CN103611169A - Immune magnetic albumin nanosphere with targeting and preparation method thereof - Google Patents

Abstract

The invention discloses a targeted gene-loaded immunomagnetic albumin nanosphere and a preparation method thereof. First, the gene-loaded magnetic albumin nanosphere is prepared by a desolvation-crosslinking method: the plasmid and the magnetic nanoparticle are incubated for 30 minutes to obtain Plasmid/nanoparticle complex, this complex is then mixed with a solution of bovine serum albumin, and dropped with absolute ethanol until the solution becomes gelatinous. The precipitate after centrifugation is the gene-loaded magnetic albumin nanosphere. Then, the monoclonal antibody cetuximab is linked to the surface of the gene-loaded magnetic albumin nanospheres by using a heterotype bifunctional cross-linking agent to obtain targeted immunomagnetic albumin nanospheres. The nanosphere prepared by the invention has immune characteristics through immunological detection, and its targeting ability to lung cancer cell GLC-82 is detected in vitro, and the result shows good targeting ability.

Description

Targeted immunomagnetic albumin nanosphere and preparation method thereof

technical field

The invention belongs to the research field of anti-tumor targeted drugs, and in particular relates to a targeted immunomagnetic albumin nanosphere and a preparation method thereof.

Background technique

(1) Application of Survivin-siRNA (interfering RNA of survivin gene) in tumor therapy

Survivin is a newly cloned apoptosis suppressor gene, survivin protein is the inhibitor of apoptosis protein (inhibitor ofapoptosis protein, IAP) with the smallest molecule and the strongest function found so far. Its expression site is very special, it is only expressed in embryo and developing fetal tissues, not in terminally differentiated normal adult tissues (except thymus and gonad); however, survivin is expressed in cancer cells cultured in vitro and in most human tumor tissues , such as lung cancer, colon cancer, pancreatic cancer, breast cancer, etc. Studies have also shown that the survivin gene can inhibit cell apoptosis and may play an important role in the occurrence and development of lung cancer; therefore, blocking or down-regulating the expression level of the survivin gene and restoring the normal apoptosis regulation mechanism has become a new method for gene therapy of lung cancer. train of thought.

RNA interference (RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution, and is a sequence-specific post-transcriptional gene silencing mechanism. In recent years, RNAi research has made a breakthrough, and was rated as one of the top ten scientific advances in 2001 by the "Science" magazine, and listed as the top ten scientific advances in 2002. Since the use of RNAi technology can specifically knock out or shut down the expression of specific genes, this technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. Compared with traditional gene therapy technology, RNA interference has many characteristics and advantages in the study of gene function and related aspects. The most notable feature is that it only causes the degradation of mRNA homologous to double-stranded RNA, and has a high degree of specificity and targeting on the expression of other unrelated genes (Cheng S Q, Wang W L, Yan W, et al. Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721 [J]. World J Gastroenterol, 2005, 11(5): 756-759.). This is exactly what is needed for the current molecularly targeted therapy for malignant tumors.

(2) Application of albumin nanospheres as carrier

Traditional antineoplastic drugs are administered through various routes to achieve a certain blood concentration and distribute throughout the body to produce therapeutic effects. The biggest defect of this treatment method is the lack of selectivity, and the drug behavior in vivo is determined by its physicochemical properties and body anatomy/physiology characteristics. Most commonly used antineoplastic drugs have low molecular weight and readily diffuse in the body, resulting in a relatively even tissue distribution. Toxic and side effects often occur during treatment, seriously affecting the antitumor therapeutic value of these drugs. Albumin is a kind of natural biodegradable polymer material, which has numerous reticular voids, which creates favorable space conditions for mosaic carrying drugs. Therefore, as a carrier of drugs, especially anti-tumor drugs, it has been favored by researchers at home and abroad. extensive attention. Albumin microspheres are prepared from human serum albumin (HSA) or bovine serum albumin (BSA), which is an ideal drug-loaded microsphere with stable chemical properties, non-toxicity, non-immunogenicity and good biocompatibility (Fan Jibo, Chen Yongbin, Zhou Jie, et al. Research progress on preparation methods and quality evaluation of albumin microspheres [J]. Tianjin Pharmaceutical, 2009, 2l(3): 58-61.). Albumin nanospheres are nanoscale particles based on albumin. Albumin micro-nanospheres have unique advantages in drug delivery: (1) can release drugs slowly and prolong the drug’s action time in the body; (2) The purpose of targeted delivery can be achieved; (3) The dosage can be reduced under the premise of ensuring the effect of the drug, thereby reducing or avoiding toxic and side effects; (4) It can improve the stability of the drug and facilitate storage; (5) It can be used to establish some new routes of administration, including local administration in vivo, mucosal absorption administration, oral administration of polypeptide drugs, etc. (Felix Kratz.Albumin as a drug carrier: Design of prodrugs, drug conjugates and nanoparticles[J]Journal of Controlled Release, 2008, 132:171–183.). Therefore, the nano-controlled release system is a very promising new drug dosage form, and its research is getting deeper and deeper, and its use will be more extensive.

(3) Targeted therapy mediated by antigen-antibody system

It is mainly a drug delivery system that uses the specific recognition mechanism between antigens and antibodies to actively target specific cells. Many antibodies can recognize the specific antigens of most tumors, such as liver cancer, ovarian cancer, colon cancer, etc. The development of recombinant cell engineering also makes the low-cost industrial production of tumor antibodies possible. Many commonly used anti-tumor drugs are used to prepare monoclonal antibody-drug-loaded particle conjugates, that is, immune particles, which are guided by tumor-specific antibodies, and drugs are adsorbed or encapsulated in albumin and other biodegradable materials. Colloidal solid particles with a particle size of nanometer scale obtained by chemical separation have a large drug loading capacity and slow-release drug properties, and can actively and selectively bind and kill cancer cells through specific antibody guidance.

Cetuximab is a human-mouse chimeric anti-EGFR (EGFR is epidermal growth factor receptor) monoclonal antibody, which was originally researched by Eli Lilly and Company of the United States, developed by Merck of Germany and Bristol-Myers Squibb of the United States, and was first launched in Switzerland in 2003. At present, the approved indications are bowel cancer, head and neck cancer and squamous cell carcinoma, and it is still in the pre-registration stage for lung cancer. Compared with some small molecule compounds that inhibit EGFR tyrosine kinase (Iressa, Erlotinib, etc.), the biggest feature of this drug is that it can be used in EGFR-positive cells regardless of monotherapy or combined with radiotherapy or chemotherapy. Cancer cells exert obvious anti-cancer effects, significantly increasing the efficacy of radiotherapy or chemotherapy. Therefore, it will be coupled to the surface of the drug carrier system to have a specific antigen-antibody recognition mechanism for cancer cells with high expression of EGFR, so as to achieve the purpose of targeted therapy.

Contents of the invention

Technical problem: The present invention provides a gene-loaded immunomagnetic albumin nanosphere with targeting properties that uses the characteristics of magnetic albumin nanometers and immune nanospheres to couple monoclonal antibodies on the surface of albumin nanospheres. A preparation method of the magnetic albumin nanosphere.

Technical solution: The preparation method of the targeted gene-loaded immunomagnetic albumin nanospheres of the present invention comprises the following steps:

1) Preparation of gene-loaded magnetic albumin nanospheres:

Weigh polyethyleneimine-modified Fe ₃ O ₄ magnetic nanoparticle suspension and plasmid survivin-siRNA according to the mass ratio of 30:1-50:1, mix the two and incubate for more than 30 minutes to obtain polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complex;

According to the mass ratio of polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complex to bovine serum albumin powder of 1:5 to 3:5, put the former into the bovine serum albumin solution, stir and mix well, and adjust the pH value to 9; while stirring, slowly drop ethanol into the above-mentioned mixed solution until the solution is gelatinous to obtain a nanoparticle solution;

According to the mass volume ratio of bovine serum albumin and 25% glutaraldehyde solution of 2 mg/μl to 4 mg/μl, slowly add 25% glutaraldehyde solution to the nanoparticle solution, and magnetically stir for 12 to 24 hours to crosslink the nanoparticles solidified to obtain a solidified gene-loaded magnetic albumin nanosphere solution;

Centrifuge the gene-loaded magnetic albumin nanosphere solution at high speed, pour off the supernatant, ultrasonically disperse, and wash more than 3 times to obtain the gene-loaded magnetic albumin nanosphere;

2) Use the SPDP cross-linking method to couple the mouse anti-human EGFR monoclonal antibody to the gene-loaded magnetic albumin microspheres. The specific steps are as follows: Take the mouse anti-human EGFR monoclonal antibody and dissolve it in 0.01-0.1mol/L phosphate In the buffer solution, the molar ratio of mouse anti-human EGFR monoclonal antibody to SPDP is 1:15, add the solution of mouse anti-human EGFR monoclonal antibody to 15-25mmol/L SPDP ethanol solution, react for more than 60min, and use the reaction mixture with Dithiothreitol was added to the obtained monoclonal antibody solution with pyridyl disulfide group, the mass ratio of dithiothreitol to SPDP was greater than 100:1, and stirred for 30 minutes. Above, then use phosphate buffer saline as the dialysate to carry out dialysis to obtain the monoclonal antibody solution of mouse anti-human epidermal growth factor receptor with sulfhydryl group;

Ultrasonically disperse the suspension of gene-loaded magnetic albumin nanospheres in an acetate solution of pH 4-5, and then add SPDP dropwise under stirring according to the mass ratio of gene-loaded magnetic albumin nanospheres to SPDP at 10:1-20:1 Ethanol solution, after high-speed centrifugation, washed with Hank's solution to obtain activated magnetic albumin nanoparticles;

According to the mass ratio of the activated magnetic albumin nanoparticles to the mouse anti-human epidermal growth factor receptor monoclonal antibody with sulfhydryl groups of 20:1 to 30:1, mix the two solutions, shake gently for more than 15 hours, and run at high speed. After centrifugation, wash with Hank's solution to obtain the monoclonal antibody-coupled gene-loaded magnetic albumin nanosphere suspension, which is the gene-loaded immunomagnetic albumin nanosphere.

The targeted gene-loaded immunomagnetic albumin nanospheres of the present invention are prepared according to the above method.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

The invention uses albumin to wrap the plasmid to play a slow release effect, and the in vitro release gene curve shows that the release of the interference plasmid from siRNA-IMANS conforms to the law of biphasic kinetics, with rapid drug release in the initial stage and slow drug release in the later stage, which meets clinical needs , so that the target site quickly reaches the therapeutic concentration and maintains the concentration for a long time. It is also one of the innovations of this subject that gene therapy can be performed continuously and efficiently. The albumin nanosphere surface is coupled with a monoclonal antibody through a cross-linking agent to achieve its active targeting effect on a specific organ or tissue. Cetuximab (C225) monoclonal antibody is a monoclonal antibody against EGFR, which can competitively inhibit the binding of ligands to EGFR and prevent EGFR activation. Therefore, we chose the commercialized and clinically used albumin nanospheres, which can not only target cancer tissues, but also competitively inhibit the binding of ligands to EGFR for therapeutic effects.

The value of the present invention lies in that the method of targeted introduction of siRNA is compared with "shooting an arrow to shoot a bull's-eye", that is, bimolecular targeted combination therapy technology. The nano-magnetic albumin nanosphere complex mediates survivin-siRNA to directly target the target of EGFR-positive lung cancer cells. While targeting the C225 molecule to treat lung cancer, it makes RNA interference therapy more effective and avoids "random shooting" damage to innocent healthy tissue. It is expected to provide a reliable theoretical and practical basis for clinical molecular targeted therapy of lung cancer.

Description of drawings

Figure 1 is a TEM image of gene-loaded immunomagnetic nanospheres.

Fig. 2 is a curve diagram of gene-loaded immunomagnetic albumin nanosphere releasing gene in vitro.

Fig. 3 is a SEM image of immune nanospheres incubated with human lung cancer cells.

Figure 4 is a SEM image of non-immune nanospheres incubated with human lung cancer cells.

Fig. 5 is a SEM image of human lung cancer cells.

Detailed ways

The scheme of the present invention will be further specifically described below by way of examples.

Example 1:

1) Preparation of gene-loaded magnetic albumin nanospheres (siRNA-MANS)

Incubate the PEI-Fe ₃ O ₄ magnetic nanoparticle suspension with the plasmid survivin-siRNA at a mass ratio of 50:1 for more than 30 minutes to obtain the PEI-Fe ₃ O ₄ /survivin-siRNA complex;

According to the mass ratio of PEI-Fe ₃ O ₄ /survivin-siRNA complex to bovine serum albumin 2:5, put the PEI-Fe ₃ O ₄ /survivin-siRNA complex into the bovine serum albumin solution, stir and mix well, Adjust the pH value to 9; while stirring, slowly drop ethanol into the above mixed solution until the solution becomes gelatinous to obtain a nanoparticle solution. This process is a desolvation process; The mass volume ratio of the aldehyde solution is 4 mg/μl, slowly add 25% glutaraldehyde solution dropwise to the above nanoparticle solution, and magnetically stir at room temperature for 12-24 hours to cross-link and solidify the nanoparticles, and obtain solidified gene-loaded magnetic albumin nanospheres (siRNA-MANS) solution;

Centrifuge the nanosphere solution at a high speed of 12000r/min, pour off the supernatant, add water to the original volume, and ultrasonically disperse. After washing for more than 3 times, the organic solvent added in the preparation is removed, and the gene-loaded magnetic albumin nanospheres are obtained.

2) Preparation of gene-loaded immunomagnetic albumin nanospheres (siRNA-IMANS)

The SPDP cross-linking method was used to couple the mouse anti-human EGFR monoclonal antibody to the gene-loaded magnetic albumin microspheres. The specific steps were as follows: take an appropriate amount of mouse anti-human EGFR monoclonal antibody C225 (monoclonal antibody, McAb), dissolve /LPBS(pH7.4), add 20mmol/LSPDP ethanol solution at a molar ratio of 1:15, react at room temperature for 60min, put the reaction mixture into a pretreated dialysis bag, and adjust pH4.5, 0.01mmol/ Dialyze against L acetate buffer to remove excess SPDP. Dithiothreitol was added to the obtained monoclonal antibody (McAb-PDP) solution with pyridyl disulfide groups. The mass ratio of dithiothreitol to SPDP was greater than 100:1. Gently stirred at room temperature for 30 minutes, and then dissolved in PBS. Dialyze the dialysate overnight to remove excess DTT, and finally obtain a mouse anti-human epidermal growth factor receptor monoclonal antibody (McAb-PDP-SH) solution with a sulfhydryl group; wherein SPDP is 3-(2-pyridyldithiol)propane Acid N-hydroxysuccinimide ester.

According to the mass ratio of gene-loaded magnetic albumin nanospheres to SPDP of 20:1, ultrasonically disperse the gene-loaded magnetic albumin nanosphere suspension in the acetate solution of pH 4-5, add SPDP ethanol solution dropwise under stirring, and centrifuge at high speed Finally, wash with Hank's solution to obtain activated magnetic albumin nanoparticles; Hank's solution is a kind of balanced salt solution, which is mainly used for rinsing of tissue blocks, rinsing of cells, and preparation of other reagents when cell culture is taken. It is mainly composed of potassium chloride, calcium chloride, sodium chloride, magnesium sulfate, magnesium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, glucose, phenol red, etc.

According to the mass ratio of 25:1, mix the activated magnetic albumin nanoparticles with the mouse anti-human EGFR monoclonal antibody solution with sulfhydryl groups, shake gently for 18 hours, centrifuge at 15000r/min, and wash with Hank's solution to obtain the monoclonal antibody The coupled gene-loaded magnetic albumin nanosphere suspension is the gene-loaded immunomagnetic albumin nanosphere (siRNA-IMANS).

Other specific examples prepared by different proportioning ratios of each material are as follows:

Embodiment 2: The basic process steps are the same as in Embodiment 1, and the differences from Embodiment 1 are:

In step 1), the mass ratio of PEI-Fe ₃ O ₄ magnetic nanoparticle suspension to plasmid survivin-siRNA was 30:1, and then incubated to prepare polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complex; according to PEI The mass ratio of -Fe ₃ O ₄ /survivin-siRNA complex to bovine serum albumin was 1:5 to prepare nanoparticle solution; according to the mass volume ratio of bovine serum albumin to 25% glutaraldehyde solution of 3 mg/μl, the solidified carrier Gene Magnetic Albumin Nanosphere Solution.

Step 2) The mouse anti-human EGFR monoclonal antibody was dissolved in 0.05mol/L phosphate buffer; the mouse anti-human EGFR monoclonal antibody solution was added to the ethanol solution of 15mmol/L SPDP; Dithiothreitol was added to the sulfide-based monoclonal antibody solution, and the mass ratio of dithiothreitol to SPDP was greater than 150:1; the activated magnetic Albumin nanoparticles: According to the mass ratio of 20::1, the activated magnetic albumin nanoparticles are mixed with the mouse anti-human epidermal growth factor receptor monoclonal antibody solution with a sulfhydryl group to prepare gene-loaded immunomagnetic albumin nanospheres.

Other proportional relationships in this embodiment are the same as those in Embodiment 1.

Embodiment 3: The basic process steps are the same as in Embodiment 1, and the differences from Embodiment 1 are:

In step 1), the mass ratio of PEI-Fe ₃ O ₄ magnetic nanoparticle suspension to plasmid survivin-siRNA was 40:1, and then incubated to prepare polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complexes; according to PEI The mass ratio of -Fe ₃ O ₄ /survivin-siRNA complex to bovine serum albumin was 3:5 to prepare nanoparticle solution; according to the mass volume ratio of bovine serum albumin to 25% glutaraldehyde solution 2 mg/μl, the solidified carrier Gene Magnetic Albumin Nanosphere Solution.

In step 2), the mouse anti-human EGFR monoclonal antibody was dissolved in 0.1mol/L phosphate buffer; the mouse anti-human EGFR monoclonal antibody solution was added to the ethanol solution of 25mmol/L SPDP; Add dithiothreitol in the monoclonal antibody (McAb-PDP) solution of disulfide group, the mass ratio of dithiothreitol and SPDP is greater than 200:1; :1 to prepare activated magnetic albumin nanoparticles; according to the mass ratio of 30::1, the activated magnetic albumin nanoparticles were mixed with the mouse anti-human epidermal growth factor receptor monoclonal antibody solution with sulfhydryl groups to prepare gene-loaded immune Magnetic albumin nanospheres.

Other proportional relationships in this embodiment are the same as those in Embodiment 1.

1. Characterization of siRNA-IMANS

1.1 Electron microscope morphology detection

Take out a small amount of prepared immunomagnetic albumin nanosphere suspension, mix it well, drop it with membrane copper mesh, make electron microscope sample, and observe it under JEM-2010 TEM.

Figure 1 shows that the self-made gene-loaded immunomagnetic nanospheres are approximately spherical under TEM detection, with a uniform size and a diameter of about 200nm, and it is clearly observed that there are magnetic particles with high electron density in the mass. The observed results of the rest of the examples are similar to this picture.

1.2 Dynamic measurement of dynamic release gene rate in vitro

The in vitro drug release of gene-loaded magnetic albumin nanospheres was investigated by dynamic dialysis. Take 15mL of sample into the treated dialysis bag, tie both ends tightly, place in a beaker filled with 100mL of acetate buffer, shake it in a constant temperature shaker at 37°C at a speed of 150r/min, Take out 5mL supernatant at a fixed time and replenish an equal amount of buffer at the same time. Measure the DNA content in the release solution, and draw the curve of time and cumulative released plasmid concentration.

The in vitro dynamic release rate and the calculation of its cumulative release rate showed that the material released 96.89% of DNA in 13 hours, with a gentle curve (Figure 2). The release curves of other examples are not significantly different from this curve.

1.3 Observation of transfection efficiency of gene-loaded immunomagnetic nanospheres

Seed GLC-82 cells in a 6-well plate, 3×10 ⁵ cells per well, after incubation for 18 hours (80% confluence of cells), replace the original cell culture medium with 2ml serum-free medium, follow the transfection Step operation, divided into siRNA-IMANS group, siRNA-MANS group and blank protein nanosphere (ANS) group. After 48 hours of transfection, the transfection efficiency was observed under an inverted fluorescence microscope.

The results showed that both siRNA-IMANS group and siRNA-MANS group could effectively transfer the plasmid survivin-shRNA-2 expressing green fluorescent protein gene into GLC-82 cells, and the expression of bright green fluorescent protein (GFP) was observed under an inverted fluorescent microscope. Expression, the expression of green fluorescent protein was not seen in the blank protein nanosphere group. However, under the same conditions, the transfection efficiency of siRNA-IMANS group was higher than that of siRNA-MANS group.

2Immune characteristic detection of siRNA-IMANS

2.1 Slide agglutination experiment

On a clean glass slide, mix equal amounts of siRNA-IMANS or siRNA-MANS suspension with rabbit anti-mouse IgG antiserum, incubate at 37°C for several minutes, and observe the state of nanospheres under a light microscope.

After the rabbit anti-mouse IgG antiserum was added, the previously evenly dispersed siRNA-IMANS formed aggregates, while the siRNA-MANS was still in a uniformly dispersed state.

2.2 Immunofluorescence staining experiment

Take siRNA-IMANS or siRNA-MANS suspension, add an equivalent amount of AlexaFluor488-labeled rabbit anti-mouse IgG, react at 4°C for 60 minutes, wash the precipitate with PBS 3 times, and obtain AlexaFluor488-labeled fluorescent immunoalbumin nanospheres, which are placed in The fluorescent staining results of the nanospheres were observed under a fluorescent microscope.

The results showed that after adding rabbit anti-mouse IgG-Alexa Fluor488, the surface of siRNA-IMANS emitted bright yellow-green fluorescence, but siRNA-MANS could not be stained by rabbit anti-mouse IgG-AlexaFluor488, and no fluorescence was seen.

3C225-siRNA-MANS Targeted Binding to Human Lung Cancer Cells

3.1 Prussian blue dyeing

GLC-82 cells in the logarithmic growth phase were inoculated in a 12-well plate, each well containing about 3×10 ⁴ cells, and after 24 hours of cell attachment, the monoclonal antibody targeting (siRNA-IMANS) and non-targeting group (ANS ) was incubated with GLC-82 for 24 hours, the iron content in GLC-82 cells was observed by Prussian blue staining, and the targeted absorption of nanospheres by cells was detected.

The steps of Prussian blue staining are as follows: ①Take out the 24-well plate, discard the culture medium, wash 3 times with PBS; ②fix with 10% formaldehyde for 30min, wash 3 times with PBS; Mix the parts, drop them on the slices, and incubate in a 37-degree incubator for 30 minutes; ④ Wash with PBS 3 times, observe and take pictures with an inverted phase-contrast microscope.

After co-incubation with GLC-82 cells in the monoclonal antibody targeting group and magnetic targeting group, a large amount of iron existed in the cells, and a large number of blue-stained particles could be seen; no obvious iron particles existed in the cells of the non-targeting group.

3.2 Immunofluorescence staining

Immunofluorescence is performed as follows:

1). GLC-82 cell slides;

2). After 24 hours, fix with cold acetone at room temperature for 10 minutes;

3). Rinse with PBS 3 times, 5 minutes each time;

4). 0.5% Triton piercing for 15 minutes;

5). Rinse twice with PBS, 5 minutes each time;

6). Block with 1% BSA for 30 minutes;

7). Add siRNA-IMANS or siRNA-MANS diluted with 1% BSA, and react overnight at 4°C;

8). Rinse with PBST 3 times, 5 minutes each time;

9). Add fluorescent secondary antibody diluted with 1% BSA, and hybridize at 37°C for 1 hour;

10). Rinse with PBST 3 times, 5 minutes each time;

11). 5ug/ml DAPI stains the nucleus for 3 minutes;

12).Seal the slide with anti-fade mounting medium, and observe the result under confocal laser.

The results showed that the monoclonal antibody targeting group could specifically bind to human lung cancer cells, and was stained by rabbit anti-mouse IgG-Alexa Fluor488, and the cells combined with immune microspheres emitted bright fluorescence under a confocal laser microscope. The non-targeted group was not stained by rabbit anti-mouse IgG-AlexaFluor488 fluorescence, and no fluorescence was observed around the cells and in the cytoplasm.

3.3 siRNA-IMANS in vitro combined with electron microscope observation of lung cancer cells

The well-grown target cell GLC-82 was digested with 5 mL of trypsin and then centrifuged. The precipitate was washed with PBS containing 5% calf serum, and the precipitate was prepared into a 2×10 ⁵ /mL cell suspension, and immunomagnetic albumin nanoparticles were added. Balls (siRNA-IMANS) or nanospheres (siRNA-MANS) were shaken at room temperature for 60 minutes, centrifuged, the pellet was washed 3 times with PBS, and the combination of cells and particles was observed under a light microscope.

Take a clean slide and add 1% polylysine dropwise, and dry at 37°C. Take the combination of immune nanospheres and target cells and drop them on the glass slide, act at 4°C for 30 minutes, immerse in PBS and wash gently, fix with 2.5% glutaraldehyde at 4°C for 30 minutes, immerse in distilled water and wash gently, successively wash with 50 %, 70%, 90%, 100% ethanol dehydration, natural drying, spraying colloidal gold under vacuum, scanning electron microscope to observe the combination of immune nanospheres and target cells.

The binding conditions of siRNA-IMANS and GLC-82 scanned by electron microscopy are shown in Figure 3 to Figure 5. Multiple siRNA-IMANS are tightly bound to the surface of human lung cancer cells, like grapes or beads (Figure 3). GLC-82 cells ( FIG. 4 ) were significantly different from human lung cancer cells GLC-82 ( FIG. 5 ) after only trypsinization.

Effect of 4siRNA-IMANS on the growth of GLC-82 cells

4.1 Cell culture

GLC-82 cells were inoculated in DMEM culture medium containing 10% fetal bovine serum, cultured in an incubator at 37°C, saturated humidity, and 5% CO ₂ , and passaged every 2-3 days. The logarithmic growth phase was used in the experiment cell.

4.2 MTT method to measure cell proliferation rate

Take the GLC-82 cells in the logarithmic growth phase, digest them with 0.25% trypsin and blow them into a cell suspension, adjust the cell concentration to 4× ¹⁰⁴ cells/ml, inoculate three 96-well plates at 100 μl per well, and divide into groups They are: negative control group (DMEM culture medium), free C225 group, magnetic targeting gene therapy (siRNA-MANS) group, monoclonal antibody targeting gene therapy (siRNA-IMANS) group, dual targeting (magnetic targeting and single anti-target) gene therapy group. Each group had 6 replicate wells. After the cells were inoculated for 24 hours, DMEM medium, C225, nanosphere siRNA-MANS and immune nanosphere siRNA-IMANS were added according to the grouping situation, and the 96 wells of the magnetic targeting group and the double targeting group were added. A 96-well magnetic plate (Magneto FCTOR plate, chemicell Germany) was placed under the plate. After continuing to culture for 24h, 48h, and 96h respectively, add MTT20μl/well, continue to culture for 4h under the same conditions as before, discard the liquid in the well, add 150μlDMSO to each well, shake and mix for 10min, take the supernatant after centrifugation and place it on a microplate reader for reading Take the OD value at 493nm. Cell proliferation rate=OD value of experimental group/OD value of control group×100%.

The cell survival rate of each group was calculated according to the measured OD value according to the formula: cell survival rate (%)=OD mean value of the experimental group/average OD value of the control group×100 and listed in (Table 1). The MTT results measured from three days showed that the free C225 group, the magnetic targeting gene therapy (siRNA-MANS) group, the monoclonal antibody targeting gene therapy (siRNA-IMANS) group and the dual targeting gene therapy group had no significant effect on the proliferation of lung cancer cells. There is an inhibitory effect, and the inhibitory effect of the dual-targeted gene therapy group is significantly higher than that of the other two single-treatment groups, and there is a significant difference. There was no significant difference with the monoclonal antibody targeted gene therapy group. The comparison of the experimental results of three days showed that the inhibitory effect of each group at 96h was the best, while that at 24h was the worst.

Table 1 GLC-82 cell viability MTT experiment results

* is P<0.05 compared with the control group, ^# is P<0.05 compared with the control group, free C225 group, and magnetic targeting gene group.

4.3 Determination of flow cytometry

HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into a cell suspension. The cell concentration was adjusted to 3×10 ⁵ cells/ml and inoculated into 50ml culture flasks. Each flask was inoculated with 2ml and cultured for 24h. The experimental grouping was the same as the MTT experimental grouping, and the magnetic targeting group and the dual targeting group were also placed on the magnetic plate and continued to culture for 48 hours. Cells were collected, washed twice with PBS, and fixed with 70% ethanol at 4°C for more than 24 h. Wash the cells twice with PBS before detection, resuspend the cells in 0.5ml PI staining solution (20mg/ml, 0.25mg/ml RNaseA), stain at room temperature in the dark for 30min, filter through a 300-mesh screen, and analyze on the machine (all data collected and analyzed by Lysis II software).

After GLC-82 cells were treated for 48 hours in each group, FCM analysis showed that: in the free C225 group, the magnetic targeting gene therapy (siRNA-MANS) group, the monoclonal antibody targeting gene therapy (siRNA-IMANS) group, the dual targeting gene therapy Before the _G1 phase of the cell cycle in the group, there was an obvious hypodiploid apoptosis peak, and the cell cycle was arrested in the S phase to varying degrees; while there was no obvious apoptosis peak in the control group. The apoptosis rate in the dual-targeted gene therapy group was 24.80%, the apoptosis rate in the monoclonal antibody-targeted gene therapy group was 17.18%, the apoptosis rate in the non-targeted gene therapy group was 7.6%, and the apoptosis rate in the free C225 group was 7.84%. The apoptosis rate in the control group was only 0.70%.

Claims (2)

1. A method for preparing a targeted gene-loaded immunomagnetic albumin nanosphere, characterized in that the method may further comprise the steps: 1) Preparation of gene-loaded magnetic albumin nanospheres: Weigh polyethyleneimine-modified Fe ₃ O ₄ magnetic nanoparticle suspension and plasmid survivin-siRNA according to the mass ratio of 30:1-50:1, mix the two and incubate for more than 30 minutes to obtain polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complex; According to the mass ratio of polyethyleneimine-modified Fe ₃ O ₄ /survivin-siRNA complex to bovine serum albumin powder of 1:5~3:5, put the former into the bovine serum albumin solution, stir and mix well, and adjust the pH value to 9; while stirring, slowly drop ethanol into the above-mentioned mixed solution until the solution is gelatinous to obtain a nanoparticle solution; According to the mass volume ratio of bovine serum albumin and 25% glutaraldehyde solution of 2 mg/μl to 4 mg/μl, 25% glutaraldehyde solution was slowly added dropwise to the nanoparticle solution, and magnetically stirred for 12 to 24 hours to make the nanoparticles Cross-linking and curing to prepare a solidified gene-loaded magnetic albumin nanosphere solution; Centrifuge the gene-loaded magnetic albumin nanosphere solution at high speed, pour off the supernatant, ultrasonically disperse, and wash more than 3 times to obtain the gene-loaded magnetic albumin nanosphere; 2) Use the SPDP cross-linking method to couple the mouse anti-human EGFR monoclonal antibody to the gene-loaded magnetic albumin microspheres. The specific steps are as follows: Take the mouse anti-human EGFR monoclonal antibody and dissolve it in 0.01~0.1mol/L phosphate In the buffer solution, the molar ratio of mouse anti-human EGFR monoclonal antibody to SPDP is 1:15, add the solution of mouse anti-human EGFR monoclonal antibody to 15-25 mmol/L ethanol solution of SPDP, react for more than 60 minutes, and dissolve the reaction mixture Dithiothreitol was added to the obtained monoclonal antibody solution with a pyridyl disulfide group, and the mass ratio of the dithiothreitol to SPDP was greater than 100:1. Stir for more than 30 minutes, and then dialyze with phosphate buffered saline as the dialysate to obtain a mouse anti-human epidermal growth factor receptor monoclonal antibody solution with a sulfhydryl group; Ultrasonically disperse the suspension of gene-loaded magnetic albumin nanospheres in an acetate solution of pH 4~5, and then add SPDP dropwise under stirring according to the mass ratio of gene-loaded magnetic albumin nanospheres to SPDP at 10:1~20:1 Ethanol solution, after high-speed centrifugation, washed with Hank's solution to obtain activated magnetic albumin nanoparticles; According to the mass ratio of activated magnetic albumin nanoparticles and mouse anti-human epidermal growth factor receptor monoclonal antibody with sulfhydryl group at 20:1~30:1, mix the two solutions, shake gently for more than 15 hours, and run at high speed. After centrifugation, wash with Hank's solution to obtain the monoclonal antibody-coupled gene-loaded magnetic albumin nanosphere suspension, which is the gene-loaded immunomagnetic albumin nanosphere. 2. A targeted gene-loaded immunomagnetic albumin nanosphere, characterized in that the magnetic albumin nanosphere is prepared according to the method of claim 1.

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