Traceable mitochondrial targeting nano system as well as preparation method and application thereof

Abstract

The invention provides a traceable mitochondrial targeting nano system as well as a preparation method and application thereof, and belongs to the field of medicines. The traceable mitochondrial targeting nano system constructed by the invention can continuously release resveratrol and maintain the photochemical stability of resveratrol. The nano system shows efficient cellular uptake and accurate mitochondrial targeting ability, and promotes removal of mitochondrial ROS and adjustment of mitochondrial autophagy, so that vascular leakage and angiogenesis are relieved, in-vivo microvascular hyperplasia and retina degeneration are remarkably inhibited, and the curative effect is improved. The distribution of the compound in fundus can be monitored in real time through clinical ophthalmological diagnosis equipment, and the compound has a good application prospect in preparation of drugs for preventing and / or treating diabetic retinopathy.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a traceable mitochondrial targeted nanosystem and a preparation method and use thereof. Background Art

[0002] Diabetic retinopathy (DR) is a widespread microvascular complication that affects approximately 22.27% of diabetic patients worldwide. In the early stages of DR, patients often develop microaneurysms, intraretinal hemorrhages, and retinal edema; in the late stages, vitreous hemorrhages, visual field defects, and even blindness may occur. DR has become one of the leading causes of blindness in adults. Although DR imposes a huge burden on diabetic patients, clinically effective treatment strategies are still limited. Common treatments include laser photocoagulation, anti-vascular endothelial growth factor (anti-VEGF) drugs, and steroid drugs. However, laser photocoagulation may cause permanent retinal damage and may induce central vision loss. Anti-VEGF drugs may impair the normal physiological function of VEGF due to their lack of selectivity. In addition, long-term use of steroid drugs may increase the risk of ocular inflammation and infection. Therefore, it is necessary to develop safe and targeted treatment strategies that can minimize side effects and improve treatment efficacy.

[0003] DR is an ophthalmic disease associated with oxidative stress, which is often characterized by excessive production of reactive oxygen species (ROS). Mitochondria are the main organelles that produce ROS in cells. Studies have shown that mitochondrial-targeted therapies can effectively alleviate the in vitro symptoms of DR by regulating oxidative stress. However, it is worth noting that excessive ROS, which is unique to oxidative stress, can disrupt the balance of mitochondrial autophagy. When damaged mitochondria cannot be effectively cleared, it not only impairs the energy supply of the cell, but also continues to produce ROS, thereby exacerbating oxidative stress. Maintaining a healthy mitochondrial population is essential for maintaining redox homeostasis.

[0004] Therefore, there is an urgent need to develop a mitochondrial-targeted delivery system that can effectively treat diabetic retinopathy and maintain a healthy mitochondrial population. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a traceable mitochondrial targeted nanosystem and a preparation method and use thereof.

[0006] The present invention provides a mitochondrial targeting nanosystem, which is prepared by using lipophilic cation-modified tetrahedral framework nucleic acid, mitochondrial regulator and polyethylene glycol-modified protein as raw materials, wherein the molar ratio of the lipophilic cation-modified tetrahedral framework nucleic acid to the mitochondrial regulator is 1:300-400, and the molar ratio of nitrogen in the polyethylene glycol-modified protein to phosphorus in the mitochondrial regulator is 1-3:1.

[0007] Furthermore, the molar ratio of the lipophilic cationic modified tetrahedral framework nucleic acid to the mitochondrial regulator is 1:350, and the molar ratio of nitrogen in the polyethylene glycol-modified protein to phosphorus in the mitochondrial regulator is 2:1.

[0008] Furthermore, the lipophilic cation is a lipophilic cation with fluorescent properties, and the lipophilic cation-modified tetrahedral framework nucleic acid is a product prepared using the lipophilic cation with fluorescent properties and the tetrahedral framework nucleic acid as raw materials;

[0009] The polyethylene glycol-modified protein is PEGylated protamine.

[0010] Furthermore, the preparation method of the tetrahedral framework nucleic acid modified with a lipophilic cation comprises the following steps: (1) reacting a lipophilic cation with a single-stranded S1 to obtain a single-stranded S1-Rh; (2) self-assembling single-stranded S1-Rh, S2, S3 and S4 to obtain a tetrahedral framework nucleic acid modified with a lipophilic cation; the sequences of the single-stranded S1, S2, S3 and S4 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively, the mass ratio of the lipophilic cation to the single-stranded S1 is 1:3-6, preferably 1:4.6; the molar ratio of the single-stranded S1-Rh, S2, S3 and S4 is 1:0.5-2, preferably 1:1.

[0011] Furthermore, the lipophilic cation is rhodamine 19; and the mitochondrial regulator is resveratrol.

[0012] The present invention also provides a method for preparing the above-mentioned mitochondrial targeting nanosystem, the method comprising the following steps: the method comprising the following steps: mixing a lipophilic cation-modified tetrahedral framework nucleic acid, a mitochondrial regulator and a polyethylene glycol-modified protein and reacting them to obtain the mitochondrial targeting nanosystem.

[0013] Furthermore, the reaction temperature is 0-10°C, and the reaction time is 3-10 hours; preferably, the reaction temperature is 4°C, and the reaction time is 6.5 hours.

[0014] The present invention also provides use of the mitochondrial targeting nanosystem in preparing drugs for preventing and / or treating ophthalmic diseases.

[0015] Furthermore, the ophthalmic disease is diabetic retinopathy.

[0016] Furthermore, the drug is a drug that inhibits microvascular proliferation and retinal degeneration.

[0017] The experimental results show that the traceable mitochondrial targeted nanosystem constructed by the present invention can continuously release resveratrol and maintain its photochemical stability. The nanosystem exhibits efficient cellular uptake and precise mitochondrial targeting capabilities, promotes the removal of mitochondrial ROS and the regulation of mitochondrial autophagy, thereby reducing vascular leakage and angiogenesis, significantly inhibiting microvascular proliferation and retinal degeneration in vivo, and can be monitored in real time by clinical ophthalmic diagnostic equipment in the fundus, and has good application prospects in the preparation of drugs for the prevention and / or treatment of diabetic retinopathy.

[0018] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0019] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The CCK8 method was used to detect the cell viability under different concentrations of glucose and H2O2. The data are presented as mean ± SD (n = 5) *p < 0.05, **p < 0.01 indicates that there is a statistically significant difference between the marker groups.

[0021] Figure 2 This is to screen the image with the best ratio when TRh and RSV reach saturated drug loading sites.

[0022] Figure 3 The CCK8 assay was used to detect the cell viability at different PP / TRh RSV ratios. Data are presented as mean ± SD (n = 5) *p < 0.05, ***p < 0.001, ****p < 0.0001 indicate statistically significant differences between the marker groups.

[0023] Figure 4 The results are from 8% polyacrylamide gel electrophoresis.

[0024] Figure 5 The results are obtained by capillary gel electrophoresis.

[0025] Figure 6 This is the detection result of UV absorbance method.

[0026] Figure 7 The results of particle size (A) and charge (B) were measured by Zeta potential and particle size.

[0027] Figure 8 The results are shown in Figure 5. These are the results of HPLC testing of the stability of RSV and PP-TRh-RSV under light irradiation.

[0028] Fig. 9 The morphology of PP and PP-TRh-RSV, as well as the stability of the morphology over time, were observed by TEM.

[0029] Fig.10 The results of the cell entry ability test of PP-TRh-RSV are as follows: (A) represents the immunofluorescence results; (B) represents the immunofluorescence statistics.

[0030] Fig.11 These are the test results for the mitochondrial targeting ability of PP-TRh-RSV.

[0031] Fig.12 The results of the lysosomal escape ability test of PP-TRh-RSV.

[0032] Fig.13 These are the results of the cell viability test of PP-TRh-RSV under oxidative stress.

[0033] Fig.14 These are the results of the Transwell experiment of PP-TRh-RSV under oxidative stress.

[0034] Fig.15 These are the qPCR experimental results of PP-TRh-RSV under oxidative stress environment.

[0035] Fig.16 These are the results of angiogenesis detection of PP-TRh-RSV under oxidative stress environment.

[0036] Fig.17 The results of the distribution test of PP-TRh-RSV in the fundus and the test of delayed material metabolism are shown.

[0037] Fig.18 The imaging results of PP-TRh-RSV in retinal vessels.

[0038] Fig.19 This is the OCT detection result of PP-TRh-RSV in the retina.

[0039] Fig. 20 The results of H&E staining of PP-TRh-RSV in the retina.

[0040] Fig.21 The results of PAS staining of the retinal vascular network. DETAILED DESCRIPTION

[0041] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0042] Rhodamine 19 was purchased from Roen Biotechnology, specification / model R089688;

[0043] The "room temperature" referred to in the present invention is 25±10°C, and "overnight" is 12±5h.

[0044] Example 1: Synthesis of PP-TRh-RSV

[0045] 1. Synthesis of S1-Rh

[0046] 1.7 mg of rhodamine 19 was dissolved in dimethyl sulfoxide (DMSO), and 7.6 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 2.6 mg of N,N-diisopropylethylamine (DIPEA) were added and shaken at room temperature for 30 minutes; then (7.8 mg) of S1 single chain (see Table 1) dissolved in PBS buffer (pH = 8.5) was added and shaken at room temperature for 1 hour to ensure sufficient reaction (confirmed by LC-MS). Then, 5 mol·L-1 NaCl aqueous solution and anhydrous ethanol were added, vortexed and mixed, stored at -20°C for 20 minutes, and centrifuged at 12k rpm for 15 minutes. Then the ethanol was discarded and the solvent was evaporated. Finally, the residue was dissolved in sterile RNase-free water, and the target product was purified by preparative high performance liquid chromatography to obtain S1-Rh (its sequence number was named: SEQ ID NO.5).

[0047] 2. Synthesis of TRh

[0048] DNA chains (S1-Rh, S2, S3 and S4, S1-S4 see Table 1) were mixed in an equimolar ratio (1 mmol L-1) and dissolved in TM buffer, and a complex of tetrahedral framework nucleic acid and rhodamine 19 (i.e., tFNAs-Rhodamine 19, abbreviated as TRh) was synthesized through a DNA self-assembly process. The specific steps were: heating at 95°C for 10 minutes and then cooling at 4°C for 20 minutes.

[0049] Table 1 ssDNA sequences

[0050]

[0051] 3. Synthesis of TRh-RSV

[0052] Subsequently, TRh prepared in Example 1 was mixed with resveratrol (RSV) at a molar ratio of 1:350 in PBS and stirred at 4°C for 6 hours to obtain TRh-RSV (containing 228 nmol·L -1 TRh, 80 μmol·L -1 RSV). Unreacted single-stranded DNA and RSV were removed by ultrafiltration using a 30 kDa molecular weight filter (Millipore, USA).

[0053] 4. Synthesis of PEGylated protamine (PP)

[0054] 1) Preparation of PEG, SE: Take 0.5g PEG (MW≈2000; i.e. 0.25mmol), add it to 20mL anhydrous DMSO solution, and then add 1mL triethylamine (CAS No.: 121-44-8). Under nitrogen protection, slowly heat up to 50℃, continue to stir fully until completely dissolved, wait for the PEG solution to cool to room temperature, add 180mg 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) (MW=300; i.e. 0.6mmol), heat up to 35℃, continue to stir fully, and react overnight. After stopping stirring, add ether to precipitate a white solid. Further wash with ether to form a white powder, filter with a microtube, and vacuum dry the precipitate to obtain 610mg PEG, SE white powder.

[0055] 2) Prepare protamine solution: Take 0.5 g of protamine, add 20 mL of anhydrous DMSO, add saturated Na2CO3 solution dropwise, adjust the pH to 9.0, use an ultrasonic dissolver and control the temperature to dissolve the protamine to obtain a protamine solution.

[0056] 3) PEG, SE and protamine binding reaction: Dissolve 610 mg of PEG, SE powder in an appropriate amount of anhydrous DMSO (about 4-5 mL). Add the above PEG, SE solution to the reaction solution (i.e., protamine solution) through a constant pressure dropping funnel, and the time is controlled to be about 10 minutes. The whole process is carried out under nitrogen protection, with continuous stirring and reaction for 4 hours.

[0057] 4) Remove impurities: Add ether to the reaction solution in 3) until a white solid precipitates, centrifuge and wash the solid precipitate with acetone, centrifuge and settle to remove impurities (PEG, SE, water, Na2CO3, etc.). Wash the solid precipitate with ethanol and centrifuge and settle to further remove impurities (mainly PEG, SE). The obtained precipitate is vacuum dried to obtain PEGylated protamine (PP).

[0058] 5. Synthesis of PP-TRh-RSV

[0059] Then, the PP prepared in step 4 was mixed with the TRh-RSV prepared in step 3 at an N / P ratio of 2:1, and the mixture was gently vortexed at 4°C for 30 minutes to obtain PP-TRh-RSV (PP:TRh-RSV (N / P) = 2:1, containing 228 nmol·L -1 TRh, 80 μmol·L -1 RSV).

[0060] In the present invention, the N / P ratio (referring to the ratio of nitrogen in protamine to phosphorus in TRh) was calculated based on the following concentrations: 1 μg of protamine provides 15 nmol L-1 of nitrogen, and 1 μg of TRh provides 3 nmol L-1 of phosphorus.

[0061] The following is a control sample preparation.

[0062] Comparative Example 1: Preparation of Tetrahedral Framework Nucleic Acids

[0063] DNA chains (S1, S2, S3 and S4, S1-S4 see Table 1) were mixed in an equimolar ratio (1 mmol L-1) and dissolved in TM buffer to synthesize tetrahedral framework nucleic acids (tFNAs) through a DNA self-assembly process. The specific steps are: heating at 95°C for 10 minutes and then cooling at 4°C for 20 minutes. Comparative Example 2, Preparation of RSV-containing tetrahedral framework nucleic acids

[0064] Specific preparation method: tFNAs prepared in Control Example 1 were mixed with RSV at a molar ratio of 1:350 in PBS and stirred at 4° C. for 6 hours. Unreacted single-stranded DNA and RSV were removed by ultrafiltration using a 30 kDa molecular weight filter to obtain tFNAs-RSV.

[0065] Comparative Example 3: Preparation of PP-RSV

[0066] Specific preparation method: PP prepared in step 4 of Example 1 was mixed with RSV, and the mixture was gently vortexed at 4°C for 30 minutes to obtain PP-RSV (containing 7.08×10 -6 μg·mL -1 PP, 80 μmol·L -1 RSV).

[0067] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0068] Experimental Example 1: Optimal Concentration Screening

[0069] 1. Experimental methods

[0070] In order to determine the optimal concentrations of therapeutic drugs RSV, TRh and PP, the present invention detected the drug loading saturation of TRh and RSV, and the survival rate of HUVEC cells damaged by glucose and H2O2 at different concentrations of RSV and PP by CCK8 method.

[0071] 2. Experimental results

[0072] (1) RSV concentration screening

[0073] result( Figure 1 ) shows that 80 μmol·L -1 RSV provided the highest cell viability in glucose- and H2O2-damaged HUVECs; therefore, the optimal concentration of RSV was 80 μmol·L -1 .

[0074] (2) PP concentration screening

[0075] result( Figure 2 ) showed that among the ratios of 1:0, 1:50, 1:100, 1:200, 1:350 and 1:500, when the ratio of TRh to RSV reached 1:350, the drug loading sites were saturated. -1 TRh was used to load 80 μmol·L -1 RSV. To determine the optimal concentration of PP, PP and 228 nmol·L -1 TRh is between 1:1 and 10:1 N / P ratio. -1 TRh with an N / P ratio of 2:1 produced the highest cell viability ( Figure 3 ).

[0076] Combined with the above experimental data, the present invention prepared the best ratio of PP-TRh-RSV (PP:TRh-RSV (N / P) = 2:1, containing 228nmol·L -1 TRh, 80 μmol·L -1 RSV).

[0077] Experimental Example 2: Synthesis Verification

[0078] 1. Experimental methods

[0079] (1) TRh-RSV synthesis verification

[0080] The successful synthesis of TRh was verified by 8% polyacrylamide gel electrophoresis (PAGE) and capillary gel electrophoresis; the successful synthesis of TRh-RSV was confirmed by ultraviolet absorbance.

[0081] The preparation method of S1-Rh+S2 is as follows: S1-Rh and S2 are mixed in an equal molar ratio (1 mmol L-1) and dissolved in TM buffer, and tetrahedral framework nucleic acids (tFNAs) are synthesized through a DNA self-assembly process. The specific steps are: heating at 95°C for 10 minutes, and then cooling at 4°C for 20 minutes;

[0082] The preparation method of S1-Rh+S2+S3 is as follows: S1-Rh, S2, and S3 are mixed and dissolved in TM buffer in an equal molar ratio (1 mmol L-1), and tetrahedral framework nucleic acids (tFNAs) are synthesized through a DNA self-assembly process. The specific steps are: heating at 95°C for 10 minutes, and then cooling at 4°C for 20 minutes.

[0083] (2) PP-TRh-RSV synthesis verification and stability verification

[0084] Charge and particle size are measured by Zeta potential and particle size;

[0085] The stability of RSV and PP-TRh-RSV under light was detected by HPLC (where Trans / Cis represents trans-resveratrol / formal resveratrol; Cis-RSV represents formal resveratrol; Trans-RSV represents trans-resveratrol);

[0086] The morphology of PP and PP-TRh-RSV, as well as the stability of morphology changes over time, were observed by TEM.

[0087] The cells were separated, washed with DPBS solution, and fixed with 2.5% glutaraldehyde solution at room temperature for 3 hours. After fixation, the cells were stored in Sorensen buffer and fixed in 2% osmium tetroxide for 60 minutes. Observation was performed using a Jeol (Tokyo, Japan) JEM-1400 transmission electron microscope at 80 kV.

[0088] 2. Experimental results

[0089] (1) TRh and TRh-RSV synthesis verification

[0090] The results of PAGE ( Figure 4 ) showed that TRh was about 200 bp, which was larger than S1-Rh, S2, S3, S4, S1-Rh+S2, and S1-Rh+S2+S3. The two channels in S1-Rh, S1-Rh+S2, S1-Rh+S2+S3 and TRh overlapped. This confirmed that Rhodamine 19 was successfully loaded onto S1 and TRh was synthesized.

[0091] The results of capillary gel electrophoresis ( Figure 5 ) showed that the peak time of TRh was significantly different from others, verifying the synthesis of TRh.

[0092] The results of UV absorbance method ( Figure 6 ) showed that the TRh-RSV peak overlapped with TRhj and RSV, verifying the synthesis of TRh-RSV.

[0093] In summary, it was confirmed that the present invention successfully synthesized TRh and TRh-RSV.

[0094] (2) PP-TRh-RSV synthesis verification and stability verification

[0095] Depend on Figure 7 It can be seen that the particle size of tFNA is 15nm, TRh is 15.71nm, TRh-RSV is 20.81nm, and the size of PP-TRh-RSV is 295nm. The potential of tFNAs is -7.18mV, TRh is -4.75mV, TRh-RSV is -5.56mV, and the potential of PP-TRh-RSV is 1.97mV. PP-TRh-RSV and RSV were exposed to natural light for 9 hours, and then the isomerization of RSV was analyzed using HPLC. Figure 8 As shown, the retention time of trans RSV is 7.76 minutes and that of cis RSV is 8.32 minutes. In PP-TRh-RSV, the ratio of trans / cis is 2:1, while in RSV it is 1:3.8. Transmission electron microscopy (TEM) results ( Fig. 9 ) showed that the size of PP was about 200nm and had an irregularly curled structure. PP-TRh-RSV was round and about 200nm in size.

[0096] Experimental Example 3: Performance Verification of PP-TRh-RSV

[0097] 1. Experimental methods

[0098] (1) Method for verification of cell-penetration ability

[0099] HUVECs were seeded into confocal culture dishes and allowed to firmly adhere to the bottom. They were then exposed to Cy5-RSV, Cy5-TRh-RSV, Cy5-tFNAs-RSV, and Cy5-PP-TRh--RSV for 8 and 10 hours. After treatment, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) and phalloidin and observed using a confocal laser microscope (FV3000 Olympus, Tokyo, Japan).

[0100] (2) Methods for verifying mitochondrial targeting ability

[0101] After incubation with Cy5-RSV, TRh-RSV, and PP-TRh-RSV for 10 h, the cells were fixed with polyformaldehyde. Serum-free ECM medium containing 100 nmol·L-1 Mitotracker Red was applied for 30 min to label mitochondria in living cells. Afterwards, the cells were stained with FITC-phalloidin and DAPI (Solarbio Corporation) for 30 min and 10 min, respectively, and observed using a confocal laser microscope (FV3000 Olympus, Tokyo, Japan).

[0102] (3) Lysosomal escape effect verification method

[0103] After incubation of cells with Cy5-RSV, TRh-RSV, and PP-TRh-RSV for 10 h, 50 nmol·L - 1 Serum-free ECM medium containing LysoTracker Green was added for 45 minutes to label lysosomes in living cells. -1 Serum-free ECM medium containing Hoechst was applied to label the nuclei for 10 min.

[0104] (4) Verification method of cell viability under oxidative stress environment

[0105] To simulate the environment of oxidative damage, a simulated culture medium was established. The simulated culture medium (high glucose culture medium) consisted of ECM, 30mmol·L -1 Glucose, 20 μmol·L -1 The HUVECs were treated with the modeling medium for 18 h to establish the in vitro model. To evaluate the treatment efficiency, the cells were cultured in the mock medium for 12 h and then in the treatment medium for another 12 h. To compare the effects of TRh, PP, RSV, PP-RSV, TRh-RSV, and PP-TRh-RSV, the TRh (228 nmol·L -1 )、PP(7.08×10 -6 μg·mL -1 )、RSV(80μmol·L -1 ), PP-RSV (containing 7.08×10 -6 μg·mL -1 PP, 80 μmol·L -1 RSV), TRh-RSV (containing 228nmol·L -1 TRh, 80 μmol·L -1 RSV) and PP-TRh-RSV (containing 7.08×10 -6μg·mL -1 PP, 228 nmol·L -1 TRh, 80 μmol·L -1 RSV) concentration, respectively, was prepared into 100 μL solution, added to the culture medium for treatment, and compared. CCK-8 was used to evaluate the survival rate of HUVEC treated with modeling medium and treatment medium. CCK-8 solution (Med Chem Express, USA) was diluted at a ratio of 1:10. The absorbance was measured at a wavelength of 450 nm to determine the cell survival rate.

[0106] (5) Verification method of cell permeability under oxidative stress environment

[0107] To evaluate the permeability of HUVEC under various stimuli, a transwell-24-well permeable support system (polycarbonate, 0.4 μm, Corning) was used. HUVECs were seeded in the upper insert of the transwell culture plate. After 12 hours of cell sedimentation, the culture medium in (4) was replaced with the mock culture medium for 12 hours, and then the culture medium was treated for another 12 hours until complete confluence was achieved. The control group was maintained in the treatment medium for 24 hours. After these treatments, FITC-BSA (2 mg / mL) was added to the upper insert of the Transwell plate, and 200 μL of culture medium was collected from the lower chamber after 1 hour to measure the fluorescence intensity.

[0108] (6) Verification method of angiogenesis under oxidative stress environment

[0109] Matrigel (Corning, USA) was coated on a pre-cooled 24-well plate and incubated at 37°C until the gel solidified. HUVEC were treated with high-glucose medium for 12 h, and RSV (80 μmol·L -1 )、TRh-RSV (containing 228nmol·L -1 TRh, 80 μmol·L -1 RSV), PP-TRh-RSV (containing 7.08×10 -6 μg·mL -1 PP, 228 nmol·L -1 TRh, 80 μmol·L - 1 RSV) was prepared into 1 mL solution and added to the culture medium for 12 hours. The pretreated HUVECs were seeded in a 24-well culture dish. After 6 hours, angiogenesis was observed under Eclipse Ti.

[0110] (7) In order to evaluate the distribution of drugs in the fundus, the present invention used Cy7 to label RSV, PP-TRh-RSV and TRh-RSV without modification of other fluorophores. 18-week-old male heterozygous mice (db / m) were used to study the distribution of RSV, TRh-RSV and PP-TRh-RSV. Cy7-RSV (80 μmol·L -1 )、TRh-RSV (containing 228nmol·L -1 TRh, 80 μmol·L - 1 RSV) and PP-TRh-RSV (containing 7.08×10 -6 μg·mL -1 PP, 228 nmol·L -1 TRh, 80 μmol·L -1 RSV) was prepared as 2 μL solution and administered to mice. The biodistribution of RSV, TRh-RSV and PP-TRh-RSV in the fundus was detected by ophthalmic laser equipment on days 1, 4, 7, 10 and 14.

[0111] 2. Experimental results

[0112] (1) Results of cell-penetration ability verification

[0113] result( Fig.10 ) showed that after 8 hours, some fluorescence appeared outside the cells of the RSV group. However, obvious intracellular fluorescence was found in the tFNAs-RSV, TRh-RSV, and PP-TRh-RSV groups at 8 hours. At 10 hours, weak intracellular fluorescence appeared in the original RSV group, while bright fluorescence appeared in the tFNAs-RSV, TRh-RSV, and PP-TRh-RSV groups. Statistical analysis was performed, and the results showed that tFNAs-RSV, TRh-RSV, and PP-TRh-RSV showed significantly larger fluorescence areas compared with the RSV group. The PP-TRh-RSV group showed the highest intracellular fluorescence intensity at 8 or 10 hours (from p<0.05 to p<0.001).

[0114] (2) Results of mitochondrial targeting capability verification

[0115] result( Fig.11 ) showed that the fluorescence of PP-TRh-RSV mostly overlapped with the fluorescence of mitochondria, indicating its accumulation in mitochondria. However, in the RSV group, RSV hardly co-localized with mitochondria. The corresponding statistical analysis results are shown in the statistical graph on the right. The mitochondrial targeting rate of the PP-TRh-RSV group was 94%, significantly higher than 47% in the RSV group and 75% in the TRh-RSV group.

[0116] (3) Verification of lysosomal escape effect

[0117] result( Fig.12 ) showed that at 10 hours, most of the RSV in the RSV group was located in the lysosome, and the lysosomal escape rate was 25%. In the TRh-RSV group, the fluorescence signal of some RSV was separated from the signal of the lysosomal tracking dye (LysoTracker), and the escape rate was 57%. In the PP-TRh-RSV group, most of the fluorescence was located outside the lysosome, and the escape rate was as high as 92%, indicating that PP-TRh-RSV has a strong ability to escape from the endolysosome.

[0118] (4) Cell viability of PP-TRh-RSV under oxidative stress

[0119] result( Fig.13 ) showed that PP-TRh-RSV significantly increased the survival rate of HUVECs (human umbilical vein endothelial cells) injured by hyperglycemia (HG) and H2O2. These findings suggest that PP-TRh-RSV has an advantage in improving the survival rate of HUVECs.

[0120] (5) Cell permeability of PP-TRh-RSV under oxidative stress

[0121] Transwell assay ( Fig.14 ) showed that the OD value of the HG+H2O2 group was the highest, indicating that more FITC-BSA permeated into the lower cavity due to impaired cell permeability. The OD value of the PP-TRh-RSV group was significantly reduced, indicating that PP-TRh-RSV restored vascular permeability after oxidative stress injury.

[0122] Table 1. Differences in RNA expression of Occludin in each experimental group

[0123]

[0124] The protein expression of Occludin was significantly increased in the PP-TRh-RSV group. qPCR results ( Fig.15 ) also showed that the RNA expression of Occludin was upregulated in the PP-TRh-RSV group. For Claudin-5, the fluorescence in the HG+H2O2 group was weak, while the fluorescence in the PP-TRh-RSV group was enhanced. This was also confirmed by the RNA expression level. Therefore, PP-TRh-RSV is believed to alleviate the increased vascular permeability under oxidative stress and play an important role in protecting vascular barrier function.

[0125] The data in Table 1 show that the treatment with RSV, TRh-RSV and PP-TRh-RSV groups could upregulate the RNA expression of Occludin after treatment with HG+H2O2 group, and the increase in RNA expression of Occludin in the PP-TRh-RSV group was greater than the sum of the effects of RSV and TRh-RSV groups alone.

[0126] (6) Angiogenesis of PP-TRh-RSV under oxidative stress

[0127] Six hours after the pretreated HUVECs were seeded on Matrigel, HUVECs began to establish connections and form vascular network structures. However, the PP-TRh-RSV group inhibited the establishment of these connections and the formation of vascular networks. Results ( Fig.16 ) showed that the vascular junctions and branches in the PP-TRh-RSV group were significantly less than those in the HG+H2O2 group. In our experiment, the angiogenesis inhibition rate in the PP-TRh-RSV group was close to 70%. These findings suggest that PP-TRh-RSV can effectively reduce angiogenesis under oxidative stress.

[0128] (7) Detection of the distribution of PP-TRh-RSV in the fundus and its ability to delay material metabolism

[0129] like Fig.17 As shown, PP-TRh-RSV and TRh-RSV were able to be detected by ophthalmic laser diagnostic equipment. Most of the RSV was cleared on day 7, while in the PP-TRh-RSV group, fluorescence could be continuously detected until day 14. Statistical analysis in the figure shows that the average fluorescence intensity of the PP-TRh-RSV group decayed to half on day 7, indicating that its decomposition rate was slower than that of the RSV and TRh-RSV groups. As the fluorescent area gradually shrank, the fluorescence of the RSV group could not be detected on day 10, and the TRh-RSV group was no longer visible on day 14. The fluorescence of the PP-TRh-RSV group was still detectable on day 14.

[0130] The above results indicate that PP-TRh-RSV has excellent intracellular entry ability, mitochondrial targeting ability and excellent lysosomal escape ability. In addition, PP-TRh-RSV can protect cell viability, cell permeability and angiogenesis under oxidative stress environment. PP-TRh-RSV can be detected by ophthalmic laser diagnostic equipment and delay material metabolism.

[0131] Experimental Example 4: Animal Experimental Study of PP-TRh-RSV

[0132] 1. Experimental methods

[0133] 18-week-old male heterozygous mice (db / m) were used as the control group, and 18-week-old female db / db mice were used as the experimental group. Inclusion criteria included fasting blood glucose level greater than 13.3mmol / L and body weight greater than 40g. After one week of adaptive feeding, 19-week-old db / db mice were randomly divided into NS, RSV, TRh-RSV, and PP-TRh-RSV groups. RSV, TRh-RSV, and PP-TRh-RSV solutions were prepared in normal saline. Compound RSV (80μmol·L -1 ), TRh-RSV (228nmol·L -1 TRh, 80 μmol·L -1 RSV)PP-TRh-RSV(7.08×10 -6 μg·mL -1 PP, 228 nmol·L -1 TRh, 80 μmol·L -1 RSV) were administered intravitreally. Each group received 2 μL of intravitreal injections weekly. The control group of db / m mice was also injected with the same volume of saline. These injections were performed once a week for a total of 5 weeks.

[0134] (1) The retinal vessels were imaged by FFA (fundus fluorescein angiography) to verify the effect of PP-TRh-RSV on microvessels.

[0135] (2) Optical coherence tomography (OCT) was used to measure the overall thickness of the retina, and H&E staining and PAS staining were used to verify the effect of PP-TRh-RSV on the retina.

[0136] 2. Experimental results

[0137] (1) PP-TRh-RSV can inhibit microvascular proliferation

[0138] Retinal vasculature was imaged by FFA (fluorescent fundus angiography). At week 19, significant microvascular proliferation was observed in db / db mice. After five weeks of treatment, microvascular proliferation was alleviated in the RSV, TRh-RSV, and PP-TRh-RSV groups. Fig.18 As shown in Figure 3, the vascular area was larger in the db / db group, while it was significantly reduced in the PP-TRh-RSV group. In addition, the number of vascular junctions was significantly reduced in the PP-TRh-RSV group compared with the db / db group and the RSV group. These results suggest that PP-TRh-RSV significantly inhibits microangiogenesis.

[0139] (2) PP-TRh-RSV can inhibit retinal degeneration

[0140] Fig.19The OCT images in Figure 4 show that the total retinal thickness of db / db mice was reduced in both the horizontal and vertical axes. Treatment of db / db mice with PP-TRh-RSV alleviated retinal degeneration.

[0141] H&E staining results ( Fig. 20 ) showed that PP-TRh-RSV treatment significantly alleviated the disorders of the nerve fiber layer (NFL) and ganglion cell layer (GCL) in db / db mice.

[0142] Results of PAS staining of retinal vascular network ( Fig.21 ) showed that the db / db group showed obvious acellular capillaries (red arrows) compared with the db / m group, while PP-TRh-RSV improved the abnormal vascular morphology and reduced the vascular density to a certain extent. The statistical analysis results of PAS staining are shown in the figure. Obviously, the number of endothelial cells, the number of vascular junctions, the vascular area and the vascular length in the PP-TRh-RSV group were significantly reduced.

[0143] The above results indicate that PP-TRh-RSV can also inhibit microvascular proliferation and retinal degeneration.

[0144] In summary, the present invention provides a traceable mitochondrial targeted nanosystem and its preparation method and use. The traceable mitochondrial targeted nanosystem constructed by the present invention can continuously release resveratrol and maintain its photochemical stability. The nanosystem exhibits efficient cellular uptake and precise mitochondrial targeting capabilities, promotes the clearance of mitochondrial ROS and the regulation of mitochondrial autophagy, thereby reducing vascular leakage and angiogenesis, significantly inhibiting microvascular proliferation and retinal degeneration in vivo, and can be monitored in real time by clinical ophthalmic diagnostic equipment in the fundus, and has good application prospects in the preparation of drugs for the prevention and / or treatment of diabetic retinopathy.

Claims

1. A mitochondrial targeting nanosystem, characterized in that: The invention relates to a mitochondrial targeting nanosystem prepared by using lipophilic cation-modified tetrahedral framework nucleic acid, mitochondrial regulator and polyethylene glycol-modified protein as raw materials, wherein the molar ratio of the lipophilic cation-modified tetrahedral framework nucleic acid and the mitochondrial regulator is 1:300-400, and the molar ratio of nitrogen in the polyethylene glycol-modified protein to phosphorus in the mitochondrial regulator is 1-3:

1.

2. The mitochondrial targeting nanosystem according to claim 1, characterized in that: The molar ratio of the lipophilic cationic modified tetrahedral framework nucleic acid to the mitochondrial regulator is 1:350, and the molar ratio of nitrogen in the polyethylene glycol-modified protein to phosphorus in the mitochondrial regulator is 2:

1.

3. The mitochondrial targeting nanosystem according to claim 1, characterized in that: The lipophilic cation is a lipophilic cation with fluorescent properties, and the lipophilic cation-modified tetrahedral framework nucleic acid is a product prepared using the lipophilic cation with fluorescent properties and the tetrahedral framework nucleic acid as raw materials; The polyethylene glycol-modified protein is PEGylated protamine.

4. The mitochondrial targeting nanosystem according to claim 3, characterized in that: The preparation method of the tetrahedral framework nucleic acid modified with a lipophilic cation comprises the following steps: (1) reacting a lipophilic cation with a single-stranded S1 to obtain a single-stranded S1-Rh; (2) self-assembling the single-stranded S1-Rh, S2, S3 and S4 to obtain a tetrahedral framework nucleic acid modified with a lipophilic cation; the sequences of the single-stranded S1, S2, S3 and S4 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively; the mass ratio of the lipophilic cation to the single-stranded S1 is 1:3-6, preferably 1:4.6; the molar ratio of the single-stranded S1-Rh, S2, S3 and S4 is 1:0.5-2, preferably 1:

1.

5. The mitochondrial targeting nanosystem according to claim 1, characterized in that: The lipophilic cation is rhodamine 19; and the mitochondrial regulator is resveratrol.

6. A method for preparing the mitochondrial targeting nanosystem according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: mixing lipophilic cation-modified tetrahedral framework nucleic acid, mitochondrial regulator and polyethylene glycol-modified protein and reacting them to obtain a mitochondrial targeting nanosystem.

7. The method according to claim 6, characterized in that The reaction temperature is 0-10°C and the reaction time is 3-10 hours; preferably, the reaction temperature is 4°C and the reaction time is 6.5 hours.

8. Use of the mitochondrial targeting nanosystem according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating ophthalmic diseases.

9. The use according to claim 8, characterized in that The ophthalmic disease is diabetic retinopathy.

10. The use according to claim 8, characterized in that The drug is a drug for inhibiting microvascular proliferation and retinal degeneration.

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