CN102397557B - Modification method for gold nanorods and gold nanorods-functional molecule composite - Google Patents

Abstract

The invention provides a method for modifying gold nanorods, the method comprising: contacting the gold nanorods with a solution of DNA or a solution of protein antigen, and the surface of the gold nanorods is positively charged. The invention also provides the gold nanorod-functional molecule complex obtained by the modification method. The gold nanorod-functional molecule complex obtained by the modification method of the invention can significantly improve the level of gene transfection, promote protein antigens to enter cells, and has good safety.

Description

Modification method of gold nanorods and gold nanorods-functional molecular complex

technical field

The invention relates to a method for modifying gold nanorods and a gold nanorod-functional molecule complex obtained by the method.

Background technique

Vaccines play an extremely important role in the prevention and treatment of diseases. Vaccine vectors are mainly divided into two types: viral vectors and non-viral vectors. Compared with viral vectors, non-viral vectors have the advantages of easy production and less toxic side effects. Therefore, research on non-viral vectors as potential vaccine vectors or adjuvants has attracted much attention in recent years. Compared with other non-viral vectors, nanomaterials as carriers have the advantages of promoting the uptake of functional molecules such as DNA by cells, prolonging the circulation time of functional molecules in the blood, and realizing the targeted delivery of functional molecules through appropriate modification and processing (Jun -ichiro Jo, Yasuhiko Tabata. Non-viral gene transfection technologies for genetic engineering of stem cells. European Journal of Pharmaceutics and Biopharmaceutics, 2008, 68: 90-104.). As the most promising carrier, nanomaterials with good biocompatibility or biodegradability have become a research hotspot in recent years. Due to their good biocompatibility, gold nanoparticles and gold nanorods have attracted much attention as potential carriers in the diagnosis and treatment of diseases. Due to its unique optical properties, gold nanorods have become a research hotspot in recent years for their applications in biomedical imaging, disease diagnosis and treatment, especially tumor hyperthermia. At the same time, gold nanomaterials are easy to carry out surface modification, which can be combined with negatively charged genetic molecules such as DNA and small interfering RNA (siRNA) through electrostatic adsorption, thereby realizing the effective transportation of genetic functional molecules and exerting their functions, prevention or treatment. related diseases. By modifying some biomolecules on the surface of gold nanorods, targeted transport can also be achieved to better play the role of functional molecules. It is also reported that gold-nickel nanorods can significantly enhance antigen-specific immune response (Megan E.Pearce, Jessica B.Melanko, Aliasger K.Salem.Multifunctional Nanorods for Biomedical Applications.PharmaceuticalResearch, 2007, 24 (12): 2335 -2352.).

Antigen-Presenting Cells (APCs) play an important role in the body's immune defense process. Dendritic cells (DCs), as professional antigen-presenting cells, play a more important role in the immune response due to their ability to ingest, process and transport antigens to lymphoid tissues for presentation to naive T cells role. For vaccines, whether they can be effectively delivered to and interacted with APCs, especially DCs, to induce a long-term and efficient immune response is related to the success or failure of disease prevention or treatment. Therefore, DCs have attracted the attention of researchers in the research and development of vaccines. Compared with micron-sized vaccine carriers, nanomaterials are easier to enter the body, and nanomaterials with a particle size of 40-70nm are more likely to enter lymphoid tissue (A.E.Hawley, S.S.Davis, L.Illum.Targeting of colloids to lymphnodes-influence of lymphatic physiology and colloidal characteristics. Advanced Drug Delivery Review, 1995, 17(1): 129-148.). The modification of APCs, especially DCs-specific surface molecules on the surface of nanomaterials will provide a more favorable guarantee for promoting the effective delivery of antigens.

Functional molecules or vaccine antigens, especially DNA vaccines, need to cross the following three barriers if they are to be successfully delivered to immune cells: (1) cell membrane; (2) degradation of endosomes or lysosomes; (3) nucleus. In other words, a successful vaccine, especially a DNA vaccine, needs to effectively deliver the antigen, protect it from degradation, and enter the nucleus in order to truly prevent and treat disease.

Contents of the invention

The object of the present invention is to provide a method for modifying gold nanorods and a gold nanorod-functional molecule complex obtained by the method.

The invention provides a method for modifying gold nanorods, the method comprising: contacting the gold nanorods with a solution of DNA or a solution of protein antigen, and the surface of the gold nanorods is positively charged.

The present invention also provides a gold nanorod-functional molecule complex, the gold nanorod-functional molecule complex is obtained by the above gold nanorod modification method.

The present invention successfully realizes the compounding of gold nanorods and DNA or protein antigens of genetic functional molecules by contacting gold nanorods with DNA solution or protein antigen solution, thereby realizing the effective transportation of genetic functional molecules and exerting their functions , to prevent or treat related diseases.

In a preferred embodiment of the present invention, gold nanorods are modified with modified polyethylene glycol (PEG), so as to avoid premature phagocytosis of gold nanorods by the reticuloendothelial system and prolong their circulation time in the body.

A preferred embodiment of the present invention sequentially modifies fusion peptides or nuclear localization signals and functional molecules (such as transferrin, folic acid, etc.) The antigen enters the cell, allowing it to function more efficiently.

A preferred embodiment of the present invention ensures more efficient presentation of DNA or protein antigens to B cells and T cells by modifying gold nanorods with specific antibodies targeting antigen-presenting cells.

Description of drawings

FIG. 1 is a TEM characterization picture of the gold nanorods described in Example 1.

2 is a SEM characterization picture of gold nanorods with different aspect ratios described in Example 1.

FIG. 3 is an ultraviolet scanning spectrum of gold nanorods with different aspect ratios described in Example 1. FIG.

FIG. 4 is the potential detection result of the gold nanorods described in Example 1. FIG.

FIG. 5 is the ultraviolet scanning spectrum of the gold nanorods described in Example 2 after pEGFP is adsorbed.

Fig. 6 is the ultraviolet scanning spectrum of the gold nanorod-pEGFP-PEG and gold nanorod-pEGFP-PEG-NLS complexes described in Examples 6 and 7.

Fig. 7 is a picture of HEK293 cells transfected by various gold nanorod-functional molecule complexes described in Example 10 (magnification 10×10).

8 is a bar graph showing the effect of gold nanorods with an aspect ratio of 4 prepared in Example 1 on the viability of HEK293 cells.

Detailed ways

The invention provides a method for modifying gold nanorods, the method comprising: contacting the gold nanorods with a solution of DNA or a solution of protein antigen, and the surface of the gold nanorods is positively charged.

In the modification method provided by the present invention, relative to 1 part by weight of the gold nanorods, the amount of DNA used is 0.001-0.2 parts by weight, the concentration of the DNA solution is 2-400 μg/ml, or the amount of protein antigen used is 0.1 -2 parts by weight, the concentration of the protein solution is 0.1-2 mg/ml, preferably, relative to 1 part by weight of the gold nanorods, the amount of DNA is 0.1-0.2 parts by weight, and the concentration of the DNA solution is 200 - 400 μg/ml, or the amount of protein antigen used is 0.5-1 parts by weight, and the concentration of the protein solution is 0.5-1 mg/ml.

The gold nanorods used in the present invention have a length of 40-70 nm and an aspect ratio of 1-6:1. Since the raw materials for the preparation of gold nanorods contain cationic surfactants such as cetyltrimethylammonium bromide (CTAB) or positively charged surfactants such as polydimethyldiallylammonium chloride (PDDAC). Electric polyelectrolyte, so the surface of the gold nanorods is positively charged, and the gold nanorods in the present invention have a positive charge of 10-50 millivolts.

Since DNA and most of the protein antigens are negatively charged, and the surface of the gold nanorods described in the present invention is positively charged, it is easy to combine together by electrostatic adsorption, so in the present invention, the DNA or protein antigens The type of DNA is not particularly limited, the DNA such as HIV Env DNA (HIV Env DNA), hepatitis B virus membrane protein encoding gene (HBV) and human papillomavirus membrane protein encoding gene (HPV) etc., the protein antigen For example, AIDS virus membrane protein (gp140), hepatitis B virus membrane protein and human papillomavirus membrane protein antigen, etc., these DNA or protein antigens can be combined with the gold nanorods in the present invention.

In order to increase the amount of DNA and protein antigens entering cells, and at the same time prevent DNA from being degraded by lysosomes, so as to ensure that DNA and protein antigens play an effective role, the modification method provided by the present invention also includes mixing gold nanorods with a solution of DNA or protein antigen solution, in the presence of a solvent, any one or more of modified polyethylene glycol, fusion peptides, nuclear localization signals, molecules that promote cell entry, and specific antibodies targeting antigen-presenting cells One or multiple contacts, each contact time is 2-12 hours, the skeleton of the modified polyethylene glycol is a polyethylene glycol skeleton, one end of the modified polyethylene glycol is a mercapto group and the other One end is amino or carboxyl. The solvent may be one of deionized water, phosphate buffered saline (PBS) or morpholinoethanesulfonic acid buffered solution (MES).

In the present invention, the weight average molecular weight of the modified polyethylene glycol may be 150-635, and the modified polyethylene glycol may be, for example, HSC ₁₁ -EG ₂ NH ₂ from Prochimia, Poland.

In the method for modifying gold nanorods of the present invention, relative to 1 part by weight of gold nanorods, the amount of the fusion peptide can be 0-2 parts by weight, preferably 0-1 parts by weight, and the nuclear localization signal The dosage can be 0-2 parts by weight, preferably 0-1 parts by weight, the dosage of the molecules promoting cell entry can be 0-2 parts by weight, preferably 0-1 parts by weight, and the targeted antigen-presenting cell The amount of specific antibody can be 0-2 parts by weight, preferably 0-1 parts by weight, the amount of modified polyethylene glycol can be 0-3 parts by weight, preferably 0-1.5 parts by weight, and the gold nano The total amount of rod and fusion peptide, nuclear localization signal, cell entry-promoting functional molecule, specific antibody targeting antigen-presenting cells and modified polyethylene glycol can be 0.01-11 parts by weight, preferably 3.5-6.5 parts by weight.

In the present invention, there is no particular limitation on the type of the fusion peptide, nuclear localization signal, cell entry-promoting function molecule, or specific antibody targeting antigen-presenting cells, such as the JTS-1 sequence small peptide ( GLEEALLFLLESLWELLLEA), INF-7 sequence small peptide (GLFEAIEGFIENGWEGMIWDYG) and small peptide containing KALA sequence, etc.; the nuclear localization signal such as SV40T antigen, two clusters of positively charged amino acid residue sequences separated by a spacer, c-MYC Nuclear localization signals of oncogenes and other types of nuclear localization signals such as ribosomal proteins, etc.; the molecules that promote cell entry can include, for example, small peptides containing arginine-glycine-aspartic acid (RGD) sequences, transferrin protein, epidermal growth factor (EGF), folic acid, etc., the specific antibody targeting antigen-presenting cells, such as the specific antibody anti-DEC205 targeting dendritic cells, etc.

The present invention also provides a gold nanorod-functional molecule complex, the gold nanorod-functional molecule complex is obtained by the above gold nanorod modification method.

The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

Example 1

Synthesis of Gold Nanorod Matrix

At 25°C, mix 5 mL of 0.2 mol/L cetyltrimethylammonium bromide (CTAB, Amersco, USA) solution with 5 mL of 0.96 mmol/L tetrachloroauric acid solution, and add Add 0.6 mL of 0.01 mol/L ice-bathed sodium borohydride solution to the system, and stir at 800 rpm for 2 min to obtain a seed crystal solution.

At 25°C, add 10mL of 25mmol/L tetrachloroauric acid solution and 250mL of water to 12.5mL of 4mmol/L silver nitrate solution and mix well, then add 250mL of 0.2mol/L CTAB solution to the mixed system and 5 mL of 0.08 mol/L ascorbic acid solution, and mix for 15-30 seconds to obtain the growth solution. When the growth liquid gradually changed from orange to colorless, 0.6 mL of seed crystal liquid was added to the growth liquid, mixed evenly, left standing at 25° C. for 16-18 h, and finally centrifuged and washed to obtain gold nanorods 2 .

Prepare gold nanorods according to the above method, the difference is that the molar ratio of silver nitrate and tetrachloroauric acid in the growth solution is adjusted to 0, 0.2, 0.3, 0.4, and the total volume of the growth solution remains unchanged, and gold nanorods 1, Gold nanorod 2, gold nanorod 3, gold nanorod 4.

The obtained gold nanorods were observed with a transmission electron microscope and a scanning electron microscope, and the results are shown in FIGS. 1 and 2 . Fig. 1A, Fig. 1B, Fig. 1C, Fig. 1D represent respectively gold nanorod 1, gold nanorod 2, gold nanorod 3, gold nanorod 4, gold nanorod 1, gold nanorod 2, gold nanorod 3, gold nanorod The average particle diameters of the rods 4 are 40nm, 20nm×40nm, 15nm×45nm, 15nm×60nm respectively, and the aspect ratios of the gold nanorods 1, 2, 3 and 4 are 1:1 respectively. , 2:1, 3:1, 4:1. Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D represent respectively gold nanorod 1, gold nanorod 2, gold nanorod 3, gold nanorod 4, gold nanorod 1, gold nanorod 2, gold nanorod 3, gold nanorod The average particle diameters of the rods 4 are 40nm, 20nm×40nm, 15nm×45nm, 15nm×60nm respectively, and the aspect ratios of the gold nanorods 1, 2, 3 and 4 are 1:1 respectively. , 2:1, 3:1, 4:1.

Utilize the ultraviolet spectrometer (UV-vis) to detect the obtained gold nanorods, the results are shown in Figure 3, the characteristic absorption peaks of gold nanorods 1, gold nanorods 2, gold nanorods 3, and gold nanorods 4 are at 530nm and 620nm respectively , 700nm and 820nm.

The obtained gold nanorods were detected by a laser particle size analyzer, and the results are shown in Figure 4. The surfaces of the gold nanorods 1, 2, 3 and 4 all had a positive charge of about 28.6 millivolts.

Example 2

Preparation of gold nanorod-DNA complex

Mix 0.2 mL of an aqueous solution of 1 mg/mL gold nanorod 4 with 0.1 mL of a 2 μg/mL pEGFP (green fluorescent protein plasmid DNA, Biovector-08, Clontech company) solution, and place it at 25 ° C for about 30 min to obtain The mixed solution containing the gold nanorod-DNA complex is finally centrifuged and washed to obtain the gold nanorod-DNA complex.

The obtained gold nanorod-DNA complex was detected by UV-vis, and the results are shown in Figure 5. It can be seen that the absorbance (OD) value of the gold nanorod-DNA complex is lower than that of the gold nanorod.

Example 3

Preparation of gold nanorod-DNA complex

Mix 0.2 mL of an aqueous solution of 1 mg/mL gold nanorod 4 with 0.1 mL of a 200 μg/mL pEGFP (green fluorescent protein plasmid DNA, Biovector-08, Clontech company) solution, and place it at 25 ° C for about 30 min to obtain The mixed solution containing the gold nanorod-DNA complex is finally centrifuged and washed to obtain the gold nanorod-DNA complex.

The obtained gold nanorods were detected by UV-vis, and the result was similar to that of Example 2. Compared with the gold nanorods, the absorbance (OD) value of the gold nanorods decreased after adsorbing DNA.

Example 4

Preparation of gold nanorod-DNA complex

Mix 0.2 mL of an aqueous solution of 1 mg/mL gold nanorod 4 with 0.1 mL of a 400 μg/mL pEGFP (green fluorescent protein plasmid DNA, Biovector-08, Clontech company) solution, and place it at 25 ° C for about 30 min to obtain The mixed solution containing the gold nanorod-DNA complex is finally centrifuged and washed to obtain the gold nanorod-DNA complex.

The obtained gold nanorods were detected by UV-vis, and the result was similar to that of Example 2. Compared with the gold nanorods, the absorbance (OD) value of the gold nanorods decreased after adsorbing DNA.

Example 5

Detection of DNA Adsorption Rate of Gold Nanorods

Mix the aqueous solution of 1 mg/mL gold nanorod 4 with pEGFP solutions of different concentrations (see Table 1) in equal volumes, let stand at 25°C for 30 min, centrifuge at 10,000 rpm for 15 min, and then use a microplate reader at 260 nm to detect The absorbance (OD) of DNA in the supernatant, and according to "when the absorbance of DNA is 1, its concentration is equivalent to 50ng/μL", calculate the residual amount of DNA in the supernatant, thereby according to the formula: adsorption rate=(DNA total amount -the amount of residual DNA)×100%/total amount of DNA to determine the adsorption rate of gold nanorods to DNA, the results are shown in Table 1.

Table 1

Concentration of pEGFP (μg/mL) 10 25 62.5 125 250 Adsorption rate(%) 100 100 100 100 80

Example 6

Preparation of gold nanorod-DNA-PEG complex

Take 150 μL of the mixed solution containing gold nanorods-DNA complexes obtained in Example 2, add 1 μL of HSC ₁₁ -EG ₆ OCH ₂ COOH (1 mmol/L, Prochimia, Poland, weight average molecular weight 635) and mix well, at 25°C Stand still for 2 hours under the condition to obtain the gold nanorod-DNA-PEG complex mixture, and finally centrifuge to obtain the gold nanorod-DNA-PEG complex. Due to the easy formation of gold-sulfur covalent bonds, SH-PEG can be easily attached to the surface of gold nanorods-DNA complexes. The obtained gold nanorod-DNA-PEG complex was detected by UV-vis, and the results are shown in FIG. 6 .

Example 7

Preparation of Gold Nanorod-DNA-PEG-NLS Complex

Mix 150 μL of the mixture containing gold nanorods-DNA-PEG complexes obtained in Example 6 with 20 μL of morpholineethanesulfonic acid buffer containing NLS (2.5 mg/mL, the sequence is PAAKRVKLD, synthesized by Xi’an Lianmei Biotechnology Co., Ltd.) Mix the solution (MES) evenly, and let stand at 25°C for 30 minutes. Then add 30 μL of MES buffer containing carbodiethylimide hydrochloride (EDC, 0.5 mg/mL) to the above mixed system, react at 4 °C for 2 h, then centrifuge at 9500 rpm for 10 min, and wash with deionized water Once, and then resuspended in 150 μL double-distilled water to obtain a mixture containing gold nanorods-DNA-PEG-NLS complexes, and finally centrifuged to obtain gold nanorods-DNA-PEG-NLS complexes.

The obtained gold nanorod-DNA-PEG-NLS complex was detected by UV-vis, and the results are shown in Figure 6. It can be seen that the absorbance of the gold nanorod-DNA-PEG-NLS complex is lower than that of the gold nanorod-DNA-PEG.

Example 8

Preparation of gold nanorod-DNA-PEG-NLS-transferrin complex

150 μL of the mixed solution containing gold nanorods-DNA-PEG-NLS complex obtained in Example 7 was mixed with 20 μL of morpholinoethanesulfonic acid buffer containing transferrin (2.5 mg/mL, apo-Transferrin, Sigma Company) ( MES) were mixed evenly, and stood at 25°C for 30min. Then add 30 μL of MES buffer containing carbodiethylimide hydrochloride (EDC, 0.5 mg/mL) to the above mixed system, react at 4 °C for 2 h, then centrifuge at 9500 rpm for 10 min, and wash with deionized water Once, and then resuspended in 150 μL double distilled water to obtain a mixture containing gold nanorods-DNA-PEG-NLS-transferrin complex, and finally centrifuged to obtain gold nanorods-DNA-PEG-NLS-transferrin Complex.

The obtained gold nanorod-DNA-PEG-NLS-transferrin complex is detected by UV-vis, and the result is similar to that of Example 2. The absorbance of the gold nanorod-DNA-PEG-NLS-transferrin complex is higher than that of gold Low for nanorod-DNA-PEG-NLS complexes.

Example 9

Transfection test of gold nanorod-functional molecule complex to HEK293 cells

Preparation of serum medium containing gold nanorods-functional molecule complexes: Take 30 μL of the mixed solution containing gold nanorods-functional molecule complexes prepared in Example 2 and 6-8 respectively, and add 10% fetal bovine serum ( FBS) in DMEM medium was diluted to 300 μL.

Transfection of HEK293 cells with gold nanorods-functional molecule complexes: First, HEK293 cells were digested with 0.25% trypsin, and centrifuged at 1000 rpm for 5 min after the digestion was terminated. A single cell suspension was prepared with DMEM medium containing 10% fetal bovine serum (FBS), and inoculated in a 24-well plate at a concentration of 5×10 ⁴ /ml, 500 μl per well, and then placed in an incubator (37°C, 5% CO ₂ ) for 24 hours. Afterwards, the original medium was aspirated, and 400 μl of serum-containing medium was added to each well. A blank control group, a naked EGFP plasmid control group, 4 gold nanorod-functional molecule complex test groups, and liposome (Lipofectamine2000, Invitrogen) transfection positive control group were set up, and three parallel wells were set up for each group. 100 μl/well of serum-containing DMEM medium was added to the blank control group, and 100 μl/hole of serum-containing DMEM medium was added to the naked EGFP plasmid control group. 8 μg/mL) 100 μl/well, 4 gold nanorods-functional molecule complexes test groups were added the serum culture medium 100 μl/well of the above-mentioned preparation containing gold nanorods-functional molecule complexes, shake well, place in incubator (37 Cultivate in 5% CO ₂ ) for 12 hours, replace the medium containing serum, continue to cultivate, and observe the transfection result with a fluorescence microscope after 48 hours.

The test results are shown in Figure 7, Figure 7A is the pEGFP plasmid DNA control group, Figure 7B is the gold nanorod-pEGFP complex, Figure 7C is the gold nanorod-pEGFP-PEG complex, Figure 7D is the gold nanorod-pEGFP- PEG-NLS complex, Figure 7E is the gold nanorod-pEGFP-PEG-NLS-Tf complex, Figure 7F is the liposome (Lipofectamine 2000) transfection positive control group. It can be seen from the figure that it is difficult to transfect HEK293 cells with naked pEGFP, and there is no fluorescent protein expression, while the gold nanorod-pEGFP complex can transfect HEK293 cells, and with a series of modifications to the gold nanorod-pEGFP complex, the gold nanorods The ability of rod-pEGFP-PEG-NLS and gold nanorod-pEGFP-PEG-NLS-Tf complex to transfect cells was significantly improved, and the transfection efficiency of gold nanorod-pEGFP-PEG-NLS-Tf complex was comparable to that of the positive control group quite.

Example 10

Preparation of gold nanorod-protein antigen complex

Mix 0.2 mL of an aqueous solution of 1 mg/mL gold nanorods 4 with 0.2 mL of an aqueous solution of 0.1 mg/mL ovalbumin (OVA, Sigma Company) and leave to stand at 25°C for 30 min to obtain a complex containing gold nanorods-protein antigen. The mixed solution of the body was finally centrifuged and washed to obtain the gold nanorod-protein antigen complex.

The obtained gold nanorod-protein antigen complex was detected by UV-vis, and the result was similar to Example 2. It can be seen that the absorbance of the gold nanorod-protein antigen complex was lower than that of the gold nanorod.

Example 11

Preparation of gold nanorod-protein antigen complex

Mix 0.2 mL of an aqueous solution of 1 mg/mL gold nanorod 4 with 0.2 mL of a 2 mg/mL ovalbumin (OVA, Sigma company) aqueous solution, and let stand at 25° C. for 30 min to obtain a gold nanorod-protein antigen complex The mixed solution was finally centrifuged and washed to obtain the gold nanorod-protein antigen complex.

The obtained gold nanorod-protein antigen complex was detected by UV-vis, and the result was similar to Example 2. It can be seen that the absorbance of the gold nanorod-protein antigen complex was lower than that of the gold nanorod.

Example 12

Preparation of Gold Nanorod-Protein Antigen-Molecule Complex for Enhancing Cell Entry

Mix 150 μL of the mixture containing gold nanorods-protein antigen complex obtained in Example 11 with 20 μL of morpholineethanesulfonic acid buffer (MES) containing transferrin (2.5 mg/mL, apo-Transferrin, Sigma Company) Evenly, let stand at 25°C for 30min. Then add 30 μL of MES buffer containing carbodiethylimide hydrochloride (EDC, 0.5 mg/mL) to the above mixed system, react at 4 °C for 2 h, then centrifuge at 9500 rpm for 10 min, and wash with deionized water Once, and then resuspended in 150 μL double-distilled water to obtain a gold nanorod-protein antigen-transferrin complex mixture, and finally centrifuged to obtain a gold nanorod-protein antigen-transferrin complex.

Utilize UV-vis to detect the obtained gold nanorod-protein antigen-transferrin complex, the result is similar to embodiment 2, the absorbance of visible gold nanorod-protein antigen-transferrin complex is higher than gold nanorod-protein Antigen complexes are low.

Example 13

Preparation of gold nanorods-protein antigen-enhancing functional molecule-PEG

Take 150 μL of the mixture containing gold nanorods-protein antigen-transferrin complex obtained in Example 12, add 1 μL of HSC ₁₁ -EG ₆ OCH ₂ COOH (1 mmol/L, Prochimia, Poland, weight average molecular weight 635) Mix evenly and let stand at 25° C. for 2 hours to obtain a mixed solution containing gold nanorods-protein antigen-transferrin-PEG complex.

Example 14

Effect of gold nanorods on the viability of HEK293 cells

Preparation of serum-containing medium containing gold nanorods: Take 6 μL of the aqueous solution of gold nanorods with an aspect ratio of 4 prepared in Example 1 and dilute to 300 μL with DMEM medium containing 10% fetal bovine serum (FBS), so that the gold The final concentration of nanorods was 20 μg/mL.

Preparation of serum-containing medium containing gold nanorods-functional molecule complexes: take 6 μL of the mixed solution containing gold nanorods-functional molecule complexes prepared in Examples 2, 6-8, and 11-13, respectively, to contain 10% Fetal bovine serum (FBS) was diluted to 300 μL in DMEM medium so that the final concentration of gold nanorods was 20 μg/mL.

The effect of gold nanorods on the viability of HEK293 cells: First, HEK293 cells were digested with 0.25% trypsin, and after the digestion was terminated, centrifuged at 1000rpm for 5min. A single cell suspension was prepared with DMEM medium containing 10% fetal bovine serum (FBS), and inoculated in a 96-well plate at a concentration of 5×10 ⁴ /ml, 100 μl per well, and then placed in an incubator (37°C, 5% CO ₂ ) for 24 hours. A blank control group, a negative control group, a gold nanorod test group, and a gold nanorod-functional molecule complex test group were divided into three parallel groups. Add serum-containing medium, the serum-containing medium containing gold nanorods prepared above, and the serum-containing medium containing gold nanorods-functional molecule complexes to the wells, 100 μl per well, and place in an incubator (37°C, 5% CO ₂ ) for 12, 24, and 48 h, then discard the medium, wash twice with PBS buffer, and finally add serum-containing medium containing 10% CCK-8, 100 μl per well, continue to culture for 2 h, then A microplate reader (detection wavelength: 450nm) was used for detection to determine the effect of the gold nanorod-functional molecule complex on the viability of HEK293 cells.

8 is a histogram showing the effect of gold nanorods with an aspect ratio of 4 on the viability of HEK293 cells prepared in Example 1. Compared with the negative control group, gold nanorods have no effect on the viability of HEK293 cells. The prepared gold Nanorods are safe. The gold nanorod-functional molecule complexes prepared in Examples 2, 6-8, and 11-13 had no obvious effect on the viability of HEK293 cells, and the results were similar to those in Example 1, showing good safety.

Claims (2)

1. the method for modifying of a gold nanorods is characterized in that, this method may further comprise the steps:

(1) the gold nanorods matrix is synthetic

Under 25 ℃ of conditions, the cetyl trimethyl ammonium bromide solution 5mL of 0.2mol/L with after the tetra chlorauric acid solution 5mL of 0.96mmol/L mixes, is added the sodium borohydride solution 0.6mL of the ice bath of 0.01mol/L in this mixed system, stir 2min with the speed of 800rpm, obtain crystal seed liquid

Under 25 ℃ of conditions, with mixing among the tetra chlorauric acid solution 10mL of 25mmol/L, the silver nitrate solution 12.5mL that water 250mL adds to 4mmol/L, in this mixed system, add the CTAB solution 250mL of 0.2mol/L and the ascorbic acid solution 5mL of 0.08mol/L then, mix 15-30 second, namely get growth-promoting media, when growth-promoting media gradually becomes colourless by crocus, namely in growth-promoting media, add 0.6mL crystal seed liquid, leave standstill 16-18h under 25 ℃ behind the mix homogeneously, last centrifuge washing namely gets gold nanorods 2

Prepare gold nanorods 4 according to the method identical with preparing gold nanorods 2, different is, the mol ratio of silver nitrate in the growth-promoting media and tetra chlorauric acid is adjusted to 0.4, and the growth-promoting media cumulative volume is constant;

(2) preparation of gold nanorods-dna complex

With the plasmid DNA solution 0.1mL mix homogeneously of the green fluorescent protein of the aqueous solution 0.2mL of the gold nanorods 4 of 1mg/mL and 2 μ g/mL, place 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-dna complex;

(3) preparation of gold nanorods-DNA-modified poly (ethylene glycol) complex

Get the mixed liquor that contains gold nanorods-dna complex that obtains in the 150 μ L steps (2), add 1 μ L modified poly (ethylene glycol) HSC ₁₁-EG ₆OCH ₂The COOH mix homogeneously leaves standstill 2h under 25 ℃ of conditions, namely gets the mixed liquor of gold nanorods-DNA-modified poly (ethylene glycol) complex;

(4) preparation of gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal complex

The mixed liquor that contains gold nanorods-DNA-modified poly (ethylene glycol) complex and 20 μ L that 150 μ L steps (3) are obtained contain the morpholino b acid buffer mix homogeneously that sequence is the nuclear localization signal 2.5mg/mL of PAAKRVKLD, leave standstill 30min under 25 ℃ of conditions, the morpholino b acid buffer that adds 30 μ L carbon containing diethyl inferior amine salt hydrochlorate 0.5mg/mL afterwards in the above-mentioned mixed system, react 2h under 4 ℃ of conditions, afterwards with the centrifugal 10min of 9500rpm, reuse deionized water centrifuge washing once, be resuspended in afterwards in the 150 μ L distilled waters, namely get the mixed liquor that contains gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal complex;

(5) preparation of gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal-transferrins complex

The morpholino b acid buffer mix homogeneously that the mixed liquor that contains gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal complex that 150 μ L steps (4) are obtained and 20 μ L contain transferrins 2.5mg/mL, leave standstill 30min under 25 ℃ of conditions, the morpholino b acid buffer that adds 30 μ L carbon containing diethyl inferior amine salt hydrochlorate 0.5mg/mL afterwards in the above-mentioned mixed system, react 2h under 4 ℃ of conditions, afterwards with the centrifugal 10min of 9500rpm, reuse deionized water centrifuge washing once, be resuspended in afterwards in the 150 μ L distilled waters, namely get the mixed liquor that contains gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal-transferrins complex, the last centrifugal gold nanorods-DNA-modified poly (ethylene glycol)-nuclear localization signal-transferrins complex that namely gets.

2. gold nanorods-functional molecular complex is characterized in that, described gold nanorods-functional molecular complex obtains by the described method of modifying of claim 1.

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