CN104840947B - AFP antibody modification PLGA loads DCN nanoparticle anti-tumor drugs targeting - Google Patents

Abstract

An AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug, which uses DCN recombinant plasmids as the core therapeutic drug, selects highly specific liver cancer-targeting AFP monoclonal antibody molecules, and is prepared using degradable polymer biomaterials The drug-loaded nanoparticle polylactic acid/glycolic acid complex (PLGA) is loaded with DCN anti-cancer gene plasmid and coupled with liver cancer-targeting AFP monoclonal antibody molecule to construct AFP mAb‑PLGA‑rhDCN therapeutic nanoparticles. The medicine of the present invention can actively combine with liver cancer cells and enter into the cells, slowly release DCN plasmid, and realize specific targeting inhibition on liver cancer cells. The medicine of the invention improves the action efficiency of the transfection gene, realizes the long-term inhibitory effect of the target gene on the target cell at the same time, is used for the targeted therapy of primary liver cell carcinoma, and has great market potential.

Description

AFP Antibody Modified PLGA-loaded DCN Nanoparticles Targeting Antitumor Drugs

technical field

The invention belongs to the technical field of biomedicine and relates to a targeted antitumor drug, in particular to a targeted therapeutic drug for primary hepatocellular carcinoma.

Background technique

Primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the most common malignant tumors with the highest mortality rate. The incidence of HCC in my country ranks first in the world and is still increasing year by year. It is a major disease that seriously threatens the health of our people. Due to the characteristics of high malignancy, early metastasis, and high recurrence rate of HCC, its curative effect is severely restricted. In addition, traditional treatment methods have a great negative impact on the immune function of the body, which accelerates and intensifies the tumor progression, and the prognosis is extremely poor. Paying attention to and vigorously developing HCC treatment research has very important and realistic social and medical significance to our country. With the continuous development of molecular biology and immunology, the understanding of the occurrence and development mechanism of HCC has been deepened, and the gene-targeted therapy of HCC has become a hot spot in clinical tumor research.

Traditional gene carrier systems are divided into two types: viral vectors and non-viral vectors. Existing viral vectors have defects in immunogenicity and tumorigenicity; non-viral vectors have the problem of low delivery efficiency. At the same time, these methods of gene transfection lack cell targeting, and the target gene carried by the carrier is often digested and destroyed by intracellular enzymes in a short period of time after it acts on tumor cells through transfection. low degree. These problems lead to the poor effect of traditional gene therapy, which has become a bottleneck restricting the further development of gene therapy.

Nano-gene carrier is a new type of gene carrier system, which uses nanoparticles as carriers, wraps therapeutic genes in nanoparticles or adsorbs them on the surface, and allows nanoparticles to enter cells and release therapeutic genes through cellular endocytosis, thereby exerting Gene therapy efficacy. Polylactic acid-polyglycolic acid (PLGA) nanoparticles are biomaterials with good biocompatibility and degradability, and have been approved by the US FDA for use in the human body. PLGA nanoparticles also have a nucleotide protection effect. By modifying the active carboxyl group at the end of PLGA, they can be coupled to ligands or antibodies of target cells to achieve active targeting of gene therapy.

Decorin (decorin, DCN) is a pleiotropic molecule of proteoglycan (proteoglycan, PG) family. It regulates cell cycle through core protein and regulates cell proliferation, differentiation and matrix formation by binding cytokines. A large amount of data in recent years shows that: 1) DCN is an effective inhibitor of tumor cell growth and migration, which can inhibit the growth of tumor cells from a variety of different tissue sources and induce their apoptosis; 2) DCN has recently been found to be a Met(HGFR) 3) DCN can also reverse the drug resistance of tumor cells.

The complex binding ability of DCN to multiple targets and the striking anti-tumor effect fully demonstrate its medicinal value in tumor therapy. DCN gene therapy studies conducted by foreign scholars using adenovirus vectors have shown that DCN has local and remote specific anti-tumor effects. There are individual reports that the reduction of DCN content can be used as an indicator to predict poor prognosis. The previous research data of our research group showed that whether DCN is overexpressed in vivo, or used as a recombinant protein or gene transfected cells, it can inhibit the growth of tumor cells from a variety of different tissue sources and induce tumor cell apoptosis, and DCN can reverse tumor cell drug resistance.

Contents of the invention

The purpose of the present invention is to solve the problems existing in traditional gene carriers, and provide a novel carrier-based AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug.

The AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug of the present invention is based on multiple anti-tumor effects such as DCN inhibiting tumor cell proliferation, metastasis, tumor angiogenesis, and reversing tumor cell drug resistance, with DCN recombinant plasmid as the core treatment Drugs, select highly specific liver cancer-targeting AFP monoclonal antibody molecules, use drug-loaded nanoparticles polylactic acid/glycolic acid complex (PLGA) prepared from degradable polymer biomaterials to load DCN anti-cancer gene plasmids and couple to liver cancer AFP mAb-PLGA-rhDCN therapeutic nanoparticles are constructed by targeting AFP monoclonal antibody molecules.

The preparation method of AFP antibody-modified PLGA-loaded DCN nanoparticles targeting anti-tumor drug of the present invention is to disperse PLGA in dichloromethane solution as oil phase, and disperse pIRES2-EGFP-DCN plasmid in 1wt% PVA aqueous solution as water phase, mix the oil phase with the water phase to make colostrum; drop the colostrum into 0.3wt% PVA aqueous solution and stir to form double emulsion; remove the dichloromethane in the double emulsion, collect nanoparticles by centrifugation, and suspend in Nanoparticle dispersion system is made in the PBS buffer solution of pH7.4; Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N to the nanoparticle dispersion system -Hydroxysuccinimide (NHS), react to obtain activated nanoparticles, add AFP monoclonal antibody, react to obtain AFP mAb-PLGA-rhDCN therapeutic nanoparticles.

Wherein, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide added is 2:1.

Further, in the above preparation method of the present invention, the activation is a stirring reaction at room temperature for 40 minutes; the antibody coupling reaction time is 6 hours.

The targeted anti-tumor drug of the present invention utilizes the characteristics of AFP secreted by liver cancer cells and the specific binding of antibodies to antigens, so that AFP mAb-PLGA-rhDCN nanoparticles can actively bind to liver cancer cells and enter the cells, slowly releasing DCN plasmids and expressing DCN genes The corresponding encoded product exerts its anti-tumor effect and realizes the specific targeted inhibition of DCN on liver cancer cells. The targeted antitumor drug of the present invention avoids the problem of rapid degradation of genes transferred into cells by liposome transfection methods, improves the efficiency of transfected genes, and realizes long-term inhibition of target cells by target genes effect.

The targeted antitumor drug prepared by the invention can be used for the targeted therapy of primary hepatocellular carcinoma, and has great market potential.

Description of drawings

Fig. 1 is a diagram of the appearance and morphology of AFP mAb-PLGA-rhDCN nanoparticles prepared in the present invention.

Fig. 2 is a particle size distribution diagram of AFP mAb-PLGA-rhDCN nanoparticles prepared in the present invention.

Fig. 3 is a Zeta potential diagram of AFP mAb-PLGA-rhDCN nanoparticles prepared in the present invention.

Fig. 4 is an in vitro release curve of AFP mAb-PLGA-rhDCN nanoparticles prepared in the present invention.

Fig. 5 shows the situation that AFP mAb-PLGA-rhDCN nanoparticles prepared by the present invention enter HepG2 cells.

Fig. 6 is the inhibition rate of proliferation of HepG2 cells by different concentrations of AFP mAb-DCN-PLGA nanoparticles.

Figure 7 shows the effect of different concentrations of AFP mAb-DCN-PLGA nanoparticles on the transfer ability of HepG2 cells.

Figure 8 shows the effect of different concentrations of AFP mAb-DCN-PLGA nanoparticles on the invasion ability of HepG2 cells.

detailed description

Example 1

Add 20mg of PLGA to 1ml of dichloromethane to disperse the polymer uniformly as the oil phase (O); take 2ml of 1wt% PVA aqueous solution dissolved with 500μg of pIRES2-EGFP-DCN plasmid as the water phase (W1). The oil phase (O) and the water phase (W1) were mixed, and the ice-water bath was ultrasonicated for 5 minutes (ultrasonic power 300W, supersonic for 1 s and stop for 2 s), to obtain colostrum (W1/O). Then the obtained colostrum was dropped into 10ml of 0.3wt% PVA aqueous solution (W2), and magnetically stirred for 5min to form double emulsion (W1/O/W2). The obtained double emulsion was rotary evaporated to remove dichloromethane, and centrifuged to obtain PLGA-rhDCN nanoparticles.

Suspend the obtained PLGA-rhDCN nanoparticles in 2 mL of PBS buffer to make a dispersion system, then add 200 μg of a mixture of EDC and NHS (the molar ratio of EDC to NHS is 20:10), stir at room temperature for 40 min, and centrifuge at 10,000 r/min for 5 min , to obtain activated PLGA-rhDCN nanoparticles precipitate. Collect the precipitate, wash twice with 2ml PBS buffer (pH 7.4), redisperse in PBS buffer, add 200 μg AFP monoclonal antibody, stir and react for 6 hours, repeat centrifugation and washing with PBS to obtain AFPmAb-DCN-PLGA nanoparticles, freeze Store dry.

Example 2

Take 10 μg of the nanoparticles prepared in Example 1 and disperse them in 1 ml of PBS buffer to make a suspension, drop them on the carbon-coated support membrane (copper grid for electron microscopy), and dry in the air. The morphology characteristics of the nanoparticles shown in Figure 1 were observed under a scanning electron microscope. In the figure, A is x4000 times, and B is x5000 times. The particle size distribution range and Zeta potential of the nanoparticles were measured with a laser particle size analyzer. The results are shown in Figure 2 and Figure 3. The particle size range of the nanoparticles is mainly distributed between 100 and 300nm, and the Zeta potential is between -20 and -13mV.

From Fig. 1, Fig. 2 and Fig. 3, it can be seen that the particle size of the prepared nanoparticles belongs to the optimal range for cell uptake. If the particle size of the prepared nanoparticles is too large, it will make it difficult for the particles to enter the cells; if the particle size is too small, it may destroy the biological structure of the contained gene, and at the same time reduce the effectiveness of the conjugated antibody and lose its targeted anti-tumor effect. The nanoparticle prepared by the present invention minimizes the particle size of the nanoparticle as far as possible on the premise of effectively wrapping the loaded target gene plasmid to ensure the integrity of the nanoparticle structure and function, so as to facilitate cell uptake.

Example 3

Take 20 μg of nanoparticles prepared in Example 1, resuspend them in a centrifuge tube filled with 3ml of PBS buffer and seal, place the centrifuge tube in a constant temperature shaker at 37°C, centrifuge at 100r/min, and separate at 1h, 12h, and 24h , 36h, 48h, 72h and after that, centrifuge every 24h, take 2ml supernatant, detect the amount of released plasmid, and supplement 2ml fresh PBS buffer at the same time, continue to shake at constant temperature until the time point of 240h. Calculate the cumulative release rate and draw the curve as shown in Figure 4.

The average value of cumulative release rate in the first 24h was 35.01%, and then released slowly, and the release rate reached 79.3% at 240h.

In the results shown in Figure 4, part of the gene-carrying plasmid was adsorbed on the nanomaterial to form an unstable combination, which led to a sudden increase in the release rate within the first 24 hours. This burst release phenomenon can make the drug quickly form a higher therapeutic concentration in the tumor tissue, and quickly exert its anti-tumor effect. Afterwards, with the gradual degradation of PLGA in the cells, the contained genes were continuously released slowly and steadily, realizing the long-term inhibitory effect of DCN on HepG2 cells, and avoiding the rapid destruction of genes entered into the cells by transfection methods such as liposomes. Degradation improves the efficiency of gene action.

Example 4

The IgG antibody was used instead of the AFP monoclonal antibody, and the other steps were exactly the same as in Example 1 to prepare IgG-DCN-PLGA nanoparticles, which were used as materials for the control test of AFP mAb-DCN-PLGA nanoparticles.

Example 5

First, the antibodies were labeled with carboxyfluorescein (FAM), the PLGA nanoparticles loaded with DCN plasmids were labeled with Rhodamine fluorescence, and the cell nuclei were labeled with Hoechst33342. Then according to the steps described in Examples 1 and 4, double fluorescently labeled nanoparticles were prepared respectively.

HepG2 cells in the logarithmic growth phase were inoculated in a six-well plate, and after 12 hours of culture, 10 μg of fluorescently labeled AFP mAb-DCN-PLGA nanoparticles were incubated with the cells as the experimental group, and 10 μg of fluorescently labeled IgG-DCN-PLGA The co-incubation of nanoparticles and cells was used as a negative control group. At 4h and 12h respectively, the co-localization of the two types of fluorescence was observed under a laser confocal microscope × 630 times (488nm excited FAM, 543nm excited rhodamine, 350nm excited Hoechst33342).

The results are shown in Figure 5, in which the blue fluorescence marks the nucleus, the green and red fluorescence double-fluorescently label the nanoparticles, and the yellow fluorescence is the color after the green and red fluorescence overlap. Experimental group (a) has a large amount of fluorescence gathered around the nucleus, indicating that AFP mAb-PLGA-rhDCN nanoparticles have a biological targeting effect, and HepG2 cells can actively take up AFP mAb-modified nanoparticles, and the longer the action time, the greater the intake ; while in the negative control group (b) there was only a small amount of fluorescence gathered around the nucleus, indicating that the non-targeting IgG could not mediate a large amount of nanoparticles into the cell.

Example 6

The inhibitory effect of AFP mAb-PLGA-rhDCN nanoparticles on HepG2 cells was evaluated by MTT assay. HepG2 cells in the logarithmic phase were inoculated on a 96-well plate, cultured in 180 µL DMEM medium for 12 hours, and 20 µL of AFP mAb-PLGA-rhDCN nanoparticles with final concentrations of 5 µg/mL, 10 µg/mL, and 15 µg/mL were added to the cultured cells As low, medium, and high dose experimental groups, 20 µL of diluted IgG-DCN-PLGA nanoparticles were added to cultured cells as a negative control group, and cultured cells without nanoparticles were used as a blank control group, and incubated for 24h, 48h, After 72 hours, discard the culture solution, add 100 µL of fresh serum-free DMEM culture solution and 20 µL MTT solution, discard the culture solution after incubation for 4 hours, add 150 µL DMSO to each well, and measure the OD value of each well at 490 nm with a microplate reader. Three replicate holes were set up for each group. The relative inhibition rate of nanoparticles to cells was calculated according to the following formula: (OD value of blank well-OD value of experimental well)/OD value of blank well×100%.

The experimental results are shown in Figure 6. Compared with the IgG non-specific antibody group, the AFP mAb-PLGA-rhDCN nanoparticles in the experimental group had a significantly higher inhibitory rate on the proliferation of HepG2 cells, and it was enhanced with the increase of the concentration of the effect. strengthened over time.

Example 6

The effect of AFP mAb-PLGA-rhDCN nanoparticles on the migration ability of HepG2 cells was determined by scratch injury assay. Cells in the logarithmic growth phase were taken and blown to form a single cell suspension, and 1×10 ⁵ cells per well were seeded in a 6-well plate. After the cells adhered to the wall the next day, use a sterile pipette tip to scratch, and ingest the corresponding nanoparticles to treat the cells according to the experiment in Example 5. After 24 hours, take pictures of the scratches, as shown in Figure 7, and perform image analysis.

The results of the scratch test in Figure 7 show that the scratch width of cells in the experimental group treated with different concentrations of AFP mAb-PLGA-rhDCN nanoparticles is significantly higher than that of the control group, suggesting that AFP mAb-PLGA-rhDCN nanoparticles have the ability to migrate HepG2 cells There is an inhibitory effect, and it shows a certain dose-dependent trend.

Example 7

Transwell chamber invasion assay was used to determine the effect of AFP mAb-PLGA-rhDCN nanoparticles on the invasion ability of HepG2 cells. After the cells in the logarithmic phase were digested with trypsin, the culture medium was suspended, and 1×10 ⁵ cells per well were seeded in a 6-well Transwell plate, grouped according to the experiment in Example 5, and stimulated with nanoparticles for 24 hours. The following operations were performed: the chamber was washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes; washed three times with PBS, 5 minutes each time; cells were permeabilized with 0.1% Trition X-100 for 15 minutes; washed three times with PBS, 5 minutes each time; stained with hematoxylin for 20 minutes, Wash with water for 10 minutes, remove the penetrating membrane of the small chamber after it turns blue, put it on a glass slide, seal the slide and examine under the microscope. Three high-power fields of view were randomly selected, and the number of cells on the lower surface of the chamber was counted, which was the number of cells that penetrated Matrigel. The experimental results are shown in Figure 8.

The results in Figure 8 show that the number of membrane-penetrating cells in the experimental group showed a downward trend compared with the untreated group and the blank group, indicating that AFP mAb-PLGA-rhDCN nanoparticles have an inhibitory effect on the invasion ability of HepG2 cells.

Claims (6)

1. A method for preparing AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drugs, using DCN recombinant plasmids as the core therapeutic drug, loading DCN plasmids with PLGA, and coupling liver cancer-targeting AFP monoclonal antibody molecules, AFP mAb-PLGA-rhDCN therapeutic nanoparticles were constructed according to the following method: PLGA was dispersed in dichloromethane solution as the oil phase, pIRES2-EGFP-DCN plasmid was dispersed in 1wt% PVA aqueous solution as the water phase, and the oil The colostrum is mixed with the water phase to make colostrum; the colostrum is dropped into a 0.3wt% PVA aqueous solution and stirred to form a double emulsion; the dichloromethane in the double emulsion is removed, and the nanoparticles are collected by centrifugation, suspended in PBS buffer of pH 7.4 A nanoparticle dispersion system is prepared in the nanoparticle dispersion system; EDC/NHS is added to the nanoparticle dispersion system to react to obtain activated nanoparticles, and AFP monoclonal antibody is added to react to obtain AFP mAb-PLGA-rhDCN therapeutic nanoparticles. 2. The preparation method of AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug according to claim 1, characterized in that the DCN recombinant plasmid is pIRES2-EGFP-DCN. 3. The preparation method of AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug according to claim 1, characterized in that the molar ratio of added EDC to NHS is 2:1. 4. The preparation method of AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug according to claim 1, characterized in that the activation is a stirring reaction at room temperature for 40 minutes. 5. The preparation method of AFP antibody-modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug according to claim 1, characterized in that the coupling reaction time of the antibody is 6h. 6. The AFP antibody modified PLGA-loaded DCN nanoparticle-targeted anti-tumor drug prepared by the preparation method described in claim 1.