RPE precise dual-targeting and high-efficiency endosome escape nano drug delivery system and preparation method and application thereof

Abstract

The invention discloses an RPE precise double-targeting and high-efficiency endosome escape nano drug delivery system as well as a preparation method and application of the RPE precise double-targeting and high-efficiency endosome escape nano drug delivery system. The preparation method of the nano drug delivery system comprises the following steps: 1) mixing an arginine solution with a dopamine solution and black phosphorus quantum dots, and adding siRNA gene drugs into the mixture for reaction to obtain PDA-Arg-BPQs wrapping the gene drugs; and 2) carrying out targeted modification on the surface of the PDA-Arg-BPQs wrapping the gene drug by using hyaluronic acid and annular RGD polypeptide, so as to obtain the RPE precise dual-targeting and high-efficiency endosome escape nano drug delivery system. According to the invention, arginine and polydopamine are doped, so that the ROS removal performance and the VEGF resistance of polydopamine are improved; in addition, the black phosphorus quantum dots are wrapped with PDA-Arg, endosome escape of the gene drug is promoted, and the black phosphorus quantum dots and PDA-Arg are successfully combined for the first time; besides, the PDA-Arg-BPQs nano drug delivery system is modified in a dual-targeting manner by selecting hyaluronic acid and annular RGD polypeptide, so that the biocompatibility and RPE targeting performance of the nano drug delivery system can be enhanced, and the anti-VEGF and CNV growth performance of the nano drug delivery system is further enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a nano drug delivery system with precise dual targeting of RPE and high-efficiency endosomal escape, as well as a preparation method and application thereof. Background Art

[0002] Retinal pigment epithelium (RPE) cells have the function of forming the blood-retina barrier and maintaining normal homeostasis in the eye. They are also one of the main sources of vascular endothelial growth factor (VEGF) in the eye. Under pathological conditions such as hypoxia, hyperglycemia, and inflammation, reactive oxygen species (ROS) in the body increase excessively, inducing VEGF overexpression in RPE cells, further promoting the formation of choroidal neovascularization (CNV). Common blinding eye diseases characterized by CNV in clinical practice include wet age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV), and CNV secondary to pathological myopia. The high incidence and high blindness of CNV-related eye diseases make it particularly important to take effective treatment measures in a timely manner. At present, the main treatment method for CNV in clinical practice is intravitreal injection of anti-VEGF drugs (ranibizumab, aflibercept, and conbercept). However, long-term injection of anti-VEGF drugs can easily lead to related side effects such as endophthalmitis, uveitis, and subretinal fibrosis.

[0003] Therefore, the high incidence and high blindness of CNV-related eye diseases make it particularly important to take effective treatment measures in a timely manner. Reducing the level of intraocular VEGF is considered to be an effective treatment option, and a variety of anti-VEGF preparations have been successfully used in clinical practice, such as Conbercept and Aflibercept. At present, the main treatment method for CNV in clinical practice is intravitreal injection of anti-VEGF drugs. However, long-term injection of anti-VEGF drugs also brings some side effects and potential risks. Although the dose of anti-VEGF drugs is very small in the treatment of eye diseases, there will be less VEGF expression in the blood, increasing the risk of cardiovascular disease in patients. For some severe CNV patients, because of their complexity and frequent other diseases such as subretinal fibrosis, they often face the problem of ineffective treatment or recurrence, requiring multiple treatments and finding the best combination of medication. Therefore, in order to improve the treatment effect of CNV diseases and improve patient compliance, it is urgent to seek safer and more effective treatment methods. Small interfering RNA (siRNA) is a double-stranded RNA-induced homologous sequence-specific post-transcriptional gene silencing process that can specifically degrade the expression of mRNA, inhibit the production of target proteins, and more efficiently and specifically inhibit the expression of target genes. At present, there are three main delivery technologies used in the field of gene therapy, namely physical transfection technology, viral vector technology, and non-viral nanocarrier system. Electroporation is the most commonly used physical delivery method for siRNA, but it is difficult to perform in vivo. Lentivirus, adenovirus, etc., as the most widely used viral vectors, have immunogenicity and insertion mutagenesis. In contrast, non-viral nanocarriers have good non-immunogenicity and safety. At present, non-viral nanocarriers mainly include cationic polymers, liposomes, dendrimers, etc. Nanomedicine has the advantages of targeted delivery, controlled release, and penetration of the blood-brain barrier. Therefore, the delivery of siRNA molecules to silence the mRNA of VEGF and its receptors provides a new breakthrough for the treatment of CNV-related eye diseases. Compared with anti-VEGF monoclonal antibodies and fusion proteins, siRNA has the advantages of specificity and high efficiency, but studies have found that in addition to inhibiting the target gene, siRNA also has off-target effects. Therefore, the successful development of RNA drugs depends on the development of gene carrier technology. How to safely, efficiently and targetedly deliver RNA drugs into cells remains a difficulty and hot spot in RNA therapy. Summary of the invention

[0004] In view of the shortcomings of existing nanocarriers in delivering siRNA, the present invention has conducted a lot of searches and related experimental verifications, and finally constructed a nano drug delivery system with precise dual targeting of RPE and high-efficiency endosomal escape. Compared with other nano drug delivery systems, the present invention combines black phosphorus quantum dots with polydopamine (PDA)-arginine nanomaterials for the first time, which has stronger reactive oxygen species (ROS) scavenging and VEGF reducing performance; at the same time, under the coordinated effect of the high ROS responsiveness of PDA-arginine and the superior degradability of black phosphorus quantum dots, the endosomal escape performance of siRNA gene drugs is effectively enhanced; the present technology provides a technical solution of hyaluronic acid (HA) and cyclic RGD polypeptide with dual targeted modification on the surface of nanocarriers. Compared with the existing nano targeted modification technology [Gan L et al, Biomaterials 34 (2013) 5978-5987; Huang D et al, Biomaterials (2018) 03.034], it can not only realize the precise dual targeting function of RPE and significantly inhibit the off-target effect, but also has higher biological stability and safety. The multifunctional nanocarriers finally provided by the present invention have highly uniform particle sizes and exhibit excellent biosafety due to their relatively negative surface potentials. They have good biomedical prospects and are expected to be used in the development of fundus siRNA injection preparations.

[0005] The preparation method of the RPE dual-targeted ROS responsive and endosomal escape-promoting nano drug delivery system provided by the present invention comprises the following steps:

[0006] 1) Mixing arginine (Arg) solution, black phosphorus quantum dots (BPQs), gene drugs and dopamine (DA) solution and reacting them to obtain PDA-Arg-BPQs encapsulating gene drugs;

[0007] 2) The surface of the PDA-Arg-BPQs encapsulating the gene drug is modified with hyaluronic acid (HA) and cyclic polypeptide RGD (cRGDfk) to obtain a nano drug delivery system that is RPE dual-targeted, ROS-responsive and promotes endosomal escape.

[0008] In step 1) of the above method, in the reaction system, the molar ratio of arginine, dopamine and black phosphorus quantum dots is 1:(0.5-1):(0.05-0.2), and the saturation concentration of the gene drug is 500nM-700nM.

[0009] In step 1) of the above method, the gene drug is an RNA drug, such as anti-VEGF-siRNA.

[0010] In step 1) of the above method, the reaction condition is stirring at room temperature for 1 hour.

[0011] The above method step 1) also includes: after the reaction is completed, centrifuging in a high-speed centrifuge, discarding the supernatant, washing with pure water, and centrifuging multiple times until the supernatant is colorless. Finally, the precipitate of PDA-Arg-BPQs encapsulating the gene drug is obtained, which is vacuum freeze-dried and stored in a 4°C refrigerator.

[0012] The specific modification method of step 2) of the above method is: dissolve HA in water, adjust the pH value to 4.0-5.0, place it in a 37°C constant temperature shaking incubator for reaction for 18-24 hours, add EDC and NHSS catalysts in sequence to activate the carboxyl group of HA, increase the HA coupling reaction, and then place it in a 37°C constant temperature incubator for further activation for 2-4 hours. After the activation is completed, take the PDA-Arg-BPQs and cRGDfk encapsulated with the gene drug and dissolve them in Trisbuffer (pH=8.5) respectively, mix them, and then mix them with an equal volume of HA activated solution, and stir at room temperature for reaction for 18-20 hours.

[0013] Wherein, the molecular weight of the HA is 30-50 kDa;

[0014] The mass ratio of HA, cyclic RGD polypeptide, EDC and NHSS catalyst is 10:10:(3-5):(3-5);

[0015] The mass ratio of HA, PDA-Arg-BPQs encapsulating gene drugs, and cRGDfk is 1:1:1;

[0016] The stirring speed of the stirring reaction may be 300-500 rpm / min.

[0017] The modification method of step 2) further includes: after the reaction is completed, centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, washing with pure water, and centrifuging for multiple times until the supernatant is colorless, and finally obtaining a precipitate of HA-cRGDfk-PDA-Arg-BPQs (i.e., a RPE dual-targeted ROS responsive and endosomal escape-promoting nanodrug delivery system) encapsulating gene drugs, and then vacuum freeze-drying and storing in a 4°C refrigerator.

[0018] In addition, the RPE dual-targeted ROS responsiveness and endosomal escape-promoting nano drug delivery system prepared by the above method also falls within the protection scope of the present invention.

[0019] The average particle size of the RPE-targeted ROS-responsive nano drug delivery system is 180-280 nm.

[0020] The present invention also protects the application of the above RPE dual-targeted ROS responsiveness and endosomal escape-promoting nano drug delivery system.

[0021] The application of the RPE dual-targeted ROS responsiveness and endosomal escape-promoting nano drug delivery system provided by the present invention is its application in the preparation of targeted anti-VEGF (vascular endothelial growth factor) drugs, or in the preparation of drugs for treating and / or preventing CNV-related eye diseases.

[0022] Furthermore, the CNV-related eye disease is a blinding eye disease characterized by CNV.

[0023] The present invention first constructs a PDA-Arg-BPQs nanocarrier modified with HA (hyaluronic acid) and cRGDfk (cyclic polypeptide RGD) to construct a dual-targeting carrier with the functions of targeting RPE cells, reducing ROS and promoting endosomal escape, delivering siRNA that can interfere with VEGF, and screening and verifying its drug encapsulation rate, RPE targeting and anti-VEGF performance in vivo, providing a potential effective treatment for blinding eye diseases characterized by CNV. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Agarose gel electrophoresis and FAM-siRNA standard curve; (A) Agarose gel electrophoresis of PDA-Arg-BPQs-siRNA loaded with different concentrations of siRNA. (0) 0ul; (1) 5μL, 20μM; (2) 10μL, 20μM; (3) 20μL, 20μM; (4) 40μL, 20μM; (5) 50μL, 20μM; (6) 60μL, 20μM; (7) 70μL, 20μM; (B) FAM-siRNA standard curve.

[0025] Figure 2 Fluorescence images of ROS produced after the oxidative stress cell model was co-incubated with different types of nanomaterials for 24 hours. DCFH-DA was the fluorescent probe (green fluorescence represents the ROS content produced in RPE cells); (a) 0mM H2O2; (b) 0.50mH2O2; (c) PDA; (d) PDA-Arg; (e) PDA-BPQs; (f) PDA-Arg-BPQs. The scale bar is 100um.

[0026] Figure 3 Lysosome co-localization images after different types of nanomaterials were co-incubated with cells for 4h and 8h, (A) PDA; (B) PDA-Arg; (C) PDA-BPQs; (D) PDA-Arg-BPQs.

[0027] Figure 4Characterization pictures of different types of nanomaterials, (A) SEM image, scale is 100 nm; (B) TEM image, scale is 50 nm, (a) PDA-Arg-BPQs-siRNA; (b) HA-PDA-Arg-BPQs-siRNA; (c) cRGDfk-PDA-Arg-BPQs-siRNA; (d) HA-cRGDfk-PDA-Arg-BPQs-siRNA; (C) Zeta potential; (D) DLS particle size diagram; (E) XPS diagram.

[0028] Figure 5 Experimental study on the cellular uptake of four nanomaterials (blue fluorescence represents DAPI-stained retinal cell nuclei; green fluorescence represents nanomaterials loaded with FAM-siRNA), (a) PDA-Arg-BPQs loaded with FAM-siRNA; (b) HA-PDA-Arg-BPQs loaded with FAM-siRNA; (c) cRGDfk-PDA-Arg-BPQs loaded with FAM-siRNA; (d) HA-cRGDfk-PDA-Arg-BPQs loaded with FAM-siRNA.

[0029] Figure 6 The ELISA method was used to detect the production of VEGF in the oxidative stress RPE cell model in each nanomaterial treatment group under the action of 0.50mM H2O2. (A) VEGF standard curve; (B) The reduction of VEGF by different nanomaterials. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0031] BPQs in the following examples were purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd.;

[0032] The cyclic RGD polypeptide in the following examples was purchased from Shanghai Jie Ni Tai Biotechnology Co., Ltd.;

[0033] The siRNA in the following examples was purchased from Guangzhou Ruibo Biotechnology Co., Ltd., product number stB0002356A.

[0034] In the following examples, fluorescent FAM-siRNA was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0035] Example 1: Study on the performance of PDA-Arg-BPQs nano drug delivery system in reducing ROS and endosome escape

[0036] 1) Preparation of siRNA-loaded PDA-Arg-BPQs

[0037] An appropriate amount of dopamine hydrochloride was weighed and dissolved in pure water to obtain a dopamine solution, and an appropriate amount of BPQs and siRNA was added to prepare a mixed solution 1 (wherein, the dopamine concentration was 0.8 mg / mL; the BPQs concentration was 0.2 mg / mL; and the siRNA concentration was 20 μM); arginine was weighed and dissolved in a certain volume of pure water to prepare a mixed solution 2 with a concentration of 0.8 mg / mL, and the mixed solutions 1 and 2 were mixed at a volume ratio of 6:1, and magnetically stirred for 1 hour. After the reaction was completed, the mixture was centrifuged in a high-speed centrifuge, the supernatant was discarded, washed with pure water, and centrifuged for multiple times until the supernatant was colorless. The PDA-Arg-BPQs-siRNA precipitate was finally obtained, and was freeze-dried in a vacuum oven and stored in a refrigerator at 4°C.

[0038] The ability of the nanoparticle complex to bind siRNA and the encapsulation efficiency were tested for the prepared PDA-Arg-BPQs loaded with siRNA. When the concentration of siRNA (20 μM) was 500 nM, saturation was achieved and the encapsulation efficiency reached 91%.

[0039] The specific method is as follows:

[0040] ① PDA-Arg-BPQs loaded with siRNA were prepared according to the above steps, and the efficiency of loading siRNA on the nanomaterials was optimized by agarose gel electrophoresis. Figure 1 As shown in A. From left to right, the siRNA bands in agarose gel electrophoresis gradually increase, and as the concentration of siRNA increases, the bands also gradually increase, and saturate when the concentration is 700nM. In order to save materials, we finally chose siRNA with a concentration of 500nM for the experiment.

[0041] ② Detect the encapsulation efficiency of PDA-Arg-BPQs by siRNA fluorescence quantitative determination. This test requires first using different concentrations of fluorescent FAM-labeled siRNA to make a standard curve, and then calculate the encapsulation efficiency of PDA-Arg-BPQs loaded with fluorescent FAM-siRNA through the standard curve.

[0042] Preparation of PDA-Arg-BPQs loaded with fluorescent Fam-siRNA: dopamine hydrochloride (0.8 mg / mL) was weighed and dissolved in an appropriate amount of pure water, BPQs (0.2 mg / ml) and FAM-siRNA (20 μM) were added to prepare a mixed solution 1 (wherein, dopamine concentration was 0.8 mg / mL; BPQs concentration was 0.2 mg / mL; FAM-siRNA concentration was 20 μM); an appropriate amount of arginine was weighed and dissolved in pure water to prepare a mixed solution 2 with a concentration of 0.8 mg / mL, mixed solutions 1 and 2 were mixed in a volume ratio of 6:1, magnetically stirred for 1 hour, and after the reaction was completed, high-speed centrifugation was performed, and the supernatant after the first centrifugation at 4°C was collected. The fluorescence intensity in the supernatant was measured using a fluorescence microplate reader, and the concentration of siRNA not encapsulated in the carrier was obtained by calculation. The encapsulation efficiency of PDA-Arg-BPQs nanoparticles loaded with siRNA was calculated according to the following formula.

[0043] Encapsulation efficiency = (total siRNA - free siRNA) / total siRNA * 100%

[0044] The fluorescence intensity and FAM-siRNA concentration showed a linear regression relationship, and the obtained linear regression was y = 131.33x-186.43, R 2 =0.9955 (e.g. Figure 1 The standard curve was calculated to show that the encapsulation efficiency of the nanoparticles PDA-Arg-BPQs loaded with siRNA was 91%.

[0045] 2) Study on the ROS scavenging performance of PDA-Arg-BPQs

[0046] The levels of ROS reduction by different nanomaterials were detected by using the DCFH-DA kit.

[0047] The assay method is as follows: ARPE-19 cells with a cell density of 80%-90% in the logarithmic growth phase are plated in 24-well plates at 1.5x10 5Cells were inoculated at a cell density of 10 μg / mL / well, and the supernatant was discarded after the cells adhered to the wall for 24 hours. PDA, PDA-Arg, PDA-BPQs, and PDA-Arg-BPQs at a concentration of 10 μg / mL were added respectively, and the cells were incubated in a cell culture incubator for 3-5 hours. The cells were washed with PBS, and a 0.50 mM H2O2 solution was added. The cells were further incubated in a cell culture incubator (37°C, 5% CO2) for 24 hours. The supernatant was discarded, the cells were washed with PBS, and 1 mL of DCFH-DA working solution (concentration of 10 μM) diluted with basal culture medium was added to each well under light-proof conditions. The cells were further incubated in a cell culture incubator (37°C, 5% CO2) for 45 minutes. After the incubation, the cells were washed 3 times with basal culture medium, and the images were observed and collected under a fluorescence microscope.

[0048] Results Figure 2 .

[0049] Under the same conditions, compared with the 0.50 mM H2O2 control group, the green fluorescence brightness of oxidative stress APPE-19 cells was reduced after treatment with the four nanomaterials, but compared with the simple PDA experimental group, the green fluorescence intensity of the PDA-Arg-BPQs experimental group was significantly reduced ( Figure 2 ), suggesting that arginine-modified PDA has a stronger ROS scavenging ability.

[0050] 3) Study on the endosomal escape performance of PDA-Arg-BPQs

[0051] The four nanomaterials used in this example, PDA, PDA-Arg, PDA-BPQs, and PDA-Arg-BPQs, are nanomaterials loaded with fluorescent FAM-siRNA.

[0052] The preparation method of the PDA loaded with fluorescent FAM-siRNA is as follows: first, 1.15 mL of anhydrous ethanol solution, 3.35 mL of pure water and 33.25 uL of sodium hydroxide solution are measured and mixed, and after stirring at room temperature for 30 min, 125 uL of FAM-siRNA (20 μM) is added. 10 mg of dopamine hydrochloride is weighed and dissolved in 0.5 mL of pure water, mixed with the above solution, stirred for 1 h in the dark, and after the reaction is completed, centrifuged in a high-speed refrigerated centrifuge, the supernatant is discarded, washed with pure water, and centrifuged several times until the supernatant is transparent and colorless, and the PDA precipitate loaded with FAM-siRNA is obtained, which is vacuum freeze-dried and stored in a refrigerator at 4°C.

[0053] The preparation method of the PDA-Arg loaded with fluorescent FAM-siRNA is as follows: 4 mg of dopamine hydrochloride is weighed and dissolved in 4.2 ml of pure water. After fully dissolved, 125 uL of FAM-siRNA (20 μM) is added to prepare solution 1; 4 mg of arginine is weighed, added to 0.8 mL of pure water, fully dissolved, and prepared into solution 2, and then solution 2 is slowly added dropwise to solution 1, stirred at room temperature in the dark for 1 hour, and after the reaction is completed, centrifuged in a high-speed refrigerated centrifuge, the supernatant is discarded, washed with pure water, and centrifuged multiple times until the supernatant is colorless to obtain a PDA-Arg precipitate loaded with FAM-siRNA, which is vacuum freeze-dried and stored in a 4°C refrigerator.

[0054] The preparation method of the PDA-BPQs loaded with fluorescent FAM-siRNA is as follows: first, 4.6 ml of anhydrous ethanol solution, 7.7 ml of pure water and 133 uL of sodium hydroxide solution are measured and mixed, and after stirring at room temperature for 30 minutes, 5.7 ml of BPQs solution and 500 uL of FAM-siRNA (20 μM) are added; 40 mg of dopamine hydrochloride is weighed and dissolved in 2 ml of pure water, and after mixing with the above solution, stirring is continued in the dark for 1 hour. After the reaction is completed, centrifuge at 4°C, discard the supernatant, wash with pure water, and centrifuge several times until the supernatant is colorless, and finally obtain the PDA-BPQs precipitate loaded with FAM-siRNA, which is vacuum freeze-dried and stored in a refrigerator at 4°C.

[0055] The preparation method of the PDA-Arg-BPQs loaded with fluorescent FAM-siRNA is as follows: the same as the preparation method of the PDA-Arg-BPQs loaded with FAM-siRNA in 1) above.

[0056] The lysosome co-localization method was used to study the endosomal escape ability of PDA, PDA-Arg, PDA-BPQs, and PDA-Arg-BPQs nanomaterials.

[0057] The specific test method is: take the adult retinal pigment epithelial cell line-19 (ARPE-19) with a cell density of 80%-90% in the logarithmic growth phase, and incubate the cells at 1.5X10 5 / well were inoculated in a 24-well plate, incubated in a 37°C, 5% CO2 incubator, until the cells grew to 80%-90% of the well plate, the complete medium was discarded, and the diluted vectors containing FAM-siRNA (concentration of 25μg / mL) were added, and incubated in a 37°C, 5% CO2 cell incubator for 4h and 8h respectively. After the time point, the original culture medium was discarded, PBS was washed, and diluted Lyso-Tracker Red (final concentration of 50nM) was added, and incubated in a cell incubator to stain lysosomes for 90min. After the incubation, the supernatant was discarded, the cell slides were washed, the cells were fixed with 4% paraformaldehyde, DAPI was stained, dried, and sealed, and stored at -20°C for later use, and finally photographed with a laser confocal scanning microscope.

[0058] Results Figure 3 .

[0059] Lyso-Tracker Red showed red fluorescence, with a maximum excitation wavelength of 577nm and a maximum emission wavelength of 590nm during detection. FAM-siRNA encapsulated in nanomaterials showed green fluorescence (maximum excitation wavelength of 492nm and maximum emission wavelength of 518nm). When the red fluorescence overlapped with the green fluorescence, it showed yellow, indicating that the nanomaterials were in the lysosomes. When the incubation time of nanoparticles and cells was 4h, the green and red colors of the four nanomaterials overlapped more, indicating that the nanomaterials were taken up by cells to form endosomes. When the cell incubation time was 8h, PDA-Arg-BPQs showed more green and less yellow than the other three nanomaterials (PDA, PDA-Arg, PDA-BPQs). Figure 3 ), suggesting that PDA-Arg-BPQs has better endosomal escape performance.

[0060] Example 2: Targeting study of HA-cRGDfk-PDA-Arg-BPQs nano-drug delivery system loaded with siRNA

[0061] 1) Preparation of HA-cRGDfk-PDA-Arg-BPQs loaded with siRNA

[0062] The specific preparation method is as follows:

[0063] The preparation method of PDA-Arg-BPQs loaded with siRNA is shown in Example 1.

[0064] Preparation of HA-cRGDfk-PDA-Arg-BPQs loaded with siRNA: Weigh an appropriate amount of hyaluronic acid (HA, molecular weight: 30-50 kDa), add pure water to dissolve, and obtain a HA solution with a concentration of 2 mg / mL; then add an appropriate amount of concentrated hydrochloric acid to adjust the pH value of the HA solution to about 4.0-5.0, place it in a 37°C constant temperature incubator to react for 24 hours, and add 6 mg EDC and 6 mg NHSS catalysts were used to activate the carboxyl group of HA, increase the HA coupling reaction, and the mixture was placed in a constant temperature incubator for further activation for 2-4 h. After the activation was completed, appropriate amounts of PDA-Arg-BPQs-siRNA (2 mg / mL) and cRGDfk (2 mg / mL) were weighed and dissolved in a certain volume of Tris buffer (pH = 8.5), and an equal volume of the above HA activation solution was added and mixed. The mixture was stirred at room temperature for 18-20 h. After the reaction was completed, the mixture was centrifuged in a high-speed refrigerated centrifuge, the supernatant was discarded, the mixture was washed with pure water, and centrifuged. The HA-cRGDfk-PDA-Arg-BPQs-siRNA precipitate was vacuum freeze-dried and stored in a refrigerator at 4°C. In addition, HA and cRGDfk were removed from the preparation of HA-cRGDfk-PDA-Arg-BPQs-siRNA to obtain HA-PDA-Arg-BPQs-siRNA and cRGDfk-PDA-Arg-BPQs-siRNA, which were also vacuum freeze-dried and stored.

[0065] Results Figure 4 .

[0066] The prepared PDA-Arg-BPQs-siRNA, HA-PDA-Arg-BPQs-siRNA, cRGDfk-PDA-Arg-BPQs-siRNA, and HA-cRGDfk-PDA-Arg-BPQs-siRNA were characterized. SEM and TEM showed that the prepared PDA-Arg-BPQs-siRNA, HA-PDA-Arg-BPQs-siRNA, cRGDfk-PDA-Arg-BPQs-siRNA, and HA-cRGDfk-PDA-Arg-BPQs-siRNA nanoparticles had uniform size distribution, regular spherical shape, and good dispersion ( Figure 4 A and B). Zeta potential results showed that after modification with HA, cRGDfk and dual-functional modification, the potential of PDA-Arg-BPQs-siRNA increased from -13.17±0.74mV to -30.33±0.68mV, -31.73±0.62mV and -26.60±1.42mV, respectively, proving that negatively charged HA and cRGDfk were successfully modified on the surface of PDA-Arg-BPQs-siRNA ( Figure 4C). The particle size DLS analysis showed that the particle size of PDA-Arg-BPQs-siRNA was 323.13±5.31nm. The average particle sizes of PDA-Arg-BPQs-siRNA modified with HA and cRGDfk were 192.40±4.73nm, 200.03±4.87nm and 268.60±7.05nm, respectively. Compared with the particle size of PDA-Arg-BPQs-siRNA, the particle size was reduced ( Figure 4 D), the possible reason is that the surface modification of HA and cRGDfk tightened the structure of PDA-Arg-BPQs-siRNA, and although the coating was increased, the overall particle size of the nanoparticles was reduced. XPS analysis showed that compared with PDA-Arg-BPQs-siRNA, CO / CN and CC increased after modification with HA and cRGDfk, indicating that HA and cRGDfk were successfully connected ( Figure 4 Middle E).

[0067] 2) Targeting study of HA-cRGDfk-PDA-Arg-BPQs nano-drug delivery system loaded with siRNA

[0068] The four siRNA-loaded nanomaterials used in this example are PDA-Arg-BPQs labeled with FAM-siRNA, HA-PDA-Arg-BPQs labeled with FAM-siRNA, cRGDfk-PDA-Arg-BPQs labeled with FAM-siRNA, and HA-cRGDfk-PDA-Arg-BPQs labeled with FAM-siRNA.

[0069] The preparation method of PDA-Arg-BPQs labeled with FAM-siRNA is as follows: the same as the preparation method of PDA-Arg-BPQs loaded with FAM-siRNA in Example 1.

[0070] The preparation method of FAM-siRNA labeled HA-PDA-Arg-BPQs, cRGDfk-PDA-Arg-BPQs and HA-cRGDfk-PDA-Arg-BPQs is as follows: the preparation method of HA-cRGDfk-PDA-Arg-BPQs loaded with siRNA in Example 2 is the same as that in Example 2, except that the siRNA therein is replaced with FAM-siRNA.

[0071] The targeting properties of the four siRNA-loaded nanomaterials were studied by cell uptake experiments. ARPE-19 cells with a cell density of 80%-90% in the logarithmic growth phase were taken and the cells were cultured at a rate of 1.5X10 5 / well density inoculated in a 24-well plate, prepared cell slides, incubated in a constant temperature incubator, incubated, discarded the complete medium, added 1mL of H2O2 (concentration of 0.50mM) solution to each well, continued to incubate in the incubator for 4-6h, discarded the supernatant, added the diluted FAM-siRNA containing each group of vectors (concentration of 25μg / mL), incubated in the cell incubator for 48h in the dark. After the incubation, discarded the supernatant, washed with PBS, fixed the cells with 4% paraformaldehyde, stained with DAPI, dried, sealed, stored at -20℃ for later use, and finally observed by laser confocal scanning microscope.

[0072] Results Figure 5 .

[0073] After incubating the FAM-siRNA-loaded PDA-Arg-BPQs, HA-PDA-Arg-BPQs, cRGDfk-PDA-Arg-BPQs and HA-cRGDfk-PDA-Arg-BPQs nanomaterials with ARPE-19 cells of oxidative stress model for 24 hours, the green fluorescence of the unmodified FAM-siRNA-loaded PDA-Arg-BPQs was weak, while the green fluorescence of the nanomaterials HA-PDA-Arg-BPQs and cRGDfk-PDA-Arg-BPQs modified with HA and cyclic RGD was strong, but the green fluorescence of the dual-targeting ligand-modified nanomaterials was the strongest ( Figure 5 ). This suggests that the RPE targeting of nanomaterials is increased after dual targeting modification with HA and cyclic RGD.

[0074] 3) HA-cRGDfk-PDA-Arg-BPQs nano-drug delivery system loaded with siRNA against VEGF

[0075] The eight siRNA-loaded nanomaterials used in this example are PDA-Arg-BPQs, HA-PDA-Arg-BPQs, cRGDfk-PDA-Arg-BPQs, HA-cRGDfk-PDA-Arg-BPQs, PDA-Arg-BPQs-siRNA, HA-PDA-Arg-BPQs-siRNA, cRGDfk-PDA-Arg-BPQs-siRNA and HA-cRGDfk-PDA-Arg-BPQs-siRNA, all of which are siRNAs.

[0076] The preparation method of PDA-Arg-BPQs is as follows: the same as the preparation method of PDA-Arg-BPQs in Example 1.

[0077] The preparation methods of HA-PDA-Arg-BPQs, cRGDfk-PDA-Arg-BPQs, and HA-cRGDfk-PDA-Arg-BPQs are as follows: the same as the preparation method of HA-cRGDfk-PDA-Arg-BPQs loaded with FAM-siRNA in Example 2.

[0078] The preparation method of PDA-Arg-BPQs loaded with siRNA is as follows: the same as the preparation method of PDA-Arg-BPQs-siRNA in Example 1.

[0079] The preparation methods of HA-PDA-Arg-BPQs, cRGDfk-PDA-Arg-BPQs and HA-cRGDfk-PDA-Arg-BPQs loaded with siRNA are as follows: the same as the preparation method of HA-cRGDfk-PDA-Arg-BPQs loaded with siRNA in Example 2.

[0080] At the cell level, ELISA was used to detect the VEGF content secreted by ARPE-19 cells in each treatment group. The determination method was as follows: ARPE-19 cells with a cell density of 80%-90% in the logarithmic growth phase were taken and the cells were plated at 1.5X10 5 / well density was inoculated in a 24-well plate, incubated in a constant temperature incubator for 24 hours, the culture medium was discarded, 1 mL of each group of vectors containing siRNA diluted (concentration of 25 μg / mL) was added to each well, incubated in a constant temperature cell incubator for a certain period of time, the supernatant was discarded, PBS was washed, 1 mL of H2O2 (concentration of 0.50 mM) solution was added, and the cells were placed in the incubator for another 24 hours. After the incubation was completed, the cell culture medium was placed in a 1.5 mL EP tube, centrifuged, and the supernatant was taken. After measuring the protein concentration in the supernatant according to the instructions of the BCA kit, the detection was performed according to the instructions of the ELISA kit.

[0081] Results Figure 6 . Figure 6 In the experiment, RPE was the blank control group, H2O2 group was the group that induced RPE cells to produce oxidative stress with a certain concentration, and the non-drug-loaded group was the group of different types of nanomaterials + oxidative stress RPE cells.

[0082] A small amount of VEGF was expressed in normal ARPE-19 cells in vitro. Compared with the ARPE-19 cell control group, the VEGF content in the oxidative stress RPE cell model group was significantly increased (P<0.0001). Compared with the H2O2 model group, the VEGF content in the unloaded nanoparticle group and the loaded nanoparticle group was significantly decreased (P<0.0001). The VEGF content in the drug-loaded HA-PDA-Arg-BPQs-siRNA group was significantly lower than that in the drug-loaded PDA-Arg-BPQs-siRNA group (P<0.01). Compared with the unloaded PDA-Arg-BPQs, HA-PDA-Arg-BPQs and cRGDfk-PDA-Arg-BPQs groups, the VEGF content in the drug-loaded cRGDfk-PDA-Arg-BPQs-siRNA group was significantly decreased (P<0.05). Compared with the unloaded group and the loaded PDA-Arg-BPQs-siRNA group, the VEGF content in the loaded HA-cRGDfk-PDA-Arg-BPQs-siRNA group was significantly decreased (P<0.0001). Compared with the cRGDfk-PDA-Arg-BPQs-siRNA group, the VEGF content in the loaded HA-cRGDfk-PDA-Arg-BPQs-siRNA group was significantly decreased (P<0.01) ( Figure 6 B), indicating that the arginine-doped nanomaterials can not only reduce the expression of VEGF by themselves, but also loaded with siRNA gene drug carriers can more effectively reduce the expression of VEGF. Therefore, our nanomaterials can not only reduce the expression of VEGF by themselves, but also the dual-targeted nanomaterials loaded with gene drugs can better reduce the expression of VEGF.

Claims

1. A method for preparing a nano drug delivery system with precise dual targeting of RPE and high-efficiency endosomal escape, comprising the following steps: 1) Mixing arginine solution, black phosphorus quantum dots, gene drugs and dopamine solution and reacting them to obtain PDA-Arg-BPQs encapsulating gene drugs; 2) The surface of the PDA-Arg-BPQs encapsulating the gene drug is modified by using hyaluronic acid and cyclic RGD polypeptide to obtain a RPE dual-targeted ROS responsive and endosomal escape-promoting nano drug delivery system, namely HA-cRGDfk-PDA-Arg-BPQs.

2. The preparation method according to claim 1, Features: In the step 1), in the reaction system of the reaction, the molar ratio of arginine, dopamine and black phosphorus quantum dots is 1:(0.5-1):(0.05-0.2), and the saturation concentration of the gene drug is 500nM-700nM; In the step 1), the reaction condition of the reaction is stirring at room temperature for 1 hour.

3. The preparation method according to claim 1 or 2, Features: In the step 1), the gene drug includes a siRNA drug, and further is an anti-VEGF siRNA.

4. The preparation method according to any one of claims 1 to 3, Features: The step 1) further comprises: after the reaction is completed, centrifuging in a high-speed centrifuge, discarding the supernatant, washing with pure water, and centrifuging for multiple times until the supernatant is colorless, and finally obtaining a precipitate of PDA-Arg-BPQs encapsulating the gene drug, vacuum freeze-drying, and storing in a refrigerator at 4°C.

5. The preparation method according to any one of claims 1 to 4, Features: The specific modification method of step 2) is as follows: dissolving hyaluronic acid in water, adjusting the pH value to 4.0-5.0, placing it in a 37°C constant temperature shaking incubator for reaction for 18-24 hours, adding EDC and NHSS catalysts in sequence to activate the carboxyl group of hyaluronic acid, and then placing it in a 37°C constant temperature incubator for further activation for 2-4 hours; after the activation is completed, taking the PDA-Arg-BPQs and cyclic RGD polypeptide encapsulating the gene drug and dissolving them in Trisbuffer respectively, mixing them, and then mixing them with an equal volume of the activated hyaluronic acid solution, stirring and reacting at room temperature for 18-20 hours.

6. The preparation method according to claim 5, Features: The molecular weight of the hyaluronic acid is 30-50 kDa; The mass ratio of the hyaluronic acid, cyclic RGD polypeptide, EDC and NHSS catalyst is 10:10:(3-5):(3-5); The mass ratio of HA, PDA-Arg-BPQs encapsulating gene drugs, and cRGDfk is 1:1:1; The stirring speed of the stirring reaction is 300-500 rpm / min.

7. The preparation method according to claim 5 or 6, Features: The modification method of step 2) further comprises: after the reaction is completed, centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, washing with pure water, and centrifuging for multiple times until the supernatant is colorless, and finally obtaining a precipitate of HA-cRGDfk-PDA-Arg-BPQs encapsulating the gene drug, and then vacuum freeze-drying and storing in a 4°C refrigerator.

8. A nano drug delivery system with precise dual targeting of RPE and high-efficiency endosomal escape prepared by the method according to any one of claims 1 to 7.

9. The RPE precise dual-targeting and high-efficiency endosomal escape nano drug delivery system according to claim 8, Features: The average particle size of the RPE precise dual-targeting and high-efficiency endosomal escape nano drug delivery system is 180-280nm.

10. Use of the RPE precise dual-targeting and high-efficiency endosomal escape nanodrug delivery system according to claim 8 or 9 in the preparation of anti-VEGF drugs, or in the preparation of drugs for treating and / or preventing CNV-related eye diseases.

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