Anti-inflammatory and anti-neovascular dual-targeting nanoparticles based on tipe2 expression regulation and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-inflammatory and anti-neovascular dual-target nanoparticle based on expression regulation of TIPE2, and a preparation method and application thereof. The application connects and modifies TAT-AT7 peptide on PBAE with acrylate at the end, endows the PBAE with neovascular targeting and enhances the tissue penetration of the PBAE, and then electrostatically adsorbs the expression plasmid containing the TIPE2 coding sequence to self-assemble in an aqueous solution, so as to obtain the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticle for regulating the expression of TIPE2. The dual-target nanoparticle prepared by the application targets the neovascularization of the fundus, regulates the expression of TIPE2, adjusts the immune response, has good penetration and safety, exhibits good anti-neovascular and anti-inflammatory effects in the body, is suitable for eye drop administration for treating the neovascularization of the fundus, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-target nanoparticle with anti-inflammatory and anti-angiogenic properties based on TIPE2 expression regulation, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Retinal neovascularization is an abnormal proliferation of blood vessels caused by retinal ischemia, hypoxia, inflammation, or genetic factors. Due to structural defects, these new blood vessels are prone to hemorrhage and exudation, ultimately leading to vision loss or even blindness. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) is currently the most common treatment for retinal neovascularization. This therapy requires repeated invasive procedures and may lead to complications such as endophthalmitis, retinal detachment, cataracts, and intraocular pressure. Some patients may experience non-response, relapse, or tolerance, and frequent injections can impose a heavy psychological and economic burden on families and society. Furthermore, VEGF is an important neurotrophic factor; long-term use of anti-VEGF treatment may lead to retinal atrophy or subretinal fibrosis, highlighting the urgency of finding new treatment methods. Research has confirmed that retinal neovascularization is not only related to VEGF overexpression but also closely related to inflammation. Under pathological conditions, microglia in the retina are activated, proliferate, and migrate, and macrophage infiltration increases. Both release large amounts of inflammatory factors, disrupting the blood-retinal barrier and causing tissue damage. Therefore, multi-target regulation beyond anti-angiogenesis is the treatment trend for this type of disease.

[0004] In the field of gene delivery, plasmid DNA struggles to cross cell membranes autonomously and is easily degraded by lysosomes after endocytosis. Viral gene vectors present challenges such as immunogenicity, packaging capacity, potential insertion risks, complex preparation, and high cost. Recent studies have shown that gene therapy using adeno-associated viruses in the eye may trigger inflammatory responses and innate or adaptive immune responses to transgenic products or vector capsids. Non-viral gene vectors such as PBAEs offer advantages such as good biocompatibility, high stability, and no immunogenicity; however, multiple barriers in the eye, including the tear film barrier, corneal barrier, conjunctival barrier, blood-aqueous humor barrier, lens barrier, and blood-retinal barrier, prevent existing PBAE vectors from delivering nucleic acids to the posterior segment of the eye via non-invasive drug delivery methods, necessitating effective solutions.

[0005] Therefore, developing a dual-target nanoparticle that combines penetrability and safety with anti-inflammatory and anti-angiogenic properties to achieve non-invasive treatment of retinal neovascularization is a key need to solve the current treatment dilemma. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a dual-target nanoparticle with anti-inflammatory and anti-angiogenic properties based on TIPE2 expression regulation, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing anti-inflammatory and anti-angiogenic dual-target nanoparticles based on TIPE2 expression regulation, comprising the following steps:

[0009] (1) 1,4-Butanediol diacrylate and 4-amino-1-butanol were mixed at a mass ratio of 1~1.5:1 and subjected to Michael addition reaction. After washing, centrifugation and freeze-drying, PBAE with acrylate groups at the end was obtained.

[0010] (2) The PBAE containing acrylate groups at the end and the TAT-AT7 peptide containing thiol groups are reacted in DMSO at a mass ratio of 1:1~2, and then washed, centrifuged and lyophilized to obtain the drug carrier PBAE-TAT-AT7; the amino acid sequence of the TAT-AT7 peptide is RKKRRQRRRCATWLPPR (SEQ ID NO.1).

[0011] (3) The PBAE-TAT-AT7 and the plasmid that regulates TIPE2 expression are vortexed in an aqueous solution at a mass ratio of 5~100:1. After standing, the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles based on TIPE2 expression regulation are obtained. The coding sequence of the plasmid regulating TIPE2 expression is shown in SEQ ID NO.2.

[0012] Secondly, the present invention provides anti-inflammatory and anti-angiogenic dual-target nanoparticles based on TIPE2 expression regulation prepared by the above preparation method.

[0013] Thirdly, this invention provides the application of the aforementioned TIPE2 expression-regulated dual-target nanoparticles for both anti-inflammatory and anti-angiogenic purposes. This application is specifically the use of these TIPE2 expression-regulated dual-target nanoparticles in the preparation of a drug for treating retinal neovascularization. The specific dosage can be determined using conventional methods in the art, depending on the intended use of the pharmaceutical composition.

[0014] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0015] 1. The TIPE2 expression-regulated dual-target nanoparticles prepared in this invention have the ability to target retinal neovascularization and have the dual functions of anti-inflammatory and anti-angiogenic effects, providing a new strategy and approach for the treatment of retinal neovascularization.

[0016] 2. The anti-inflammatory and anti-angiogenic dual-target nanoparticles based on TIPE2 expression regulation prepared in this invention have good tissue penetration and safety. They can be administered via eye drops, achieving non-invasive treatment of fundus neovascularization. They are highly safe, have good compliance, and have high clinical translational value.

[0017] 3. The nanoparticle preparation method of the present invention is simple and mild, suitable for large-scale production, and has good reproducibility. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 This is a schematic diagram of the preparation process of the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles in Example 1 of the present invention.

[0020] Figure 2 This is the structural formula of the TAT-AT7 peptide in Example 1 of the present invention.

[0021] Figure 3 It is the structural formula of PBAE-TAT-AT7 in Embodiment 1 of the present invention.

[0022] Figure 4 This is the proton nuclear magnetic resonance spectrum of PBAE in Example 2 of the present invention.

[0023] Figure 5 This is the proton NMR spectrum of PBAE-TAT-AT7 in Example 2 of the present invention.

[0024] Figure 6 This is an agarose gel electrophoresis result of the dual-target nanoparticles of the PBAE-TAT-AT7 / TIPE2 plasmid in Example 3 of the present invention.

[0025] Figure 7 This is a transmission electron microscope image of the dual-target nanoparticles of the PBAE-TAT-AT7 / TIPE2 plasmid in Example 3 of the present invention.

[0026] Figure 8This is a particle size distribution diagram of the dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid in Example 3 of the present invention.

[0027] Figure 9 This is an in vitro transfection effect diagram of the dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid in Example 4 of the present invention.

[0028] Figure 10 This is an in vitro cytotoxicity diagram of the dual-target nanoparticles of the PBAE-TAT-AT7 / TIPE2 plasmid in Example 5 of the present invention.

[0029] Figure 11 This is an in vivo penetration effect diagram of the dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid in Example 6 of the present invention.

[0030] Figure 12 This is a diagram showing the safety evaluation results of the dual-target nanoparticles of the PBAE-TAT-AT7 / TIPE2 plasmid in Example 7 of the present invention.

[0031] Figure 13 This is a diagram showing the angiogenesis inhibition results of the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles in Example 8 of the present invention.

[0032] Figure 14 This is a diagram showing the inflammation regulation results of the dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid in Example 8 of the present invention. Detailed Implementation

[0033] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The TAT-AT7 peptide is composed of the cell-penetrating peptide TAT and the vascular-targeting peptide AT7 linked together. The AT7 peptide specifically recognizes the neurocilia 1 receptor and inhibits its binding to VEGF. The TAT peptide effectively enhances tissue permeability, thus the TAT-AT7 peptide exhibits excellent tissue penetration and angiogenesis targeting. This invention utilizes a thiol-alkene click reaction between a carbon-carbon double bond and a thiol group to link a terminal acrylate-containing PBAE with a thiol-containing TAT-AT7 peptide. This allows the nanoparticles to overcome multiple physiological barriers of the eye, delivering the drug to the posterior segment lesion site and inhibiting angiogenesis.

[0035] Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is a key immunomodulatory factor that negatively regulates the function of Toll-like receptors, inhibits signaling pathways such as STAT3, NF-κB, JNK, and p38MAPK, suppresses macrophage and microglia polarization, and reduces the expression of inflammatory cytokines and VEGF. In in vitro cell experiments, under inflammatory or hyperglycemic stimulation, TIPE2 expression in retinal microvascular endothelial and pigment epithelial cells is downregulated, while the levels of VEGF, TNF-α, and IL-1β are increased. Furthermore, TIPE2 deficiency leads to retinal hypoperfusion and retinal thinning in mice, suggesting that TIPE2 plays an important role in maintaining retinal homeostasis.

[0036] The technical solution of the present invention will be further described below with reference to specific embodiments. In the following embodiments, the plasmid regulating TIPE2 expression (pcDNA3.1(+)-TNFAIP8L2-3xFLAG-P2A-EGFP, 6838bp) and the control empty vector plasmid (pcDNA3.1(+)-3xFLAG-P2A-EGFP, 6283bp) were purchased from Shanghai Heyuan Biotechnology (Group) Co., Ltd., and the coding sequence for regulating TIPE2 expression is shown in SEQ ID NO.2. The TAT-AT7 peptide was purchased from Nanjing Detai Biotechnology Co., Ltd. All other reagents were commercially available.

[0037] Example 1: Preparation of dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid

[0038] 0.89 g of 4-amino-1-butanol and 2.002 g of 1,4-butanediol diacrylate were mixed and stirred in an oil bath at 90 °C for 24 hours to carry out a Michael addition reaction. The product was washed three times with 5-10 times its volume of cold ether and then lyophilized to obtain PBAE. 60 mg of PBAE was dissolved in DMSO, and 60 mg of TAT-AT7 peptide was added. The mixture was stirred at room temperature for 12 hours. The product was washed three times with 5-10 times its volume of cold acetone / ether mixture and then lyophilized to obtain PBAE-TAT-AT7. 50 mg of PBAE-TAT-AT7 was dissolved in 100 μL of DMSO, and pTIPE2 was added at mass ratios of 5:1, 25:1, 50:1, 75:1, and 100:1, respectively. The mixture was vortexed and allowed to stand for 20 minutes to obtain PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles based on TIPE2 expression regulation. The preparation process is as follows. Figure 1 As shown, the structural formula of the TAT-AT7 peptide is as follows: Figure 2 As shown, the structural formula of PBAE-TAT-AT7 is as follows:Figure 3 As shown.

[0039] Example 2: Characterization of PBAE-TAT-AT7 nanocarriers

[0040] The prepared PBAE and PBAE-TAT-AT7 were dissolved in deuterated methanol and characterized using nuclear magnetic resonance spectroscopy. The 1H NMR spectra are shown below. Figure 4 and Figure 5 As shown, the peak at δ 5.89–6.42 ppm corresponds to the proton signal of the acrylate double bond, confirming the successful synthesis of PBAE. The disappearance of the peak at δ 5.89–6.42 ppm after reaction with TAT-AT7, and the peak at δ 7.01–7.59 ppm corresponding to the aromatic proton signal, confirm the successful synthesis of PBAE-TAT-AT7.

[0041] Example 3: Characterization of dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid

[0042] A 1% agarose gel was prepared, and the compressive encapsulation ability of PBAE-TAT-AT7 on the TIPE2 plasmid was detected by electrophoresis. The results are as follows: Figure 6 As shown, PBAE-TAT-AT7 at different mass ratios significantly inhibited plasmid migration in an electric field. The morphology of nanoparticles with a mass ratio of 75:1 was observed using transmission electron microscopy, and the results are as follows. Figure 7 As shown, the particles have a regular morphology, are nearly spherical, and their size meets the requirements for nanomaterials. The particle size distribution of nanoparticles with a mass ratio of 75:1 was determined using a particle size analyzer, and the results are as follows. Figure 8 As shown.

[0043] Example 4: In vitro transfection efficiency of dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid

[0044] The in vitro transfection efficiency of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles was evaluated using commonly used cell transfection reagents PEI and Lipo 2000 as controls. The main experimental steps were as follows: Human retinal epithelial cell line (ARPE-19) was uniformly seeded in 6-well plates and cultured for 24 hours. The original culture medium was discarded, and 10% FBS complete medium without antibiotics was added. Wells containing equal amounts of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles (75:1), PEI gene mixture, and Lipo 2000 gene mixture were added. The medium in wells containing Lipo 2000 was changed after 4 hours. After 48 hours of culture, green fluorescence signals were observed under a fluorescence microscope. Results are as follows: Figure 9As shown, the transfection efficiency of the dual-target nanoparticles of PBAE-TAT-AT7 / TIPE2 plasmid is significantly higher than that of PEI and lipo 2000.

[0045] Example 5: Cytotoxicity of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles

[0046] Using commonly used cell transfection reagents PEI and lipo 2000 as controls, the cytotoxicity of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles against ARPE-19 cells was evaluated by a CCK-8 cytotoxicity assay. The main experimental steps were as follows: ARPE-19 cells were uniformly seeded in 96-well plates and cultured for 24 hours. The original culture medium was discarded, and 10% FBS complete medium without antibiotics was added. Wells containing equal amounts of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles (75:1), PEI gene mixture, and lipo 2000 gene mixture were added. The medium in wells containing lipo 2000 was changed after 4 hours. After 48 hours of culture, the original culture medium was discarded, and complete culture medium and CCK-8 solution were added. The plates were incubated at 37°C, and the absorbance was measured using a microplate reader. Results are as follows: Figure 10 As shown, the PBAE-TAT-AT7 / pTIPE2 dual-target nanoparticles exhibit less cytotoxicity than commonly used transfection reagents PEI and lipo 2000.

[0047] Example 6: In vivo penetration ability of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles

[0048] The in vivo penetration ability of nanoparticles was assessed by observing the green fluorescence signal in mouse eyeball sections. The main experimental steps were as follows: Male C57BL / 6J mice aged 6 to 8 weeks were randomly divided into a control group and a treatment group. The treatment group was given eye drops containing PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles at a mass ratio of 75:1, 5 μl each time (containing 0.5 μg TIPE2 plasmid), four times daily. The control group was given eye drops in the corresponding solvent (1.5% DMSO). After 7 consecutive days of eye drops, the mice were sacrificed, and the eyeballs were removed and prepared into 8 μm thick frozen sections. These sections were then fixed with 4% paraformaldehyde at room temperature for 10 minutes, stained with DAPI, and finally observed using a confocal microscope. The results are as follows: Figure 11 As shown, the green fluorescence signal is mainly concentrated in the retinal pigment epithelium. Weak green fluorescence can also be observed in various layers of the retina, indicating that the nanoparticles have successfully delivered the TIPE2 plasmid to the retinal pigment epithelium and the retina and expressed it.

[0049] Example 7: In vivo safety of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles

[0050] To evaluate the in vivo safety of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles, 6- to 8-week-old male C57BL / 6J mice were randomly divided into a control group, a solvent group, an empty vector group, and a TIPE2 group. The empty vector group used a control empty vector plasmid to exclude the influence of the plasmid itself on the results. Except for the control group, the other three groups were administered solvent eye drops (1.5% DMSO), PBAE-TAT-AT7 / empty vector nanoparticle eye drops (containing 0.5 μg of empty vector plasmid) at a mass ratio of 75:1, and PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticle eye drops (containing 0.5 μg of TIPE2 plasmid), respectively, 5 μl four times daily. Corneal damage in mice was observed by fluorescein staining after 7 consecutive days of eye drops. Results are as follows: Figure 12 As shown, the corneal structures of the four groups of mice were transparent, and the corneal epithelium showed no fluorescent staining, indicating that the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles did not have significant corneal toxicity and had high biocompatibility.

[0051] Example 8: In vivo pharmacodynamic evaluation of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles against angiogenesis

[0052] The therapeutic effect of PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles on retinal neovascularization was evaluated using a laser-induced choroidal neovascularization mouse model. The main experimental steps were as follows: Mice were anesthetized with 1.25% tribromoethanol and their pupils were fully dilated with 1% tropicamide eye drops. Subsequently, the pigment epithelium was destroyed by laser (wavelength 532 nm, power 200 mW, spot diameter 50 μm, duration 50 ms), ultimately forming four laser spots uniformly distributed around the optic nerve. After modeling, mice were randomly divided into three groups: solvent group, empty vector group, and TIPE2 group. Starting 24 hours after laser photocoagulation, mice were administered solvent eye drops (1.5% DMSO), PBAE-TAT-AT7 / empty vector nanoparticle eye drops (containing 0.5 μg empty vector nanoparticles) at a mass ratio of 75:1, and PBAE-TAT-AT7 / TIPE2 dual-target nanoparticle eye drops (containing 0.5 μg TIPE2 plasmid), 5 μl each time, four times daily. Mice that did not receive laser photocoagulation or any other treatment served as the control group. After 7 consecutive days of eye drops, the lesions in the mice were assessed by fluorescence angiography and optical coherence tomography. Results are as follows: Figure 13As shown, the lesion height, extent, fluorescence intensity, and area size were similar in the solvent group and the empty vector group, while the lesion height, extent, fluorescence intensity, and area size were significantly reduced in the TIPE2 group.

[0053] Mice were sacrificed after receiving eye drops for 7 consecutive days. The eyeballs were removed, and the retinas were dissected. Total protein was extracted from the mouse retinal tissue using RIPA lysis buffer. The expression levels of TNF-α, IL-1β, IL-6, TIPE2, and β-actin in the mouse retina were detected by Western blot. The main experimental steps were as follows: The four groups of mouse retinal proteins were separated by 10% SDS-PAGE electrophoresis. The proteins were then transferred to 0.22 μm PVDF membranes at 400 mA, blocked with blocking buffer, and incubated overnight at 4°C with primary antibody. After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, followed by chemiluminescence analysis. The results are as follows: Figure 14 As shown, the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles significantly upregulated TIPE2 expression, confirming the efficient delivery and expression of the TIPE2 plasmid in the eye. Inflammatory factors TNF-α, IL-1β, and IL-6 were significantly elevated in both the solvent and empty vector groups, but significantly decreased in the TIPE2 group, indicating that the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles can inhibit the expression of these three inflammatory factors. These results demonstrate that the PBAE-TAT-AT7 / TIPE2 plasmid dual-target nanoparticles have a significant inhibitory effect on retinal neovascularization.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing dual-target nanoparticles with anti-inflammatory and anti-angiogenic effects based on TIPE2 expression regulation, characterized in that, Includes the following steps: (1) 1,4-Butanediol diacrylate and 4-amino-1-butanol were mixed at a mass ratio of 1~1.5:1 and subjected to Michael addition reaction. After washing, centrifugation and freeze-drying, PBAE with acrylate groups at the end was obtained. (2) The PBAE containing acrylate groups at the end is reacted with the TAT-AT7 peptide containing thiol groups in DMSO at a mass ratio of 1:1~2. Then, after washing, centrifugation and freeze-drying, PBAE-TAT-AT7 is obtained as a drug carrier. The amino acid sequence of the TAT-AT7 peptide is RKKRRQRRRCATWLPPR (SEQ ID NO.1). (3) Mix the PBAE-TAT-AT7 with the plasmid that regulates TIPE2 expression in an aqueous solution at a mass ratio of 5~100:1 and let stand to obtain anti-inflammatory and anti-angiogenic dual-target nanoparticles based on TIPE2 expression regulation.

2. The preparation method according to claim 1, characterized in that, In step (1), the reaction time is 12~24h and the reaction temperature is 90~95℃.

3. The preparation method according to claim 1, characterized in that, In step (2), the reaction time is 12~24h and the reaction temperature is 10~30℃.

4. The preparation method according to claim 1, characterized in that: The coding sequence for plasmid regulation of TIPE2 expression in step (3) is shown in SEQ ID NO.

2.

5. The application of the TIPE2 expression-regulated anti-inflammatory and anti-angiogenic dual-target nanoparticles according to any one of claims 1-4 in the preparation of drugs for treating retinal neovascularization.

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