CN112007016B

CN112007016B - Nano slow-release preparation, preparation method thereof and application thereof in preparing medicine for treating mitochondrial dysfunction diseases

Info

Publication number: CN112007016B
Application number: CN202010927175.2A
Authority: CN
Inventors: 李宽钰; 徐莉
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2021-07-23
Anticipated expiration: 2040-09-07
Also published as: CN112007016A

Abstract

The invention provides a nano sustained-release preparation, belonging to the technical field of nano materials. The nano sustained release preparation provided by the invention comprises dextran-modified cerium oxide nanoparticles and silk fibroin coated on the outer layer of the dextran-modified cerium oxide nanoparticles. In the invention, the dextran-coated cerium oxide nanoparticles have good water-soluble dispersibility, excellent antioxidant activity and oxygen carrying capacity, can remarkably improve neuron loss caused by hypoxia and oxidative damage, and recover the form and structure of mitochondria; the silk fibroin has good biocompatibility, and the dextran-modified cerium oxide nanoparticles are coated by the silk fibroin, so that the effect of slowly releasing the nanoparticles can be achieved, and the defects of instantaneous overhigh drug concentration and rapid metabolism are avoided; meanwhile, the silk fibroin has biodegradability, and degradation products of the silk fibroin are safe and pollution-free.

Description

Nano slow-release preparation, preparation method thereof and application thereof in preparing medicine for treating mitochondrial dysfunction diseases

Technical Field

The invention relates to the technical field of nano materials, in particular to a nano sustained-release preparation, a preparation method thereof and application thereof in preparing a medicament for treating mitochondrial dysfunction diseases.

Background

Mitochondria are organelles coated by the mitochondrial outer membrane OMM and the mitochondrial inner membrane IMM, and are sites where eukaryotic organisms undergo oxidative metabolism. Mitochondria are one of the most vulnerable organelles, and inhibition of the respiratory chain, reduction of enzymatic activity, damage to mitochondrial DNA, and damage to the mitochondrial membrane all cause Mitochondrial Dysfunction (MD). MD is generally characterized by changes in the number, size and morphology of mitochondria, which quantitatively undergoes proliferation, an adaptive response to chronic nonspecific cellular injury, and a lesser number in the case of acute injury or autophagy and lysis. The most common change in cell damage is mitochondrial enlargement. In size, mitochondrial swelling, which is the most common form of cell damage, is adaptive hypertrophy with increased organ functional load, while mitochondria shrink when organs shrink. Morphologically, the mitochondrial cristae is destroyed to a different extent in MD, becomes shorter, becomes smaller, and even disappears, while most enzymes that function in mitochondria are distributed in the mitochondrial cristae.

Many diseases related to mitochondrial dysfunction are known at present, for example, friedreich's ataxia (FRDA), Alzheimer's Disease (AD), Parkinson's Disease (PD), and the like. In the case of friedreich ataxia (FRDA), the pathogenesis of frataxin is the high-fold repetitive extension of the GAA sequence in the first intron of the FXN gene, resulting in a reduction in frataxin protein levels, and studies have shown that defects in frataxin protein lead to a secondary deficiency of the iron-sulfur cluster in mitochondria, which is a key component of a series of enzymes in the tricarboxylic acid cycle and mitochondrial electronic respiratory chain enzymatic reactions, such as mitochondrial complexes I, II and III, aconitase, hepcidin I, etc. Meanwhile, iron accumulation caused by iron-sulfur cluster synthesis defects enables free ferrous ions in mitochondria to be unable to be balanced and to be in a high-concentration level state, Reactive Oxygen Species (ROS) can be increased under the action of Fenton reaction, and oxidative stress damage of mitochondria is aggravated. The final pathology is therefore represented by a degeneration of the nerve and the cardiac muscle cells, which is followed by a mitochondrial dysfunction.

The current drugs for treating diseases related to mitochondrial dysfunction include cyclosporin A (CSA), cyclosporin A analogues, and mitochondrial coenzyme Q₁₀Alpha-lipoic acid, etc., but cyclosporin a has a poor ability to penetrate the blood-brain barrier, limiting its therapeutic effect; mitochondrial coenzyme Q₁₀Can eliminate the active oxygen ROS level in the mitochondria, but only can relieve diseases and can not radically cure the diseases; alpha-lipoic acid is a mitochondrial antioxidant, but it also has the drawback of being only alleviating it, which cannot be radical.

Disclosure of Invention

In view of the above, the present invention aims to provide a nano sustained release preparation, a preparation method thereof and an application thereof in preparing a medicament for treating mitochondrial dysfunction diseases. The nano sustained-release preparation provided by the invention can recover the form and structure of mitochondria and has obvious curative effect on mitochondrial dysfunction diseases.

In order to achieve the purpose of the invention, the invention provides the following technical scheme:

the invention provides a nano sustained release preparation, which comprises dextran-modified cerium oxide nano-particles and silk fibroin coated on the outer layer of the dextran-modified cerium oxide nano-particles.

Preferably, the size of the dextran-modified cerium oxide nanoparticles is 1-30 nm; the hydrodynamic size of the nano sustained-release preparation is 80-120 nm.

The invention provides a preparation method of the nano sustained-release preparation, which comprises the following steps:

(1) providing an alcoholic dispersion of dextran-modified cerium oxide nanoparticles;

(2) mixing silk fibroin and a lithium bromide solution, sequentially carrying out hydrolysis and first dialysis, and obtaining a silk fibroin solution in a dialysis bag;

(3) mixing the dextran-modified cerium oxide nanoparticle alcohol dispersion liquid with a silk fibroin solution, and performing ultrasonic treatment to obtain a suspension;

(4) freezing, melting and carrying out second dialysis on the suspension to obtain a nano sustained-release preparation;

the steps (1) and (2) are not limited in chronological order.

Preferably, the mass concentration of cerium element in the alcohol dispersion liquid of the dextran-modified cerium oxide nanoparticles is 1-5 mg/mL.

Preferably, the molar concentration of the lithium bromide solution is 8.5-10 mol/L; the mass ratio of the silk fibroin to the lithium bromide solution is 1 g: 1-5 mL.

Preferably, the hydrolysis temperature is 20-70 ℃, and the hydrolysis time is 30-360 min;

the first dialysis bag retains a molecular weight of 12000-14000.

Preferably, the volume ratio of the mass of cerium element in the dextran-modified cerium oxide nanoparticles to the silk fibroin solution is 1mg: 1-5 mL;

the power of the ultrasonic wave is 100-300W, and the time is 10-60 min.

Preferably, the freezing temperature is-80 to-20 ℃, and the time is 24 to 48 hours;

the molecular weight intercepted by the second dialysis bag is 5000-10000.

The invention provides application of the nano sustained-release preparation in preparing a medicament for treating mitochondrial dysfunction diseases.

Preferably, the mitochondrial dysfunction disease comprises one or more of friedreich's ataxia, alzheimer's disease, parkinson's disease or amyotrophic lateral sclerosis.

The invention provides a nano sustained release preparation, which comprises dextran-modified cerium oxide nano-particles and silk fibroin coated on the outer layer of the dextran-modified cerium oxide nano-particles. In the invention, the dextran-coated cerium oxide nanoparticles have good water-soluble dispersibility, excellent antioxidant activity and oxygen carrying capacity, can remarkably improve neuron loss caused by hypoxia and oxidative damage, and recover the form and structure of mitochondria; the silk fibroin has good biocompatibility, and the dextran-modified cerium oxide nanoparticles are coated by the silk fibroin, so that the effect of slowly releasing the nanoparticles can be achieved, and the defects of instantaneous overhigh drug concentration and rapid metabolism are avoided; meanwhile, the silk fibroin has biodegradability, and degradation products of the silk fibroin are safe and pollution-free. The results of the examples show that the nano sustained-release preparation provided by the invention has sustained-release effect, and has obvious curative effect on mitochondrial dysfunction diseases when being used for preparing the medicine for treating the mitochondrial dysfunction diseases, and the specific expression is as follows: (1) the number and the form of Purkinje cells in the cerebellum of the FRDA mouse can be recovered, and the phenomenon of iron accumulation in the cardiac muscle cells disappears; (2) the morphology and the function of mitochondria of an FRDA mouse can be recovered, the size and the morphology of the mitochondria are recovered to be normal, the inner membrane ridges of the mitochondria are orderly arranged, and the function of generating ATP by the mitochondria is recovered; (3) the protein expression level of the mitochondrial complex I, II and III subunits can be recovered to be normal, and the activity of the mitochondrial complex I and II can also be recovered to be normal.

The invention provides a preparation method of the nano sustained release preparation, wherein alcohol dispersion liquid of dextran-modified cerium oxide nano particles is mixed with silk fibroin solution, and ultrasonic treatment is carried out to wrap the cerium oxide nano particles by silk fibroin, so that the composite nano particles are self-assembled; the suspension after ultrasonic treatment is frozen to achieve the effect of stabilizing the composite nano particles, and the nano sustained-release preparation is obtained after melting and dialysis. The preparation method provided by the invention is simple and is easy to realize industrial mass production.

Drawings

FIG. 1 shows the sustained release formulation SF @ CeO obtained in example 1₂SEM pictures of NPs;

FIG. 2 shows the sustained release formulation SF @ CeO obtained in example 1₂Hydrodynamic size of NPs;

FIG. 3 shows SF @ CeO₂The drug release rate of NPs;

FIG. 4 shows SF @ CeO₂Weight change of mice during 1 month of intraperitoneal administration of NPs;

FIG. 5 is a graph showing the results of the behavioral-wooden beam experiment tests of mice administered with SF @ CeO2 NPs;

FIG. 6 shows SF @ CeO₂The results of the behavioral-rotational fatigue tester test of the mice after NPs administration are shown;

FIG. 7 shows SF @ CeO₂Test result chart of ethology-suspension experiment of mice after NPs administration;

FIG. 8 shows SF @ CeO₂Section H of mouse cerebellum after NPs administration&E, staining pattern;

FIG. 9 is SF @ CeO₂Prussian blue staining pattern of mouse heart after NPs administration;

FIG. 10 shows SF @ CeO₂ATP content in mouse tissues after NPs administration;

FIG. 11 shows SF @ CeO₂Protein expression levels of mitochondrial complex I/II/III subunits in mouse tissues after NPs administration;

FIG. 12 shows SF @ CeO₂The activity of mitochondrial complexes I in various tissues of mice after NPs administration;

FIG. 13 is SF @ CeO₂Mouse tissue midline after NPs administrationActivity of mitochondrial complex ii.

Detailed Description

In the invention, the size of the dextran-modified cerium oxide nanoparticle is preferably 1-30 nm, more preferably 5-20 nm, and further preferably 10-15 nm; in the invention, the hydrodynamic size of the nano sustained-release preparation is preferably 80-120 nm, more preferably 90-110 nm, and further preferably 100 nm.

In the invention, the dextran-modified cerium oxide nanoparticles preferably have a core-shell structure, wherein the core is cerium oxide nanoparticles, and the modified shell is dextran; in the present invention, the dextran is very thin and its thickness is negligible.

In the invention, the dextran-coated cerium oxide nanoparticles have good water-soluble dispersibility, excellent antioxidant activity and oxygen carrying capacity, can remarkably improve neuron loss caused by hypoxia and oxidative damage, and recover the form and structure of mitochondria; the silk fibroin has good biocompatibility, and the dextran-modified cerium oxide nanoparticles are coated by the silk fibroin, so that the effect of slowly releasing the nanoparticles can be achieved, and the defects of instantaneous overhigh drug concentration and rapid metabolism are avoided; meanwhile, the silk fibroin has biodegradability, and degradation products of the silk fibroin are safe and pollution-free.

the steps (1) and (2) are not limited in chronological order.

The present invention provides an alcohol dispersion of dextran-modified cerium oxide nanoparticles. The present invention has no special requirement for the preparation method of the Dextran modified Cerium Oxide Nanoparticles, and the preparation method known to those skilled in the art can be used, and as a specific example of the present invention, the preparation method of the Dextran modified Cerium Oxide Nanoparticles refers to English literature, Dextran-coated Cerium Oxide Nanoparticles: A computer Tomography content Agent for Imaging the gastric structural polypeptide and Influmimator Bowell diseases.

In the present invention, the method for preparing the alcohol dispersion of the dextran-modified cerium oxide nanoparticles preferably comprises the steps of:

mixing the dextran-modified cerium oxide nanoparticles with an alcohol solvent to obtain an alcohol dispersion of the dextran-modified cerium oxide nanoparticles.

In the present invention, the alcohol solvent is preferably one or more of methanol, ethanol and propanol, and more preferably methanol. In the invention, the mass concentration of cerium element in the alcohol dispersion liquid of the dextran-modified cerium oxide nanoparticles is preferably 1-5 mg/mL, and more preferably 2-4 mg/mL.

The silk fibroin solution is obtained by mixing silk fibroin and a lithium bromide solution, sequentially carrying out hydrolysis and first dialysis, and carrying out a dialysis bag. In the invention, the preparation method of the silk fibroin is preferably prepared by alkali degumming of natural silk fiber. In the invention, the molar concentration of the lithium bromide solution is preferably 8.5-10 mol/L, and more preferably 9-9.5 mol/L; the mass ratio of the silk fibroin to the lithium bromide solution is preferably 1 g: 1-5 mL, preferably 1 g: 2-4 mL.

In the invention, the hydrolysis temperature is preferably 20-70 ℃, and more preferably 30-60 ℃; the time is preferably 30 to 360min, and more preferably 60 to 240 min. The invention can make the silk fibroin uniformly dispersed in the water solution by the hydrolysis. The material of the first dialysis bag is not particularly required, and a dialysis bag made of a material well known to those skilled in the art can be used; the molecular weight cut off by the first dialysis bag is preferably 12000-14000, and more preferably 13000. According to the invention, impurity molecules can be removed through the first dialysis, so that pure silk fibroin is obtained.

After the alcohol dispersion liquid of the dextran-modified cerium oxide nano-particles and the silk fibroin solution are obtained, the alcohol dispersion liquid of the dextran-modified cerium oxide nano-particles and the silk fibroin solution are mixed and subjected to ultrasonic treatment to obtain a suspension. The invention preferably performs the mixing under ultrasonic conditions; in the present invention, the mixing is preferably performed by slowly adding the silk fibroin solution dropwise to the alcohol dispersion of the dextran-modified cerium oxide nanoparticles.

In the invention, the mass ratio of cerium element in the dextran-modified cerium oxide nanoparticles to the volume ratio of the silk fibroin solution is preferably 1mg: 1-5 mL, and more preferably 1mg: 2-4 mL; the power of the ultrasonic wave is preferably 100-300W, more preferably 200W, and the time is preferably 10-60 min, more preferably 20-40 min; the present invention preferably performs the sonication at room temperature. According to the invention, silk fibroin can wrap cerium oxide nanoparticles through the ultrasound, and the cerium oxide nanoparticles can be self-assembled into composite nanoparticles.

After the suspension is obtained, the suspension is subjected to freezing storage, melting and second dialysis, and a nano sustained-release preparation is obtained in a dialysis bag. In the invention, the freezing temperature is preferably-80 to-20 ℃, more preferably-60 to-40 ℃, and the time is preferably 24 to 48 hours, more preferably 30 to 36 hours. The invention can achieve the effect of stabilizing the composite nano particles by freezing. The present invention has no special requirement on the melting mode, and the melting mode known to those skilled in the art can be used, such as room temperature melting or water bath melting. In the present invention, the dialysis is preferably performed in water. The material of the second dialysis bag is not particularly required, and a dialysis bag made of a material well known to those skilled in the art can be used; the molecular weight intercepted by the second dialysis bag is preferably 5000-10000, and more preferably 6000-8000.

After the nano sustained-release preparation is obtained, the nano sustained-release preparation is preferably subjected to freeze-drying preservation. The present invention does not require any particular method for lyophilization, and lyophilization methods well known to those skilled in the art may be used.

The invention provides application of the nano sustained release reagent in preparing a medicament for treating mitochondrial dysfunction diseases. In the present invention, the mitochondrial dysfunction disease comprises one or more of friedreich's ataxia (FRDA), Alzheimer's Disease (AD), Parkinson's Disease (PD), or Amyotrophic Lateral Sclerosis (ALS). In the invention, when the nano slow release reagent is used for preparing a medicament for treating mitochondrial dysfunction diseases, the concentration of the nano slow release reagent is preferably 0.2-1 mg/mL; calculated by the mass of cerium element in the nano sustained-release reagent, the administration dosage of the nano sustained-release preparation is preferably 1-5 mg/kg, and more preferably 2-4 mg/kg.

The nano sustained release preparation provided by the present invention, the preparation method thereof and the application thereof in the preparation of the drugs for treating mitochondrial dysfunction diseases are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

(1) The remaining cotton-like bulky solid is silk fibroin after degumming natural silk fiber (purchased from Nantong Xin Yuan Co., Ltd.) with alkali; 20g of silk fibroin was hydrolyzed with 50mL of 9M lithium bromide (from Aladdin) for 240min at 60 ℃. Removing impurity molecules through a dialysis bag with the molecular weight cutoff of 12000-14000 to obtain a silk fibroin solution;

(2) mixing Dextran-modified Cerium Oxide Nanoparticles (prepared by a method referred to as "Dextran-coated Cerium Oxide Nanoparticles: A computer aided biology Contrast Agent for Imaging the organic matter track and Influmatory Bowell diseases") with a diameter of 1-10 nm with an absolute ethanol solution to obtain an alcohol dispersion of the Dextran-modified Cerium Oxide Nanoparticles with a Cerium element concentration of 1 mg/mL;

(3) slowly adding the silk fibroin solution into the dextran-modified cerium oxide nanoparticle alcohol dispersion liquid drop by drop under the ultrasonic condition of 200W, performing ultrasonic treatment at room temperature for 60min, and stirring to obtain a suspension;

(4) freezing the suspension at-20 deg.C for 48h, thawing at room temperature or in water bath, dialyzing in water with dialysis bag with cut-off molecular weight of 5000-10000kDa to obtain sustained-release nano preparation SF @ CeO₂ NPs。

For the obtained sustained-release nano preparation SF @ CeO₂NPs are tested by a Scanning Electron Microscope (SEM), and the parameters of the SEM are as follows:

the model is as follows: JEM-2100, Japan;

detection conditions are as follows: the detection was carried out after palladium sputter coating at an acceleration voltage of 15 kV.

The obtained scanning electron micrograph is shown in FIG. 1, and it can be seen from FIG. 1 that the inner layer particles are CeO modified by dextran₂And the outer layer of the nano-particles is wrapped with silk fibroin.

The obtained SF @ CeO was measured using a Nano ZS90 nanometer particle size instrument₂The hydrodynamic size of the NPs was tested and the results are shown in figure 2. As can be seen from FIG. 2, SF @ CeO₂The hydrodynamic size of NPs is around 80 nm.

Test example 1 SF @ CeO₂Drug release rate test of NPs sustained-release preparation

200. mu.L of SF @ CeO obtained in example 1 was aspirated₂Nano-sustained release preparation, put into 10mL protease XIV (Sigma UK). Incubate in a 37 ℃ water bath with fresh enzyme solution every two days. The content of Ce element was measured by ICP (inductively coupled plasma mass spectrometry) at time points 6h, 12h, 24h, 48h, 96h and 196h, and the rate of Ce element release was shown in fig. 3. As can be seen from FIG. 3, the nano sustained release preparation prepared by the present invention has sustained release effect.

Test example 2 weight Change test of mice

The SF @ CeO obtained in example 1 was used₂The NPs nano sustained-release preparation is used for carrying out intraperitoneal administration on YG8R (#012253) of an FRDA transgenic mouse, and the administration mouse is marked as YG8R + NPs; in nanometerThe dosage of the cerium element in the sustained release reagent is 5mg/kg by mass calculation, the cerium element is injected once in 5 days, and the administration period is one month.

The FRDA transgenic mouse YG8R was administered intraperitoneally (dose 5mg/kg, 5 days with one injection, administration period one month) with silk fibroin as a control group, which was designated as YG8R + SF; FRDA transgenic mouse YG8R without drug injection was used as the lesion group; control mouse Y47(#024097) not injected with the drug was used as a normal group for comparison. FRDA transgenic mouse YG8R (#012253) and control mouse Y47(#024097) used during the experiment were purchased from Jackson laboratories.

The weight change profile of the obtained mice is shown in FIG. 4, and it can be seen from FIG. 4 that SF @ CeO provided by the present invention₂NPs do not influence the body weight of mice, which shows that the SF @ CeO provided by the invention₂The nanometer sustained release preparation has in vivo biological safety.

Test example 3 SF @ CeO₂Behavioral scoring of mice after NPs administration

FRDA disease affects both the balance of the mouse and the paw grip.

The behaviours of the mice to be dosed are tested by adopting a wood beam experiment, a rotary fatigue instrument test and a suspension experiment. FRDA transgenic mouse YG8R (#012253) and control mouse Y47(#024097) used during the experiment were purchased from Jackson laboratories.

Nanometer sustained release preparation SF @ CeO adopting abdominal cavity administration method₂NPs were injected into YG8R abdominal cavity of FRDA transgenic mouse (calculated by the mass of cerium element in the nano sustained release reagent, the administration dose was 5mg/kg, the administration period was one month after 5 days of injection), and the comparative test was performed on the NPs in the control group, the injured group and the normal group. The behavioral testing method is as follows:

wooden beam experiment: the wood beam length was 1m, the width was 12mm and 22mm, and the time elapsed from the wood beam was recorded to evaluate the coordination ability. Mice received 3 training sessions and were tested in triplicate with at least 5 minutes between each test.

Testing by a rotary fatigue instrument: a test was performed using a rotary rod fatigue tester (Bi angsico.ltd.) to evaluate motor coordination. At least four experiments were performed with an interval of no less than 200 seconds between each test. The speed of the rotating shaft was gradually increased from 4rpm to 400rpm, and the maximum time was set to 400 seconds. The time the mouse dropped off the spinning wand was recorded.

Suspension experiment: a metal mesh knitted with 1 mm diameter wire was fixed 1.5 m from the ground and a protective article was placed directly under it to prevent the mouse from falling down. The test was started after the mice had gripped the wire mesh and the time the mice fell was recorded. Four tests were run with at least 5 minutes between each test.

The obtained test result graph of the wood beam experiment is shown in fig. 5, the test result graph of the rotary fatigue meter is shown in fig. 6, and the test result graph of the suspension experiment is shown in fig. 7. The data in fig. 5 to 7 are all expressed by Mean SEM, and n is 10.

As can be seen from figures 5 to 7, the nano sustained release preparation SF @ CeO provided by the invention₂NPs can fully restore the behavioural score of diseased mice.

Test example 4 SF @ CeO₂Staining of tissue sections after NPs administration

SF@CeO₂One month after intraperitoneal administration of NPs, mice were vector-sliced throughout the brain and subjected to H&E staining (hematoxylin and eosin dye), purkinje cells (largest neurons in cerebellar cortex) in mice cerebellum were observed, with damaged and normal groups as controls. The result is shown by the arrows in fig. 8. As can be seen in fig. 8, the number and morphology of purkinje cells were significantly restored after administration.

SF@CeO₂After NPs are administrated in the abdominal cavity for one month, the heart of the mouse is sliced, Prussian blue staining is carried out, the staining method refers to the national standard (GB/T17816-1999, 4.10), the injured group and the normal group are used as comparison, the staining result is shown in figure 9, as can be seen from figure 9, the heart iron accumulation phenomenon of the mouse disappears after the administration, and the nano sustained-release preparation SF @ CeO provided by the invention is illustrated₂NPs are capable of exerting an iron redistribution effect in vivo.

Test example 5 SF @ CeO₂ATP levels in mouse tissues following NPs administration

SF@CeO₂ NPs intraperitoneal administration for one month, mice were taken from the lower central system tissues of brain, cerebellum, brainstem, hippocampus and spinal cord, and non-central system tissues of heart, liver, spleen, lung, kidney and skeletal muscle, each tissue was completely lysed with a tissue lysate (Biyunyan P0013), a clear supernatant was taken, and the measurement of ATP content was performed strictly according to the instructions of an ATP detection kit (Sigma company kit), with the injured group and the normal group as a comparison.

The ATP content in each tissue of the mouse is shown in figure 10, and as can be seen from figure 10, the nano sustained release preparation SF @ CeO provided by the invention₂NPs can restore mitochondrial ATP-producing function.

Test example 6 SF @ CeO₂Protein expression levels of mitochondrial complex I/II/III subunits following NPs administration

The protein expression level is detected by a Western blotting method, and the operation steps are as follows: cells were harvested and centrifuged (1000rpm, 5min) to remove supernatant and washed once with PBS. According to the cell amount, adding a proper amount of NP40 cell lysate, resuspending the cells, placing on ice for lysis for 10min, and shaking for 2-3 times in the period. Centrifuge at 15000rpm, 4 ℃ for 10 min. The centrifuged supernatant was transferred to a fresh, precooled Eppendorf (EP) tube and the protein concentration was determined using Bradford's working solution to ensure consistent loading of each sample, and a suitable amount of 5 XSDS-loading buffer was added and heated at 100 ℃ for 5min to fully denature the protein. After the sample was centrifuged for a short time, the sample was applied to SDS-PAGE gel application wells by a microsyringe and electrophoresed at a voltage of 100V. And (3) after the electrophoresis is finished, taking the gel off the device, removing the concentrated gel, and transferring the protein on the gel to an NC membrane by using an electrotransfer under the condition of stable current of 250 mA. After the membrane transfer is finished, the membrane is placed into ponceau staining solution for staining for 2min, the staining solution which is not combined with protein is washed away by acid water with pH of 5.5, and the stained NC membrane is photographed. The membrane was then blocked in Tris-PM containing 5% skimmed milk powder for 1h at room temperature. After blocking, add the appropriate dilution of primary antibody, 4 degrees C were incubated overnight. After the incubation is finished, the primary antibody is recovered, and the membrane is washed by Tris-PM for 10min for 4 times. The membrane was incubated with the appropriate diluted secondary antibody for 1h at room temperature. After incubation, the cells were washed with Tris-PM for 10min 4 times. The protein on the membrane is combined with a chemiluminescence substrate, exposed by a Western blot chemiluminescence imaging system, and photographed.

FIG. 11 shows SF @ CeO₂The protein expression level of mitochondrial complex I/II/III subunit in each tissue of mice after NPs administration is expressed by Mean SEM and n is 3. As can be seen from FIG. 11, the nano sustained release preparation SF @ CeO provided by the invention₂NPs can restore normal expression levels of proteins in the I, II and III subunits of mitochondrial complexes.

Test example 7 SF @ CeO₂Activity of mitochondrial complexes I and II after NPs administration

SF@CeO₂After NPs are administrated in an abdominal cavity for one month, central system tissues such as brain, cerebellum, brainstem, hippocampus and spinal cord, and non-central system tissues such as heart, liver, spleen, lung, kidney and skeletal muscle are taken down, the tissues are completely cracked by using a tissue cracking solution (Biyunyan P0013), clear supernatant is taken, the activity of the complexes I and II is detected by using a mitochondrial complex I activity detection kit (Abcam) and a mitochondrial complex II activity detection kit (Suzhou Keming Biotechnology Co., Ltd.), the activity of the mitochondrial complexes I and II is determined strictly according to kit specifications, and an injury group and a normal group are used as comparison.

SF@CeO₂The activity of mitochondrial complex i in each tissue of mice after NPs administration is shown in fig. 12, which data are expressed as Mean SEM, where n is 3.

SF@CeO₂The activity of mitochondrial complex ii in each tissue of mice after NPs administration is shown in fig. 13, which data are expressed as Mean SEM, where n is 3.

As can be seen from FIGS. 12 and 13, the nano sustained release preparation SF @ CeO provided by the invention₂NPs can restore normal mitochondrial complex I and II activity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A nano sustained release preparation is characterized by comprising dextran-modified cerium oxide nanoparticles and silk fibroin coated on the outer layer of the dextran-modified cerium oxide nanoparticles;

the preparation method of the nano sustained-release preparation comprises the following steps:

the steps (1) and (2) are not limited in chronological order.

2. The nano sustained release preparation according to claim 1, wherein the dextran-modified cerium oxide nanoparticles have a size of 1 to 30 nm; the hydrodynamic size of the nano sustained-release preparation is 80-120 nm.

3. The method for preparing a nano sustained release preparation according to claim 1, comprising the steps of:

the steps (1) and (2) are not limited in chronological order.

4. The method of claim 3, wherein the mass concentration of cerium in the alcoholic dispersion of the dextran-modified cerium oxide nanoparticles is 1-5 mg/mL.

5. The preparation method according to claim 3, wherein the molar concentration of the lithium bromide solution is 8.5-10 mol/L; the mass ratio of the silk fibroin to the lithium bromide solution is 1 g: 1-5 mL.

6. The preparation method according to claim 3, wherein the hydrolysis temperature is 20-70 ℃ and the hydrolysis time is 30-360 min;

the first dialysis bag retains a molecular weight of 12000-14000.

7. The preparation method of claim 3, wherein the mass ratio of cerium element in the dextran-modified cerium oxide nanoparticles to the volume of the silk fibroin solution is 1mg: 1-5 mL;

the power of the ultrasonic wave is 100-300W, and the time is 10-60 min.

8. The preparation method according to claim 3, wherein the freezing temperature is-80 to-20 ℃ and the time is 24 to 48 hours;

the molecular weight intercepted by the second dialysis bag is 5000-10000.

9. Use of the nano sustained release preparation according to claim 1 or 2 or the nano sustained release preparation prepared by the preparation method according to any one of claims 3 to 8 in the preparation of a medicament for treating mitochondrial dysfunction.

10. Use according to claim 9, wherein the mitochondrial dysfunction disease comprises one or more of friedreich's ataxia, alzheimer's disease, parkinson's disease or amyotrophic lateral sclerosis.