CN115708813A - Multifunctional manganese-based nanoparticles and preparation method and medical application thereof - Google Patents
Multifunctional manganese-based nanoparticles and preparation method and medical application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a multifunctional manganese-based nanoparticle, a preparation method and medical application thereof, wherein the multifunctional manganese-based nanoparticle comprises manganese dioxide, dihydroartemisinin, poly-metformin and hyaluronic acid, the manganese dioxide loads the dihydroartemisinin to form a manganese-based nano-core, the poly-metformin is coated on the surface of the manganese-based nano-core to form a manganese-based nano-composite, and the hyaluronic acid is combined on the surface of the manganese-based nano-composite. According to the invention, the multifunctional manganese-based nanoparticles have the functions of exerting the anti-tumor effect and stabilizing the nano-carrier, hyaluronic acid can be specifically combined with a CD44 receptor highly expressed on the surface of a tumor cell, so that the multifunctional manganese-based nanoparticles are actively targeted to the tumor part, manganese dioxide is cooperated with dihydroartemisinin to improve the ROS level in the tumor cell, thereby accelerating the apoptosis of the tumor cell induced by the ROS approach, and finally exerting the synergic anti-tumor effect of chemotherapy and chemodynamic therapy.
Description
Technical Field
The invention belongs to the field of nano material technology and biomedicine, and particularly relates to a multifunctional manganese-based nanoparticle and a preparation method and medical application thereof.
Background
The tumor microenvironment has low pH (6.5-7.0) and high hydrogen peroxide (H) 2 O 2 ) High Glutathione (GSH), and H in tumor cells 2 O 2 The concentration of the drug is higher (100. Mu.M to 1 mM) than that of normal cells, and the tumor cells are up-regulated by H 2 O 2 Is suitable for the oxidative stress state. Therefore, if a new anti-tumor drug delivery platform interacts with the tumor microenvironment, a large amount of OH is generated to destroy the original oxidation balance of the tumor microenvironment, and the anti-tumor drug delivery platform has important significance for enhancing the tumor treatment.
Chemokinetic therapy (CDT) is a type of treatment that utilizes the Fenton or Fenton-like reaction by reacting endogenous H 2 O 2 Emerging therapies that convert to OH to induce apoptosis and necrosis. CDT has high tumor specificity and selectivity, and does not need external stimulation in the tumor treatment process. In recent years, manganese dioxide nanomaterial has been one of popular research materials for CDT due to its catalase activity and good acid response behavior. In the process of mediating CDT, manganese dioxide first catalyzes H + /H 2 O 2 Production of Mn 2+ And O 2 (equation 1), and manganese dioxide degradation product Mn 2+ Continued catalysis of H 2 O 2 Strong oxidizing OH is generated (equation 2), thereby killing tumor cells.
MnO 2 +H 2 O 2 +2H + ＝Mn 2+ +2H 2 O+O 2 (equation 1)
Mn 2+ +H 2 O 2 ＝Mn 3+ +·OH+OH - (equation 2)
Although the manganese dioxide nano material shows excellent CDT effect, the application of the manganese dioxide nano material in preparing tumor drugs still faces many challenges, such as poor water dispersibility, no tumor targeting property and the like, and the targeted drug delivery and the high-efficiency low-toxicity combined treatment are difficult to realize.
Artemisinin and its derivatives are a class of sesquiterpene lactones containing a peroxy bridge and are well known for their excellent action in the treatment of malaria. In recent years, artemisinin and its derivatives have been studied to have anticancer effects and have good safety, among which the anticancer activity of Dihydroartemisinin (DHA) is the best. Among the anticancer mechanisms of DHA, the most widely accepted is the metal ion or transition metal ion mediated cleavage of peroxide bonds to form carbon radicals, which promote apoptosis. However, DHA is poorly water soluble, has a short half-life in vivo, and is difficult to accumulate in tumor sites.
Metformin (Metformin, met) is a first-line drug for treating type II diabetes, and recent epidemiological and clinical studies show that Metformin has great potential in tumor prevention, treatment and prognosis, and researches show that Metformin can independently inhibit tumor growth and can remarkably improve the curative effects of tumor radiotherapy, chemotherapy, biological treatment and the like. However, metformin is a water-soluble small-molecule drug, and targeted drug delivery is difficult to realize through conventional nano-carrier loading. The poly-metformin (PolyMet, PM) is based on natural high molecular polymer Chitosan (Chitosan, CH), and a large amount of biguanidino is grafted on a molecular structure, so that the anti-tumor activity of the metformin is inherited. Meanwhile, the PM has the characteristics of good water solubility, strong stability and the like, and can effectively modify the nano-carrier and enhance the stability of the nano-carrier in water.
Hyaluronic Acid (HA) molecule is an essential polysaccharide in the human body and HAs good biocompatibility.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a multifunctional manganese-based nanoparticle, which can effectively solve the problems of poor water dispersibility of manganese dioxide/poor water solubility of dihydroartemisinin, weak drug targeting property and the like, can realize the combination of chemotherapy and chemodynamic therapy and play a role in enhancing the anti-tumor effect of the chemodynamic therapy.
The technical scheme is as follows: the multifunctional manganese-based nanoparticle comprises manganese dioxide, dihydroartemisinin, metformin and hyaluronic acid, wherein the manganese dioxide loads the dihydroartemisinin to form a manganese-based nano inner core, the metformin is coated on the surface of the manganese-based nano inner core to form a manganese-based nano composite, and the hyaluronic acid is combined to the surface of the manganese-based nano composite.
The manganese dioxide has a honeycomb cell structure.
The poly-metformin is prepared by addition reaction of a hydrochloride macromolecule containing primary amino group or secondary amino group and a dicyandiamide monomer, wherein the hydrochloride macromolecule containing primary amino group or secondary amino group is one of chitosan, linear polyethyleneimine, polylysine, polyvinylamine and polyallylamine.
The preparation method of the multifunctional manganese-based nanoparticles is characterized by comprising the following steps of:
(1) Dicyandiamide is grafted to a hydrochloride macromolecule containing primary amino or secondary amino through a chemical reaction to obtain the poly-metformin;
(2) Preparing honeycomb manganese dioxide by potassium permanganate and oleic acid by using a soft membrane plate method; placing the obtained honeycomb manganese dioxide in a carrier dispersion medium for dispersion, and freeze-drying to obtain honeycomb manganese dioxide nanoparticles;
(3) The manganese-based nano-core is obtained by loading dihydroartemisinin on the honeycomb-shaped manganese dioxide nano-particles, the surface of the manganese-based nano-core is coated with the metformin to form a manganese-based nano-composite, and the hyaluronic acid is further combined to the outer layer of the drug-loaded manganese-based nano-composite.
In the preparation method of the multifunctional manganese-based nanoparticle, in the step (2), the carrier dispersion medium is one or more of acetone, dimethyl sulfoxide, water, ethanol, methanol or N, N-dimethylformamide.
In the preparation method of the multifunctional manganese-based nanoparticles, dihydroartemisinin is loaded into a honeycomb-shaped porous structure of manganese dioxide by a physical adsorption method in the step (3).
According to the preparation method of the multifunctional manganese-based nanoparticle, the metformin is coated on the surface of a manganese-based nano kernel through electrostatic adsorption in the step (3).
In the preparation method of the multifunctional manganese-based nanoparticle, the method for binding the hyaluronic acid to the surface of the manganese-based nanocomposite in the step (3) comprises electrostatic adsorption and chemical bond connection.
The multifunctional manganese-based nanoparticles are used for preparing antitumor drugs.
The multifunctional manganese-based nanoparticles are used for preparing a medicine for treating breast cancer.
The multifunctional manganese-based nanoparticle is characterized in that dicyanodiamine is grafted onto chitosan containing primary amino groups through a chemical reaction to obtain poly (metformin) PM, potassium permanganate is adopted for reduction to prepare cellular manganese dioxide, the cellular manganese dioxide physically adsorbs dihydroartemisinin to obtain a manganese-based nano kernel, the PM is wrapped on the surface of the manganese-based nano kernel to form a manganese-based nano composite with good water dispersibility, and hyaluronic acid is further combined to the outer layer of the drug-loaded manganese-based nano composite through electrostatic adsorption or chemical bond connection to construct the multifunctional manganese-based nanoparticle with CD44 receptor targeting-PM stabilization.
The preparation method of the multifunctional manganese-based nanoparticle comprises the following steps:
(1) Preparation of PM:
adding the polymer powder containing primary amino group or secondary amino group into hydrochloric acid solution, stirring into paste, performing hydrochloric acid treatment overnight to obtain hydrochloric acid polymer solution, and freeze-drying to obtain hydrochloric acid polymer solid. Dissolving the hydrochloric acid macromolecule solid and dicyanodiamine in a hydrochloric acid solution, heating a reaction system, cooling to room temperature after the reaction is finished, filtering, dialyzing, collecting dialysate, and freeze-drying to obtain the PM solid.
In the step (1), the macromolecule containing primary amino group or secondary amino group is one of chitosan, linear polyethyleneimine, polylysine, polyvinylamine and polyallylamine; dissolving the polymer powder in 75-150mL 0.5-1.5% (m/m) hydrochloric acid; dissolving the hydrochloride polymer solid in 15-50mL0.5-1.5% (m/m) hydrochloric acid; the mass ratio of the hydrochlorinated high-molecular solid to the dicyanodiamide is (1).
Preferably, the primary amino group-containing polymer is chitosan; dissolving the polymer powder in 100mL of 1% (m/m) hydrochloric acid; dissolving the hydrochloric acid macromolecule solid in 20mL of 0.75% (m/m) hydrochloric acid; the mass ratio of the acidified polymer solid to dicyanodiamine is 1.
(2) Preparing the honeycomb manganese dioxide nanoparticles:
adding potassium permanganate into ultrapure water, stirring and uniformly mixing at normal temperature, slowly adding oleic acid into the potassium permanganate solution under the stirring condition, and after the room-temperature reaction is finished, changing the original solution from purple red into a dark brown turbid liquid state. And (3) collecting a product through centrifugation, washing the product with ethanol and ultrapure water for several times respectively, and finally drying the product to obtain the honeycomb-shaped manganese dioxide solid. And (3) placing the cellular manganese dioxide in a proper amount of carrier dispersion medium, performing ultrasonic dispersion by using a probe, and freeze-drying the aqueous dispersion to obtain cellular manganese dioxide nanoparticles (M).
In the step (2), the volume ratio of the potassium permanganate aqueous solution to the oleic acid is 150-600; the mass ratio of potassium permanganate to oleic acid is (1); the reaction time of potassium permanganate and oleic acid is 12-36 h; the carrier dispersion medium is one or more solvents of acetone, dimethyl sulfoxide, water, ethanol, methanol and N, N-dimethylformamide; the ultrasonic treatment time of M is 5-30 min.
Preferably, the volume ratio of the potassium permanganate solution to the oleic acid is 500; the mass ratio of potassium permanganate to oleic acid is 1; the reaction time of potassium permanganate and oleic acid is 24 hours; the carrier dispersion medium is water; the sonication time for M was 10min.
(3) Preparing a manganese-based nano inner core:
dissolving a certain amount of DHA in an organic solvent, adding the honeycomb-shaped manganese dioxide nanoparticles prepared in the step (2) into the DHA solvent, ultrasonically dispersing by using a probe, then placing in a vacuum drier, and slowly evaporating the organic solvent until the organic solvent is almost completely evaporated; washing with ethanol for several times, and lyophilizing to obtain manganese-based nanometer core (MD).
In the step (3), the organic solvent is one or more of acetone, dimethyl sulfoxide, ethanol, methanol and N, N-dimethylformamide; the ultrasonic mixing treatment time of manganese dioxide and DHA is 5-60 min; the mass ratio of the manganese dioxide to the DHA is 2-1;
preferably, the organic solvent is acetone; the mass ratio of manganese dioxide to DHA is 1; the ultrasonic mixing treatment time of the manganese dioxide and the DHA is 30min;
(4) Preparation of PM-modified manganese-based nanocomposite:
and (4) dissolving a proper amount of PM in pure water, uniformly stirring, adding into the manganese-based nano kernel aqueous dispersion prepared in the step (3), and stirring in a dark place. And removing redundant PM by a centrifugal method to obtain the PM modified drug-loaded manganese-based nano composite (MDP).
In the step (4), the mass ratio of PM to manganese dioxide is (1) - (1); the concentration of the PM aqueous solution is 0.5 mg/mL-4 mg/mL; stirring in dark place for 0.5-5 h.
Preferably, the mass ratio of PM to manganese dioxide is 1; the concentration of the PM aqueous solution is 2mg/mL; the reaction was stirred for 3h in the dark.
(5) Preparing the hyaluronic acid modified multifunctional manganese-based nanoparticles:
and (5) dissolving a proper amount of hyaluronic acid in pure water, uniformly stirring, adding the hyaluronic acid into the manganese-based nano composite prepared in the step (4) under the condition of magnetic stirring, and stirring in a dark condition. And after the reaction is finished, performing centrifugal treatment to remove free HA to obtain the multifunctional manganese-based nanoparticles (MDPH).
In the step (5), the molecular weight of the hyaluronic acid is between 10 and 200 KDa; the mass ratio of the hyaluronic acid to the PM is 1-20; the concentration of the HA water solution is 1 mg/mL-5 mg/mL; stirring in a dark place for reaction for 0.5-12 h; methods of binding of hyaluronic acid to PM include, but are not limited to, electrostatic adsorption and chemical bonding.
Preferably, the molecular weight of the hyaluronic acid is 35KDa; the mass ratio of the hyaluronic acid to the PM is 1.5; the concentration of the HA water solution is 4mg/mL; stirring in dark place for 6h; the method of binding hyaluronic acid to PM is electrostatic adsorption.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages: (1) The MDPH prepared by the invention has small size, uniform distribution, good stability and biological safety. (2) In the present invention, the manganese dioxide has fenton-like activity, and can directly decompose a large amount of hydrogen peroxide accumulated in a tumor tissue region to generate OH, thereby killing tumors by CDT.The manganese-based nanometer inner core is collapsed by the acidity and high hydrogen peroxide microenvironment of the tumor part, and is used for tumor chemotherapy along with the release of DHA and PM; mn obtained by degrading manganese-based nano-core 2+ Can crack the peroxy bond of DHA to generate carbon free radical, and continuously increase the intracellular ROS level, thereby obviously amplifying the curative effect of CDT. (3) The MDPH prepared by the invention is a multifunctional targeting manganese-based nanoparticle, has an active targeting function for tumors, and can exert the synergistic anti-tumor effect of chemotherapy and chemo-kinetic therapy. (4) HA can be specifically combined with an overexpression receptor CD44 on tumor cells, and the HA-modified nanoparticles can improve the hydrophilicity and stability of the nanoparticles, prolong the blood circulation time, realize the active targeting of the tumor of the nanoparticles, reduce the toxic and side effects of the nanoparticles on normal cells and realize the active targeted treatment of cancer.
Drawings
FIG. 1 is a representation of multifunctional manganese-based nanoparticles, wherein (a) Transmission Electron Microscope (TEM) morphology of cellular manganese dioxide nanoparticles; (b) A Transmission Electron Microscope (TEM) morphology of the multifunctional manganese-based nanoparticles; (c) An ultraviolet-visible light absorption wavelength analysis chart of the multifunctional manganese-based nanoparticles under the condition of hydrogen peroxide along with time; (d) An ultraviolet-visible light absorption wavelength analysis chart of the PM water solution;
FIG. 2: in-vitro pharmacodynamic evaluation of the multifunctional manganese-based nanoparticles, wherein (a) the uptake of the nanoparticles by tumor cells; (b) Investigating the ROS generating capacity of the nanoparticles by adopting a flow cytometry; (c) Cell survival histograms of MDPH treated breast cancer cells 4T1 at different concentrations; (d) IC50 of free drug and formulation group for 24h of 4T1 cells;
FIG. 3: in vivo pharmacodynamic evaluation of the multifunctional manganese-based nanoparticles, wherein (a) a curve graph of the change of the tumor volume of mice of different experimental groups along with time; (b) weight change curve chart of mice of different experimental groups.
Detailed Description
The method is characterized by means such as Zeta potential and dynamic light scattering analysis (DLS), ultraviolet spectrum (UV-Vis), transmission Electron Microscope (TEM) and the like; then, evaluating the cytotoxicity of the nanoparticles by using an MTT method, detecting the uptake condition of cells to the nanoparticles by using a flow cytometry, and detecting the generation capacity of cell ROS by using an inverted fluorescence microscope; and finally, establishing a mouse in-situ breast cancer model for an anti-tumor experiment. The specific test results are as follows:
example 1 preparation of multifunctional manganese-based nanoparticles
(1) Preparing PM:
adding chitosan into 1% hydrochloric acid solution, stirring to obtain paste, performing hydrochloric acid treatment overnight to obtain hydrochloric acid chitosan solution, and lyophilizing to obtain hydrochloric acid chitosan solid. Dissolving the chitosan hydrochloride solid and dicyanodiamine in 0.75% hydrochloric acid, reacting for 5h at 100 ℃, cooling to room temperature, performing suction filtration, dialyzing, collecting dialysate, and freeze-drying to obtain PM solid.
(2) Preparing honeycomb manganese dioxide nanoparticles:
0.1g of potassium permanganate was placed in a 100mL beaker, followed by the addition of 50mL of ultrapure water. The solution was stirred on a magnetic stirrer for 30min at room temperature. After potassium permanganate is completely dispersed in ultrapure water, 0.4mL of oleic acid is slowly added into the potassium permanganate solution, and the reaction is continued for 24h at room temperature. After the reaction is finished, the original solution is changed into a dark brown turbid liquid state from purple red. And (3) collecting a product through centrifugation, washing the product with ethanol and ultrapure water for several times respectively, and finally drying the product to obtain the honeycomb-shaped manganese dioxide solid. And placing the honeycomb manganese dioxide in a proper amount of carrier dispersion medium, performing ultrasonic dispersion by using a probe, and freeze-drying the aqueous dispersion to obtain the honeycomb manganese dioxide nanoparticles (M).
(3) Preparing a manganese-based nano inner core:
dissolving 5mg of DHA in 5mL of acetone, adding the honeycomb-shaped manganese dioxide nanoparticles into the acetone solution of DHA, ultrasonically dispersing for 30min by using a probe, then placing the mixture into a vacuum drier, and slowly evaporating the organic solvent until the organic solvent is almost completely evaporated; and washing with ethanol for several times, and freeze-drying to obtain manganese-based nano-core (MD).
(4) Preparing a PM modified manganese-based nano composite:
and (3) according to the mass ratio of the PM to the manganese dioxide of 1. And removing redundant PM by a centrifugal method to obtain the PM modified drug-loaded manganese-based nano composite (MDP).
(5) Preparing the HA-modified multifunctional manganese-based nanoparticles:
and (3) according to the mass ratio of the HA to the PM of 1.5, dissolving a certain amount of HA in pure water, uniformly stirring, adding the mixture into the manganese-based nanocomposite aqueous dispersion prepared in the step (4) under the action of magnetic stirring, and stirring for 6 hours in a dark condition, wherein the HA is adsorbed on the surface of the MDP due to the strong electrostatic interaction of cationic amino and anionic carboxyl. And after the reaction is finished, performing centrifugal treatment to remove free HA, thus obtaining the multifunctional targeted manganese-based nanoparticles (MDPH).
Example 2 determination of basic Properties of multifunctional manganese-based nanoparticles
(1) TEM test:
and (3) taking 100 mu L of resuspended manganese dioxide nanoparticles M and multifunctional manganese-based nanoparticles MDPH, adding ultrapure water for dilution, dripping a drop of diluted manganese dioxide nanoparticles M and multifunctional manganese-based nanoparticles MDPH on a copper net, airing, and observing the morphology of the nanoparticles by using a transmission electron microscope. The TEM image of FIG. 1 (a) shows that the manganese nanoparticles prepared by the method of the present invention are honeycomb-shaped, uniformly dispersed, uniform in size and about 107.97nm in particle size. The TEM chart in fig. 1 (b) indicates that the particle size of the multifunctional manganese-based nanoparticles prepared by the method of the present invention is about 171.06nm, and the morphology is cellular, which indicates that after the manganese dioxide is modified by drug loading and outer polymer, the particle size of the obtained nanoparticles is increased compared to the original manganese-based nanocore, but the drug loading and outer modification in the nanoparticle preparation process do not affect the cellular shape of the manganese-based nanocore.
(2)H 2 O 2 And (3) sensitivity test:
preparing 2mL of 10. Mu.g/mL MDPH solution and adding a certain amount of H 2 O 2 The nano-particles are at 100 mu MH 2 O 2 And in the environment, testing the ultraviolet spectrum at intervals of certain time (0 min, 1h, 2h, 4h and 8 h). The ultraviolet spectrum test results are shown in FIG. 1 (c), and it can be found that MDPH purple can be obtained over time under the condition of simulating the high hydrogen peroxide in the tumor microenvironmentThe external absorption gradually decreased, indicating that the amount of MDPH in the solution was degraded and gradually decreased under the hydrogen peroxide condition.
(3) UV-Vis test of PM:
according to the invention, the prepared PM is characterized by a UV-Vis test, as shown in figure 1 (d), the metformin has obvious ultraviolet absorption at 233nm, and the PM prepared on the basis of chitosan has the same strong ultraviolet absorption at 233nm due to the fact that the PM has the same ultraviolet absorption functional group biguanidino as the metformin, which shows that biguanidino is successfully grafted on the chitosan after the chitosan and dicyanodiamine are subjected to addition reaction, thereby proving the successful synthesis of the PM.
(4) Zeta potential and hydrodynamic diameter test:
the prepared nanoparticles were diluted to 50 μ g/mL with ultrapure water and used for measuring surface potential and hydrodynamic diameter. As shown in Table 1, the average Zeta potential of the synthesized manganese nanoparticles was-19.6 mV as measured by DLS particle sizer; when the M is loaded with the hydrophobic chemical therapy DHA, the potential is basically unchanged, which shows that the potential of the manganese nano-particles is basically not influenced after the hydrophobic DHA is loaded into the manganese nano-particles; the average Zeta potential of MDP is 23.6mV, and the potential changes from negative to positive prove that PM is successfully wrapped outside the inner core of the manganese-based nanoparticle; when HA is loaded on the multifunctional manganese-based nanoparticle with stable PM, the average Zeta potential is changed into a negative value, which proves that HA is successfully coated on the nanoparticle.
Table 1.
| Sample(s) | Hydrodynamic diameter (nm) | Polydispersity index (PDI) | Surface potential (mV) |
| M | 158.02±13.04 | 0.403±0.048 | -19.6±1.28 |
| MD | 155.88±1.75 | 0.144±0.051 | -19.2±0.839 |
| MDP | 187.84±2.97 | 0.142±0.029 | 23.6±2.44 |
| MDPH | 180.28±1.03 | 0.074±0.015 | -15.5±1.70 |
Example 3 cellular uptake of multifunctional manganese-based nanoparticles investigation:
in order to examine the uptake condition of the multifunctional nanoparticles by cells, a hydrophobic fluorescent dye Coumarin6 (Coumarin 6) is used as a fluorescent probe, DHA in the step (3) in the embodiment 1 is replaced by the Coumarin6 to prepare corresponding fluorescent labeled nanoparticles, and MCP is prepared in the step (4) in the embodiment 1; MCPH was prepared from example 1, step (5). The cellular uptake portion was operated as follows: adherently growing 4T1 cells were removed from the medium and diluted with serum-containing 1640 medium after trypsinization. 4T1 cells were subsequently plated at 2X 10 5 Cell density per well seeded in six well plates, 37 ℃,5% CO 2 The cultivation in the incubator of (1) is carried out for 24 hours. MCP, MCPH and HA + MCPH are set in three groups, and the HA + MCPH treatment group treats tumor cells through HA (2 mg/mL)The cells were pre-saturated for 2h. Then, 1mL of each sample was added to each well, and 6 hours after the administration, cells of all the well plates were digested, centrifuged, collected, and the fluorescence intensity of the sample was measured by flow cytometry. The results are shown in FIG. 2 (a). By comparison, MCPH HAs higher fluorescence intensity than MCP, probably due to modification of the HA in the MCPH outer layer, nanoparticles enter cells in large amounts through CD44 receptor-mediated endocytosis. In addition, by the MCPH treatment group after presaturating the receptor on 4T1 cells with HA, the MCPH fluorescence intensity of the resulting intracellular nanoparticles was reduced, indicating that MCPH is inhibited by the CD44 receptor-mediated endocytosis pathway. From the above analysis, it can be concluded that HA can promote phagocytosis of MCPH by cells via the cellular CD44 receptor pathway.
Example 4. Cytotoxicity assay:
selecting 4T1 mouse mammary gland cancer cells in logarithmic phase by MTT method, adjusting cell number to 8000/well, inoculating to 96-well culture plate, and culturing until cell confluency reaches 80%. Discarding a culture medium, respectively adding free medicines DHA and PM with gradient concentration and MDPH nanoparticles into each hole, setting 5 multiple holes, each hole is 100 mu L, after 24h incubation, adding 10 mu L MTT (5 mg/mL) and 90 mu L serum-free 1640 culture medium into each hole for 4h, discarding a supernatant, then discarding the supernatant, adding 150 mu L LDMSO for dissolution, shaking at a low speed for 10min, measuring absorbance at 490nm by using a microplate reader, and measuring the toxicity of the nanoparticles to 4T1 cells. The results of the experiment in FIG. 2 (c) show that the survival rate of MDPH decreases with increasing concentration, indicating concentration-dependent cytotoxicity, i.e., a concentration-effect relationship. Toxicity results (FIG. 2 (d)) show that free drug DHA is less toxic to 4T1 cells, while free drug PM is more cytotoxic, free MnO 2 The MDPH has stronger toxicity to tumor cells due to better Fenton-like reaction activity, and shows the strongest tumor killing effect on the tumor cells due to the combined action of chemotherapy and chemodynamic therapy.
Example 5 Effect of multifunctional manganese-based nanoparticles on ROS levels in 4T1 cells
4T1 cells in logarithmic growth phase at 2X 10 5 Cell density per well seeded into six well platesAnd culturing until the confluence degree of the cells reaches 80%. The control group, DHA, PM, MP, MDP, and MDPH were set for 6 groups, and MP was obtained using the preparation methods of step (2) and step (4) in example 1 (i.e., no DHA load). Each sample was added to each well and after incubation for 6h, the media was removed, the cells were washed twice with PBS, the cells from all the wells were digested, the cells were collected by centrifugation and incubated in serum free media containing 10 μ M DCFH-DA for 20min at 37 ℃ in the absence of light. Cells were washed twice with PBS and fluorescence intensity was measured by flow cytometry. The results are shown in FIG. 2 (b). Free PM can generate a small amount of ROS; free DHA does not increase the ROS content in 4T1, while MDP has a stronger induction effect on ROS in 4T1 cells than MP, which shows that DHA in combination with manganese dioxide can significantly increase the ROS content in 4T1 cells. And the MDPH nanoparticle induces the generation of ROS in 4T1 cells to be stronger than that of MDP, which probably results in that more nanoparticles enter tumor cells due to the targeting affinity effect of HA on the outer layer of the nanoparticles to CD44 receptors of the tumor cells, so that a large amount of ROS are generated in the cells.
Example 6 in vivo antitumor Studies of multifunctional manganese-based nanoparticles
Establishing an in-situ breast cancer mouse model: taking 4T1 cells in logarithmic growth phase and suspending in serum-free medium, adjusting the concentration of cell suspension to 1x10 6 cel is 1s/50mL and is kept at 4 ℃ for later use. Female Balb/c mice were anesthetized by intraperitoneal injection of 200. Mu.L of 5% chloral hydrate, the body hair near the third and fourth pairs of milk fat pads on the left side of the mice was washed off with depilatory cream, and the cell suspension was carefully inoculated into the milk fat pads on the fourth pair of left sides of the mice; when the tumor volume reaches 150mm 3 On the left and right, experimental mice were randomly divided into 6 groups (n = 3). Five experimental groups of free DHA, PM, MP, MDP and MDPH were set with the saline group as a control, and MP was obtained by the preparation method of step (2) and step (4) in example 1 (i.e., no loading of DHA). The administration was performed once every two days from day 0 for a total of 5 times (DHA dose of 1mg/kg, PM dose of 0.5mg/kg, other formulation dose equivalent was the same), while the body weight of the mice was recorded every two days. Mice were sacrificed on day 15, groups of tumors were taken, weighed, tumor inhibition rate was calculated, and pharmacodynamic evaluation was performed, and the results are shown in fig. 3. Curve of tumor volume over time for mice of different experimental groupsAs shown in fig. 3 (a), both free DHA and PM failed to significantly inhibit tumor growth compared to the control group; through stabilizing the manganese-based nanoparticles by PM, the manganese-based nanoparticles have stronger inhibition effect on tumor growth by influencing oxidative stress balance of tumor cells; MDP combines chemotherapy and chemodynamics therapy, and has enhanced tumor growth inhibition effect, while MDPH has tumor active targeting effect, can reduce toxic and side effects, improve antitumor effect, and exhibit strongest tumor inhibition effect.
The result shows that the multifunctional manganese-based nanoparticles can be safely and effectively used as a drug delivery platform, and can achieve the effects of chemotherapy in cooperation with chemokinetic therapy and inhibition of proliferation of tumor cells by combining with the characteristics of a tumor microenvironment.
Claims (10)
1. The multifunctional manganese-based nanoparticle is characterized by comprising manganese dioxide, dihydroartemisinin, metformin and hyaluronic acid, wherein the manganese dioxide loads the dihydroartemisinin to form a manganese-based nano inner core, the polydimethybiguanide wraps the surface of the manganese-based nano inner core to form a manganese-based nano composite, and the hyaluronic acid is combined to the surface of the manganese-based nano composite.
2. The multi-functional manganese-based nanoparticles according to claim 1, wherein the manganese dioxide has a honeycomb cell structure.
3. The multifunctional manganese-based nanoparticle according to claim 1, wherein the metformin is prepared by an addition reaction of a primary or secondary amino group-containing hydrochloride polymer and a dicyanodiamine monomer, and the primary or secondary amino group-containing hydrochloride polymer is one of chitosan, linear polyethyleneimine, polylysine, polyvinylamine and polyallylamine.
4. The method for preparing multifunctional manganese-based nanoparticles according to claim 1, comprising the steps of:
(1) Dicyandiamide is grafted to a hydrochloride macromolecule containing primary amino or secondary amino through a chemical reaction to obtain the poly-metformin;
(2) Preparing cellular manganese dioxide by potassium permanganate and oleic acid by using a soft membrane plate method; placing the obtained honeycomb manganese dioxide in a carrier dispersion medium for dispersion, and freeze-drying to obtain honeycomb manganese dioxide nanoparticles;
(3) The manganese-based nano-core is obtained by loading dihydroartemisinin on the cellular manganese dioxide nano-particles, the surface of the manganese-based nano-core is wrapped by the metformin to form a manganese-based nano-composite, and the hyaluronic acid is further combined to the outer layer of the drug-loaded manganese-based nano-composite.
5. The preparation method of the multifunctional manganese-based nanoparticle according to claim 4, wherein the carrier dispersion medium in step (2) is one or more selected from acetone, dimethyl sulfoxide, water, ethanol, methanol or N, N-dimethylformamide.
6. The method for preparing multifunctional manganese-based nanoparticles according to claim 4, wherein dihydroartemisinin is loaded into the honeycomb pore-like structure of manganese dioxide by physical adsorption in step (3).
7. The preparation method of the multifunctional manganese-based nanoparticles according to claim 4, wherein the poly-metformin in step (3) is coated on the surface of the manganese-based nanocore by electrostatic adsorption.
8. The method of preparing multifunctional manganese-based nanoparticles according to claim 4, wherein the binding of hyaluronic acid to the surface of manganese-based nanocomposites in step (3) comprises electrostatic adsorption and chemical bonding.
9. The use of the multifunctional manganese-based nanoparticles of claim 1 in the preparation of antitumor drugs.
10. Use of the multifunctional manganese-based nanoparticles of claim 1 for the preparation of a medicament for the treatment of breast cancer.
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