CN103623417A - Application of functionalized polyamide-amine dendrimer nanocomposite - Google Patents
Application of functionalized polyamide-amine dendrimer nanocomposite Download PDFInfo
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Abstract
The invention relates to application of a functionalized polyamide-amine dendrimer nanocomposite, and particularly relates to application of a functionalized polyamide-amine dendrimer nanocomposite which carries hBMP-2pDNA and induces hMSCs to be differentiated into a carrier of osteoblast. The preparation process of the functionalized polyamide-amine dendrimer nanocomposite is simple, the experiment conditions are mild, and the experiment is easy to operate. The functionalized polyamide-amine dendrimer nanocomposite carrier is a good gene carrier which can successfully carry hBMP-2pDNA and induce mesenchymal stem cells to be differentiated into osteoblast, and has good application prospect in repair and rebuilding of bone tissues.
Description
Technical field
The invention belongs to high molecular nanometer carrier target gene transfection field, particularly a kind of nano-complex of functionalization Polyamidoamine Dendrimers is as load hBMP-2pDNA(human bone morphogenesis protein plasmid DNA) induction hMSCs(mesenchymal stem cells MSCs) be divided into the application of osteoblastic carrier.
Background technology
Gene therapy combines as a kind of modern medicine and molecular biology and the new technique that is born, is that external source normal gene is imported to target cell, to correct or compensator gene disappearance or the disease that extremely causes, thereby reaches the new hand of disease treatment.As far back as nineteen sixty-eight, American scientist Michael Bu Laize, has proposed the concept of gene therapy first in medical circle.But until nineteen ninety, the mankind have just carried out relevant clinical trial first.The nearly more than ten years, a lot of clinical trials are the medicinal strategy using gene therapy as a kind of novelty all, is applied to cancer, hereditary, cardiovascular disease, immunodeficiency and many other relevant genetic flaw or loss of obstacle disease.
Yet the topmost obstacle of gene therapy lacks a kind of expression vector that transmits safely and effectively exactly at present, and hereditary material is delivered to desired position in organism.In gene vector system, mainly by virus and non-virus carrier, exogenous gene is imported to target cell.Viral vector is because its efficient transfection efficiency is applied to gene therapy clinically.But when being applied in clinical experiment using viral vector as gene delivery vehicle, but found a lot of potential safety hazards, such as gene mutation, immunogenicity, these have all further restricted its extensive use as carrier.And non-virus carrier, due to features such as its low cytotoxicity, non-immunogenicity, easy to operate and high gene useful loads, more and more receives the concern of researcheres.Non-virus carrier mainly comprises: cationic-liposome, cationic polymer, chitin carrier polymer and inorganic nano-particle carrier etc.Wherein, the dendrimer in cationic polymer is as a kind of novel, and the macromolecule with unique highly branched 3-D solid structure is widely used in gene delivery.
The transfection ability of dendrimer depends on the number of amino groups on its algebraical sum surface, and the dendrimer of different algebraically has different surface amino groups numbers, therefore selects the dendrimer of suitable algebraically to seem most important.The 5th generation daiamid (PAMAM) dendrimer, surface has enriches amino group and can carry out multifunction modification, the cavity of inside configuration can packaging medicine molecule or other little nano-particle, the structure of monodispersity makes it easily enter cell, these excellent characteristics impel us to utilize the 5th generation PAMAM dendrimer as template, at its finishing RGD and PEG, wrap up gold nano grain simultaneously, be expected to prepare and there is dendrimer and the nano-particle thereof that high efficiency gene transmits the functionalization of performance, for exploitation and the medical application thereof of novel gene therapy system provides new thinking and reference value.
Retrieving domestic and international pertinent literature and patent results shows: dendrimer and the nano-particle thereof of functionalization of take is carrier, and the method for mesenchymal stem cells MSCs gene transfection, there is not yet report.
Summary of the invention
The nano-complex that technical problem to be solved by this invention is to provide a kind of functionalization Polyamidoamine Dendrimers is divided into the application of osteoblastic carrier as load hBMP-2pDNA induction hMSCs, the method preparation process is simple, experiment condition is gentle, easy to operate, there is good gene transfection expression of results, in the repair and reconstruction of bone tissue engineer, have good application prospect.
The nano-complex of a kind of functionalization Polyamidoamine Dendrimers of the present invention is divided into the application of osteoblastic carrier as load hBMP-2pDNA induction hMSCs;
The nano-complex of described functionalization Polyamidoamine Dendrimers is specially: { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10; Its preparation method is as follows:
(1) by NH
2-PEG-COOH solution reacts 6-8h with 6-MAL solution stirring, obtain MAL-PEG-COOH solution, then add RGD-SH solution, stirring reaction 10-12h, obtain RGD-PEG-COOH solution, with activating by EDC and NHS solution, then add in the 5th PAMAM dendrimer solution, reaction 12-30h, obtains G5.NH
2-(PEG-RGD)
10solution, then joins above-mentioned G5.NH by the mPEG-COOH solution through EDCHCl and NHS activation
2-(PEG-RGD)
10in solution, reaction 12-30h, obtains G5.NH
2-(PEG-RGD)
10-mPEG
10solution;
(2) at above-mentioned G5.NH
2-(PEG-RGD)
10-mPEG
10in, add HAuCl
4solution, stirring reaction 20-30min, then add NaBH
4solution, stirs 2-4h, obtains { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10solution;
(3) solution of step (2) gained is dialysed, lyophilization processes, the { (Au that obtains being dried
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10.
The described nano-complex of functionalization Polyamidoamine Dendrimers and the N/P of hBMP-2pDNA be than being 1:1～5:1, and described N/P is than being phosphate group mol ratio on the primary amino radical of dendrimer and pDNA skeleton.
The described nano-complex of functionalization Polyamidoamine Dendrimers and the N/P of hBMP-2pDNA are than being 1:1,2.5:1 or 5:1.
The method that the nano-complex of described functionalization Polyamidoamine Dendrimers and hBMP-2pDNA are compound is: by { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10with sterilized water dilution, sterilized water dilution plasmid then, then will after the two mix homogeneously, hatch 30min in 37 ℃.
NH in described step (1)
2the concentration of-PEG-COOH solution, 6-MAL solution, RGD-SH solution, mPEG-COOH solution is 5.8mmol/mL; The concentration of the 5th PAMAM dendrimer solution is 0.58mmol/mL; Solvent is DMSO.
HAuCl in described step (2)
4the concentration of solution is 30mg/mL, NaBH
4the concentration of solution is 10mg/mL.
In described step (3), dialysis is the 2-3d that dialyses with deionized water, every day 3 times, and each 2L deionized water, wherein bag filter molecular cut off is 14000.
The preparation method of functionalization Polyamidoamine Dendrimers and nano-complex thereof, comprising:
(1) at the DMSO(dimethyl sulfoxide of the 5th PAMAM dendrimer) in solution, add respectively through EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) and NHS(N-N-Hydroxysuccinimide) the mPEG-COOH(end group of activation is respectively the Polyethylene Glycol of carboxyl and methoxyl group) solution, stirring reaction 12-30h, obtains G5.NH
2-mPEG
20solution; Wherein the mol ratio of EDC and NHS is 1:1, and the mol ratio of EDC and NHS and mPEG-COOH is 1.8:1, and the mol ratio of mPEG-COOH and dendrimer is 20:1.
(2) at above-mentioned G5.NH
2-mPEG
20in solution, add HAuCl
44H
2o(gold chloride) solution, stirring reaction 20-30min, then add NaBH
4(sodium borohydride) solution, stirs 2-4h, obtains { (Au
0)
25-G5.NH
2-mPEG
20solution; HAuCl wherein
44H
2the mol ratio of O and dendrimer is 25:1, NaBH
4with HAuCl
44H
2the mol ratio of O is 5:1.
(3) by NH
2-PEG-COOH(end group is respectively the Polyethylene Glycol of amino and carboxyl) solution and 6-MAL(6-(dimaleoyl imino) caproic acid succinimide ester) solution stirring reacts 6-8h, obtain MAL-PEG-COOH solution, then the arginine-glycine-aspartic acid aminoacid sequence peptide that adds RGD-SH(sulfydryl end ring shape) (Qiang Yao bio tech ltd, Shanghai) solution, stirring reaction 10-12h, obtain RGD-PEG-COOH solution, with activating by EDC and NHS solution, add again in the 5th PAMAM dendrimer solution, reaction 12-30h, obtain G5.NH
2-(PEG-RGD)
10solution, then by the process in step (1), obtains the mPEG-COOH of activation, joins G5.NH
2-(PEG-RGD)
10in, reaction 12-30h, obtains G5.NH
2-(PEG-RGD)
10-mPEG
10solution, NH wherein
2the mol ratio of-PEG-COOH, 6-MAL, RGD-SH is 1:1:1, the mol ratio of EDC and NHS is 1:1, the mol ratio of EDC and NHS sum and mPEG-COOH is 1.8:1, the mol ratio of EDC and NHS sum and RGD-PEG-COOH is 1.8:1, and the mol ratio of mPEG-COOH and dendrimer is 20:1.
(4) at above-mentioned G5.NH
2-(PEG-RGD)
10-mPEG
10in, the process according to step (2), obtains { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10solution; HAuCl wherein
44H
2the mol ratio of O and dendrimer is 25:1, NaBH
4with HAuCl
44H
2the mol ratio of O is 5:1.
(5) solution of step (1) (2) (3) (4) gained is dialysed, lyophilization processes, and obtains dry G5.NH
2-mPEG
20, { (Au
0)
25-G5.NH
2-mPEG
20, G5.NH
2-(PEG-RGD)
10-mPEG
10, { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10; Respectively by G5.NH
2, G5.NH
2-mPEG
20, { (Au
0)
25-G5.NH
2-mPEG
20, G5.NH
2-(PEG-RGD)
10-mPEG
10, { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10to they called after K0, K1, K2, K3, K4.
(6) according to different N/P than (phosphate group mol ratio on the primary amino radical of dendrimer and pDNA skeleton), get K1, K2, K3, K4 dilutes with sterilized water, and dilutes plasmid with sterilized water, then mix homogeneously, hatch 30min for 37 ℃, obtain functionalization Polyamidoamine Dendrimers and the nano-complex/hBMP-2pDNA thereof of different N/P ratio.
In described step (1), the concentration of mPEG-COOH solution is 11.6mmol/mL; The concentration of EDC and NHS solution is 20.88mmol/mL; The concentration of the 5th PAMAM dendrimer solution is 0.58mmol/mL; Solution solvent is dimethyl sulfoxide DMSO.
HAuCl in described step (2)
4concentration of aqueous solution is 30mg/mL, NaBH
4the concentration of solution is 10mg/mL.
NH in described step (3)
2the concentration of-PEG-COOH solution, 6-MAL solution, RGD-SH solution, mPEG-COOH solution is 5.8mmol/mL; The concentration of EDC and NHS solution is 10.44mmol/mL; The concentration of the 5th PAMAM dendrimer solution is 0.58mmol/mL; Solvent is DMSO.
HAuCl in described step (4)
44H
2the concentration of O solution is 30mg/mL; NaBH
4the concentration of solution is 10mg/mL; Solvent is water.
In described step (5) dialysis for bag filter molecular cut off be 14000; With the deionized water 2-3d that dialyses, every day 3 times, each 2L deionized water.
N/P ratio in described step (6) is respectively 1:1,2.5:1,5:1.
In the present invention, hydrophilic reagent PEG is grafted to end for the 5th amino PAMAM dendrimer surface, can in and part surface amino to reduce the toxicity of material to cell, thereby improve the biocompatibility of material.
The present invention modifies the 5th PAMAM dendrimer surface by targeting agent RGD, make material can with the α on mesenchymal stem cells MSCs surface
vβ
3integrin combines, and realizes the selectively targeted effect of material to stem cell.
The present invention is by HAuCl
44H
2after O joins in Polyamidoamine Dendrimers solution, rapid stirring 20-30min, can make HAuCl
44H
2o fully mixes with dendrimer, enters into dendrimer internal cavities.Add fast again NaBH
4reduction obtains gold nano grain, has avoided the gathering of gold nano grain.
It is carrier that functionalization Polyamidoamine Dendrimers and nano-complex thereof are take in the present invention, and load hBMP-2pDNA, usings hMSCs as target cell, through gene transfection, induces it to be divided into osteoblast.The present invention analyzes particle diameter and the electromotive force of carrier/hBMP-2pDNA complex by hydrodynamics particle diameter and surface potential; Toxicity by mtt assay test material to cell; By the while, the pDNA with luciferase reporter gene (Luc) and reinforcement green fluorescence protein gene (EGFP) measures the gene transfection ability of carrier; By carrying the pDNA of hBMP-2 gene, the gene transfection ability of carrier loaded hBMP-2 is measured; By quantitative alkali phosphatase (ALP) activity, Bone Gla protein deposition, calcium ion secretion and Feng Kusa dyeing qualitatively, hMSCs being divided into osteoblastic ability analyzes.
Hydrodynamics particle diameter and surface potential, cell toxicity test, luciferase gene and Transfection of Enhanced Green Fluorescent experiment, ALP activity, Bone Gla protein deposit, calcium ion is secreted and Feng Kusa coloration result is as follows respectively qualitatively:
(1) hydrodynamics particle diameter and surface potential test result:
Hydrodynamics particle diameter and surface potential test result are used for characterizing the ability that functionalization Polyamidoamine Dendrimers and nano-complex/hBMP-2pDNA thereof enter cell, and test result is as shown in Figure of description 1.Result shows, the hydrodynamics particle diameter of functionalization Polyamidoamine Dendrimers and nano-complex/hBMP-2pDNA thereof is in 200nm left and right, and surface potential is in 20mV left and right, illustrates that the size of complex and electromotive force are all within the scope of suitable transfection.
(2) cell toxicity test test result:
Cell toxicity test is used for characterizing functionalization Polyamidoamine Dendrimers and nano-complex and functionalization Polyamidoamine Dendrimers and the toxicity of nano-complex/hBMP-2pDNA to cell thereof, referring to Figure of description 2.MTT result of the test surface, along with the concentration increase of carrier and complex, material also increases the toxicity of cell thereupon.And under same concentration, complex decreases to the toxicity of cell than independent carrier to the toxicity of cell, illustrate hBMP-2pDNA with carrier function in, neutralized the amino of carrier surface part, thereby reduced its biology toxicity.
(3) luciferase reporter gene expression of results:
Luciferase expression experiment is for the transfection ability of Study of Support to gene, with the plasmid with luciferase gene and to have express alpha
vβ
3the mesenchymal stem cells MSCs hMSCs of integrin is that model cell is checked the transfection effect of five kinds of materials to stem cell, referring to Figure of description 3.Result shows, the transfection efficiency of material all with N/P than relevant, and at N/P than when being 2.5, luciferase transfection efficiency is the highest.Contrast five kinds of materials, discovery is at N/P than when being 2.5, and the transfection efficiency of K4 is the highest, compares the difference all with significance with other material.Illustrate that hydrophilic molecules PEG, the modification of targeted molecular RGD and the parcel of gold nano grain effectively raise the transfection efficiency of material.
(4) strengthen Green Fluorescent Protein Gene Expression result:
Strengthen egfp expression experiment for the transfection ability of Study of Support to gene, with the plasmid with enhanced green fluorescent protein gene and to there is express alpha
vβ
3the mesenchymal stem cells MSCs hMSCs of integrin is that model cell is checked the transfection effect of five kinds of materials to stem cell, referring to Figure of description 4.Result shows, at N/P than when being 2.5, green fluorescent protein intensity expressed after K4 load plasmid is the strongest, this result is surface also, and the material that has shown to modify the material of RGD and wrapped up gold nano grain is higher than not modifying the material of RGD and not wrapping up the material transfection efficiency of gold nano grain.
(5) hBMP-2 gene expression results:
HBMP-2 gene expression experiment is for the transfection ability to stem cell after Study of Support load hBMP-2pDNA, referring to Figure of description 5.Result shows, at N/P, than when being 2.5, than the cell of untransfected, the cell of transfection has obvious hBMP-2 expresses.Wherein, the gene expression concentration after K4 load is the highest, compares the difference all with significance with K0 with K1, and hBMP-2 expression of results is K4>K2>K3>K1Gre atT.GreaT.GTK0.This is consistent with above-mentioned luciferase reporter gene and reinforcement Green Fluorescent Protein Gene Expression result.
(6) the active result of ALP:
The active result of ALP is divided into osteoblastic ability for quantitative Study of Support load hBMP-2pDNA induction hMSCs, referring to Figure of description 6.Result shows: at N/P ratio, be 2.5 o'clock, ALP activity increases along with the prolongation of time, and the cell of transfection is than the cell of untransfected, and ALP expression obviously gets a promotion, and wherein the ALP expression of K4 is the highest, consistent with hBMP-2 gene expression results.
(7) osteocalcin secretion result:
Osteocalcin secretion result is divided into osteoblastic ability for quantitative research material as carrier loaded hBMP-2pDNA induction hMSCs, referring to Figure of description 7.Result shows: osteocalcin secretion amount is in the 14th day, secrete less, prolongation along with the time, in the 21st day, osteocalcin secretion amount significantly increases, and wherein osteocalcin secretion amount is: K4>K2>K3>K1Gre atT.GreaT.GTK0.This result has embodied outstanding Targeting Performance and the efficient gene transfection performance of K4 material equally.
(8) calcium ion deposition result:
Calcium ion deposition result is divided into osteoblastic ability for quantitative research material as carrier loaded hBMP-2pDNA induction hMSCs, referring to Figure of description 8.Result shows: the calcium ion content in transfectional cell increases along with the prolongation of time, in 14 days and 21 days, the cell of K4 material processed all has the highest calcium ion concentration, and this has embodied equally surface-functionalized being modified with of dendrimer and has been beneficial to gene transfection.
(9) Feng Kusa coloration result:
Feng Kusa coloration result is used for research material qualitatively and is divided into osteoblastic ability as carrier loaded hBMP-2pDNA induction hMSCs.Referring to Figure of description 9.Result shows: the cell after transfection, obvious change has all occurred for their color and form, illustrates that stem cell is successfully divided into osteoblast.And cell after K4 transfection, the density that osteoblast is colored is maximum.This and ALP are active, osteocalcin secretion, and calcium ion deposition result is all consistent, has embodied K4 material to the specific target tropism of hMSCs and high-efficiency transfection efficiency.
Beneficial effect:
(1) nano-complex of the functionalization Polyamidoamine Dendrimers that prepared by the present invention has good gene transfection effect, for the gene transfection of mesenchymal stem cells MSCs lays the foundation;
(2) functionalization Polyamidoamine Dendrimers and the nano-complex thereof that prepared by the present invention have targeting to mesenchymal stem cells MSCs, can be used for the targeted of gene;
(3) to have preparation process simple for material of the present invention, and experiment condition is gentle, easy to operate, has good application prospect in the repair and reconstruction of osseous tissue.
Accompanying drawing explanation
Fig. 1 is hydrodynamics particle diameter (a) and electromotive force (b) figure of carrier/hBMP-2pDNA complex of preparing of the present invention;
Fig. 2 is cytotoxicity result figure (a) carrier of the material prepared of the present invention to hMSCs; (b) carrier/hBMP-2pDNA complex;
Fig. 3 is carrier/hBMP-2pDNA complex luciferase gene transfection efficiency figure to hMSCs under different N/P prepared by the present invention;
Fig. 4 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, the shows fluorescent microscopy images to the Transfection of Enhanced Green Fluorescent of hMSCs;
Fig. 5 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, the hBMP-2 efficiency gene transfection figure to hMSCs;
Fig. 6 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, the active result figure of ALP;
Fig. 7 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, Bone Gla protein deposition results figure;
Fig. 8 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, calcium ion secretion result figure;
Fig. 9 be carrier/hBMP-2pDNA complex of preparing of the present invention when N/P=2.5, Feng Kusa coloration result figure;
Figure 10 is the preparation principle schematic diagram of functionalization Polyamidoamine Dendrimers and nano-complex thereof.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Take NH
2-PEG-COOH11.58mg, is dissolved in 5mL DMSO.Dropwise drip while stirring and be dissolved in the 6-MAL that the dry weight of 1mLDMSO solution is 1.785mg, reaction 8h, obtains MAL-PEG-COOH solution.By the DMSO solution (4mg/mL) of 1mLRGD-SH, dropwise join in above-mentioned MAL-PEG-COOH solution, reaction 12h, obtains RGD-PEG-COOH solution.Take 4.0mg EDC and 2.4mg NHS, be dissolved in respectively in 1mL DMSO, successively EDC solution and NHS solution are dropwise dripped in RGD-PEG-COOH solution to stirring reaction 3h.Take the 5th PAMAM dendrimer (G5.NH
2) 15.06mg, be dissolved in 5mL DMSO.The RGD-PEG-COOH solution that above-mentioned activation is good dropwise joins G5.NH
2in solution, room temperature lower magnetic force stirs, and reaction 3d, obtains G5.NH
2-(PEG-RGD)
10.Weighing m PEG-COOH11.58mg, is dissolved in 5mL DMSO.Take 2.0mg EDC and 1.2mg NHS, be dissolved in respectively in 1mL DMSO, successively EDC solution and NHS solution are dropwise added drop-wise in mPEG-COOH solution to stirring reaction 3h.The mPEG-COOH solution that above-mentioned middle activation is good dropwise joins G5.NH
2-(PEG-RGD)
10in solution, room temperature lower magnetic force stirs, and reaction 3d, obtains G5.NH
2-(PEG-RGD)
10-mPEG
10solution.By G5.NH
2-(PEG-RGD)
10-mPEG
10be transferred to molecular cut off and be in 14000 bag filter, in distilled water, dialyse three days (2L * 3), then carry out lyophilization processing, finally by the dry G5.NH obtaining
2-(PEG-RGD)
10-mPEG
10be dissolved in 5mL water, more dropwise add 198.67 μ L HAuCl
4aqueous solution (30mg/mL) mix and blend 30min, then adds the NaBH of the 10mg/mL of 27.4 μ L fast
4solution, reaction 3h, obtains { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10solution.After reaction finishes, by product { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10to be transferred to molecular cut off be in 14000 bag filter, in distilled water, dialyse three days (2L * 3).Then carry out lyophilization processing, obtain final desciccate { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10.
By G5.NH
2, comparative example 1, comparative example 2, the G5.NH that the method for comparative example 3 and embodiment 1 prepares
2-mPEG
20, { (Au
0)
25-G5.NH
2-mPEG
20, G5.NH
2-(PEG-RGD)
10-mPEG
10, { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10at different N/P, than (1:1,2.5:1,5:1) under condition, by electrostatic interaction, form carrier/hBMP-2pDNA complex with 5 μ g hBMP-2pDNA respectively, and make final volume be fixed on 100 μ L, under room temperature, hatch 20min, then add 1mL PBS.By Ma Erwen laser particle analyzer (Malvern, Μ K, 633nm laser), its hydrodynamics particle diameter and surface potential are characterized, result is as shown in Figure of description 1.Result shows, along with the increase of N/P ratio, the hydrodynamics particle diameter of complex has reduced, nearly all below 200nm; And the surface potential of most of complex increases along with the increase of N/P ratio, but also all in 20mV left and right.These illustrative material are conducive to absorption and the endocytosis of cell, are also just conducive to the transmission of carrier to genes of interest.
Embodiment 3
To have
v 3the mesenchymal stem cells MSCs hMSCs of relative association of integrins expression is that model cell is checked G5.NH
2, comparative example 1, comparative example 2, the cytotoxicity of the material of comparative example 3 and embodiment 1 preparation.With 1.5 * 10
4the density in/hole in 96 orifice plate, is incubated at hMSCs kind to add 100U/mL penicillin, in 100 μ L α-MEM culture fluid of 100U/mL streptomycin and 10%FBS, 37 ℃, under 5% gas concentration lwevel, cultivates 24h.Then culture medium is changed into the G5.NH that contains that concentration is respectively 0nM, 50nM, 100nM, 500nM, 1000nM, 2000nM and 3000nM
2, G5.NH
2-mPEG
20, { (Au
0)
25-G5.NH
2-mPEG
20, G5.NH
2-(PEG-RGD)
10-mPEG
10{ (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10cell culture medium and co-culture of cells 24h, add subsequently 20 μ LMTT(5.0mg/mL) solution, continue to cultivate 4h.Remove the culture fluid in hole, every hole adds 150 μ L DMSO, shaking table vibration 30min, and with multi-functional microplate reader test light absorption value, test wavelength 570nm, reference wavelength 630nm, result is as shown in Figure of description 2a.Subsequently, under above-mentioned same experiment condition of culture, at material, change G5.NH into
2, G5.NH
2-mPEG
20, { (Au
0)
25-G5.NH
2-mPEG
20, G5.NH
2-(PEG-RGD)
10-mPEG
10{ (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10with the complex of 1 μ g pDNA, then measure light absorption value, result is as shown in Figure of description 2b.Result shows, along with the increase of material concentration, cell survival rate declines, but the toxicity of four kinds of materials is all than G5.NH
2toxicity low.Illustrate that the modification of PEG and RGD has all reduced the number of amino groups on dendrimer surface, thereby reduced the cytotoxicity of material.Meanwhile, under identical concentration conditions, the toxicity of complex will decrease than the toxicity of independent carrier.PDNA is when combining by electrostatic interaction with carrier in this explanation, and the part that has neutralized dendrimer surface is amino, thereby has reduced the cytotoxicity of material.
Embodiment 4
To carry the plasmid of luciferase reporter gene and to have
v 3the mesenchymal stem cells MSCs hMSCs of relative association of integrins expression is that model cell is checked G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is the efficiency gene transfection to stem cell as carrier.With 1.5 * 10
4the density in/hole in 24 orifice plate, is incubated at hMSCs kind to add 100U/mL penicillin, in 1mL α-MEM culture fluid of 100U/mL streptomycin and 10%FBS, 37 ℃, under 5% gas concentration lwevel, cultivates 24h.According to N/P ratio=1:1,2.5:1 and 5:1, prepare carrier/hBMP-2pDNA complex subsequently, and wherein the amount of the hBMP-2pDNA in each hole is 1 μ L.Culture medium is changed into the α-MEM culture medium that does not contain FBS, then add above-mentioned complex and co-culture of cells 4h.Then change the fresh α-MEM culture medium containing 10%FBS, continue to cultivate 24h.By lysis, and detect uciferase activity by the luciferase assay of Promega company, the results are shown in Figure of description 3.From figure, can significantly find out, material is when N/P ratio is 2.5:1, uciferase activity is the highest, the uciferase activity of five kinds of materials is respectively: K4>K2>K3>K1Gre atT.GreaT.GTK0, and K4 compares with front four kinds of materials and has significant difference, the modification of dendrimer surface PEG and RGD is described, and the parcel of inner gold nano grain all significance raising the efficiency gene transfection of material.
Embodiment 4
To carry, strengthen the plasmid of green fluorescence protein gene and to have
v 3the mesenchymal stem cells MSCs hMSCs of relative association of integrins expression is that model cell is checked G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is the efficiency gene transfection to stem cell as carrier.The cultural method of cell and the preparation method of complex as described in Example 3, have adopted and have carried the plasmid of strengthening green fluorescence protein gene, at N/P ratio, are cell to be carried out to transfection at 2.5 o'clock.After transfection 24h, adopt fluorescence microscope result, the results are shown in Figure of description 4.As can be seen from the figure, the expression intensity of the reinforcement fluorescence protein gene of K4 is the strongest, and K0 is the most weak, and this result is consistent with luciferase reporter gene expression of results.
To carry the plasmid of hBMP-2 gene and to have
v 3the mesenchymal stem cells MSCs hMSCs of relative association of integrins expression is that model cell is checked G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is the efficiency gene transfection to stem cell as carrier.The cultural method of cell and the preparation method of complex as described in Example 3, have adopted the plasmid that carries hBMP-2 gene, at N/P ratio, are cell to be carried out to transfection at 2.5 o'clock.After transfection 48h, culture medium is changed into the α-MEM culture medium that does not contain FBS, then cultivate 24h.Collect subsequently 100 μ L supernatant, with hBMP-2ELISA test kit, detect the expression of hBMP-2 gene, the results are shown in Figure of description 5.Test result shows: at N/P ratio, be 2.5 o'clock, K4 material tests goes out the expression of the strongest hBMP-2 gene, than K0 and K1, all there is significant difference, this may be owing to the Targeting Performance of rgd peptide, and the facilitation of the gold nano grain rigidity stereoscopic three-dimensional structure of maintaining to gene transfection.
With the active result of ALP, check G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is divided into osteoblastic ability as carrier loaded hBMP-2pDNA induction hMSCs.The cultural method of cell and the preparation method of complex as described in Example 3, have adopted the plasmid that carries hBMP-2 gene, at N/P ratio, are cell to be carried out to transfection at 2.5 o'clock.Transfection the 7th day, in the time of the 14th day and the 21st day, collect the cell after transfection, by lysis, adopt cell colorimetric analysis method to detect the ALP activity of cell.Known by Figure of description 6, ALP activity increases along with the prolongation of time, but in the 14th day to the 21st day, gathering way of ALP activity decreases.This may be due to along with differentiation of stem cells becomes osteoblast, and ALP expression rises thereupon, but forms gradually along with osteoblastic, and ALP expression decreases.From figure, also can find out, at three time points, the cell of transfection is than the cell of untransfected, and ALP expression obviously gets a promotion, and wherein the ALP expression of K4 is the highest, consistent with hBMP-2 gene expression results.
With osteocalcin secretion amount, check G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is divided into osteoblastic ability as carrier loaded hBMP-2pDNA induction hMSCs.Bone Gla protein is by cell, produced and secreted when osteoblast forms, therefore can be used as a kind of critical index that osteoblast forms.The cultural method of cell and the preparation method of complex as described in Example 3, have adopted the plasmid that carries hBMP-2 gene, at N/P ratio, are cell to be carried out to transfection at 2.5 o'clock.Because osteoblast formed in the later stage, supernatant when being collected in transfection the 14th day and the 21st day, by people's Bone Gla protein enzyme linked immunological kit detection osteocalcin secretion, the results are shown in Figure of description 7.From figure, can learn, in 14 days, osteocalcin secretion is less, and be described this period, and stem cell is in the osteoblast initial stage, still in atomization.And in 21 days, osteocalcin secretion measures the raising of significance, illustrate that stem cell has started to be divided into osteoblast, wherein osteocalcin secretion amount is: K4>K2>K3>K1Gre atT.GreaT.GTK0.This result has embodied the outstanding gene delivery performance of K4 material equally.
With calcium ion deposition result, check G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is divided into osteoblastic ability as carrier loaded hBMP-2pDNA induction hMSCs.Calcium ion deposition is also one of important indicator of a kind of osteoblast differentiation, and the cultural method of cell and the preparation method of complex as described in Example 3, have adopted the plasmid that carries hBMP-2 gene, at N/P ratio, is cell to be carried out to transfection at 2.5 o'clock.According to the result of above-mentioned osteocalcin secretion, we collect the cell of 14 days and 21 days equally, by lysis, by calcium ion, measure the content that test kit detects calcium ion in cell, the results are shown in Figure of description 8.Test result shows: along with the time is surveyed prolongation, the calcium ion content in transfectional cell significantly increases.In 14 days and 21 days, the cell of K4 material processed all has the highest calcium ion concentration, and this has verified the modification of PEG and RGD equally, and the parcel of gold nano grain has improved material transfection performance greatly, embodies efficient efficiency gene transfection.
Embodiment 8
With Feng Kusa coloration result, check qualitatively G5.NH
2, comparative example 1, comparative example 2, the material of comparative example 3 and embodiment 1 preparation is divided into osteoblastic ability as carrier loaded hBMP-2pDNA induction hMSCs.Feng Kusa dyeing is can lead to hyperchromatic method the substrate of mineralising is dyed to black, thereby verifies qualitatively osteoblastic formation.The cultural method of cell and the preparation method of complex as described in Example 3, have adopted the plasmid that carries hBMP-2 gene, at N/P ratio, are cell to be carried out to transfection at 2.5 o'clock.According to the result of embodiment 5,6,7, stem cell was successfully divided into osteoblast in 21 days, and therefore, we carry out Feng Kusa dyeing to the cell of 21 days, the results are shown in Figure of description 9.Test result shows: after dyed, for the cell of untransfected, from color and the form of cell, obviously find out that cell remains stem cell; And for the cell of transfection, all there is the change of significance in the color of cell and form, illustrates that stem cell is successfully divided into osteoblast.For the cell after K4 transfection, the density that cell is dyed to black is maximum, this result and ALP are active, osteocalcin secretion, calcium ion deposition result is all consistent, illustrates that the PEG on dendrimer modifies the transfection efficiency that has improved material, and RGD modifies and improved the Targeting Performance of material to stem cell, the parcel of gold nano grain has improved the solid rigid structure of material, has promoted the endocytosis of cell to material.
Comparative example 1
Weighing m PEG-COOH23.16mg, is dissolved in 5mL DMSO.Take 4.0mg EDC and 2.4mg NHS, be dissolved in respectively in 1mL DMSO, successively EDC solution and NHS solution are dropwise added drop-wise in mPEG-COOH solution to stirring reaction 3h.Take the 5th PAMAM dendrimer (G5.NH
2) 15.06mg, be dissolved in 5mL DMSO.The mPEG-COOH solution that above-mentioned middle activation is good dropwise joins G5.NH
2in solution, room temperature lower magnetic force stirs, and reaction 3d, obtains sample G5.NH
2-mPEG
20solution.After reaction finishes, by product G5.NH
2-mPEG
20be transferred to molecular cut off and be in 14000 bag filter, in distilled water, dialyse three days (2L * 3).Then carry out lyophilization processing, obtain dry G5.NH
2-mPEG
20product.
Comparative example 2
By the G5.NH obtaining in comparative example 1
2-mPEG
20be dissolved in 5mL water, more dropwise add 198.67 μ L HAuCl
4aqueous solution (30mg/mL) mix and blend 30min, then adds the NaBH of the 10mg/mL of 27.4 μ L fast
4solution, reaction 3h, obtains sample { (Au
0)
25-G5.NH
2-mPEG
20solution.By product { (Au
0)
25-G5.NH
2-mPEG
20to be transferred to molecular cut off be in 14000 bag filter, in distilled water, dialyse three days (2L * 3).Then carry out lyophilization processing, the { (Au that obtains being dried
0)
25-G5.NH
2-mPEG
20product.
Comparative example 3
Take NH
2-PEG-COOH11.58mg, is dissolved in 5mL DMSO.Dropwise drip while stirring and be dissolved in the 6-MAL that the dry weight of 1mLDMSO solution is 1.785mg, reaction 8h, obtains MAL-PEG-COOH solution.By the DMSO solution (4mg/mL) of 1mLRGD-SH, dropwise join in above-mentioned MAL-PEG-COOH solution, reaction 12h, obtains RGD-PEG-COOH solution.Take 4.0mg EDC and 2.4mg NHS, be dissolved in respectively in 1mL DMSO, successively EDC solution and NHS solution are dropwise dripped in RGD-PEG-COOH solution to stirring reaction 3h.Take the 5th PAMAM dendrimer (G5.NH
2) 15.06mg, be dissolved in 5mL DMSO.The RGD-PEG-COOH solution that above-mentioned activation is good dropwise joins G5.NH
2in solution, room temperature lower magnetic force stirs, and reaction 3d, obtains G5.NH
2-(PEG-RGD)
10.Weighing m PEG-COOH11.58mg, is dissolved in 5mL DMSO.Take 2.0mg EDC and 1.2mg NHS, be dissolved in respectively in 1mL DMSO, successively EDC solution and NHS solution are dropwise added drop-wise in mPEG-COOH solution to stirring reaction 3h.The mPEG-COOH solution that above-mentioned middle activation is good dropwise joins G5.NH
2-(PEG-RGD)
10in solution, room temperature lower magnetic force stirs, and reaction 3d, obtains G5.NH
2-(PEG-RGD)
10-mPEG
10solution.After reaction finishes, by product G5.NH
2-(PEG-RGD)
10-mPEG
10be transferred to molecular cut off and be in 14000 bag filter, in distilled water, dialyse three days (2L * 3).Then carry out lyophilization processing, obtain dry G5.NH
2-(PEG-RGD)
10-mPEG
10product.
Claims (7)
1. an application for the nano-complex of functionalization Polyamidoamine Dendrimers, is characterized in that: the nano-complex of described functionalization Polyamidoamine Dendrimers is divided into osteoblastic carrier as load hBMP-2pDNA induction hMSCs;
The nano-complex of described functionalization Polyamidoamine Dendrimers is specially: { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10; Its preparation method is as follows:
(1) by NH
2-PEG-COOH solution reacts 6-8h with 6-MAL solution stirring, obtain MAL-PEG-COOH solution, then add RGD-SH solution, stirring reaction 10-12h, obtain RGD-PEG-COOH solution, with activating by EDC and NHS solution, then add in the 5th PAMAM dendrimer solution, reaction 12-30h, obtains G5.NH
2-(PEG-RGD)
10solution, then joins above-mentioned G5.NH by the mPEG-COOH solution through EDCHCl and NHS activation
2-(PEG-RGD)
10in solution, reaction 12-30h, obtains G5.NH
2-(PEG-RGD)
10-mPEG
10solution;
(2) at above-mentioned G5.NH
2-(PEG-RGD)
10-mPEG
10in, add HAuCl
4solution, stirring reaction 20-30min, then add NaBH
4solution, stirs 2-4h, obtains { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10solution;
(3) solution of step (2) gained is dialysed, lyophilization processes, the { (Au that obtains being dried
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10.
2. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 1, it is characterized in that, the described nano-complex of functionalization Polyamidoamine Dendrimers and the N/P of hBMP-2pDNA be than being 1:1～5:1, and described N/P is than being phosphate group mol ratio on the primary amino radical of dendrimer and pDNA skeleton.
3. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 2, it is characterized in that, the described nano-complex of functionalization Polyamidoamine Dendrimers and the N/P of hBMP-2pDNA are than being 1:1,2.5:1 or 5:1.
4. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 1, is characterized in that, the method that the nano-complex of functionalization Polyamidoamine Dendrimers and hBMP-2pDNA are compound is:
By { (Au
0)
25-G5.NH
2-(PEG-RGD)
10-mPEG
10with sterilized water dilution, sterilized water dilution plasmid then, then will after the two mix homogeneously, hatch 30min in 37 ℃.
5. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 1, is characterized in that, NH in described step (1)
2the concentration of-PEG-COOH solution, 6-MAL solution, RGD-SH solution, mPEG-COOH solution is 5.8mmol/mL; The concentration of the 5th PAMAM dendrimer solution is 0.58mmol/mL; Solvent is DMSO.
6. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 1, is characterized in that, HAuCl in described step (2)
4the concentration of solution is 30mg/mL, NaBH
4the concentration of solution is 10mg/mL.
7. the application of the nano-complex of a kind of functionalization Polyamidoamine Dendrimers according to claim 1, it is characterized in that, in described step (3), dialysis is the 2-3d that dialyses with deionized water, every day 3 times, each 2L deionized water, wherein bag filter molecular cut off is 14000.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004196A (en) * | 2014-05-04 | 2014-08-27 | 健雄职业技术学院 | Preparation method and application of degradable hyperbranched polyamidoamine |
| CN106046387A (en) * | 2015-04-15 | 2016-10-26 | 韩冰 | Novel high-efficiency gene vector, and preparation and application thereof |
| CN106620701A (en) * | 2016-12-30 | 2017-05-10 | 东华大学 | Preparation method of G5-MoS2/Bcl-2 siRNA compound |
| CN106754673A (en) * | 2016-12-09 | 2017-05-31 | 北京雨泽瑞清生物科技有限公司 | The method of modifying and mescenchymal stem cell at a kind of culture medium bottom separate amplification method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235084A (en) * | 2007-01-31 | 2008-08-06 | 浙江工业大学 | Method for preparing bone morphogenic protein BMP-2 mature peptide |
| CN102883773A (en) * | 2009-09-30 | 2013-01-16 | 麻省理工学院 | Phototriggered nanoparticles for cell and tissue targeting |
| CN103435815A (en) * | 2013-07-11 | 2013-12-11 | 东华大学 | Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection |
-
2013
- 2013-12-18 CN CN201310699637.XA patent/CN103623417B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235084A (en) * | 2007-01-31 | 2008-08-06 | 浙江工业大学 | Method for preparing bone morphogenic protein BMP-2 mature peptide |
| CN102883773A (en) * | 2009-09-30 | 2013-01-16 | 麻省理工学院 | Phototriggered nanoparticles for cell and tissue targeting |
| CN103435815A (en) * | 2013-07-11 | 2013-12-11 | 东华大学 | Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004196A (en) * | 2014-05-04 | 2014-08-27 | 健雄职业技术学院 | Preparation method and application of degradable hyperbranched polyamidoamine |
| CN104004196B (en) * | 2014-05-04 | 2016-08-31 | 健雄职业技术学院 | A kind of preparation method and applications of degradable over-branched polyamidoamine |
| CN106046387A (en) * | 2015-04-15 | 2016-10-26 | 韩冰 | Novel high-efficiency gene vector, and preparation and application thereof |
| CN106754673A (en) * | 2016-12-09 | 2017-05-31 | 北京雨泽瑞清生物科技有限公司 | The method of modifying and mescenchymal stem cell at a kind of culture medium bottom separate amplification method |
| CN106620701A (en) * | 2016-12-30 | 2017-05-10 | 东华大学 | Preparation method of G5-MoS2/Bcl-2 siRNA compound |
| CN108743962A (en) * | 2018-06-19 | 2018-11-06 | 东华大学 | A kind of preparation method of double load medicine targeted nano platforms of the dendrimer based on fluorescent carbon point modification |
| CN108743962B (en) * | 2018-06-19 | 2021-07-02 | 东华大学 | Preparation method of double-drug-loading targeting nano platform based on fluorescent carbon dot modified dendrimer |
| CN113456674A (en) * | 2021-07-06 | 2021-10-01 | 中山大学附属第三医院（中山大学肝脏病医院） | Stem cell-based nano-drug and preparation method and application thereof |
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