A biomimetic liposome nanoreactor, preparation method and application thereof

Abstract

The present invention provides a biomimetic liposome nanoreactor, a preparation method and its application, and relates to the field of biomedical engineering technology. The biomimetic liposome nanoreactor comprises liposomes, thylakoid membrane fragments and electron donors. The present invention integrates thylakoid fragments (Tk) into liposomes (Lip) and loads electron donors into the liposomes to prepare a biomimetic liposome nanoreactor. The biomimetic liposome nanoreactor of the present invention can be taken up by renal epithelial cells and catalyze NAD + Generate; and under the drive of ultrasound, the electron transport chain on it transfers a large number of electrons to the cytoplasm, converting NAD + Reduction to NADH promotes ATP production, rapidly alleviating AKI in the early stages. Furthermore, in the presence of electrons, the effective generation of NADPH fundamentally prevents the generation of oxidative stress such as ROS, effectively preventing the progression of AKI to CKD.

Description

Technical Field

[0001] The present invention relates to the technical field, in particular to a bionic liposome nanoreactor, a preparation method and application thereof. Background Art

[0002] Acute kidney injury (AKI) is a common condition characterized by a rapid decline in renal function. It affects nearly every system in the body and, if left untreated, can rapidly progress to chronic kidney disease (CKD), causing long-term damage and increasing mortality. Unfortunately, there is currently no effective clinical treatment for AKI.

[0003] The kidney is the most important organ for energy metabolism and requires a large amount of ATP. In the early stage of AKI, a sharp drop in ATP can lead to severe dysfunction of the renal tubules. For the proximal tubules, ATP is usually produced by nicotinamide adenine dinucleotide (NAD + ) as an electron acceptor. Unfortunately, recent studies have shown that catalyzing NAD + Quinolinate phosphoribosyltransferase (QPRT), an enzyme essential for de novo synthesis, is significantly reduced in AKI kidney tissue, thereby hindering ATP replenishment.

[0004] In addition, NADPH plays a key role in the antioxidant defense of cells and has multiple reducing functions. + Can be phosphorylated to produce NADP + , while NADP + In the presence of electrons, it is reduced to NADPH. + Supplementing QPRT with renal cells is a key factor in the comprehensive treatment of AKI. However, only when sufficient electrons coexist with QPRT can NAD simultaneously affect the synthesis of ATP and NADPH, which is difficult to achieve artificially.

[0005] We know that NAD + It is widely present in plant cells. Therefore, plant cells may have evolved a large number of QPRTs in the thylakoid membrane. At the same time, a typical electron transport chain exists in the thylakoid membrane, which transfers electrons from water to NADH under light irradiation. Therefore, thylakoids are ideal materials for constructing a powerful AKI treatment system. However, when thylakoids are separated from plant cells, there is a problem of reduced electron transfer efficiency. Therefore, it is necessary to provide additional electron donors to enable the conversion of NAD catalyzed by QPRT to NADH. + Biosynthesis is effectively coupled to the electron transfer process in thylakoids.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a biomimetic liposome nanoreactor, a preparation method and its application. The biomimetic liposome nanoreactor of the present invention can be taken up by renal epithelial cells and catalyze NAD + Generate; and under the drive of ultrasound, the electron transport chain on it transfers a large number of electrons to the cytoplasm, converting NAD + Reduction to NADH promotes ATP production, rapidly alleviating AKI in the early stages. Furthermore, in the presence of electrons, the effective generation of NADPH fundamentally prevents the generation of oxidative stress such as ROS, effectively preventing the progression of AKI to CKD.

[0008] The technical solutions provided by the present invention are as follows:

[0009] In one aspect, the present invention provides a biomimetic liposome nanoreactor, which comprises a liposome, a thylakoid membrane, and an electron donor.

[0010] In one embodiment, the electron donor comprises L-ascorbic acid. The electron donor is preferably L-ascorbic acid (AA), which is a classic electron donor.

[0011] In one embodiment, the encapsulation concentration of the L-ascorbic acid in the biomimetic liposome nanoreactor is 1.5-2.5 mM, preferably 2 mM.

[0012] In one embodiment, the biomimetic liposome nanoreactor contains proteins in the thylakoid membrane, including PSI-A, LHC-II and QPRT.

[0013] In another aspect, the present invention also provides a method for preparing the biomimetic liposome nanoreactor, comprising adding thylakoid membranes and electron donors into liposomes, ultrasonically hydrating the liposomes in an ice bath, and dialyzing the liposomes with a buffer solution to remove the free electron donors.

[0014] In one embodiment, the method for preparing the liposome comprises dissolving dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) and cholesterol in an organic solvent, and then removing the organic solvent; preferably, the molar ratio of the distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol is in the range of 4-6:1:2-3:1; more preferably 5:1:3:1. In one embodiment, the added amount of the liposome, L-ascorbic acid and thylakoid membrane is 13-18 mg:1.0-2.0 mg:0.2-1 mg; preferably, the added amount is 14 mg:2 mg:0.8 mg.

[0015] The average particle size of the biomimetic liposome nanoreactor is 90 to 110 nm; preferably, the average particle size of the biomimetic liposome nanoreactor is 100 nm.

[0016] In one embodiment, the thylakoid membrane is extracted from leaves of photosynthetic plants or photosynthetic bacteria, for example, thylakoids extracted from spinach.

[0017] In another aspect, the present invention provides the use of the biomimetic liposome nanoreactor in one or more of the following:

[0018] (a) Catalyzes NAD + generate;

[0019] (b) Transfer and replenishment of electrons under ultrasonic conditions;

[0020] (c) Promote the synthesis of NAD in renal epithelial cells;

[0021] (d) Increased intracellular ATP levels under ultrasound conditions;

[0022] (e) promoting NADPH synthesis and / or improving oxidative stress;

[0023] (f) improving mitochondrial quality, restoring damaged mitochondria and / or significantly reducing mitochondrial fragmentation;

[0024] (g) enhancing renal epithelial cell activity and / or reducing renal tubular injury index.

[0025] The biomimetic liposome nanoreactor prepared by the present invention, such as the biomimetic liposome nanoreactor (LipTk-AA) prepared from L-ascorbic acid (AA), thylakoid fragments (Tk), and liposomes (Lip), can be taken up by renal epithelial cells (TECs). The QPRT carried by the biomimetic liposome nanoreactor can catalyze the generation of NAD+. Under ultrasonic drive, with AA as the electron donor, the electron transport chain on the LipTk-AA transfers a large amount of electrons to the cytoplasm, reduces NAD+ to NADH in the presence of electrons, and promotes the production of ATP.

[0026] In addition, DHA generated by AA after donating electrons is released into the cytoplasm, promoting the shift of sugar metabolism to the antioxidant pentose phosphate pathway (PPP). In the presence of electrons, NADPH is efficiently produced, which fundamentally prevents the generation of oxidative stress such as ROS.

[0027] In another aspect, the present invention provides use of the biomimetic liposome nanoreactor in preparing the claimed medicine for treating acute kidney injury.

[0028] The biomimetic liposome nanoreactor prepared by the present invention can quickly alleviate AKI in the early stage, not only can treat early AKI, but also can effectively prevent the progression of AKI to CKD.

[0029] In one embodiment, the treating acute kidney injury comprises one or more of halting the progression of acute kidney injury, avoiding the transition of acute kidney injury to chronic kidney disease, and preventing early kidney damage.

[0030] In the present invention, the term thylakoid fragment has the same meaning as thylakoid membrane.

[0031] Beneficial effects:

[0032] The biomimetic liposome nanoreactor of the present invention retains proteins in the thylakoid membrane, integrates thylakoid fragments into the liposomes, and simultaneously loads classic electron donors into the liposomes. Under ultrasound, it maintains complete electron transfer capacity and can promote the synthesis of NAD in renal epithelial cells, improving mitochondrial quality. Furthermore, after ultrasound treatment, the biomimetic liposome nanoreactor significantly increases intracellular ATP levels, preventing the progression of AKI. It also significantly increases the relative abundance of NADPH in TECs, and the metabolite DHA can improve oxidative stress in damaged epithelial cells by promoting the PPP pathway, thereby preventing the transition from AKI to CKD. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 To explore the amount of thylakoids on liposomes, different ratios of Tk and Lip were added.

[0035] Figure 2 To add different concentrations of AA, we explored the results of the amount of AA encapsulated in the liposomes;

[0036] Figure 3 The test results of the liposome nanoreactor (LipTk-AA) provided by the present invention, wherein (A) is the transmission electron microscopy analysis of LipTk-AA; (B) is the concentration and stability of AA coated with LipTk-AA; (C) is the analysis of the whole protein of LipTk-AA by SDS-PAGE; (D) is the immunoblot analysis of the characteristic proteins of LipTk-AA, especially QPRT;

[0037] Figure 4 Provided herein are (A) analysis of the UV absorption of DCPIP in different ultrasound treatment groups and (B) UV absorption spectra of AA in LipTk-AA as ultrasound time increases;

[0038] Figure 5 Immunofluorescence analysis of QPRT expression levels in TECs and renal tubules after different treatments provided by the present invention;

[0039] Figure 6 The present invention provides (A) to (B) analysis of quinolinic acid concentration in TECs and mouse kidney tissue after different treatments; (C) to (D) analysis of NAD in TECs and mouse kidney tissue after different treatments. + Level of analysis;

[0040] Figure 7 Quantitative statistics of mitochondrial fragmentation rates in ETCs of different treatment groups provided by the present invention;

[0041] Figure 8 Analysis of ATP levels in ETCs of different treatment groups provided by the present invention;

[0042] Figure 9Provided herein are (A) the relative abundance of NADPH in ETCs of different treatment groups; (B) the relative lipid peroxidation levels in ETCs of different treatment groups; (C) the GSSG / GSH ratio statistics in ETCs of different treatment groups;

[0043] Figure 10 Provided herein are (A) cell activity of ETCs in different treatment groups; (B) analysis of KIM-1 expression levels in mouse kidney tissue after different drug administrations. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] Example 1. Preparation of liposome nanoreactor (LipTk-AA)

[0046] Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) and cholesterol were dissolved in a chloroform:methanol mixture (3:1 v / v) at a molar ratio of 5:1:3:1 and placed on a rotary evaporator to remove the organic solvent. (During the exploration process, AA 500μg / mL, 1000μg / mL, 1500μg / mL, 2000μg / mL and 2500μg / mL were added respectively. It was found that starting from the addition of 1500μg / mL, the drug loading of AA reached saturation, that is, 350μg / mL, and the drug loading rate was 23%); the mass ratio of liposomes to thylakoids was optimized as follows: 140:1, 70:1, 35:1, 17.5:1; 8.75:1. It was found that 17.5:1 was the best ratio. At this ratio, the amount of thylakoids on the liposomes reached a maximum of 100μg / mL. The results are shown in the figure. Figure 1-Figure 2 .

[0047] The free AA was removed by dialysis with PBS for 3 days, and finally a biomimetic liposome nanoreactor (LipTk-AA) was obtained.

[0048] The results showed that the average particle size of the prepared LipTk-AA was about 100 nm. The encapsulation concentration of AA in LipTk was determined to be stable at around 2 mM (about 350 μg / mL) by UV-visible absorption.

[0049] All Tk proteins were detected in LipTk-AA. Western blotting analysis also confirmed the obvious presence of typical Tk proteins in LipTk-AA, including PSI-A and LHC-II, especially the abundant presence of QPRT, which was absent in Lip-AA.

[0050] Figure 3 This is the detection result of liposome nanoreactor (LipTk-AA).

[0051] Example 2. Under ultrasound, LipTk-AA has complete electron transfer ability.

[0052] DCPIP is an artificial electron acceptor that can capture electrons, causing DCPIP to be reduced, resulting in a decrease in UV absorbance. We found that the absorbance of DCPIP remained almost unchanged in the LipTk and LipTk-AA groups after ultrasonic treatment, while the absorbance of DCPIP decreased significantly in the LipTk-AA combined with ultrasonic treatment group. As the ultrasonication time increased, the absorbance value further decreased, but the addition of the electron transport chain inhibitor DCMU (dichlorophenyl dimethyl urea) could effectively prevent this. If the ultrasonication was repeated, the content of AA in LipTk gradually decreased with the increase in the number of ultrasonications. These results collectively confirmed the integrity of the electron transport chain in LipTk-AA, and that electron transfer and replenishment could be achieved using AA as a donor under ultrasonic conditions.

[0053] Figure 4 (A) Analysis of the UV absorption of DCPIP in different ultrasonic treatment groups; (B) UV absorption spectra of AA in LipTk-AA with the extension of ultrasonic time.

[0054] Example 3. LipTk-AA has typical QPRT activity, thereby promoting the synthesis of NAD in renal epithelial cells and improving mitochondrial quality

[0055] We found that QPRT was significantly decreased in cisplatin-induced injured renal tubular epithelial cells (TECs) and AKI tubules compared with normal and healthy tubules.

[0056] Interestingly, treatment with LipTk or LipTk-AA significantly supplemented this decrease, while Lip-AA had no significant effect on this point. This is because LipTk and LipTk-AA contain QPRT. Quinolinic acid (QA) is a NAD + QA is a direct precursor for biosynthesis, and QPRT is one of the essential enzymes. Therefore, an increase in QA should be a highly specific indicator of a reduced QPRT effect.

[0057] The results showed that QA levels in cisplatin-injured TECs and AKI kidneys were 6-fold and 2-fold higher than those in normal cells and kidney tissue, respectively. LipTk and LipTk-AA significantly reduced QA to levels similar to those in healthy cells and kidneys.

[0058] In contrast, Lip-AA treatment did not reduce QA levels. + Since LipTk-AA plays an important role in NAD biosynthesis, we then evaluated the effect of LipTk-AA on NAD biosynthesis in injured TECs and kidneys. + It can be seen that the NAD + The content of NAD increased significantly, while the NAD + The content did not change much.

[0059] We found that mitochondria were significantly damaged in cisplatin-treated TECs. + The results were consistent. LipTk or LipTk-AA treatment could significantly restore damaged mitochondria and significantly reduce mitochondrial fragmentation, while Lip-AA could not.

[0060] Figure 5 Immunofluorescence analysis of QPRT expression levels in TECs and renal tubules after different treatments.

[0061] Figure 6 (AB) Analysis of quinolinic acid concentrations in TECs and mouse kidney tissues after different treatments; (CD) Analysis of NAD+ levels in TECs and mouse kidney tissues after different treatments. Figure 7 Quantitative statistics of mitochondrial fragmentation rates in ETCs of different treatment groups.

[0062] Example 4. LipTk-AA plus ultrasound treatment significantly increases ATP levels

[0063] We then investigated the ATP concentration in injured renal epithelial cells after various treatments. The results showed that LipTk-AA plus ultrasound treatment (5 minutes at 0.5W power) increased intracellular ATP levels by 7-fold compared to the control, while the other treatments failed to significantly elevate intracellular ATP levels. This suggests that increasing electron supply through ultrasound and supplementing with QPRT can effectively promote ATP synthesis, thereby preventing the progression of AKI. Figure 8 Analysis of ATP levels in ETCs of different treatment groups.

[0064] Example 5. LipTk-AA promotes NADPH synthesis and improves oxidative stress

[0065] On the other hand, we found that LipTk-AA treatment for 48 h could significantly increase the relative abundance of NADPH in TECs, and ultrasonic treatment could further enhance this function of LipTk-AA.

[0066] Therefore, representative indicators of oxidative stress, such as the GSSG / GSH ratio and lipid peroxidation, were reduced, confirming that DHA, a metabolite of LipTk-AA, can improve the oxidative stress of damaged epithelial cells by promoting the PPP pathway, thereby avoiding the transition from AKI to CKD.

[0067] Figure 9 (A) Relative abundance of NADPH in ETCs of different treatment groups; (B) Relative lipid peroxidation levels in ETCs of different treatment groups; (C) Statistics of GSSG / GSH ratios in ETCs of different treatment groups.

[0068] Example 6. LipTk-AA enhances renal epithelial cell activity and reduces renal tubular injury index

[0069] Results showed that LipTK-AA significantly reduced apoptosis in ETCs. After 48 hours of LipTK-AA plus ultrasound treatment, cell viability remained consistent with that of normal ETCs due to the combination of early ATP generation and the antioxidant effects of LipTK-AA. Furthermore, severe renal tubular damage led to a significant increase in kidney injury molecule-1 (KIM-1) expression. LipTk-AA plus US treatment rapidly reduced KIM-1 levels within 24 hours, with a greater reduction after 48 hours. This suggests that LipTk-AA plus US treatment can prevent early kidney damage in AKI.

[0070] Figure 10 (A) Cell activity of ETCs in different treatment groups; (B) Analysis of KIM-1 expression levels in mouse kidney tissue after different drug administrations.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of a biomimetic liposome nanoreactor in the preparation of a drug for treating acute kidney injury, characterized in that: The biomimetic liposome nanoreactor comprises liposomes, thylakoid membranes and electron donors; the electron donors comprise L-ascorbic acid.

2. The use of the biomimetic liposome nanoreactor according to claim 1 in preparing a medicine for treating acute kidney injury, characterized in that The encapsulation concentration of the L-ascorbic acid in the bionic liposome nanoreactor is 1.5-2.5 mM.

3. The use of the biomimetic liposome nanoreactor according to claim 2 in preparing a medicine for treating acute kidney injury, characterized in that The encapsulation concentration of the L-ascorbic acid in the biomimetic liposome nanoreactor is 2 mM.

4. The use of the biomimetic liposome nanoreactor according to any one of claims 1 to 3 in the preparation of a medicament for treating acute kidney injury, characterized in that The biomimetic liposome nanoreactor contains proteins in the thylakoid membrane, including PSI-A, LHC-II and QPRT.

5. The use of the biomimetic liposome nanoreactor according to any one of claims 1 to 3 in the preparation of a medicament for treating acute kidney injury, wherein the preparation method of the biomimetic liposome nanoreactor is as follows: adding thylakoid membranes and electron donors to liposomes, ultrasonically hydrating in an ice bath, and dialyzing with a buffer solution to remove free electron donors.

6. Use of the biomimetic liposome nanoreactor according to claim 5 in preparing a medicament for treating acute kidney injury, characterized in that: The preparation method of the liposome comprises the steps of dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in an organic solvent, and then removing the organic solvent.

7. Use of the biomimetic liposome nanoreactor according to claim 6 in preparing a medicament for treating acute kidney injury, characterized in that: The molar ratio of the dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol is in the range of 4-6:1:2-3:

1.

8. Use of the biomimetic liposome nanoreactor according to claim 7 in preparing a medicament for treating acute kidney injury, characterized in that: The molar ratio of the dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol is 5:1:3:

1.

9. Use of the biomimetic liposome nanoreactor according to claim 8 in preparing a medicament for treating acute kidney injury, characterized in that: The added amounts of the liposome, L-ascorbic acid and thylakoid membrane are 13-18 mg: 1.0-2.0 mg: 0.2-1 mg.

10. Use of the biomimetic liposome nanoreactor according to claim 9 in preparing a medicament for treating acute kidney injury, characterized in that: The added amounts of the liposome, L-ascorbic acid and thylakoid membrane are 14 mg: 2 mg: 0.8 mg.

11. Use of the biomimetic liposome nanoreactor according to claim 1 in preparing a medicament for treating acute kidney injury, characterized in that: The treatment of acute kidney injury includes one or more of preventing the progression of acute kidney injury, preventing the transformation of acute kidney injury to chronic kidney disease, and alleviating early kidney damage in AKI.

Images