CN111407742B - Anti-tumor nano-particles and preparation method and application thereof - Google Patents
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Abstract
An anti-tumor nanoparticle and a preparation method and application thereof, belonging to the field of novel medical preparations. The anti-tumor nanoparticle includes a cell vesicle including a loading substance therein, the loading substance including quantum dots having a photo-thermal effect. The cell vesicle with large grain diameter is provided with a cavity, the cavity is used for loading a loading substance, the loading substance comprises quantum dots with photothermal action, the cell vesicle can be used as a carrier to prolong the circulation time in vivo and is enriched at a tumor part, the loading substance in the cavity is also enriched at the tumor part, at the moment, the cell vesicle can be disintegrated to release the loading substance with small grain diameter by applying light stimulation to the tumor nanoparticles, and the loading substance with small grain diameter can enter the tumor part to be treated more deeply. The preparation method of the anti-tumor nanoparticle comprises the steps of introducing a loading substance into a cell vesicle through an electroporation technology, culturing for more than 30min, and centrifuging to prepare the anti-tumor nanoparticle. The preparation method is simple and convenient, and the prepared anti-tumor nano-particles have stable performance.
Description
Technical Field
The application relates to the field of novel medical preparations, in particular to an anti-tumor nanoparticle and a preparation method and application thereof.
Background
Chemotherapy, as the main cancer treatment method at present, has great harm to normal organs of human bodies, and is a problem that researchers want to solve, and nanoparticles loaded with antitumor drugs are produced. The tumor part has the characteristics of high interstitial fluid pressure, closely packed tumor cells, compact extracellular matrix, incomplete vascular system and the like, and brings challenges to penetration and diffusion of nanoparticles.
To address the penetration problem at the tumor site, there are two broad categories of improvements. One of these approaches focuses on dynamic interactions against components of the tumor microenvironment, i.e., normalizing the tumor site microenvironment. In particular, various parameters of tumor pathophysiology are altered by pre-treating the tumor prior to or simultaneously with nanoparticle administration. This treatment modality promotes the systemic delivery of the agent into the tumor and into deeper layers of the tumor tissue, which can be subdivided into physical methods and physiologically active agent treatments, wherein physical methods include local heating, local ionizing radiation, ultrasound treatment, etc.; the physiological activity regulator mainly comprises a vascular permeability medium, a matrix modifier, a vasoconstrictor and the like. In addition, another way to effectively enhance the accumulation and penetration of nanoparticles at the tumor site can be achieved by adjusting the characteristics of the nanoparticles. Among these, because of the high interstitial fluid pressure of tumors, diffusion remains the primary driving force for tumor penetration. However, too small a particle size of the nanoparticles for loading the anti-tumor drug may result in inefficient enrichment of the nanoparticles at the tumor site, and too large a particle size of the nanoparticles for loading the anti-tumor drug may result in inefficient penetration of the tumor.
Disclosure of Invention
The application provides an anti-tumor nanoparticle, a preparation method and an application thereof, the nanoparticle can be enriched at a tumor part, and meanwhile, a loading substance in the nanoparticle is released to enter the tumor part for treatment at a deeper level through cell vesicle disintegration.
The embodiment of the application is realized as follows:
in a first aspect, the present examples provide an anti-tumor nanoparticle comprising a cell vesicle including a loading substance therein, wherein the loading substance comprises quantum dots having a photothermal effect.
In the technical scheme, the cell vesicle with large particle size is provided with a cavity, the cavity is used for loading a loading substance, the loading substance comprises quantum dots with photothermal action, the cell vesicle can be used as a carrier to prolong the in vivo circulation time of the cell vesicle and enrich the cell vesicle at a tumor part, the loading substance in the cavity is also enriched at the tumor part, at the moment, the cell vesicle can be disintegrated to release the loading substance with small particle size by applying light stimulation to anti-tumor nanoparticles, the loading substance with small particle size can enter the tumor part to be treated at a deeper level, and the quantum dots with photothermal action have photothermal treatment effect.
With reference to the first aspect, in a first possible example of the first aspect of the present application, the particle size of the anti-tumor nanoparticle is 100 to 1000nm, and the particle size of the quantum dot is less than or equal to 10 nm.
In the above example, the particle size of the anti-tumor nanoparticles is beneficial to enrich the anti-tumor nanoparticles in the tumor site, and the particle size of the quantum dots is beneficial to the quantum dots to enter the tumor site for deeper treatment.
In a second possible example of the first aspect of the present application in combination with the first aspect, the quantum dots comprise Ag2And (4) S quantum dots.
In the above examples, Ag2The S quantum dots have a good photo-thermal effect, and can be heated and heated after being stimulated by light so as to disintegrate cell vesicles and release loaded substances with small particle sizes.
In a third possible example of the first aspect of the present application in combination with the first aspect, the above-mentioned carrier further comprises an anti-tumor drug.
In the above example, the cell vesicle can also be loaded with anti-tumor drugs, so that the anti-tumor drugs are brought to and enriched in the tumor site, the cell vesicle can be disintegrated to release the small-particle-size loaded substance after the anti-tumor nanoparticles are applied with light stimulation, and meanwhile, the anti-tumor drugs in the cell vesicle are also released to enter the tumor site for further treatment.
In a second aspect, the present application provides a method for preparing the above anti-tumor nanoparticles, which comprises introducing a loading substance into a cell vesicle by an electroporation technique, culturing for more than 30min, and centrifuging to obtain the anti-tumor nanoparticles.
In the above technical solution, a pore of 1 to 10nm can be formed on the surface of the cell vesicle by electroporation, the loading substance enters the cavity in the cell vesicle through the pore formed on the surface of the cell vesicle by electroporation, the pore formed by electroporation on the surface of the cell vesicle can disappear by culturing for more than 30min, and the loading substance is stably loaded in the cell vesicle, and the cell vesicle is centrifuged to remove the excess loading substance that has not entered the cell vesicle. The preparation method is simple and convenient, and the prepared anti-tumor nano-particles have stable performance.
In combination with the second aspect, in a first possible example of the second aspect of the present application, 25 to 150 μ L of a solution of cell vesicles with a protein mass concentration of 100 to 400 μ g/mL, 25 to 200 μ L of an aqueous solution of quantum dots with a concentration of 0.5 to 2.5mg/mL, and 25 to 100 μ L of phosphate buffered saline solution are added to an electroporation cuvette, and the electroporation is started by setting the process parameters of electroporation.
Optionally, the process parameters of the electroporation are voltage 100-350V, resistance 50-200 Ω and capacitance 100-500 μ F.
In combination with the second aspect, in a second possible example of the second aspect of the present application, the centrifuging includes centrifuging at 8000-20000 g for 5-20 min.
In the above example, the centrifugation conditions are favorable to remove excess loading that does not enter the cell vesicles and retain the anti-tumor nanoparticles.
In a third possible example of the second aspect of the present application in combination with the second aspect, the quantum dot includes Ag2S quantum dots, Ag2The S quantum dot is prepared by the following method:
reacting the mixed solution with the pH value of 6.0-8.0 and containing silver nitrate and reduced glutathione at the temperature of 80-120 ℃ for 6-20 h, and centrifuging to prepare Ag2An aqueous solution of S quantum dots.
Optionally, the mass concentration of the reduced glutathione in the mixed solution is 1.57-6.27 mg/mL, and the mass concentration of the silver nitrate is 0.22-0.87 mg/mL.
Optionally, the centrifugation comprises centrifugation in an ultrafiltration tube of 15-50 mL at 2000-6000 g for 5-20 min.
In the above examples, the present application uses reduced glutathione as a ligand for providing elemental sulfur, the above preparation method is simple, and the prepared Ag2The S quantum dots have the photothermal effect and stable performance.
Centrifugation is used to remove unreacted molecules and ions.
In a fourth possible example of the second aspect of the present application in combination with the second aspect, the above-described cell vesicle is prepared by:
culturing cells, stimulating the cells to secrete cell vesicles, centrifuging, and collecting the cell vesicles.
Alternatively, the cells comprise mouse monocyte macrophage RAW 264.7.
Optionally, the centrifuging includes sequentially centrifuging for 10-30 min under 2000-6000 g and for 1.5-3 h under 30000-100000 g respectively.
In the above example, the above method for preparing the cell vesicle is simple and convenient, and the particle size of the prepared cell vesicle is 100 to 1000 nm.
The first centrifugation is used to remove dead cells and large impurities and the second centrifugation is used to collect cell vesicles.
In a third aspect, the present application provides the use of an anti-tumor nanoparticle in the treatment of a tumor.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a schematic structural diagram of an anti-tumor nanoparticle of the present application;
FIG. 2 shows Ag in test example 1 of the present application2S, a temperature change curve of the quantum dots within 10 min;
FIG. 3 shows Ag in test example 1 of the present application2S, performing infrared thermal imaging on the quantum dots within 10 min;
FIG. 4 is a transmission electron micrograph of the anti-tumor nanoparticles of Experimental example 2 of the present application before irradiation with light;
FIG. 5 is a transmission electron microscope image of the anti-tumor nanoparticles of Experimental example 2 of the present application after illumination;
fig. 6 is a comparison graph of the cell ball penetration of the anti-tumor nanoparticles of example 2 that did not respond to light stimulation, the anti-tumor nanoparticles of example 2 that did respond to light stimulation, and the anti-tumor nanoparticles of comparative example 1 that were light-stimulated in experimental example 3 of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The inventors have found that the minimization of size is still an important factor in enhancing the effective penetration of the nanoparticles to the tumor, achieving a uniform distribution at the tumor site, compared to the surface charge, hydrophilicity and hydrophobicity, etc., of the nanoparticles. The quantum dots and the anti-tumor drugs can enter the tumor part to be treated more deeply.
Although small-sized nanoparticles have better tumor penetration, they have a short circulation time in vivo and cannot be concentrated at the tumor site. Although the large-sized nanoparticles have longer circulation time in vivo and can be concentrated at tumor sites, the large-sized nanoparticles cannot effectively penetrate tumors.
The following description specifically describes an anti-tumor nanoparticle, a preparation method and an application thereof in the embodiments of the present application:
the application provides an anti-tumor nanoparticle, the structure of which is shown in figure 1, and the anti-tumor nanoparticle comprises a cell vesicle and a loading substance, wherein the cell vesicle is provided with a cavity, and the loading substance is loaded in the cavity of the cell vesicle and is carried by the cell vesicle.
Wherein the loading substance comprises quantum dots with photo-thermal effect, and the quantum dots can generate heat after being stimulated by light.
The particle size of the prepared anti-tumor nano particles is 100-1000 nm, and the particle size of the quantum dots with the photothermal effect is less than or equal to 10 nm.
The application provides an anti-tumor nanoparticle to cell vesicle prolongs its internal circulation time and enriches in tumour position as the carrier, after anti-tumor nanoparticle enriches in tumour position, applies light stimulation to anti-tumor nanoparticle, and the quantum dot that has the photothermal effect in the cell vesicle heats up after receiving light stimulation to make cell vesicle disintegrate and release little graininess's load, little graininess's load can enter into tumour position more deep level and treat this moment.
Optionally, the carrier further comprises an anti-tumor drug.
The anti-tumor drug has extremely small particle size, can be loaded in a cavity of the cell vesicle as a loading substance, is carried by the cell vesicle to be conveyed and enriched to a tumor part, and is decomposed to release the loading substance with small particle size after the anti-tumor nano particles are applied with light stimulation, and meanwhile, the anti-tumor drug is also released to enter the tumor part for deeper treatment.
It is noted that the loading substance can only comprise the quantum dots with the photothermal effect, the quantum dots with the photothermal effect have the photothermal treatment effect, and can be independently used as the loading substance to prepare the anti-tumor nanoparticles for treating tumors. The load can also comprise quantum dots with photothermal effect and anti-tumor drugs, and the quantum dots with photothermal effect and the anti-tumor drugs have the synergistic effect and are used for treating tumors.
The application also provides a preparation method of the anti-tumor nanoparticle, which comprises the steps of introducing a loading substance into a cell vesicle through an electroporation technology, culturing for more than 30min, and centrifuging to prepare the anti-tumor nanoparticle.
The method comprises the steps of forming holes with the size of 1-10 nm on the surface of a cell vesicle through an electroporation technology, enabling a loading substance to enter a cavity in the cell vesicle through the holes formed on the surface of the cell vesicle through electroporation, enabling the holes formed on the surface of the cell vesicle through electroporation to disappear after culturing for more than 30min, enabling the loading substance to be stably loaded in the cell vesicle, and centrifuging to remove the redundant loading substance which is not inserted into the cell vesicle. The preparation method is simple and convenient, and the prepared anti-tumor nano-particles have stable performance.
Taking 25-150 mu L of cell vesicle solution with protein mass concentration of 100-400 mu g/mL and 25-200 mu L of Ag with concentration of 0.5-2.5 mg/mL2Adding an aqueous solution of S quantum dots and 25-100 mu L of Phosphate Buffered Saline (PBS) into an electroporation cuvette with a width of 0.2-0.4 mm, and setting electroporationThe process parameters of the hole are 100-350V of voltage, 50-200 omega of resistance and 100-500 muF of capacitance, and the electroporation is started.
When the loaded substance further comprises an anti-tumor drug, 25-200 mu L of anti-tumor drug solution with the concentration of 5-30 mu g/mL needs to be added into the electroporation cuvette.
The pH of the PBS buffer solution used in the present application is 7.4, and the present application provides a method for preparing the PBS buffer solution, which includes:
0.24g of monopotassium phosphate, 1.44g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride are taken and mixed with 800mL of deionized water uniformly, and hydrochloric acid is added to adjust the pH value to 7.4.
Antineoplastic drugs include, but are not limited to, water-soluble chemotherapeutic drugs such as doxorubicin, gemcitabine, and hydroxycamptothecin.
The temperature of the culture was 37 ℃ and the atmosphere was 5% carbon dioxide and 95% air.
Centrifuging at 8000-20000 g for 5-20 min, and collecting precipitate.
Wherein the loading substance comprises quantum dots with a photothermal effect.
It is to be noted that quantum dots having a photothermal action can be applied to the present application.
For example, quantum dots having photothermal effects include Ag2S quantum dots, CuS quantum dots, and the like.
The present application provides an Ag2The preparation method of the S quantum dot, which takes reduced Glutathione (GSH) as a ligand for providing sulfur element, comprises the following steps:
mixing a mixture of 1: 1-4: dissolving reduced glutathione and silver nitrate of 1 in purified water to prepare a mixed solution, wherein the mixed solution is white and turbid, the mass concentration of the reduced glutathione in the mixed solution is 1.57-6.27 mg/mL, and the mass concentration of the silver nitrate is 0.22-0.87 mg/mL; adjusting the pH value of the mixed solution to 6.0-8.0 by adopting an alkali solution, wherein the mixed solution is changed from a white turbid solution into a clear solution; introducing inert gas and stirring for more than 5min until the air in the mixed solution is removed; heating the mixture to 80-120 ℃,stopping the reaction after 6-20 h to obtain a reaction solution, and centrifuging the reaction solution to obtain Ag with the concentration of 1-4 mg/mL2An aqueous solution of S quantum dots.
The alkali solution comprises a sodium hydroxide solution with the concentration of 0.5-2 mol/L.
Centrifuging for 5-20 min under the condition of 2000-6000 g in an ultrafiltration tube of 15-50 mL for more than four times, and washing with purified water for more than two times. Centrifuging to remove unreacted molecules and ions to obtain concentrated Ag2An aqueous solution of S quantum dots.
The present application also provides a method for preparing a cell vesicle, comprising the steps of:
adding the cells into a DMEM high-glucose medium added with 5-15% V/V fetal calf serum, wherein the culture temperature is 37 ℃, and the gas environment comprises 5% carbon dioxide and 95% air; adding serum-free DMEM high-glucose medium after the cells are full, and continuously culturing for 48h at 37 ℃ in an environment with 5% carbon dioxide and 95% air as gas environment; starvation is used for stimulating cells to secrete a large number of cell vesicles for collection, cell culture fluid is taken for centrifugation, and the cell vesicles are collected.
The cells can be selected from mouse mononuclear macrophage RAW264.7, mouse melanoma cell B16 and mouse breast cancer cell 4T 1.
Centrifuging, namely taking a cell culture solution into a centrifuge tube, balancing, centrifuging for 10-30 min at the temperature of 4-37 ℃ at 2000-6000 g to remove dead cells and cell debris, and collecting supernatant into a clean centrifuge tube; and centrifuging at 30000-100000 g and 4-37 ℃ for 1.5-3 h, and collecting and concentrating the cell vesicles. And continuously washing the separated cell vesicle precipitate with sterile PBS buffer solution, and further centrifuging for 1.5-3 h at 30000-100000 g and 4-37 ℃ to collect the cell vesicle. The collected cell vesicles can be resuspended in phosphate buffered saline solution for direct use or stored in liquid nitrogen for storage, and then taken out when necessary.
The application also provides an application of the anti-tumor nano-particles in treating tumors.
An anti-tumor nanoparticle of the present application, a method for preparing the same, and applications thereof are further described in detail with reference to examples below.
Example 1
The embodiment of the application provides an anti-tumor nanoparticle and a preparation method thereof, and the preparation method comprises the following steps:
(1) preparation of Ag2S quantum dot
Adding 94.05mg of reduced glutathione, 17.4mg of silver nitrate and 40mL of purified water into a three-neck round-bottom flask, uniformly mixing to obtain a white turbid mixed solution, adjusting the pH value of the mixed solution to 6.5 by adopting a sodium hydroxide solution with the concentration of 1mol/L, clarifying the mixed solution, introducing Ar gas into the mixed solution, opening magnetic stirring, introducing the gas for 10min, heating to 100 ℃, reacting for 8h to stop the reaction to obtain a reaction solution, centrifuging the reaction solution for 5 times by using a 50mL ultrafiltration centrifugal tube under the condition of 4000 g for 5min, centrifuging for 5 times, and washing for 2 times by using the purified water to obtain stable Ag with the concentration of 2mg/mL2An aqueous solution of S quantum dots.
(2) Preparation of cell vesicles
Selecting cells as mouse mononuclear macrophage RAW264.7, adding the mouse mononuclear macrophage RAW264.7 into a DMEM high-glucose type culture medium added with 10% V/V fetal calf serum, and culturing at 37 ℃ in an air environment with 5% carbon dioxide and 95% air; adding serum-free DMEM high-glucose medium after the cells are full, and continuously culturing for 48h at 37 ℃ in an environment with 5% carbon dioxide and 95% air as gas environment; hunger stimulating cells to secrete a large amount of cell vesicles for collection, taking cell culture solution to a centrifuge tube, balancing, centrifuging for 10-30 min at 2000g and 4 ℃ to leave dead cells and cell debris, and collecting supernatant to a clean centrifuge tube; then, the cells were centrifuged at 80000g at 4 ℃ for 1.5 hours, and the vesicles were collected and concentrated. The separated cell vesicle pellet is washed with sterile PBS buffer solution, and is further centrifuged for 1.5h at 80000g and 4 ℃, and the cell vesicle pellet can be resuspended in 50 μ L of phosphate buffered saline solution for later use.
(3) Preparation of antitumor nanoparticles
50. mu.L of the cell vesicle solution prepared above and 100. mu.L of the Ag solution prepared above were taken2Adding an aqueous solution of S quantum dots and 100 mu L of PBS buffer solution into an electroporation cuvette with the width of 0.4mm, setting the electroporation process parameters to be voltage 200V, resistance 50 omega and capacitance 300 mu F, and starting electroporation; culturing the mixture subjected to electroporation in an atmosphere of 5% carbon dioxide and 95% air at 37 deg.C for 40 min; and (3) putting the cultured mixture into a centrifuge tube, centrifuging for 15min under the condition of 10000g, and collecting the precipitate to prepare the anti-tumor nano-particles.
Example 2
The embodiment of the application provides an anti-tumor nanoparticle and a preparation method thereof, and the preparation method comprises the following steps:
50. mu.L of the cell vesicle solution prepared in example 1 and 100. mu.L of Ag prepared in example 1 were collected2Adding an aqueous solution of S quantum dots, 50 mu L of doxorubicin aqueous solution with the concentration of 20 mu g/mL and 100 mu L of PBS buffer solution into an electroporation cuvette with the width of 0.4mm, setting the electroporation process parameters to 200V of voltage, 50 omega of resistance and 300 mu F of capacitance, and starting electroporation; culturing the mixture subjected to electroporation in an atmosphere of 5% carbon dioxide and 95% air at 37 deg.C for 40 min; and (3) putting the cultured mixture into a centrifuge tube, centrifuging for 15min under the condition of 10000g, and collecting the precipitate to prepare the anti-tumor nano-particles.
Example 3
The embodiment of the application provides an anti-tumor nanoparticle and a preparation method thereof, and the preparation method comprises the following steps:
50. mu.L of the cell vesicle solution prepared in example 1 and 100. mu.L of Ag prepared in example 1 were collected2Adding an aqueous solution of S quantum dots, 50 mu L of gemcitabine aqueous solution with the concentration of 20 mu g/mL and 100 mu L of PBS buffer solution into an electroporation cuvette with the width of 0.4mm, setting the technological parameters of electroporation to be voltage 200V, resistance 50 omega and capacitance 300 mu F, and starting electroporation; the mixture after electroporation was heated at 37 ℃ in a gaseous atmosphere to 5% carbon dioxide concentrationCulturing in 95% air environment for 40 min; and (3) putting the cultured mixture into a centrifuge tube, centrifuging for 15min under the condition of 10000g, and collecting the precipitate to prepare the anti-tumor nano-particles.
Example 4
The embodiment of the application provides an anti-tumor nanoparticle and a preparation method thereof, and the preparation method comprises the following steps:
50. mu.L of the cell vesicle solution prepared in example 1 and 100. mu.L of Ag prepared in example 1 were collected2Adding an aqueous solution of S quantum dots, 50 mu L of hydroxycamptothecin aqueous solution with the concentration of 20 mu g/mL and 100 mu L of PBS buffer solution into an electroporation cuvette with the width of 0.4mm, setting the technological parameters of electroporation as voltage 200V, resistance 50 omega and capacitance 300 mu F, and starting electroporation; culturing the mixture subjected to electroporation in an atmosphere of 5% carbon dioxide and 95% air at 37 deg.C for 40 min; and (3) putting the cultured mixture into a centrifuge tube, centrifuging for 15min under the condition of 10000g, and collecting the precipitate to prepare the anti-tumor nano-particles.
Example 5
The embodiment of the application provides application of anti-tumor nanoparticles.
According to the proportion of 1-3 multiplied by 10 for each mouse6Inoculating the tumor cell suspension to the fat pad of the experimental mouse at a dose of 100 mu L, when the tumor volume is about 50-100 mm3Meanwhile, the preparation of anti-tumor nanoparticles prepared in example 2 was injected for treatment three times every 2 days. The anti-tumor nano particle preparation prepared in the example 2 of the Balb/c intravenous injection of the lotus tumor is 10-30 mg of QDs/Kg of body weight. After 12-36 h of injection, the power density is 1W/cm2808nm near infrared laser irradiates the tumor for 10 min.
Comparative example 1
The application provides an anti-tumor nanoparticle and a preparation method thereof, and the preparation method comprises the following steps:
adding 50 μ L of the cell vesicle solution prepared in example 1, 50 μ L of doxorubicin aqueous solution with a concentration of 20 μ g/mL, and 100 μ L of PBS buffer solution into an electroporation cuvette with a width of 0.4mm, and starting electroporation by setting electroporation process parameters to 200V of voltage, 50 Ω of resistance, and 300 μ F of capacitance; culturing the mixture subjected to electroporation in an atmosphere of 5% carbon dioxide and 95% air at 37 deg.C for 40 min; and (3) putting the cultured mixture into a centrifuge tube, centrifuging for 15min under the condition of 10000g, and collecting the precipitate to prepare the anti-tumor nano-particles.
Test example 1
Respectively prepare Ag with the concentration of 2mg/mL, 1.5mg/mL, 1mg/mL and 0.5mg/mL2An aqueous solution of S quantum dots, using 1W/cm2Irradiating the 808nm near-infrared laser, and simultaneously recording the temperature change in real time by using an infrared thermal imager. Taking the temperature value every 15s, recording the temperature change condition in 10min and drawing a temperature change curve, wherein the temperature change curve in 10min is shown in figure 2, an infrared thermal imaging graph is shot every 2 minutes, and the infrared thermal imaging graph in 10min is shown in figure 3.
As can be seen from FIGS. 2 and 3, Ag2After the S quantum dots are illuminated, the temperature of the S quantum dots is obviously increased.
Test example 2
Dropwise adding the anti-tumor nanoparticle solution prepared in the example 2 onto an ultrathin carbon film, carrying out negative staining by using 1% phosphotungstic acid, putting the ultrathin carbon film on a sample rod after the solvent is volatilized to be dry, and putting the sample rod into a transmission electron microscope for observation to obtain a graph 4; then the anti-tumor nano particles pass through 1W/cm2And (3) performing illumination for 10min, dropwise adding the illuminated nanoparticle solution onto the ultrathin carbon film, performing negative staining by using 1% phosphotungstic acid, after the solvent is volatilized to be dry, putting the ultrathin carbon film on a sample rod, and putting the sample rod into a transmission electron microscope for observation to obtain a graph 5.
As can be seen from fig. 4 and 5, the quantum dots can be effectively loaded into the cell vesicles after electroporation, and after light treatment, the cell vesicles are significantly disintegrated, and the quantum dots are released.
The anti-tumor nanoparticles can realize particle size conversion under light stimulation, so that the aims of prolonging blood circulation time, ensuring that the particle size is changed from large to small after the tumor part has enough enrichment amount through light irradiation and realizing deep penetration of the tumor are fulfilled.
Test example 3
Add 2ml of agarose to 6 well plates, after coagulation add 2ml of medium and 2 x 10 to each well4B16 cell of (1). And cultured in an incubator at 37 ℃ in an atmosphere of 5% carbon dioxide and 95% air for 4-7 days to allow assembly of Multicellular Tumor Spheres (MTS).
When the diameter of the cell ball is increased to about 300 μm, the anti-tumor nanoparticles which are not subjected to the light stimulation response in the embodiment 2, the anti-tumor nanoparticles which are subjected to the light stimulation response in the embodiment 2 and the anti-tumor nanoparticles which are subjected to the light stimulation in the proportion 1 are added, and after 6h of incubation, a CLSM (CLSM color) graph is taken, so that the CLSM graph with the penetration depth is obtained as shown in fig. 6, and the conditions that the cell vesicle after NIR irradiation releases quantum dots and the anti-tumor drug penetrates into the cell ball are illustrated, wherein the light irradiation condition is 1W/cm2And (5) irradiating for 10 min.
As can be seen from fig. 6, the cell vesicles in the anti-tumor nanoparticle prepared in comparative example 1 (second row in fig. 6) have no quantum dots with photothermal effect, and after illumination, the cell vesicles cannot be disintegrated to release the anti-tumor drug in the cell cavities, and the cell penetration ability is poor. When the anti-tumor nanoparticles prepared in example 2 do not respond to light stimulation (the first row in fig. 6), the cell vesicles cannot be disintegrated to release the anti-tumor drugs and quantum dots in the cavities, and the cell penetration capability is poor. After the anti-tumor nanoparticles prepared in example 2 respond to the light stimulus (third row in fig. 6), the cell vesicles can be disintegrated to release the anti-tumor drugs and the quantum dots in the cavities, and the cell penetration capability of the anti-tumor nanoparticles is good and can reach the penetration depth of 80 μm.
Further proves that the anti-tumor nano-particles can realize the conversion of the particle size under the stimulation of light, the particle size can be reduced from large to small, and the aim of deep penetration of the tumor is fulfilled.
The foregoing is illustrative of the present application and is not to be construed as limiting thereof, as numerous modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (11)
1. An anti-tumor nanoparticle comprising a cell vesicle including a loading substance therein;
wherein the loading substance comprises Ag having a photothermal effect2S quantum dots and antitumor drugs;
applying a light stimulus to the anti-tumor nanoparticles enables the cell vesicles to disintegrate to release the loading substance;
the cell vesicle is prepared by the following method:
culturing cells, stimulating the cells to secrete cell vesicles, centrifuging, and collecting the cell vesicles;
the cells include mouse monocyte macrophage RAW 264.7.
2. The anti-tumor nanoparticle according to claim 1, wherein the particle size of the anti-tumor nanoparticle is 100-1000 nm, and the particle size of the quantum dot is less than or equal to 10 nm.
3. The method of claim 1 or 2, wherein the loading substance is introduced into the cell vesicle by electroporation, cultured for more than 30min, and centrifuged to obtain the anti-tumor nanoparticle.
4. The method for preparing the anti-tumor nanoparticle according to claim 3, wherein 25 to 150 μ L of the solution of the cell vesicle with a protein mass concentration of 100 to 400 μ g/mL, 25 to 200 μ L of the aqueous solution of the quantum dot with a concentration of 0.5 to 2.5mg/mL, 25 to 200 μ L of the solution of the anti-tumor drug with a concentration of 5 to 30 μ g/mL, and 25 to 100 μ L of the phosphate buffered saline solution are added to an electroporation cuvette, and electroporation is started by setting the process parameters of the electroporation.
5. The method for preparing anti-tumor nanoparticles according to claim 4, wherein the process parameters of the electroporation are voltage of 100-350V, resistance of 50-200 Ω and capacitance of 100-500 μ F.
6. The method for preparing anti-tumor nanoparticles according to claim 3, wherein the centrifugation comprises centrifugation at 8000-20000 g for 5-20 min.
7. The method of any one of claims 3 to 6, wherein the quantum dots comprise Ag2S quantum dot, said Ag2The S quantum dot is prepared by the following method:
reacting a mixed solution with a pH value of 6.0-8.0 and containing silver nitrate and reduced glutathione at 80-120 ℃ for 6-20 h, and centrifuging to prepare the Ag2An aqueous solution of S quantum dots.
8. The method for preparing the antitumor nanoparticle according to claim 7, wherein the mass concentration of reduced glutathione in the mixed solution is 1.57-6.27 mg/mL, and the mass concentration of silver nitrate is 0.22-0.87 mg/mL.
9. The method for preparing the anti-tumor nanoparticles according to claim 7, wherein the centrifugation comprises centrifugation in an ultrafiltration tube of 15-50 mL at 2000-6000 g for 5-20 min.
10. The method for preparing the anti-tumor nanoparticle according to claim 1, wherein the centrifugation comprises centrifugation for 10-30 min at 2000-6000 g and centrifugation for 1.5-3 h at 30000-100000 g respectively.
11. Use of the anti-tumor nanoparticle of claim 1 or 2 for the preparation of a medicament for the treatment of tumors.
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