Targeted mitochondrial nano hydrogel delivery system as well as preparation method and application thereof

Abstract

The invention discloses a targeted mitochondrial nano-hydrogel delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Potassium ferricyanide and Zn < 2 + > are used as raw materials to prepare Prussian blue-Zn < 2 + >, phenylboronic acid and silk fibroin are used as raw materials to prepare a carrier material boric acid ester-silk fibroin microgel, and the Prussian blue-Zn < 2 + > and a mitochondrial targeting substance are jointly loaded on the carrier material to obtain the mitochondrial targeting and ROS responsive nano hydrogel delivery system. And a drug delivery system with a therapeutic effect is obtained by further loading a drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a mitochondrial targeted nano hydrogel delivery system and a preparation method and application thereof. Background Art

[0002] Sarcopenia is an age-related disease characterized by decreased skeletal muscle mass, decreased muscle strength, and loss of physical motor function, accompanied by serious adverse health consequences such as falls, fractures, increased risk of hospitalization, and high disability rates. Currently, the prevalence of sarcopenia in people aged 60-70 is 5-13%, and the prevalence in people over 80 is 11-50%.

[0003] Intervention for sarcopenia in the elderly is a global public issue. In addition to exercise and nutrition, there is currently a lack of other effective intervention measures. Current studies have found that skeletal muscle homeostasis is maintained by the balance of interactions between myofiber cells and different cell types in their environment, including muscle satellite cells (MuSCs), motor neurons, and various interstitial cells, involving intercellular dialogue, immune niches, metabolic regulation, and protein post-transcriptional modification. Using multi-omics technologies such as proteomics, metabolomics, and transcriptomics to explore key targets in the process of skeletal muscle atrophy will help to study new intervention technologies for delaying skeletal muscle atrophy in the elderly from a new perspective.

[0004] In order to delay skeletal muscle atrophy in the elderly, drug delivery systems with good biocompatibility, sustained release and degradability are prepared, and key drugs are combined to intervene in key targets, which can provide a certain basis for the further development of drugs for the treatment of sarcopenia. Summary of the invention

[0005] The purpose of the present invention is to provide a mitochondrial targeted nano hydrogel delivery system and its preparation method and application. 2+ Preparation of Prussian blue-Zn 2+ Boric acid and silk fibroin were used as raw materials to prepare the carrier material borate@silk fibroin microgel. 2+ The nano-hydrogel delivery system with mitochondrial targeting substances is loaded on the carrier material to obtain a mitochondrial targeting and ROS responsive nano-hydrogel delivery system, which can be further loaded with drugs to obtain a drug delivery system with therapeutic effects.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a method for preparing a mitochondrial targeted nanohydrogel delivery system, comprising the following steps:

[0008] Potassium ferrocyanide and Zn 2+Preparation of Prussian blue-Zn 2+ ; Boric acid (PBA) and silk fibroin (SF) were used as raw materials to prepare the carrier material borate@silk fibroin microgel; the Prussian blue-Zn 2+ The mitochondrial targeting substance elamipine (SS-31) is co-loaded on the carrier material to obtain the mitochondrial targeting nanohydrogel delivery system (PB-Zn 2+ @PBA@SF@SS-31).

[0009] Preferably, the mass ratio of the silk fibroin to the phenylboric acid is 2-10:0.5-2.

[0010] Preferably, the amount of the elamitide added is 1-10% of the mass of the borate@silk fibroin microgel.

[0011] Preferably, the specific steps of the preparation method of the mitochondrial nanohydrogel delivery system include:

[0012] Potassium ferrocyanide and polyvinyl pyrrolidone (PVP) are dissolved in water, and a soluble zinc salt is added to react to obtain Prussian blue-Zn 2+ ;

[0013] The silk fibroin is dissolved in water, phenylboronic acid and an activator are added, the mixture is evenly mixed, the pH is adjusted to 4-6.5 with a MES buffer solution, and the borate@silk fibroin microgel is obtained after stirring;

[0014] The Prussian blue-Zn 2+ The aqueous solution of the borate@silk fibroin microgel is mixed and activated, the pH is adjusted to 5.5-6, and elamitrin is added to obtain the mitochondrial targeted nanohydrogel delivery system after reaction.

[0015] Preferably, the weight average molecular weight of the polyvinyl pyrrolidone is 10,000-40,000.

[0016] Polyvinyl pyrrolidone is used as a stabilizer and dispersant and can be added in conventional amounts.

[0017] Preferably, the soluble zinc salt is zinc acetate.

[0018] More preferably, the mass ratio of the potassium ferrocyanide to the zinc acetate is 20:(1-16).

[0019] Preferably, the activator is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0020] More preferably, the activator DMTMM may be added and dimethyl sulfoxide (DMSO) may be added to help dissolution.

[0021] Preferably, the concentration of the MES buffer solution is 1 M, and the pH is 7.4.

[0022] Preferably, the activation is performed using ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0023] More preferably, the molar ratio of ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride to N-hydroxysuccinimide is 1:5.

[0024] The second technical solution of the present invention is to provide a mitochondrial targeted nanohydrogel delivery system prepared according to the preparation method of the mitochondrial targeted nanohydrogel delivery system.

[0025] The third technical solution of the present invention is to provide a drug-loaded mitochondrial targeted nanohydrogel, which is obtained by mixing the above-mentioned mitochondrial targeted nanohydrogel delivery system with a drug.

[0026] The fourth technical solution of the present invention is to provide a CSN5 inhibitor mitochondrial targeted nanohydrogel, which is obtained by mixing the above-mentioned mitochondrial targeted nanohydrogel delivery system with a CSN5 inhibitor.

[0027] Optionally, the CSN5 inhibitor is CSN5i-3.

[0028] The fifth technical solution of the present invention: provides an application of the above-mentioned mitochondrial targeted nanohydrogel delivery system in the preparation of drugs for the treatment of sarcopenia.

[0029] Technical solution six of the present invention: Provides an application of the above-mentioned CSN5 inhibitor mitochondrial targeted nanohydrogel in the preparation of a drug for the treatment of sarcopenia.

[0030] The beneficial technical effects of the present invention are as follows:

[0031] The present invention uses potassium ferrocyanide and Zn 2+ Preparation of Prussian blue-Zn 2+ Boric acid and silk fibroin were used as raw materials to prepare the carrier material borate@silk fibroin microgel. 2+ The nano hydrogel delivery system with mitochondrial targeting substances is loaded on the carrier material to obtain a mitochondrial targeting and ROS responsive nano hydrogel delivery system. By further loading drugs, a drug delivery system with therapeutic effects is obtained. For example, if the loaded CSN5 inhibitor is CSN5i-3, Zn 2+ Drugs for the treatment of sarcopenia in combination with CSN5i-3 dual drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 PB-Zn in Example 1 2+TEM image (a), SEM image (b) of PBA@SF, and dried PB-Zn 2+ SEM image of @PBA@SF@CSN5i-3@SS-31 (c), PB-Zn 2+ Particle size distribution diagram (d), PB-Zn 2+ Particle size distribution diagram of @PBA@SF@CSN5i-3@SS-31 (e), PB-Zn 2+ PB-Zn 2+ @PBA@SF and PB-Zn 2+ Zeta potential diagram of @PBA@SF@CSN5i-3@SS-31 (f), PB-Zn 2+ Particle size change of @PBA@SF@CSN5i-3@SS-31 in PBS buffer within 56 days (g), PB-Zn 2+ , PBA@SF and PB-Zn 2+ FT-IR spectrum of @PBA@SF@CSN5i-3@SS-31 (h) and picture of PBA@SF prepared with different SF:PBA mass ratios in step (2) of Example 1 before freeze-drying (i).

[0033] Figure 2 PB-Zn in Example 1 2+ Drug release curves of CSN5i-3 (a) and Zn@PBA@SF@CSN5i-3@SS-31 2+ Release curve of (b).

[0034] Figure 3 (a) is PB-Zn in Example 1 2+ @PBA@SF@CSN5i-3@SS-31 and PB-Zn in Example 2 2+ Uptake behavior of @PBA@SF@CSN5i-3 in C2C12 cells after co-incubation for 6 h; (b) PBS, PBA@SF in Example 1, CSN5i-3, PB-Zn in Example 3 2+ @PBA@SF@SS-31, PB-Zn in Example 2 2+ @PBA@SF@CSN5i-3 and PB-Zn in Example 1 2 + @PBA@SF@CSN5i-3 affects the activity of C2C12 cells (left) and atrophic myotube cells (right); (c) PBS, PBA@SF in Example 1, CSN5i-3, PB-Zn in Example 3 2+ @PBA@SF@SS-31, PB-Zn in Example 2 2+@PBA@SF@CSN5i-3 and PB-Zn in Example 1 2+ Quantitative analysis results of reactive oxygen species determined by flow cytometry after DEX-induced myotube atrophy was treated with @PBA@SF@CSN5i-3@SS-31. (d) Western blot analysis of PBS, PBA@SF in Example 1, CSN5i-3, PB-Zn in Example 3 2+ @PBA@SF@SS-31, PB-Zn in Example 2 2+ The apoptosis of atrophic myotube cells was determined by flow cytometry after the atrophic myotube cells were treated with @PBA@SF@SS-31 and PB-Zn2+@PBA@SF@CSN5i-3@SS-31 in Example 1.

[0035] Figure 4 PBS, PBA@SF in Example 1, CSN5i-3, PB-Zn in Example 3 2+ @PBA@SF@SS-31, PB-Zn in Example 2 2+ @PBA@SF@CSN5i-3 and PB-Zn in Example 1 2+ @PBA@SF@CSN5i-3@SS-31 affects the expression of Cul 3, MuRF-1, Atrogin, Parkin and PINK1 in atrophic myotubes.

[0036] Figure 5 PBS, PBA@SF in Example 1, CSN5i-3 and PB-Zn in Example 1 2+ @PBA@SF@CSN5i-3@SS-31The movement distance, resting time and average speed of the forced swimming test on 18-month-old mice were plotted, with young mice as negative controls.

[0037] Figure 6 (a) to (e) are PBS, PBA@SF in Example 1, CSN5i-3 and PB-Zn in Example 1 2+ @PBA@SF@CSN5i-3@SS-31 affects the expression of Cul 3, MuRF-1, Atrogin, Parkin and PINK1 in the muscle tissue of 18-month-old mice, with young mice as negative controls; (f) H&E staining results of the muscle tissue of mice in each treatment group in the forced swimming experiment, with young mice as negative controls. DETAILED DESCRIPTION

[0038] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0039] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] (1) Prussian Blue-Zn 2+ (PB-Zn 2+ )

[0044] To a 1M hydrochloric acid solution (20 mL) was added potassium ferrocyanide (K) 3 [Fe(CN) 6 ](0.1mmol, 32.924mg), polyvinylpyrrolidone PVP (MW: 10000, 0.3g), stirred for 1h (speed 640rpm). Then 13.208mg of zinc acetate was added to the above mixed solution, and the reactants were kept stirred for 1 hour (speed 640rpm). The resulting solution was placed in an oven at 80℃ for 3h to obtain Prussian blue-Zn 2+ .

[0045] (2) Preparation of borate@silk fibroin (PBA@SF)

[0046] First, 125 mg of silk fibroin (SF) was dissolved in 15 mL of deionized water under stirring. 21 mg of phenylboronic acid (PBA), DMSO (600 μL) and DMTMM (182.2 mg) were added to the SF solution respectively. After sufficient dissolution, the pH of the solution was adjusted to 6.5 using MES buffer (1 M). The reaction mixture was then stirred at room temperature for 3 days. The final mixture was purified by dialyzing deionized water (molecular weight cutoff of 10 kDa) at room temperature for 2 days to remove DMSO, and the water was changed twice a day. The dialyzed solution was freeze-dried using a desktop freeze dryer (Labconco) to obtain PBA@SF microgels.

[0047] (3)PB-Zn 2+ Preparation of @PBA@SF@CSN5i-3@SS-31

[0048] To prepare a hydrogel with targeted effect, a certain amount of 7.5 mg (346.7 μL) of PB-Zn 2+ 1.45 mL (100 mg / mL) of PBA@SF hydrogel was added, followed by 13 mg of EDC and 39 mg of NHS (the molar ratio of EDC to NHS was 1:5), and sodium hydroxide solution was added dropwise to adjust the solution pH to 5.5-6, and then the carboxyl group was activated for 2 h (600 rpm), and then a certain amount of SS-31 (2 mg) was added to react at room temperature for 5 h, and dialyzed for 4 h at 10 kDa (dialysate was 50 mL of water). After the dialysis, CSN5i-3 was added and vortexed quickly, and then dialyzed for 4 h (molecular weight cutoff was 10 kDa) to remove excess impurities (EDC, NHS and uncoupled SS-31), and finally the hydrogel sample was obtained and stored in a refrigerator at 4 °C.

[0049] (4) Cy5-labeled nanoparticles

[0050] MES buffer (pH 7.4) was adjusted to pH 5.5 with HCl solution, 2.8 mg of sulfonyl Cy5 carboxylic acid (Cy5) was added to 200 μL of MES buffer (pH 5.5), and EDC (3.64 mg) and NHS (3.64 mg) were added, and the mixture was stirred in an ice bath for 30 min. 2+ 500 μL of @PBA@SF@CSN5i-3@SS-31 solution was added to 71.4 μL of the above solution, vortexed for 60 s, incubated at 2-8 °C for 24 h, and finally dialyzed for 75 min (3500 kDa, dialysate was 50 mL of aqueous solution with pH 7.4).

[0051] (6) CSN5i-3 drug release experiment

[0052] The hydrogel was concentrated to increase the concentration of CSN5i-3. 1 mL of the hydrogel was placed in a 3500 Da dialysis bag and the dialysate was PBS, H 2 O 2 20 mL of PBS (pH = 7.4) buffer (1 mM) was added, and 1 mL of the dialyzed solution was taken into a centrifuge tube every 0.5, 1, 2, 4, 8, 24, 48, and 72 hours, and then the dialyzed solution was replenished with an equal volume of solution. The dialyzed solution was used as a control group with 30 mL of PBS (pH = 7.4) buffer, and the steps were the same as above.

[0053] Example 2

[0054] (1) Prussian Blue-Zn 2+ (PB-Zn 2+ )

[0055] To a 1M hydrochloric acid solution (20 mL) was added potassium ferrocyanide (K) 3 [Fe(CN) 6 ](0.1mmol, 32.924mg), polyvinylpyrrolidone PVP (MW: 10000, 0.3g), stirred for 1h (speed 640rpm). Then 13.208mg of zinc acetate was added to the above mixed solution, and the reactants were kept stirred for 1 hour (speed 640rpm). The resulting solution was placed in an oven at 80℃ for 3h to obtain Prussian blue-Zn 2+ .

[0056] (2) Preparation of borate@silk fibroin (PBA@SF)

[0057] First, 125 mg of silk fibroin (SF) was dissolved in 15 mL of deionized water under stirring. 21 mg of phenylboronic acid (PBA complex), DMSO (600 μL) and DMTMM (182.2 mg) were added to the SF solution respectively. After sufficient dissolution, the pH of the solution was adjusted to 6.5 using MES buffer (1 M). The reaction mixture was then stirred at room temperature for 3 days. The final mixture was purified by dialyzing deionized water (molecular weight cutoff of 10 kDa) at room temperature for 2 days to remove DMSO, and the water was changed twice a day. The dialyzed solution was freeze-dried using a desktop freeze dryer (Labconco) to obtain PBA@SF microgels.

[0058] (3)PB-Zn 2+ Preparation of @PBA@SF@CSN5i-3

[0059] To prepare a hydrogel with targeted effect, a certain amount of 7.5 mg (346.7 μL) of PB-Zn 2+The above-mentioned PBA@SF (2 mL, 73 mg / mL) hydrogel was added and stirred for 2 h (rotation speed 600 rpm), and then CSN5i-3 (60 μM, 71.28 μg) was added and vortexed quickly for 60 s, and then dialyzed for 2 h (molecular weight cutoff value was 10 kDa, dialysate was 50 mL aqueous solution) to remove excess impurities, and finally stored in a refrigerator at 4 °C.

[0060] (4) Cy5-labeled nanoparticles

[0061] MES buffer (pH 7.4) was adjusted to pH 5.5 with HCl solution, 2.8 mg of sulfonyl Cy5 carboxylic acid (Cy5) was added to 200 μL of MES buffer (pH 5.5), and EDC (3.64 mg) and NHS (3.64 mg) were added, and the mixture was stirred in an ice bath for 30 min. 2+ 200 μL of @PBA@SF@CSN5i-3 solution was added to 28.6 μL of the above solution, vortexed for 60 s, incubated at 2-8 °C for 24 h, and finally dialyzed for 75 min (molecular weight cutoff value was 3500 kDa, and the dialysate was 50 mL of an aqueous solution with a pH of 7.4).

[0062] Example 3

[0063] The steps are the same as those in Example 1, except that in step (3), CSN5i-3 is not added to the solution to obtain PB-Zn 2+ @PBA@SF@SS-31.

[0064] Effect verification

[0065] 1.TEM and SEM observation

[0066] The PB-Zn prepared in Example 1 2+ Transmission electron microscopy observations showed that Figure 1 (a) - scale bar 200 nm, from which the obtained carrier PB-Zn can be clearly seen 2+ It has a cubic structure and a particle size of about 80nm.

[0067] Figure 1 (b) - scale bar 1 μm is the SEM spectrum of the cross-linked hydrogel (PBA@SF) formed by mixing phenylboronic acid and silk fibroin in Example 1. It can be seen from the figure that PBA@SF has a certain spatial network structure. 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel, its SEM morphology in dry state Figure 1 (c)-scale bar 1μm is shown, Figure 1(i) is a picture of PBA@SF prepared with different mass ratios of SF and PBA in step (2) of Example 1 before freeze-drying, wherein the mass ratios of SF and PBA are 2:1, 5:1 and 15:1, respectively. The figure shows that hydrogels can be prepared with different mass ratios of SF and PBA.

[0068] 2. Particle size distribution and Zeta potential

[0069] The particle size of the nanoparticles was measured using dynamic light scattering (DLS) to evaluate the size and stability of the nanoparticles in Example 1. The results are shown in the figure. Figure 1 Middle (d) shows PB-Zn 2+ The particle size of is 86.65nm and its PI is 0.232, indicating that it has good dispersibility; Figure 1 (e) is PB-Zn 2+ The particle size of @PBA@SF@CSN5i-3@SS-31 indicates that the nanohydrogel has increased in size after modification. Figure 1 (g) The particle size change of the nanohydrogel in PBS within 56 days was measured, which further demonstrated the stability of the hydrogel.

[0070] Figure 1 (f) is the surface charge of the nano-hydrogel before and after drug loading and targeting modification. 2+ PB-Zn 2+ @PBA@SF (sample before compounding with SS-31) and PB-Zn 2+ The Zeta potentials of @PBA@SF@CSN5i-3@SS-31 are -9.66, +4.28, and -18.92 mV, respectively. The larger potential indicates that PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel has high stability in aqueous solution.

[0071] 3. FT-IR detection

[0072] Figure 1 (h) is PB-Zn in Example 1 2+ The structural changes of @PBA@SF@CSN5i-3@SS-31 nanohydrogel before and after compounding are shown in the figure. 2+ Zn in the structure 2+ and successful cross-linking of hydrogels.

[0073] 4. Drug Release Experiment

[0074] Figure 2 (a) is PB-Zn in Example 1 2+Drug release curve of CSN5i-3 in @PBA@SF@CSN5i-3@SS-31 nanohydrogel. In PBS, the release rate of CSN5i-3 is about 40%; in PBS+H 2 O 2 Under the microenvironment, the release rate of CSN5i-3 reached nearly 95% in 72h. 2+ The release of Figure 2 (b)). This result shows that the nano-hydrogel has good ROS response performance.

[0075] 5. Animal Experiments

[0076] Fifteen C57BL / 6J male aged mice (18-24 months old) and five young mice (6-8 weeks old) were purchased from Phinok Biotech (Shanghai) Co., Ltd. All mice were approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine, and all mice were adaptively fed for 7 days. Young mice were used as the positive control group. For the treatment group, each drug group was injected intramuscularly into two sites of the unilateral quadriceps muscle. The injection was given intramuscularly once every 2 days, with an injection volume of 50 μL (for CSN5i-3 and / or Zn 2+ The concentration of CSN5i-3 in the sample was about 15 μg / mL, and Zn 2+ The concentration of the PBA@SF sample was about 20 μg / mL; the concentration of the PBA@SF sample was 100 mg / mL) for 28 days, and then the C57 / 6J mice were killed for subsequent experiments. The control group was intramuscularly injected with the same 50 μL of normal saline in a similar part of the unilateral quadriceps muscle.

[0077] 6. Cellular uptake effect

[0078] To evaluate the mitochondrial targeting ability of the hydrogel, Cy5-labeled PB-Zn 2+ @PBA@SF@CSN5i-3 (Example 2) and PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 (Example 1) was used as the research object, and the method was as follows: cells in the logarithmic growth phase and in good growth state were taken at 1.5×10 5 12-well plate, inoculated with 100 μL PB-Zn 2+ @PBA@SF@CSN5i-3 and PB-Zn 2+@PBA@SF@CSN5i-3@SS-31 was treated for 4 hours. The Mito-Tracker Green staining working solution was prepared according to the manufacturer's instructions (1mM Mito-Tracker Green stock solution was added to the cell culture medium (100nM), and then an appropriate amount of Hoechst33342 was added to make the final concentration 1μg / mL). The culture medium was removed and Mito-Tracker Green was incubated with the cells at 37°C for 20 minutes. The staining solution was removed, fresh cell culture medium was added, and photos were taken under a laser confocal microscope.

[0079] Figure 3 (a) - scale bar 50 μm shows the in vitro uptake effect of C2C12. As can be seen from the figure, the cells are very sensitive to Cy5-labeled PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 has a better uptake effect, and the red fluorescence and green fluorescence are highly overlapped, while Cy5-labeled PB-Zn 2+ @PBA@SF@CSN5i-3 nanohydrogels are mostly free outside the mitochondria (most of the fluorescence does not overlap), indicating that the modification of SS-31 significantly improved its in vitro targeting performance.

[0080] 7. Cell Viability

[0081] In vitro myotube atrophy model: C2C12 cells in the logarithmic growth phase and in good growth condition were taken and 2×10 5 Cells were inoculated into 6-well plates and incubated at 37°C with 5% CO 2 The cells were cultured overnight in an incubator; after the cells adhered, DMEM medium containing 2% horse serum was used to continue culturing the cells until they formed myotubes, and then treated with differentiation medium containing 50 μM DEX for 24 h. The drug stock solution was diluted 15 times and 200 μL was added to each of the above groups for 24 h to induce myotube atrophy. Subsequently, the cells were treated according to the following groups: PBS (I), PBA@SF in Example 1 (II), CSN5i-3 (III), PB-Zn in Example 3 2+ @PBA@SF@SS-31(IV), PB-Zn in Example 2 2+ @PBA@SF@CSN5i-3(V), PB-Zn in Example 1 2+ @PBA@SF@CSN5i-3@SS-31(VI). Finally, the absorbance value at OD 450nm in each well was measured by an ELISA reader, and the percentage of live cells was plotted using GraphPadPrism software.

[0082] Figure 3 (b) is the same concentration (CSN5i-3: 15 μg / mL; Zn 2+:20μg / mL, when CSN5i-3 and Zn 2+ The effects of different nanomedicines on the activity of C2C12 cells (left) and atrophic myotube cells (right) at the same concentration of CSN5i-3. The results show the safety of the hydrogel. At the same concentration, PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel effectively improved its cell activity.

[0083] 8. Quantitative Analysis by Flow Cytometry

[0084] Figure 3 (c) shows the quantitative analysis results of reactive oxygen species measured by flow cytometry after DEX-induced myotube atrophy and treatment with different drugs. 2+ The concentration of CSN5i-3 in the sample was about 15 μg / mL, and Zn 2+ The concentration of the PBA@SF sample was about 20 μg / mL; the concentration of the PBA@SF sample was 100 mg / mL) and the different nanoparticles were co-incubated for 6 h, followed by trypsin digestion, centrifugation at 1200 rpm for 5 min, and the precipitate was resuspended in PBS according to the instructions of the DCFH-DAROS detection kit. After washing the cells three times, the cells were resuspended in 200 μL PBS, and finally the changes in the concentration of intracellular ROS were measured by flow cytometry. Figure 3 In (c), we can see that PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel has a good ability to reverse apoptosis and can improve muscle atrophy symptoms.

[0085] 9. Western blot detection

[0086] Western blot detection is a method of transferring proteins to a membrane and then using antibodies for detection. The ultimate result of a gene expression is the production of the corresponding protein (or enzyme). Western Blot uses polyacrylamide gel electrophoresis, the object to be detected is protein, the "probe" is the antibody, and the "color development" uses a labeled secondary antibody.

[0087] Figure 3 (d) The same concentration (for CSN5i-3 and / or Zn 2+ The concentration of CSN5i-3 in the sample was about 15 μg / mL, and Zn 2+ The concentration of the PBA@SF sample was about 20 μg / mL; the concentration of the PBA@SF sample was 100 mg / mL) The apoptosis of atrophic myotubes was determined by flow cytometry after treatment with different drugs.2+ The apoptosis in the @PBA@SF@CSN5i-3@SS-31 nanohydrogel group was significantly reduced to 3.51%.

[0088] 10.qPCR detection

[0089] Different nanomedicines were used to stimulate atrophic myotubes, and the expression of ubiquitination and autophagy-related genes was measured by qPCR (e.g. Figure 4 Compared with the PBS group, the ubiquitin ligase Cul 3, Atrogin and MuRF-1 genes in other groups were decreased to varying degrees, and their corresponding Parkin and PINK1 were upregulated to varying degrees. This may be because the drug and nano-hydrogel regulate the activity of ubiquitin ligase, thereby effectively promoting mitochondrial autophagy in atrophic myotube cells and improving apoptosis.

[0090] After the mice were treated with each drug group (for methods, see 5. Animal experiments), they were euthanized, and the hind leg muscle tissue was obtained and ground, lysed, centrifuged and the supernatant discarded. According to the manufacturer's instructions, the expression of Cul 3, Atrogin, MuRF-1, PINK1 and Parkin was determined by qPCR.

[0091] Figure 6 (ae) is the relationship between ubiquitination and improved autophagy after nanohydrogel treatment of mice. The expression of genes in tissues was observed by immunohistochemistry. Obviously, PB-Zn 2+ After 28 days of treatment with @PBA@SF@CSN5i-3@SS-31 nanohydrogel, its ubiquitin ligase Cul3 was significantly inhibited, indicating that the drug and nanohydrogel may effectively enhance mitochondrial autophagy by regulating the activity of ubiquitin ligase.

[0092] 11. Forced swimming test

[0093] The experimental method was to divide 18-month-old mice into 4 groups. C57BL / 6 male aged mice were injected with PBS (Control), CSN5i-3, PBA@SF and PB-Zn every two days. 2+@PBA@SF@CSN5i-3@SS-31 nano drug (see 5. Animal experiment for injection volume), and 8-week-old C57BL / 6 male mice were injected with an equal volume of PBS as a negative control (Young). After the drug was treated for 14 days and 28 days, the mice were placed in a swimming bucket with a water depth of about 15 cm and a water temperature of 23-25°C. The suspended limbs could not touch the bottom of the bucket. The mice were taken out after 15 minutes to adapt to the environment. The next day, the same conditions were used to force swimming, the swimming time was 6 minutes, and the recording started after 2 minutes. The movement trajectory of the mice was recorded for 4 minutes using the forced swimming experiment video analysis system (Beijing Zhongshi, ZS-QPT).

[0094] Figure 5 The figure shows the movement distance, resting time and average speed of mice in the forced swimming test. 2+ The movement speed and total distance of the @PBA@SF@CSN5i-3@SS-31 group increased significantly, and the total distance and average speed were even comparable to those of young mice. The above results show that PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel has good therapeutic effect.

[0095] The muscle tissue of mice in each treatment group in the forced swimming test was analyzed, and the results are shown in Figure 6 (f) - scale bar 50 μm. The H&E staining results in the figure showed that the tissue was uneven, the nuclei were severely aggregated, and there were large gaps between the muscle fibers. Compared with the Control group, PB-Zn 2+ The muscle fibers in the @PBA@SF@CSN5i-3@SS-31 group were tightly distributed and had smooth tissue, indicating that PB-Zn 2+ @PBA@SF@CSN5i-3@SS-31 nanohydrogel can effectively reverse muscle atrophy in the elderly.

[0096] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a mitochondrial targeted nanohydrogel delivery system, characterized in that: The following steps are involved: Potassium ferrocyanide and Zn 2+ Preparation of Prussian blue-Zn 2+ ; Boric acid and silk fibroin were used as raw materials to prepare the carrier material borate@silk fibroin microgel; the Prussian blue-Zn 2+ The mitochondrial targeting substance eramitriptyline is loaded on the carrier material to obtain the mitochondrial targeting nano hydrogel delivery system.

2. The method for preparing the mitochondrial targeted nanohydrogel delivery system according to claim 1, characterized in that: The mass ratio of the silk fibroin to the phenylboronic acid is 2-10:0.5-2; and / or, the amount of the elamitide added is 1-10% of the mass of the borate@silk fibroin microgel.

3. The method for preparing the mitochondrial targeted nanohydrogel delivery system according to claim 1, characterized in that: The specific steps of the preparation method of the mitochondrial nanohydrogel delivery system include: Potassium ferrocyanide and polyvinyl pyrrolidone are dissolved in water, and soluble zinc salt is added to obtain Prussian blue-Zn after reaction. 2+ ; The silk fibroin is dissolved in water, phenylboronic acid and an activator are added, the mixture is evenly mixed, the pH is adjusted to 4-6.5 with a MES buffer solution, and the borate@silk fibroin microgel is obtained after stirring; The Prussian blue-Zn 2+ The aqueous solution of the borate@silk fibroin microgel is mixed and activated, the pH is adjusted to 5.5-6, and ilamitriptyline is added to obtain the mitochondrial nanohydrogel delivery system after reaction.

4. The method for preparing the mitochondrial targeted nanohydrogel delivery system according to claim 3, characterized in that: The activating agent is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

5. The method for preparing the mitochondrial targeted nanohydrogel delivery system according to claim 3, characterized in that: The activation is performed by adding ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide for activation.

6. A mitochondrial targeted nanohydrogel delivery system prepared according to the preparation method of the mitochondrial targeted nanohydrogel delivery system according to any one of claims 1 to 5.

7. A drug-loaded mitochondrial-targeted nanohydrogel, characterized in that: The method is obtained by mixing the mitochondrial targeted nanohydrogel delivery system according to claim 6 with a drug.

8. A CSN5 inhibitor mitochondrial targeted nanohydrogel, characterized in that: The mitochondrial targeted nanohydrogel delivery system according to claim 6 is mixed with a CSN5 inhibitor.

9. Use of the mitochondrial targeted nanohydrogel delivery system according to claim 6 in the preparation of a drug for treating sarcopenia.

10. Use of the CSN5 inhibitor mitochondrial-targeted nanohydrogel according to claim 8 in the preparation of a drug for treating sarcopenia.

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