Antisense peptide nucleic acid nanoparticle delivery system for targeting C1GALT1 gene as well as preparation method and application of antisense peptide nucleic acid nanoparticle delivery system

Abstract

The invention relates to an antisense peptide nucleic acid nanoparticle delivery system of a targeted C1GALT1 gene as well as a preparation method and application of the antisense peptide nucleic acid nanoparticle delivery system, and belongs to the technical field of biological medicine and nanotechnology. The nanoparticle delivery system takes a PLGA-PEG copolymer as a carrier material to load antisense gamma peptide nucleic acid, and the antisense gamma peptide nucleic acid is effectively delivered to cells so as to inhibit C1GALT1 gene expression in a targeted manner. The preparation method disclosed by the invention is simple and convenient in preparation process, and has good biodegradability and biological safety; the antisense gamma peptide nucleic acid can be efficiently delivered, the C1GALT1 gene in a B cell is knocked down, the stability of combination with RNA is high, the off-target effect is low, an effective tool is provided for research on related molecular diseases, and good clinical application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine and nanotechnology, and relates to a nanoparticle delivery system and a preparation method thereof, in particular to a method for delivering antisense gamma peptide nucleic acid by using PLGA-PEG nanoparticles to inhibit the expression of C1GALT1 gene, and application thereof in gene therapy and related disease research. BACKGROUND

[0002] C1GALT1 (core 1 beta 1, 3-galactosyltransferase 1) gene plays a key role in the glycosylation process, and its abnormal expression is related to various diseases such as IgA nephropathy and tumor. IgA nephropathy is a common primary glomerular disease, and its characteristic is the lack of galactose in IgA1 molecules, which leads to immune complex deposition and kidney damage. IgA nephropathy is prone to occur in children and young adults, and usually shows a chronic progressive course. The kidney function of 20% of children (followed up to adulthood) and 30% of adults will decrease by 50% or develop into kidney failure after 10 years, which causes a serious burden to the society and family. At present, the regulation strategies for C1GALT1 include antisense oligonucleotide technology, but the traditional antisense oligonucleotide has problems such as poor stability, easy degradation by nucleases, low cell delivery efficiency, and off-target effect.

[0003] Nanoparticle delivery systems such as PLGA-PEG (poly (lactic-co-glycolic acid)-polyethylene glycol) nanoparticles have been widely used in drug delivery due to their biodegradability, good biocompatibility and controllable release characteristics. However, there is no report on the use of PLGA-PEG for delivering peptide nucleic acid, especially antisense gamma peptide nucleic acid, to target C1GALT1 gene. Antisense gamma peptide nucleic acid is an artificially synthesized nucleic acid analogue, which has the characteristics of resisting proteases and nucleases, and has strong DNA / RNA binding ability, but its cell membrane penetration ability is poor, and a high-efficiency delivery system is needed. Therefore, it is of great clinical and research value to develop a nanoparticle system that can efficiently deliver antisense gamma peptide nucleic acid to achieve effective inhibition of C1GALT1 gene. SUMMARY

[0004] In order to solve the problems of low delivery efficiency and poor stability of antisense oligonucleotide in the prior art, the present application provides a method for delivering peptide nucleic acid by using nanoparticles. The present application uses PLGA-PEG nanoparticles to deliver specific antisense gamma peptide nucleic acid, realizes efficient inhibition of C1GALT1 gene expression, and is applied to the research and treatment of related diseases.

[0005] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0006] The application discloses an antisense peptide nucleic acid nanoparticle delivery system for targeting C1GALT1 genes.

[0007] The sequence of the antisense gamma peptide nucleic acid is H-TAMRA-KKK-CC#TCT#CGC#CCC#TCC#T-KKK-NH2 (SEQ ID NO: 1), wherein # represents lys-gamma modification, TAMRA is a fluorescent label, and KKK is a lysine sequence for enhancing cell penetration.

[0008] The pure water is used as an aqueous phase; the aqueous phase is added into the oil phase, ultrasonic emulsification is carried out, and an initial emulsion is formed; the initial emulsion is added into a polyvinyl alcohol solution, high-speed homogenization is carried out to form a multiple emulsion; organic solvents are evaporated, nanoparticles are collected through centrifugation, and then the nanoparticles are washed with deionized water and freeze-dried to obtain nanoparticle powder.

[0009] The molar ratio of PLGA to PEG in the PLGA-PEG copolymer is 70: (20-40), and preferably 70:30, so as to ensure the stability and drug loading capacity of the nanoparticles.

[0010] The concentration of the PLGA-PEG is 5-15 mg / ml, and preferably 10 mg / ml. The concentration of the antisense gamma peptide nucleic acid is 15-25 nmol / ml, and preferably 20 nmol / ml.

[0011] The ultrasonic emulsification and high-speed homogenization method is as follows: ultrasonic treatment is carried out for 2-3 s and then stopped for 1-2 s, and the whole process lasts for 6-10 min, and the ultrasonic power is 300-500 W. Preferably, the ultrasonic treatment is carried out for 2 s and then stopped for 1 s, and the whole process lasts for 8 min, and the ultrasonic power is 400 W.

[0012] The mass percentage concentration of the polyvinyl alcohol is 0.5-2.0%, and preferably 1%. The volume percentage of the initial emulsion to the polyvinyl alcohol is 1: (2.5-3.5), and preferably 1:2.9.

[0013] The particle size of the nanoparticles is 175-185 nm, and the polydispersity index (PDI) is less than 0.2.

[0014] Further, the antisense peptide nucleic acid nanoparticle delivery system for targeting C1GALT1 genes is used for preparing a drug for treating C1GALT1 gene related diseases.

[0015] Further, the application of the antisense peptide nucleic acid nanoparticle delivery system targeting C1GALT1 gene in the preparation of a drug for treating IgA nephropathy.

[0016] Compared with the prior art, the application has the following advantages:

[0017] 1. High delivery efficiency: PLGA-PEG nanoparticles can effectively encapsulate antisense gamma peptide nucleic acid and promote intracellular nuclear delivery, and laser confocal microscopy shows that the intracellular fluorescence signal is enhanced.

[0018] 2. High knockdown efficiency: RT-qPCR and Western Blot confirm that the delivery system can significantly reduce the mRNA and protein expression levels of C1GALT1 gene.

[0019] 3. Biological safety: PLGA-PEG is a biodegradable material with no toxic side effects; antisense gamma peptide nucleic acid is resistant to enzymatic degradation and has high stability.

[0020] 4. Strong targeting: antisense gamma peptide nucleic acid has high specificity in binding to C1GALT1 mRNA and low off-target rate, and is positively correlated with the production of galactose-deficient IgA1 (Gd-IA1) in B lymphocytes, and is suitable for the study of IgA nephropathy and other diseases.

[0021] 5. Wide application: the application can be used as a tool for studying C1GALT1 related diseases or developed as a gene therapy drug. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The expression amount of ST6GALNAC2 gene mRNA (A) and the level of Gd-IgA1 in cell supernatant (B) described in the specific embodiments.

[0023] Figure 2 The expression amount of C1GALT1 gene mRNA (A) and the level of Gd-IgA1 in cell supernatant (B) described in the specific embodiments.

[0024] Figure 3 The expression amount of COSMC gene mRNA (A) and the level of Gd-IgA1 in cell supernatant (B) described in the specific embodiments.

[0025] Figure 4 The expression amount of GALNT2 gene mRNA (A) and the level of Gd-IgA1 in cell supernatant (B) described in the specific embodiments.

[0026] Figure 5 The PLGA-PEG particle size distribution histogram described in the specific embodiments. Figure A is a blank nanoparticle histogram, and Figure B is a gamma PNA-loaded nanoparticle histogram.

[0027] Figure 6 PLGA-PEG scanning electron microscopy figure described in the detailed description. Figure A is a blank nanoparticle scanning electron microscopy figure, and Figure B is a γPNA-loaded nanoparticle scanning electron microscopy figure.

[0028] Figure 7 B lymphocyte fluorescence figure of nanoparticle intervention described in the detailed description. Figure A is a γPNA-loaded nanoparticle figure, and Figure B is a blank nanoparticle figure. Blue represents the cell nucleus, and red represents γPNA.

[0029] Figure 8 C1GALT1 gene mRNA expression (A), protein expression (B), and Gd-IgA1 level (C) described in the detailed description.

[0030] Figure 9 CCK-8 detection of cell viability at different times in each group described in the detailed description.

[0031] Figure 10 Pattern figure of C1GALT1 gene knockdown by γPNA-loaded PLGA-PEG nanoparticles described in the detailed description. DETAILED DESCRIPTION

[0032] To explain the technical content of the technical scheme, the purpose and effect achieved, the following will be described in detail in conjunction with the specific examples and the accompanying drawings.

[0033] Example 1: Screening of genes related to Gd-IgA1 production

[0034] (1) Cell line: human B lymphocyte cell line.

[0035] (2) Method:

[0036] ① Co-culture the siRNA of genes ST6GALNAC2, C1GALT1, COSMC, and GALNT2 related to Gd-IgA1 production with B lymphocytes, and the siRNA dosage is 100 umol / L.

[0037] ② Explore the regulatory effect of ST6GALNAC2, C1GALT1, COSMC, and GALNT2 genes on B lymphocyte secretion of Gd-IgA1 through siRNA interference technology, and set up siRNA gene group, blank control group, and siRNA negative control group.

[0038] ③ Detect the mRNA expression amount of related genes by RT-qPCR. The reaction system is shown in Table 1, the reaction conditions are shown in Table 2, and the primer sequences of each gene are shown in Table 3.

[0039] Table 1 RT-qPCR reaction system

[0040]

[0041] Table 2 RT-qPCR reaction conditions

[0042]

[0043] Table 3 Gene primer sequences

[0044]

[0045] IV. ELISA was used to detect the level of Gd-IgA1 in the cell supernatant (Human Gd-gA1 ELISA KIT, Xiamen Lunchangshuo, item number 16081).

[0046] (3) Results:

[0047] RT-qPCR analysis showed that 24 h after transfection, the expression of ST6GALNAC2 mRNA in the siRNA ST6GALNAC2 group was significantly lower than that in the blank control group and the siRNA negative control group (A, P<0.0001), while there was no statistically significant difference between the blank control group and the siRNA negative control group (A, P>0.05). Further detection of the level of Gd-IgA1 in the cell supernatant by ELISA found that there was no difference in the level of Gd-IgA1 among the three groups after 24 h, 48 h and 72 h of intervention (B, P>0.05). Figure 1 Figure 1 Figure 1

[0048] RT-qPCR analysis showed that 24 h after transfection, the expression of C1GALT1 mRNA in the siRNA C1GALT1 group was significantly lower than that in the blank control group and the siRNA negative control group (A, P<0.0001), while there was no statistically significant difference between the blank control group and the siRNA negative control group (A, P>0.05). Further detection of the level of Gd-IgA1 in the cell supernatant by ELISA found that after 48 h of intervention, the level of Gd-IgA1 in the siRNA C1GALT1 group was significantly higher than that in the blank control group and the siRNA negative control group, and the difference was statistically significant (B, P<0.05); but after 24 h and 72 h of intervention, there was no statistically significant difference in the level of Gd-IgA1 among the three groups (B, P>0.05). Figure 2 Figure 2 Figure 2 Figure 2

[0049] ​​​​​​​RT-qPCR analysis showed that 24 h after transfection, the expression level of COSMC mRNA in the siRNA COSMC group was significantly lower than that in the blank control group and the siRNA negative control group. Figure 3 A, P<0.0001), while there was no statistically significant difference between the blank control group and the siRNA negative control group (A, P<0.0001). Figure 3 A, P>0.05). ELISA dynamic monitoring revealed that the level of Gd-lgA1 in the cell supernatant of the siRNA COSMC group decreased at 24 h and 48 h post-transfection, and increased at 72 h, but the differences between groups at each time point were not statistically significant. Figure 3 B, P>0.05).

[0050] RT-qPCR analysis showed that, 24 h after transfection, compared with the blank control group and the siRNA negative control group, the expression of GALNT2 mRNA in the siRNAGALNT2 group was significantly reduced. Figure 4 A, P < 0.0001), while there was no statistically significant difference between the blank control group and the siRNA negative control group (A, P < 0.0001). Figure 4 A, P > 0.05. Further ELISA analysis of Gd-IgA1 levels in cell supernatant revealed that at 24 h and 48 h after intervention, Gd-IgA1 levels in the siRNA GALNT2 group were lower than those in the blank control group and the siRNA negative control group. After 72 h of intervention, the levels showed an increasing trend. There were no statistically significant differences at any time point. Figure 5 B, P > 0.05).

[0051] Conclusion: Compared with the blank control group and the siRNA negative control group, knockdown of the C1GALT1 gene significantly increased the level of Gd-IgA1 in the cell supernatant. Knockdown of the ST6GALNAC2, COSMC, and GALNT2 genes did not result in statistically significant differences in the level of Gd-IgA1 in the cell supernatant. Therefore, C1GALT1 was selected as the target gene.

[0052] Example 2: Preparation and optimization of PLGA-PEG nanoparticles

[0053] (1) Materials: PLGA-PEG (molar ratio 70:30, molecular weight 10kDa), antisense γ-peptide nucleic acid (sequence see SEQ ID NO:1), dichloromethane, polyvinyl alcohol (PVA).

[0054] (2) Steps:

[0055] ① Dissolve different masses of PLGA-PEG in 1 mL of dichloromethane to form the oil phase.

[0056] ② 20 nmol antisense γ-peptide nucleic acid was dissolved in 60.8 uL deionized water as the water phase.

[0057] ③ The water phase was added to the oil phase, and ultrasonic emulsification was performed (ultrasonic 2 s and stop 1 s for 2 min, power 400 w) to form the primary emulsion.

[0058] ④ The primary emulsion was added to 2.9 mL of different concentrations of PVA solution, and high-speed homogenization was performed (ultrasonic 2 s and stop 1 s for 8 min, power 400 w) to form the re-emulsion.

[0059] ⑤ The dichloromethane was evaporated under a rotary evaporator for 10 minutes (rotation speed 70 rpm, temperature 40°C), and the nanoparticles were collected by centrifugation at 12000 rpm (4°C, 10 min) and washed with deionized water 3 times.

[0060] ⑥ Freeze-drying was performed to obtain nanoparticle powder.

[0061] (3) Results:

[0062] ① Determination of PVA concentration: 10 mg PLGA-PEG was used to prepare nanoparticles with 0.5%, 1% and 2% PVA, with deionized water instead of antisense γ-peptide nucleic acid as the water phase. Dynamic light scattering results showed that as the PVA concentration increased (0.5%, 1%, 2%), the nanoparticle size decreased significantly from 256.66±2.78 nm to 161.40±21.88 nm, accompanied by a decrease in the absolute value of Zeta potential from -31.07±0.20 mV to -25.72±0.68 mV (the lowest was -12.17±2.84 mV in the 1% PVA group), and the PDI was ≤0.10, as shown in Table 4. The results showed that 10 mg PLGA-PEG prepared with 2% PVA had the smallest nanoparticle size, and smaller nanoparticle size was more conducive to cell uptake, so 2% PVA was selected as the optimization scheme.

[0063] Table 4 Characterization of nanoparticles prepared with different concentrations of PVA (x̄±s, n=3)

[0064]

[0065] ② Determination of PLGA-PEG mass: 10 mg, 30 mg and 50 mg PLGA-PEG were dissolved in 1 mL dichloromethane, combined with 2% PVA to prepare nanoparticles, with deionized water instead of antisense γ-peptide nucleic acid as the water phase. Dynamic light scattering results are shown in Table 5. The results showed that 10 mg PLGA-PEG prepared with 2% PVA had the smallest nanoparticle size, and smaller nanoparticle size was more conducive to cell uptake, so 10 mg PLGA-PEG was selected as the optimization scheme.

[0066] Table 5 Characterization of nanoparticles prepared with different quality of PLGA-PEG (x±s, n=3)

[0067]

[0068] Based on the above optimization screening, 10 mg of PLGA-PEG was dissolved in 1 mL of dichloromethane, and 2% PVA was used to prepare nanoparticles by double emulsification-solvent evaporation method. Dynamic light scattering analysis showed that the particle size of blank PLGA-PEG nanoparticles was 178.80±1.37 nm, PDI was 0.09±0.02, and Zeta potential was -26.45±4.52 mV; after loading γPNA, the particle size of the nanoparticles increased slightly to 179.50±2.42 nm, PDI decreased significantly to 0.02±0.01, and the absolute value of Zeta potential increased to -36.01±0.36 mV, as shown in Table 6 and Figure 5 Scanning electron microscopy showed that the nanoparticles were uniformly distributed in spherical shape, and the particle size was consistent with the results of dynamic light scattering, as shown in Figure 6 , indicating that blank nanoparticles and γPNA-loaded nanoparticles were successfully prepared.

[0069] Table 6 Particle size, polydispersity index and potential characterization of nanoparticles (x±s, n=3)

[0070]

[0071] Example 3: Cell experiment of C1GALT1 gene γPNA delivered by nanoparticles

[0072] (1) Cell line: human B lymphocyte cell line.

[0073] (2) Methods and results:

[0074] ① The nanoparticles loaded with antisense γPNA (concentration 1 mg / mL) were co-cultured with cells, and the cell supernatant was collected at 24 hours, 48 hours and 72 hours. The experiment set up blank control group, blank nanoparticle control group and γPNA-loaded nanoparticle group.

[0075] ② Laser confocal microscope observation: the nanoparticles loaded with antisense γPNA (concentration 1 mg / mL) were co-cultured with cells for 24 hours, and TAMRA fluorescence showed that the nanoparticles successfully entered the cytoplasm, as shown in Figure 7

[0076] ​③ RT-qPCR: Nanoparticles loaded with antisense γPNA (concentration 1 mg / mL) were co-cultured with cells for 24 hours, total RNA was extracted, and C1GALT1 specific primers were used to detect mRNA levels (method same as Example 1). Compared with the blank nanoparticle control group and the blank control group, the C1GALT1 mRNA expression in the γPNA-loaded nanoparticle group was significantly decreased (p<0.05), as shown in Figure 8 A.

[0077] ④ Western Blot: Nanoparticles loaded with antisense γPNA (concentration 1 mg / mL) were co-cultured with cells for 48 hours, cell proteins were extracted, and C1GALT1 antibodies were used to detect protein levels. Compared with the blank nanoparticle control group and the blank control group, the C1GALT1 protein expression in the γPNA-loaded nanoparticle group was decreased (p<0.05), as shown in Figure 8 B.

[0078] ⑤ ELISA experiment: Compared with the blank nanoparticle control group and the blank control group, the Gd-IgA1 level in the supernatant of the cells in the γPNA-loaded nanoparticle group was significantly increased after 48 hours (p<0.05), as shown in Figure 8 C. It is indicated that the γPNA-loaded nanoparticles cause an increase in the Gd-IgA1 level in the supernatant of the cells by knocking down the C1GALT1 gene.

[0079] ⑥ CCK-8: CCK-8 detection of cell viability at 24 hours, 48 hours, and 72 hours showed no significant difference between the blank nanoparticle control group, the blank control group, and the γPNA-loaded nanoparticle group (p>0.05), as shown in Figure 9 . It is indicated that the nanoparticles have no significant toxic side effects on the cells.

[0080] The above examples show that the nanoparticles prepared in the present application can efficiently deliver antisense γ peptide nucleic acids, effectively knock down the C1GALT1 gene, and increase the Gd-IgA1 level (as shown in Figure 10 ), thereby providing a new strategy for the research and treatment of IgA nephropathy.

[0081] It should be noted that although the above embodiments have been described in this paper, the patent protection scope of the present application is not limited thereby. Therefore, based on the innovative idea of the present application, changes and modifications to the embodiments described in this paper, or equivalent structures or equivalent process transformations made using the contents of the present application specification and drawings, directly or indirectly apply the above technical solutions to other related technical fields, are all included in the patent protection scope of the present application.

Claims

1. An antisense peptide nucleic acid nanoparticle delivery system targeting the C1GALT1 gene, characterized by: The nanoparticle delivery system uses PLGA-PEG copolymer as a carrier material to load antisense gamma peptide nucleic acid and effectively deliver into cells to target and inhibit C1GALT1 gene expression.

2. The antisense peptide nucleic acid nanoparticle delivery system targeted to the ClGALTl gene of claim 1, wherein: The sequence of the antisense gamma peptide nucleic acid is H-TAMRA-KKK-CC#TCT#CGC#CCC#TCC#T-KKK-NH2 (SEQ ID NO: 1), wherein # represents lys-gamma modification, TAMRA is a fluorescent label, and KKK is a lysine sequence.

3. The antisense peptide nucleic acid nanoparticle delivery system targeting the C1GALT1 gene according to claim 1, characterized in that: The preparation method of the nanoparticle delivery system is as follows: dissolving PLGA-PEG in dichloromethane as an oil phase; dissolving antisense gamma peptide nucleic acid in deionized water as an aqueous phase; adding the aqueous phase into the oil phase, ultrasonic emulsification to form a primary emulsion; adding the primary emulsion into a polyvinyl alcohol solution, high-speed homogenization to form a multiple emulsion; evaporating organic solvents, centrifuging to collect nanoparticles, washing with deionized water, and then freeze-drying to obtain nanoparticle powder.

4. The antisense peptide nucleic acid nanoparticle delivery system targeted to the ClGALTl gene of claim 3, wherein: The molar ratio of PLGA to PEG in the PLGA-PEG copolymer is 70: (20-40).

5. The antisense peptide nucleic acid nanoparticle delivery system targeting the C1GALT1 gene according to claim 3, characterized in that: The concentration of the PLGA-PEG is 5-15 mg / ml, and the concentration of the antisense gamma peptide nucleic acid is 15-25 nmol / mL.

6. The antisense peptide nucleic acid nanoparticle delivery system targeting the C1GALT1 gene according to claim 3, characterized in that: The ultrasonic emulsification and high-speed homogenization method is ultrasonic 2-3 s, stop 1-2 s, the whole process is 6-10 min, and the ultrasonic power is 300-500 W.

7. The antisense peptide nucleic acid nanoparticle delivery system targeted to the ClGALTl gene of claim 3, wherein: n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each R is independently H, OH, or OCH3. The mass percentage concentration of the polyvinyl alcohol is 0.5%-2%, and the volume percentage of the primary emulsion to polyvinyl alcohol is 1: (2.5-3.5).

8. The antisense peptide nucleic acid nanoparticle delivery system targeted to the ClGALTl gene of claim 1 or 3, wherein: The particle size of the nanoparticles is 175-185 nm, and the polydispersity index is less than 0.

2.

9. Use of the antisense peptide nucleic acid nanoparticle delivery system targeting C1GALT1 gene according to any one of claims 1-7 in the preparation of a drug for treating C1GALT1 gene related diseases.

10. Use of the antisense peptide nucleic acid nanoparticle delivery system targeting C1GALT1 gene according to any one of claims 1-7 in the preparation of a drug for treating IgA nephropathy.

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