CN111939138B - Method for preparing taxol nano-drug by taking apoferritin as carrier and recovering taxol - Google Patents

Abstract

The invention relates to a method for preparing a taxol nano-drug taking apoferritin as a carrier and recovering taxol. Paclitaxel is the most excellent natural broad-spectrum and highly effective antitumor drug which is found at present, but the clinical application of paclitaxel is limited due to the poor water solubility and the lack of targeting property. The apoferritin is hollow cage-shaped protein formed by self-assembly of 24 protein subunits, and the nano cage-shaped protein can load paclitaxel into the nano cage-shaped protein through depolymerization and recombination of the protein subunits to form the paclitaxel nano drug taking the apoferritin as a carrier. When preparing paclitaxel nano-drugs, expensive paclitaxel is wasted because it cannot be recovered due to low loading rate. The invention provides a method for recovering paclitaxel after preparing a paclitaxel nano-drug, which can greatly reduce the preparation cost and realize the recovery and reuse of the paclitaxel.

Description

Method for preparing taxol nano-drug by taking apoferritin as carrier and recovering taxol

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for preparing a taxol nano-drug by taking apoferritin as a carrier and recovering taxol.

Background

Cancer remains a significant cause of human death, while chemotherapy is one of the most effective means for treating cancer in the clinic, and the use of excellent chemotherapeutic drugs is the key to the success of chemotherapy. Paclitaxel is a broad-spectrum and efficient anticancer drug, is the most excellent natural antitumor drug found at present, and is widely used for treating cancers such as breast cancer, ovarian cancer, partial head and neck cancer, lung cancer and the like in clinic.

However, because the water solubility of the paclitaxel is very poor, the commercially available paclitaxel injection mainly adopts the volume ratio of the polyoxyethylene castor oil to the ethanol to be miscible and dissolved, so that the water solubility of the paclitaxel is increased, but the polyoxyethylene castor oil can generate serious toxic and side effects in a human body; and because the paclitaxel lacks targeting, the injection can also cause serious adverse effects on other normal cells, organs and tissues. The apoferritin is a nano hollow cage-shaped structure formed by self-assembly of 24 protein subunits, the nano cage has good water solubility, strong biocompatibility, good in-vivo stability, uniform and ideal size, and the apoferritin has targeting property and can be specifically combined with transferrin receptor 1 (TfR 1) overexpressed in tumor cells. In addition, the protein can load paclitaxel into a nano cage to form a paclitaxel nano medicament taking apoferritin as a carrier by regulating depolymerization and recombination of protein subunits, the paclitaxel nano medicament greatly improves the water solubility of the paclitaxel, enhances the targeting property of the paclitaxel, and is an ideal nano medicament.

However, when preparing the taxol nano-drug using apoferritin as a carrier, the loading rate of taxol is low, generally 12% -18%, most of taxol is wasted because of not being utilized, which results in an increase in the cost of preparing the taxol nano-drug. Recovery of unused paclitaxel is therefore a key to cost reduction.

Disclosure of Invention

The invention aims to improve the water solubility of paclitaxel, increase the targeting property of paclitaxel, relieve the adverse reaction on human body, and reduce the toxic and side effects of paclitaxel on normal cells, organs and tissues as much as possible, and provides a paclitaxel nano-drug taking apoferritin as a carrier, which not only improves the water solubility of paclitaxel to 100%, but also can be specifically combined with TfR1 and enter tumor cells under the mediation of TfR1, effectively inhibits the growth, migration and diffusion of the tumor cells, can greatly improve the treatment effect of cancer, and realizes the targeted treatment and accurate treatment of cancer. In addition, in the process of preparing the taxol nano-drug, the unused taxol is recovered, the cost for preparing the nano-drug is greatly reduced, and the recovery and the reutilization of the taxol are realized.

To solve the above technical problems, the present invention provides the following technical solutions:

the invention provides a preparation method of a taxol nano-drug taking apoferritin as a carrier, which comprises the following steps:

(1) Cloning and expressing apoferritin;

(2) Separating and purifying the apoferritin;

(3) Preparing taxol nanometer medicine with apoferritin as carrier and recovering unused taxol

Preferably, the ferritin preparation method in step (1) comprises the following steps:

a. constructing an apoferritin cage expression engineering bacterium: the apoferritin encoding gene was cloned into a plasmid vector and transformed into competent cells. Screening positive monoclonal transformant through antibiotic resistance to obtain engineering bacteria expressing apoferritin;

b. expression of apoferritin: inoculating engineering bacteria expressing apoferritin into a culture medium, adding IPTG (isopropyl-beta-D-thiogalactoside) to induce expression when the OD600 value of the bacteria liquid reaches a certain value, and centrifugally collecting bacteria.

Preferably, the engineering bacteria in the step a heterologously express apoferritin through escherichia coli;

preferably, the plasmid vector in step a is pET-20b (+);

preferably, the antibiotic in step a is ampicillin.

Preferably, the OD600 value in step b is 0.6-0.8;

preferably, the final concentration of IPTG in step b is 0.5mM;

preferably, the induction temperature in step b is 30 ℃;

preferably, the induction time in the step b is 6-8h;

preferably, the centrifugal speed in the step b is 10000rpm;

preferably, the centrifugation time in step b is 30min.

Preferably, the separation and purification of apoferritin in step (2) mainly comprises the following steps:

c. crushing the collected engineering bacteria expressing the target protein, collecting supernatant, thermally treating the supernatant, and centrifuging again to obtain the supernatant which is crude apoferritin;

d. and purifying the crude protein to obtain refined protein.

Preferably, the disruption in step c is ultrasonication;

preferably, the crushing time in step c is 30min;

preferably, the temperature of the heat treatment in the step c is 60 ℃;

preferably, the heat treatment time in step c is 10min;

preferably, the centrifugal rotation speed of the step c is 10000rpm;

preferably, the centrifugation time in step c is 30min.

Preferably, the chromatography in step d is affinity chromatography or gel filtration chromatography;

preferably, the chromatographic column in step d is a nickel column or a Superdex 200 gel filtration chromatographic column.

Preferably, the preparation of the taxol nano-drug with the apoferritin as the carrier in the step (3) mainly comprises the following steps:

e. mixing the apoferritin solution and paclitaxel solution according to a certain molar ratio, adjusting pH of the solution to 2.5-3, slowly stirring, adjusting pH of the solution to 7.0-7.5 again to obtain mixed solution of apoferritin-paclitaxel, free paclitaxel, denatured apoferritin, etc.;

f. and e, averagely dividing the mixed solution finally obtained in the step e into a plurality of groups, respectively putting the groups into dialysis bags for dialysis for different times, taking out the mixed solution, continuously dialyzing the supernatant after centrifugation, accumulating and dialyzing for a certain time, detecting the amount of the paclitaxel in the paclitaxel nano-drug, and calculating the encapsulation efficiency. Encapsulation efficiency = amount of paclitaxel encapsulated into apoferritin cage/total amount of paclitaxel 100%

g. Dissolving the precipitate obtained by centrifugation in organic solvent, shaking, mixing thoroughly, centrifuging to obtain supernatant, and detecting the amount of paclitaxel in the supernatant. Removing organic solvent from the supernatant to obtain pure paclitaxel.

Preferably, all processes in step e are carried out at 4 ℃ and all processes are stirred slowly;

preferably, the paclitaxel solution in step e is a solution of paclitaxel dissolved in absolute ethanol;

preferably, the molar ratio in the step e is 1:200;

preferably, the stirring time in step e is 10min.

Preferably, in step f, the supernatant is adjusted to pH 2.5-3 again, centrifuged again, and part of the supernatant is subjected to HPLC to detect the amount of paclitaxel in the apoferritin-paclitaxel;

preferably, the cut-off value of the dialysis bag of step f is 6-8kDa;

preferably, the dialysis time of step f is 18h;

preferably, the centrifugal speed of step f is 6000rpm;

preferably, the centrifugation time in step f is 10min.

Preferably, the organic solvent of step g is absolute ethanol;

preferably, the centrifugal speed in the step g is 10000rpm;

preferably, the centrifugation time of step g is 10min;

preferably, the method for removing the organic solvent in the step g is vacuum drying.

As a preferred technical scheme, the preparation method of the taxol nano-drug with the apoferritin as the carrier comprises the following steps:

cloning and expressing apoferritin: the apoferritin encoding gene was cloned into a pET-20b (+) plasmid vector and transformed into E.coli competent cells. Screening positive monoclonal transformant through ampicillin to obtain engineering bacteria expressing apoferritin; and (3) expressing the recombinant protein, inoculating the engineering bacteria expressing the apoferritin into an LB culture medium containing ampicillin, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression when the OD600 value of the bacteria liquid reaches 0.6-0.8, and centrifugally collecting bacteria.

Separating and purifying the apoferritin: crushing engineering bacteria expressing apoferritin, centrifuging, collecting supernatant, heat treating the supernatant at 60 deg.C for 10min, centrifuging again, collecting supernatant, and performing affinity chromatography with nickel column and gel filtration chromatography to obtain pure apoferritin.

Mixing the apoferritin solution with paclitaxel dissolved in solvent such as anhydrous alcohol at a molar ratio of 1:200, adjusting pH of the mixed solution to 2.5, slowly stirring for 10min, and adjusting pH of the mixed solution to 7.0-7.5 to obtain mixed solution containing apoferritin as carrier, paclitaxel nanometer drug, free paclitaxel, denatured apoferritin, etc. And (3) putting the mixed solution into a dialysis bag, dialyzing to remove free paclitaxel, taking out after dialyzing for 12-18h, centrifuging at 6000rpm for 10min to obtain supernatant, namely the paclitaxel nano-drug taking ferritin as a carrier, and detecting the content of paclitaxel in the paclitaxel nano-drug by HPLC. And (3) resuspending the precipitate obtained by dialysis by using an organic solvent such as absolute ethyl alcohol, fully shaking, centrifugally collecting supernatant, and removing the organic solvent in the supernatant to obtain a pure paclitaxel product.

The invention has the beneficial effects that: (1) The invention provides a method for preparing taxol nano-drugs; (2) The paclitaxel nano-drug prepared by the invention has the effect of targeting tumor cells, and provides a precondition for realizing accurate treatment of the drug; (3) The invention provides a simple and feasible method for recovering unused paclitaxel, and the production cost is reduced. Therefore, the invention has great potential in the field of biological medicine and has good application prospect.

Drawings

FIG. 1: in example 2 apoferritin was purified by nickel column affinity chromatography of the fractions eluted.

FIG. 2: characterization of the pure apoferritin obtained in example 2. FIG. 2A is a size exclusion chromatogram of apoferritin; FIG. 2B is a transmission electron micrograph of apoferritin; figure 2C is a circular dichroism map of apoferritin.

FIG. 3: characterization of paclitaxel nano-drug with ferritin as carrier was prepared in example 3. FIG. 3A is a size exclusion chromatogram of a nanoparticulate paclitaxel drug carried with apoferritin; FIG. 3B is a circular dichroism spectrum of a paclitaxel nano-drug using apoferritin as a carrier; fig. 3C is a dynamic light scattering characterization particle size distribution diagram of the paclitaxel nano-drug using apoferritin as a carrier.

FIG. 4: the flow chart of the preparation of the taxol nano-drug taking ferritin as a carrier and the recovery of the taxol in the embodiment 3 is shown.

FIG. 5: the release profile of paclitaxel in the paclitaxel nano-drug using apoferritin as a carrier in example 4.

FIG. 6: the survival rate of SMMC-7721 cells in example 5 is determined by the effect of paclitaxel nano-drugs with different concentrations and apoferritin as a carrier.

FIG. 7: the flow cytometer in example 6 examined the binding ability of SMMC-7721 cells to HFtn, HFtn-PTX nanoparticles.

FIG. 8: in example 7, after the action of paclitaxel and a paclitaxel nano-drug using apoferritin as a carrier, the SMMC-7721 cells are detected by a flow cytometer, wherein A is an apoptosis characteristic diagram of each group, and B is apoptosis rate statistics.

Examples of the embodiments

The invention will be further explained and illustrated with reference to specific examples, which are, however, to be understood as being given by way of illustration only and not in any way limitative of the invention.

Example 1. Cloning and expression of apoferritin;

the coding gene of the apoferritin is cloned into a pET-20b (+) plasmid vector and transformed into an escherichia coli BL21 competent cell. Screening positive monoclonal transformant by ampicillin to obtain engineering bacteria for expressing apoferritin; inoculating engineering bacteria expressing apoferritin into LB culture medium containing ampicillin, adding IPTG to induce expression when OD600 value of bacterial liquid reaches 0.6-0.8, adding IPTG with final concentration of 0.5mM to induce expression for 6-8h, centrifuging at 10000rpm for 5-8min, and collecting thallus.

Example 2. Isolation and purification of apoferritin;

ultrasonically crushing engineering bacteria expressing apoferritin, centrifuging, collecting supernatant, thermally treating the supernatant at 60 deg.C for 10min, centrifuging again, collecting supernatant, and performing nickel column affinity chromatography and gel filtration chromatography to obtain pure apoferritin.

Collecting engineering bacteria expressing the apoferritin, carrying out ultrasonication on the engineering bacteria, carrying out 30-40min, centrifuging at 10000rpm for 30min, taking a supernatant, carrying out heat treatment on the supernatant at 60 ℃ for 10min, centrifuging at 10000rpm for 30min, taking the supernatant, carrying out nickel column affinity chromatography, eluting impurity proteins and target recombinant proteins by using gradient imidazole solutions (5 mM, 10mM, 20mM and 250 mM) in sequence, collecting an elution liquid, analyzing protein components in the eluate by an SDS-PAGE method, and purifying the target protein eluate again by gel filtration chromatography to obtain a pure product apoferritin.

Example 3 preparation of Desferritin-Supported paclitaxel Nanomedicine and recovery of paclitaxel

Mixing the apoferritin solution and paclitaxel dissolved in solvents such as absolute ethanol according to a molar ratio of 1:200 to form a mixed solution, adjusting the pH of the mixed solution to 2.5, slowly stirring for 10min, and then adjusting the pH of the solution to 7.0-7.5 again to obtain a mixed solution of a paclitaxel nano-drug, free paclitaxel, denatured apoferritin and the like with apoferritin as a carrier. Putting the mixed solution into a dialysis bag with a cut-off value of 6-8kDa, dialyzing to remove free paclitaxel, taking out after dialyzing for 12-18h, centrifuging at 6000rpm for 10min, taking part of the supernatant to be the paclitaxel nano-medicament with ferritin as a carrier, taking part of the supernatant, re-adjusting the pH to 2.5 to re-release the paclitaxel in the ferritin into the solution, and detecting the content of the paclitaxel in the paclitaxel nano-medicament by HPLC. And (3) resuspending the precipitate obtained by dialysis by using an organic solvent such as absolute ethyl alcohol, fully shaking, centrifugally collecting supernatant, and removing the organic solvent in the supernatant to obtain a pure paclitaxel product.

Example 4 stability and in vitro Release of Taxol Nandrugcompounds with apoferritin as Carrier

Placing paclitaxel nanometer medicine into dialysis bag (molecular weight cut-off is 6-8 kDa), placing into PBS buffer solution with pH of 7.4, and incubating at 37 deg.C. Samples were taken at incubation times 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, 60 hours respectively and the released paclitaxel was quantified by HPLC. Paclitaxel release = total amount of paclitaxel/paclitaxel released. The results are shown in fig. 5, and the paclitaxel nano-drug using apoferritin as the carrier is relatively stable at pH 7.4. However, paclitaxel in the paclitaxel nano-drug is easier to release at pH 5.0, and the result shows that the release form of the paclitaxel nano-drug is pH-dependent.

Example 5 in vitro cytotoxicity study of Taxol Nandrugs with apoferritin as Carrier

In order to study the in vitro cytotoxicity of the taxol nano-drug taking the apoferritin as the carrier, SMMC-7721 was seeded in a 96-well plate at a density of 5000 cells per well, and after 24 hours of culture at 37 ℃, the taxol with the taxol content of 1-5000ng/mL and the taxol nano-drug taking the apoferritin as the carrier were added into the same well for 48 hours of culture. Wash 2 times with PBS, incubate 4h after addition of MTT, centrifuge carefully remove supernatant, add dimethyl sulfoxide, and measure uv absorbance at 570 nm. The result shows that the toxicity of the taxol nano-drug taking the apoferritin as the carrier is lower than that of the taxol naked drug. The same phenomenon exists in other drug-loaded systems, which may be related to paclitaxel entering cells through passive diffusion, while the paclitaxel nano-drug taking apoferritin as a carrier enters cells through an endocytosis mechanism mediated by transferrin receptor 1 (TfR 1).

Example 6 targeting of Taxol Nanomedicines with apoferritin as vehicle

SMMC-7721 cells were seeded in well plates to grow to 1X 10 ⁵ About one cell, trypsinized, washed 3 times with pre-chilled PBS, added with 50 μ g/mL FITC-labeled HFtn and HFtn-PTX nanoparticles, incubated at 4 ℃ for 45min, washed 3 times with 0.3% BSA/PBS, resuspended in PBS, and the fluorescence intensity was detected by flow cytometry. The results are shown in the figure, the fluorescence intensity of the FITC-labeled HFtn and HFtn-PTX nanoparticles are substantially equivalent in both groups, much higher than the PBS blank control group. This suggests that HFtn and HFtn-PTX nanoparticles can rapidly bind to receptors and enter cells, and this result also demonstrates that whether the HFtn nanocages are loaded with drugs does not affect the binding of nanoparticles to receptors.

Example 8 in vitro apoptosis study of Desferritin-Supported paclitaxel Nanomedicine

In order to study the in vitro apoptosis characteristics of the taxol nano-drug taking apoferritin as a carrier, an Annexin V-FITC apoptosis kit is used for testing. SMMC-7721 at 4X 10 per well ⁵ The density of each cell was seeded in 6-well plates, and HFtn-PTX having a paclitaxel content of 6. Mu.g/mL was cultured at 37 ℃ for 24 hours,PTX was added to different wells for 24h to add a blank culture group as a control. Cells were collected and suspended in binding buffer provided by Annexin V-FITC apoptosis kit, then 5. Mu.L of Annexin V-FITC was added to each cell suspension and incubated at room temperature for 15min, followed by addition of 5. Mu.L of propidium iodide. Double-stained cells were immediately analyzed by flow cytometry. The results show that: apoptosis was evident in the PTX group and HFtn-PTX group compared to the control group, with apoptosis more evident in the HFtn-PTX group, which may be related to factors such as the HFtn-PTX nanoparticles binding to cells, entering cells and releasing drugs at a faster rate than the PTX particles.

Claims (6)

1. A method for preparing taxol nano-drugs with apoferritin as a carrier and recovering taxol is characterized in that the preparation method comprises the following steps: mixing the apoferritin solution with paclitaxel solution, adjusting pH to 2.5-3.0, stirring, adjusting pH to 7.0-7.5 to obtain mixed solution of apoferritin-paclitaxel, free paclitaxel, and denatured apoferritin; putting the obtained mixed solution into a dialysis bag for dialysis, taking out a dialysis sample and centrifuging, wherein the supernatant is the taxol nano-drug taking the apoferritin as a carrier; resuspending and precipitating with anhydrous ethanol, shaking sufficiently, centrifuging again, and removing organic solvent in supernatant to obtain paclitaxel; the apoferritin is human heavy chain ferritin; the molar ratio of the apoferritin to the paclitaxel is 1; the dialysis time is 12-18 h.

2. The method for preparing the paclitaxel nano-drug using apoferritin as a carrier and recovering paclitaxel according to claim 1, wherein the amino acid sequence of human heavy chain ferritin is shown in SEQ ID No.2, and the nucleic acid sequence is shown in SEQ ID No. 1.

3. The method for preparing the apoferritin-carried paclitaxel nano drug and recovering paclitaxel according to claim 1, wherein the preparation process is performed at 4 ℃.

4. The method for preparing the apoferritin-loaded paclitaxel nanopharmaceutical and recovering paclitaxel according to claim 1 wherein said dialysis bag has a cut-off of 6-8 kDa.

5. The method for preparing the apoferritin-carried paclitaxel nano drug and recovering paclitaxel according to claim 1, wherein the method for removing organic solvent is vacuum drying.

6. The paclitaxel nano-drug using apoferritin as a carrier prepared by the method of any one of claims 1 to 5.

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