CN113018456A - Preparation method of multiple-response nano-drug capable of size conversion - Google Patents

Preparation method of multiple-response nano-drug capable of size conversion Download PDF

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CN113018456A
CN113018456A CN202110301480.5A CN202110301480A CN113018456A CN 113018456 A CN113018456 A CN 113018456A CN 202110301480 A CN202110301480 A CN 202110301480A CN 113018456 A CN113018456 A CN 113018456A
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吴波震
李明沛
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides a preparation method of a multi-response nano-drug capable of size conversion, which comprises the steps of loading adriamycin by graphene oxide, integrating the adriamycin and gelatin by glutaraldehyde in a covalent bond manner, and finally coating the adriamycin and gelatin by bovine serum albumin; according to the invention, the graphene oxide is coated by the bovine serum albumin and the gelatin in a double-layer manner, so that the protein adsorption resistance of the nano-drug is enhanced, the size conversion function is realized, the nano-drug is highly enriched at a tumor part and effectively penetrates deep tumors, and meanwhile, chemotherapy combined photo-thermal treatment is realized by using the adriamycin and the graphene oxide, so that the anti-tumor effect is remarkably improved.

Description

Preparation method of multiple-response nano-drug capable of size conversion
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a multiple-response anti-tumor nano-drug capable of size conversion.
Background
The traditional cancer treatment modes, such as radiotherapy, chemotherapy, surgical excision and the like, have poor curative effect and obvious side effect. For example, the treatment effect of the traditional chemotherapy small molecule drugs (such as adriamycin) is greatly limited due to the problems that the drugs cannot be specifically distributed in normal tissues and the tumor has multidrug resistance. Therefore, nano-drug delivery systems are produced, and common nano-drug carriers include liposomes, micelles, hydrogels, and the like. However, the single treatment mode and the nano-drug with fixed size are difficult to realize the effective inhibition of tumor cells, and especially have larger short plates in the aspect of deep tumor cell treatment. The combination therapy-based multi-response nano-drug and the nano-drug with the size transformation capability are expected to realize better cancer treatment effect and are also the contents of the key points of the invention.
The combination of chemotherapy and photothermal therapy (PTT) is a typical technique for treating cancer. PTT is a method for realizing conversion of light energy into heat energy by using near infrared light (NIR) to excite a photothermal conversion agent so as to induce thermal ablation of cancer cells. The commonly used photothermal conversion agents include nanocarbon materials, noble metal nanoparticles, organic dyes, semiconductor nanoparticles, and the like. For example, CN 112093233 a discloses a graphene oxide drug carrier with magnetic targeting ability, and CN 111821436 a discloses a compound with targeting penetration and diagnosis and treatment integrated. In the above studies, authors propose and prepare a graphene oxide drug carrier with a targeting function, and have made beneficial work in the aspects of combination therapy and diagnosis and treatment integration. However, how to achieve high concentration of the nano-drug at the tumor site and effective penetration to the deep tumor becomes a problem to be solved by those skilled in the art.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of a size-convertible multiple-response nano-drug.
The method can solve the problem of poor single chemotherapy effect by combining chemotherapy with photothermal therapy by using the graphene oxide loaded adriamycin. The adriamycin is modified to a gelatin molecular chain by using glutaraldehyde in a covalent bond mode, so that the selective release capacity of the adriamycin is greatly improved, the adriamycin delivery efficiency is improved, and the side effect is reduced. And bovine serum albumin is integrated on the surface of the nano-drug, so that the stability of the nano-drug is improved, the size conversion function is realized, and the enrichment of the nano-drug at the tumor part and the effective penetration to deep tumors are enhanced.
The nano-drug provided by the invention can realize the fixed-point release of the adriamycin in tumor treatment, and penetrate into the deep part of a tumor, and finally, the chemotherapy is combined with photothermal therapy to improve the anti-tumor effect, thereby achieving the purpose of solving the problem of the drug resistance of tumor multidrug.
The technical scheme of the invention is as follows:
a method for preparing a size-convertible multi-responsive nano-drug, the method comprising the steps of:
(1) dissolving a medicine in a solvent, dripping the medicine solution into a glutaraldehyde aqueous solution, stirring for 3 min-3 h (preferably 1-2 h) at 5-45 ℃ (preferably 30-38 ℃), then mixing with a graphene oxide aqueous dispersion, stirring for 3 min-2 h (preferably 0.5-1 h) at 5-45 ℃ (preferably 30-38 ℃), then centrifuging, washing, and re-dispersing with the solvent for later use;
(2) dropwise adding a gelatin aqueous solution into the dispersion liquid obtained in the step (1), heating to 35-40 ℃, stirring for 1-2 h, centrifuging, washing, and dispersing again by using a solvent for later use;
(3) and (3) dropwise adding a bovine serum albumin aqueous solution into the dispersion liquid obtained in the step (2), heating to 35-40 ℃, stirring for 0.5-3 h, centrifuging, collecting precipitates, washing, and dispersing by using a solvent to prepare the size-convertible multi-response type nano-drug.
In the above-mentioned preparation method, the first step,
suitable drugs are, for example: doxorubicin hydrochloride, wherein the amount of the doxorubicin hydrochloride is 10-50 wt% of the mass of the glutaraldehyde, and the amount of the doxorubicin hydrochloride is 5-50 wt% of the mass of the graphene oxide;
the concentration of the glutaraldehyde aqueous solution is 0.1-5 wt%;
the mass ratio of the gelatin to the graphene oxide is 2-50: 1;
the concentration of the gelatin water solution is 0.1-4 wt%;
the mass ratio of bovine serum albumin to graphene oxide is 1-25: 1;
the concentration of the bovine serum albumin aqueous solution is 0.1-2 wt%;
the solvent is water or phosphate buffer solution;
the stirring speed is 500-1500 r/min;
the conditions of centrifugation were: 5000-15000 r/min, 5-30 min, preferably 9000-10000 r/min, 8-12 min.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the covalent bond of the adriamycin to the gelatin molecular chain is modified by glutaraldehyde, so that the selective release capacity of the nano-drug is greatly improved, the side effect on normal tissues is effectively reduced, and the dilemma of low drug utilization rate of the existing system is improved.
2. According to the invention, the graphene oxide is coated by two layers of bovine serum albumin and gelatin, so that the biocompatibility of the nano particles is obviously improved, and the protein adsorption resistance of the nano particles is enhanced. Meanwhile, the small-size bovine serum albumin released after the gelatin is degraded has the capability of penetrating deep tumors, and a large number of disulfide bonds in the molecular chain of the bovine serum albumin can greatly improve the response of the nano-drug to over-expressed GSH in the tumors and solve the problem of poor penetrating capability of the existing antitumor drug.
3. The invention utilizes the synergistic enhancement effect of chemotherapy and photothermal therapy to effectively improve the anti-tumor effect and has practical application value in solving the problem of tumor multidrug resistance.
Drawings
Fig. 1 is a TEM photograph of blank nanoparticles prepared in example 1.
Fig. 2 is a TEM photograph of the nano-drug a prepared in example 2.
Fig. 3 is a TEM photograph of the nano-drug B prepared in example 3.
Fig. 4 is a TEM photograph of the nano-drug C prepared in example 4.
Fig. 5 is a temperature rise curve of the nano-drug C prepared in example 4 by NIR excitation.
Fig. 6 is a cumulative release profile of doxorubicin after gelatinase pretreatment of nano-drug C prepared in example 4.
FIG. 7 is a cumulative release profile of doxorubicin under 10mM glutathione condition for Nanopadry C prepared in example 4.
Detailed Description
The technical solution of the present invention is further specifically described below by specific examples, but the scope of the present invention is not limited thereto.
Example 1:
3mL of gelatin aqueous solution (20mg/mL) was slowly dropped into 6mL of graphene oxide aqueous dispersion (1mg/mL), and the reaction was carried out at 37 ℃ with stirring at 1000r/min for 30 min. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of bovine serum albumin aqueous solution (10mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 1 hour. And after the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and redispersing by using 6mL of deionized water to prepare blank nano particles.
Example 2:
dissolving 3mg of doxorubicin hydrochloride in 3mL of deionized water, slowly dropping the solution into 1mL of glutaraldehyde solution (1 wt%), reacting at 37 ℃, and stirring at the rotation speed of 1000r/min for 2 hours. After the reaction, the mixture is dropped into 6mL of graphene oxide aqueous dispersion (1mg/mL), and the reaction temperature is 37 ℃, and the reaction is carried out for 30min by stirring at the rotating speed of 1000 r/min. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of gelatin aqueous solution (20mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 2 hours. And after the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing, and finally re-dispersing by using 6mL of deionized water to prepare the nano-drug A.
Example 3:
dissolving 3mg of doxorubicin hydrochloride into 3mL of deionized water, and slowly dropping the solution into 6mL of graphene oxide aqueous dispersion (1mg/mL), wherein the reaction temperature is 37 ℃, and the reaction is carried out for 30min by stirring at the rotation speed of 1000 r/min. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of gelatin aqueous solution (20mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 2 hours. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of bovine serum albumin aqueous solution (10mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 1 hour. And after the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing, and finally re-dispersing by using 6mL of deionized water to prepare the nano-drug B.
Example 4:
dissolving 3mg of doxorubicin hydrochloride in 3mL of deionized water, slowly dropping the solution into 1mL of glutaraldehyde solution (1 wt%), reacting at 37 ℃, and stirring at the rotation speed of 1000r/min for 2 hours. After the reaction, the mixture is dropped into 6mL of graphene oxide aqueous dispersion (1mg/mL), and the reaction temperature is 37 ℃, and the reaction is carried out for 30min by stirring at the rotating speed of 1000 r/min. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of gelatin aqueous solution (20mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 2 hours. After the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting precipitates, washing, and re-dispersing by using 6mL of deionized water. Then, 3mL of bovine serum albumin aqueous solution (10mg/mL) was slowly added dropwise thereto, and the mixture was stirred at a reaction temperature of 37 ℃ and a rotation speed of 1000r/min for 1 hour. And after the reaction is finished, centrifuging for 10min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing, and finally re-dispersing by using 6mL of deionized water to prepare the nano-drug C.
Example 5:
in order to determine the photothermal conversion performance of the nano-drug provided by the invention, near infrared light with the wavelength of 808nm is selected and respectively used at 2.0W/cm and 1.0W/cm2The nano-drug C dispersion was irradiated at a power density of (graphene oxide concentration, 600 μ g/mL) for 10 minutes, sampled every 30 seconds and a temperature rise curve was drawn. At 1.0W/cm2At a power density of 51.7 ℃ in 10 minutes, and when the temperature reaches 45 ℃, effective thermal ablation of cancer cells is achieved. Thus, it is possible to provideThe nano-drug provided by the invention can realize better photo-thermal treatment effect.
Example 6:
in order to determine the response capability of the nano-drug provided by the invention to gelatinase, a nano-drug C dispersion (graphene oxide concentration, 1mg/mL) was prepared and set as a control group and a gelatinase group, respectively. Control group: 1mL of the dispersion was placed in a dialysis bag with a cut-off of 8-14kDa and the in vitro drug release behavior studies were performed in 10mL of PBS buffer (pH 6.5) at 37 ℃. Samples were taken at 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 hours, 3mL each time with 3mL PBS buffer. Gelatinase group: on the basis of a control group, the nano-drug C is pretreated by 2 mug MMP-2 at 37 ℃ for 2 h. Then, samples were taken at 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 hours, respectively, and 3mL of PBS buffer was added to each sample while 3mL of the buffer was added. Each group was made in triplicate. And measuring the absorbance at 480nm by using a visible spectrophotometer, and drawing an in vitro drug release curve. Compared with a control group, after the nano-drug is pretreated by the gelatinase, the cumulative release amount of the adriamycin is improved by about 1.5 times, which shows that the nano-drug has better gelatinase responsiveness.
Example 7:
in order to determine the response capability of the nano-drug provided by the invention to glutathione, a nano-drug C dispersion solution (graphene oxide concentration, 1mg/mL) is prepared and respectively set as a control group and a glutathione group. Control group: 1mL of the dispersion was placed in a dialysis bag with a cut-off of 8-14kDa and the in vitro drug release behavior studies were performed in 10mL of PBS buffer (pH 5.0) at 37 ℃. Samples were taken at 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 hours, 3mL each time with 3mL PBS buffer. Glutathione group: 1mL of the dispersion was placed in a dialysis bag with a cut-off of 8-14kDa and subjected to in vitro drug release behavior studies at 37 ℃ in 10mL of PBS buffer (pH 5.0) containing 10mM glutathione. Samples were taken at 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 hours, respectively, 3mL each of which was supplemented with 3mL of PBS buffer containing 10mM glutathione. Each group was made in triplicate. And measuring the absorbance at 480nm by using a visible spectrophotometer, and drawing an in vitro drug release curve. Compared with a control group, the accumulative release amount of the adriamycin is improved by about 1.6 times under the condition of 10mM glutathione, which shows that the nano-drug has better redox responsiveness.
Comparative example 1:
according to the patent literature: a graphene targeted drug carrier, a preparation method and application thereof, and a graphene targeted drug carrier prepared by the method of a transportation box (CN 112093233A) embodiment.
The method comprises the following steps:
1) preparing a biotinylation target substance, namely dissolving and diluting the target substance by using a carbonate buffer solution to obtain a target substance diluent, dissolving N-hydroxysuccinimide biotin by using dimethyl sulfoxide to obtain an N-hydroxysuccinimide biotin diluent, mixing the target substance diluent and the N-hydroxysuccinimide biotin diluent, continuously stirring for 1-4h, dialyzing by using a phosphate buffer solution, and purifying by using a molecular sieve to obtain the biotinylation target substance;
2) preparing magnetic aminated graphene oxide, namely oscillating and resuspending streptavidin and carboxyl modified nano Fe3O4, adding aminated graphene oxide, rotationally mixing for 1-3h, and washing for 2-4 times by adopting a phosphate buffer solution to obtain the magnetic aminated graphene oxide;
3) magnetic target graphene, adding the biotinylation target substance obtained in the step 1) to the magnetic amination graphene oxide obtained in the step 2), rotating and uniformly mixing for 50-100min, performing magnetic separation, removing supernatant, and washing for 3-5 times by adopting phosphate buffer solution to obtain the magnetic target graphene;
4) preparing a graphene targeted drug carrier, namely oscillating and resuspending the magnetic targeted graphene obtained in the step 3), adding a loaded drug, rotating and uniformly mixing for 2-3h, magnetically separating, removing supernatant, and performing interactive dialysis by adopting a phosphate buffer solution and a borate buffer solution to obtain the graphene targeted drug carrier.
Comparing the nano-drug prepared in the embodiments 2, 3 and 4 of the present invention with the graphene-targeted drug carrier prepared in the comparative example 1, it can be seen that the product of the present invention has the following advantages:
(1) compared with NHS active ester, the glutaraldehyde has higher reaction efficiency, and avoids the use of organic solvent.
(2) The graphene oxide is coated with the bovine serum albumin and the gelatin in a double-layer manner, so that the protein adsorption resistance of the nano-drug is obviously enhanced, and the stability of the nano-drug in the blood circulation process is effectively improved. And secondly, the size conversion function of the nano-drug can be realized through the degradation of the gelatin, so that the capacity of effectively accumulating at a tumor part and penetrating to a deep tumor is achieved.
The above embodiments are merely representative examples of the present invention. It is obvious that the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. Variations that would be directly derivable by a person of ordinary skill in the art from the present disclosure are to be considered within the scope of the present invention.

Claims (8)

1. A preparation method of a size-convertible multiple-response nano-drug is characterized by comprising the following steps:
(1) dissolving a medicine in a solvent, dripping the medicine solution into a glutaraldehyde aqueous solution, stirring for 3 min-3 h at 5-45 ℃, then mixing with a graphene oxide aqueous dispersion, stirring for 3 min-2 h at 5-45 ℃, centrifuging, washing, and re-dispersing with the solvent for later use;
(2) dropwise adding a gelatin aqueous solution into the dispersion liquid obtained in the step (1), heating to 35-40 ℃, stirring for 1-2 h, centrifuging, washing, and dispersing again by using a solvent for later use;
(3) and (3) dropwise adding a bovine serum albumin aqueous solution into the dispersion liquid obtained in the step (2), heating to 35-40 ℃, stirring for 0.5-3 h, centrifuging, collecting precipitates, washing, and dispersing by using a solvent to prepare the size-convertible multi-response type nano-drug.
2. The method for preparing the size-convertible multi-response type nano-drug according to claim 1, wherein the drug in the step (1) is doxorubicin hydrochloride, the amount of the doxorubicin hydrochloride is 10-50 wt% of the mass of the glutaraldehyde, and the amount of the doxorubicin hydrochloride is 5-50 wt% of the mass of the graphene oxide.
3. The method for preparing size-convertible multi-responsive nano-drug according to claim 1, wherein the concentration of the glutaraldehyde aqueous solution in step (1) is 0.1 to 5 wt%.
4. The method for preparing the size-convertible multi-response type nano-drug according to claim 1, wherein the mass ratio of the gelatin in the step (2) to the graphene oxide in the step (1) is 2-50: 1.
5. the method for preparing size-convertible multi-responsive nano-drug according to claim 1, wherein the concentration of the aqueous solution of gelatin in the step (2) is 0.1 to 4 wt%.
6. The method for preparing the size-convertible multi-response type nano-drug according to claim 1, wherein the mass ratio of the bovine serum albumin in the step (3) to the graphene oxide in the step (1) is 1-25: 1.
7. the method for preparing size-convertible multi-responsive nano-drug according to claim 1, wherein the concentration of the aqueous solution of bovine serum albumin in the step (3) is 0.1-2 wt%.
8. The method for preparing a size-convertible multi-responsive nano-drug according to claim 1, wherein in the steps (1) to (3), the solvent is water or a phosphate buffer.
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