CN117883409A - Medicine-carrying calcium-based nano generator wrapped by plant-derived exosomes, preparation method and anti-tumor application thereof - Google Patents
Medicine-carrying calcium-based nano generator wrapped by plant-derived exosomes, preparation method and anti-tumor application thereof Download PDFInfo
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Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention relates to a medicine-carrying calcium-based nano generator wrapped by plant source exosomes and a preparation method thereofThe preparation method and the application thereof, the nano generator comprises a medicine carrying inner core, and a plant source exosome shell layer is coated on the outer side of the medicine carrying inner core; wherein the plant source exosome shell layer is selected from one or more of Curcuma rhizome source exosome, broccoli source exosome, herba Artemisiae Annuae source exosome, and tea flower source exosome. The nano generator of the invention has the functions of enhancing the overload of calcium and amplifying the oxidative stress of cells, and can improve CaO 2 Overcomes the oxidation resistance defense mechanism of tumor cells while simultaneously overcoming the stability and biocompatibility of the tumor cells. By wrapping CaO around 2 The exosome of turmeric source is used as a protective layer to solve CaO 2 The problem of poor stability and dispersibility of blood circulation in vivo increases the efficiency of the nano-generator in entering tumor cells.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a medicine-carrying calcium-based nano generator wrapped by plant-derived exosomes, a preparation method thereof and an anti-tumor application thereof.
Background
Cancer is a serious public health concern. According to the data in the world health organization and the International cancer research institute, the 2020 Global cancer report shows that 1929 ten thousand new cancer cases are global and 996 ten thousand cancer death cases are global in 2020. 457 ten thousand people with new cancer in China in 2020 account for 23.7% of the world; the number of deaths is 300 ten thousand, accounting for about 30% of the world. Cancer is the first in new cases and death number in China worldwide, and the malignant tumor burden in China is increasing weight gain at present, so breakthrough in tumor treatment is urgently needed. Currently, clinical treatments for tumors mainly include surgical therapies, radiotherapy and chemotherapy. However, the risk of cancer recurrence following surgical therapy is high; the lack of specific selectivity of radiotherapy for tumor cells and normal cells leads to various adverse reactions in patients; the chemotherapeutic drugs have the problems of poor solubility, insufficient enrichment of tumor parts, large side effects and the like. Therefore, the development of new and effective tumor treatment techniques is particularly important.
Calcium peroxide nanoparticles (CaO) 2 ) Is a common calcium ion nano generator which can respond to the weak acid environment of tumor to degrade so as to release Ca 2+ And hydrogen peroxide (H) 2 O 2 ) Released H 2 O 2 Can increase the sensitivity of tumor cells to calcium overload, consume glutathione in cells, and further kill tumor cells more effectively. CaO in neutral pH environment 2 H produced by slow decomposition 2 O 2 Can be rapidly decomposed into oxygen and water by catalase, so that oxidative stress cannot be induced in normal cells. In addition, caO has been reported in studies 2 The surface has higher specific surface area and mesoporous structure, and has good potential for loading medicines. Thus CaO 2 As calcium-based nanoparticles, they are widely used in anti-tumor research. But CaO 2 The long-term exposure to aqueous solutions is slow to degrade, and the administration into the blood through the tail vein leads to an increase in blood calcium concentration, and the dispersibility of the particle size is poor, so that a sufficient accumulation of tumor sites cannot be ensured. Furthermore, caO 2 The surface of the material is positively charged, and hemolysis reaction is caused, so that certain damage to other organs is unavoidable. Therefore, how to increase CaO 2 In vivo circulatory stability is a critical issue that must be addressed to achieve effective tumor treatment.
The existing nano generator based on calcium peroxide can be used for inducing the overload of tumor cell calcium so as to play an anti-tumor role, but has poor in vivo stability, can be slowly degraded after long-term exposure in water, and can cause the problems of hemolysis and the like to influence the further application. Although it can be made by the method of adding CaO to the mixture 2 Surface further modified material isolating CaO 2 Contact with the solution, thereby enhancing CaO 2 Is stable. However, these modified nanoreactors are treated by overloading the Fenton reaction, photodynamic or chemodynamic in combination with calcium to produce higher concentrations of ROS. However, excessive ROS risks damaging surrounding normal tissues, and, as ROS increases, tumor cells develop antioxidant defense mechanisms to accommodate high levels of ROS, rendering tumor cells resistant to ROS and unable to apoptosis. This inherent mechanism of tumor cell DNA damage repair greatly reduces the efficacy of the calcium-based nanoparticles already developed in tumor therapy, and thus there is a strong need to develop a drug that can effectively treat the tumor without damaging the body tissuesMethods of treating tumors.
Disclosure of Invention
In order to solve the problems, the nano generator is prepared by loading target drugs in the drug-loaded inner core and then coating the drug-loaded inner core and the target drugs by using plant-derived exosomes. The nano generator is found to be capable of responding to degradation in weak acid environment and producing a large amount of Ca in tumor cells 2+ And H 2 O 2 Synergizing CUR causes mitochondrial calcium overload and ROS oxidative stress. In addition, the released target medicine can overcome the problem of ROS damage tolerance in tumor cells, improve the killing power of ROS on the tumor cells, and realize the effect of killing the tumor cells by synergizing calcium overload. Animal experiment results show that after the plant source exosomes are wrapped, the hemolytic reaction of the drug-loaded calcium-based nano generator is obviously reduced, and the nano generator is verified to be capable of effectively inhibiting the growth of tumors and improving the survival rate of mice. The drug-loaded calcium-based nano generator wrapped by plant-derived exosomes is expected to be used as an effective anti-tumor nano preparation for enhancing calcium overload to amplify oxidative stress.
The invention aims to provide a medicine-carrying calcium-based nano generator wrapped by plant-derived exosomes, which comprises a medicine-carrying inner core, wherein target medicines are loaded in mesopores of the medicine-carrying inner core, and the outer side of the medicine-carrying inner core is wrapped with a plant-derived exosome shell layer;
wherein the plant-derived exosome shell layer is selected from one or more of Curcuma rhizome exosomes (TDNPs), broccoli exosomes, herba Artemisiae Annuae exosomes, and tea flower exosomes.
Preferably, the plant-derived exosome shell layer is a turmeric-derived exosome.
Specifically, curcumin (CUR) is an antitumor active substance. CUR can promote Ca 2+ Release from the endoplasmic reticulum to the cytoplasm and inhibition of Ca 2+ Increasing Ca in mitochondria from cytoplasmic discharge to extracellular 2+ Concentration, thereby synergistically inducing calcium-based nanomaterials to undergo mitochondrial calcium overload, which in turn causes mitochondrial dysfunction, caspase-3 upregulation and subsequent apoptosis. Calcium-based nanoparticle-loaded CUR (compute unified device architecture) realization synergyCalcium overload, but poor water solubility of CUR affects its bioavailability. The plant-derived exosomes extracted from the edible turmeric have CUR, have a structure similar to that of animal-derived exosomes, have a structure of a phospholipid-containing bilayer membrane with negative charges on the surface, and can be used as a delivery carrier to encapsulate drugs or nanoparticles inside. Compared with liposome carrier, the liposome carrier has the characteristics of good stability, good biocompatibility and low immunogenicity, and can obtain higher cell uptake rate through plasma membrane fusion; compared with an exosome of animal origin, the preparation method has the characteristics of easily obtained raw materials, mass preparation and good water solubility. More importantly, the turmeric-derived exosomes containing CUR can simultaneously improve the water solubility of CUR. Based on the characteristics, the turmeric-derived exosomes are expected to be used as a promising novel carrier and applied to tumor treatment in combination with a calcium-based nano generator.
Further, the drug-loaded inner core is selected from one or more of calcium peroxide, calcium carbonate and calcium fluoride.
Further, the target drug is selected from one or more of TH588, TH287,(s) -Crizotinib, 3-lsomangostin, MTH 1-IN-2.
Further, the particle size of the nano generator is 90-150nm.
Further, the mass ratio of the drug-carrying inner core to the target drug to the plant-derived exosome shell layer is 1:0.07-0.09:1.1-1.3.
Particularly, on the premise of ensuring stability and safety, the calcium-based nano generator delivers target drugs into tumor cells, solves the problem of tolerance of the tumor cells to ROS in the existing calcium-based nano material, and greatly improves the killing power of ROS to the tumor cells. Finally, the experiment proves that the nano generator has excellent anti-tumor treatment effect in vitro and in vivo and has higher biological safety in vivo. Therefore, the drug-loaded calcium-based nano generator wrapped by the turmeric-derived exosomes provides a means with development prospect for tumor treatment.
The invention also provides a preparation method of the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes, which comprises the following steps: and coating the medicine-carrying inner core by the plant-derived exosome shell layer to obtain the nano generator.
Further, the time of the coating is 25-30min.
The invention also provides application of the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes in preparation of antitumor drug administration.
The invention also provides application of the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes in synergistic anti-tumor.
The invention has the following beneficial effects:
1. the nano generator has the functions of enhancing the calcium overload and amplifying the oxidative stress of cells, and can overcome the anti-oxidative defense mechanism of tumor cells while improving the stability and biocompatibility of the medicine carrying inner core. The plant-derived exosome wrapped outside the drug-carrying inner core is used as a protective layer, so that the problem that the nano generator is poor in-vivo blood circulation stability and dispersibility is solved, and the efficiency of the nano generator entering tumor cells is increased. Meanwhile, on the premise of ensuring stability and safety, the plant source exosome simultaneously delivers the drug-carrying inner core, the target drug and the plant source exosome into the tumor cells, overcomes the challenge of repairing the DNA damage of the tumor cells under the condition of not generating excessive ROS, and finally realizes the curative effect of killing the tumor cells with high efficiency.
2. The invention designs the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes, and can be applied to anti-tumor treatment without damaging organism tissues. The technical method has the advantages of simple preparation method, cheap raw materials and lower cost. The solubility of the antitumor active substances contained in the plant-derived exosomes can be improved by using the plant-derived exosomes as a shell layer, so that the bioavailability of the plant-derived exosomes in vivo is improved, and the calcium overload of tumor cells can be caused by the cooperation of drug-carrying inner cores, thereby providing a ingenious idea for the application of the plant-derived exosomes in tumor treatment.
Drawings
FIG. 1 (a) is CaO 2 A transmission electron microscope image of the nanoparticle;
FIG. 1 (b) is a transmission electron microscope image of TDNPs nanoparticles;
FIG. 1 (c) is TDNPs@CaO 2 A transmission electron microscope image of the nanoparticle, wherein black arrows represent exosome membrane structure diagrams;
FIG. 1 (d) is CaO 2 Nanoparticles, TDNPs nanoparticles and TDNPs@TH588@CaO 2 SDS-PAGE gel electrophoresis of the nano-generator;
FIG. 1 (e) is TDNPs@TH588@CaO 2 Element distribution diagram of nano generator;
FIG. 1 (f) is TDNPs@CaO 2 CaO in nanoparticle systems 2 And TDNPs;
FIG. 1 (g) is CaO 2 Nitrogen adsorption-desorption isothermal curves of nanoparticles;
FIG. 1 (h) is CaO 2 Nanoparticles and TDNPs@CaO 2 Particle size distribution diagram of the nano generator;
FIG. 1 (i) is CaO 2 Pore size distribution map of the nanoparticles;
FIG. 1 (j) is TDNPs nanoparticle, caO 2 Nanoparticles, CUR@CaO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 Zeta potential diagram of the nano-generator;
FIG. 1 (k) is TDNPs nanoparticle, caO 2 Nanoparticles, CUR@CaO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 The hydrated particle size measurement of the nano-generator, (n=3, data expressed as mean ± standard deviation);
FIG. 1 (l) is the dispersion of CUR nanoparticles and TDNPs nanoparticles in aqueous solution, respectively;
FIG. 2 (a) is TDNPs@CaO 2 The nanoparticles were incubated in phosphate buffer at pH 7.4 and pH 6.0 for different times (0 h and 12 h) for transmission electron microscopy images.
FIG. 2 (b) is CaO 2 Nanoparticles and TDNPs@CaO 2 Calcium release profile of nanoparticles after incubation in phosphate buffer at pH 7.4 and pH 6.0 for different times;
FIG. 2 (c) is CaO 2 Nanoparticles and TDNPs@CaO 2 The nanoparticles were incubated in phosphate buffer at pH 7.4 and pH 6.0H after different incubation times 2 O 2 Is a curve generated by the method;
FIG. 2 (d) is TDNPs@TH588@CaO 2 Incubating the nano generator in phosphate buffer solution with pH of 7.4 and pH of 6.0 for different time to obtain a drug release curve;
FIG. 3 (a) is CaO 2 Nanoparticles and TDNPs@CaO 2 Fluorescent intensity quantitative analysis of endocytosis of the nanoparticles after incubation with 4T1 tumor cells for different times;
FIG. 3 (b) is TH588, TDNPs nanoparticle, caO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 Cell activity of the nano generator after 24h incubation with 4T1 tumor cells;
FIG. 3 (c) is a TDNPs nanoparticle, caO 2 Nanoparticles, TDNPs@CaO 2 Intracellular calcium ion content after 8h incubation of the nanoparticles with 4T1 tumor cells;
FIG. 3 (d) is TH588, TDNPs nanoparticle, caO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 Confocal imaging images of the nano-generator stained with ROS probe after 8h incubation with 4T1 tumor cells;
FIG. 3 (e) is TH588, TDNPs nanoparticle, caO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 Confocal imaging images of the nano-generator stained with JC-1 probe after 8h incubation with 4T1 tumor cells;
FIG. 3 (f) is TH588, TDNPs nanoparticle, caO 2 Nanoparticles, TDNPs@CaO 2 Nanoparticles, TDNPs@TH588@CaO 2 Apoptosis after 24h incubation of the nano generator and the 4T1 tumor cells;
FIG. 4 (a) is a schematic illustration of an experimental protocol for in vivo anti-tumor studies;
FIG. 4 (b) is a graph showing tumor growth of BALB/c mice after tail vein injection of various groups of nanoparticles;
FIG. 4 (c) is an image of an anatomic tumor after 18 days of treatment with each group of nanoparticles by tail vein injection in BALB/c mice;
FIG. 4 (d) is a graph showing the average tumor weight statistics of BALB/c mice;
FIG. 4 (e) is a spleen average weight statistic of BALB/c mice;
FIG. 4 (f) is an image of H & E, TUNEL, ki67, ROS and MTH1 stained sections of tumors from BALB/c mice treated with each group of nanoparticles by tail vein injection for 18 days;
FIG. 4 (g) is a statistical plot of survival of BALB/c mice treated with each group of nanoparticles by tail vein injection;
FIG. 5 (a) is an H & E slice staining image of heart, liver, spleen, lung and kidney of BALB/c mice after 18 days of treatment with each group of nanoparticles by tail vein injection;
FIGS. 5 (b) - (f) are concentration analyses of ALT, ALP, AST, BUN, CREA in serum;
FIG. 5 (g) is a monitor of body weight of mice after administration;
FIG. 5 (h) is CaO 2 Nanoparticles, TDNPs@CaO 2 Hemolysis rate analysis after in vitro incubation of nanoparticles and erythrocytes for 1 h;
FIG. 6 is a schematic diagram of the preparation and therapeutic mechanism of the nano-generator of the present invention;
FIG. 7 is a standard curve of TH588 in methanol;
FIG. 8 is a standard curve of TH588 in PBS containing 1.5wt% Tween 80 and 5wt% ethanol;
FIG. 9 is a standard curve of turmeric-derived exosomes in PBS.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the following examples are set forth. The starting materials, reactions and workup procedures used in the examples are those commonly practiced in the market and known to those skilled in the art unless otherwise indicated.
The words "preferred," "more preferred," and the like in the present disclosure refer to embodiments of the present disclosure that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
It should be understood that all numbers expressing, for example, amounts of ingredients used in the specification and claims, except in any operating example or otherwise indicated, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.
TH588, available from Shanghai Tao Shu biosystems, inc (TargetMol);
curcumin (CUR), purchased from Shanghai microphone company;
anhydrous methanol, hydrogen peroxide (30%), ammonia (25% -30%), purchased from guangzhou chemical reagent factories;
anhydrous calcium chloride, available from ala Ding Shenghua limited;
cell lines: the 4T1 breast cancer cell number used in this experiment was ATCCRL-2539, derived from American Type Culture Collection (ATCC, USA);
experimental animals: the study used BALB/c mice (6-8 weeks, female, body weight 16-18 g, SPF grade) as animal subjects purchased from zhujiang baitong biotechnology limited, animal production license number: SCXK (guangdong) 2020-0051, animal eligibility number: no.44822700019542. Mice were kept in clean areas with free water during the experiment.
Example 1
CaO 2 Preparation of nanoparticles
CaCl is added with 2 ·2H 2 O aqueous solution (1 mL,2 mol/L) was added to 60mL of anhydrous methanol, and after vigorous stirring at room temperature for 5min, 300. Mu.L of 30wt% H was added dropwise 2 O 2 Stirring for 5min, and adding NH 3 ·H 2 O (400. Mu.L, 25 wt%) was used to trigger the reaction, and after stirring for 2min, the resulting milky solution was centrifuged (20000 g,4 ℃ C., 10 min) to collect the precipitate and washed three times with anhydrous methanol to give CaO 2 Nanoparticles of CaO 2 Dispersing the nanoparticles in 10mL of absolute methanol, and preserving at 4 ℃ for later use;
FIG. 1 (a) is CaO 2 A transmission electron microscope image of the nanoparticles can observe CaO with an average diameter of about 80nm 2 In a relatively uniform spherical morphology, FIG. 1 (g) is CaO 2 Nitrogen adsorption-desorption isotherms of nanoparticles.
Example 2
Preparation of turmeric-derived exosome (TDNPs) nanoparticles
Collecting 500g of fresh and cleaned turmeric, spraying alcohol, placing into a biosafety cabinet, irradiating for 30min, turning over, continuously irradiating for 30min for sterilization, peeling the biosafety cabinet, weighing, adding sterile PBS (poly-butylene glycol) in a proportion of 1g/mL for juicing, filtering with gauze for removing residues, collecting filtrate, loading into a 50mL centrifuge tube for centrifuging (3000 g,4 ℃ for 20 min), collecting supernatant, continuously centrifuging (10000 g,4 ℃ for 40 min), taking supernatant again into an ultra-high speed centrifuge tube for ultra-high speed centrifuging (150 g,4 ℃ for 1 h), removing supernatant, adding 5mL of sterile PBS into the precipitate for blowing heavy suspension, performing ultra-dispersion uniformly, obtaining turmeric source exosomes (TDBCA) dispersed in the sterile PBS, diluting the prepared turmeric exosomes solution by 20 times with PBS, detecting protein concentration contained in turmeric source exosomes with a protein quantification kit, quantifying the concentration of turmeric source exosomes, and storing the turmeric source exosomes solution at-80 ℃;
FIG. 1 (b) is a transmission electron microscope image of TDNPs, and TDNPs having a clear film structure with a particle size of 60-180nm can be observed.
Example 3
TH588@CaO 2 Preparation of nanoparticles
A nano-generator comprising a drug-loaded inner core, wherein the surface of the drug-loaded inner core is loaded with target drugs;
wherein the average diameter of the nano-generator is about 90nm; the target medicine is TH588, and the medicine carrying inner core is CaO 2 And (3) nanoparticles.
The preparation method of the nano generator comprises the following steps:
will contain 0.2mL of 5mg/mL CaO at room temperature 2 Diluting with methanol to 1mg/mL, adding 0.2mg of TH588 for drug loading, centrifuging after 24h, and washing with methanol for 2 times to obtain TH588@CaO 2 And measuring with ultraviolet spectrophotometerAnd the drug loading rate is 7.742 percent, and finally 1mL of absolute methanol is added for resuspension and preservation at 4 ℃ for standby.
Wherein the drug loading= (M TH588 feeding -M TH588 washing supernatant )/(M TH588 feeding -M TH588 washing supernatant +M CaO2 )×100％。
Example 4
TDNPs@CaO 2 Preparation of nano-generators
A drug-loaded calcium-based nano generator wrapped by plant-derived exosomes, the nano generator comprises a drug-loaded inner core, and a plant-derived exosome shell layer is wrapped on the outer side of the drug-loaded inner core;
wherein, as shown in fig. 1 (c), the particle size of the nano generator is 90nm; the medicine carrying inner core is CaO 2 Nanoparticles; the plant source exosome shell layer is a turmeric source exosome;
the preparation method of the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes comprises the following steps:
0.2mL of CaO at a concentration of 5mg/mL was taken 2 Is centrifuged to collect the precipitate, 2mg of TDNPs is added, and CaO is treated with PBS 2 The concentration is regulated to 1mg/mL, and the film is coated to obtain TDNPs@CaO 2 The supernatant was taken to measure the UV absorbance, the supernatant concentration was calculated from the standard curve of turmeric-derived exosomes in PBS (FIG. 9), and TDNPs@CaO was used 2 Adding sterile PBS, and storing at 4deg.C for no more than 24 hr, and applying to test in time; wherein, the step of the coating is as follows: ice-bath ultrasound at 10% power for 2s, closing for 3s, repeating for 1min, incubating at 4deg.C for 15min, centrifuging (10000 g, 4deg.C for 10 min);
according to the formula: coating ratio= (M Initial TDNPs -M Supernatant TDNPs )/(M Initial TDNPs -M Supernatant TDNPs +M CaO2 )×100％，
Obtaining the TDNPs@CaO 2 The coating rate is 54.55%;
FIG. 1 (h) is CaO 2 Nanoparticles and TDNPs@CaO 2 Particle size distribution of the nano-generator, (n=3, data expressed as mean ± standard deviation).
Example 5
TDNPs@TH588@CaO 2 Preparation of nano-generators
Referring to fig. 6, a drug-loaded calcium-based nano-generator wrapped by plant-derived exosomes, the nano-generator comprising a drug-loaded inner core, the surface of the drug-loaded inner core being loaded with a target drug;
The outer side of the medicine carrying inner core is coated with a shell layer of a plant-derived exosome;
wherein the particle size of the nano generator is 90nm; the target medicine is TH588, and the medicine carrying inner core is CaO 2 Nanoparticles; the shell layer of the plant-derived exosome is TDNPs;
the preparation method of the drug-loaded calcium-based nano generator wrapped by the plant-derived exosomes comprises the following steps:
take 0.2mL TH588@CaO of example 3 2 Is centrifuged to collect the precipitate, 2mg of TDNPs is added and TH588@CaO is treated with PBS 2 The concentration is regulated to 1mg/mL, and the film is coated to obtain TDNPs@TH588@CaO 2 The supernatant was taken to measure the ultraviolet absorbance, and the supernatant concentration was calculated from the standard curve of turmeric-derived exosomes in PBS (fig. 9);
wherein, the step of the coating is as follows: ice-bath sonication at 10% power for 2s, shut off for 3s, repeated for 1min, and after incubation at 4℃for 15min, centrifugation (10000 g,4℃for 10 min).
According to the formula: coating ratio= (M Initial TDNPs -M Supernatant TDNPs )/(M Initial TDNPs -M Supernatant TDNPs +M CaO2 )×100％，
Obtaining the TDNPs@CaO 2 The coating rate is 54.55%;
FIG. 1 (e) is TDNPs@TH588@CaO 2 The elemental distribution of the nano-generator can be seen as TDNPs@TH588@CaO 2 The nano generator has a uniformly distributed Ca, O, cl, C and N element structure, and the nano generator prepared by the method comprises CaO 2 And entrapped drug TH588 (Cl element with characteristics).
FIG. 1 (i) is CaO 2 Pore size distribution map of the nanoparticles; it can be seen that CaO is prepared 2 Having a diameter of 109.7589m 2 The specific surface area per g and pore size of 21.6745nm, gives it drug-loading potential, and the drug-loading of TH588 is 7.742% calculated from the standard curve of TH588 (as shown in fig. 7). The experimental results jointly prove that TDNPs@TH588@CaO 2 Successful construction of the nano-generator.
Comparative example 1
CUR@CaO 2 Preparation of nanoparticles
A nanoparticle comprising a drug-loaded core, the drug-loaded core having a mesoporous loading of curcumin.
Wherein, the particle size of the nanoparticle is 814nm;
the preparation method of the nanoparticle comprises the following steps:
1mg of CaO of example 1 was added at room temperature 2 Diluting with methanol to 1mg/mL, adding 0.2mg CUR for drug loading, centrifuging after 24 hr, and washing with methanol for 2 times to obtain CUR@CaO 2 And (3) nanoparticles.
FIG. 1 (j) is TDNPs nanoparticle, caO 2 Nanoparticles, CUR@CaO 2 Nanoparticles, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 Zeta potential diagram of the nano-generator; FIG. 1 (k) is TDNPs nanoparticle, caO 2 Nanoparticles, CUR@CaO 2 Nano generator, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 The hydrated particle size measurement of the nano-generator, (n=3, data expressed as mean ± standard deviation); further validation of TDNPs@TH588@CaO was achieved by measuring zeta potential and hydrated particle size 2 Successful construction of the nanogenerator, caO before coating 2 Has a zeta potential of +20.5mv and a hydrated particle size of 556.6nm, and TDNPs@CaO after encapsulation of turmeric-derived exosomes 2 The zeta potential of the nano-generator was converted to-15.8 mv and the hydrated particle size was 171nm. The zeta potential is changed from positive charge to negative charge, and the improvement of the dispersibility of the hydrated particle size indicates the success of the coating. Furthermore, caO is treated 2 Cur@cao obtained after drug loading CUR 2 Particle size 814nm, indicating that CUR@CaO is due to the very strong hydrophobicity of CUR 2 The dispersibility of (3) is greatly reduced, however TDNPs@CaO 2 The hydration particle size of the nano generator is far smallerAt CUR@CaO 2 This demonstrates that TDNPs are more beneficial for reducing CaO than CUR in direct drug delivery 2 。
FIG. 1 (l) shows the dispersion of CUR and TDNPs in aqueous solution, respectively, and shows that CUR is low in solubility in water, and the phenomenon that CUR is settled at the bottom of a centrifuge tube is observed, while TDNPs are uniformly dispersed to show the phenomenon that the solution is clear. The TDNPs are good in water solubility, and can improve the solubility of CUR contained in the TDNPs.
Test example 1
For TDNPs@CaO of example 4 2 Nano-generator, TDNPs nano-particle of example 2 and CaO of example 1 2 Nanoparticle electrophoresis analysis test
Respectively subjecting the TDNPs@CaO to 2 Nano-generator, TDNPs nano-particle and CaO 2 Adding a sample solution to be detected of the nanoparticles into a loading buffer solution loading buffer, enabling the volume ratio of the sample solution to be detected to be 4:1, heating and denaturing at 97 ℃ for 10min, adding the sample into a 12wt% SDS-PAGE gel for electrophoresis, and then imaging by a multifunctional molecular imaging instrument;
FIG. 1 (d) is CaO 2 Nanoparticles, TDNPs nanoparticles and TDNPs@CaO 2 SDS-PAGE gel electrophoresis of the nano generator shows that TDNPs@CaO are obtained by analysis of SDS-PAGE gel electrophoresis 2 The protein band of the nano-generator is similar to that of the simple TDNPs.
TDNPs@CaO 2 Co-location testing of nano-generators
TDNPs@CaO using a laser confocal microscope (CLSM, zeiss LSM 800, germany) and a Beckman Cytoflex flow cytometer 2 The nano-generator was subjected to co-localization test, the test results are shown in FIG. 1 (e), wherein the turmeric-derived exosome membrane was labeled with DiO (green), whereas CaO 2 Marked with Nile Red (Red).
FIG. 1 (f) is TDNPs@CaO 2 CaO in a nano-generator system 2 And TDNPs, it can be seen that CaO was labeled with nile red 2 DiO-marked TDNPs were examined by laser confocal microscopy for CaO under excitation at 528nm and 484nm, respectively 2 Fluorescence co-localization with TDNPs, results indicate thatConstructed TDNPs@CaO 2 The nano-generator can observe obvious red and green fluorescent signals at the same time, and has good co-localization.
Test example 2
Examine the drug release performance of TDNPs@TH588@CaO2 nano generator under neutral and acidic conditions
The experimental method comprises the following steps: the standard curve for TH588 was determined in PBS solution containing 1.5wt% Tween 80 and 5wt% ethanol;
2.5mg of TDNPs@TH588@CaO 2 The nano generator is respectively dispersed in PBS (phosphate buffered saline) solution containing 1.5wt% Tween 80 and 5wt% ethanol and having pH value of 6 and pH value of 7.4, 0.6mL of the solution is taken for centrifugation after shaking incubation for 0h,1.5h,3h,6h,9h and 12h at 37 ℃, the supernatant is taken for ultraviolet absorbance, 0.6mL of PBS buffer solution is supplemented, and a standard curve of TH588 between 1.5wt% Tween 80 and 5wt% ethanol is obtained, and the standard curve is shown in figure 8.
Test example 3
Investigation of acid response degradation and drug release Capacity
After determining TDNPs@TH588@CaO 2 After successful fabrication of the nano-generator, the study then explores its pH responsive release properties.
The testing method comprises the following steps:
observing TDNPs@CaO by adopting transmission electron microscope 2 The nano generator is incubated for 0h and 12h in 1mL of 0.5mg/mL phosphate buffer solution with pH 7.4 and pH 6.0 respectively, and then TDNPs@CaO is added 2 Degradation of the nano-generator. As can be seen from FIG. 2 (a), TDNPs@CaO 2 Significant degradation was observed after incubation of the nano-generator in an acidic solution (pH 6.0) for 12 hours, whereas TDNPs@CaO 2 The nano-generator remained essentially unchanged after 12h incubation at neutral conditions (pH 7.4). Under acidic condition, the final nano generator TDNPs@CaO 2 Degradation of the nano-generator releases Ca 2+ (FIG. 2 (b));
as shown in FIG. 2 (c) and FIG. 2 (d), TDNPs@CaO after encapsulation by turmeric-derived exosomes 2 Nano generator and CaO 2 Ca under acidic and neutral conditions compared to nanoparticles 2+ Release amount, H 2 O 2 And TH588 releaseThe amount of the exosomes is slightly reduced, which is probably due to the fact that the exosomes are wrapped to isolate the CaO to a certain extent 2 Contact with phosphate buffer solution enhances CaO 2 Is stable. After 12h incubation with phosphate buffer at pH 6.0, TDNPs@CaO 2 Ca of nano generator 2+ The release amount reaches 57.23%, H 2 O 2 The yield was 95.2. Mu.M, and the TDNPs@TH588@CaO was then used 2 In the drug release experiment with the nano-generator, the amount of TH588 released was 75.94% as measured according to the standard curve of TH588 (see fig. 8). It can be seen that the constructed nano-generator has better weak acid environment response degradation characteristics, as shown in fig. 2 (d). Compared with other calcium-based nano-device preparation technologies, the curcumin-derived exosome used in the invention does not need the step of drug loading CUR, so that the time cost is greatly saved.
Test example 4
TDNPs@CaO has been previously verified 2 The nano generator has better weak acid response degradation characteristic, and then the TDNPs@CaO is researched 2 Uptake of the nanoscaler by 4T1 tumor cells;
the testing method comprises the following steps:
first, 50ug/mL of CaO labeled with nile red dye 2 And TDNPs@CaO 2 Nano generator (CaO contained) 2 50 μg/mL) were incubated with 4T1 tumor cells for 2h,4h,8h, and then the cells were collected, and the fluorescence intensity quantitative analysis was performed using flow cytometry to examine the uptake of nanoparticles by the cells.
FIG. 3 (a) is CaO 2 And TDNPs@CaO 2 Fluorescence intensity quantitative analysis of endocytosis of the nanoscaler after incubation with 4T1 tumor cells for different times (n=3, data expressed as mean ± standard deviation); as shown in FIG. 3 (a), with CaO 2 In contrast, TDNPs@CaO 2 The nano-generator uptake exhibited a significant increase, with the time-dependent increase in uptake characteristic, probably due to the increased CaO by encapsulation of turmeric-derived exosomes 2 And the ability of the exosomes to fuse with the cell membrane thereby promoting cellular uptake of the nanoparticle.
Test example 5
In the demonstration of TDNPs@CaO 2 After good cellular uptake by the nano-generator we used the MTT method to evaluate CaO 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 Cytotoxicity of the nano-generator;
the testing method comprises the following steps:
4T1 tumor cells were plated in 96-well plates at 7X 10 per well 3 The density of individual cells was cultured at 37℃for 18 hours. Thereafter, 100. Mu.L of CaO at 200. Mu.g/mL was added, respectively 2 Nanoparticles, 100. Mu.L of TDNPs nanoparticles of 240. Mu.g/mL, 100. Mu.L of TH588 nanoparticles of 16.78. Mu.g/mL, 100. Mu.L of TDNPs@CaO nano generator (CaO contained) 2 200. Mu.g/mL), 100. Mu.L TDNPs@TH588@CaO 2 (CaO contained) 2 DMEM medium containing 200. Mu.g/mL) of the nanosensor was incubated at 37℃for 24h in a cell incubator. The solution was carefully discarded, 100. Mu.L of blank DMEM containing 10. Mu.L of MTT was added to each well and incubated for 4 hours at 37℃in a cell incubator protected from light. Subsequently, 100 μl of formazan solution was added and incubated in the dark for at least 4 hours to dissolve the resulting purple crystals, and absorbance of each well was measured at 570nm using an enzyme-labeled instrument;
FIG. 3 (b) is CaO 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 Cell activity after 24h incubation of the nanogenerator with 4T1 tumor cells (n=3, data expressed as mean ± standard deviation); as shown in FIG. 3 (b), after 24h incubation with cells, TDNPs@CaO 2 Nano generator and TDNPs@TH588@CaO 2 The cell viability of the nano-generator treated group was 38.62% and 13.42%, respectively, with CaO 2 Compared with the TH588 treatment group, the nanoparticle, the TDNPs nanoparticle and the TH588 treatment group have obviously improved capability of inhibiting the growth activity of the 4T1 tumor cells.
Test example 6
We used Fluo 4-AM probe to study intracellular calcium ion content for TDNPs@CaO 2 And TDNPs@TH588@CaO 2 Is researched by an anti-tumor mechanism of the formula (I);
the testing method comprises the following steps:
4T1 tumor cells were 1X 10 per well 5 The density of individual cells was seeded in confocal dishes and incubated for 18h. Then each was treated with 1mL of CaO at 100. Mu.g/mL 2 Nanoparticles, 1mL of TDNPs nanoparticles of 120 mug/mL, 1mL of TDNPs@CaO 2 Nano generator (CaO contained) 2 At 100. Mu.g/mL) was incubated with 4T1 tumor cells for 8h at 37℃in a cell incubator. Subsequently, the cells were washed three times with PBS and stained with 1mL of Fluo 4-AM (4. Mu.M) solution for 30min in the dark. Cells were collected after pancreatin digestion for flow cytometry testing.
FIG. 3 (c) is CaO 2 Nanoparticles, TDNPs nanoparticles, TDNPs@CaO 2 Intracellular calcium ion content after 8h incubation of the nano-generator with 4T1 tumor cells (n=3, data expressed as mean ± standard deviation); as shown in FIG. 3 (c), TDNPs@CaO was studied 2 Intracellular Ca after 8h incubation of nanoparticles with 4T1 tumor cells 2+ The content is as follows. Experimental results show that TDNPs@CaO 2 Nano generator treated intracellular Ca 2+ The content is obviously higher than that of TDNPs nano particles and CaO 2 Nanoparticle treated cells. This is probably due to TDNPs@CaO 2 Good endocytosis of the nano-generator and the CUR carried in the TDNPs nano-particles can inhibit Ca 2+ The cells are excreted, thus together resulting in a significant increase in intracellular Ca 2+ The content is as follows.
Test example 7
Detecting intracellular ROS levels using DCFH-DA fluorescent probes;
the testing method comprises the following steps:
4T1 tumor cells were plated at 2X 10 per well 5 The density of individual cells was seeded in confocal dishes and incubated for 18h. Then 1mL of CaO with a concentration of 100 mug/mL is used 2 Nanoparticles, 1mL of TDNPs nanoparticles of 120 mug/mL, 1mL of TH588 nanoparticles of 8.39 mug/mL, 1mL of TDNPs@CaO 2 Nano generator (CaO contained) 2 At 100. Mu.g/mL), 1mL TDNPs@TH588@CaO 2 Nano generator (CaO contained) 2 At 100 μg/mL) were incubated with 4T1 tumor cells at 37 ℃ in a cell incubator for 8h, the cells were washed three times with PBS, nuclei were stained with Hoechst 33342, and intracellular fluorescence was observed using CLSM.
FIG. 3 (d) is CaO 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 Confocal imaging images of the nano-generator stained with ROS probe after 8h incubation with 4T1 tumor cells; as shown in FIG. 3 (d), it was found that TDNPs@CaO 2 Nano generator and TDNPs@TH588@CaO 2 The strongest green fluorescence was observed in the nano-generator treated 4T1 tumor cells, indicating that both nanoparticles produced large amounts of ROS after treatment of the cells and that the entrapped drug TH588 did not affect the ability of the nanoparticles to produce ROS.
Test example 8
Since both an increase in intracellular calcium content and the generation of large amounts of ROS lead to mitochondrial calcium overload, we subsequently used JC-1 probes to detect changes in mitochondrial membrane potential.
The testing method comprises the following steps: 4T1 tumor cells were plated at 2X 10 per well 5 The density of individual cells was seeded in confocal dishes and incubated for 18h, followed by 1mL of 100. Mu.g/mL CaO 2 Nanoparticles, 1mL of TDNPs nanoparticles of 120 mug/mL, 1mL of TH588 nanoparticles of 8.39 mug/mL, 1mL of TDNPs@CaO 2 Nano generator (CaO contained) 2 At 100. Mu.g/mL), 1mL TDNPs@TH588@CaO 2 Nano generator (CaO contained) 2 At 100 μg/mL) were incubated with 4T1 tumor cells at 37 ℃ in a cell incubator for 8h, the cells were washed three times with PBS, stained with JC-1 kit, followed by staining the nuclei with Hoechst 33342, and intracellular fluorescence was observed using CLSM.
FIG. 3 (e) is CaO 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 Confocal imaging images of the nano-generator stained with JC-1 probe after 8h incubation with 4T1 tumor cells; as shown in FIG. 3 (e), TDNPs@TH588@CaO 2 The strongest green fluorescence was observed in the nano-generator treated 4T1 tumor cells, probably due to CaO coating the turmeric-derived exosomes 2 Can enhance intracellular calcium ion content and ROS level, while entrapped TH588 can inhibit MTH1 protein activity, block DNA oxidative repair, thereby enhancing mitochondrial DNA sensitivity to ROS, and furtherAffecting mitochondrial function and resulting in a significant drop in mitochondrial membrane potential.
Test example 9
Further verifying TDNPs@TH588@CaO 2 We measured CaO by Annexin-V FITC/PI kit 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator and TDNPs@TH588@CaO 2 Apoptosis after 24h incubation of the nano generator and the 4T1 tumor cells;
the testing method comprises the following steps:
4T1 tumor cells were plated at 2X 10 per well 5 The density of individual cells was seeded in 6-well plates and incubated for 18h. Then 2mL of CaO at 200. Mu.g/mL was added 2 Nanoparticles, 2mL of TDNPs nanoparticles of 240 mug/mL, 2mL of TH588 nanoparticles of 16.78 mug/mL, 2mL of TDNPs@CaO 2 Nano generator (CaO contained) 2 At 100. Mu.g/mL) and 2mL of TDNPs@TH588@CaO 2 Nano generator (CaO contained) 2 At 100. Mu.g/mL) was incubated with 4T1 tumor cells for 24h, respectively. After incubation with an Annexin V-FITC/PI apoptosis detection kit for 15min under the dark condition, the apoptosis condition is analyzed by a flow cytometer.
FIG. 3 (f) is CaO 2 Nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO 2 Nano generator and TDNPs@TH588@CaO 2 Apoptosis after 24h incubation of the nanogenerator with 4T1 tumor cells. P value was calculated by t-test: * P is p<0.05,**p<0.01,***p<0.001；
As shown in FIG. 3 (f), TDNPs@TH588@CaO 2 The apoptosis rate of the 4T1 tumor cells is obviously increased after the treatment of the nano generator, which shows that TDNPs@TH588@CaO 2 The synergistic effect of the calcium overload amplified oxidative stress caused by the nano generator and the inhibition of DNA damage repair exerts excellent anti-tumor effect.
Test example 10
To evaluate TDNPs@TH588@CaO 2 In vivo anti-tumor efficacy, we established a 4T1 breast cancer tumor cell subcutaneous tumor vaccination model using BALB/c mice;
the specific method comprises the following steps: the right lower abdomen of each mouse was dehaired before day 6 of administration, withA1 mL sterile syringe was used to subcutaneously administer 100. Mu.L of 1X 10 to the right lower abdomen of a subcutaneous dehairing 6 PBS solution of individual 4T1 breast cancer tumor cells was then fed for observation, as shown in fig. 4 (a).
When the tumor volume reaches 80mm 3 After that, BALB/c mice were randomly divided into PBS, TH588, caO 2 Nanoparticles, TDNPs nanoparticles, TDNPs@CaO 2 Nano generator and TDNPs@TH588@CaO 2 The nano-generator groups, 6 per group, were administered by tail vein injection on days 0,2,4,6,8, and tumor volumes were recorded and calculated (L/2 x w) and body weights of mice were measured.
Wherein, the dosage calculated according to the drug loading and the coating rate: the TH588 dose was: 0.05 mg/CaO 2 The dosage is as follows: the dose of TDNPs was 0.6 mg/dose: 0.72 mg/min;
TDNPs@CaO 2 nano generator, TDNPs@TH588@CaO 2 Nano generator with CaO contained therein 2 The calculated dosage of the content is as follows: 0.6 mg/dose.
FIG. 4 (b) is a tumor growth curve of BALB/c mice injected with each group of nanoparticles via tail vein (n=6, data expressed as mean.+ -. Standard deviation); FIG. 4 (c) is an image of an anatomic tumor after 18 days of treatment with each group of nanoparticles by tail vein injection in BALB/c mice; FIG. 4 (d) is a graph showing the average tumor weight statistics of BALB/c mice;
the results are shown in FIGS. 4 (b) - (d), TH588, TDNPs nanoparticle, caO, compared to PBS blank after 18 days of treatment 2 The nanoparticle treated group had a weaker therapeutic effect, while TDNPs@CaO 2 The nanometer generator treatment group has obvious anti-tumor curative effect, the tumor inhibition rate reaches 67.55 percent, and importantly, the TDNPs@TH588@CaO 2 The nanometer generator treatment group shows the best treatment effect, and the tumor inhibition rate reaches 87.3%, which indicates that TDNPs@TH588@CaO 2 The nano generator has obvious in vivo synergistic anti-tumor effect.
Fig. 4 (e) is a spleen mean weight statistic plot of BALB/c mice (n=6, data expressed as mean ± standard deviation); when tumor growth induced splenomegaly in tumor model, as shown in fig. 4 (e), compared to PBS blank,TDNPs@CaO 2 the weight of spleen of the nano generator treatment group is reduced to 59.07% of that of PBS group, so that the nano generator treatment group has a certain effect of relieving splenomegaly; and TDNPs@TH588@CaO 2 The spleen weight of the nano-generator treated group was reduced to 37.07% of that of the PBS group, and the effect of relieving splenomegaly was most obvious, which also verifies that TDNPs@TH588@CaO 2 Tumor treatment effect of the nano generator.
FIG. 4 (f) is H of tumors of BALB/c mice after 18 days of treatment with each group of nanoparticles by tail vein injection&E, TUNEL, ki67, ROS and MTH1 stained slice images; as shown in FIG. 4 (f), tumor section H&E and immunofluorescence staining results showed TDNPs@TH588@CaO 2 Tumor cells treated by the nano generator have the most obvious phenomena of cavitation, nuclear shrinkage, cytoplasmic permeabilization and the like, and TUNEL immunofluorescence staining shows that TDNPs@TH588@CaO 2 Green fluorescence of the nano generator treated tumor cells is most obvious, and Ki67 immunofluorescence staining shows TDNPs@TH588@CaO 2 The red fluorescence decrease was also most pronounced in the nano-generator treated group of tumor cells, and these results further indicate that TDNPs@TH588@CaO 2 The treatment of the nano-generator causes the tumor cells to undergo the most obvious apoptosis and necrosis.
In addition, TDNPs@CaO was found by observing tumor ROS fluorescent sections 2 Nano-generator treatment group and TDNPs@TH588@CaO 2 The nanoscope treated groups exhibited the strongest green fluorescent signal, indicating that the oxygen stress levels of tumor cells were higher after both groups of treatments, which could be the cause of tumor cell apoptosis. Tumor tissue MTH1 immunofluorescence section shows that TDNPs@CaO 2 The nano-generator treatment group exhibited the strongest red fluorescence, probably because of the TDNPs@CaO 2 Nano-generator enhanced intracellular calcium overload and H 2 O 2 Is generated, thereby causing extensive DNA damage in tumor cells, and thereby exciting the tumor cells to overexpress MTH1 protein to prevent DNA from being damaged by oxidation, thus TDNPs@CaO is observed in MTH1 immunofluorescence sections 2 The strongest MTH1 expression signal of the nano-generator set (as shown in the fifth row of fig. 4 (f)). While TDNPs@TH588@CaO 2 The weakest red fluorescence was observed in the nano-generator treated group due toTH588 released by the nano generator inhibits the expression of MTH1 protein and blocks DNA damage repair of tumor cells, so that DNA is intolerant to ROS, DNA oxidative damage induced by ROS is amplified, and then the inhibition effect of calcium overload and oxidative stress on tumors is improved. We also counted the survival rate of treated mice, passing through tdnps@th588@cao 2 The highest survival rate (71.43%) was obtained for the nano-generator group treated mice, which was far higher than for the other treatment groups (as shown in fig. 4 (g)).
From the above, it is clear that TDNPs@TH588@CaO constructed in the present study 2 The nano generator has excellent in vivo synergic anti-tumor therapeutic capability.
Test example 11
To verify the biosafety of the nano-generators in mice, the study analyzed mice treated with different treatment groups.
The testing method comprises the following steps:
A4T 1 tumor cell subcutaneous tumor inoculation model was established with BALB/c mice prior to treatment (for inoculation model please refer to test case 10). The right lower abdomen of the mice was dehaired before day 6 of administration, 100. Mu.L of PBS containing 100 ten thousand 4T1 breast cancer tumor cells was subcutaneously injected into the dehaired right lower abdomen with a 1mL sterile syringe until the tumor volume reached 80mm 3 The mice with successful seed tumor are randomly divided into PBS, TH588 and CaO 2 Nanoparticles, TDNPs nanoparticles, TDNPs@CaO 2 Nano generator, TDNPs@TH588@CaO 2 The tail vein injection was performed on days 0,2,4,6,8 for 6 of the nano-generator groups, and tumor volumes were recorded and calculated (L/2×w×w) to measure body weight of the mice.
Wherein, the dosage calculated according to the drug loading and the coating rate: the TH588 dose was: 0.05 mg/CaO 2 The dosage is as follows: the dose of TDNPs was 0.6 mg/dose: 0.72 mg/min;
TDNPs@CaO 2 nano generator, TDNPs@TH588@CaO 2 Nano generator with CaO contained therein 2 The calculated dosage of the content is as follows: 0.6 mg/dose.
After the end of the administration, the tumor volume was recorded and calculated continuously every other day, and the body weight of the mice was detected. Blood was collected from the eyeball after 18 days for biochemical evaluation. Tumors and heart, liver, spleen, lung and kidney were dissected out, tumors and spleen were photographed and weighed, and then the tumors and vital organs were stained with H & E, TUNEL and Ki67 sections, MTH1 immunofluorescence labeled. To examine the formation of ROS at the tumor site after the administration, 80. Mu.M DCFH-DA probe was injected into the tumor after the end of the tail vein injection twice for 24 hours, the animals were sacrificed after 1 hour, the dissected tumor was placed in a-80℃refrigerator for 15min, and then frozen section preparation and fluorescence scanning were performed.
FIG. 5 (a) is an H & E slice staining image of the major organs (heart, liver, spleen, lung and kidney) after 18 days of treatment with each group of nanoparticles by tail vein injection in BALB/c mice; as shown in fig. 5 (a), no significant lesions and necrosis were observed in H & E sections of the major organs (heart, liver, spleen, lung and kidney) of all treatment groups relative to the PBS group.
Fig. 5 (b-f) is a concentration analysis of ALT, ALP, AST, BUN, CREA in serum (n=6, data expressed as mean ± standard deviation).
As shown in FIG. 5 (b-f), in the biochemical blood test, TDNPs@TH588@CaO 2 No significant differences were observed between aspartate Aminotransferase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN) and Creatinine (CREA) in the serum of the mice from the nano-generator treated group and healthy mice, indicating that the treatment did not cause hepatotoxicity and nephrotoxicity in the mice.
Fig. 5 (g) is a monitoring of the body weight of mice after administration (n=6, data expressed as mean ± standard deviation); as shown in FIG. 5 (g), the body weight of mice in each treatment group showed a healthy increase trend during the treatment period, indicating that TDNPs@TH588@CaO 2 Has better biological safety in vivo.
Test example 12
Hemolysis test
0.5mL of TDNPs@CaO having a concentration of 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL, 800. Mu.g/mL, 1000. Mu.g/mL was added 2 Nanometer generator, 0.5mL CaO 1000 mug/mL 2 After incubation of the nanoparticles with 0.5mL of red blood cell suspension at 37 ℃ for 1h, the samples were centrifuged (1000 g,10 min) and the enzyme was usedThe absorbance of the supernatant at 570nm was measured by a label meter.
Wherein 0.5mL of PBS and 0.5mL of a 0.1wt% Triton X-100 solution were used to incubate with 0.5mL of the red blood cell suspension, respectively, as negative and positive controls.
The hemolysis rate was calculated according to the following formula: hemolysis ratio (%) = (a) Sample of -A Negative of )/(A Positive and negative -A Negative of )×100％。
A Sample of 、A Negative of And A Positive and negative Absorbance of the sample, negative control and positive control are shown, respectively.
Fig. 5 (h) is a hemolysis analysis (n=3, data expressed as mean ± standard deviation) of different nanoparticles after in vitro incubation with erythrocytes for 1 h. P value was calculated by t-test: * p <0.05, < p <0.01, < p <0.001.
As shown in FIG. 5 (h), the hemolysis experiment shows that TDNPs@CaO 2 The nano generator can obviously improve CaO 2 Is probably due to CaO being encapsulated by turmeric-derived exosomes 2 The reduction of the positive potential on the surface improves the biocompatibility, thereby reducing the adsorption and toxicity of the nanoparticle to blood cells. In conclusion, the nano generator constructed by the method has better in-vivo biosafety, and can not cause obvious hepatotoxicity or nephrotoxicity after treatment.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (9)
1. The medicine-carrying calcium-based nano generator wrapped by the plant-derived exosomes is characterized by comprising a medicine-carrying inner core, wherein target medicines are loaded in mesopores of the medicine-carrying inner core, and the outer side of the medicine-carrying inner core is wrapped with a plant-derived exosome shell layer;
Wherein the plant source exosome shell layer is selected from one or more of Curcuma rhizome source exosome, broccoli source exosome, herba Artemisiae Annuae source exosome, and tea flower source exosome.
2. The plant-derived exosome-wrapped drug-loaded calcium-based nano-generator according to claim 1, wherein the drug-loaded core is selected from one or more of calcium peroxide, calcium carbonate, and calcium fluoride.
3. The plant-derived exosome-coated drug-loaded calcium-based nano-generator according to claim 1, wherein the target drug is selected from one or more of TH588, TH287,(s) -Crizotinib, 3-lsomangostin, MTH 1-IN-2.
4. A drug-loaded calcium-based nano-generator encapsulated by plant-derived exosomes according to claim 3, wherein the particle size of the nano-generator is 90-150nm.
5. The plant-derived exosome-wrapped drug-loaded calcium-based nano-generator according to claim 4, wherein the mass ratio of the drug-loaded inner core, the target drug and the plant-derived exosome shell is 1:0.07-0.09:1.1-1.3.
6. A method for preparing the drug-loaded calcium-based nano generator wrapped by plant-derived exosomes as claimed in any one of claims 1 to 5, comprising the steps of: and coating the medicine-carrying inner core by the plant-derived exosome shell layer to obtain the nano generator.
7. The method for preparing a drug-loaded calcium-based nano generator wrapped by plant-derived exosomes according to claim 6, wherein the time of the coating is 25-30min.
8. Use of a plant-derived exosome-coated drug-loaded calcium-based nano-generator according to any one of claims 1-5 for the preparation of an anti-tumor drug-delivery agent.
9. Use of the plant-derived exosome-encapsulated drug-loaded calcium-based nano-generator of any one of claims 1-5 in synergistic antineoplastic.
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