CN115708813B - Multifunctional manganese-based nanoparticle and preparation method and medical application thereof - Google Patents
Multifunctional manganese-based nanoparticle and preparation method and medical application thereof Download PDFInfo
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- 239000011572 manganese Substances 0.000 title claims abstract description 101
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 229910052748 manganese Inorganic materials 0.000 title claims abstract description 95
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- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims abstract description 100
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 41
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Classifications
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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
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, metformin and hyaluronic acid, the manganese dioxide is loaded with the dihydroartemisinin to form a manganese-based nano core, the metformin is wrapped 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. In the invention, the multifunctional manganese-based nanoparticle has the functions of playing an anti-tumor drug effect and stabilizing a nano carrier, hyaluronic acid can be specifically combined with a CD44 receptor expressed on the surface of a tumor cell, so that the multifunctional manganese-based nanoparticle can be actively targeted to a tumor part, manganese dioxide is cooperated with dihydroartemisinin, and the ROS level in the tumor cell is improved, thereby accelerating the apoptosis of the tumor cell induced by ROS (reactive oxygen species) pathway, and finally playing a synergistic anti-tumor effect of chemotherapy and chemical kinetics therapy.
Description
Technical Field
The invention belongs to the field of nano material technology and biological medicine, and in particular relates to a multifunctional manganese-based nanoparticle, a preparation method and medical application thereof.
Background
The tumor microenvironment has low pH (6.5-7.0), high hydrogen peroxide (H) 2 O 2 ) High Glutathione (GSH) and the like, and H in tumor cells 2 O 2 The concentration (100. Mu.M to 1 mM) was higher than that of normal cells, and tumor cells were purified by up-regulating H 2 O 2 Adapt to the oxidative stress state. Therefore, if a large amount of OH is generated to destroy the original oxidation balance of the tumor microenvironment through the interaction of a novel anti-tumor drug delivery platform and the tumor microenvironment, the method has important significance for enhancing tumor treatment.
Chemotherapy (Chemodynamic therapy, CDT) is a type of treatment that utilizes the Fenton or Fenton-like response by converting endogenous H 2 O 2 Emerging therapies that convert to OH to induce apoptosis and necrosis. CDT has higherTumor specificity and selectivity, and no external stimulus is required during tumor treatment. In recent years, manganese dioxide nanomaterial has catalase activity and good acid response, and thus has become one of the materials for the intensive research of CDT. 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 degradation product Mn of manganese dioxide 2+ Continue to catalyze H 2 O 2 Producing a strong oxidizing OH (equation 2) to kill 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 manganese dioxide nano-materials show excellent CDT effects, a plurality of challenges are still faced to realize the application of the manganese dioxide nano-materials in preparing tumor medicaments, such as poor water dispersibility, no characteristics of tumor targeting and the like, and the targeted transportation of the medicaments 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, which are well known for their excellent effect in the treatment of malaria. In recent years, artemisinin and its derivatives have been studied to demonstrate anticancer effects and have better safety, with the anticancer activity of Dihydroarteannuin (DHA) being the best. Among the anticancer mechanisms of DHA, the most widely accepted is the cleavage of peroxide bonds mediated by metal ions or transition metal ions, forming carbon radicals, promoting apoptosis. However, DHA has poor water solubility and short half-life in vivo, and is difficult to accumulate at tumor sites.
Metformin (Metformin, met) is a first-line drug for treating type II diabetes, epidemiology and clinical studies in recent years show that Metformin has great potential in tumor prevention, treatment and prognosis, and studies find that Metformin not only can independently inhibit tumor growth, but also 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 achieve 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 biguanide groups are grafted on the molecular structure, so that the antitumor activity of the poly (metformin) is inherited. Meanwhile, PM has the characteristics of good water solubility, strong stability and the like, can effectively modify the nano-carrier, and enhances the stability of the nano-carrier in water.
Hyaluronic Acid (HA) molecules are an indispensable polysaccharide in the human body, and have better biocompatibility.
Disclosure of Invention
The invention aims to: 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 and the like, can realize the combination of chemotherapy and chemical kinetics therapy and play a role in enhancing the anti-tumor effect of the chemical kinetics therapy.
The technical scheme is as follows: the multifunctional manganese-based nanoparticle comprises manganese dioxide, dihydroartemisinin, metformin and hyaluronic acid, wherein the manganese dioxide is loaded with the dihydroartemisinin to form a manganese-based nano core, the metformin is wrapped 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.
The manganese dioxide has a honeycomb cell structure.
The poly-metformin is prepared by adding a primary or secondary amino group-containing hydrochloride polymer and dicyandiamide monomer, wherein the primary or secondary amino group-containing hydrochloride polymer is one of chitosan, linear polyethylenimine, polylysine, polyvinylamine and polyallylamine.
The preparation method of the multifunctional manganese-based nanoparticle is characterized by comprising the following steps of:
(1) Grafting dicyandiamide onto a hydrochlorinated polymer containing primary amino or secondary amino through a chemical reaction to obtain the metformin;
(2) Preparing honeycomb manganese dioxide from potassium permanganate and oleic acid by using a soft membrane plate method; dispersing the obtained honeycomb manganese dioxide in a carrier dispersion medium, and freeze-drying to obtain honeycomb manganese dioxide nano particles;
(3) Loading dihydroartemisinin on honeycomb manganese dioxide nano particles to obtain a manganese-based nano core, wrapping the surface of the manganese-based nano core with the metformin to form a manganese-based nano composite, and further combining hyaluronic acid to the outer layer of the drug-loaded manganese-based nano composite.
In the preparation method of the multifunctional manganese-based nanoparticle, the carrier dispersion medium in the step (2) is one or more of acetone, dimethyl sulfoxide, water, ethanol, methanol or N, N-dimethylformamide.
In the preparation method of the multifunctional manganese-based nanoparticle, dihydroartemisinin is loaded into a honeycomb pore structure of manganese dioxide in the step (3) through a physical adsorption method.
According to the preparation method of the multifunctional manganese-based nanoparticle, in the step (3), the metformin is wrapped on the surface of the manganese-based nanoparticle inner core through electrostatic adsorption.
The method for preparing the multifunctional manganese-based nanoparticle comprises the step (3) that hyaluronic acid is combined on the surface of the manganese-based nanocomposite, wherein the method comprises the steps of electrostatic adsorption and chemical bond connection.
The multifunctional manganese-based nanoparticle is applied to the preparation of antitumor drugs.
The multifunctional manganese-based nanoparticle is applied to the preparation of medicaments for treating breast cancer.
The method comprises the steps of grafting dicyandiamide onto chitosan containing primary amino groups through chemical reaction to obtain the metformin PM, reducing by potassium permanganate to prepare cellular manganese dioxide, obtaining manganese-based nano-cores through physical absorption of dihydroartemisinin by the cellular manganese dioxide, wrapping the surfaces of the manganese-based nano-cores by the PM to form manganese-based nano-composites with good water dispersibility, and further combining hyaluronic acid to the outer layers of the drug-loaded manganese-based nano-composites through electrostatic absorption or chemical bond connection to construct the CD44 receptor targeting-PM stable multifunctional manganese-based nano-particles.
The preparation method of the multifunctional manganese-based nanoparticle comprises the following steps:
(1) Preparation of PM:
adding polymer powder containing primary amino or secondary amino into hydrochloric acid solution, stirring to paste, and carrying out overnight hydrochloride to obtain a hydrochloride polymer solution, and freeze-drying to obtain the hydrochloride polymer solid. Dissolving the hydrochloride high molecular solid and dicyandiamide in hydrochloric acid solution, heating a reaction system, cooling to room temperature after the reaction is finished, dialyzing after suction filtration, collecting dialysate and freeze-drying to obtain PM solid.
In the step (1), the polymer containing primary amino or secondary amino is one of chitosan, linear polyethylenimine, polylysine, polyvinylamine and polyallylamine; dissolving the polymer powder in 75-150mL of 0.5-1.5% (m/m) hydrochloric acid; dissolving the hydrochloride macromolecule solid in 15-50ml of 0.5-1.5% (m/m) hydrochloric acid; the mass ratio of the hydrochlorinated polymer solid to dicyandiamide is 1:2-1:20.
Preferably, the polymer containing primary amino groups is chitosan; the polymer powder was dissolved in 100mL of 1% (m/m) hydrochloric acid; the hydrochloride macromolecule solid is dissolved in 20mL of 0.75% (m/m) hydrochloric acid; the mass ratio of the hydrochloride macromolecular solid to dicyandiamide is 1:15.
(2) Preparation of honeycomb manganese dioxide nano-particles:
adding potassium permanganate into ultrapure water, stirring and mixing uniformly at normal temperature, slowly adding oleic acid into the potassium permanganate solution under stirring, and changing the original solution from purple red to a dark brown turbid liquid state after the room temperature reaction is finished. And collecting the product through centrifugation, washing the product with ethanol and ultrapure water for several times respectively, and finally drying the product to obtain the honeycomb manganese dioxide solid. And (3) placing the honeycomb manganese dioxide in a proper amount of carrier dispersion medium, ultrasonically dispersing by a probe, and freeze-drying the aqueous dispersion liquid to obtain the honeycomb manganese dioxide nano particles (M).
In the step (2), the volume ratio of the potassium permanganate aqueous solution to the oleic acid is 150:4-600:4; the mass ratio of the potassium permanganate to the oleic acid is 1:7-1:1; the reaction time of the potassium permanganate and the oleic acid is 12-36 hours; the carrier dispersion medium is one or more solvents selected from 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 aqueous solution to the oleic acid is 500:4; the mass ratio of potassium permanganate to oleic acid is 1:2; the reaction time of the potassium permanganate and the oleic acid is 24 hours; the carrier dispersion medium is water; the sonication time for M was 10min.
(3) Preparation of manganese-based nanocores:
dissolving a certain amount of DHA in an organic solvent, then adding the honeycomb manganese dioxide nano particles prepared in the step (2) into the DHA solvent, ultrasonically dispersing by a probe, then placing in a vacuum dryer, and slowly evaporating the organic solvent until the organic solvent is almost completely evaporated; and washing with ethanol for several times, and lyophilizing to obtain manganese-based nanokernel (MD).
In the step (3), the organic solvent is one or more solvents selected from 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 manganese dioxide to DHA is 2:1-1:5;
preferably, the organic solvent is acetone; the mass ratio of manganese dioxide to DHA is 1:1; the ultrasonic mixing treatment time of manganese dioxide and DHA is 30min;
(4) Preparation of PM-modified manganese-based nanocomposites:
dissolving a proper amount of PM in pure water, uniformly stirring, then adding the mixture into the manganese-based nano-core aqueous dispersion prepared in the step (3), and stirring in a dark place. And removing excessive 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 1:1-1:8; the concentration of the PM aqueous solution is 0.5 mg/mL-4 mg/mL; the reaction time is 0.5 to 5 hours after light shielding stirring.
Preferably, the mass ratio of PM to manganese dioxide is 1:4; the concentration of the PM aqueous solution is 2mg/mL; the reaction was stirred for 3h in the dark.
(5) Preparation of hyaluronic acid modified multifunctional manganese-based nanoparticles:
dissolving a proper amount of hyaluronic acid in pure water, stirring uniformly, adding the mixture into the manganese-based nanocomposite prepared in the step (4) under the magnetic stirring condition, and stirring under the light-shielding condition. And after the reaction is finished, centrifuging to remove free HA, and obtaining the multifunctional manganese-based nanoparticle (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 1:1-20:1; the concentration of the HA aqueous solution is 1 mg/mL-5 mg/mL; the reaction time of light-shielding stirring is 0.5 to 12 hours; methods of hyaluronic acid binding to PM include, but are not limited to, electrostatic adsorption and chemical bond attachment.
Preferably, the hyaluronic acid has a molecular weight of 35KDa; the mass ratio of hyaluronic acid to PM is 1:1.5; the concentration of the HA aqueous solution is 4mg/mL; stirring and reacting for 6 hours in dark; the method of binding hyaluronic acid to PM is electrostatic adsorption.
The beneficial effects are that: compared with the prior art, the invention has the following advantages: (1) The MDPH prepared by the invention has smaller size, uniform distribution and good stability and biological safety. (2) In the invention, the manganese dioxide has Fenton-like activity, and can directly decompose a large amount of hydrogen peroxide accumulated at tumor tissue sites to generate OH, thereby killing tumors through CDT. The acidity and high hydrogen peroxide microenvironment of the tumor part cause the manganese-based nanokernel to collapse, and the release of DHA and PM is accompanied, so that the manganese-based nanokernel is used for tumor chemotherapy; and Mn is obtained by degradation of the manganese-based nano-core 2+ The peroxy bond of DHA is cracked to generate carbon free radical, and the intracellular ROS level is continuously increased, so that the curative effect of CDT is obviously amplified. (3) The MDPH prepared by the invention is a multifunctional targeted manganese-based nanoparticle, has the active targeting function of tumor, and can exert the synergistic anti-tumor effect of chemotherapy and chemical kinetic therapy. (4) HA can be specifically combined with an over-expression receptor CD44 on tumor cells, and the HA modified nanoparticles can improve the hydrophilicity and stability of the tumor cells, prolong the blood circulation time, realize the active targeting of the tumor cells, reduce the toxic and side effects of the tumor cells on normal cells and realize the active targeting treatment of cancers.
Drawings
FIG. 1 is a representation of a multifunctional manganese-based nanoparticle wherein (a) a Transmission Electron Microscope (TEM) topography of a cellular manganese dioxide nanoparticle; (b) A Transmission Electron Microscope (TEM) topography of the multifunctional manganese-based nanoparticle; (c) An ultraviolet-visible light absorption wavelength analysis chart of the multifunctional manganese-based nanoparticle under the hydrogen peroxide condition along with time; (d) An ultraviolet-visible absorption wavelength analysis chart of the PM aqueous solution;
fig. 2: in vitro pharmacodynamics evaluation of multifunctional manganese-based nanoparticles, wherein (a) uptake of the nanoparticles by tumor cells; (b) Taking into account ROS-producing capacity of nanoparticles by flow cytometry; (c) Cell survival histogram of breast cancer cells 4T1 treated with different concentrations of MDPH; (d) 24h IC50 of free drug and formulation group to 4T1 cells;
fig. 3: in vivo pharmacodynamics evaluation of multifunctional manganese-based nanoparticles, wherein, (a) graphs of tumor volume changes over time for different experimental groups of mice; (b) graph of body weight change in mice of different experimental groups.
Detailed Description
The invention uses Zeta potential and dynamic light scattering analysis (DLS), ultraviolet spectrum (UV-Vis), transmission Electron Microscope (TEM) and other means to characterize; then evaluating cytotoxicity of the nanoparticles by using an MTT method, detecting uptake condition of the nanoparticles by cells by using a flow cytometry, and detecting generation capacity of the ROS by using an inverted fluorescence microscope; and finally, establishing a mouse in-situ breast cancer model to perform an anti-tumor experiment. The specific test results are as follows:
example 1 preparation of multifunctional manganese-based nanoparticles
(1) Preparation of PM:
adding chitosan into 1% hydrochloric acid solution, stirring to paste, and performing overnight hydrochloric acid to obtain a hydrochloric acid chitosan solution, and lyophilizing to obtain a solid of chitosan hydrochloride. Dissolving the chitosan hydrochloride solid and dicyandiamide in 0.75% hydrochloric acid, reacting for 5 hours at 100 ℃, cooling to room temperature, dialyzing after suction filtration, collecting dialysate and freeze-drying to obtain PM solid.
(2) Preparation of honeycomb manganese dioxide nano-particles:
0.1g of potassium permanganate was taken in a 100mL beaker, and then 50mL of ultrapure water was added. The above solution was stirred on a magnetic stirrer at room temperature for 30min. After the potassium permanganate is completely dispersed in the ultrapure water, 0.4mL of oleic acid is slowly added into the potassium permanganate solution, and the reaction is continued for 24 hours at room temperature. After the reaction, the original solution is changed from purple to dark brown turbid liquid. And collecting the product through centrifugation, washing the product with ethanol and ultrapure water for several times respectively, and finally drying the product to obtain the honeycomb manganese dioxide solid. And (3) placing the honeycomb manganese dioxide in a proper amount of carrier dispersion medium, ultrasonically dispersing by a probe, and freeze-drying the aqueous dispersion liquid to obtain the honeycomb manganese dioxide nano particles (M).
(3) Preparation of manganese-based nanocores:
dissolving 5mgDHA in 5mL of acetone, adding honeycomb manganese dioxide nano particles into the acetone solution of DHA, ultrasonically dispersing for 30min by a probe, then placing in a vacuum dryer, and slowly evaporating the organic solvent until the organic solvent is almost completely evaporated; and washing with ethanol for several times, and lyophilizing to obtain manganese-based nanokernel (MD).
(4) Preparation of PM-modified manganese-based nanocomposites:
and (3) dissolving a certain amount of PM in pure water according to the mass ratio of PM to manganese dioxide of 1:4, stirring uniformly, adding into the manganese-based nanoparticle inner core aqueous dispersion prepared in the step (3), and stirring for 3 hours in a dark place. And removing excessive PM by a centrifugal method to obtain the PM modified drug-loaded manganese-based nano-composite (MDP).
(5) Preparation of HA modified multifunctional manganese-based nanoparticles:
according to the mass ratio of HA to PM of 1.5:1, a certain amount of HA is taken and dissolved in pure water, after being stirred uniformly, the mixture is added into the manganese-based nanocomposite aqueous dispersion prepared in the step (4) under the action of magnetic stirring, and the mixture is stirred for 6 hours under the condition of avoiding light, and HA is adsorbed on the surface of MDP due to the strong electrostatic interaction of cationic amino and anionic carboxyl. And after the reaction is finished, centrifuging to remove free HA, and obtaining the multifunctional targeted manganese-based nanoparticle (MDPH).
Example 2 determination of essential Properties of multifunctional manganese-based nanoparticles
(1) TEM test:
taking 100 mu L of resuspended manganese dioxide nano particles M and multifunctional manganese-based nano particles MDPH, adding ultrapure water for dilution, then dripping the diluted manganese dioxide nano particles M and the multifunctional manganese-based nano particles MDPH on a copper mesh, and finally, observing the morphology of the nano particles by using a transmission electron microscope after airing. The TEM (transmission electron microscope) graph of FIG. 1 (a) shows that the manganese nano particles prepared by the method are honeycomb-shaped, uniformly dispersed, uniform in size and about 107.97nm in particle size. The TEM image of fig. 1 (b) shows that the particle size of the multifunctional manganese-based nanoparticle prepared by the method of the present invention is about 171.06nm, and the morphology of the multifunctional manganese-based nanoparticle is honeycomb, which indicates that after the manganese dioxide is modified by the drug loading and the outer polymer, the particle size of the obtained nanoparticle is increased compared with that of the original manganese-based nanoparticle core, but the drug loading and the outer modification in the nanoparticle preparation process do not affect the honeycomb shape of the manganese-based nanoparticle core.
(2)H 2 O 2 Sensitivity test:
2mL of MDPH solution with the concentration of 10 mug/mL is prepared and a certain amount of H is added 2 O 2 The nanoparticle is subjected to 100 mu MH 2 O 2 In the environment, ultraviolet spectra are tested at intervals of a 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 under the condition of simulating the tumor microenvironment with high hydrogen peroxide, the ultraviolet absorption of MDPH gradually decreases along with the time, which indicates that the MDPH amount in the solution is degraded and gradually decreases under the condition of hydrogen peroxide.
(3) UV-Vis test of PM:
according to the invention, the prepared PM is characterized by a UV-Vis test, as shown in a figure 1 (d), the metformin has obvious ultraviolet absorption at 233nm, and the PM prepared based on chitosan has the same strong ultraviolet absorption at 233nm due to the biguanide group which has the same ultraviolet absorption functional group as the metformin, so that the successful synthesis of the PM is proved by successfully grafting the biguanide group on the chitosan after the addition reaction of the chitosan and the dicyandiamide.
(4) Zeta potential and hydrodynamic diameter test:
each of the prepared nanoparticles was diluted to 50. Mu.g/mL with ultrapure water and used for measuring the surface potential and hydrodynamic diameter thereof. As shown in Table 1, the DLS particle sizer measures the average Zeta potential of the synthesized manganese nanoparticles to be-19.6 mV; when M is loaded with hydrophobic chemotherapeutic drug DHA, the potential is basically unchanged, which indicates that the hydrophobic DHA is loaded into the manganese nanoparticles and basically has no influence on the potential of the manganese nanoparticles; the average Zeta potential of MDP is 23.6mV, and the change from negative to positive in potential proves that PM is successfully wrapped outside the manganese-based nanoparticle core; after HA is loaded onto the PM-stabilized multifunctional manganese-based nanoparticles, the average Zeta potential is turned negative, proving that HA is successfully encapsulated onto the nanoparticles.
Table 1.
| Sample of | 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 cells on the multifunctional nanoparticles, hydrophobic fluorescent dye Coumarin6 (Coumarin 6) is selected as a fluorescent probe, DHA in the step (3) of the embodiment 1 is replaced by Coumarin6 to prepare corresponding fluorescent-labeled nanoparticles, and MCP is prepared in the step (4) of the embodiment 1; MCPH was prepared from step (5) of example 1. The cell uptake moiety was operated as follows: the adherent 4T1 cells were removed from the medium, digested with pancreatin and diluted with serum-containing 1640 medium. The 4T1 cells were then plated at 2X 10 5 Cell density of wells/cell density of wells was seeded into six well plates at 37℃with 5% CO 2 Is cultured in an incubator for 24 hours. MCP, MCPH, HA+MCPH groups were set, and the HA+MCPH treated groups were pre-saturated with HA (2 mg/mL) for 2h. Then, 1mL of each sample was added to each well, and after 6 hours of administration, cells of all the well plates were digested, centrifuged, and collected, and the fluorescence intensity of the samples was measured by a flow cytometer. The results are shown in FIG. 2 (a). By comparison, MCPH HAs a higher fluorescence intensity than MCP, probably due to modification of MCPH outer HA, nanoparticles enter the cells in large amounts by CD44 receptor mediated endocytosis. In addition, by treating the group with MCPH after presaturation of the receptor on 4T1 cells with HA, the resulting intracellular nanoparticles showed a decrease in MCPH fluorescence intensity, indicating that MCPH was inhibited by CD44 receptor-mediated endocytosis pathway. From the above analysis, it can be concluded that HA can promote phagocytosis of MCPH by cells through the cellular CD44 receptor pathway.
Example 4 cytotoxicity assay:
selecting 4T1 mouse breast cancer cells in logarithmic phase by MTT method, and adjusting cell number to 8000/well was inoculated into 96-well plates and cultured until cell confluency reached 80%. The culture medium is discarded, free drugs DHA and PM with gradient concentration and MDPH nanoparticles are respectively added into each hole, 5 compound holes are arranged, 100 mu L of each hole is incubated for 24 hours, 10 mu L of MTT (5 mg/mL) and 90 mu L of serum-free 1640 culture medium are added into each hole for 4 hours, the supernatant is discarded, 150 mu L of LDMSO is added for dissolution, the absorbance is measured at 490nm after shaking for 10 minutes at low speed, and the toxicity of the nanoparticles to 4T1 cells is measured. FIG. 2 (c) shows that the survival rate of MDPH decreases with increasing concentration, indicating concentration-dependent cytotoxicity, i.e., concentration-effect relationship. The toxicity results (FIG. 2 (d)) show that the free drug DHA has weaker toxicity to 4T1 cells, whereas the free drug PM has stronger cytotoxicity, free MnO 2 The MDPH has better Fenton-like reaction activity, stronger toxicity to tumor cells, and the MDPH has the strongest tumor killing effect on the tumor cells due to the combined action of chemotherapy and chemical kinetics therapy.
Example 5 Effect of multifunctional manganese-based nanoparticles on ROS levels in 4T1 cells
4T1 cells in logarithmic growth phase were grown at 2X 10 5 Cell density of/well was seeded into six well plates and cultured until cell confluence reached 80%. The control group, DHA, PM, MP, MDP and MDPH were set to 6 groups and MP was obtained using the preparation method of step (2) and step (4) of example 1 (i.e., no DHA loading). After 6h incubation, the medium was removed and the cells were washed twice with PBS, cells from all well plates were digested, collected by centrifugation and incubated in serum-free medium containing 10 μm 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 produce small amounts of ROS; free DHA does not increase ROS content in 4T1, whereas MDP has a stronger ROS-inducing effect on 4T1 cells than MP, which suggests that DHA in combination with manganese dioxide can significantly increase ROS content in 4T1 cells. And MDPH nanoparticle induces ROS to generate more in 4T1 cells than MDP, probably due to targeting affinity of outer nanoparticle layer HA to tumor cell CD44 receptor, resulting in more nanoparticle in-takeInto tumor cells, thereby generating a large amount of ROS in the cells.
EXAMPLE 6 multifunctional manganese-based nanoparticle in vivo anti-tumor Studies
Establishment of in situ breast cancer mouse model: taking 4T1 cells in logarithmic growth phase and re-suspending in serum-free culture medium, and adjusting the concentration of cell suspension to 1x10 6 cel1s/50mL was placed at 4deg.C for use. Female Balb/c mice were anesthetized by intraperitoneal injection of 200 μl of 5% chloral hydrate, the body hair near the third and fourth pairs of creamy pads on the left side of the mice was removed with depilatory cream, and the cell suspension was carefully inoculated into the fourth pair of creamy pads on the left side 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, free DHA, PM, MP, MDP and MDPH, were set up using the saline group as a control, and MP was obtained (i.e., no DHA loading) using the preparation methods of step (2) and step (4) of example 1. The dosing was performed once every two days from day 0 (DHA dose 1mg/kg, PM dose 0.5mg/kg, equivalent of other formulation doses was the same) and the mice weights were recorded every two days. Mice were sacrificed on day 15, each group of tumors was weighed, tumor inhibition rates were calculated, and pharmacodynamic evaluation was performed, and the results are shown in fig. 3. The curves of the tumor volume changes of mice in different experimental groups with time are shown in fig. 3 (a), and compared with the control group, free DHA and PM can not obviously inhibit tumor growth; through PM stabilized manganese-based nanoparticles, the oxidation stress balance of tumor cells is influenced, so that the growth of tumors is inhibited more strongly; MDP has enhanced tumor growth inhibition effect by combining chemotherapy and chemical kinetics therapy, and MDPH has tumor active targeting effect, so that toxic and side effects can be reduced, anti-tumor efficacy can be improved, and the strongest tumor inhibition effect can be presented.
The result shows that the multifunctional manganese-based nanoparticle can be safely and effectively used as a drug delivery platform, and combines the micro-environmental characteristics of tumors to achieve the effects of chemotherapy, cooperative chemical kinetics therapy and tumor cell proliferation inhibition.
Claims (7)
1. The multifunctional manganese-based nanoparticle is characterized by comprising manganese dioxide, dihydroartemisinin, metformin and hyaluronic acid, wherein the manganese dioxide is loaded with the dihydroartemisinin to form a manganese-based nano-core, the metformin is wrapped on the surface of the manganese-based nano-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 pore structure; the metformin is prepared by an addition reaction of a hydrochlorinated chitosan and dicyandiamide monomer.
2. The method for preparing the multifunctional manganese-based nanoparticle according to claim 1, comprising the steps of:
(1) Grafting dicyandiamide onto chitosan hydrochloride through chemical reaction to obtain the poly-metformin;
(2) Preparing honeycomb manganese dioxide from potassium permanganate and oleic acid by using a soft membrane plate method; dispersing the obtained honeycomb manganese dioxide in a carrier dispersion medium, and freeze-drying to obtain honeycomb manganese dioxide nano particles;
(3) Loading dihydroartemisinin on honeycomb manganese dioxide nano particles to obtain a manganese-based nano core, wrapping the surface of the manganese-based nano core with the metformin to form a manganese-based nano composite, and further combining hyaluronic acid to the outer layer of the drug-loaded manganese-based nano composite.
3. The method for preparing multifunctional manganese-based nanoparticles according to claim 2, wherein the carrier dispersion medium in step (2) is one or more of acetone, dimethyl sulfoxide, water, ethanol, methanol or N, N-dimethylformamide.
4. The method for preparing multifunctional manganese-based nanoparticles according to claim 2, wherein dihydroartemisinin is loaded into the cellular structure of manganese dioxide in step (3) by physical adsorption.
5. The method for preparing the multifunctional manganese-based nanoparticle according to claim 2, wherein the metformin is coated on the surface of the manganese-based nanokernel through electrostatic adsorption in the step (3).
6. The method of preparing a multifunctional manganese-based nanoparticle according to claim 2, wherein the method of binding hyaluronic acid to the surface of the manganese-based nanocomposite in step (3) comprises electrostatic adsorption and chemical bond connection.
7. Use of the multifunctional manganese-based nanoparticle of claim 1 in the preparation of a medicament for treating breast cancer.
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