Preparation method of tubular epithelial cell targeted anti-oxidative peptide and application thereof

Abstract

The application discloses a preparation method of a tubular epithelial cell-targeted anti-oxidative peptide and application thereof, and specifically comprises the following steps: 1) first, N-Boc protected serine (Ser) is dissolved in N,N-dimethylformamide (DMF), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are sequentially added, stirring is carried out at room temperature for 2-4 hours to obtain an active ester solution; 2) polypeptide CTGFVAVR is added into the active ester solution, stirring is continuously carried out at room temperature for 2-4 hours, trifluoroacetic acid (TFA) is added after the reaction is completed, and a reaction product is purified to obtain a tubular epithelial cell-targeted anti-oxidative peptide. The prepared Ser-CTG can break through the double bottleneck of targeting efficiency and curative effect in AKI treatment, provides technical support for the precise medical development of kidney diseases, and has significant clinical transformation value and market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a preparation method of a renal tubular epithelial cell targeted anti-oxidative peptide and application thereof. BACKGROUND

[0002] Acute kidney injury (AKI) is a syndrome of acute decline of renal function caused by ischemia, toxins or inflammation, etc. It has a high incidence, high mortality and a risk of transformation to chronic kidney disease. According to statistics, among the about 13.3 million AKI patients in the world every year, 1.7 million people die from it, and the proportion of high-risk groups such as the elderly and diabetics is increasing year by year. The core pathological mechanism of AKI involves oxidative stress and mitochondrial damage of renal tubular epithelial cells, and the abnormal accumulation of reactive oxygen species (ROS) leads to apoptosis and inflammatory cascade, eventually causing renal failure. Although antioxidant therapy shows the potential to protect mitochondria and reduce damage in animal models, there are two major limitations in traditional drug delivery methods: first, it is difficult for drugs to accurately target damaged renal tubules, and the systemic distribution leads to insufficient effective concentration; second, the high metabolic characteristics of the kidney cause rapid drug excretion, which cannot be retained in the lesion for a long time, greatly limiting the therapeutic effect. Therefore, developing a delivery system with both targeting and high-efficiency antioxidant functions is a key direction to break through the current bottleneck of AKI treatment.

[0003] Kidney injury molecule-1 (KIM-1) as a sensitive biomarker and therapeutic target for AKI, shows a unique pathological targeting advantage. KIM-1 is almost not expressed in normal kidney tissue, but is specifically highly expressed on the cell membrane surface after damage to renal tubular epithelial cells, and its extracellular IgV domain provides a high-affinity binding site for targeted delivery, which makes it an ideal target for precise drug delivery. In recent years, certain progress has been made in KIM-1-based targeting strategies, for example: polypeptides selected by phage display technology can specifically bind to KIM-1 and be used for targeted delivery of siRNA or exosomes; black phosphorus nanosheets modified by KIM-1 ligands can enhance the enrichment of drugs in damaged kidneys and have antioxidant function; and serine-modified chitosan carriers achieve targeted release of drugs through KIM-1-mediated endocytosis. However, the existing technology still has significant limitations: first, some targeted polypeptides can achieve kidney enrichment, but their affinity is still not high enough to form high-concentration accumulation at the lesion site; in addition, some synthetic carriers (such as viral vectors or non-natural polypeptides) may cause immune reactions, and although biological materials such as chitosan have higher safety, their complex assembly process limits large-scale production. Therefore, it is urgent to develop a new delivery system with high-affinity targeting, high-efficiency antioxidant and good biocompatibility to break through the bottleneck of existing technology and meet the urgent need for precise treatment of AKI in clinical practice. SUMMARY

[0004] The application aims at solving the problems in the prior art and providing a preparation method of a tubular epithelial cell targeted antioxidant peptide with high affinity targeting, high efficiency antioxidant and good biocompatibility and application thereof.

[0005] The technical scheme of the application is as follows: a preparation method of a tubular epithelial cell targeted antioxidant peptide, specifically comprising the following steps:

[0006] 1) First, dissolve N-Boc protected serine (Ser) in N,N-dimethylformamide (DMF), and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) in sequence, and stir at room temperature for 2-4 hours to obtain an active ester solution;

[0007] 2) Add polypeptide CTGFVAVR to the active ester solution, continue to stir at room temperature for 2-4 hours, and then add trifluoroacetic acid (TFA) after the reaction is completed, and purify the reaction product to obtain a tubular epithelial cell targeted antioxidant peptide, also known as a serine-antioxidant peptide conjugate (Ser-CTG).

[0008] The chemical structural formula of the CTGFVAVR is as follows:

[0009] .

[0010] The chemical synthesis equation of the application is as follows:

[0011] .

[0012] The application further provides a tubular epithelial cell targeted antioxidant peptide and application thereof, which is used for preparing an active targeted treatment drug for treating acute kidney injury.

[0013] The beneficial effects of the present application are: the serine-antioxidant peptide conjugate (Ser-CTG) prepared in the present application can use natural amino acid serine (Ser) as a KIM-1 targeting group, utilize its high affinity binding characteristics with the IgV domain of KIM-1, realize the active targeting delivery of drugs to damaged renal tubules, and significantly improve the drug concentration at the lesion site based on the low immunogenicity of serine and the verified kidney enrichment ability of existing research; secondly, the core therapeutic molecule CTGFVAVR as a high-efficiency antioxidant peptide can specifically remove ROS and repair mitochondrial function, and cooperates with the targeting module to block the AKI pathological process from the source of oxidative stress; in addition, the serine and CTGFVAVR are directly coupled through covalent grafting technology, avoiding the introduction of a complex carrier system, not only simplifying the preparation process, but also prolonging the drug half-life and improving the kidney distribution efficiency by optimizing the pharmacokinetic characteristics. In view of the complexity of AKI etiology and patient heterogeneity, its targeted delivery characteristics can minimize systemic side effects, and are particularly suitable for critically ill patients with multiple organ dysfunction; based on the design strategy of natural ingredients and small molecule peptides, the risk of immune rejection is significantly reduced and the controllability of synthesis is improved, laying a foundation for large-scale production; there is no approved KIM-1 targeted drug in the world at present, and the present application not only fills the gap of AKI active targeted therapy, but also provides an expandable technology for the intervention of chronic kidney diseases such as diabetic nephropathy. In summary, the Ser-CTG prepared in the present application can break through the dual bottleneck of targeting efficiency and efficacy in AKI treatment, provide technical support for the precision medicine development of kidney diseases, and has significant clinical translation value and market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The synthesis route of Ser-CTG in the examples.

[0015] Figure 2 The nuclear magnetic hydrogen spectrum of CTG in the examples.

[0016] Figure 3 The nuclear magnetic hydrogen spectrum of Ser-CTG in the examples.

[0017] Figure 4 The nuclear magnetic hydrogen spectrum comparison chart of CTG and Ser-CTG in the examples.

[0018] Figure 5 The mass spectrum of Ser-CTG in the examples.

[0019] Figure 6 The DPPH and ABTS free radical scavenging rate of Ser-CTG in the examples.

[0020] Figure 7Chemical structure of the fluorescent probe FITC-labeled Ser-CTG (Ser-CTG(F)) in the examples.

[0021] Figure 8 Active transport of Ser-CTG(F) in normal and oxidative stress damaged renal tubular epithelial cells in the examples.

[0022] Figure 9 Fluorescence imaging results of the distribution of Ser-CTG(F) in the main organs of the acute kidney injury model mice in the examples.

[0023] Figure 10 Effect of Ser-CTG on superoxide dismutase and malondialdehyde changes in oxidative stress damaged cells in the examples.

[0024] Figure 11 Effect of Ser-CTG on reactive oxygen species production in oxidative stress damaged cells in the examples.

[0025] Figure 12 Anti-apoptotic effect of Ser-CTG in the examples.

[0026] Figure 13 Changes in serum creatinine and urea nitrogen levels in acute kidney injury model mice in the examples.

[0027] Figure 14 Kidney tissue pathological section results of acute kidney injury model mice in the examples. DETAILED DESCRIPTION

[0028] The technical solutions in the examples of the present application will be described clearly and completely below in combination with the drawings in the examples of the present application. Obviously, the described examples are only some of the examples of the present application, not all the examples.

[0029] The renal tubular epithelial cell targeting antioxidant peptide, referred to as Ser-CTG, was prepared according to the following steps:

[0030] First, the N-Boc protected serine (12 mg, 0.06 mmol) was dissolved in 10 mL DMF, and EDCI (11.5 mg, 0.06 mmol) and NHS (6.9 mg, 0.06 mmol) were added in sequence, and stirred at room temperature for 2 hours to generate an active ester; then the polypeptide CTGFVAVR (0.05 mmol, 42.6 mg) was added. After the reaction was completed, 0.1 mL trifluoroacetic acid was added to hydrolyze the Boc protecting group. The crude product was purified by preparative liquid chromatography to obtain 36 mg of white solid Ser-CTG with a yield of 76%, and the synthetic route is shown in Figure 1 CTG and Ser-CTG were characterized by nuclear magnetic resonance hydrogen spectrum, and the results are as follows:Figures 2-4 Figure 6 shows the NMR spectra of CTG and Ser-CTG. Figure 4 Figure 6 shows the NMR spectra of CTG and Ser-CTG. Figure 5 Figure 7 shows the ESI-MS spectrum of Ser-CTG.

[0031] Ser-CTG and the control peptide CTG were dissolved in PBS buffer (pH 7.4) to prepare 0, 10, 50, 100, and 200 μM concentration gradient solutions. 100 μL of the sample solution was mixed with 100 μL of 0.2 mM DPPH ethanol solution (prepared in the dark), and incubated at 37°C in the dark for 30 minutes. Then, the precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. The absorbance (OD value) of the supernatant was measured at a wavelength of 517 nm. The free radical scavenging rate was calculated according to the formula: scavenging rate (%) = [1 - (OD sample - OD blank) / OD control] x 100%.

[0032] ABTS solution (7 mM) and potassium persulfate solution (2.45 mM) were mixed in equal amounts, and reacted at room temperature in the dark for 12-16 hours to generate ABTS stock solution. 20 μL of the sample to be tested (concentration gradient same as in the DPPH experiment) was mixed with 180 μL of ABTS working solution, and reacted at room temperature in the dark for 6 minutes. The absorbance was immediately measured at 734 nm. The dose-effect curve was plotted using Trolox as the standard, and the scavenging rate was calculated according to the formula: scavenging rate (%) = [1 - (OD sample / OD blank)] x 100%.

[0033] The antioxidant activity of Ser-CTG was evaluated by DPPH free radical scavenging experiment and ABTS free radical scavenging experiment, with the uncoupled antioxidant peptide CTG as the control. The results are shown in Figure 6As shown, within the concentration ranges of 0, 10, 50, 100, and 200 μM, both Ser-CTG and CTG exhibited significant dose-dependent free radical scavenging capabilities, and their scavenging rates increased linearly with increasing concentration. Furthermore, the scavenging rates of Ser-CTG and CTG were not statistically significant. These data indicate that the chemical coupling process between serine and CTG did not affect the conformation of the active group of the antioxidant peptide, and Ser-CTG fully retained the free radical scavenging capability of CTG. This confirms the rationality of its structural modification strategy and provides crucial experimental evidence for subsequent targeted delivery and synergistic therapy with antioxidant function.

[0034] Using FITC-labeled CTG as a model drug (CTG(F)), such as Figure 7 As shown, Ser-CTG(F) was synthesized using the same method to investigate the active transport of Ser-CTG(F) and CTG(F) in normal and oxidative stress-damaged renal tubular epithelial cells. Human renal tubular epithelial cells (HK-2) were divided into a normal group and an oxidative stress-damaged group. The damaged group was pretreated with 200 μM hydrogen peroxide (H2O2) for 2 hours to induce high expression of KIM-1 receptor. Both groups of cells were then co-incubated with 50 μM Ser-CTG(F) or CTG(F) for 2 hours, washed three times with PBS buffer, and finally the intracellular fluorescence distribution was observed using a fluorescence inverted microscope to compare the differences in Ser-CTG(F) and CTG(F) uptake between normal and damaged cells. Fluorescence microscopy imaging is shown below. Figure 8 As shown, in H2O2-induced oxidative stress-damaged HK-2 cells, the Ser-CTG (F) group exhibited significantly enhanced intracellular fluorescence signals, concentrated in the cytoplasm and near-membrane region, consistent with the membrane localization characteristics of KIM-1. In contrast, no significant difference in fluorescence intensity was observed between the two probes in normal HK-2 cells. These results demonstrate that Ser-CTG can actively target and accumulate at oxidative stress lesions by binding with high affinity to KIM-1 on the surface of damaged renal tubular cells via serine residues, while free CTG cannot specifically accumulate due to the lack of a targeting group. This fully validates the biological effectiveness of the Ser-CTG design strategy.

[0035] C57BL / 6J mice were used as animal models to establish an acute kidney injury mouse model by bilateral renal artery clipping. Mice were injected with either Ser-CTG(F) or CTG(F), and sacrificed by cervical dislocation 2 hours after administration. Heart, liver, spleen, lung, and kidney were harvested, fixed in 4% paraformaldehyde, mounted, and their fluorescence distribution was observed under a fluorescence inverted microscope. Results are as follows: Figure 9As shown, the fluorescence intensity of Ser-CTG(F) in the kidney of AKI mice was significantly higher than that in the kidney of normal mice, and the fluorescence signal was concentrated in the renal cortical area (the area rich in renal tubular epithelial cells), which was highly consistent with the pathological expression location of KIM-1; in contrast, the fluorescence intensity of free CTG(F) in the kidney of AKI and normal mice was low and almost invisible. In addition, Ser-CTG(F) was not found in the heart, liver, spleen, lung and other non-target organs, further reducing the risk of non-specific organ accumulation. The above data confirmed that Ser-CTG could achieve efficient enrichment in damaged kidneys based on the pathological targeting characteristics of KIM-1, while maintaining a low interaction level with other organs, providing a key basis for reducing systemic toxicity and improving the therapeutic window.

[0036] The cells were divided into a normal control group, an H2O2 injury model group, an H2O2+Ser-CTG (50 μM) treatment group, and an H2O2+CTG (50 μM) treatment group. After 200 μM H2O2 was continuously incubated for 2 hours, the drugs were added for 12 hours. After the cells were lysed, the oxidative damage level was determined by the WST-8 method (SOD activity detection) and the thiobarbituric acid method (MDA content detection), respectively. The results are shown in Figure 10 As shown, the SOD activity of the Ser-CTG treatment group was significantly higher than that of the model group and the CTG group, indicating that serine-targeted modification could enhance the repair ability of endogenous free radical scavenging system of the antioxidant peptide; at the same time, the MDA content of the Ser-CTG group was significantly lower than that of the model group and the CTG group, proving that Ser-CTG could more effectively inhibit the lipid peroxidation chain reaction.

[0037] The cells were divided into a normal control group, an H2O2 injury model group, an H2O2+Ser-CTG (50 μM) treatment group, and an H2O2+CTG (50 μM) treatment group. After 200 μM H2O2 was continuously incubated for 2 hours, the drugs were added for 12 hours. The culture medium was discarded and 10 μM DCFH-DA probe was added (37°C, dark incubation for 30 minutes). After being washed with PBS for three times, the ROS fluorescence intensity in the cells was observed by fluorescence inverted microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The results are shown in Figure 11 As shown, the ROS fluorescence intensity of the H2O2 injury model group was higher than that of the normal group, and the ROS level of the Ser-CTG treatment group was significantly lower than that of the model group, which was better than that of the CTG group; fluorescence microscopic imaging further showed that the fluorescence signal of the cells in the Ser-CTG group was weak and scattered positive, while the strong fluorescence in the CTG group was still visible, indicating that serine-targeted modification significantly improved the enrichment efficiency of the antioxidant peptide at the lesion site, thereby more efficiently removing excess ROS and reversing the oxidative stress damage process.

[0038] Cells were divided into normal control group, H2O2 injury model group, H2O2 + Ser-CTG (50 μM) treatment group and H2O2 + CTG (50 μM) treatment group. After 200 μM H2O2 was incubated for 2 hours, drugs were added for 12 hours. Cells were collected, and Annexin V-FITC and propidium iodide (PI) were added for 15 minutes of light-free staining according to the kit instructions. Flow cytometry was used to detect the apoptosis rate (Annexin V + / PI - for early apoptosis, and Annexin V + / PI + for late apoptosis). The results are shown in Figure 12 . The total apoptosis rate of the H2O2 injury model group was increased to 53% compared with the normal group, while the total apoptosis rate of the Ser-CTG treatment group was significantly reduced to 5.4%, which was better than the 32.6% of the CTG group. Further subgroup analysis showed that Ser-CTG could inhibit both early apoptosis (model group 37.8%→treatment group 2.9%) and late apoptosis (model group 15.2%→treatment group 2.5%), and its anti-apoptotic effect was significantly negatively correlated with the ROS clearance level. This data confirms that Ser-CTG enhances the enrichment of antioxidant peptides in damaged cells by targeted delivery, effectively blocks the ROS-mediated mitochondrial apoptosis pathway, and thus significantly reverses oxidative stress-induced cell death, providing key mechanistic evidence for reducing the loss of renal tubular epithelial cells in acute kidney injury.

[0039] C57BL / 6 male mice (body weight 25-30 g) were selected, and they were fasted for 8-12 hours before surgery. After intraperitoneal injection of sodium pentobarbital anesthesia, they were fixed on a 37°C constant temperature operating table. The bilateral renal pedicles were exposed through a dorsal incision, and atraumatic arterial clamps were used to clamp for 40 minutes. The successful ischemia was confirmed by observing the color of the kidney changing from bright red to purple black. The clamps were removed to restore blood flow (reperfusion), the incision was sutured layer by layer, and warm normal saline was injected intraperitoneally for fluid replacement. Two hours after reperfusion, drugs (Ser-CTG or CTG) were administered via the tail vein. Twelve hours after administration, serum creatinine (Scr) and blood urea nitrogen (BUN) were detected, and the kidney tissue was subjected to H&E staining.

[0040] As Figure 13As shown, the serum creatinine (Scr) and urea nitrogen (BUN) levels of the acute kidney injury model were significantly increased (Scr: 43.3 ± 2.9 μmol / L vs. 262.3 ± 22.4 μmol / L; BUN: 8.2 ± 1.2 mmol / L vs. 25.1 ± 3.0 mmol / L) compared with the sham operation group, indicating that the kidney function was severely impaired. The Scr and BUN levels of the Ser-CTG treatment group were significantly lower than those of the model group (Scr: 71.5 ± 11.9 μmol / L vs. model group; BUN: 9.9 ± 1.7 mmol / L vs. model group), and were significantly better than those of the CTG group (Scr: 164.9 ± 18.1 μmol / L; BUN: 18.9 ± 2.2 mmol / L), indicating that the serine modification can enhance the renal targeted delivery and therapeutic effect of the antioxidant peptide CTG. As shown, Figure 14 As shown, further examination of the pathological changes of the kidney tissues of the acute kidney injury mice showed that the renal tubular epithelial cells of the model group were widely necrotic, the brush border was shed, and a large number of protein casts were visible in the lumen; while the pathological damage of the Ser-CTG treatment group was significantly reduced, only focal tubular dilation and slight vacuolization were observed, and the improvement effect was better than that of the free drug group; the kidney tissue structure of the sham operation group was complete. This result verified the treatment advantage of Ser-CTG for acute kidney injury from the morphological level.

[0041] The above is only an exemplary embodiment of the present application, and does not constitute any limitation on the protection scope of the present application. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present application.

Claims

1. A method for preparing an anti-oxidative peptide targeted to renal tubular epithelial cells, characterized by, Specifically comprising the following steps: 1) First, dissolve N-Boc protected serine (Ser) in N, N-dimethylformamide (DMF), and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) in sequence, and stir at room temperature for 2-4 hours to obtain an active ester solution; 2) Add polypeptide CTGFVAVR to the active ester solution, continue to stir at room temperature for 2-4 hours, and then add trifluoroacetic acid (TFA) after the reaction is completed, and purify the reaction product to obtain a tubular epithelial cell-targeted anti-oxidative peptide.

2. The method for preparing renal tubular epithelial cell-targeting antioxidant peptides according to claim 1, characterized in that, The chemical structural formula of the CTGFVAVR is as follows: 。 3. The method for preparing renal tubular epithelial cell-targeting antioxidant peptides according to claim 1, characterized in that, The chemical synthesis equation is as follows: 。 4. The use of a tubular epithelial cell targeted arotomoi peptide prepared according to any one of claims 1 to 3, characterized in that, It is used for preparing a proactively targeted therapeutic drug for treating acute kidney injury.

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