CN112138156A - Preparation method of multiple-response nano-drug based on combination therapy - Google Patents
Preparation method of multiple-response nano-drug based on combination therapy Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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Abstract
The invention provides a design and preparation method of a multi-response type nano-drug based on combined therapy, which comprises the steps of loading adriamycin and indocyanine green on graphene oxide, coating the graphene oxide with gelatin, obtaining nano-particles with uniform size by a secondary emulsification method, and finally crosslinking with N, N' -bis (acryloyl) cystamine to obtain the multi-response type nano-drug; according to the invention, gelatin is used as a nanoparticle shell layer, so that the biocompatibility is improved, the size conversion capability is realized, the penetration effect of the nano-drug on a tumor part is improved, the combined treatment can be realized through the adriamycin, the indocyanine green and the graphene oxide, and the anti-tumor effect is remarkably improved.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a design and a preparation method of an anti-tumor drug delivery system.
Background
Conventional cancer treatment methods include surgical treatment, chemotherapy, radiation therapy, and the like. However, surgical resection has a high recurrence rate and chemotherapy and radiotherapy have significant side effects on normal tissues. Therefore, drug delivery systems are developed, and liposomes, micelles, hydrogels, and the like are common. However, a single treatment mode cannot achieve ideal effects, and at present, a combined treatment method is established mainly by integrating multiple treatment modes into one system, so that better anti-tumor effect is achieved.
Emerging cancer treatment modalities are photothermal therapy (PTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and immunotherapy, among others. Among them, PTT and PDT are being studied more and more, and PTT induces thermal ablation of cancer cells by converting light energy into thermal energy under Near Infrared (NIR) triggering using photothermal conversion agents. Commonly used photothermal conversion agents are carbonyl nanoparticles, noble metal nanoparticles, organic dyes, semiconductor nanoparticles, and the like. PDT mainly utilizes photosensitizer to absorb photon and then jump to excited state, and then transfers energy to molecular oxygen to generate singlet oxygen to kill cancer cells. The photosensitizer mainly comprises porphyrin, anthraquinone, xanthine, anthocyanidin and curcuminoid. For example, CN108371714A discloses a GO-gelatin-FA drug carrier with targeting ability, and CN104306325A discloses an anti-tumor hydrogel of glutaraldehyde-crosslinked graphene oxide and collagen. In the above research, although the graphene oxide and gelatin drug carrier or hydrogel is proposed and prepared, and beneficial work is done on the aspects of drug targeting ability and biocompatibility, the used cross-linking agent has certain toxicity, and the problems of controlled release of the drug, size conversion of the drug, insufficient responsiveness of a tumor microenvironment and the like exist.
In order to solve the technical problems, the gelatin and graphene oxide are used for preparing the gelatin-coated graphene oxide nano particles to form the core-shell structure, and matrix metalloproteinases (MMP-2 and MMP-9) overexpressed in a tumor microenvironment are used for degrading gelatin shell layers, so that the multi-level size conversion of the nano particles is realized, and the capacity of effectively accumulating at tumor parts and penetrating deep tumors is realized. And then crosslinking N, N' -di (acryloyl) cystamine (BAC) to prepare the reduced Glutathione (GSH) responsive nano particles, thereby realizing the reductive response and release of the drug and effectively consuming GSH in the tumor environment to improve the chemotherapeutic effect. The graphene oxide can effectively load medicines such as adriamycin and indocyanine green to realize combined treatment of chemotherapy, photothermal therapy and photodynamic therapy, and the excellent photothermal conversion capacity of the graphene oxide can further promote the release of the adriamycin to enhance the effect of the chemotherapy.
Disclosure of Invention
In order to overcome the defects of obvious toxic and side effects and poor treatment effect of the conventional cancer treatment method, the invention provides a novel drug delivery system, which integrates chemotherapy, photothermal therapy and photodynamic therapy into one system to realize the controllable release of drugs on time and space and the synergistic effect of multiple treatment modes.
When the nanoparticles are accumulated at the tumor part, the gelatin shell layer can be degraded by MMP-2 and the like, so that the size conversion of the nanoparticles is realized, GO with smaller size is released to effectively penetrate to the deep tumor, and the problem of poor penetrating capability of common antitumor drugs is solved. In addition, the BAC crosslinking enables the nanoparticles to have reductive response and effectively consume GSH, so that the chemotherapeutic effect is improved. Compared with the traditional cross-linking agent with disulfide bonds, the BAC cross-linking avoids a complex group activation process and simplifies the experimental steps.
The technical scheme of the invention is as follows:
a preparation method of a multi-response type nano-drug based on combination therapy comprises the following steps:
(1) dissolving a medicine in a solvent, mixing the medicine with the graphene oxide aqueous dispersion, stirring for 3 min-2 h (preferably 0.5-1 h) at 5-45 ℃ (preferably 35-38 ℃), centrifuging (5000-15000 r/min, 5-30 min), washing, and re-dispersing with the solvent for later use;
the drugs are specifically exemplified by: doxorubicin hydrochloride and indocyanine green, wherein the dose of the doxorubicin hydrochloride is 5-50 wt% of graphene oxide, and the dose of the indocyanine green is 5-50 wt% of graphene oxide;
the solvent is water or phosphate buffer solution;
(2) mixing the dispersion liquid prepared in the step (1) with a gelatin aqueous solution, heating to 35-45 ℃, and stirring at a rotating speed of 500-1500 r/min for 0.5-4 h to obtain a drug delivery system precursor;
the mass ratio of the gelatin to the graphene oxide is 5-50: 1, the type of gelatin is type a gelatin;
(3) dropping the drug delivery system precursor obtained in the step (2) into a dichloromethane solution of dioctyl sodium sulfosuccinate, stirring and emulsifying at a rotation speed of 500-1500 r/min for 1-30 min (preferably 10min), dropping the obtained emulsion into an aqueous solution of polyvinyl alcohol (Mw: 600-205000, alcoholysis degree: 72.5-99 mol%), stirring and emulsifying at a rotation speed of 500-1500 r/min for 1-30 min (preferably 10min), adding N, N' -bis (acryloyl) cystamine, reacting at a temperature of 25-40 ℃ (preferably 38 ℃) and a stirring speed of 500-1500 r/min for 6-18 h (preferably 12h), centrifuging (10000r/min, 20min), collecting precipitates, washing, and dispersing with water or a multiple phosphate buffer solution to prepare the responsive nano-drug;
the mass ratio of the dioctyl sodium sulfosuccinate to the polyvinyl alcohol to the N, N' -bis (acryloyl) cystamine to the graphene oxide is 25-2500: 50-5000: 1-25: 1;
the concentration of the dichloromethane solution of dioctyl sodium sulfosuccinate is 1-3 wt%;
the concentration of the aqueous solution of the polyvinyl alcohol is 1-3 wt%.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the graphene oxide is used for loading the anti-cancer drug, pH response drug release is achieved, and the drug release is effectively triggered while the photothermal therapy is realized through NIR.
2. The invention obviously improves the biocompatibility of the nano particles by utilizing the characteristics that the gelatin can be degraded and the degradation product has no side effect on organisms. Meanwhile, the graphene oxide with smaller size released after the gelatin is degraded has stronger tumor penetrating capability, can act on deep tumors, and solves the problem of poor penetrating capability of the existing common antitumor drugs.
3. The invention designs and prepares a drug delivery system with multiple treatment modes for synergistic enhancement and multiple responses of a tumor microenvironment, and compared with the prior art, the drug delivery system has lower toxic and side effects, higher stability and higher drug delivery efficiency.
Drawings
Fig. 1 is a TEM photograph of the nano-delivery system prepared in example 1.
Fig. 2 is a TEM photograph of the nano-delivery system prepared in example 2.
Fig. 3 is a TEM photograph of the nano-delivery system prepared in example 3.
Fig. 4 is a TEM photograph of the nano-delivery system prepared in example 4.
Figure 5 is a temperature ramp curve under NIR triggering for the nano delivery system prepared in example 2.
Fig. 6 is a cumulative release profile of doxorubicin from the nano-delivery system prepared in example 2.
Detailed Description
The technical solution of the present invention is further specifically described below by way of examples, but the scope of the present invention is not limited thereto.
Example 1: 2mL of gelatin aqueous solution (25mg/mL) was added to a sample bottle containing 1mL of graphene oxide aqueous dispersion (1mg/mL), and the mixture was stirred at a reaction temperature of 45 ℃ and a rotation speed of 1000r/min for 2 hours. After the reaction is finished, dripping the mixture into 10mL of dichloromethane solution (25mg/mL) of dioctyl sodium sulfosuccinate, stirring and emulsifying for 10min at the rotating speed of 1000r/min, then dripping the emulsion into 45mL of polyvinyl alcohol (1788 type) aqueous solution (20mg/mL), stirring and emulsifying for 10min at the rotating speed of 1000r/min, and finally adding 5mg of N, N' -bis (acryloyl) cystamine, wherein the reaction temperature is 38 ℃, the stirring speed is 800r/min, and the reaction time is 12 h. And after the reaction is finished, centrifuging for 20min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing for 3 times by using pure water, and finally dispersing by using 10mL of pure water to prepare the nano delivery system.
Example 2: 0.5mg of doxorubicin hydrochloride was dissolved in 1mL of deionized water, and then slowly dropped into a sample bottle containing 1mL of an aqueous dispersion of graphene oxide (1mg/mL), and the mixture was stirred at a rotation speed of 500r/min at a reaction temperature of 37 ℃ for 1 hour. After the reaction, the reaction mixture was centrifuged at 10000r/min for 20min by a high-speed centrifuge, the supernatant was removed, and the precipitate was dispersed with pure water, centrifuged again (5 mL. times.3), and finally dispersed with 2mL of pure water. Then, 1mL of an aqueous gelatin solution (25mg/mL) was added to the dispersion, and the mixture was stirred at a rotation speed of 1000r/min at a reaction temperature of 45 ℃ for 2 hours. After the reaction is finished, dripping the mixture into 10mL of dichloromethane solution (25mg/mL) of dioctyl sodium sulfosuccinate, stirring and emulsifying for 10min at the rotating speed of 1000r/min, then dripping the emulsion into 45mL of polyvinyl alcohol (1788 type) aqueous solution (20mg/mL), stirring and emulsifying for 10min at the rotating speed of 1000r/min, and finally adding 5mg of N, N' -bis (acryloyl) cystamine, wherein the reaction temperature is 38 ℃, the stirring speed is 800r/min, and the reaction time is 12 h. And after the reaction is finished, centrifuging for 20min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing for 3 times by using pure water, and finally dispersing by using 10mL of pure water to prepare the nano delivery system.
Example 3: 0.5mg of doxorubicin hydrochloride and 0.5mg of indocyanine green are dissolved in 1mL of deionized water respectively, and then slowly dropped into a sample bottle containing 1mL of graphene oxide aqueous dispersion (1mg/mL), and the mixture is stirred at the rotation speed of 500r/min, the reaction temperature is 37 ℃, and the reaction time is 1 hour. After the reaction, the reaction mixture was centrifuged at 10000r/min for 20min by a high-speed centrifuge, the supernatant was removed, and the precipitate was dispersed with pure water, centrifuged again (5 mL. times.3), and finally dispersed with 2mL of pure water. Then, 1mL of an aqueous gelatin solution (25mg/mL) was added to the dispersion, and the mixture was stirred at a rotation speed of 1000r/min at a reaction temperature of 45 ℃ for 2 hours. After the reaction is finished, dripping the mixture into 10mL of dichloromethane solution (25mg/mL) of dioctyl sodium sulfosuccinate, stirring and emulsifying for 10min at the rotating speed of 1000r/min, then dripping the emulsion into 45mL of polyvinyl alcohol (1788 type) aqueous solution (20mg/mL), stirring and emulsifying for 10min at the rotating speed of 1000r/min, and finally adding 5mg of N, N' -bis (acryloyl) cystamine, wherein the reaction temperature is 38 ℃, the stirring speed is 800r/min, and the reaction time is 12 h. And after the reaction is finished, centrifuging for 20min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing for 3 times by using pure water, and finally dispersing by using 10mL of pure water to prepare the nano delivery system.
Example 4: 0.2mg of doxorubicin hydrochloride and 0.2mg of indocyanine green are dissolved in 0.5mL of deionized water respectively, and then slowly dropped into a sample bottle containing 1mL of graphene oxide aqueous dispersion (1mg/mL), and the mixture is stirred at the rotation speed of 500r/min, the reaction temperature is 37 ℃, and the reaction time is 1 hour. After the reaction, the reaction mixture was centrifuged at 10000r/min for 20min by a high-speed centrifuge, the supernatant was removed, and the precipitate was dispersed with pure water, centrifuged again (5 mL. times.3), and finally dispersed with 2mL of pure water. Then, 2mL of an aqueous gelatin solution (25mg/mL) was added to the dispersion, and the mixture was stirred at a rotation speed of 1000r/min at a reaction temperature of 45 ℃ for 2 hours. After the reaction is finished, dripping the mixture into 10mL of dichloromethane solution (25mg/mL) of dioctyl sodium sulfosuccinate, stirring and emulsifying for 10min at the rotating speed of 1000r/min, then dripping the emulsion into 45mL of polyvinyl alcohol (1788 type) aqueous solution (10mg/mL), stirring and emulsifying for 10min at the rotating speed of 1000r/min, finally adding 25mg of N, N' -bis (acryloyl) cystamine, reacting at the temperature of 38 ℃, stirring at the speed of 800r/min, and reacting for 12 h. And after the reaction is finished, centrifuging for 20min at the rotating speed of 10000r/min by using a high-speed centrifuge, collecting the precipitate, washing for 3 times by using pure water, and finally dispersing by using 10mL of pure water to prepare the nano delivery system.
Example 5: in order to detect the photo-thermal property of the nano-drug provided by the invention, laser with the wavelength of 808nm is selected and used at the rate of 1.0W/cm2The nano-drug dispersion of 10mg/mL was irradiated for 10 minutes at the power density of (1), the temperature change was recorded every 30 seconds and the temperature rise curve was plotted. Under NIR triggering, the samples reach 62.3 ℃ in 10 minutes and above 50 ℃ in 2 minutes. It takes 15-60 minutes to kill the tumor cells at 42 deg.C, while the duration can be shortened to 4-6 minutes when the temperature reaches 50 deg.C. Therefore, the nano-drug provided by the invention can realize better photo-thermal treatment effect.
Example 6: in order to detect the laser-triggered drug release capacity of the nano-drug provided by the invention, 10mg/mL nano-drug dispersion liquid is prepared and respectively set as a control group and a laser group. Control group: 1mL of the dispersion was placed in a dialysis bag with a cut-off of 8-14kDa in 10mL of PBS buffer (pH 6.5) and drug release was performed in the phantom at 37 ℃. At 1, 2, 3, 4, 6, 8, 10, 12, 24 and 48 hoursSamples were taken 3mL each time and supplemented with 3mL PBS buffer. Laser group: on the basis of the control group, a laser with a wavelength of 808nm was used at a wavelength of 1.0W/cm2The power density of (a) was irradiated for 10 minutes before sampling at 1 st, 2 th, 4 th and 8 th hours, respectively. 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 NIR triggering, the release rate and the accumulated release amount of DOX are obviously improved, which shows that DOX can be further released by laser fixed-point triggering, and the anti-tumor effect is better.
Comparative example 1: according to the patent: a graphene oxide nano drug delivery process method and an application (patent number: CN 108371714A) thereof to prepare an adriamycin-graphene oxide nano drug delivery carrier.
The method comprises the following steps:
(1) acidification of Graphene Oxide (GO): preparing graphene oxide by using a Hummers method, adding 100mL of nitric acid, refluxing for 12h at 120 ℃, cooling and filtering, repeatedly washing filter residues by using distilled water, and drying to obtain acidified graphene oxide;
(2) synthesis of folate-activated lipid (NHS-FA): adding 1mol of folic acid into ethanol, sequentially adding 3mol of DCC and 2mol of NHS into a condensing agent, reacting at 60 ℃ in a dark place for 12 hours, stopping the reaction, and performing column chromatography to obtain NHS-FA;
(3) synthesis of gelatin-FA conjugate: dissolving 1.5mol of gelatin in a PB buffer solution with the pH value of 4.5-5.0, dropwise adding 1mol of ethanol solution of NHS-FA, adding 1mol of EDC, and reacting overnight in a dark place to obtain a gelatin-FA conjugate;
(4) synthesis of GO-gelatin-FA vector: adding 1mol of acidified graphene oxide into water, performing ultrasonic treatment at 500W for 20min to uniformly disperse the oxidized graphene oxide, dropwise adding 1mol of ethanol solution of gelatin-FA conjugate, adding 1mol of EDC, stirring to react for 12h, filtering, and repeatedly washing filter residues with distilled water to obtain the white GO-gelatin-FA carrier.
Comparing the multi-response nano-drug prepared in the embodiments 2, 3 and 4 of the present invention with the adriamycin-graphene oxide nano-drug delivery carrier prepared in the comparative example 1, it can be seen that the product of the present invention has the following advantages:
(1) the use of drugs with certain cytotoxicity such as EDC, DCC and NHS is avoided.
(2) By gelatin coating GO, an effective size conversion is achieved, thereby more effectively accumulating at the tumor site and penetrating to the deep tumor.
(3) The high medicine-loading capacity and good photo-thermal conversion capacity of GO are fully utilized, and through NIR triggering, the medicine can be effectively released at fixed points while photo-thermal treatment is realized, so that the cell compatibility is improved, and the anti-tumor effect is enhanced.
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 (7)
1. A preparation method of a multi-response type nano-drug based on combination therapy is characterized by comprising the following steps:
(1) dissolving a drug in a solvent, mixing the drug with the graphene oxide aqueous dispersion, stirring the mixture for 3min to 2h at the temperature of 5 to 45 ℃, centrifuging the mixture, washing the mixture, and re-dispersing the mixture by using the solvent for later use;
(2) mixing the dispersion liquid prepared in the step (1) with a gelatin aqueous solution, heating to 35-45 ℃, and stirring at a rotating speed of 500-1500 r/min for 0.5-4 h to obtain a drug delivery system precursor;
(3) dropping the drug delivery system precursor obtained in the step (2) into a dichloromethane solution of dioctyl sodium sulfosuccinate, stirring and emulsifying at a rotating speed of 500-1500 r/min for 1-30 min, dropping the obtained emulsion into a water solution of polyvinyl alcohol, stirring and emulsifying at a rotating speed of 500-1500 r/min for 1-30 min, adding N, N' -bis (acryloyl) cystamine, reacting at a temperature of 25-40 ℃ and a stirring speed of 500-1500 r/min for 6-18 h, centrifuging, collecting precipitates, washing, and dispersing with water or a phosphate buffer solution to obtain the multi-response type nano drug.
2. The method for preparing a multi-response type nano-drug based on a combination therapy according to claim 1, wherein in the step (1), the drugs are doxorubicin hydrochloride and indocyanine green, and the doxorubicin hydrochloride is 5-50 wt% of the graphene oxide, and the indocyanine green is 5-50 wt% of the graphene oxide.
3. The method for preparing a multiple-response type nano-drug based on combination therapy according to claim 1, wherein the solvent is water or phosphate buffer in step (1).
4. The preparation method of the combination therapy-based multi-response type nano-drug according to claim 1, wherein in the step (2), the mass ratio of the gelatin to the graphene oxide is 5-50: 1, the type of gelatin is type a gelatin.
5. The method for preparing the multi-response type nano-drug based on the combination therapy according to claim 1, wherein in the step (3), the mass ratio of the dioctyl sodium sulfosuccinate, the polyvinyl alcohol, the N, N' -bis (acryloyl) cystamine and the graphene oxide is 25-2500: 50-5000: 1-25: 1.
6. the method for preparing a multi-responsive nano-drug based on combination therapy according to claim 1, wherein in the step (3), the concentration of the dioctyl sodium sulfosuccinate in dichloromethane is 1-3 wt%.
7. The method for preparing a multi-responsive nano-drug based on combination therapy according to claim 1, wherein in the step (3), the concentration of the aqueous solution of the polyvinyl alcohol is 1 to 3 wt%.
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