CN114668771A

CN114668771A - Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together

Info

Publication number: CN114668771A
Application number: CN202210273421.6A
Authority: CN
Inventors: 张瑜; 宁晨; 王飞; 李迅; 董亦馨; 杨昆
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-06-28
Anticipated expiration: 2042-03-18
Also published as: CN114668771B

Abstract

The invention provides a preparation method and application of a ferritin drug-loaded nanoparticle co-loaded with two drugs, the nanoparticle effectively delivers the two drugs by targeting on a tumor cell transferrin receptor 1(TfR1), compared with the delivery of a single drug, the method can further enhance the anti-tumor curative effect and reduce the generation of drug resistance, and the invention co-loads the two drugs by ferritin, has simple steps, keeps the original structure of the ferritin complete and has good application prospect.

Description

Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together

Technical Field

The invention belongs to the technical field of anti-tumor, and particularly relates to a preparation method and application of a ferritin nanoparticle co-loaded with adriamycin and ursolic acid.

Background

Cancer is a serious threat to human life and health, and is the second leading disease of death in the human body worldwide. Although chemotherapy is one of the most effective methods for treating cancer in clinic, chemotherapy drugs often kill tumor cells and human normal cells. Therefore, chemotherapy may produce strong toxic side effects that may lead to adverse reactions. In order to solve the above problems, nano-drug delivery systems such as liposomes, polymeric micelles, lipid-based nano-carriers, inorganic nano-carriers, and the like have been developed and utilized. In addition, the protein nanoparticles have the characteristics of good biocompatibility and the like, so that the protein nanoparticles become a very potential drug delivery system.

Human heavy chain ferritin (HFtn) is a globular cage-shaped protein nanomaterial with an outer diameter of 12nm and an inner diameter of 8 nm. Ferritin as a nano carrier has the characteristics of low immunogenicity, biodegradability, specific targeting of TfR1 and the like. Therefore, ferritin plays an important role in the field of cancer therapy. The ferritin loaded single drug has undesirable anti-tumor effect and can suffer from various problems such as drug resistance and the like. Therefore, in order to improve the anti-tumor effect and reduce the toxic and side effects of the drugs, the ferritin inner cavity is simultaneously loaded with the two drugs, and the two drugs have synergistic effect to enhance the anti-tumor effect, so that the method is a promising strategy.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

In order to improve the therapeutic effect and reduce the generation of drug resistance, most cancer treatments in clinic use anticancer drugs with different mechanisms in combination. The invention provides a preparation method of a ferritin drug-loaded nanoparticle carrying two drugs together, the ferritin nanoparticle effectively delivers the two drugs by targeting on a tumor cell transferrin receptor 1(TfR1), compared with delivering a single drug, the method can further enhance the anti-tumor curative effect and reduce the generation of drug resistance, and has good application prospect.

The inventor has tried various methods to load the drug required for the experiment, and there are three main methods for loading ferritin with the drug: pH method, temperature method, and urea method. Compared with the pH method, the temperature method can greatly reduce the loss of ferritin and improve the loading capacity, and the pH method only has a very small amount of drugs to be loaded; compared with the low-concentration urea treatment, the temperature method can load two drugs simultaneously and further enhance the quantity of the loaded drugs, the pH method only has a very small number of drugs to be loaded, the protein loss is too large to be suitable for further research, and the low-concentration urea method only can load one drug but cannot load two drugs of adriamycin-ursolic acid simultaneously. Therefore, the invention mainly provides a preparation method and application of the ferritin nanoparticles carrying both adriamycin and ursolic acid by a temperature method.

Therefore, the invention aims to provide a preparation method and application of a ferritin nanoparticle loaded with adriamycin and ursolic acid together.

In order to solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions: a method for preparing ferritin nanoparticles co-loaded with adriamycin and ursolic acid comprises,

The ferritin nanoparticles load two drugs, namely hydrophilic drug and hydrophobic drug.

As a preferable scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the invention also comprises the following steps,

adding the medicines into ferritin solution respectively, incubating, centrifuging, separating supernatant, and dialyzing in PBS; and (4) carrying out centrifugal filtration to obtain the load sharing drug nanoparticles.

As a preferable scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the invention comprises the following steps: the medicine is hydrophilic adriamycin and hydrophobic ursolic acid.

As a preferred scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the method comprises the following steps: the adriamycin is PBS solution of adriamycin; the ursolic acid is ethanol solution of ursolic acid.

As a preferred scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the method comprises the following steps: the ferritin solution is Tris-HCl solution of human heavy chain ferritin, the concentration is 20mM, and the pH value is 8.0.

As a preferred scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the method comprises the following steps: the molar ratio of the adriamycin to the ferritin in the co-loaded drug nanoparticles is 50-300: 1, the molar ratio of the ursolic acid to the ferritin is 450: 1.

As a preferred scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the method comprises the following steps: the incubation is carried out in a constant temperature water bath kettle at 60 ℃ for 4-5 hours.

As a preferable scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the invention comprises the following steps: and centrifuging after incubation at the centrifugal rotation speed of 12000rpm for 10-15 min. As a preferred scheme of the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid, the method comprises the following steps: dialyzing in PBS for 24-48 h at 4 ℃ and changing PBS every 6 hours; and (4) performing centrifugal filtration under the centrifugal condition of 5000 Xg for 5-10 min.

The co-drug-loaded nanoparticle prepared by the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid is applied to delivery of antitumor drugs.

The invention has the beneficial effects that:

the invention provides a preparation method and application of a ferritin drug-loaded nanoparticle carrying two drugs together, the nanoparticle effectively delivers the two drugs by targeting on a tumor cell transferrin receptor 1(TfR1), compared with delivering a single drug, the method can further enhance the anti-tumor curative effect and reduce the generation of drug resistance, and the invention loads the two drugs together through ferritin, has simple steps, does not change the original structure of ferritin, and can greatly reduce the loss of ferritin and improve the loading capacity compared with a pH method; compared with low-concentration urea treatment, the method can load two drugs simultaneously, can further enhance the quantity of the loaded drugs, and has good application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive labor. Wherein:

FIG. 1 is a schematic diagram showing the loading of the medicines Doxorubicin (DOX) and Ursolic Acid (UA) by ferritin in example 1 and example 2; wherein (A) adding low concentration urea to expand the channel on the protein shell, allowing the drug to enter the protein cavity, dialyzing to remove urea and free drug, and contracting the pore channel to fix the drug in the cavity; (B) adding DOX and UA into protein solution, incubating at 60 deg.C for 4 hr to enlarge protein channel, introducing the medicine into the inner cavity, and dialyzing to remove free medicine.

FIG. 2 is a characterization of drug-loaded ferritin and ferritin nanoparticles in example 1; wherein, the recombinant protein (A) is expressed in colon bacillus, and an expected gel band is obtained by nickel column and SEC purification, which indicates that the required protein is successfully purified; (B) low concentration urea treatment and heat treatment of the prepared samples; (C) the circular dichroism chromatogram of four kinds of nano particles of HFtn, HFtn-DOX/UA, HFtn-DOX and HFtn-UA shows that the prepared nano particles have obvious characteristic absorption peaks near 208nm and 222 nm. The encapsulation of DOX and UA drugs in the ferritin lumen has no influence on the ferritin secondary structure and is still alpha-helix; (D) SEC polymerization state analysis of the four kinds of nanoparticles can obtain that the prepared nanoparticles are still 24-polymer, which proves that the drug is encapsulated in the protein cage, and the polymerization state of the protein carrier is not influenced by heat treatment; (E) in transmission electron microscope images before and after drug encapsulation, the four nanoparticles still have a cage-shaped structure with uniform particle size, which shows that the stability of the cage-shaped structure is not influenced by the loading of DOX and UA drugs; (F) the DLS characterization of the nanoparticles shows that the hydrodynamic diameter of the ferritin nanoparticles loaded with one drug is slightly higher than that of ferritin, and the hydrodynamic diameter of the ferritin nanoparticles loaded with two drugs is slightly higher than that of the ferritin nanoparticles loaded with one drug.

FIG. 3 is the drug release of example 3, wherein (A) the drug release of HFtn-DOX and HFtn-UA, (B) the drug release of HFtn-DOX/UA;

FIG. 4 is a cytotoxicity assay and drug synergy assay of examples 4 and 5; wherein, the activity of the in vitro cells of the free drug and the MCF-7 cells after 24 hours of incubation; (B) the drug-loaded nanoparticles and MCF-7 cells are incubated for 24 hours to ensure the in vitro cell activity; (C) drug Combination Index (CI) of doxorubicin and ursolic acid drug synergy in MCF-7 cells;

FIG. 5 shows the FITC-labeled ferritin uptake by MCF-7 cells as measured by confocal laser and flow cytometry in example 6; wherein, (A) MCF-7 cells were incubated with free FITC and FITC-HFtn for 4h fluorescence images, white lines representing a size of 20 μm; (B) measuring uptake of free FITC, FITC-HFtn by MCF-7 cells by flow cytometry; (C) mean fluorescence intensity (n-3) analyzed according to panel B.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, the references herein to "one embodiment" or "an embodiment" refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

In order to improve the therapeutic effect and reduce the generation of drug resistance, most cancer therapies in clinic use anticancer drugs with different mechanisms. The invention provides a preparation method of a ferritin drug-loaded nanoparticle carrying two drugs together, the ferritin nanoparticle effectively delivers the two drugs by targeting on a tumor cell transferrin receptor 1(TfR1), compared with delivering a single drug, the method can further enhance the anti-tumor curative effect and reduce the generation of drug resistance, and has good application prospect.

The chemical reagents used in the examples of the present invention were all ordinary commercially available analytical reagents unless otherwise specified.

The adriamycin and the ursolic acid used in the embodiment of the invention are purchased from: adriamycin (97% or more) was purchased from Shanghai Aladdin Biotechnology GmbH; ursolic acid (94% or more) was purchased from Sahn chemical technology (Shanghai) Co., Ltd; human heavy chain ferritin (HFtn) was purchased from south kyin smith biotechnology limited; MCF-7 cells were obtained from the China academy of sciences (CCTCC) cell bank.

Example 1:

preparing the ferritin nanoparticles loaded with the adriamycin and the ursolic acid medicaments together by a temperature method:

dissolving the adriamycin medicine with PBS solution, and dissolving the ursolic acid medicine with ethanol solution.

Doxorubicin and ursolic acid drug were added to 4ml of ferritin solution in Tris-HCl (20mM, pH 8.0). The input molar ratio of the ferritin to the adriamycin to the ursolic acid is 1: 150. 1: 450, after incubation at 60 ℃ for 4h, centrifugation at 12000rpm for 10min, the supernatant was separated and dialyzed against PBS for 36 h. After the dialysis is finished, centrifuging for 5min under the condition of 5000 Xg, filtering the supernatant by using a filter membrane of 0.45 μm to obtain the ferritin drug-loaded nanoparticles loaded with the adriamycin and the ursolic acid drugs (the method for loading the adriamycin and the ursolic acid drugs independently is the same as the method for loading the adriamycin and the ursolic acid drugs together). The preparation method is shown in figure 1, and the characterization result is shown in figure 2.

Through detection, each ferritin nanoparticle can independently load 100 ursolic acid drugs and 90 adriamycin drugs, and the ferritin nanoparticles jointly loaded with the adriamycin-ursolic acid drugs provided by the invention contain 90 ursolic acid drugs and 18 adriamycin drugs, which are higher than those independently loaded with one drug.

Example 2:

1. the process for preparing the drug-loaded nanoparticles by treating the low-concentration urea comprises the following steps:

dissolving ursolic acid in ethanol solution;

ferritin solution was present in GFC (50mM Na)₂HPO₄150mM NaCl, pH 7.0), urea was added to a final concentration of 50 mM. Incubating the mixture at 4 deg.C for 2h, adding ethanol-dissolved ursolic acid medicine, and slowly stirring at 4 deg.C for 40min, wherein the molar ratio of ferritin to ursolic acid is 1: 450, then dialyzing in a PBS solution for 24 hours; after the dialysis is finished, centrifuging (5000 Xg, 5min) and filtering the protein liquid in the dialysis bag in the above steps to obtain the nanoparticle loaded with the medicine, wherein the preparation method is shown in figure 1;

the protein nanoparticles loaded with the adriamycin and the ursolic acid are prepared by using the low-concentration urea treatment process, and only the ursolic acid drug is detected, but the adriamycin drug cannot be detected, which indicates that only the ursolic acid drug is loaded. And (4) carrying 65 ursolic acid medicaments per ferritin nanoparticle by low-concentration urea treatment.

2. The process for preparing the drug-loaded nanoparticles by pH method treatment comprises the following steps:

ferritin solution was present in GFC (50mM Na)₂HPO₄150mM NaCl, pH 7.0), adding 1M HCl dropwise into the solution, adjusting the pH of the solution to 2-3, adding a PBS-dissolved adriamycin medicament and an ethanol-dissolved ursolic acid medicament, slowly stirring the mixture for 40min at the temperature of 4 ℃ in a chromatography cabinet, then adding 1M NaOH dropwise to adjust the pH of the solution to about 7 again, and finally dialyzing the solution in the PBS for 24 h; after the dialysis is finished, eggs in the dialysis bag obtained in the above step are dialyzed The white liquid is centrifuged (5000 Xg, 5min) and filtered to obtain the nanoparticles carrying the drug which are required by people. Both drug loadings were single digit by pH.

Example 3:

the research on the in-vitro drug release of the ferritin nanoparticles co-loaded with the adriamycin and the ursolic acid comprises the following steps:

1ml of ferritin-doxorubicin/ursolic acid solution prepared by the temperature method was placed in a dialysis bag (MWCO 6-8kDa), placed in PBS buffer at pH 5.0 and pH 7.4, respectively, and dialyzed at 37 ℃ for 60 hours at a stirring speed of 100 rpm. At set time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48 and 60h), 300 μ L of dialysate was removed and replaced with an equal volume of PBS. The release amount of DOX was measured by fluorescence, and the release amount of UA was measured by high performance liquid chromatography, and the results are shown in FIG. 3. It can be seen from the figure that all three nanoparticles maintain a relatively low release at pH 7.4; the release rates of all three nanoparticles reached higher levels at pH 5.0, indicating that the nanoparticles are unstable at this pH and DOX and UA are released continuously in lysosomes, so HFtn can be an effective drug delivery vehicle.

Example 4:

in vitro cytotoxicity study of ferritin nanoparticles co-loaded with doxorubicin and ursolic acid drugs:

In order to study the in vitro cytotoxicity of the ferritin nanoparticles co-loaded with adriamycin and ursolic acid, MCF-7 cells were seeded in a 96-well plate at a density of 5000 cells per well, after 24 hours of culture at 37 ℃, DOX, UA, HFtn-DOX, HFtn-UA, HFtn-DOX/UA with adriamycin and ursolic acid contents of 0.01-10 μ M/mL and 0.05-50 μ M/mL were added into the same well to culture for 24 hours, PBS was washed twice, MTT was added into the culture medium to incubate for 4 hours, centrifugation was performed, dimethyl sulfoxide (DMSO) was added after carefully removing the supernatant, ultraviolet absorption was measured at 490nm, the survival rate of MCF-7 cells under the action of adriamycin and ursolic acid drugs with different concentrations and drug-loaded nanoparticles is shown in FIG. 4, which indicates that the ferritin nanoparticles co-loaded with adriamycin and ursolic acid drugs show stronger cytotoxicity, indicating that better therapeutic efficacy can be achieved by the delivery of both drugs by ferritin.

Example 5:

the research on the synergistic effect of the adriamycin and the ursolic acid medicaments comprises the following steps:

to demonstrate the synergistic effect of ferritin loaded DOX and UA, visual expression can be based on drug Combination Index (CI). The CI value is calculated by the following equation:

CI_X＝D_a/Dx_a+D_b/Dx_b

wherein D_aAnd D_bIs the concentration of drug a and drug b that act together to achieve a particular drug effect. Dx _aAnd Dx_bIs the concentration of the single drug counterpart that achieves the same effect. CI values greater than, equal to, and less than 1 reflect antagonism, additivity, and synergy, respectively. The extent of drug interaction can be obtained by plotting the CI value versus the inhibition rate (Fa value). From FIG. 4 we can see that the CI values for all data points are less than 1, indicating a strong interaction between the two drugs DOX and UA; according to the fitted curve, the Fa value is increased and the CI value is decreased to be stable along with the increase of the concentration of the drug, which shows that the synergistic effect of the drug is stronger but cannot be infinitely enhanced by a certain limit when the inhibition rate is increased.

Example 6:

the study on the in vitro cell uptake of the ferritin nanoparticles co-loaded with the adriamycin and the ursolic acid comprises the following steps:

to study the uptake of ferritin co-loaded with both doxorubicin and ursolic acid by cells, MCF-7 cells were plated at 3X 10 per well⁴Inoculating the cells into a 24-hole plate in which a glass slide is placed, culturing for 24h, adding 20 mu g/mL FITC-labeled HFtn nanoparticles, incubating for 4h, washing the cells for 3 times by PBS, and fixing for 30min at room temperature by using 4% paraformaldehyde; and then PBS is used for cleaning twice, then DAPI is added for cell nucleus staining, and a laser confocal microscope is used for imaging cells, and the result is shown in figure 5, the FITC marked ferritin group shows strong fluorescence, which indicates that ferritin can be taken up by cells and has targeting property.

The ferritin drug-loaded nanoparticles prepared by the temperature method in example 1 can be loaded with two drugs, doxorubicin-ursolic acid. Compared with the pH method in the embodiment 2, the temperature method can greatly reduce the loss of ferritin and improve the loading capacity, and the pH method only has a very small amount of two medicines loaded, basically the proteins are accumulated and precipitated, and subsequent experiments cannot be carried out; compared with the urea treatment with low concentration, the invention not only can load two drugs at the same time, but also can further enhance the amount of the loaded drugs. In summary, the temperature method provided by the present invention: the medicine can be loaded with two medicines; secondly, the loss of protein is reduced; and the quantity of the loaded medicine is further enhanced.

The invention provides a preparation method and application of a ferritin drug-loaded nanoparticle jointly loaded with two drugs, the nanoparticle effectively delivers the two drugs by targeting on a tumor cell transferrin receptor 1(TfR1), compared with the delivery of a single drug, the method can further enhance the anti-tumor curative effect and reduce the generation of drug resistance, and the invention loads the two drugs together through ferritin, has simple steps, keeps the original structure of the ferritin complete and has good application prospect.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A preparation method of a ferritin nanoparticle co-loaded with adriamycin and ursolic acid is characterized by comprising the following steps: comprises the steps of (a) preparing a substrate,

2. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 1, wherein the method comprises the following steps: also comprises a step of adding a new type of additive,

3. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 1 or 2, wherein the method comprises the following steps: the medicine is hydrophilic adriamycin and hydrophobic ursolic acid.

4. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 3, wherein the method comprises the following steps: the adriamycin is PBS solution of adriamycin; the ursolic acid is ethanol solution of ursolic acid.

5. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 1 or 2, wherein the method comprises the following steps: the ferritin solution is Tris-HCl solution of human heavy chain ferritin, the concentration is 20mM, and the pH value is 8.0.

6. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 1 or 2, wherein the method comprises the following steps: the molar ratio of the adriamycin to the ferritin in the co-loaded drug nanoparticles is 50-300: 1, the molar ratio of the ursolic acid to the ferritin is 450: 1.

7. the method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 2, wherein the method comprises the following steps: the incubation is carried out in a constant temperature water bath kettle at 60 ℃ for 4-5 hours.

8. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 2, wherein the method comprises the following steps: and centrifuging after incubation at the centrifugal rotation speed of 12000rpm for 10-15 min.

9. The method for preparing the ferritin nanoparticles co-loaded with adriamycin and ursolic acid according to claim 2, wherein the method comprises the following steps: dialyzing in PBS for 24-48 h at 4 ℃ and changing PBS every 6 hours; and (4) performing centrifugal filtration under the centrifugal condition of 5000 Xg for 5-10 min.

10. The application of the co-drug-loaded nanoparticle prepared by the preparation method of the ferritin nanoparticle co-loaded with adriamycin and ursolic acid according to claims 1-9 in delivering anti-tumor drugs.