CN102397557B - Modification method for gold nanorods and gold nanorods-functional molecule composite - Google Patents
Modification method for gold nanorods and gold nanorods-functional molecule composite Download PDFInfo
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Abstract
The invention provides a modification method for gold nanorods, which is characterized in that the gold nanorods are contacted with a DNA solution or a protein antigen solution, the surface of the gold nanorods carries a positive charge. The invention also relates to a gold nanorods-functional molecule composite obtained by the modification method. The gold nanorods-functional molecule composite is capable of obviously raising gene transfection level and promoting protein antigen for entering in cells, and has good security.
Description
Technical field
Gold nanorods-functional molecular the complex that the present invention relates to a kind of method of modifying of gold nanorods, obtained by this method.
Background technology
In the prevention and treatment of diseases process, vaccine is being brought into play important role.Vaccine carrier mainly is divided into two kinds of viral vector and non-virus carriers.With respect to viral vector, non-virus carrier has advantages such as easy production, toxic and side effects is little.Therefore, non-virus carrier enjoys people to pay close attention to as the research of potential vaccine carrier or adjuvant in recent years.Compare with other non-virus carrier, nano material has the promotion cell as carrier absorption, the prolongation functional molecular of functional molecular such as DNA etc. are processed advantage (the Jun-ichiro Jo such as targeting transportation that realize functional molecular at the circulation time of blood and by suitable modification, Yasuhiko Tabata.Non-viral gene transfection technologies for geneticengineering of stem cells.European Journal of Pharmaceutics andBiopharmaceutics, 2008,68:90-104.).The nano material of good biocompatibility or biodegradability becomes hot research in recent years as the most promising carrier.And just owing to its excellent biological compatibility, gold nano grain and gold nanorods receive much concern in the diagnosis of disease and treatment as potential carrier.Gold nanorods is also because its unique optical property, in biomedical imaging, medical diagnosis on disease with treat the application in fields such as tumor thermotherapy especially and become the focus of Recent study.Gold nano-material carries out finishing easily simultaneously, it can be by electrostatic adsorption and electronegative genetic molecule such as DNA, siRNA combinations such as (siRNA), and then realize hereditary functional molecular effectively transport and bring into play its function, prevention or treatment relevant disease.By in some biomolecule of gold nanorods finishing, can realize that also targeting transports, and better brings into play the effect of functional molecular.Other reports the significantly specific immunoreation of enhancement antigen (Megan E.Pearce of gold-nickel nanometer rods, Jessica B.Melanko, Aliasger K.Salem.Multifunctional Nanorods for Biomedical Applications.PharmaceuticalResearch, 2007,24 (12): 2335-2352.).
(Antigen-Presenting Cells APCs) is bringing into play important effect to antigen-presenting cell in the immune defence process of body.(Dendritic Cells is DCs) as the antigen-presenting cell of sole duty, because it is taken in, handles antigen and be transported to lymphoid tissue and then is the ability of passing inmature T cell and is bringing into play even more important effect in immunoreation for dendritic cell.For vaccine, can effectively be passed to APCs especially among the DCs and with its interaction, thereby induce for a long time, success or failure that immunoreation efficiently is related to prevention or treatment disease.Therefore DCs also enjoys the concern of research worker in the research and development of vaccine.With respect to the micron order vaccine carrier, the easier body that enters of nano material, and particle diameter is the easier lymphoid tissue (A.E.Hawley that enters of the nano material of 40-70nm, S.S.Davis, L.Illum.Targeting of colloids to lymphnodes-influence of lymphatic physiologyand colloidal characteristics.Advanced Drug Delivery Review, 1995,17 (1): 129-148.).And modify the especially antibody of DCs specific surfaces molecule of APCs at nano-material surface, will provide more favorably for the effective transmission that promotes antigen to ensure.
Functional molecular or vaccine antigen especially dna vaccination need pass through following three kinds of barriers: (1) cell membrane if will successfully be passed to immunocyte; (2) endocytosis body or lysosomal degraded; (3) nucleus.In other words, a kind of successful vaccine especially dna vaccination need realize antigen is effectively transmitted, protects it to avoid degraded and enters in the nucleus could real prevention and treatment disease.
Summary of the invention
Gold nanorods-functional molecular the complex that the object of the present invention is to provide a kind of method of modifying of gold nanorods, obtained by this method.
The invention provides a kind of method of modifying of gold nanorods, this method comprises: gold nanorods is contacted with the solution of DNA or the solution of proteantigen, and described gold nanorods surface is positively charged.
The present invention also provides a kind of gold nanorods-functional molecular complex, and described gold nanorods-functional molecular complex is that the method for modifying by above-mentioned gold nanorods obtains.
The present invention is by contacting gold nanorods with the solution of DNA or the solution of proteantigen, successfully realized the compound of the DNA of gold nanorods and hereditary functional molecular or proteantigen, and then realize hereditary functional molecular effectively transport and bring into play its function, prevention or treatment relevant disease.
Preferred implementation of the present invention is by modifying gold nanorods with modified poly (ethylene glycol) (PEG), thus avoid gold nanorods too early by reticuloendothelial system phagocytic, prolong its circulation time in body.
Preferred implementation of the present invention is by modifying the functional molecular (as transferrins, folic acid etc.) that born of the same parents are gone in fusogenic peptide or nuclear localization signal, promotion successively on positively charged gold nanorods surface, thereby improve the gene transfection level, promote that proteantigen enters cell, it is played a role more effectively.
Preferred implementation of the present invention is modified gold nanorods by the specific antibody that is delivery cell with targeting antigen, thereby assurance DNA or proteantigen are presented more effectively to B cell and T cell.
Description of drawings
Fig. 1 is that the TEM of gold nanorods described in the embodiment 1 characterizes picture.
Fig. 2 is that the SEM of different draw ratio gold nanorods described in the embodiment 1 characterizes picture.
Fig. 3 is the UV scanning collection of illustrative plates of the different draw ratio gold nanorods described in the embodiment 1.
Fig. 4 is the current potential testing result of gold nanorods described in the embodiment 1.
Fig. 5 is the UV scanning collection of illustrative plates behind the gold nanorods absorption pEGFP described in the embodiment 2.
Fig. 6 is the UV scanning collection of illustrative plates of the gold nanorods-pEGFP-PEG described in the embodiment 6,7 and gold nanorods-pEGFP-PEG-NLS complex.
Fig. 7 is the picture (amplification 10 * 10) of various gold nanorods described in the embodiment 10-functional molecular complex transfection HEK293 cell.
Fig. 8 is that the draw ratio of expression embodiment 1 preparation is that 4 gold nanorods is to the block diagram of the influence of HEK293 cell viability.
The specific embodiment
The invention provides a kind of method of modifying of gold nanorods, this method comprises: gold nanorods is contacted with the solution of DNA or the solution of proteantigen, and described gold nanorods surface is positively charged.
In method of modifying provided by the invention, described gold nanorods with respect to 1 weight portion, the consumption of DNA is the 0.001-0.2 weight portion, the concentration of the solution of DNA is 2-400 μ g/ml, or the consumption of proteantigen is the 0.1-2 weight portion, the concentration of the solution of protein is 0.1-2mg/ml, under the preferable case, described gold nanorods with respect to 1 weight portion, the consumption of DNA is the 0.1-0.2 weight portion, the concentration of the solution of DNA is 200-400 μ g/ml, or the consumption of proteantigen is the 0.5-1 weight portion, and the concentration of the solution of protein is 0.5-1mg/ml.
The length of the gold nanorods that uses in the present invention is 40-70nm, and draw ratio is 1-6: 1.Owing in the preparation raw material of gold nanorods, contain just like cationic surfactant such as cetyl trimethyl ammonium bromide (CTAB) or as the polyelectrolyte of PDDA positively chargeds such as (PDDAC), therefore described gold nanorods surface is positively charged, and the described gold nanorods among the present invention has the positive charge of 10-50 millivolt.
Because DNA and most proteantigen are all electronegative, and the gold nanorods surface described in the present invention is positively charged, it easily combines by electrostatic adsorption, therefore in the present invention, kind to described DNA or proteantigen has no particular limits, described DNA is HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) memebrane protein encoding gene (HIV Env DNA) for example, hepatitis B virus memebrane protein encoding gene (HBV) and human papillomavirus's memebrane protein encoding gene (HPV) etc., described proteantigen is HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) memebrane protein (gp140) for example, hepatitis B virus memebrane protein and human papillomavirus's memebrane protein antigen etc., these DNA that enumerate or proteantigen all can with the present invention in gold nanorods mutually combine.
In order to improve the amount that DNA and proteantigen enter cell, make DNA avoid lysosomal degraded simultaneously, thereby guarantee that DNA and proteantigen effectively play a role, also comprise in the method for modifying provided by the present invention, after the solution of the solution of gold nanorods and DNA or proteantigen contacted, again in the presence of solvent with modified poly (ethylene glycol), fusogenic peptide, nuclear localization signal, promote to go into born of the same parents' functional molecular, targeting antigen is any one or the once multiple or contact several times in the specific antibody of delivery cell, the time of each contact is 2-12 hour, the skeleton of described modified poly (ethylene glycol) is polyethylene glycol backbone, and an end of described modified poly (ethylene glycol) is that sulfydryl and the other end are amino or carboxyl.Described solvent can be in deionized water, phosphate buffer (PBS) or the morpholino b acid buffer (MES) etc. a kind of.
In the present invention, the weight average molecular weight of described modified poly (ethylene glycol) can be 150-635, and described modified poly (ethylene glycol) can use for example HSC of Polish Prochimia company
11-EG
2NH
2
In the method for modifying of gold nanorods of the present invention, gold nanorods with respect to 1 weight portion, the consumption of described fusogenic peptide can be the 0-2 weight portion, be preferably the 0-1 weight portion, the consumption of described nuclear localization signal can be the 0-2 weight portion, be preferably the 0-1 weight portion, the consumption that born of the same parents' functional molecular is gone in described promotion can be the 0-2 weight portion, be preferably the 0-1 weight portion, the consumption that described targeting antigen is the specific antibody of delivery cell can be the 0-2 weight portion, be preferably the 0-1 weight portion, the consumption of described modified poly (ethylene glycol) can be the 0-3 weight portion, be preferably the 0-1.5 weight portion, and gold nanorods and fusogenic peptide, nuclear localization signal, promote to go into born of the same parents' functional molecular, targeting antigen is the specific antibody of delivery cell and total consumption of modified poly (ethylene glycol) can be the 0.01-11 weight portion, is preferably the 3.5-6.5 weight portion.
In the present invention, the kind that born of the same parents' functional molecular or targeting antigen be the specific antibody of delivery cell is gone in described fusogenic peptide, nuclear localization signal, promotion have no particular limits, described fusogenic peptide is the little peptide of JTS-1 sequence (GLEEALLFLLESLWELLLEA), the little peptide of INF-7 sequence (GLFEAIEGFIENGWEGMIWDYG) and contain little peptide of KALA sequence etc. for example; Described nuclear localization signal is nuclear localization signal and other types of nuclear framing signals such as the ribosomal protein etc. of SV40T antigen, the amino acid residue sequence that is spaced apart two bunches of positively chargeds that distinguish, c-MYC proto-oncogene for example; Born of the same parents' functional molecular is gone in described promotion can enumerate the little peptide that for example contains arginine-glycine-aspartic acid (RGD) sequence, transferrins, epidermal growth factor (EGF), folic acid etc., and the specific antibody that described targeting antigen is delivery cell is the specific antibody anti-DEC205 etc. of targeting dendritic cell for example.
The present invention also provides a kind of gold nanorods-functional molecular complex, and described gold nanorods-functional molecular complex is that the method for modifying by above-mentioned gold nanorods obtains.
The present invention is described in detail below in conjunction with embodiment and accompanying drawing.
Synthesizing of gold nanorods matrix
Under 25 ℃ of conditions, cetyl trimethyl ammonium bromide (CTAB with 0.2mol/L, Amersco company, the U.S.) solution 5mL is with after the tetra chlorauric acid solution 5mL of 0.96mmol/L mixes, the sodium borohydride solution 0.6mL that adds the ice bath of 0.01mol/L in this mixed system, speed with 800rpm stirs 2min, obtains 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 according to the method described above, different is, and the mol ratio of silver nitrate and tetra chlorauric acid is adjusted to 0,0.2,0.3,0.4 in the growth-promoting media with regulating, and the growth-promoting media cumulative volume is constant, obtains gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4.
Utilize transmission electron microscope, scanning electron microscope that resulting gold nanorods is observed, the results are shown in illustrated in figures 1 and 2.Figure 1A, Figure 1B, Fig. 1 C, Fig. 1 D represent gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 respectively, the mean diameter of gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 is respectively 40nm, 20nm * 40nm, 15nm * 45nm, 15nm * 60nm, and the draw ratio of gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 was respectively 1: 1,2: 1,3: 1,4: 1.Fig. 2 A, Fig. 2 B, Fig. 2 C, Fig. 2 D represent gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 respectively, the mean diameter of gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 is respectively 40nm, 20nm * 40nm, 15nm * 45nm, 15nm * 60nm, and the draw ratio of gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 was respectively 1: 1,2: 1,3: 1,4: 1.
Utilize ultraviolet spectrometer (UV-vis) that resultant gold nanorods is detected, the results are shown in Figure 3, the characteristic absorption peak of gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 is respectively at 530nm, 620nm, 700nm and 820nm.
Utilize laser particle analyzer that resultant gold nanorods is detected, the results are shown in Figure 4, gold nanorods 1, gold nanorods 2, gold nanorods 3, gold nanorods 4 surfaces all have about 28.6 millivolts positive charge.
Embodiment 2
The preparation of gold nanorods-dna complex
With the aqueous solution 0.2mL of the gold nanorods 4 of 1mg/mL and the pEGFP (plasmid DNA of green fluorescent protein of 2 μ g/mL, Biovector-08, Clontech company) solution 0.1mL mix homogeneously, place about 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-dna complex, last centrifuge washing namely gets gold nanorods-dna complex.
Utilize UV-vis that resultant gold nanorods-dna complex is detected, the results are shown in Figure 5, as seen with respect to gold nanorods, the absorbance of gold nanorods-dna complex (OD) value reduces.
Embodiment 3
The preparation of gold nanorods-dna complex
With the aqueous solution 0.2mL of the gold nanorods 4 of 1mg/mL and the pEGFP (plasmid DNA of green fluorescent protein of 200 μ g/mL, Biovector-08, Clontech company) solution 0.1mL mix homogeneously, place about 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-dna complex, last centrifuge washing namely gets gold nanorods-dna complex.
Utilize UV-vis that resultant gold nanorods is detected, the result is similar to Example 2, compares with gold nanorods, and behind the gold nanorods adsorption of DNA, its absorbance (OD) value reduces.
Embodiment 4
The preparation of gold nanorods-dna complex
With the aqueous solution 0.2mL of the gold nanorods 4 of 1mg/mL and the pEGFP (plasmid DNA of green fluorescent protein of 400 μ g/mL, Biovector-08, Clontech company) solution 0.1mL mix homogeneously, place about 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-dna complex, last centrifuge washing namely gets gold nanorods-dna complex.
Utilize UV-vis that resultant gold nanorods is detected, the result is similar to Example 2, compares with gold nanorods, and behind the gold nanorods adsorption of DNA, its absorbance (OD) value reduces.
Embodiment 5
Gold nanorods is to the detection of DNA adsorption rate
With the aqueous solution of 1mg/mL gold nanorods 4 and the pEGFP solution equal-volume mix homogeneously of variable concentrations (seeing Table 1), after leaving standstill 30min under 25 ℃ of conditions, with the centrifugal 15min of 10000rpm, utilize the absorbance (OD) of microplate reader DNA in 260nm detection supernatant afterwards, and calculate the residual quantity of DNA in the supernatant according to " when the absorbance of DNA is 1; its concentration is equivalent to 50ng/ μ L ", thereby according to formula: adsorption rate=(amount of DNA total amount-residual DNA) * 100%/DNA total amount, determine that gold nanorods to the adsorption rate of DNA, the results are shown in Table 1.
Table 1
| PEGFP concentration (μ g/mL) | 10 | 25 | 62.5 | 125 | 250 |
| Adsorption rate (%) | 100 | 100 | 100 | 100 | 80 |
Embodiment 6
The preparation of gold nanorods-DNA-PEG complex
Get the mixed liquor that contains gold nanorods-dna complex that obtains among the 150 μ L embodiment 2, add 1 μ L HSC
11-EG
6OCH
2COOH (weight average molecular weight is 635 for 1mmol/L, Polish Prochimia company) mix homogeneously leaves standstill 2h under 25 ℃ of conditions, namely gets the mixed liquor of gold nanorods-DNA-PEG complex, the last centrifugal gold nanorods-DNA-PEG complex that namely gets.Because gold-sulfur covalent bond very easily forms, so SH-PEG can be connected gold nanorods-dna complex surface easily.Utilize UV-vis that resultant gold nanorods-DNA-PEG complex is detected, the results are shown in Figure 6.
Embodiment 7
The preparation of gold nanorods-DNA-PEG-NLS complex
The mixed liquor that contains gold nanorods-DNA-PEG complex and 20 μ L that 150 μ L embodiment 6 are obtained contain NLS (2.5mg/mL, sequence is PAAKRVKLD, it is synthetic that Xi'an joins U.S. bio tech ltd) morpholino b acid buffer (MES) mix homogeneously, leave standstill 30min under 25 ℃ of conditions.In above-mentioned mixed system, add 30 μ L carbon containing diethyl inferior amine salt hydrochlorate (EDC afterwards, 0.5mg/mL) the MES buffer, 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-PEG-NLS complex, the last centrifugal gold nanorods-DNA-PEG-NLS complex that namely gets.
Utilize UV-vis that resultant gold nanorods-DNA-PEG-NLS complex is detected, the results are shown in Figure 6, the absorbance of visible gold nanorods-DNA-PEG-NLS complex is low than gold nanorods-DNA-PEG.
Embodiment 8
The preparation of gold nanorods-DNA-PEG-NLS-transferrins complex
The mixed liquor that contains gold nanorods-DNA-PEG-NLS complex and 20 μ L that 150 μ L embodiment 7 are obtained contain transferrins (2.5mg/mL, apo-Transferrin, Sigma company) morpholino b acid buffer (MES) mix homogeneously leaves standstill 30min under 25 ℃ of conditions.In above-mentioned mixed system, add 30 μ L carbon containing diethyl inferior amine salt hydrochlorate (EDC afterwards, 0.5mg/mL) the MES buffer, 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-PEG-NLS-transferrins complex, the last centrifugal gold nanorods-DNA-PEG-NLS-transferrins complex that namely gets.
Utilize UV-vis that resultant gold nanorods-DNA-PEG-NLS-transferrins complex is detected, the result is similar to Example 2, and gold nanorods-DNA-PEG-NLS-transferrins complex absorbance is low than gold nanorods-DNA-PEG-NLS complex.
Embodiment 9
Gold nanorods-functional molecular complex is to the transfection test of HEK293 cell
Contain the preparation of the blood serum medium of gold nanorods-functional molecular complex: get the mixed liquor that contains gold nanorods-functional molecular complex for preparing among 30 μ L embodiment 2, the 6-8 respectively, be diluted to 300 μ L with the DMEM culture medium that contains 10% hyclone (FBS).
Gold nanorods-functional molecular complex transfection HEK293 cell: at first with 0.25% trypsinization HEK293 cell, after stopping digesting, with the centrifugal 5min of 1000rpm.Make single cell suspension with the DMEM culture medium that contains 10% hyclone (FBS), and with 5 * 10
4/ ml concentration is inoculated in 24 orifice plates, and every hole 500 μ l place incubator (37 ℃, 5%CO afterwards
2) the middle 24h that cultivates.The former culture medium of sucking-off afterwards, every hole add the culture medium that 400 μ l contain serum.Set up 1 blank group separately, 1 naked EGFP plasmid matched group, 4 gold nanorods-functional molecular complex test group, liposome (Lipofectamine2000, Invitrogen company) transfection positive controls, establish three parallel holes for every group, the blank group adds and contains serum DMEM culture medium 100 μ l/ holes, naked EGFP plasmid matched group adds and contains the EGFP plasmid (plasmid DNA of green fluorescent protein, Biovector-08, Clontech company) contain serum DMEM culture medium (concentration of EGFP plasmid is 8 μ g/mL) 100 μ l/ holes, 4 gold nanorods-functional molecular complex test group add the blood serum medium that the contains gold nanorods-functional molecular complex 100 μ l/ holes of above-mentioned preparation, shake up, place (37 ℃ of incubators, 5%CO
2) the middle 12h that cultivates, change the culture medium that contains serum, continue to cultivate, behind 48h, utilize the fluorescence microscope transfection results.
Result of the test as shown in Figure 7, Fig. 7 A is that pEGFP plasmid DNA matched group, Fig. 7 B are that gold nanorods-pEGFP complex, Fig. 7 C are that gold nanorods-pEGFP-PEG complex, Fig. 7 D are that gold nanorods-pEGFP-PEG-NLS complex, Fig. 7 E are that gold nanorods-pEGFP-PEG-NLS-Tf complex, Fig. 7 F are liposome (Lipofectamine 2000) transfection positive controls.As seen from the figure, naked pEGFP is difficult to transfection HEK293 cell, no fluorescent protein expression, and gold nanorods-pEGFP complex can transfection HEK293 cell, and along with a series of modifications to gold nanorods-pEGFP complex, the ability that is gold nanorods-pEGFP-PEG-NLS, gold nanorods-pEGFP-PEG-NLS-Tf complex transfectional cell obviously improves, and transfection efficiency and the positive controls of gold nanorods-pEGFP-PEG-NLS-Tf complex are suitable.
Embodiment 10
The preparation of gold nanorods-proteantigen complex
Aqueous solution 0.2mL and 0.1mg/mL ovalbumin (OVA with 1mg/mL gold nanorods 4, Sigma company) aqueous solution 0.2mL mix homogeneously, leave standstill 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-proteantigen complex, last centrifuge washing namely gets gold nanorods-proteantigen complex.
Utilize UV-vis that resultant gold nanorods-proteantigen complex is detected, the result is similar to Example 2, and visible gold nanorods-proteantigen complex absorbance is low than gold nanorods.
Embodiment 11
The preparation of gold nanorods-proteantigen complex
Aqueous solution 0.2mL and 2mg/mL ovalbumin (OVA with 1mg/mL gold nanorods 4, Sigma company) aqueous solution 0.2mL mix homogeneously, leave standstill 30min under 25 ℃ of conditions, namely get the mixed liquor that contains gold nanorods-proteantigen complex, last centrifuge washing namely gets gold nanorods-proteantigen complex.
Utilize UV-vis that resultant gold nanorods-proteantigen complex is detected, the result is similar to Example 2, and the absorbance of visible gold nanorods-proteantigen complex is low than gold nanorods.
Embodiment 12
The preparation of born of the same parents' functional molecular complex is gone in gold nanorods-proteantigen-promotion
The mixed liquor that contains gold nanorods-proteantigen complex and 20 μ L that 150 μ L embodiment 11 are obtained contain transferrins (2.5mg/mL, apo-Transferrin, Sigma company) morpholino b acid buffer (MES) mix homogeneously leaves standstill 30min under 25 ℃ of conditions.In above-mentioned mixed system, add 30 μ L carbon containing diethyl inferior amine salt hydrochlorate (EDC afterwards, 0.5mg/mL) the MES buffer, 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 of gold nanorods-proteantigen-transferrins complex, the last centrifugal gold nanorods-proteantigen-transferrins complex that namely gets.
Utilize UV-vis that resultant gold nanorods-proteantigen-transferrins complex is detected, the result is similar to Example 2, and the absorbance of visible gold nanorods-proteantigen-transferrins complex is low than gold nanorods-proteantigen complex.
Embodiment 13
The preparation of born of the same parents functional molecular-PEG is gone in gold nanorods-proteantigen-promotion
Get the mixed liquor that contains gold nanorods-proteantigen-transferrins complex that obtains among the 150 μ L embodiment 12, add 1 μ L HSC
11-EG
6OCH
2COOH (weight average molecular weight is 635 for 1mmol/L, Polish Prochimia company) mix homogeneously leaves standstill 2h under 25 ℃ of conditions, namely gets the mixed liquor that contains gold nanorods-proteantigen-transferrins-PEG complex.
Embodiment 14
Gold nanorods is to the influence of HEK293 cell viability
Contain the preparation that contains blood serum medium of gold nanorods: the draw ratio of getting 6 μ L embodiment, 1 preparation is 4 gold nanorods aqueous solution, is diluted to 300 μ L with the DMEM culture medium that contains 10% hyclone (FBS), makes that the final concentration of gold nanorods is 20 μ g/mL.
Contain the preparation that contains blood serum medium of gold nanorods-functional molecular complex: the mixed liquor that contains gold nanorods-functional molecular complex of getting 6 μ L embodiment 2,6-8,11-13 preparation respectively, be diluted to 300 μ L with the DMEM culture medium that contains 10% hyclone (FBS), make that the final concentration of gold nanorods is 20 μ g/mL.
Gold nanorods is to the influence of HEK293 cell viability: at first with 0.25% trypsinization HEK293 cell, stop digestion after, 1000rpm, centrifugal 5min.Make single cell suspension with the DMEM culture medium that contains 10% hyclone (FBS), and with 5 * 10
4/ ml concentration is inoculated in 96 orifice plates, and every hole 100 μ l place incubator (37 ℃, 5%CO afterwards
2) the middle 24h that cultivates.Set up blank group, negative control group, gold nanorods test group, gold nanorods-functional molecular complex test group separately, establish 3 parallel group for every group.To contain the blood serum medium that contains that contains blood serum medium and contain gold nanorods-functional molecular complex that blood serum medium and above-mentioned preparation contain gold nanorods respectively and add in the hole, every hole 100 μ l place incubator (37 ℃, 5%CO
2) in cultivate 12,24,48h, discard culture medium afterwards, with PBS buffer washing 2 times, add the blood serum medium that contains that contains 10%CCK-8 at last, every hole 100 μ l, continue to cultivate 2h, utilize microplate reader (the detection wavelength is 450nm) to detect afterwards, determine that gold nanorods-functional molecular complex is to the situation that influences of HEK293 cell viability.
Fig. 8 be the draw ratio of expression embodiment 1 preparation be 4 gold nanorods to the block diagram of the influence of HEK293 cell viability, compare with negative control group, gold nanorods is to the not influence of cell viability of HEK293, prepared gold nanorods safety is good.Embodiment 2,6-8, the prepared gold nanorods-functional molecular complex of 11-13 also do not have obvious influence to the HEK293 cell viability, and its result and embodiment 1 result are similar, demonstrate 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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