maximized at a pH of 9.0.

Example 3

5 parts of 1.0mg of YMS were weighed, washed first by the method (1) in example 1, and then 1.0mL of a buffer solution (C) having a pH of 9.0 was added₆H₈O₇·H₂O and Na₂HPO₄·12H₂O blending), ultrasonic dispersing for 0.5 hour, then adding 0.2mg of adriamycin, uniformly mixing, placing on an infrared heating electromagnetic stirrer, reacting for 0.5 hour, 1 hour, 4 hours, 12 hours and 20 hours at room temperature in a dark place, after the reaction is finished, centrifuging at 12000rpm for 5 minutes at high speed, washing with ultrapure water for three times until the supernatant is nearly colorless, and after the washing is finished, placing the Y-type molecular sieve-adriamycin (YMS-DOX) nano-drug in a freeze dryer for drying and standby. All washes were collected with a brown reagent bottle, stored away from light, and used to determine the mass of DOX adsorbed on YMS nanoparticles. And detecting the absorbance of the adriamycin at 480nm by using an ultraviolet-visible light spectrophotometric method, and calculating the load capacity of the DOX under different action times.

The results are shown in fig. 3A and 3B, and the insets are the nano-drug supernatant images. The DOX adsorption amount increases with time, and the adsorption amount reaches the maximum after 20 h.

Example 4

6 parts of 1.0mg of YMS were weighed out, washed first by the method (1) in example 1, and then 1.0ml of a buffer solution (C) having a pH of 9.0 was added₆H₈O₇·H₂O and Na₂HPO₄·12H₂O blending), ultrasonic dispersing for 0.5 hour, then respectively adding 0.1mg, 0.2mg, 0.4mg, 0.5mg, 0.8mg and 1.0mg of adriamycin, uniformly mixing, placing on an infrared heating electromagnetic stirrer for reaction for 20 hours in a dark place at room temperature, after the reaction is finished, centrifuging at 12000rpm for 5 minutes at high speed and washing with ultrapure water for three times until the supernatant is nearly colorless, after the washing is finished, placing the Y-type molecular sieve-adriamycin (YMS-DOX) nano-drug in a freeze dryer for drying for later use, collecting all washing liquid by using a brown reagent bottle, storing in a dark place, and determining the mass of adsorbed DOX on the YMS nano-particle. And detecting the absorbance of the adriamycin at 480nm by using an ultraviolet-visible light spectrophotometric method, and calculating the load capacity of DOX under different adriamycin addition amounts.

The results are shown in fig. 4A and 4B, and the insets are the nano-drug supernatant images. The DOX loading increased with increasing DOX addition, but at a DOX addition of 200. mu.g, the DOX loading was as high as 99.61%.

Example 5

To confirm success of YMS-loaded DOX, YMS and YMS-DOX were added to PBS, dispersed ultrasonically for 30 minutes, and then the UV-visible absorption spectrum was measured. While DOX was used as a control.

FIG. 5 is a UV-VIS absorption spectrum of the prepared nano-drug, and a DOX absorption peak appears at 480nm in the absorption curve of YMS-DOX, indicating that DOX is successfully loaded on the YMS surface.

Example 6

To further confirm the successful preparation of YMS-DOX, YMS and YMS-DOX were taken and added to PBS, ultrasonically dispersed for 30 minutes, and then fluorescence excitation and emission spectra were measured. While DOX was used as a control.

FIG. 6 is a fluorescence spectrum of the prepared nano-drug, where the excitation wavelength is set to 480nm, and the maximum fluorescence emission peak of YMS-DOX appears at 560nm, and the maximum fluorescence emission peak of DOX appears at 590 nm. In contrast to DOX, due to fluorescence energy resonance transfer. The YMS-DOX maximum emission wavelength is blue-shifted and the fluorescence intensity decreases, indicating that DOX is successfully loaded onto YMS.

Example 7

To confirm whether doxorubicin was supported on the surface of the Y-type molecular sieve, lyophilized YMS-DOX and KBr were mixed, ground and tableted, and the infrared spectrum was measured.

FIG. 7 is an infrared spectrum of the prepared nano-drug, wherein 3456cm^-1Stretching vibration peak at-OH, 1487cm^-1Is represented by-NH₂The stretching vibration peak of (1). 1471cm in infrared spectrum of YMS-DOX in the figure^-1A characteristic stretching vibration peak of DOX occurs, thereby indicating that DOX has been successfully loaded on YMS.

Example 8

Detecting items: effect of YMS on Activity of MM-231 and DC cells cultured in vitro

Thinning human breast cancer in logarithmic growth phaseCell (MM-231), Dendritic Cell (DC) cells (10% FBS/DMEM medium, 5% CO)₂37 ℃ C.) according to 5 × 10 per well³After the cells are attached to the wall, 200 mu L of each experimental group (5.0 mu g/mL, 10.0 mu g/mL, 25.0 mu g/mL, 50.0 mu g/mL, 100.0 mu g/mL, 200.0 mu g/mL, 400.0 mu g/mL, 800.0 mu g/mL and 1000.0 mu g/mL) prepared by 10% FBS/DMEM culture medium is replaced, untreated MM-231 and DC cells are used as blank control groups for each group, 6 duplicate wells are arranged for each group, 20 mu L of 5.0mg/mL MTT PBS solution is added into each well after 72 hours of culture, the culture is continued for 4 hours, then the old culture solution in the wells is removed, 150 mu L DMSO is added into each well, the mixture is uniformly shaken for 10 minutes, and the detection is carried out on a microplate reader.

FIG. 8A shows the effect of different concentrations of YMS and DC on proliferation after 24h, 48h, 72h and 96h, respectively, and FIG. 8B shows the effect of different concentrations of YMS and MM-231 on proliferation after 24h, 48h, 72h and 96h, respectively. Compared with a blank cell group, the Y-type molecular sieve YMS hardly influences the activities of MM-231 cells and DC cells under different concentration conditions, and has no obvious toxicity when the action time is prolonged to 96 hours, which indicates that the YMS carrier has biocompatibility;

example 9

Detecting items: effect of nano-drug YMS-DOX on in vitro culture of MM-231 and DC cell activity

Human breast cancer cells (MM-231) and Dendritic Cell (DC) cells (10% FBS/DMEM medium, 5% CO) in logarithmic growth phase₂37 ℃ C.) according to 5 × 10 per well³Inoculating the cells to a 96-well culture plate, replacing 200 mu L of each experimental group (YMS-DOX 0.35 mu g/mL, 0.7 mu g/mL, 1.4 mu g/mL, 2.8 mu g/mL and 6.0 mu g/mL) prepared by 10% FBS/DMEM culture medium after the cells are attached to the wall, using untreated MM-231 and DC cells in each group as a blank control group, setting 6 multiple wells in each group, adding 20 mu L of 5.0mg/mL MTT PBS solution into each well after culturing for 72h, continuing culturing for 4h, then removing old culture solution in each well, adding 150 mu L of LDMSO into each well, uniformly shaking for 10min, and carrying out on-machine detection by using an enzyme labeling instrument.

FIG. 9A is the effect of each drug group on MM-231 cell proliferation in vitro, and FIG. 9B is the effect of each drug group on DC cell proliferation in vitro. As can be seen from the results, the nanomedicine YMS showed various degrees of killing on MM-231 cells, and was concentration-and time-dependent, while being less toxic to DC cells, about 2-fold less than MM-231 cells. The YMS-DOX nano-drug can effectively kill tumor cells and reduce the toxic and side effects on normal cells.

In conclusion, the YMS-DOX nano-drug is successfully prepared by physically adsorbing the adriamycin on the surface of the nano-carrier Y-type molecular sieve under the action of pH9.0. MTT is used for testing the effect of YMS-DOX, MM-231 and DC cells, and the nano-drug has the characteristic of slow-release drug, can effectively kill tumor cells, and has gradually enhanced killing power along with the prolongation of time and the increase of concentration. Therefore, YMS-DOX can reduce the toxic and side effects of the chemotherapeutic drug on normal cells, can improve the killing power of the chemotherapeutic drug on tumor cells, and can be applied to the preparation of anti-breast cancer and anti-tumor drugs.

Claims

1. A preparation method of a high-load Y-type molecular sieve-adriamycin nano-drug is characterized in that the Y-type molecular sieve is used as a carrier, and the adriamycin is adsorbed by hydrogen bonds and Van der Waals force under the condition of pH 9.0.

2. The preparation method of the high-load Y-type molecular sieve-adriamycin nano-drug of claim 1 is characterized by comprising the following steps:

(1) respectively adding 0.5-1.0mL of ethanol solution and ultrapure water into each mg of Y-type molecular sieve in turn, mixing, carrying out high-speed centrifugal washing at 12000rpm for 2 times, then adding 0.5-1.0mL of buffer solution (prepared by citric acid and disodium hydrogen phosphate) with the pH of 3.0-12.0 into each mg of Y-type molecular sieve, and carrying out ultrasonic dispersion for 0.5h to form suspension;

3. The method for preparing the high-load Y-shaped molecular sieve-adriamycin nano-medicament of claim 2, wherein the weight ratio of the Y-shaped molecular sieve to the adriamycin is as follows: 5:1.

4. The method for preparing the high-load Y-shaped molecular sieve-adriamycin nano-drug according to claim 2, wherein the pH value of the buffer solution is 9.0.

5. The method for preparing the high-load Y-shaped molecular sieve-adriamycin nano-medicament according to claim 2, wherein the high-speed centrifugal rotation speed is more than or equal to 12000 rpm.

6. The use of the high-loading Y-type molecular sieve-doxorubicin nanometer medicament according to claim 1 in the preparation of a breast cancer tumor resisting medicament.