CN109589402B - Preparation method and application of multi-effect nano material with targeted photothermal therapy and controllable drug release - Google Patents
Preparation method and application of multi-effect nano material with targeted photothermal therapy and controllable drug release Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a multi-action nano material with targeted photo-thermal treatment and controllable drug release. The multi-action nano material with targeted photothermal therapy and controllable drug release in the invention takes glucose as a targeted molecule, is connected to the polydopamine nano material with photothermal conversion capacity through a covalent bond, and carries an anti-tumor drug through the polydopamine nano material. The multi-action nano material prepared by the invention has multiple functions of targeting, photo-thermal and pH response drug release, can realize specific accumulation and drug release of tumor tissues, reduce the toxic and side effects of chemotherapeutic drugs on human bodies, can aim at different weaknesses of the tumor tissues from multiple angles, achieve the effect of efficiently treating tumors in a combined manner, has high biocompatibility, no toxicity, no pollution, simple preparation process and lower cost, and is suitable for mass production and clinical medical use.
Description
Technical Field
The invention relates to a preparation method and application of a multi-action nano material with targeted photo-thermal treatment and controllable drug release, belonging to the technical field of medicaments for treatment.
Background
Tumors are the second leading cause of death worldwide, and traditional surgical treatment, radiotherapy and chemotherapy have the defects of high invasiveness, high side effects and the like, so that new treatment means and strategies need to be developed urgently. In the tumor treatment, aiming at the weak points of tumor tissues, a therapeutic agent with multiple functions is developed, the anti-tumor effect is improved, and the toxic and side effects are reduced, so that the method is an important strategy for tumor treatment. The main characteristic of tumor cells is sustained, rapid proliferation, which increases glucose uptake for glycolysis to provide the energy required by the tumor cells themselves. Studies have shown that tumor cells often overexpress Glucose Transporters (Gluts), a transmembrane Glucose receptor on the cell membrane to accomplish uptake of large amounts of Glucose. Therefore, the Gluts can be used as a target spot for tumor treatment, and drugs and materials for tumor detection and treatment can be targeted to tumor parts through the targeting capability of glucose and analogues thereof to the Gluts, so that the treatment effect can be greatly improved, and the toxic and side effects can be remarkably reduced.
Due to abnormal vascularity within the tumor tissue, the tumor tissue is more sensitive to hyperthermia than normal tissue. Photothermal therapy (Photothermal therapy) is a minimally invasive tumor therapy technology developed in recent years, and near-infrared light energy is converted into heat energy by using a Photothermal conversion reagent, so that coagulation necrosis or apoptosis of tumor cells caused by local temperature rise of tumors can be realized, and specific inhibition of tumors can be realized, so that Photothermal therapy is regarded as one of the most potential tumor therapy technologies. Polydopamine (PDA) is a biomimetic material derived from marine mussel mucin, has the advantages of high biocompatibility, thermal stability and the like, has good biocompatibility in vivo, is non-toxic and can be biodegraded. Recently, research reports that polydopamine has excellent photothermal conversion efficiency under near infrared conditions, and can be used as a photothermal conversion agent for treating tumors in vivo. Polydopamine also carries reactive groups, such as catechol groups. The group can be oxidized into a quinone group under the condition of alkaline oxygen introduction, and can perform Schiff base reaction or Michael addition reaction with a primary amino group to form a C-N covalent bond so as to connect molecules to which the primary amino group belongs. The property of the polydopamine enables the polydopamine to form covalent connection with molecules with primary ammonia under mild conditions, and the polydopamine is used for further chemical modification.
The traditional anti-tumor chemotherapeutic drugs often lack selectivity, are widely distributed in vivo after administration, have large toxic and side effects on normal tissues and organs under the treatment dosage, bring great pain to patients and have poor curative effect. There is therefore an urgent need to develop more efficient methods and routes for targeted delivery of drugs to the target site. By adopting a targeted drug delivery system, the drug can be specifically delivered to a target site, the drug concentration of the target site is improved, the systemic toxic and side effects of the chemotherapeutic drug are effectively reduced, the drug effect is improved, and the dosage is reduced. Due to the high-speed metabolism of the tumor, the pH of the tumor microenvironment is lower than that outside normal tissue cells, and the controllable drug release of pH response is designed aiming at the characteristic of low pH outside tumor cells, so that the accurate effect of a targeted drug delivery system can be further improved, and the efficient release of the drug at tumor parts is ensured.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and application of a multi-action nano material with targeted photothermal therapy and controllable drug release. The multi-action nano material with targeted photothermal therapy and controllable drug release is characterized in that glucose is used as a targeted molecule, the targeted molecule is connected to a polydopamine nano material with photothermal conversion capacity through a covalent bond, and an anti-tumor drug is loaded through the polydopamine nano material, so that the pH response drug release of a tumor microenvironment is realized.
The technical scheme of the invention is as follows:
a preparation method of a multi-action nano material with targeted photo-thermal treatment and controllable drug release comprises the following steps:
(1) preparing a dopamine hydrochloride aqueous solution with the concentration of 0.01-5mg/mL, adding the dopamine hydrochloride aqueous solution into an ammonia water/ethanol/deionized water solution, wherein the volume ratio of the dopamine hydrochloride aqueous solution to the ammonia water/ethanol/deionized water solution is 1 (2-30), stirring and reacting for 6-48h at the temperature of 10-60 ℃, and after the reaction is finished, separating, washing and freeze-drying to obtain a poly-dopamine nano material;
(2) adding the polydopamine nano material prepared in the step (1) and glucosamine hydrochloride into a sodium phosphate buffer solution with the concentration of 1-50mM, wherein the concentration of the polydopamine nano material in the sodium phosphate buffer solution is 2-20mg/mL, and the mass ratio of the polydopamine nano material to the glucosamine hydrochloride is 1: (1-100), stirring at 10-60 ℃ for reaction for 4-36h, and after the reaction is finished, separating, washing and freeze-drying to obtain a poly-dopamine nano material connected with glucose;
(3) resuspending the glucose-linked polydopamine nano-material prepared in the step (2) in 1-50mM Tris buffer solution to prepare a nano-material system with the concentration of 0.1-500 mg/mL; dissolving the anti-tumor drug bortezomib in 1-50mM Tris buffer solution to prepare a bortezomib solution with the concentration of 0.05-100 mg/mL; and dropwise adding the bortezomib solution into the nano material system under the stirring condition, wherein the mass ratio of the bortezomib to the poly-dopamine nano material connected with glucose is 1 (5-50), stirring and reacting for 8-24h at 25-45 ℃, and separating and washing after the reaction is finished to obtain the multi-action nano material with targeted photothermal therapy and controllable drug release.
According to the invention, the ammonia water/ethanol/deionized water solution in the step (1) is obtained by mixing ammonia water, absolute ethyl alcohol and deionized water, and stirring for 10-90 minutes at 10-60 ℃, wherein the volume ratio of the ammonia water to the absolute ethyl alcohol to the deionized water is 1: (2-320): (3-600).
According to the invention, the ammonia water/ethanol/deionized water solution in the step (1) is obtained by mixing ammonia water, absolute ethyl alcohol and deionized water, and stirring for 20-60 minutes at 20-45 ℃, wherein the volume ratio of the ammonia water to the absolute ethyl alcohol to the deionized water is 1: (5-80): (11-150).
More preferably, the mass concentration of the ammonia water is 25-28%.
According to the invention, the stirring reaction condition in the step (1) is that the stirring reaction is carried out for 10-24h at the temperature of 20-45 ℃.
Preferably, the concentration of the sodium phosphate buffer solution in the step (2) is 5-25mM, and the pH value is 7.0-9.5.
According to the invention, the mass ratio of the polydopamine nano-material to the glucosamine hydrochloride in the step (2) is 1: (1-50); further preferably, the mass ratio of the polydopamine nano-material to the glucosamine hydrochloride is 1 (5-50).
According to the invention, the stirring reaction condition in the step (2) is preferably 20-60 ℃ for 8-24 h.
Preferably, the concentration of the Tris buffer in the step (3) is 5-25mM, and the pH value is 7.8-10.
Preferably, according to the present invention, the concentration of the nanomaterial system in step (3) is 0.16-8 mg/mL.
Preferably, according to the present invention, the concentration of the bortezomib solution in step (3) is 0.5-40 mg/mL.
According to the invention, the rotation speed of the stirring reaction in the steps (1), (2) and (3) is preferably 50-300 rpm.
Preferably according to the present invention, the separation in steps (1), (2) and (3) is performed by high-speed centrifugation at 12000rpm for 10-60min at 6000-.
Preferably, according to the invention, the washing described in steps (1) and (2) is carried out 2 to 5 times with 1 to 50mM sodium phosphate buffer.
According to the invention, the washing in the step (3) is preferably performed 2 to 5 times by using deionized water.
The multi-action nano material with targeted photothermal therapy and controllable drug release prepared by the preparation method is applied to preparing a tumor targeted drug for combined therapy of photothermal therapy and chemotherapy.
The invention has the technical characteristics and beneficial effects that:
1. the invention connects glucosamine to the surface of the polydopamine nano material through a covalent bond for the first time, and utilizes the targeting capability of the glucose to deliver the polydopamine nano material to a tumor part in a targeted manner; the polydopamine is used as a photothermal conversion agent, can efficiently convert near infrared light into heat energy, and achieves the purpose of increasing the local temperature of the tumor to inhibit the tumor.
2. The glucose-linked polydopamine nano material prepared by the invention has a catechol structure, and can be combined with a medicament containing boric acid in a weakly alkaline environment to form boric acid ester; the boric acid ester bond is unstable in a weak acid environment, such as a tumor microenvironment, and can hydrolyze and release the medicament containing boric acid, so that the glucose-linked polydopamine nano-material loaded anti-tumor medicament bortezomib can release the medicament in the tumor microenvironment with slightly acidic pH, and the tumor is inhibited through the inhibition effect of the bortezomib on tumor cell proteasome.
3. The prepared multi-action nano material with targeted photothermal therapy and controllable drug release has multiple functions of targeted drug release, photothermal drug release and pH response drug release, can realize specific accumulation and drug release of tumor tissues, reduce the toxic and side effects of chemotherapeutic drugs on human bodies, and can aim at different weaknesses of the tumor tissues from multiple angles to achieve the effect of efficiently treating tumors in a combined manner. At present, no relevant report exists for connecting glucose with polydopamine with photothermal conversion capability and carrying an antitumor drug bortezomib. The prepared multi-action nano material targets a photothermal conversion agent polydopamine to a tumor part by utilizing the targeting capability of glucose to carry out photothermal treatment; meanwhile, the poly-dopamine carries the bortezomib, the pH response release is realized in the acidic microenvironment of the tumor, the tumor is treated in a targeted combination manner, and the tumor can be effectively inhibited.
4. The multi-action nano material with targeted photothermal therapy and controllable drug release, prepared by the invention, has the advantages of high biocompatibility, no toxicity, no pollution, simple preparation process and lower cost, and is suitable for large-scale production and clinical medical treatment.
Drawings
FIG. 1 is a transmission electron micrograph of a glucose-linked polydopamine nanomaterial;
FIG. 2 is a Fourier transform infrared spectrum of glucose-linked polydopamine nanomaterials;
in the figure, the ordinate represents transmittance (%), and the abscissa represents wavelength (cm)-1)；
FIG. 3 is the efficiency of glucose-linked polydopamine nanomaterials to convert near infrared light to heat;
in the figure, the ordinate is temperature (. degree. C.) and the abscissa is time(s);
FIG. 4 shows the release of the multi-functional nanomaterial with targeted photothermal therapy and controlled release under different pH conditions;
in the figure, the ordinate represents cumulative release rate (%), and the abscissa represents time (h);
FIG. 5 is a histogram of the survival rate of tumor cells treated with glucose-linked polydopamine nanomaterials under near-infrared illumination;
FIG. 6 is a live image of the tumor targeting ability of the glucose linked polydopamine nanomaterials in vivo;
FIG. 7 shows the therapeutic effect of the multi-functional nanomaterial with targeted photothermal therapy and controlled drug release on tumor-bearing mice;
in the figure, A is a treatment group and B is a control group.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
The materials and reagents used in the following examples are commercially available biological and chemical laboratory materials unless otherwise specified.
Example 1 preparation of Multi-acting nanomaterials with Targeted photothermal therapy and controlled drug Release
A preparation method of a multi-action nano material with targeted photo-thermal treatment and controllable drug release comprises the following steps:
(1) preparation of poly-dopamine nano material
1.5mL of ammonia water (with the mass concentration of 25-28%), 45mL of absolute ethyl alcohol and 80mL of deionized water are put in a round-bottom flask and stirred for 20 minutes at 35 ℃ to obtain an ammonia water/ethyl alcohol/deionized water solution; weighing 30mg of dopamine hydrochloride (purchased from Sigma Aldrich, H8502-5G) and dissolving in 10mL of deionized water to obtain a dopamine hydrochloride aqueous solution; adding the dopamine hydrochloride aqueous solution into the ammonia water/ethanol/deionized water solution, stirring at 35 ℃ and 150rpm for reaction for 30 hours, centrifuging at 8000rpm for 30 minutes after the reaction is finished, collecting the precipitate, washing the obtained precipitate for 3 times by using 15mM sodium phosphate buffer solution, and freeze-drying and storing by using a freeze-dryer to obtain the poly-dopamine nano material;
(2) preparation of glucose-linked polydopamine nano-material
Weighing 1G of the polydopamine nano material prepared in the step (1) and 5G of glucosamine hydrochloride (purchased from Sigma Aldrich, G4875-25G) (the proportion is 1: 5), adding the polydopamine nano material and the glucosamine hydrochloride into 100mL of 15mM sodium phosphate buffer solution with the pH value of 8.5, stirring and reacting at 40 ℃ and 150rpm for 20 hours, after the reaction is finished, centrifuging at 8000rpm for 30 minutes, collecting precipitate, washing the precipitate for 3 times by using the 15mM sodium phosphate buffer solution, and freeze-drying the precipitate in a freeze-dryer to obtain the glucose-linked polydopamine nano material;
the Transmission Electron Microscope (TEM) detection of the glucose-linked polydopamine nano-material is shown in figure 1, and the average particle size is about 200 nm; the infrared spectrum of the sample is detected by a Fourier infrared spectrometer, and the result is shown in figure 2, and a characteristic peak at about 1034nm represents that glucosamine and polydopamine are connected through a C-N bond;
(3) preparation of multi-action nano material with targeted photo-thermal treatment and controllable drug release
Weighing 300mg of the glucose-linked polydopamine nanomaterial prepared in the step (2), and resuspending the polydopamine nanomaterial by using 100mL of 10mM Tris buffer solution with the pH of 8.0 to obtain a nanomaterial system; weighing 20mg of bortezomib, and dissolving the bortezomib in 10mL of 10mM Tris buffer solution with the pH value of 8.0 to obtain a bortezomib solution; dropwise adding the bortezomib solution into the nano material system under the stirring condition, stirring at 35 ℃ and 150rpm for reaction for 10h, centrifuging at 8000rpm for 20 min after the reaction is finished, collecting precipitate and supernatant, washing the precipitate with deionized water for 3 times to obtain the multi-action nano material with targeted photothermal therapy and controllable drug release, and collecting washing liquid.
Detecting the drug loading rate of the glucose-linked polydopamine nano material to the anti-tumor drug bortezomib:
and (3) combining the washing liquid and the supernatant liquid collected in the step (3) into a liquid to be detected, calculating the volume of the liquid, analyzing the bortezomib standard substance by using HPLC (high performance liquid chromatography), making a standard curve, analyzing the content of the bortezomib in the liquid to be detected by referring to the standard curve, and calculating the loading amount of the poly-dopamine nano material connected with glucose on the bortezomib. Meanwhile, 3 parallel experiments are designed, the average value and the standard deviation are calculated, the result is shown in table 1, and the average drug loading of the glucose-linked polydopamine nano-material for resisting the tumor drug bortezomib is 10.96%.
TABLE 1 drug loading of glucose covalently linked polydopamine nanomaterials against the anti-tumor drug bortezomib
Wherein: drug loading (LC%) was evaluated using the following equation:
LC％＝(WBTZ on NPs)/(W NPs)×100％
wherein, WBTZ on NPsIs (the mass of bortezomib added in the initial reaction-the mass of bortezomib remaining in the solution to be tested); wNPsMass of glucose-linked polydopamine nanomaterial added for initial reaction.
Example 2 photothermal conversion Properties of glucose-linked Polydopamine nanomaterials
(1) Configuration of reaction System
10mg of the glucose-linked polydopamine nanomaterial prepared in step (2) of example 1 was weighed, dispersed in 10mL of Phosphate (PBS) buffer, and diluted in a gradient to obtain suspensions of 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, and 200. mu.g/mL. The experimental group is that more than 200 mu L of glucose-connected poly-dopamine nano-material suspension is respectively added into a single hole of a 96-hole plate; the control group is that 200 mu L PBS buffer solution is added into a single hole of a 96-hole plate;
(2) testing of photothermal conversion Performance
The near infrared light emitted by the BST808-5-F laser in the experimental group and the control group is 3.6W/cm2The temperature change was measured and recorded every 30 seconds with an electronic thermometer under the condition of 5 minutes irradiation, and the result is shown in fig. 3, and the higher the concentration of the glucose-linked polydopamine nanomaterial, the more the amount of heat generated. Taking a 100 μ g/mL glucose-linked polydopamine nanomaterial as an example, the system temperature can be raised by 56 ℃ after 5 minutes of near-infrared illumination, and the system temperature can be raised by 64 ℃ after 5 minutes of near-infrared illumination of a 200 μ g/mL nanomaterial.
Example 3 Multi-acting nanomaterials with Targeted photothermal therapy and controlled drug Release
20mg of the multiple-acting nanomaterial with targeted photothermal therapy and controlled drug release prepared in example 1 was weighed, resuspended in 4mL of PBS buffer (10mM, pH7.4, containing 0.1% Tween20), and 3 repeated experiments were set, wherein 1mL of the multiple-acting nanomaterial with targeted photothermal therapy and controlled drug release suspension was placed in a dialysis bag (MW: 7000) and dialyzed in 15mL of 10mL of centrifuge tube and 10mM of dialysate. Set 4 different sets of conditions, including: 1) the dialyzate was PBS buffer solution with pH 7.4; 2) the dialysate is PBS buffer solution with pH 6.5, 3) the dialysate is PBS buffer solution with pH 5.0; 4) the dialysate was PBS buffer pH 5.0 and was treated with near infrared light (2W/cm) emitted by BST808-5-F laser2) Irradiation was carried out for 3 minutes. The four groups were each subjected to HPLC analysis by taking 0.5mL of the respective dialysate at different time points, and adding 0.5mL of PBS buffer at the respective pH to make up the dialysate at time points of 0 hour, 0.3 hour, 0.5 hour, 1 hour, 1.5 hour, 2 hours, 2.5 hours, and 3 hours. Conditions for HPLC analysis: agilent HPLC1260, reverse phase C18 column (150x4.6mm, 5 μm, C18; Agilent Technologies, CA, USA), mobile phase acetonitrile: 80 parts of water: 20, flow rate of 1 mL/min, UV detection wavelength of 270 nm.
The detection result is shown in fig. 4, the loaded antitumor drug bortezomib is released to the lowest extent under the condition that the pH is the pH of normal body fluid of a human body (pH 7.4), and the release amount of bortezomib is gradually increased along with the reduction of the pH, which shows that the multi-action nano material with targeted photothermal therapy and controllable drug release can responsively release drugs along with the reduction of the environmental pH, and the characteristic can be used for the controllable drug release in response to the pH of a tumor microenvironment. In addition, near-infrared light irradiation is carried out simultaneously in a slightly acid pH environment, the release amount of bortezomib is increased, and the multiple-action nano material of near-infrared light irradiation targeted photo-thermal treatment and controllable drug release not only has the photo-thermal treatment effect, but also can promote the nano material to accelerate the drug release, thereby achieving the purpose of jointly treating tumors.
Example 4 specific killing of Breast cancer cells MDA-MB-231 by glucose-linked Polydopamine nanomaterials
The MTT method is adopted to detect the activity of the cells, and the specific steps are as follows:
(1) culturing breast cancer cells MDA-MB-231 according to conventional operation, culturing cells to 70% fusion degree, digesting, collecting and counting, and diluting cell density to 1 × 105Per/mL, 100. mu.L of the above cell dilution was added to each well of a 96-well plate, and the mixture was incubated at 37 ℃ with 5% CO2Culturing in an incubator for 24 hours;
(2) after replacing the complete medium with a new one, the glucose-linked polydopamine nanomaterial prepared in step (2) of example 1 was added to final concentrations of 10. mu.g/mL, 25. mu.g/mL, and 50. mu.g/mL, respectively, and the treated group supplemented with PBS buffer was used as a control group at 37 ℃ with 5% CO2Culturing for 4 hours in an incubator;
(3) near infrared light (3.6W/cm) emitted by BST808-5-F laser per hole2) After 3 minutes of irradiation, 5% CO at 37 ℃2Culturing in an incubator for 12h, discarding the old culture medium, and adding a fresh culture medium;
(4) the 96-well plate was stained by adding 10. mu.L of thiazole blue (MTT) (5mg/mL) per well of the 96-well plate, 37 ℃ and 5% CO2Culturing in incubator for 4h, removing culture medium, adding 100 μ L dimethyl sulfoxide (DMSO) into each well, mixing gently by shaking table for 10 min, dissolving MTT, and determining OD490Value, calculate inhibition rate.
Under the condition of external near infrared light, after the breast cancer cell MDA-MB-231 is treated by glucose-linked polydopamine nano-materials with different concentrations, the survival rate of the cell is shown in figure 5. The result shows that the glucose-linked polydopamine nano material has a high cell survival rate when used for treating the breast cancer cell MDA-MB-231 under the condition of no external near infrared light, the nano material has a greatly enhanced inhibition effect on the breast cancer cell MDA-MB-231 under the condition of near infrared light, the inhibition rate of the 50 microgram/mL glucose-linked polydopamine nano material on the tumor cell can reach over 60 percent, and the cell survival rate is only 39 percent at the moment.
Example 5 targeting ability of glucose-linked Polydopamine nanomaterials to tumors in vivo
(1) Quantum dot-labeled glucose-linked polydopamine nano material
Weighing 2.5mg of the glucose-linked polydopamine nanomaterial prepared in step (2) of example 1, adding 100. mu.L of PBS buffer for suspension, sequentially adding 10. mu.L of 8. mu.M of carboxyl quantum dots (from GmbH Quantum dot technologies, GmbH, Q3625), 300. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (from Sigma Aldrich, E6383), 90. mu.L of 1mM of hydroxythiosuccinimide (from Sigma Aldrich, 56485), shaking for 4h at 20 ℃ in a 1.5mL EP tube, adding 20. mu.L of 15mM ethanolamine (from Sigma Aldrich, 411000), shaking for 4h at 20 ℃, centrifuging the tube with a cut-off molecular weight of 10kD after the reaction is finished, centrifuging at 8000rpm for 10 min to remove unreacted small molecules and chemical reagents, then washing with 10mM PBS buffer solution with pH7.4 for 5 times, and storing at 4 deg.C;
(2) establishing tumor-bearing animal model, and detecting in vivo tumor targeting ability of material
Adopting BALB/c-nu nude mouse to construct animal tumor-bearing model, subculturing and amplifying human tumor cell MDA-MB-231, collecting MDA-MB-231 cell growing to logarithmic phase, digesting and counting, and preparing into 2 × 107one/mL of the cell suspension was injected subcutaneously into the axilla of the forelimb of BALB/c-nu nude mice to inoculate the nude mice with tumor cells at a rate of 5X 106Single/single, SPF class environmentCulturing a nude mouse, and observing the growth condition of the tumor;
when the tumor diameter of a BALB/c-nu nude mouse tumor-bearing model is 5mm, an in-vivo imaging experiment is carried out: injecting 50 mu L of glucose-linked polydopamine nano-material labeled by quantum dots with 1mg/mL into tail veins of a naked mouse (n is 2), observing fluorescence changes in the mouse body in 0h, 1h, 2h, 4h, 8h and 24h respectively by using a small animal living body imager (IVIS Kinetic, Caliper Life Sciences) after injection, judging the distribution of the nano-material in the mouse body according to the position and the intensity of fluorescence, and further researching the targeting effect of the nano-material in the body, wherein the result is shown in figure 6, the injecting of the glucose-linked polydopamine nano-material labeled by quantum dots into the tail veins of a BALB/c-nu naked mouse shows that the tumor part shows fluorescence after 1h, the fluorescence intensity reaches the maximum after 8h, the tumor part still has fluorescence after 24h, the fluorescence intensity at other parts of the body is far smaller than that of the tumor part, and only the kidney part shows fluorescence at the position of the naked mouse when 8-24h, indicating that the nanomaterial is metabolized by the animal kidney. The results show that the poly-dopamine nano material connected with glucose can efficiently target breast cancer tumors in vivo.
Example 6 treatment of tumors in vivo with Multi-acting nanomaterials with Targeted photothermal therapy and controlled drug Release
(1) Weighing 1mg of the multi-acting nanomaterial with targeted photothermal therapy and controllable drug release prepared in the embodiment 1, dispersing the multi-acting nanomaterial with targeted photothermal therapy and controllable drug release in 1mL of PBS buffer solution, and preparing 1mg/mL of multi-acting nanomaterial suspension with targeted photothermal therapy and controllable drug release;
(2) inoculating a mouse breast cancer cell 4T1 tumor cell on the back of a BALB/c mouse for 6 weeks, constructing a tumor-bearing mouse model, and carrying out a test after keeping the mouse normally fed for one week;
(3) dividing the tumor-bearing mice in the step (2) into two groups, and respectively carrying out intraperitoneal injection of 100 mu L/10g chloral hydrate (5%) for anesthesia; the following operations were performed on two groups of tumor-bearing mice, respectively: group A is a treatment group, 100 muL and 1mg/mL multiple-action nano material suspension with targeted photothermal treatment and controllable drug release is injected into tail vein of mouse, and the wavelength is 808nm and the power is 2W/cm after 8 hours of injection2The near infrared light irradiation treatment is carried out for 10 minutes, the nano material suspension is injected once in the whole process, the near infrared light irradiation is carried out once, and the change of the tumor is recorded; group B is control group, mice tail vein is injected with 100 μ L physiological saline and adopts wavelength 808nm and power 2W/cm2The near infrared light irradiation treatment is carried out for 10 minutes, the normal saline is injected once in the whole process, the near infrared light irradiation is carried out once in the whole process, and the change of the tumor is recorded.
The results are shown in fig. 7, after the near-infrared irradiation treatment for 15 days, the tumor was peeled off, the group a treatment was performed only by one drug injection and near-infrared irradiation treatment, and the tumor size was significantly reduced compared to the group B control group, demonstrating that the multi-acting nanomaterial with targeted photothermal treatment and controlled drug release of the present invention has high tumor treatment capacity.
Claims (12)
1. A preparation method of a multi-action nano material with targeted photo-thermal treatment and controllable drug release is characterized by comprising the following steps:
(1) preparing a dopamine hydrochloride aqueous solution with the concentration of 0.01-5mg/mL, adding the dopamine hydrochloride aqueous solution into an ammonia water/ethanol/deionized water solution, wherein the volume ratio of the dopamine hydrochloride aqueous solution to the ammonia water/ethanol/deionized water solution is 1 (2-30), stirring and reacting for 6-48h at the temperature of 10-60 ℃, and after the reaction is finished, separating, washing and freeze-drying to obtain a poly-dopamine nano material;
(2) adding the polydopamine nano material prepared in the step (1) and glucosamine hydrochloride into a sodium phosphate buffer solution with the concentration of 1-50mM, wherein the concentration of the polydopamine nano material in the sodium phosphate buffer solution is 2-20mg/mL, and the mass ratio of the polydopamine nano material to the glucosamine hydrochloride is 1: (1-100), stirring at 10-60 ℃ for reaction for 4-36h, and after the reaction is finished, separating, washing and freeze-drying to obtain a poly-dopamine nano material connected with glucose;
(3) resuspending the glucose-linked polydopamine nano-material prepared in the step (2) in 1-50mM Tris buffer solution to prepare a nano-material system with the concentration of 0.1-500 mg/mL; dissolving the anti-tumor drug bortezomib in 1-50mM Tris buffer solution to prepare a bortezomib solution with the concentration of 0.05-100 mg/mL; and dropwise adding the bortezomib solution into the nano material system under the stirring condition, wherein the mass ratio of the bortezomib to the poly-dopamine nano material connected with glucose is 1 (5-50), stirring and reacting for 8-24h at 25-45 ℃, and separating and washing after the reaction is finished to obtain the multi-action nano material with targeted photothermal therapy and controllable drug release.
2. The preparation method of claim 1, wherein the ammonia/ethanol/deionized water solution in step (1) is obtained by mixing ammonia, absolute ethanol and deionized water, and stirring at 10-60 ℃ for 10-90 minutes, wherein the volume ratio of ammonia, absolute ethanol and deionized water is 1: (2-320): (3-600).
3. The preparation method of claim 1, wherein the ammonia/ethanol/deionized water solution in step (1) is obtained by mixing ammonia, absolute ethanol and deionized water, and stirring at 20-45 ℃ for 20-60 minutes, wherein the volume ratio of ammonia, absolute ethanol and deionized water is 1: (5-80): (11-150); wherein the mass concentration of the ammonia water is 25-28%.
4. The method according to claim 1, wherein the stirring reaction in the step (1) is carried out at 20 to 45 ℃ for 10 to 24 hours.
5. The method according to claim 1, wherein the concentration of the sodium phosphate buffer in the step (2) is 5 to 25mM, and the pH is 7.0 to 9.5.
6. The preparation method according to claim 1, wherein the mass ratio of the polydopamine nano-material to the glucosamine hydrochloride in the step (2) is 1: (1-50).
7. The preparation method of claim 1, wherein the mass ratio of the polydopamine nano-material to the glucosamine hydrochloride in the step (2) is 1 (5-50).
8. The method according to claim 1, wherein the stirring reaction in the step (2) is carried out at 20 to 60 ℃ for 8 to 24 hours.
9. The method of claim 1, wherein step (3) is performed under conditions that one or more of the following conditions are satisfied:
i. the concentration of the Tris buffer solution is 5-25mM, and the pH value is 7.8-10;
the concentration of the nanomaterial system is 0.16-8 mg/mL;
the concentration of the bortezomib solution is 0.5-40 mg/mL.
10. The production method according to claim 1, wherein the rotation speed of the stirring reaction in steps (1), (2) and (3) is 50 to 300 rpm; the separation is 6000-12000rpm high-speed centrifugation for 10-60 min.
11. The method according to claim 1, wherein the washing in steps (1) and (2) is performed 2 to 5 times with 1 to 50mM sodium phosphate buffer; and (4) washing for 2-5 times by using deionized water in the step (3).
12. The application of the multi-acting nano material with targeted photothermal therapy and controllable drug release prepared by the preparation method of claim 1 in preparing tumor targeted drugs for photothermal and chemotherapy combined therapy.
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