A method for preparing a co-loaded dual-gene targeted delivery system and its application

Abstract

This invention discloses a method for preparing a co-loaded dual-gene targeted delivery system and its application, involving the construction and preparation of a dual-gene (pHNF4α, pFOXA3) delivery system. This delivery system can target myofibroblasts (MFs) and activated hepatic stellate cells (aHSCs) and can be used for gene therapy of liver fibrosis. This invention synthesizes the targeting carrier material PEI. 25K -PEG 2K -pPB, through electrostatic adsorption, combines with pHNF4α and pFOXA3 to form a co-loaded dual-gene targeted delivery system, F4α / A3@PP-pPB. This delivery system, through the targeting group pPB, binds to MFs (metacellular matrix cells) that lead to excessive accumulation of extracellular matrix in fibrotic livers and to PDGFR (polydimethylformamide) highly expressed on the surface of aHSCs. On the one hand, it inhibits the activation of aHSCs; on the other hand, it transdifferentiates MFs into hepatocyte-like cells (iHep). These two modes, while combating liver fibrosis, can also achieve a certain degree of liver function reconstruction through transdifferentiated iHep cells, providing new ideas and methods for the effective treatment of liver fibrosis.

Description

Technical Field

[0001] This invention relates to a method for preparing a co-loaded dual-gene targeted delivery system and its application. This type of delivery system can target therapeutic genes to myofibroblasts (MFs) and activated hepatic stellate cells (aHSCs) for the treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a prevalent chronic liver disease worldwide. It is estimated that at least 800 million people worldwide suffer from chronic liver disease, and more than 2 million die from liver-related diseases each year [Journal of Hepatology. 2019, 70 (1), 151-171.]. Liver fibrosis is a pathological process accompanying various chronic liver diseases and is considered the beginning of the deterioration of liver disease. Liver fibrosis is an early, reversible pathological stage of liver disease, and its treatment is of great significance in the prevention and treatment of liver diseases [Gastroenterol. Hepatology. 2021, 18(3): 151-166.]. However, there are currently no effective drugs for its treatment in clinical practice, therefore, the development of effective drugs for the treatment of liver fibrosis is urgently needed.

[0003] When the liver is damaged, cytokines secreted and released by hepatocytes stimulate the activation of hepatic stellate cells (HSCs) and their further transformation into myofibroblasts (MFs) [Gut, 2019, 68(12):2214-2227.]. HSCs have been confirmed as the main source of ECM, and their activation and transformation into MFs are key cellular events in liver fibrosis [Hepatology, 2019, 70(6): 2107-2122]. Simultaneously targeting both aHSCs and MFs, a multi-pathway combined therapeutic strategy was developed. This strategy aims to inhibit the activation of aHSCs while inducing MFs to transdifferentiate into hepatocyte-like cells (iHep) through cell transdifferentiation. Both approaches can treat liver fibrosis while simultaneously achieving liver function reconstruction to some extent through the transdifferentiated iHep cells, providing new ideas and methods for the effective treatment of liver fibrosis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a co-loaded dual-gene targeted delivery system and its application. The constructed nanodelivery system can target and deliver therapeutic genes to myofibroblasts (MFs) and activated hepatic stellate cells (aHSCs). On the one hand, it inhibits the activation of aHSCs; on the other hand, it transdifferentiates MFs into hepatocyte-like cells (iHep). While effectively eliminating the two key effector cells of liver fibrosis, it can also reconstruct liver parenchymal function to a certain extent through cell transdifferentiation, thereby achieving a more effective therapeutic effect on liver fibrosis.

[0005] The technical solutions adopted by this invention to solve its technical problems include the following: Step (1): The targeting peptide pPB and 3-(2-pyridyl)propionic acid N-hydroxysuccinimide ester (SPDP) were stirred at room temperature until completely dissolved in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution. The SPDP solution was added to the pPB solution according to the corresponding molar ratio, and stirred at a certain reaction temperature and reaction time to obtain a dimethyl sulfoxide solution containing pPB-SPDP. Step (2): Add PEI 25K -PEG 2K -SH was stirred at room temperature until completely dissolved in pure water. The dimethyl sulfoxide solution containing pPB-SPDP obtained in step (1) was added. The mixture was stirred at a certain reaction temperature and reaction time. After the reaction was completed, the reaction solution was transferred to a dialysis bag (with a molecular weight cutoff of 3500 Da) and dialyzed in pure water. The dialysis solution was collected and freeze-dried to obtain the target carrier material PP-pPB. Step (3): Dissolve the targeting vector material PP-pPB in DEPC water, and mix the above solution with pHNF4α and pFOXA3 aqueous solution according to the experimental dosage. Mix well by pipetting and let stand at room temperature for 30 minutes to obtain the co-loaded dual gene targeting delivery system F4α / A3@PP-pPB. In the specific implementation process, in step (1), the molar ratio of pPB to SPDP is 1:0.1 ~ 1:10, the reaction temperature is 0℃ ~ 100℃, and the reaction time is 0.1 ~ 48 h.

[0006] In step (2), PEI 25K -PEG 2K The molar ratio of -SH to pPB-SPDP is 0.5:40 ~ 40:1, the reaction temperature is 0℃ ~ 100℃, and the reaction time is 0.1 ~ 48 h.

[0007] In step (3), the concentration of PP-pPB is 0.5 ~ 2 mg / mL solution, the concentration of pHNF4α and pFOXA3 is 0.1 ~ 2 mg / mL, and the mass ratio of the total amount of pHNF4α and pFOXA3 to PP-pPB when mixed is 1:50 ~ 10:1.

[0008] The co-loaded dual-gene targeted delivery system F4α / A3@PP-pPB prepared by the aforementioned method can be applied to the treatment of liver fibrosis.

[0009] The activity evaluation results of the co-loaded dual-gene targeted delivery system F4α / A3@PP-pPB showed that this type of delivery system can achieve targeted gene delivery to activated hepatic stellate cells and myofibroblasts. While specifically inhibiting the activation of hepatic stellate cells, it can also transdifferentiate myofibroblasts into hepatocyte-like cells, thus exerting a synergistic anti-hepatic fibrosis effect. It showed a highly effective anti-hepatic fibrosis treatment effect without causing damage to other major organs, and has good prospects for clinical application. Attached Figure Description

[0010] Figure 1 In the image, Figure A is a scanning electron microscope image of F4α / A3@PP-pPB (scale bar: 200 nm), and Figure B is a particle size distribution map of F4α / A3@PP-pPB.

[0011] Figure 2 This is a targeting experiment of F4α / A3@PP-pPB (scale bar: 20 μm). Figure A is a fluorescence diagram of competitive uptake of aHSCs and MFs, and Figure B is a quantitative analysis diagram of Figure A.

[0012] Figure 3 This is an in vitro experiment on the inhibition of hepatic stellate cell activation and the induction of myofibroblast transdifferentiation by F4α / A3@PP-pPB (scale bar: 20 μm). Figure A is an evaluation of the effect of nanoparticles on inhibiting hepatic stellate cell activation in vitro. Figure B is a semi-quantitative analysis of Collagen I in Figure A. Figure C is a semi-quantitative analysis of α-SMA in Figure A. Figure D is an effect of nanoparticles on inducing myofibroblast transdifferentiation.

[0013] Figure 4 These are liver photographs of mice in different treatment groups, with HE, Sirius red, and Masson staining and immunohistochemical staining images (10×).

[0014] Figure 5 These are HE staining images of major tissues and organs in a liver fibrosis model mouse after treatment in various groups. Detailed Implementation

[0015] The specific embodiments of the present invention are further described below. The specific embodiments described below provide a more detailed explanation of the technical problems solved and the technical safeguards provided by the present invention. It should be understood that the following descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. 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. Example 1

[0016] Preparation of Targeted Carrier Material PP-pPB Step (1): Take 10 mg of the target peptide (0.01 mmol) pPB and 6.7 mg of 3-(2-pyridyl)propionic acid N-hydroxysuccinimide ester (SPDP, 0.02 mmol) and stir them at room temperature until they are completely dissolved in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution. Add the SPDP solution to the pPB solution according to the corresponding molar ratio and stir the reaction at 25℃ for 24 h to obtain a dimethyl sulfoxide solution containing pPB-SPDP. Step (2): 37.6 mg (1.4 mmol) of PEI 25K -PEG 2K -SH was stirred at room temperature until completely dissolved in 40 mL of pure water. The mixture containing pPB-SPDP obtained in step (1) was added. The reaction was stirred at 25 °C for 24 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed in pure water. The dialysis solution was collected and freeze-dried to obtain the target carrier material PP-pPB, a white solid with a yield of 75.4%. Example 2

[0017] Preparation and characterization of co-loaded dual-gene targeted delivery system pHNF4α, pFOXA3, and PP-pPB were prepared into solutions with concentrations of 0.5 µg / µL and 1 mg / mL using DEPC-treated water. The two solutions were mixed in the desired experimental concentrations according to the specified ratio, and thoroughly mixed by pipetting. The mixtures were then incubated at room temperature for 30 minutes to obtain F4α / A3@PP-pPB nanoparticles. The optimal loading ratio of PP-pPB to genes (pHNF4α and pFOXA3) was screened using agarose gel electrophoresis. The particle size of F4α / A3@PP-pPB was determined using a laser particle size analyzer, and its morphology was observed using scanning electron microscopy.

[0018] Experimental Results: Based on gel electrophoresis, PP-pPB was screened and combined with genes (pHNF4α and pFOXA3) at a mass ratio of 2:1 to prepare nanoparticles. The particle size and morphology were then investigated. The results are as follows: Figure 1 As shown. The results indicate that the particle size of F4α / A3@PP-pPB nanoparticles is 77.7 ± 2.4 nm, and the PDI is 0.22 ± 0.01 (…). Figure 1 (Figure A). Scanning electron microscopy results of the nanoparticles show that they are spherical and uniformly dispersed. Figure 1 (Figure B in the middle) Example 3

[0019] Targeting evaluation of co-loaded dual-gene targeted delivery system Targeting efficacy was evaluated using a pPB competitive uptake inhibition assay. aHSCs or MFs cells were incubated for 2 h with DMEM complete culture medium containing pPB (without pPB) and pPB (containing 2 μg / mL pPB), respectively, to allow the platelet-derived growth factor receptor on the cell surface to fully bind to pPB. Cells were then incubated with F4α / A3@PP and F4α / A3@PP-pPB for 4 h, respectively. The cell surface was washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and the nuclei were stained with 1 μg / mL DAPI for 15 min. The cell surface was then washed twice with PBS, and cell uptake was observed using a laser confocal microscope.

[0020] The results of the pPB competitive uptake experiment showed that ( Figure 2 Upon addition of free pPB, the uptake of F4α / A3@PP-pPB nanoparticles by aHSCs and MFs cells was significantly reduced, but the uptake of F4α / A3@PP nanoparticles without the pPB target was unaffected. This indicates that pPB competitively inhibits the uptake of F4α / A3@PP-pPB nanoparticles, validating that the constructed nanodelivery system can achieve active targeted delivery and therapy to aHSCs and MFs cells. Example 4

[0021] In vitro anti-hepatic fibrosis activity evaluation of co-loaded dual-gene targeted delivery system HSCs were seeded into 24-well cell culture plates, 1 × 10⁶ cells per well. 5 After adhesion, each group was activated with 10 ng / mL TGF-β1 for 24 h, and then incubated with F4α / A3@PP-pPB and F4α / A3@PP (plasmid concentration 2 μg / mL, 0.25 mL / well) for 48 h. After washing three times with PBS, fixation with 4% paraformaldehyde for 20 min, washing three times with PBS, and permeabilization with 0.1% Triton-X-100 at room temperature for 10 min, followed by three more washes with PBS. After blocking with 5% serum at room temperature for 30 min, primary antibody blocking overnight at 4°C (α-SMA and Collagen I antibody), and secondary antibody blocking at room temperature for 1 h, protein expression was observed using a laser confocal microscope. Simultaneously, MFs cells were seeded in 6-well plates, 1 × 10⁶ cells per well. 5 After cell adhesion, each group was incubated with F4α / A3@PP-pPB and F4α / A3@PP (plasmid concentration 2 μg / mL, 0.25 mL / well) for 48 h, followed by subculturing and seeding in plates for 72 h. This process was repeated four times. The morphological changes of iHeps in the resulting cells were observed and compared with those of MFs and Heps.

[0022] The in vitro anti-liver fibrosis experiment results of F4α / A3@PP-pPB showed that ( Figure 3 After treatment with various drugs, the expression levels of fibrosis-associated proteins α-SMA and Collagen I were reduced, with the F4α / A3@PP-pPB group showing the best inhibitory effect on hepatic stellate cell activation. Simultaneously, the morphology of iHep cells obtained after four consecutive transfections of MFs with F4α / A3@PP-pPB nanoparticles showed significant changes compared to the initial MFs morphology, becoming more similar to hepatocytes (Hep), indicating that the nanoparticles can induce MFs to transdifferentiate into hepatocytes. Example 5

[0023] In vivo anti-hepatic fibrosis activity evaluation of co-loaded dual-gene targeted delivery system A liver fibrosis mouse model was established by intraperitoneal injection of thioacetamide every other day for 4 weeks in 6-8 week old BALB / c mice. The mice were divided into 6 groups (n=6 per group) and treated with saline (II), F4α@PP (III), A3@PP (IV), F4α / A3@PP (IV), and F4α / A3@PP-pPB (VI) via tail vein injection every other day (plasmid dosage 1 mg / kg, for 4 weeks). The in vivo anti-liver fibrosis activity of the F4α / A3@PP-pPB gene delivery system was evaluated using a healthy mouse group (I) as a control. Twenty-four hours after the last administration, heart, liver, spleen, lung, and kidney tissues were collected from the mice. The heart, liver, spleen, lung, and kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and processed into 4 mm thick sections. Paraffin sections of the heart, spleen, lungs, and kidneys were dewaxed, rehydrated, and stained with hematoxylin and eosin (HE). The liver was stained with HE, Sirius red, Masson's red, and immunohistochemically with α-SMA and Collagen I. The stained sections were observed using an upright microscope.

[0024] In vivo anti-hepatic fibrosis activity evaluation experiments of F4α / A3@PP-pPB showed that ( Figure 4 After treatment with F4α / A3@PP-pPB nanoparticles, a significant reduction in liver fibrosis was observed, with intact liver lobule structure and no obvious fibrous septa. Compared with other treatment groups, it showed the best anti-liver fibrosis effect. At the same time, no obvious organ damage was found in the HE staining results of the heart, spleen, lungs and kidneys. Figure 5 Therefore, the co-loaded dual-gene targeted delivery system F4α / A3@PP-pPB constructed in this invention not only possesses excellent anti-liver fibrosis therapeutic effects but also exhibits good safety, providing important clinical guidance for the research of drugs for the treatment of liver fibrosis.

Claims

1. A method for preparing a co-loaded dual-gene targeted delivery system, characterized in that: Polyethylene imine (PEI) PEGylated with polyethylene glycol 25K -PEG 2K Using SH and platelet-derived growth factor PDGFR targeting peptide pPB as raw materials, the two are chemically reacted to synthesize the targeting carrier material PEI. 25K -PEG 2K -pPB, or PP-pPB; the obtained targeting carrier material PP-pPB is combined with pHNF4α and pFOXA3 through electrostatic adsorption to form a co-loaded dual-gene targeted delivery system F4α / A3@PP-pPB; this delivery system binds to MFs that cause excessive accumulation of extracellular matrix in fibrotic liver and PDGFR highly expressed on the surface of aHSCs through the targeting group pPB. On the one hand, it inhibits the activation of aHSCs, and on the other hand, it transdifferentiates MFs into hepatocytes. The two modes achieve liver function reconstruction through transdifferentiated hepatocytes while resisting liver fibrosis.

2. The method for preparing the co-loaded dual-gene targeted delivery system according to claim 1, characterized in that, The specific steps are as follows: Step (1): The targeting peptide pPB and 3-(2-pyridyl)propionic acid N-hydroxysuccinimide ester SPDP were stirred at room temperature until completely dissolved in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution. The SPDP solution was added to the pPB solution and stirred at a certain reaction temperature and reaction time to obtain a dimethyl sulfoxide solution containing pPB-SPDP. Step (2): Add PEI 25K -PEG 2K -SH was stirred at room temperature until completely dissolved in pure water. The dimethyl sulfoxide solution containing pPB-SPDP obtained in step (1) was added. The mixture was stirred at a certain reaction temperature and reaction time. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed in pure water. The dialysis solution was collected and freeze-dried to obtain the target carrier material PP-pPB. Step (3): Dissolve the targeting vector material PP-pPB in DEPC water. According to the experimental dosage, mix the above solution with pHNF4α and pFOXA3 aqueous solution in proportion. Mix well by pipetting and let stand at room temperature for 30 minutes to obtain the co-loaded dual gene targeting delivery system F4α / A3@PP-pPB.

3. The method for preparing the co-loaded dual-gene targeted delivery system according to claim 2, characterized in that: In step (1), the molar ratio of pPB to SPDP is 1:0.1 ~ 1:10, the reaction temperature is 0℃ ~ 100℃, and the reaction time is 0.1 ~ 48 h.

4. The method for preparing the co-loaded dual-gene targeted delivery system according to claim 2, characterized in that: In step (2), PEI 25K -PEG 2K The molar ratio of -SH to pPB-SPDP is 0.5:40 ~ 40:1, the reaction temperature is 0℃ ~ 100℃, and the reaction time is 0.1 ~ 48 h.

5. The method for preparing the co-loaded dual-gene targeted delivery system according to claim 2, characterized in that: In step (3), the concentration of PP-pPB is 0.5 ~ 2 mg / mL solution, the concentration of pHNF4α and pFOXA3 is 0.1 ~ 2 mg / mL, and the mass ratio of the total amount of pHNF4α and pFOXA3 to PP-pPB is 1:50 ~ 10:

1.

6. The application of the co-loaded dual-gene targeted delivery system prepared by the preparation method according to claims 1-5 in the preparation of drugs for treating liver fibrosis.

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