CN112755195A - Preparation method and application of hydrogen power micro motor - Google Patents
Preparation method and application of hydrogen power micro motor Download PDFInfo
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- CN112755195A CN112755195A CN202110082841.1A CN202110082841A CN112755195A CN 112755195 A CN112755195 A CN 112755195A CN 202110082841 A CN202110082841 A CN 202110082841A CN 112755195 A CN112755195 A CN 112755195A
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- hydrogen
- polylactic acid
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pain & Pain Management (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Rheumatology (AREA)
- Dermatology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method and application of a hydrogen power micro motor, wherein the hydrogen power micro motor comprises a magnesium element core and a polylactic acid-glycollic acid copolymer shell coated outside the magnesium element core, and the polylactic acid-glycollic acid copolymer shell is provided with at least 1 notch, so that the magnesium element core can be communicated with the external environment. The hydrogen powered micromotor has a Janus junctionStructure capable of stably releasing H in an aqueous medium2And the material has the motion characteristic, and the infarct area can be effectively reduced by injecting the material in vivo, so that the material has excellent effect on relieving inflammation caused by acute ischemic stroke.
Description
Technical Field
The invention relates to the field of biological materials, in particular to a preparation method and application of a hydrogen power micromotor.
Background
Stroke is an acute cerebrovascular disease characterized by high mortality and disability rate. The incidence of ischemic stroke is rapidly increasing compared to hemorrhagic stroke. In the related technology, the clinical cerebral ischemic stroke mainly adopts thrombolytic therapy combined with nerve protection measures, and the thrombolytic therapy comprises intravascular therapy technologies such as venous thrombolysis, arterial thrombus removal and the like, so that blood at ischemic positions in the brain is reflowed. However, after blood recirculation in the blood vessels, the improvement of the ischemic and hypoxic state of brain tissue can rather further exacerbate tissue damage and inflammatory response, a phenomenon known as Cerebral Ischemia Reperfusion Injury (CIRI).
Artificial micro-and nanomotors (MNMs) are a technology that can convert chemical or external energy into mechanical motion to accomplish complex tasks, but it has been challenging to manufacture micro-and nanomotor systems that are completely biocompatible and biodegradable and can be accurately delivered to target tissues, especially for hydrogen therapy, which has difficulty in achieving a good therapeutic effect due to the difficulty in efficiently accumulating hydrogen gas at the focal site. Moreover, how to realize the slow release of hydrogen while storing hydrogen in large quantities to perform hydrogen therapy remains a challenge.
Therefore, it is urgently needed to develop an artificial micro/nano motor technology capable of effectively solving the above problems, and put it into the treatment of accurately treating ischemic stroke, so as to further achieve the purposes of providing a new research direction for the development of anti-inflammatory carrier drugs and promoting the development of functionalized micro/nano motors.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. For this purpose, the invention provides a hydrogen power material which has a Janus structure and can stably release H in an aqueous medium2And the material has the motion characteristic, and the infarct area can be effectively reduced by injecting the material in vivo, so that the material has excellent effect on relieving inflammation caused by acute ischemic stroke.
In a first aspect of the invention, hydrogen-powered materials (HPMs) are provided that comprise a magnesium core and a polylactic-co-glycolic acid (PLGA) shell surrounding the magnesium core.
Wherein, the shell of the polylactic acid-glycollic acid copolymer has at least 1 gap, so that the magnesium element core can be communicated with the external environment.
According to a specific embodiment of the invention, at least the following advantages are achieved:
the HPMs of the present invention have a notch on one side of their structure that can be considered as H2Resulting in a magnesium-water reaction interface. At the same time, H is generated2The HPMs are pushed to generate a helical or linear motion pattern with a long coil tail, and the speeds of the HPMs in ACSF and PBS reach 51.1 mu m s-1And 61.12 μm s-1Can rapidly provide a large amount of H for the area on the motion trail2. Due to H2Can easily diffuse across cell membranes, enter cells and organelles, and react with Reactive Oxygen Species (ROS) to effectively and selectively scavenge excess reactive oxygen species and proinflammatory cytokines from cells. Moreover, the HPMs are non-cytotoxic, and have excellent biodegradability and biocompatibility. The inventor also finds that the HPMs can be accurately injected in vivo, so that the nerve can be effectively protected, the infarct size is reduced, the in vivo oxidative stress is lightened, and the ischemia-induced spatial memory injury is improved, and the application prospect is extremely high.
In some preferred embodiments of the present invention, the polylactic acid-glycolic acid copolymer shell has only 1 notch.
According to a first aspect of the invention, in some embodiments of the invention, the hydrogen kinetic material has a Janus structure.
The Janus nano material is a nano material with an asymmetric structure in material composition, morphology and surface chemical properties, and has the advantages of asymmetric structure, easy surface modification, capability of self-assembly to form more complex composite nano particles and the like compared with other nano materials.
According to a first aspect of the invention, in some embodiments of the invention, the hydrogen kinetic material has a diameter of 10 to 25 μm, the percentage concentration by mass of magnesium in the hydrogen kinetic material is 38.4 to 52.54%, and the core of magnesium has a diameter of 10 to 17 μm.
In some more preferred embodiments of the invention, the magnesium core has a diameter of 17 μm and the percentage concentration by mass of magnesium in the hydrogen kinetic material is 45.47%.
According to a first aspect of the invention, in some embodiments of the invention, the indentations have a diameter of 5-15 μm.
In some preferred embodiments of the invention, the diameter of the indentations is 8-12 μm.
In some more preferred embodiments of the invention, the indentations are 10 μm in diameter.
The notch on the PLGA shell of the HPMs in the invention is a reaction interface of the magnesium element core and water, and water in the environment can react with the magnesium element core through the notch, thereby generating H2And because the area of the gap is limited, the contact area of the magnesium element core and water is limited, the reaction speed is reduced, and H2The generation speed of (b) is controlled. Moreover, only the gap can discharge H2The direction of the airflow discharged is consistent, thrust is generated, and the HPMs obtain the motion capability.
In a second aspect of the present invention, there is provided a method for producing the hydrogen power material according to the first aspect of the present invention, comprising the steps of:
placing the magnesium material on a substrate material, coating polylactic acid-glycolic acid copolymer, and scraping the magnesium material coated with the polylactic acid-glycolic acid copolymer from the substrate material to obtain the magnesium-based material.
According to a specific embodiment of the invention, at least the following advantages are achieved:
the preparation method of the HPMs is simple and rapid, does not need instrument assistance, and the prepared HPMs have stable effect and small difference, can be produced in mass and have extremely high application value.
According to a second aspect of the invention, in some embodiments of the invention, the substrate material comprises polyvinylpyrrolidone.
In some more preferred embodiments of the invention, the substrate material is a polyvinylpyrrolidone coating, which is coated on the glass. Of course, the polyvinylpyrrolidone coating may be applied to other suitable materials according to the actual needs of the art.
According to a second aspect of the present invention, in some embodiments of the present invention, the polylactic acid-glycolic acid copolymer is dissolved in ethyl acetate, and the mass to volume ratio of the polylactic acid-glycolic acid copolymer to the ethyl acetate is 1% (w/v%).
According to a second aspect of the invention, in some embodiments of the invention, the polyvinylpyrrolidone has a mass concentration percentage of 1-3% and a molecular weight of 38-42 kDa.
In some more preferred embodiments of the invention, the polyvinylpyrrolidone has a mass concentration percentage of 2% and a molecular weight of 40 kDa.
According to a second aspect of the present invention, in some embodiments of the present invention, the polylactic acid-glycolic acid copolymer has a mass concentration percentage of 0.5-1.5% and a molecular weight of 48-52 kDa.
In some more preferred embodiments of the present invention, the polylactic acid-glycolic acid copolymer has a mass concentration percentage of 1% and a molecular weight of 50 kDa.
In a third aspect of the invention, there is provided a formulation comprising a hydrokinetic material according to the first aspect of the invention and a pharmaceutically acceptable adjuvant.
According to a third aspect of the invention, in some embodiments of the invention, the formulation is in dosage forms including, but not limited to, injections, tablets and capsules.
In a third aspect of the invention, there is provided a device prepared from a feedstock comprising the hydrogen kinetic material of the first aspect of the invention.
In a fourth aspect of the present invention, there is provided a use of the hydrogen kinetic material according to the first aspect of the present invention in the preparation of an inflammation inhibitory preparation or a medicament.
According to a specific embodiment of the invention, at least the following advantages are achieved:
the HPMs of the present invention can be in vivoInside and outside generate a large amount of H2And the expression of inflammatory cytokines TNF-alpha, IL-1 beta and IL-6 in the cells is inhibited, so that the inflammation can be effectively slowed or avoided.
In a fifth aspect of the present invention, there is provided a use of the hydrogen kinetic material according to the first aspect of the present invention in the preparation of an antioxidant agent or medicament.
According to a specific embodiment of the invention, at least the following advantages are achieved:
HPMs of the present invention are capable of producing large amounts of H in vitro and in vivo2And its motion ability is enhanced H2Due to H2Can easily diffuse across cell membranes, enter cells and organelles (such as nucleus and mitochondria), and has neutral electrical property and volume ratio of O2Small, so that it can react with Reactive Oxygen Species (ROS) to effectively and selectively scavenge excessive reactive oxygen species in cells, thereby exerting a certain antioxidant effect.
In a sixth aspect of the present invention, there is provided a use of the hydrogen power material according to the first aspect of the present invention in the preparation of an agent or a medicament for preventing or treating stroke.
According to a sixth aspect of the invention, in some embodiments of the invention, the stroke comprises ischemic stroke.
In the invention, research on HPMs with Janus structures to be prepared discovers that the HPMs can relieve in-situ oxidative stress and down-regulate inflammatory factors on an LPS-induced RAW264.7 cell inflammation model on a cell level, and have excellent in-vivo treatment effect on acute ischemic stroke on an animal level, and the technical principle is shown in figure 1. The HPMs are composed of biocompatible and biodegradable magnesium-based micromotors and a stereotaxic device, and in actual work, the micromotors with asymmetric polymer PLGA coatings can be used as H2Generator and active releaser, implementing H2So that it can be subjected to in situ H without any hazardous components2And (5) treating. The HPMs can be administered through a stereotaxic system, which is convenient for brain microsurgery. Precise injection of HPMs to a target under the direction of a stereotactic instrument systemArea to treat acute ischemic stroke in vivo without inflammatory reaction. Furthermore, with H2The HPMs are gradually exhausted and disappear after the treatment of the brain injury caused by oxidative stress is finished, and the prominent neuroprotective and ischemic stroke treatment effects are shown.
Drawings
FIG. 1 is a technical schematic diagram of HPMs in the treatment of acute ischemic stroke in an embodiment of the present invention;
FIG. 2 is a diagram of steps in the preparation of HPMs in an example of the present invention;
FIG. 3 is a particle size histogram of magnesium particles (A) and HPMs (B) in an example of the present invention;
fig. 4 is an SEM image (a) and a C element energy spectrum (B), a Mg element energy spectrum (C) and an O element energy spectrum (D) of the HPMs in the example of the present invention.
FIG. 5 is a Nile Red stain image (A) of HPMs and a corresponding 2.5D signal plot (B) of an embodiment of the present invention, including images of the red fluorescence channel, white light field channel, and Merge;
FIG. 6 is a chromatogram of a Mg standard solution (A), a standard curve of a Mg solution (B), and chromatograms of HPMs (C) in an example of the present invention;
FIG. 7 is a methylene blue absorption spectrum (A) of HPMs in an example of the invention; concentration-absorbance standard curve (B); methylene blue absorption spectrum (C) of magnesium microparticles in ACSF; methylene blue absorption spectra (D) of HPMs in ACSF; h generated by magnesium particles in ACSF2A concentration (E); h generated by HPMs in ACSF2A concentration (F);
FIG. 8 illustrates two different types of motion patterns of HPMs in an embodiment of the present invention;
FIG. 9 shows the helix (B) and linear motion mode (A) with long coil tails of HPMs in an embodiment of the present invention;
FIG. 10 shows the motion trajectories of HPMs in ACSF (A) and PBS (B) in an embodiment of the present invention;
FIG. 11 shows the use of SiO in an embodiment of the present invention2Cell activity histograms after @ PLGA, magnesium microparticles and HPMs;
FIG. 12 shows an embodiment of the present inventionSiO2Gene expression levels of TNF- α (A), IL-1 β (B) and IL-6(C) after @ PLGA, magnesium microparticles and HPMs;
FIG. 13 shows the use of SiO in an embodiment of the present invention2The levels of TNF- α (A), IL-1 β (B) and IL-6(C) after @ PLGA, magnesium microparticles and HPMs;
FIG. 14 shows the use of SiO in an embodiment of the present invention2Fluorescence images of TNF- α and IL-1 β after @ PLGA, magnesium microparticles and HPMs;
FIG. 15 shows the use of SiO in an embodiment of the present invention2Relative DCF fluorescence intensity after @ PLGA, magnesium microparticles and HPMs;
FIG. 16 shows the use of SiO in an embodiment of the present invention2Fluorescence images of CellROX Green staining after @ PLGA, magnesium microparticles and HPMs;
FIG. 17 is a schematic representation of a rat MCAO/R model wire-tying method in an example of the present invention;
FIG. 18 is a photograph showing an actual operation of the rat MCAO/R model in the example of the present invention;
FIG. 19 is a photograph of a pathological section of the heart, liver, spleen, lung, kidney and brain in an example of the present invention;
FIG. 20 is a graph of a neurological score (A) and a cross-bar balance score (B) of an experimental rat in an example of the present invention;
FIG. 21 shows the use of SiO in an embodiment of the present invention2Magnetic resonance imaging of the brain after @ PLGA, magnesium microparticles and HPMs;
FIG. 22 shows the use of SiO in an embodiment of the present invention2Statistical plot of rat cerebral infarction volume after @ PLGA, magnesium microparticles and HPMs;
FIG. 23 shows the use of SiO in an embodiment of the present invention2Material plot of brain sections after @ PLGA, magnesium microparticles and HPMs
FIG. 24 shows the use of SiO in an embodiment of the present invention2The levels of TNF- α (A), IL-1 β (B) and IL-6(C) after @ PLGA, magnesium microparticles and HPMs;
FIG. 25 shows the use of SiO in an embodiment of the present invention2Path length in the water maze for rats after @ PLGA, magnesium microparticles and HPMs (a), escape latency (B) and time in target quadrant (C) statistical plot;
FIG. 26 shows the use of SiO in an embodiment of the present invention2Graph of the pathway in the water maze for rats after @ PLGA, magnesium microparticles and HPMs.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Preparation of Hydrogen-Powered micro motors (HPMs)
The method comprises the following steps:
(1) and (3) sieving the Mg balls by a screen of 800-1000 meshes to obtain Mg ball particles with the size of about 10 mu m.
(2) A layer of 2% polyvinylpyrrolidone solution (PVP, MW 40000, in an amount of about 120. mu.L), appropriate amount of Mg spheres and 1% polylactic acid-glycolic acid copolymer (PLGA, in an amount of about 70. mu.L, in ethyl acetate) was coated on the slide glass in this order and dried overnight at room temperature. And (3) scraping the Mg @ PLGA particles obtained by reaction on the glass slide in a single layer under a body type microscope to obtain the Janus structure Mg @ PLGA micro-nano motor (namely HPMs).
Structural characterization of HPMs:
using the method of making the hydrogen powered micromotor of the above example, PVP (40kD) was sprayed onto a glass plate, Mg particles (17 μm in diameter) were fixed onto the PVP layer, and then a biodegradable PLGA solution (50000 molecular weight, 1 w/v% in ethyl acetate) was applied to the surface of the Mg particles to form a thin polymer layer. After the polymer layer was scraped off from the glass plate, a perfect asymmetric spherical structure, i.e. Mg @ PLGA micro-nano motor HPMs, was obtained (the preparation steps are shown in fig. 2).
The prepared Mg @ PLGA micro-nano motor is respectively subjected to electron microscope Scanning (SEM), and Back Scattered Electrons (BSE) and Secondary Electrons (SE) are used for detection to obtain an energy spectrum (EDX). Merge will be performed using the energy spectrum of C, O etc. elements in PLGA.
Electron Microscopy (SEM) of the prepared Mg @ PLGA micro-micromotors (HPMs) was found to exhibit larger dimensions than Mg microparticles (HPMs about 24 microns in diameter, see FIG. 3), with leaks on one side of the structure (about 10 microns in diameter, FIG. 4) as H2Resulting in a magnesium-water reaction interface. The obtained HPMs were further element mapped for visualization of Janus structures, and the results showed that the elemental distribution of magnesium from the magnesium microparticles and the distribution of carbon and oxygen from the PLGA outer shell could be observed inside the HPMs, demonstrating the successful formation of Janus structures.
To further verify the asymmetric PLGA distribution, HPMs were stained with nile red, which would be loaded into the PLGA shell due to the hydrophobicity of the PLGA coating, resulting in red fluorescence. The nile red stained HPMs were photographed using an inverted fluorescence microscope (fig. 5A) and converted to a 2.5D signal map according to a microscopic stereo scan (fig. 5B).
ICP-OES detection of Mg content in HPMs
Mg standard solutions were prepared in a series of concentrations and tested for injection (inductively coupled plasma emission spectroscopy) at 285.213nm and a standard curve was plotted. And dissolving the HPMs by nitric acid, fixing the volume and then measuring by using an inductively coupled plasma emission spectrometry.
As a result, as shown in fig. 6, fig. 6A is a chromatogram of the Mg standard solution, fig. 6B is a standard curve drawn based on the chromatogram of the Mg standard solution, and fig. 6C is a chromatogram of the HPMs prepared in the example. It can be calculated that the percentage concentration by mass of magnesium in the HPMs is 45.47 + -7.07%.
Methylene blue method for detecting H2Is generated in relation to time
Measurement of H released from HPMs according to Lambert-beer's law with methylene blue-platinum Probe solution2And (4) concentration.
The experimental principle is as follows:
methylene Blue (MB) may be combined with an equimolar amount of H in the presence of platinum2The reaction generates colorless reduced methylene blue (leuco methylene blue, leucoMB), and the reaction formula is as follows:
MB (blue) +2H++2e-→ leucoMB (colorless);
the method comprises the following specific steps:
(1) drawing a methylene blue standard curve by an ultraviolet spectrophotometer to quantify the released H2Metrological relationship to methylene blue reduction:
10mL of methylene blue probe solutions (1.875, 3.75, 7.5, 15, and 30 μ M) containing 10 μ L of platinum nanoparticles (2 w/w%, diameter about 50nm) were prepared and absorbance was measured with an ultraviolet-visible spectrophotometer at a wavelength of 664 nm.
(2) HPMs (final concentration 50. mu.g/mL, 100. mu.g) and Mg microparticles (final concentration 50. mu.g/mL, 100. mu.g) were added to a methylene blue-platinum probe solution (final volume 2mL) containing 0.3M sodium bicarbonate, respectively, and the change in absorbance was monitored with a UV-visible spectrophotometer at a wavelength of 664 nm. According to the released H2The quantitative relationship with methylene blue reduction, H is calculated2The amount of production of (c). Among them, the preparation method of the methylene blue probe can be referred to the preparation method of the methylene blue probe which is conventional in the art.
The results are shown in FIG. 7. It can be seen from FIGS. 7A and 7B that methylene blue can accurately detect H released from HPMs2And (4) concentration. In fig. 7C, the absorbance of methylene blue drops sharply within 20 minutes, indicating that a large amount of H is rapidly released due to the rapid degradation of magnesium in Artificial Cerebrospinal Fluid (ACSF)2. While the methylene blue faded more slowly than the magnesium particles compared to the HPMs (FIGS. 7D and 7E), indicating that H in HPMs is unique due to Janus structure2The release is stable, controllable and continuous.
Movement of HPMs and intracellular active H2Transfer of
The HPMs micromotors prepared in the above examples were placed in a container containing NaHCO at final concentrations of 0.5mol/L and 0.3mol/L, respectively3In the ACSF or PBS, the motion situation is observed in an inverted microscope, and the motion track, the motion speed, the directness and other data are tracked and calculated by image J software and Chemotaxis software.
The results are shown in FIGS. 8 to 11. The above embodiments can be observed in ACSFThe HPMs prepared in (a) have two different types of motion, including spiral and linear modes with long coil tails (fig. 8 and 9), which reflects that the motion behavior of HPMs is closely related to medium viscosity, and the various motion capabilities in biological fluids are mainly limited by H in different media2Diffusion and transport capabilities. Of course, HPMs can also exhibit similar motor behavior in PBS. To further analyze the trajectory path of the HPMs, the average velocity, directionality and cumulative travel distance of the HPMs in ACSF and PBS were calculated using image J software, which found H due to constant generation2The speeds of HPMs in ACSF and PBS reached 51.1 μm s, respectively-1And 61.12 μm s-1(FIG. 10). While the directionality of HPMs in ACSF and PBS is 0.495 and 0.351, respectively.
Use of HPMs for in vitro scavenging reactive oxygen species and reducing inflammation
Due to H2Can easily diffuse across cell membranes, enter cells and organelles (such as nucleus and mitochondria), and has neutral electrical property and volume ratio of O2Small, and therefore, it can react with Reactive Oxygen Species (ROS) to effectively and selectively scavenge excess reactive oxygen species and proinflammatory cytokines in cells. While motion of the HPMs will enhance the generation of H2Further diffusion and dissolution in aqueous solution promote the elimination of intracellular reactive oxygen species and the reduction of inflammation.
SiO with asymmetric PLGA coating was prepared according to the above HPMs preparation method2@ PLGA (average diameter 9.9 microns) was used as a control.
(1) And (3) detecting cytotoxicity:
at 5X 104Individual cells/well RAW264.7 cells were transferred to 96-well plates and incubated for 24 hours (DMEM + 10% FBS medium), followed by the addition of SiO2@ PLGA (0.4mg/mL), magnesium microparticles (0.15mg/mL) and HPMs prepared in the above examples (0.4mg/mL), incubation was continued for 6, 12 and 24 hours and cell viability was quantified using the CCK-8 assay kit.
The results are shown in FIG. 11, SiO at a concentration of 0.4mg/mL2Neither the @ PLGA and HPMs, nor the magnesium microparticles at a concentration of 0.15mg/mL were cytotoxic.
(2) Effects of HPMs on the cytokines IL-1 β, TNF- α and IL-6:
the expression of the cell inflammatory factors IL-1 beta, TNF-alpha and IL-6 is detected by ELISA method and immunofluorescence method respectively.
The ELISA method for detecting the expression of IL-1 beta, TNF-alpha and IL-6 in RAW264.7 cells comprises the following specific steps: treating RAW264.7 cells with Lipopolysaccharide (LPS, 1 μ g/mL) to construct an inflammation induction model, and adding SiO2@ PLGA, magnesium microparticles and HPMs prepared in the above examples (magnesium content in HPMs being the same as magnesium microparticles), after 6 hours of incubation, cells were lysed in immunoprecipitation lysis buffer and centrifuged at 13000 rpm for 10 minutes at 4 ℃. Supernatants were collected using ELISA kits and analyzed to assess expression levels of IL-1 β, TNF- α and IL-6.
② the immunofluorescence method for observing IL-1 beta, TNF-alpha and IL-6 in RAW264.7 cells, the concrete steps are: treating RAW264.7 cells with Lipopolysaccharide (LPS, 1 μ g/mL) to construct an inflammation induction model, and adding SiO2@ PLGA, magnesium microparticles and HPMs prepared in the above examples (magnesium content in HPMs being the same as magnesium microparticles) after 6 hours of incubation, cells were washed in PBS and then fixed in 4% paraformaldehyde. The cells were blocked with 5% goat serum for 1 hour at 37 ℃ and then incubated with 5% goat serum (containing monoclonal antibodies IL-1 β, TNF- α and IL-6(Abcam) overnight at 4 ℃ then the cells were washed again and incubated with fluorescent secondary antibody for 2 hours at 37 ℃.
The results are shown in FIGS. 12-14, and are obtained by SiO2Both HPMs and magnesium microparticles significantly reduced the levels of TNF-alpha, IL-1 beta and IL-6 gene expression (P) compared to cells treated with @ PLGA<0.05), indicating HPMs and H produced by the magnesium particles2Can reduce excessive secretion of TNF-alpha, IL-1 beta and IL-6 induced by LPS. Intracellular TNF-. alpha. (green) and IL-1. beta. (red) expression was observed by CLSM as compared to DAPI staining (blue). After being stimulated by LPS, Raw264.7 cells simultaneously show deepened green fluorescence and red fluorescence, which shows that the levels of TNF-alpha, IL-1 beta and IL-6 are rapidly increased,is highly correlated with inflammatory responses in diseased tissues. Inflammatory cytokines in LPS-induced raw264.7 cells were significantly reduced after incubation with HPMs, while the inflammatory clearance of magnesium microparticles was relatively weak. Due to movement enhance H2Delivery of TNF-alpha, IL-1 beta and IL-6 levels in HPMs-treated cells compared to H2Passively diffused magnesium particle treated cells were 0.45, 0.52 and 0.37 times lower (P)<0.05). In contrast to SiO2@ PLGA, HPMs systems with kinematic Properties have higher H2The efficiency is actively reduced, so that the inflammation clearance is stronger.
(3) And (3) ROS detection:
treating RAW264.7 cells with Lipopolysaccharide (LPS, 1 μ g/mL) to construct an inflammation induction model, and adding SiO2@ PLGA, magnesium microparticles and HPMs prepared in the above examples (magnesium content in HPMs being the same as magnesium microparticles) after 6 hours incubation at 37 ℃ and 5% CO2Under the conditions of 20M DCFH-DA (2',7' -dichloro-fluoro-xanthate and Reactive Oxygen Species (ROS) probe (5 μ M, Cellrox) treatment of cells for 30 minutes, then immediately using the multifunctional plate reader (excitation wavelength 488nm, emission wavelength 525nm) analysis, using laser scanning confocal microscope (CLSM) observation in LPS stimulated RAW264.7 cells in the LSP treated RAW264.7 cells without any reagent as a positive control.
The results are shown in FIGS. 15-16, with HPMs at completion H2After release, the magnesium content is depleted, leaving only the outer PLGA shell, which can be gradually degraded at ACSF (fig. 15), with by-product Mg2+Is an element beneficial to human body, and makes HPMs not have cytotoxicity, and has excellent biodegradability and biocompatibility. DCFH-DA is a cell-permeable active oxygen fluorescent probe, mainly used for evaluating the level of active oxygen in cells. After the cells are incubated with the magnesium particles or the HPMs, the fluorescence intensity is obviously reduced compared with the LPS positive control, which indicates that the content of active oxygen in the cells is also reduced correspondingly. HPMs show the highest efficiency for scavenging reactive oxygen species compared to the other groups, mainly due to their enhanced mobility with H2Active transfer of (2). Another active oxygen fluorescent probe-CellROX Green is also usedAnd detecting active oxygen. When RAW264.7 cells were stimulated with LPS, relatively high reactive oxygen species levels were observed, whereas HPMs showed minimal intracellular fluorescence, indicating excellent ability to scavenge reactive oxygen species. Without H2SiO of release character2The fluorescence of the @ PLGA group did not change significantly.
Application of HPMs (HPMs) in-situ treatment of acute ischemic stroke
The protection effect of the HPMs micromotors on cerebral ischemia-reperfusion of rats is evaluated from the aspects of nerve function score, weight loss rate, tissue Reactive Oxygen Species (ROS) level detection, cerebral infarction rate, cytokine level detection, pathology detection, MRI imaging detection, Morris water maze and the like.
(1) Establishing a rat cerebral ischemia reperfusion injury model (MCAO/R) and designing an HPMs micro-nano motor dosing scheme:
rat MCAO/R models were established using the wire-embolus method (ligation of the proximal left Common Carotid Artery (CCA) and External Carotid Artery (ECA), schematic diagrams of the procedures are shown in fig. 17 and 18). After 7 days of acclimation, 40 male rats were randomly divided into 6 groups (Mg-sphere group, SiO)2The @ PLGA group, HPMs group, MCAO/R model group, Sham (Sham) group), 8 per group. Performing lateral ventricle administration 2h after MCAO/R operation by using a brain stereotaxic instrument, wherein the specific method comprises the following steps: rats were anesthetized (isoflurane, where 4% isoflurane was used for induction and 2% isoflurane for maintenance) and placed on a stereotaxic apparatus. A small hole is drilled at the proper part of the skull. 30. mu.L of physiological saline, 26.7mg mL of mL were injected with 3. mu.L/min at coordinates (X) -1.6mm, (Y) -1.0mm and (Z) -3.5mm using a 100. mu.L Hamilton syringe, respectively-1SiO2@PLGA、10mg mL-1Mg or 26.7Mg mL-1HPMs, withdraw the syringe 5 minutes after injection. According to the different experimental groups, rats were injected with normal saline, 26.7mg mL, continuously on days 2, 4 and 6-1SiO2@PLGA、10mg mL-1Mg or 26.7Mg mL-1HPMs. After reperfusion for 2h after cerebral ischemia, rat blood and urine are taken for biochemical index analysis.
(2) And (3) in vivo safety detection:
after 7 days, blood samples from the sham and MCAO groups were collected and serologically analyzed using a poincare M3 biochemical analyzer to assess the potential toxicity of HPMs. Assays include renal and hepatic function assays, such as Total Protein (TP), Total Bilirubin (TBIL), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Gamma Glutamyl Transferase (GGT), urea (urea) and Creatinine (CREA).
Serum biochemical results indicate that no abnormal indicators of liver and kidney function (levels of total protein, TBIL, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, GGT, urea, and CREA) were observed.
Further H & E staining of major organs including heart, liver, spleen, lung, kidney and brain, comprising the specific steps of: and after 2h of ischemia reperfusion for 24h, anesthetizing the rat by 10% chloral hydrate, slowly perfusing by 200mL of 4% neutral paraformaldehyde, quickly cutting the head and taking out the brain after perfusion, removing olfactory bulb and cerebellum, and placing in 10% neutral buffered formalin for continuous fixation for 24 h. The brain tissue block with the thickness of about 2mm before and after the optic chiasm nerve is cut from the coronal section, dehydrated by a conventional method and embedded by paraffin. Other organs were serially coronal sliced (thickness 4-5 μm) and baked overnight in an incubator at 37 ℃.
No significant changes were found in the histopathological sections of the HPMs group (fig. 19), indicating that the HPMs were viable and safe.
(3) And (3) nerve function scoring:
the most common neurological scale in stroke animal studies includes a modified neurological severity score (mNSS, table 1). The full score of mNSS was 18, and the rats in the above examples were classified into 0 to 18 grades, wherein 0 score indicates normal performance and 18 total scores indicate severe defects. Among them, rats with a neurological score higher than 6 were extracted for the subsequent examples. The MCAO (neurological score higher than 6) rats were randomly divided into 4 groups (MCAO group, Mg sphere group, SiO group) in addition to the normal control group2@ PLGA and HPMs), 8 per group.
TABLE 1 severity of nerve damage scoring table
Injecting; if one task cannot be completed or one reflection is missing, 1 point is given, and 13-18 points suggest serious damage; 7-12 points, moderate damage; 1-6 points, mild damage.
As shown in figure 20, MCAO rats had significant neurological deficits, whereas Sham (Sham) did not. Notably, with MCAO and SiO2The @ PLGA group showed a significant reduction in neurological function and cross-bar balance scores in MCAO rats treated with magnesium microparticles and HPMs (P)<0.01), indicating H produced2Can provide effective neuroprotection. In each group, the HPMs were most effective in reducing nerve function and cross-bar balance scores, suggesting H delivered by HPMs2The active substance can effectively alleviate brain injury and accelerate the recovery of MACO rats.
(4) Analysis of cerebral infarction rate:
on day 7 of treatment, the cerebral infarct volume of MCAO rats was measured using a 3.0T magnetic resonance imaging system, with the animals fixed in a supine position with an advanced head. Coronal routine T1WI, T2WI, T2WI FLAIR, DWI, DTI examinations were performed with the view intersection as the scan center. On day 8, the rats were anesthetized and scanned, and the imaging protocol included axial T1-weighted and T2-weighted Fast Spin Echo (FSE) sequences. In addition, when b is 1000s/mm2The infarct volume of the rats was evaluated with Image-J using an STIR (short τ inversion recovery) -Diffusion Weighted Image (DWI) sequence on the coronal plane to segment and quantify the number of dark voxels in the ischemic region.
Infarct volume was calculated according to the following formula:
after the treatment is finished, after the rat is subjected to blood sampling, decapitation and brain sampling, removing the olfactory bulb and the cerebellum, absorbing moisture on the surface of the brain by using filter paper, weighing the moisture, immediately putting the rat into a refrigerator at the temperature of 20 ℃ below zero, and taking the rat out after 20 min. Serial coronal sections were made from front to back for a total of 5 slices. Placing the brain slice into 0.2% TTC (2,3, 5-triphenyltetrazolium chloride) solution, dyeing in constant temperature water bath at 37 deg.C in dark place for 30min, and shaking the beaker during the water bath to make the brain slice fully contact with TTC solution. And calculating the cerebral infarction rate by using Image-J.
3.0T magnetic resonance imaging on day 7 of treatment showed minimal cerebral infarct volume in the HPMs groups (FIGS. 21-22). In the TTC staining results, the white area was the infarct zone. As shown in FIG. 23, the area of brain infarction was greatest in TTC-stained brain sections from the MCAO and SiO2@ PLGA groups. While using H provided by HPMs2In the HPMs after treatment, infarct size was greatly reduced.
(4) Analysis of inflammatory factors:
after blood is taken from a rat, the head is broken and the brain is taken, the olfactory bulb and the cerebellum are removed, the moisture on the surface of the brain is absorbed by filter paper, the brain tissue is quickly separated from bilateral cerebral hemispheres (ischemic side and contralateral side) along the median line on ice, the brain on the injured side is weighed and then put into a homogenizer for homogenization, 0.9 percent NS (normal saline) is used as a homogenization medium to prepare 10 percent brain tissue homogenate, 3000r/min and centrifugation is carried out for 10min, and the supernatant is taken and subpackaged in an EP tube and stored in a refrigerator at minus 80 ℃ for standby. TNF-alpha, IL-1 beta and IL-6 contents were determined according to the method of ELISA kit instructions.
The ELISA detection result shows that SiO2There was no statistical difference in inflammatory factor concentration between the @ PLGA and MCAO groups. While the concentration of TNF- α from HPMs was significantly reduced to 85.01 + -9.45 pg/mL (FIG. 24A, P)<0.05), similar results were also observed for IL-1. beta. and IL-6 concentrations, indicating that HPMs have potent efficacy in relieving inflammation caused by acute ischemic stroke.
(5) Morris water maze behavioural study:
to further validate the in situ therapeutic effect of HPMs on acute ischemic stroke, the Morris water maze was used to assess the learning and memory abilities of rats.
On day 8 of treatment, rats treated with each of the groups of drugs in the example above were placed in a pool at one of the four starting points (east, west, south or north) of the Morris water maze and were allowed to swim for up to 90 seconds. When the rats found a platform, they were allowed to rest on the platform for 15 seconds and then returned to the test cage with the heat. If no platform is found within 90 seconds, the rat is placed on the platform for 30 seconds. Swimming traces and time were recorded using video tracking.
Spatial learning is evaluated according to the time it takes for the rat to find the platform (escape latency), memory capacity is evaluated according to the time it takes for the rat to spend in the target quadrant and crossing the platform (time in the target quadrant), and analyzed by SuperMaze software.
The results are shown in FIGS. 25 and 26. The pathlength of HPMs-treated rats was significantly reduced compared to magnesium microparticles (P <0.05), indicating improved learning and memory capacity. Similar trends were also found in the escape latency of the rats and in the time of the target quadrant (fig. 26). After injection of HPMs, the escape latency of MCAO rats decreased significantly, while the time in the target quadrant increased dramatically (P <0.05 compared to magnesium microparticles), indicating that rats have a stronger latent memory of the removed platform. The above results show that oxidative stress in vivo can be alleviated by precise injection of HPMs, greatly improving ischemia-induced spatial memory impairment.
In summary, the HPMs prepared in the above examples have a Janus structure, and the magnesium element contained in the HPMs does not generate H too quickly2. And the stable and controllable and persistent H2The motion characteristics of the release-fit HPMs can reach higher H2The efficiency of active delivery, and the concentration of IL-1 beta and IL-6 in the body is reduced, thereby showing stronger inflammation clearance capability. And H2Has effective neuroprotective effect, can reduce infarct size, and has effective effect in relieving inflammation caused by acute ischemic apoplexy. By in vivo precise injection, HPMs can relieve in vivo oxidative stress and improve ischemia-induced spatial memory injury, and have extremely high application prospects.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The hydrogen power material is characterized by comprising a magnesium element core and a polylactic acid-glycolic acid copolymer shell coated outside the magnesium element core;
the shell of the polylactic acid-glycollic acid copolymer is provided with at least 1 gap, so that the magnesium element core can be communicated with the external environment;
the polylactic acid-glycolic acid copolymer shell is preferably provided with 1 notch.
2. The hydrogen kinetic material of claim 1, wherein the hydrogen kinetic material has a Janus structure.
3. The hydrogen power material of claim 1, wherein the hydrogen power material has a diameter of 10-25 μm, and the percentage of the mass concentration of magnesium element in the hydrogen power material is 38.4-52.54%;
the diameter of the magnesium element core is preferably 10 to 17 μm.
4. The hydrogen kinetic material of claim 1, wherein the gaps have a diameter of 5-15 μ ι η; the diameter of the gap is preferably 8 to 12 μm.
5. A method of producing the hydrogen kinetic material of any of the claims 1 to 4, comprising the steps of:
placing a magnesium material on a substrate material, coating polylactic acid-glycolic acid copolymer, and scraping the magnesium material coated with the polylactic acid-glycolic acid copolymer from the substrate material to obtain the magnesium-based material;
wherein the base material comprises polyvinylpyrrolidone;
the mass concentration percentage of the polyvinylpyrrolidone is preferably 1-3%, and the molecular weight is preferably 38-42 kDa; the mass concentration percentage of the polylactic acid-glycolic acid copolymer is preferably 0.5-1.5%, and the molecular weight is preferably 48-52 kDa.
6. A formulation comprising a hydrokinetic material as defined in any one of claims 1 to 4 and pharmaceutically acceptable adjuvants.
7. A device prepared from a material comprising the hydrogen kinetic material of any one of claims 1 to 4.
8. Use of the hydrogen kinetic material of any one of claims 1 to 4 for the preparation of an inflammation suppressing agent or medicament.
9. Use of the hydrogen kinetic material of any one of claims 1 to 4 in the preparation of an antioxidant preparation or medicament.
10. Use of the hydrogen kinetic material of any one of claims 1 to 4 for the preparation of an agent or medicament for the prevention or treatment of stroke; wherein the stroke includes ischemic stroke.
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