A polypeptide-dexamethasone conjugate and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and relates to a polypeptide-dexamethasone conjugate and its preparation method and application. It has a chemical structure shown in formula I; wherein POI is a polypeptide, the polypeptide includes a short peptide and a cysteine ​​residue, the short peptide includes one or more of RGD, TAT, and AT7, and the POI is connected to a maleimide group by a thioether bond through a cysteine ​​residue. The polypeptide-dexamethasone conjugate provided by the present invention has both targeting, penetrability, anti-inflammatory and anti-angiogenic activity, and can be better used for targeted treatment of neovascular eye diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a polypeptide-dexamethasone conjugate and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Neovascular eye disease is a class of eye diseases defined as excessive neovascularization and abnormal vascular remodeling in the eye. Epidermal growth factor (VEGF) is widely recognized as one of the important therapeutic targets for neovascular eye disease. Almost all anti-VEGF biologics used in tumor treatment have been clinically tested in ophthalmic diseases, such as bevacizumab, ranibizumab, and aflibercept. However, anti-VEGF therapy also has some limitations. First, its duration of action is short and the response is insufficient, and a large proportion of patients have poor visual recovery after treatment. In addition, intravitreal injection of anti-VEGF drugs is often accompanied by inflammation-related adverse reactions. It has been observed that the pathogenesis of ocular neovascularization-related diseases is multifactorial. In addition to VEGF, other factors may be involved. Studies have shown that inflammation plays an important role in the pathogenesis of neovascular eye disease. As one of the highly effective anti-inflammatory drugs, glucocorticoids have broad application prospects in the treatment of inflammation-related diseases. Intravitreal implants of glucocorticoids have played a role in fundus diseases as an alternative to anti-VEGF therapy. Clinical studies have found that compared with single anti-VEGF therapy, patients with refractory diabetic macular edema (DME) have more significant improvements in foveal thickness and visual acuity after receiving combined anti-VEGF drugs and glucocorticoids. Therefore, it is necessary to provide drugs with good adaptability based on multiple factors to treat neovascular diseases.

[0004] Peptide-drug conjugates (PDC) have attracted more and more attention as a targeted drug that can be designed with multiple targets. PDC is a type of drug formed by covalent bonding of peptides and other bioactive molecules (such as drugs, antibodies, fluorescent dyes, etc.). Its advantage is that the targeting of peptides can be used to deliver drugs to specific cell or tissue targets, thereby improving the efficacy of drugs and reducing side effects. In the previous study, the inventors of the present invention designed an anti-angiogenic dual-target penetrating peptide TAT-AT7, which was lipid-modified and made into a hydrogel preparation, and applied to the eye in the form of eye drops. TAT-AT7 showed excellent activity in penetrating the eye barrier and inhibiting retinal neovascularization. However, the inventors further found that although the powerful penetrating ability of the peptide TAT-AT7 enables it to penetrate the eye barrier, it cannot concentrate its effect on the lesion site well. Therefore, it is necessary to further modify the drug. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a polypeptide-dexamethasone conjugate and a preparation method and application thereof. The polypeptide-dexamethasone conjugate has both targeting, penetrating, anti-inflammatory and anti-angiogenic activity, and can be better used for targeted treatment of neovascular eye diseases.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, a polypeptide-dexamethasone conjugate has a chemical structure shown in Formula I;

[0008]

[0009] Among them, POI is a polypeptide, which includes a short peptide and a cysteine ​​residue, and the short peptide includes one or more of RGD (amino acid residue sequence is GRGDS, as shown in SEQ ID NO.1), TAT (amino acid residue sequence is RKKRRQRRR, as shown in SEQ ID NO.2), and AT7 (amino acid residue sequence is ATWLPPR, as shown in SEQ ID NO.3), and the POI is connected to the maleimide group by a thioether bond through cysteine.

[0010] TAT is a representative class of cell-penetrating peptides. Due to its strong penetrating ability, it has unique advantages in transmembrane transduction and penetration of physiological barriers such as the blood-brain barrier (BBB) ​​and the blood-retinal barrier (BRB). It has been proven that it can carry drug molecules and has shown great advantages in the treatment of brain gliomas, central nervous system diseases, and ocular neovascular diseases. The peptide AT7 is a vascular targeting peptide screened from a phage library. The amino acid residue sequence is ATWLPPR. It can produce dual targeting effects by simultaneously binding to epidermal growth factor receptor-2 (VEGFR-2) and neuropilin-1 (NRP-1), inhibiting the angiogenic effect of VEGF, and has higher selectivity for the ocular neovascularization system. The targeted penetrating peptide TAT-AT7 constructed by coupling the peptide TAT and the peptide AT7 has significant anti-angiogenic activity and blood-brain barrier penetration.

[0011] Integrins are cell adhesion proteins that play an important role in promoting the adhesion of endothelial cells to the extracellular matrix. Integrins are composed of different subtypes, among which integrin αvβ3 has the strongest regulatory effect on angiogenesis and is highly expressed on the surface of endothelial cells of new blood vessels. Ruoslahti et al. first discovered that the peptide RGD is the binding sequence of the extracellular matrix and integrin αvβ3, and has strong selectivity and affinity for integrin αvβ3.

[0012] Based on this, the present invention connects the anti-angiogenesis penetrating peptide TAT-AT7 and / or the integrin targeting peptide RGD and the small molecule anti-inflammatory drug dexamethasone through ester bonds, amide bonds and maleimido thioether bonds to construct a new type of polypeptide-dexamethasone conjugate with targeting, penetrating, anti-inflammatory and anti-angiogenic activity.

[0013] In some embodiments, the short peptide is RGD, or a peptide composed of TAT and AT7, or a peptide composed of RGD, TAT and AT7.

[0014] In some embodiments, the short peptide is a peptide composed of RGD, TAT and AT7. Studies have shown that when the short peptide is a peptide composed of RGD, TAT and AT7, the conjugate has a better effect.

[0015] Specifically, TAT and AT7 constitute a TAT-AT7 polypeptide, and the cysteine ​​residue is located between TAT and AT7.

[0016] Specifically, the amino acid residue sequence of POI is: GRGDSRKKRRQRRRCATWLPPR, as shown in SEQ ID NO.4.

[0017] In a second aspect, a method for preparing the above-mentioned polypeptide-dexamethasone (DEX) conjugate comprises esterifying dexamethasone with succinic anhydride to obtain DEX-SA, and reacting DEX-SA with N -(2-aminoethyl)maleimide is subjected to an amidation condensation reaction to obtain DEX-MAL, and DEX-MAL is subjected to an addition reaction with POI-SH (thiolated polypeptide) to obtain the obtained product;

[0018] Among them, the chemical structure formula of DEX-SA is: ; The chemical structure of DEX-MAL is: .

[0019] In some embodiments, dexamethasone is reacted with succinic anhydride and 4-dimethylaminopyridine (DMAP) at room temperature to obtain DEX-SA. Specifically, the solvent is acetone. Specifically, the mass ratio of dexamethasone to succinic anhydride and 4-dimethylaminopyridine is 1.3-1.7:0.70-0.80:0.15-0.20. The room temperature described in the present invention refers to the temperature of the indoor environment, which is generally 15-30 °C.

[0020] In some embodiments, 2-(7-azabenzotriazole)- N , N , N ', N '-Tetramethyluronium hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT) and N , N -Diisopropylethylamine (DIPEA) was used to activate the carboxylic acid in DEX-SA, and then added N -(2-aminoethyl)maleimide is subjected to an amidation condensation reaction to obtain DEX-MAL. Specifically, the reaction temperature is room temperature.

[0021] In some embodiments, the addition reaction of DEX-MAL and POI-SH is carried out under the following conditions: reaction at room temperature for 1.5 to 3 h.

[0022] In a third aspect, a pharmaceutical composition comprises the above-mentioned polypeptide-dexamethasone conjugate and pharmaceutical excipients.

[0023] The pharmaceutical excipients of the present invention include pharmaceutical carriers and / or excipients. The pharmaceutical carriers include but are not limited to serum protein, water, physiological saline, phosphate buffer solution, etc. The excipients include but are not limited to antioxidants, buffers, pH regulators, fillers, emulsifiers, emulsifier aids, adhesives, preservatives, spices, etc.

[0024] In a fourth aspect, a pharmaceutical preparation comprises nanomicelles, wherein the nanomicelles are formed by the above-mentioned polypeptide-dexamethasone conjugate.

[0025] The dexamethasone end is hydrophobic and the polypeptide end is hydrophilic, so the conjugate can self-assemble into nanomicelles in aqueous solution.

[0026] In some embodiments, the conjugate is prepared into nanomicelles by vortex sonication.

[0027] In some embodiments, the particle size of the nanomicelles is 100-200 nm, and the Zeta potential is positive.

[0028] A fifth aspect is the use of the above-mentioned polypeptide-dexamethasone conjugate, pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for treating neovascular eye disease.

[0029] In the aqueous humor in vitro, most of the peptide-dexamethasone conjugates were metabolized within 3 hours by HPLC monitoring, breaking from the connecting arm to dissociate the dexamethasone prototype drug and the peptide. Therefore, the peptide-drug conjugate designed by the present invention can be broken in vivo through the connecting arm designed by the present invention, and dexamethasone and the peptide can be released respectively to exert their respective therapeutic effects.

[0030] In in vitro experiments, it was preliminarily confirmed at the molecular and cellular levels that the conjugate nanomicelle DEX-RGD-TAT-AT7 has targeting properties on integrin protein αvβ3; in in vitro penetration experiments, the conjugate nanomicelle DEX-RGD-TAT-AT7 can enter cells smoothly and has the activity of penetrating cell barriers; DEX-RGD-TAT-AT7 has anti-inflammatory and anti-angiogenic activities.

[0031] In the in vivo experiment, DEX-RGD-TAT-AT7 showed low hemolysis and could be used in the in vivo experiment; in the mouse ear swelling model, the DEX-RGD-TAT-AT7 conjugate micelles inhibited mouse ear swelling and had an in vivo anti-inflammatory effect; in the rat choroidal neovascularization model, DEX-RGD-TAT-AT7 could penetrate the eye barrier and act on the retina and choroid, and had the activity of inhibiting choroidal angiogenesis, thus playing a certain role in anti-neovascular eye diseases. The peptide-drug conjugate DEX-RGD-TAT-AT7 designed by the present invention can penetrate into the fundus in a targeted manner by eye drops, and has dual anti-inflammatory and anti-neovascular effects, providing a promising therapeutic strategy for the treatment of clinical neovascular eye diseases.

[0032] The beneficial effects of one or more of the above technical solutions are:

[0033] Compared with the prior art, the present invention covalently links the TAT-AT7 polypeptide with penetrating and anti-angiogenic activity, the RGD polypeptide with integrin targeting activity and dexamethasone into a new polypeptide-drug conjugate, which continues the anti-inflammatory effect of dexamethasone, has better activity in inhibiting endothelial cell proliferation and angiogenesis, and has targeting of new blood vessels. In addition, the designed connecting arm can be broken under the action of esterase, which provides a guarantee for the active effects of dexamethasone and polypeptide molecules.

[0034] The present invention constructs a novel polypeptide-drug conjugate nano-micelle, which has anti-inflammatory and anti-angiogenic dual activities compared with the prior art, reduces the side effects of single-factor treatment of fundus neovascularization diseases, and can be used to treat choroidal neovascularization eye diseases in the form of eye drops, reducing the burden of poor eye compliance of patients with vitreous injection in the past. By utilizing the hydrophobicity of dexamethasone and the hydrophilicity of polypeptides, spherical nano-micelles are formed in the form of self-assembly, which improves the water solubility of dexamethasone. At the same time, the enrichment and tissue penetration ability of dexamethasone in the ocular neovascularization site can be enhanced, so that it can also be delivered to the fundus lesion site by eye drops to exert its activity. Combining the above advantages, the conjugate nano-micelles show strong cross-ocular barrier and targeted anti-inflammatory and anti-angiogenic activities in in vivo experiments, and can inhibit the course of neovascularization eye diseases by inhibiting the formation of fundus choroidal neovascularization, providing a promising treatment strategy for the clinical treatment of anti-neovascular eye diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 This is the synthetic route of DEX-SA in Example 1.

[0037] Figure 2 This is the synthetic route of DEX-MAL in Example 1.

[0038] Figure 3 This is the synthetic route of DEX-RGD, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 in Example 1.

[0039] Figure 4 is the ESI-MS (A) of DEX-SA in Example 1 and 1 H NMR (B) identification results.

[0040] Figure 5 is the ESI-MS (A) of DEX-MAL in Example 1 and1 H NMR (B) identification results.

[0041] Figure 6 These are the ESI-Q-TOF identification results of DEX-RGD (A), DEX-TAT-AT7 (B) and DEX-RGD-TAT-AT7 (C) in Example 1.

[0042] Figure 7 is the critical micelle concentration of DEX-RGD (A), DEX-TAT-AT7 (B) and DEX-RGD-TAT-AT7 (C) in Example 2.

[0043] Figure 8 The particle size distribution diagrams of DEX-RGD (A), DEX-TAT-AT7 (B) and DEX-RGD-TAT-AT7 (C) in Example 3 are shown.

[0044] Fig. 9 These are the potential distribution diagrams of DEX-RGD (A), DEX-TAT-AT7 (B) and DEX-RGD-TAT-AT7 (C) in Example 3.

[0045] Fig.10 These are transmission electron micrographs of DEX-RGD (A), DEX-TAT-AT7 (B) and DEX-RGD-TAT-AT7 (C) in Example 3.

[0046] Fig.11 This is the HPLC detection of the changes in DEX-RGD-TAT-AT7 nanomicelles before and after being placed in aqueous humor for 3 hours in Example 4 (A); after 3 hours of metabolism, the mass spectrometry characterization of the effluent at HPLC at 7.78 min (B), 14.9 min (C), and 16.08 min (D).

[0047] Fig.12 DEX-RGD-TAT-AT7 and integrin α in Example 5 v Binding response curve of β3.

[0048] Fig.13 DEX-RGD-TAT-AT7 in Example 5 targets and binds to integrin α expressed by HUVECs v β3.

[0049] Fig.14 The uptake of DEX-RGD-TAT-AT7 by HCEC cells in Example 6. A is a fluorescence graph; B is a cell number curve; C is a fluorescence intensity bar graph, compared with the blank group, **** P <0.0001; compared with DEX-RGD group, ####P <0.0001; compared with RGD-TAT-AT7 group, &&&& P <0.0001.

[0050] Fig.15 The uptake of DEX-RGD-TAT-AT7 by ARPE-19 cells in Example 6. A is a fluorescence graph; B is a cell number curve; C is a fluorescence intensity bar graph, compared with the blank group, **** P <0.0001; compared with DEX-RGD group, #### P <0.0001; compared with RGD-TAT-AT7 group, &&&& P <0.0001.

[0051] Fig.16 This is the penetration of FITC-DEX-RGD-TAT-AT7 through the simulated ocular barrier in vitro in Example 7.

[0052] Fig.17 The effect of DEX-RGD-TAT-AT7 on the proliferation of HUVECs in Example 8 (compared with the DEX group, *** P <0.001, **** P <0.0001; compared with DEX-RGD group, #### P <0.0001; compared with DEX-TAT-AT7 group, & P <0.05, &&& P <0.001, &&&& P <0.0001; compared with RGD-TAT-AT7, %%%% P <0.0001).

[0053] Fig.18 The effect of DEX-RGD-TAT-AT7 on HUVECs invasion in Example 8. A is a micrograph, with a scale of 50 μm; B is the statistical result of cell invasion rate. Compared with the blank group, ** P <0.01, *** P <0.001; compared with the DEX group, ## P <0.01, #### P <0.0001Compared with DEX-RGD group,&&&& P <0.0001, compared with the DEX-TAT-AT7 group, %% P <0.01.

[0054] Fig.19 The effect of DEX-RGD-TAT-AT7 on HUVECs tubule formation in Example 8. A is a micrograph, with a scale of 50 μm; B is the statistical result of the total length of tubule branches. Compared with the blank group, * P <0.05,** P <0.01,*** P <0.001; compared with the DEX group, ## P <0.01, compared with the DEX-RGD group, && P <0.01, compared with the DEX-TAT-AT7 group, % P <0.05.

[0055] Fig. 20 The release of inflammatory factors inhibited by DEX-RGD-TAT-AT7 in Example 9. A is the standard curve of TNF-α, B is the standard curve of IL-6, and C is the standard curve of iNOS. The release of TNF-α (D), IL-6 (E), and iNOS (F) of BV2 after incubation with different drugs for 24 h while LPS stimulation. Compared with the LPS group, *** P <0.001 **** , P <0.0001; compared with the DEX-RGD group, #### P <0.0001; compared with the DEX-TAT-AT7 group, && P <0.01, &&& P <0.001; compared with the RGD-TAT-AT7 group, %%% P <0.001, %%%% P <0.0001.

[0056] Fig.21 The inhibition of NO release by DEX-RGD-TAT-AT7 in Example 9. A is the standard curve of NO concentration and absorbance; B is the NO release of BV2 after incubation for 24 h with different drugs while stimulated by LPS. Compared with the LPS group, **** P <0.0001; compared with the DEX group,## P <0.01; compared with the DEX-RGD group, &&&& P <0.0001, compared with the DEX-TAT-AT7 group, %%% P <0.001; compared with the RGD-TAT-AT7 group, $$$$ P <0.0001.

[0057] Fig. 22 This is the hemolytic study of DEX-RGD-TAT-AT7 in Example 10. In A, from left to right, the drug concentrations are 1000, 500, 250, 100, 50, 25, 10, 5 and 2.5 μg / mL, "-" is the negative control group, and "+" is the positive control group. B is the absorbance of the supernatant detected by an ELISA instrument, and the hemolysis rate is calculated.

[0058] Fig.23 The effect of DEX-RGD-TAT-AT7 on mouse ear swelling in Example 11. Compared with the blank group, **** P <0.0001; compared with RGD-TAT-AT7 group, #### P <0.0001( n =8).

[0059] Fig.24 This is the ocular penetration of DEX-RGD-TAT-AT7 in Example 12.

[0060] Fig.25 The effect of DEX-RGD-TAT-AT7 on CNV thickness in Example 12. A is the OCT image of the fundus structure, and the OCT image comparison of the posterior segment of the normal eye and the posterior segment of the CNV model group; B is the OCT image of different drugs administered at 7 and 14 days after CNV modeling; C is the statistical analysis of OCT on CNV thickness. Compared with the Avastin group, * P <0.05, ** P <0.01, *** P <0.001, ns means no significant difference (n=3).

[0061] Fig.26 This is a scanned image of the RPE-choroidal flat mount of the rat tested for the effect of DEX-RGD-TAT-AT7 on CNV leakage area in Example 12.

[0062] Fig. 27This is a statistical graph of the effect of DEX-RGD-TAT-AT7 on CNV leakage area in Example 12. Compared with the blank group, * P <0.05, **** P <0.0001; compared with the DEX-RGD group, ## P <0.01, ### P <0.001, #### P <0.0001; ns indicates no significant difference between the Avastin group and the DEX-RGD-TAT-AT7 eye drop group (n=3). DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0064] Example 1 Synthesis and characterization of the conjugate DEX-RGD-TAT-AT7

[0065] (1) Synthesis and characterization of dexamethasone succinate (DEX-SA)

[0066] like Figure 1 As shown, 1.5 g of dexamethasone was dissolved in 20 mL of acetone and stirred until dissolved. 750 mg of succinic anhydride and 170 mg of DMAP (4-dimethylaminopyridine) were dissolved in the reaction system and stirred at room temperature for 4 h. The reaction was monitored by thin layer chromatography (TLC) with a developing solvent ratio of dichloromethane: methanol = 9:1. After monitoring that the reaction was basically complete, the reaction solution was dried by a rotary evaporator, and a light yellow oily viscous substance was observed to be attached to the bottom of the bottle.

[0067] Add anhydrous ethanol to the spin-dried product, shake and add until the oily viscous material is redissolved, then add 60°C distilled water until the precipitate is regenerated and suspended in the solution. At this time, the ratio of distilled water to anhydrous ethanol is approximately 29:71. Heat the solution with a spherical condenser and reflux until the precipitate is completely dissolved, and immediately transfer to 4°C for 24 hours.

[0068] The solution was taken out, and a white precipitate was observed. The liquid was filtered out using a suction filter, and the precipitate was washed three times with 3 mL of 25% ethanol and dried overnight in a 50°C oven. The obtained white solid powder was the product of this step. The yield was calculated by weighing, and its purity was tested by high performance liquid chromatography (HPLC). 1H-NMR and ESI-MS were used to calculate the molecular weight and confirm the structure. Figure 4 As shown in A and B.

[0069] (2) Synthesis and characterization of dexamethasone-maleimide conjugate (DEX-MAL)

[0070] like Figure 2 As shown, take 250 mg of DEX-SA, 2-(7-azabenzotriazole)- N,N,N',N Dissolve 290 mg of '-tetramethyluronium hexafluorophosphate (HATU) and 70 mg of 1-hydroxybenzotriazole (HOBT) in 10 mL of dichloromethane. N,N -Diisopropylethylamine (DIPEA) 450 μL was added to the reaction system and stirred at room temperature for 1 h to dissolve. N 125 mg of -(2-aminoethyl)maleimide trifluoroacetate (MAL) was added to the reaction system and stirred at room temperature for 12 h. After the reaction was basically complete as monitored by TLC, the reaction solution was dried using a rotary evaporator.

[0071] The dried product was redissolved in 3 mL of dichloromethane and purified by silica gel chromatography. The eluent used was methanol: dichloromethane = 1:25. The product was collected and lyophilized to obtain dexamethasone-maleimide conjugate DEX-MAL. The yield was calculated by weighing. The purity was tested by HPLC and the product was purified by 1 H-NMR and ESI-MS were used to calculate the molecular weight and confirm the structure. Figure 5 As shown in A and B.

[0072] (3) Synthesis and characterization of peptide-dexamethasone conjugates

[0073] DEX-RGD: Dissolve 20 mg of dexamethasone-maleimide conjugate (DEX-MAL) in 2 mL of acetonitrile, and dissolve 25 mg of peptide CGRGDS (as shown in SEQ ID NO.1, where C is thiolated cysteine) in 2 mL of PBS. Mix the two solutions and stir at room temperature for 2 h. Figure 3As shown. The reaction was monitored by analytical HPLC, the mobile phase was deionized water + 0.1% TFA, the mobile phase B was acetonitrile + 0.1% TFA, and the elution gradient was: 0 min, 90% A, 10% B; 10 min, 60% A, 40% B; 20min, 40% A, 60% B; 25 min, 20% A, 80% B. The UV detection wavelength was 220 nm, the injection volume was 50 μL, and the flow rate was 1mL / min. Confirm that the raw material peak has basically disappeared and the peak area of ​​the new product accounts for more than 90% of the total peak area, that is, the reaction is basically completed. The reaction solution was purified by preparative HPLC, the mobile phase A was deionized water + 0.1% TFA, the mobile phase B was acetonitrile + 0.1% TFA, and the elution gradient was: 0 min. 90% A, 10% B; 30 min, 60% A, 40% B; 60 min, 40% A, 60% B; 80 min, 20% A, 80% B. The UV detection wavelengths were 220 nm and 240 nm, and the flow rate was 6 mL / min. The main peak eluate collected in the preparative HPLC analysis was subjected to ESI-Q-TOF mass spectrometry to obtain the molecular weight of the eluate. The results are as follows: Figure 6 As shown in A.

[0074] DEX-TAT-AT7: Dissolve 12 mg of dexamethasone-maleimide conjugate (DEX-MAL) in 1 mL of acetonitrile, and dissolve 40 mg of peptide TAT-AT7 (RKKRRQRRRCATWLPPR, as shown in SEQ ID NO.5, where C is thiolated cysteine) in 2 mL of PBS. Mix the two solutions and stir at room temperature for 2 h. Figure 3 The reaction monitoring analysis and product purification preparation methods are the same as DEX-RGD. The obtained solution is freeze-dried and weighed to calculate the yield. The molecular weight is determined by ESI-Q-TOF, and the results are as follows Figure 6 As shown in B.

[0075] DEX-RGD-TAT-AT7: Dissolve 12 mg of dexamethasone-maleimide conjugate (DEX-MAL) in 1 mL of acetonitrile, and dissolve 50 mg of peptide RGD-TAT-AT7 (GRGDSRKKRRQRRRCATWLPPR, as shown in SEQ ID NO.4, where C is thiolated cysteine) in 2 mL of PBS. Mix the two solutions and stir at room temperature for 2 h. Figure 3 The reaction monitoring analysis and product purification preparation methods are the same as DEX-RGD. The obtained solution is freeze-dried and weighed to calculate the yield. The molecular weight is determined by ESI-Q-TOF, and the results are as follows Figure 6 As shown in C.

[0076] Example 2 Determination of critical micelle concentration of peptide-dexamethasone conjugate

[0077] The critical micelle concentration of the prepared nanomicelles was determined by the conductivity method. The three coupled nanomicelles were prepared into nanomicelle solutions with concentration gradients of 1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, 0.005, 0.0025, 0.001 and 0.0005 mg / mL, and 20 mL of each concentration solution was prepared. The conductivity meter probe was inserted into the solution, and the conductivity value was recorded when the reading was stable. The conductivity mutation point was measured as the critical micelle concentration, and the results are shown in Figure 7 As shown in A, B and C in Figure 3, the critical micelle concentrations of nanomicelles DEX-RGD, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 are 0.0494, 0.0808 and 0.0650 mg / mL, respectively, indicating that the three conjugates can self-assemble into nanomicelles in aqueous solution at lower concentrations.

[0078] Example 3 Preparation and characterization of physicochemical properties of peptide-dexamethasone conjugate nanomicelles

[0079] The freeze-dried DEX-RGD, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 powders were dissolved in deionized water to prepare aqueous solutions with concentration gradients of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.016 and 0.008 mg / mL. Nanomicelles were prepared by vortex ultrasound. The conjugate was first vortexed at a low speed for 10-30 s and then ultrasounded for 10 min. The prepared solutions were filtered with a 0.22 μm microporous membrane.

[0080] The morphology of the prepared nanomicelles was observed by transmission electron microscopy. Prepare a 4 mg / mL solution of DEX-RGD, DEX-TAT-AT7, and DEX-RGD-TAT-AT7 nanomicelles, then use tweezers to pick up the copper mesh and carefully immerse it in the nanomicelle solution so that the sample is loaded on both sides of the copper mesh. After the solution on the copper mesh membrane is fully dried, place the copper mesh covered with the sample at the front end of the sample rod and observe its morphology under a transmission electron microscope to evaluate whether the nanomicelles are successfully prepared.

[0081] The DEX-RGD, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 nanomicelle solutions with a concentration of 4 mg / mL were filtered through a 0.22 μm microporous membrane and added into the ZETA potential sample cell. The particle size and potential of the nanomicelle solutions were measured using a Malvern particle size analyzer.

[0082] The results of particle size analysis and Zeta potential determination are as follows Figure 8 As shown in A, B, and C, Fig. 9 As shown in A, B, C and Fig.10 As shown in A, B, and C in the figure, the particle sizes of the three nanomicelles are all around 100~200 nm and they are positively charged.

[0083] Example 4 Dissociation of Nanomicelles in Aqueous Humor in Vitro

[0084] (1) Establishment of standard curve

[0085] DEX-RGD-TAT-AT7 was prepared into a concentrated solution of 50 mmol / L, and then diluted into solutions with a concentration gradient of 50, 40, 30, 20, 10, 8, 6, 4, 2 and 1 mmol / L. 10 μL of each concentration of DEX-RGD-TAT-AT7 was placed in a 1.5 mL EP tube, 90 μL of rabbit aqueous humor was added, and then 300 μL of protein precipitant (acetonitrile: methanol = 9:1) was added immediately. The EP tube was transferred to a -80℃ refrigerator and frozen for 30 min to precipitate the protein in the aqueous humor. The solution was centrifuged at 12000 r / min for 30 min, the supernatant was taken, 800 μL of dichloromethane was added for extraction, the upper layer of liquid was taken, filtered through a 0.22 μm filter membrane, and detected by analytical HPLC. The content of the drug solution was quantified by the peak area of ​​the drug solution at different concentrations on HPLC.

[0086] (2) HPLC determination of the dissociation of DEX-RGD-TAT-AT7 nanomicelles in aqueous humor in vitro

[0087] DEX-RGD-TAT-AT7 was prepared into a 2 mmol / L aqueous solution. 100 μL of DEX-RGD-TAT-AT7 was placed in a 1.5 mL EP tube and 900 μL of aqueous humor was added. 100 μL of sample was taken at each time point: 0, 2, 5, 10, 20, 30, 45, 60, 90 and 120 min. 300 μL of protein precipitant was added immediately after sampling. The subsequent detection method was the same as (1). The main peak eluate of the DEX-RGD-TAT-AT7 sample taken at different time points was characterized by ESI-MS, and the peak time and peak area were analyzed.

[0088] The results are as follows Fig.11As shown in A, B, C and D in the figure, most of the drugs were metabolized within 3 hours after HPLC monitoring, and DEX-RGD-TAT-AT7 was broken from the connecting arm to dissociate the dexamethasone prototype drug and the peptide. This shows that the DEX-RGD-TAT-AT7 prepared in Example 1 can be broken in vivo through the connecting arm designed by us, and dexamethasone and peptide are released respectively to exert their respective therapeutic effects.

[0089] Example 5 DEX-RGD-TAT-AT7 against integrin α v β3(Integrin α v β3) targeting

[0090] (1) DEX-RGD-TAT-AT7 and integrin α v Affinity for β3

[0091] Microscale thermophoresis (MST) was used to detect the interaction between DEX-RGD-TAT-AT7 and integrin protein. First, the protein sample was prepared. The 10×HBS-EP buffer concentrate was diluted 10 times with double distilled water to 1×HBS-EP buffer, and an appropriate amount of integrin α v Add 1×HBS-EP buffer to the β3 protein solution to dilute the protein concentration to 100 nmol / L. Take 2 μL of the dye His-Tag Labeling Kit RED-tris-NTA, add 98 μL of HBS-EP and mix well to obtain a final volume of 100 μL of His-Tag Labeling Kit RED-tris-NTA. v Mix 90 μL of β3 solution and dye solution, and let stand at room temperature for 30 min to allow the protein and dye to fully combine. Centrifuge at 15000×g for 10 min, transfer the supernatant to a new EP tube, and store at -20℃ for later use.

[0092] DEX-RGD-TAT-AT7 was prepared into a 5 mmol / L concentrated solution using HBS-EP and then diluted to 2.5, 1.25, 6.25×10 -1 ,3.125×10 -1 , 1.563×10 -1 ,7.813×10 -2 ,3.906×10 -2 , 1.953×10 -2 ,9.766×10 -3 4.883×10 -3 , 2.441×10 -3 , 1.221×10-3 , 6.104×10 -4 ,3.052×10 -4 and 1.526×10 -4 mmol / L. Take 10 μL of each concentration of DEX-RGD-TAT-AT7 and place it in a 0.1 mL EP tube. Add integrin α labeled with RED-tris-NTA to each tube. v 10 μL of β3 solution was gently pipetted 10 times to mix thoroughly, incubated at room temperature for 20 min, the capillary was inserted into the EP tube solution, the capillary was gently tilted to take samples to avoid the formation of bubbles in the tube, and placed on the Monolith instrument for detection.

[0093] (2) DEX-RGD-TAT-AT7 and integrin α expressed by HUVECs v β3 binding

[0094] HUVECs cells in the logarithmic growth phase were taken, digested and centrifuged, resuspended in complete medium and counted, and the cell density was adjusted to 2×10 5 / mL, spread into the confocal dish, and incubate at 37℃ overnight. After the cells adhere to the wall, aspirate the original culture medium, add the culture medium containing FITC-labeled DEX-RGD-TAT-AT7 conjugate nanomicelles to make the drug concentration 20 μmol / L, and set up a control group, replace the original culture medium with a medium without drug, and incubate at 37℃ for 30 min. Take out the small dish and operate at room temperature and avoid light. Discard the culture medium, add 1 mL of PBS and gently wash 3 times, add 800 μL of 4% paraformaldehyde fixative, fix the cells at room temperature for 30 min, and gently wash again with PBS 3 times. Prepare a PBS solution containing 1% BSA and 0.5% Triton X-100 for blocking nonspecific binding, take 1 mL and add it to the small dish, and block at room temperature for 30 min. Dilute the APC-coupled antibody 25 times with the blocking solution, add 250 μL of the diluted antibody to each small dish, and incubate overnight at 4℃ in the dark. The next day, discard the liquid in the small dish, wash carefully with PBS three times, add 400 μL of DAPI staining solution to each small dish, stain at room temperature in the dark for 10 min, wash carefully with PBS again three times, finally add 100 μL of PBS to the dish to infiltrate the cells, and drop a drop of anti-fluorescence quencher. Observe the binding sites of fluorescent drugs on cells under a laser confocal microscope and take pictures.

[0095] DEX-RGD-TAT-AT7 and integrin α v The binding response curve of β3 is shown in Fig.12 As shown, it shows that the conjugate nanomicelle DEX-RGD-TAT-AT7 has an effect on integrin α vβ3 has targeting affinity, and its affinity (Kd) is 7.946×10 -4 mol.

[0096] DEX-RGD-TAT-AT7 and integrin α expressed in HUVECs v The result of β3 binding is as follows Fig.13 As shown, FITC-DEX-RGD-TAT-AT7 and integrin α expressed on HUVECs v β3 had a certain degree of colocalization, while FITC-DEX-TAT-AT7 without RGD coupling was partially directly transfected into the cell and did not bind to the cell membrane surface. The above results indicate that DEX-RGD-TAT-AT7 can bind to the receptor integrin α on the surface of HUVECs membrane. v β3 binding.

[0097] Example 6 The intracellular ability of DEX-RGD-TAT-AT7

[0098] (1) Observation of cellular uptake using laser confocal microscopy

[0099] HCEC cells and ARPE-19 cells in the logarithmic growth phase were digested and centrifuged, resuspended in complete medium, and the cell density was adjusted to 3×10 5 / mL, 1 mL of cell suspension per well was added to the confocal dish and incubated overnight at 37℃. After the cells adhered to the wall, a medium containing conjugate nano-micelles was prepared and grouped into FITC-DEX-RGD, FITC-DEX-TAT-AT7 and FITC-DEX-RGD-TAT-AT7. The original medium in the dish was aspirated and replaced with drug-containing medium. Another control group was set up and replaced with basal medium. Incubate at 37℃ for 4 h, remove the dish, and operate at room temperature and away from light. Discard the culture medium, add 1 mL of PBS and wash gently 3 times, add 800 μL of 4% paraformaldehyde fixative, fix the cells at room temperature for half an hour, and wash gently again with PBS 3 times after fixation. Add 400 μL of DAPI staining solution to each dish and stain for 10 min. After staining, wash gently with PBS 3 times, and add about 100 μL of PBS to soak the bottom of the dish. Take pictures under a laser confocal microscope, and analyze the fluorescence intensity with Image J software.

[0100] The qualitative observation results of DEX-RGD-TAT-AT7's ability to enter the cell are as follows Fig.14 A and Fig.15As shown in A, after the FITC-conjugated drugs were co-incubated with the cells for 1 h, HCEC cells and ARPE-19 cells both had obvious uptake of DEX-TAT-AT7 and DEX-RGD-TAT-AT7, with the DEX-RGD-TAT-AT7 group having the highest uptake, while the fluorescence intensity of the DEX-RGD group was very weak.

[0101] (2) Flow cytometry to detect cellular uptake

[0102] HCEC cells and ARPE-19 cells in the logarithmic growth phase were digested and centrifuged, resuspended in complete medium, and the cell density was adjusted to 5×10 5 / mL, added to a six-well plate, and incubated at 37°C overnight. The drug administration part is the same as the quantitative uptake experiment. After drug administration and incubation for 4 hours, the six-well plate was taken out, 400 μL of trypsin was added to each well for digestion for 2~3 minutes, and the digested cells were transferred to a 15 mL centrifuge tube and centrifuged at 2000 r / min for 3 minutes. After centrifugation, 2 mL of PBS was added to the cell pellet, and after gently vortexing, it was continued to be centrifuged at 2000 r / min for 3 minutes. After repeating the above PBS washing steps twice, 400 μL of PBS was finally added to resuspend the cell pellet, and the cells were filtered through a 70 μm cell mesh and transferred to a flow tube. The difference in the amount of fluorescence carried by each group of cells was detected by flow cytometry. The cell counting event was 10,000 and the flow rate was 10 μL / s. The results were analyzed using flow jo software.

[0103] The quantitative results of the intracellular ability of DEX-RGD-TAT-AT7 are shown in Fig.14 B and Fig.15 As shown in Figure B, the fluorescence intensity of the FITC-DEX-RGD-TAT-AT7 group in HCEC and ARPE-19 cells reached 60.64 times and 24.95 times that of the FITC-DEX-RGD group, respectively, which was significantly different from the DEX-TAT-AT7 group and the DEX-RGD group. The above results show that DEX-RGD-TAT-AT7 has a stronger ability to enter cells, and DEX-RGD-TAT-AT7 may have the best permeability among the three conjugate nanomicelles.

[0104] Example 7 DEX-RGD-TAT-AT7 penetrates cell barriers in vitro

[0105] HCEC cells and ARPE-19 cells in the logarithmic growth phase were digested and centrifuged, and then resuspended in MEM and DMEM / F12K complete medium to a cell density of 2×10 5 / mL. Prepare two 24-well plates, place the 24-well plate cell slides in the wells with tweezers, and inoculate the two types of cells in the 24-well plates at a volume of 400 μL per well, and incubate at 37°C for 24 h. Take out the 24-well plate with HCEC cells, aspirate the original culture medium, add 400 μL of FITC-labeled conjugate nanomicelles, group them into FITC-DEX-RGD, FITC-DEX-TAT-AT7 and FITC-DEX-RGD-TAT-AT7, so that the final drug concentration is 20 μmol / L, incubate at 37°C for 4 h, take out the slides of HCEC cells and ARPE-19 cells incubated with FITC conjugates, put them in a new six-well plate, add DMEM / F12K culture medium for mixed culture, so that each well has a HCEC cell slide after drug administration and an ARPE-19 cell slide without drug administration. Continue to culture at 37°C for 4 h. Aspirate the original culture medium in the six-well plate, wash gently 3 times with PBS, add 1 mL of 4% paraformaldehyde fixative, and fix the cells at room temperature for 30 min. Wash gently with PBS again 3 times, add 500 μL of DAPI staining solution to each well, and stain at room temperature in the dark for 10 min. Wash gently with PBS 3 times, carefully pick out the cell slide with tweezers, with the side with cells facing down, and slowly cover it on the slide with anti-fluorescence quencher to avoid bubbles. Observe the drug fluorescence intensity on the two cell slides under a laser confocal microscope.

[0106] The results of DEX-RGD-TAT-AT7 penetrating the cell barrier in vitro Fig.16 As shown, laser confocal microscopy showed that after 4 h of co-incubation of FITC-coupled drugs with cell slides covered with HCEC cells, both the DEX-TAT-AT7 group and the DEX-RGD-TAT-AT7 group showed good intracellular ability. After further co-incubation with ARPE-19 cells for 4 h, the uptake of the DEX-RGD-TAT-AT7 group was higher than that of the DEX-TAT-AT7 group. The above results show that DEX-RGD-TAT-AT7 can successfully enter cells through endocytosis and penetrate into other cells after exocytosis to achieve cell barrier penetration, and has better permeability than DEX-TAT-AT7, which suggests that DEX-RGD-TAT-AT7 may have a certain ability to penetrate the eye barrier.

[0107] Example 8 Evaluation of the anti-angiogenic activity of DEX-RGD-TAT-AT7 in vitro

[0108] (1) Effect of DEX-RGD-TAT-AT7 on endothelial cell proliferation

[0109] The CCK-8 method was used to determine the cell survival rate after cell administration. After cell digestion, the cells were resuspended in complete medium, and 10 μL of the cell suspension was placed on a cell counting plate. Cells were counted under a microscope. Following the rule of "recording the top but not the bottom, and counting the left but not the right", the total number of cells in the four large grids of the cell counting plate was divided by four to obtain the cell concentration (10 4 The cell concentration was diluted to 5×10 4 / mL, spread into a 96-well plate, and set up a blank group, without cells, only adding basal medium without cells. After 24 hours, the medium containing conjugate nanomicelles was prepared with F12K basal medium, and grouped into DEX, DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7, and DEX-RGD-TAT-AT7 groups. The concentration gradient of conjugate nanomicelles in each group was set to 320, 160, 80, 40, 20, and 10 μmol / L, filtered through a 0.22 μm microporous filter, and a control group was set, and basal medium without drug was added to the well. The medium in the 96-well plate was aspirated and replaced with 100 μL of drug-containing medium, and incubated in a 37°C incubator for another 24 hours. The original medium was aspirated, and a medium containing 10% CCK-8 was prepared with F12K basal medium, 100 μL per well, and incubated at 37°C for 1-4 hours. Take out the 96-well plate, measure its absorbance (OD value) at 450 nm with a microplate reader, and read the value. Calculate the cell viability using the following formula:

[0110] Cell inhibition rate (%) = 1-(OD of drug group-OD of blank group) / (OD of control group-OD of blank group) × 100%

[0111] The results of the effects of DEX-RGD-TAT-AT7 on endothelial cell proliferation are shown in Fig.17 As shown in the results, DEX and DEX-RGD groups had no significant effect on HUVECs cell proliferation, and the cell growth inhibition rate was about 10%. However, RGD-TAT-AT7, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 groups all showed a certain concentration-dependent anti-proliferation activity on HUVECs, and their cell proliferation inhibition rate showed an increasing trend with the increase of drug concentration, and the DEX-RGD-TAT-AT7 group had a higher cell proliferation inhibition rate. The above results show that compared with DEX-RGD, DEX-TAT-AT7 and RGD-TAT-AT7, DEX-RGD-TAT-AT7 has better activity in inhibiting endothelial cell proliferation.

[0112] (2) Effect of DEX-RGD-TAT-AT7 on cell invasion

[0113] Place the matrix gel at 4°C to melt one day in advance and place it on an ice box before the experiment. Prepare the matrix gel in a clean bench, dilute the matrix gel with F12K basal medium at a ratio of 5:1, and mix well. Take a 24-well plate, use tweezers to pick up the small chamber and place it in the 24-well plate, aspirate the diluted matrix gel, add 50 μL per well to the upper chamber, and oscillate while adding to make the matrix gel evenly cover the bottom of the small chamber. Transfer the 24-well plate with matrix gel to a 37°C incubator and incubate for 1~2 h until the matrix gel solidifies. Add 20% serum to the basal medium to obtain 20% complete medium, take HUVECs in the logarithmic growth phase, digest and resuspend, count the cells, and adjust the cell density to 3×10 5 / mL. Take out the 24-well plate, use a 10 μL pipette to aspirate the unsolidified matrix gel, add 600 μL of 20% complete medium to the lower chamber of Transwell, and then spread 100 μL of cell suspension into the chamber, and then transfer the 24-well plate to a 37°C incubator for further culture for 24 h. Add 100 μL of medium containing conjugate nanomicelles to each well of the chamber, and group them into DEX, DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7 and DEX-RGD-TAT-AT7 groups, so that the final concentration of nanomicelles is 40 μmol / L. Set up a control group, add 100 μL of basal medium to the chamber, and continue to culture for 24 h. Prepare the fixative solution with a ratio of methanol: glacial acetic acid = 3:1, take out the chamber, and rinse the chamber three times with pre-cooled PBS. Add 600 μL of fixative to a clean 24-well plate, transfer the chamber to the well with fixative, and fix at room temperature for 30 min. Rinse the chamber three times with PBS, add 600 μL of 0.1% crystal violet to the lower chamber, and stain at room temperature for 30 min. Wash with PBS three times again, gently wipe off the cells and matrix gel on the inside of the chamber with a cotton swab, take pictures under bright field with an inverted fluorescence microscope, analyze the number of cells that penetrate the chamber, and calculate the cell migration rate using the following formula:

[0114] Cell migration rate (%) = number of cells in the drug group / number of cells in the control group × 100%

[0115] The results of the effect of DEX-RGD-TAT-AT7 on cell invasion are as follows Fig.18 As shown in A and B, there was no significant difference in the cell invasion rate of the DEX group compared with the control group, while the differences between the other experimental groups and the control group were significant. The cell invasion rates of RGD-TAT-AT7 and DEX-RGD-TAT-AT7 were only 13.06% and 13.28%, respectively, which were significantly lower than those of the other groups. The above results show that compared with DEX-RGD and DEX-TAT-AT7 nanomicelles, DEX-RGD-TAT-AT7 has the best activity in inhibiting HUVECs cell invasion, and its inhibitory ability is equivalent to that of RGD-TAT-AT7.

[0116] (3) Effect of DEX-RGD-TAT-AT7 on tubule formation

[0117] One day in advance, 200 μL yellow pipette tips and 96-well plates were precooled at -20°C, and the matrix gel was melted at 4°C. The matrix gel was spread on ice in a clean bench. 50 μL of matrix gel melted at 4°C was added to each well of the 96-well plate, gently mixed, and the bubbles on the matrix gel were punctured with a sterile syringe needle. The plate was placed in a 37°C incubator and incubated for 2 h to wait for the matrix gel to solidify. HUVECs in the logarithmic growth phase were digested and resuspended to a cell concentration of 1×10 5 / mL. Take out the 96-well plate from the incubator, use a pipette to suck out the unsolidified matrix gel, and add 50 μL of cell suspension to each well. At the same time, add 150 μL of culture medium containing conjugate nanomicelles and 1 μL of VEGF A165 protein, and group them into DEX, DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7 and DEX-RGD-TAT-AT7 groups, so that the final concentration of nanomicelles is 40 μmol / L. Set up a control group, each well contains 50 μL of cell suspension, 150 μL of F12K basal culture medium and 1 μL of VEGF A165 protein. Continue to incubate in a 37℃ incubator for 4 h, take out the 96-well plate, take pictures under an inverted fluorescence microscope using bright field mode, and use Image J to calculate the number of new tubular structures.

[0118] The results of the effect of DEX-RGD-TAT-AT7 on tubule formation are shown in Fig.19 As shown in A and B in Figure 1, compared with the control group, the total branch length of blood vessels in DEX and DEX-RGD was slightly reduced, while the total branch length of blood vessels in the DEX-RGD-TAT-AT7 group was the lowest, and the differences with other drug-treated groups were significant. The above results indicate that DEX-RGD-TAT-AT7 can more significantly inhibit the angiogenesis of HUVECs.

[0119] Example 9 In vitro anti-inflammatory activity assay

[0120] (1) Establishment of cellular inflammation model

[0121] BV2 cells were cultured to the logarithmic growth phase and the cell density was adjusted to 6 × 10 5 / mL, spread into six-well plates, and incubate at 37℃ for 24 h. The original culture medium was aspirated, and LPS solution prepared with DMEM basal medium was added to each well to induce inflammation in the cells. At the same time, the culture medium containing the conjugate nanomicelles was added to make the final concentration of the nanomicelles 40 µmol / L and the final concentration of LPS 1 µg / mL. They were divided into blank group, LPS group, DEX group, RGD-TAT-AT7 group, DEX-RGD group, DEX-TAT-AT7 group and DEX-RGD-TAT-AT7 group. The LPS group was not added with drug-containing culture medium, only LPS was given, and 3 replicates were set up and cultured for 24 h. After the culture was completed, the cell supernatant was removed for further ELISA inflammatory factor detection and NO detection.

[0122] (2) ELISA to detect the release of inflammatory factors

[0123] 1) Collect the cell culture medium, centrifuge it at 1000×g for 20 min at 4°C to remove impurities and cell debris, and take the supernatant for detection.

[0124] 2) Take out the reagent kit from the refrigerator 20 min in advance and equilibrate to room temperature.

[0125] 3) Preparation of washing solution: dilute the concentrated washing solution with double distilled water at a ratio of 1:24.

[0126] 4) Preparation of standard working solution: Centrifuge the standard at 10,000 × g for 1 min, add 1 mL of standard & sample diluent to the lyophilized standard, tighten the tube cap, let it stand for 10 min, invert it several times, and after it is fully dissolved, mix gently to avoid foaming. Then dilute the standard by serial dilution to prepare the following concentrations: IL-6: 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL; TNF-α: 500, 250, 125, 62.5, 1.25, 15.63, 7.81, and 0 pg / mL; iNOS: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0 ng / mL.

[0127] 5) Set up standard wells, blank wells, and sample wells respectively. Add 100 μL of the diluted standard to the standard wells, 100 μL of the standard & sample diluent to the blank wells, and 100 μL of the sample to be tested to the remaining wells. When adding samples, add the samples to the bottom of the ELISA plate, try not to touch the well wall, and gently shake to mix to avoid bubbles. Cover the ELISA plate with film and incubate at 37°C for 90 min.

[0128] 6) Preparation of biotinylated antibody working solution / HRP enzyme conjugate working solution: Calculate the required amount for the experiment before the experiment (calculated as 100 μL / well). When preparing, prepare 100~200 μL more. 15 minutes before use, centrifuge the concentrate at 800×g for 1 minute, and dilute the 100× concentrate to 1× working concentration with diluent (for example: 10 μL concentrate + 990 μL diluent). Prepare and use immediately.

[0129] 7) Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37℃ for 1 h.

[0130] 8) Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing solution to each well, soak for 1 min, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times.

[0131] 9) Add 100 μL of HRP conjugate working solution to each well, cover the plate with film, and incubate at 37°C for 30 min.

[0132] 10) Shake off all liquid in the wells and wash the plate 5 times using the same method as step (8).

[0133] 11) Add 90 μL of substrate solution (TMB) to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for 15 min.

[0134] 12) Add 50 μL of stop solution to each well to terminate the reaction. The order of adding the stop solution should be the same as that of adding the substrate solution.

[0135] 13) Immediately measure the absorbance (OD value) of each well at 450 nm using an ELISA reader. Calculate the concentrations of TNF-α, IL-6, and INOS in the sample based on the standard curve.

[0136] (3) Detection of NO concentration

[0137] 1) Take out Griess Reagent I and II and return them to room temperature.

[0138] 2) Dilute the standard with DMEM basal medium to the following concentrations: 0, 1, 2, 5, 10, 20, 40, 60 and 100 μmol / L.

[0139] 3) Add standards and samples to a 96-well plate at 50 μl / well.

[0140] 4) Add 50 μl / well of room temperature Griess Reagent I to each well.

[0141] 5) Add 50 μl / well of room temperature Griess Reagent II to each well.

[0142] 6) Measure the absorbance (OD value) at a wavelength of 540 nm using an enzyme-labeled instrument.

[0143] 7) Calculate the concentration of nitric oxide in the sample based on the standard curve.

[0144] The results of the effects of DEX-RGD-TAT-AT7 on the release of inflammatory factors are as follows Fig. 20 A, B, C, D, E, F and Fig.21 As shown in A and B in the figure, in the determination of TNF-α, IL-6, and iNOS, there was almost no release of inflammatory factors in the blank control group, while after LPS stimulation, the release of inflammatory factors increased significantly, indicating that the LPS-stimulated inflammatory model was successfully constructed. The release of inflammatory factors in the drug-treated groups was lower than that in the LPS group, among which the DEX group as the positive control group had a more obvious trend of inhibiting the release of inflammatory factors, and the concentrations of TNF-α, IL-6, and iNOS in DEX-RGD-TAT-AT7 were reduced by 94.6%, 92.46%, and 99.15% respectively compared with the LPS stimulation group, and there was no significant difference with the positive control DEX group. In the measurement of NO release, the NO release in the DEX-RGD-TAT-AT7 group decreased by 79.85% compared with the LPS stimulation group, which was better than other drug-treated groups and the DEX group. The above results show that DEX-RGD-TAT-AT7 can inhibit the release of inflammatory factors in BV2 cells stimulated by LPS in vitro, and has more significant activity in inhibiting the release of cellular inflammatory factors compared with DEX-RGD, DEX-TAT-AT7 and RGD-TAT-AT7, and retains anti-inflammatory activity similar to DEX.

[0145] Example 10 Study on the hemolytic activity of DEX-RGD-TAT-AT7

[0146] Take 1 mL of fresh rabbit blood, add it to an EP tube containing glass beads and shake for 10 min, add 10 mL of physiological saline (0.9% NaCl solution), shake well, centrifuge at 2000 r / min for 10 min, discard the supernatant, keep the red blood cell pellet, continue to add physiological saline, repeat washing 2~3 times until the supernatant is no longer red. Resuspend the obtained red blood cell pellet with 50 mL of physiological saline to prepare a 2% red blood cell suspension.

[0147] The three peptide-dexamethasone conjugate nanomicelles were formulated into saline solutions with a concentration gradient of 1000, 500, 250, 100, 50, 25, 10, 5 and 2.5 μg / mL. Double distilled water was used as the positive control group and saline was used as the negative control group. Take a 1.5 mL EP tube, add 500 μL of the prepared drug solution and 500 μL of 4% red blood cell suspension to each tube, and mix well. Place the EP tube in a 37°C water bath and incubate for 1 h, and then observe the hemolysis phenomenon. Take 100 μL of the supernatant from each tube into a 96-well plate, and use an enzyme reader to measure the absorbance value (OD value) at a wavelength of 540 nm. Calculate the hemolysis rate using the following formula:

[0148] Hemolysis rate = (OD of drug administration group - OD of negative control group) / (OD of drug administration group - OD of positive control group) × 100%

[0149] The hemolytic test results of DEX-RGD-TAT-AT7 are as follows Fig. 22 As shown in A and B in Figure 1, DEX-TAT-AT7, RGD-TAT-AT7 and DEX-RGD-TAT-AT7 between 100 and 1000 μg / mL had slight hemolysis, but the hemolysis rate was less than 5%. The above results show that DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7 and DEX-RGD-TAT-AT7 have weak hemolytic activity and good in vivo safety, and can be further used in in vivo experiments.

[0150] Embodiment 11 In vivo anti-inflammatory activity of DEX-RGD-TAT-AT7

[0151] Forty-eight Kunming mice aged 6 to 8 weeks were prepared, with a male-female ratio of 1:1, and randomly divided into 6 groups, 8 mice in each group. Each group was gavaged with normal saline, DEX, DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7, and DEX-RGD-TAT-AT7 for 7 consecutive days. One hour after the last administration on the 7th day, 20 μL of TPA dissolved in acetone was applied to the right ear of each mouse to induce inflammation, and acetone was applied to the left ear as a control. After 4 hours, the mice were killed, the ears were cut off along the auricle, and a disc with a diameter of 8 mm was punched in the same position of the left and right ears with a puncher, and weighed. The swelling degree was calculated using the following formula:

[0152] Swelling degree (mg) = right ear piece weight - left ear piece weight.

[0153] In vivo anti-inflammatory activity results of DEX-RGD-TAT-AT7 Fig.23As shown, compared with the average swelling of 11.99 mg in the control group, the swelling of the mouse ears in the RGD-TAT-AT7 group did not decrease, while the swelling of the mouse ears in the DEX-RGD, DEX-TAT-AT7 and DEX-RGD-TAT-AT7 groups decreased by 92.18%, 68.2% and 79.67% respectively compared with the control group, which was close to 81.96% in the DEX group. This indicates that the peptide-dexamethasone conjugate micelles can play an anti-inflammatory role similar to dexamethasone, among which DEX-RGD and DEX-RGD-TAT-AT7 have the most significant effect in inhibiting mouse ear swelling.

[0154] Example 12 In vivo anti-neovascular ocular activity of DEX-RGD-TAT-AT7

[0155] (1) Construction of rat choroidal neovascularization model

[0156] Twenty-four brown Norwegian rats weighing 180-200 g were prepared and randomly divided into eight groups. They were anesthetized by intraperitoneal injection of 2 mL of Avertin injection, and a drop of compound tropicamide eye drops was dripped into the right eye to dilate the pupil. The whiskers on both sides were cut off. After the right eye of the rat was observed to be dilated under strong light, it was placed on the mandibular support of the laser therapy device so that the pupil of the right eye was facing the laser emission port. A cover glass was placed against the cornea on the ocular surface, and a krypton laser transmitter was used to photocoagulate 8 points around the optic disc of the fundus. The laser power was 200 mW, the exposure time was 50 ms, and the spot diameter was 80 μm. Focusing on the Bruch's membrane, bubbles were seen or accompanied by slight bleeding, indicating that the Bruch's membrane was broken and was an effective photocoagulation point. No intervention was performed on the left eye.

[0157] (2) Ocular penetration of DEX-RGD-TAT-AT7

[0158] After the CNV model was established, FITC-labeled nanomicelles were used for drug administration and the random groups were divided into groups for administration as follows:

[0159] 1) Negative control group: normal saline, eye drops, 20 μL, 6 times / d, for 2 days;

[0160] 2) Positive control group: subconjunctival injection of FITC-RGD-TAT-AT7, 2 μL, single administration;

[0161] 3) FITC-DEX-RGD: eye drops, 20 μL, 6 times / d, for 2 days;

[0162] 4) FITC-DEX-TAT-AT7: eye drops, 20 μL, 6 times / d, for 2 days;

[0163] 5) FITC-DEX-RGD-TAT-AT7: eye drops, 20 μL, 6 times / d, for 2 days;

[0164] After the last administration, the right eye of the rat was immediately removed and immersed in an EP tube containing eyeball fixative for 24 h at room temperature. The eyeball was treated with sucrose solution. First, the eyeball was placed in a 15% sucrose solution, then transferred to a 30% sucrose solution, and continued to dehydrate and sink to the bottom at 4°C. The eyeball was rinsed twice with PBS, transferred to a slice box, and placed on ice. The embedding agent OCT was dripped around the eyeball. After the OCT turned white and hardened, a freezing slicer was used for frozen sectioning with a thickness of 8 μm. The slide with the slice was placed in PBS, the OCT embedding agent was washed off, and it was fixed with 4% paraformaldehyde at room temperature for 25 min. The slices were washed 3 times with PBS. DAPI staining solution was dripped on the slices and stained at room temperature in the dark for 10 min. The slices were washed 3 times with PBS for 10 min each time, and anti-fluorescence quenching sealing agent was dripped on the slides, and the slides were covered and sealed. The fluorescence intensity of the sections was observed using a laser confocal microscope, and the fluorescence was quantitatively analyzed using Image J software.

[0165] The ocular penetration results of DEX-RGD-TAT-AT7 are as follows Fig.24 As shown in the figure, DEX-RGD, DEX-TAT-AT7, RGD-TAT-AT7 and DEX-RGD-TAT-AT7 all have certain eye penetration, can penetrate from the anterior segment of the eye into the posterior segment of the eye, and after being delivered to the choroid, i.e. the outer side of the retina, continue to penetrate the RPE layer to reach the inner retinal layer. Compared with the DEX-RGD, DEX-TAT-AT7 and RGD-TAT-AT7 groups, the fluorescence of the posterior segment of the eye in the DEX-RGD-TAT-AT7 group with eye drops was the strongest, indicating that the amount of drug reaching the posterior segment of the eye was the highest; the subconjunctival injection of the DEX-RGD-TAT-AT7 group also showed strong fluorescence in the posterior segment of the eye. The above results show that DEX-RGD-TAT-AT7 has certain eye penetration, and the penetration effect of eye drops is not inferior to direct subconjunctival injection, and further subsequent pharmacodynamic studies can be carried out.

[0166] (3) Anti-angiogenic activity of DEX-RGD-TAT-AT7

[0167] The drug administration started from the next day after the CNV model was established, and the drugs were randomly divided into groups and administered as follows:

[0168] 1) Negative control group: normal saline, eye drops, 20 μL, 6 times / d, for 14 days;

[0169] 2) Positive control group: intravitreal injection, bevacizumab, 2 μL, 7 days / time;

[0170] 3) DEX-RGD: eye drops, 20 μL, 6 times / d, for 14 days;

[0171] 4) DEX-TAT-AT7: eye drops, 20 μL, 6 times / d, for 14 days;

[0172] 5) RGD-TAT-AT7: eye drops, 20 μL, 6 times / d, for 14 d;

[0173] 6) DEX-RGD-TAT-AT7: eye drops, 20 μL, 6 times / d, for 14 days;

[0174] 7) DEX-RGD-TAT-AT7: subconjunctival injection, 2 μL, 7 days / time.

[0175] On the 7th and 14th days of administration, optical coherence tomography (OCT) was performed on the rats' eyes. The specific operation was as follows: 0.2 mL of Shutai was injected intramuscularly to anesthetize the rats, and compound tropicamide eye drops were dripped into the left and right eyes to dilate the pupils. After the mydriasis was completed, sodium hyaluronate eye drops were dripped to moisten the cornea, and the eyeballs were rubbed to remove excess reagents. The rat pupil was aligned with the OCT lens, and the OCT software was used to perform linear scans of the CNV lesion in the right eye and the normal retina in the left eye to calculate the CNV thickness.

[0176] After the last administration, the rats were killed and choroidal flat sheets were prepared. First, the left and right eyeballs of the rats were removed with forceps, and about 5 mm of the optic nerve was enclosed. They were immersed in eyeball fixative and fixed at room temperature for 20-30 min. The eyeballs were rinsed twice with PBS, immersed in a small dish containing PBS, and transferred to a stereomicroscope for operation. The connective tissue and optic nerve around the eyeball were carefully cut with ophthalmic scissors and ophthalmic forceps. The ophthalmic scissors were used to cut a circle around the posterior side of the ciliary body, and the lens and cornea were pushed out with ophthalmic forceps. The retina on the choroid of the remaining posterior half of the eye cup was carefully removed with ophthalmic forceps to obtain the choroid-sclera tissue complex. The ophthalmic scissors were used to cut four times radially along the center of the optic disc to make it unfold into a petal shape. The removed choroid-sclera tissue was transferred to 5% BSA blocking solution and blocked at 4℃ for 24 h. The choroid was rinsed with PBS, shaken for 10 min, and repeated 3 times. Dilute FITC-conjugated isolectin B4 to 20 μg / mL with PBS, transfer the choroid-sclera tissue to the staining solution, and stain at room temperature for 24 h. Rinse again with PBS for 3 times as in the previous steps, add 10 μL of anti-fluorescence quencher on a clean slide, absorb excess water on the choroid-sclera tissue, transfer to a slide, cover with a coverslip, and seal with an anti-fluorescence quencher to minimize bubbles in the slice. The slices can be stored at -20°C for one month. Use a panoramic scanning microscope to perform fluorescence scanning on the slices, and use Image J software to calculate the CNV area.

[0177] The results of anti-angiogenic activity of DEX-RGD-TAT-AT7 are as follows Fig.25 A, B, C and Fig.26 , Fig. 27 As shown in the figure, after 14 days of administration, the CNV lesions in each administration group were slightly improved compared with the CNV lesions at 7 days of administration. Among them, the CNV thickness of the DEX-RGD-TAT-AT7 group and the subconjunctival injection DEX-RGD-TAT-AT7 group was significantly reduced compared with the control group and the DEX-RGD group. At the same time, there was no significant difference between the DEX-RGD-TAT-AT7 group and the bevacizumab group. In the statistics of the neovascularization area, the neovascularization area of ​​all administration groups was reduced compared with the control group. Among them, the DEX-RGD-TAT-AT7 group and the subconjunctival injection DEX-RGD-TAT-AT group had more significant improvement in CNV leakage than other groups, and the CNV area was 14.13 mm higher than that of the control group. 2The results showed that the eye drops of DEX-RGD-TAT-AT7 can improve the thickness of CNV lesions and the area of ​​choroidal neovascularization, and have a good anti-angiogenic effect on CNV, and the efficacy is almost equivalent to that of the subconjunctival injection group.

[0178] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide-dexamethasone conjugate, characterized in that: Select from any of the following chemical formulas: 、 、 。 2. The method for preparing a polypeptide-dexamethasone conjugate according to claim 1, characterized in that: Dexamethasone was esterified with succinic anhydride to obtain DEX-SA. N -(2-aminoethyl)maleimide is subjected to an amidation condensation reaction to obtain DEX-MAL, and DEX-MAL is subjected to an addition reaction with POI-SH to obtain the obtained product; Among them, the chemical structure formula of DEX-SA is: ; The chemical structure of DEX-MAL is: POI-SH is selected from , , .

3. The method for preparing a polypeptide-dexamethasone conjugate according to claim 2, characterized in that: Dexamethasone was reacted with succinic anhydride and 4-dimethylaminopyridine at room temperature to obtain DEX-SA.

4. The method for preparing a polypeptide-dexamethasone conjugate according to claim 2, characterized in that: Using 2-(7-azabenzotriazole)- N,N,N',N' -Tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N -Diisopropylethylamine was used to activate the carboxylic acid in DEX-SA, and then added N -(2-aminoethyl)maleimide was subjected to amidation condensation reaction to obtain DEX-MAL.

5. The method for preparing a polypeptide-dexamethasone conjugate according to claim 2, characterized in that: The conditions for the addition reaction of DEX-MAL and POI-SH are: reaction at room temperature for 1.5 to 3 h.

6. A pharmaceutical composition, characterized in that: The invention comprises the polypeptide-dexamethasone conjugate according to claim 1, and pharmaceutical excipients.

7. A pharmaceutical preparation, characterized in that: It comprises nano micelles, wherein the nano micelles are formed by the polypeptide-dexamethasone conjugate according to claim 1.

8. Use of the polypeptide-dexamethasone conjugate according to claim 1, the pharmaceutical composition according to claim 6 or the pharmaceutical preparation according to claim 7 in the preparation of a drug for treating neovascular eye diseases.

Images