CN104195176A - Supramolecular hybrid peptide dendric macromolecule self-assembly and preparation method and applications thereof - Google Patents
Supramolecular hybrid peptide dendric macromolecule self-assembly and preparation method and applications thereof Download PDFInfo
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- CN104195176A CN104195176A CN201410441436.4A CN201410441436A CN104195176A CN 104195176 A CN104195176 A CN 104195176A CN 201410441436 A CN201410441436 A CN 201410441436A CN 104195176 A CN104195176 A CN 104195176A
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Abstract
The invention discloses a supramolecular hybrid peptide dendric macromolecule self-assembly and a preparation method and applications thereof. The supramolecular hybrid peptide dendric macromolecule self-assembly comprises an inorganic nanoparticle core and peptide dendritic macromolecules, wherein the peptide dendritic macromolecules are assembled at the periphery of inorganic nanoparticles by coordination interaction. Through the introduction of the coordination interaction, the peptide dendritic macromolecules are assembled on the surfaces of the inorganic nanoparticles. The coordination interaction is a relatively strong weak interaction, and an obtained supramolecular hybrid peptide dendric macromolecule self-assembly is stable in structure and good in dispersibility, and enriches the self-assembly strategy of dendritic macromolecules. By using the supramolecular self-assembly strategy, the spontaneous, rapid, green and efficient function integration of low-algebra dendritic macromolecules is realized, so that the highly branched structure of dendritic macromolecules is effectively amplified, and the function effect of the supramolecular hybrid peptide dendric macromolecule self-assembly reaches and is even higher than the function effect of classic high-algebra dendritic macromolecules.
Description
Technical field
The invention belongs to technical field of biological material, relate to a kind of supramolecule hydridization peptide class dendrimer self-assembly.
Technical background
Peptide class dendrimer is due to its good biocompatibility and peripheral have the abundant features such as functional group, at biomedical sector, has a wide range of applications.In its numerous application direction, the quantity of the peripheral functional group of peptide class dendrimer is all one of key factor affecting its effect conventionally, in order to obtain more peripheral functional group, common way is the algebraically that increases peptide class dendrimer, yet, increase peptide class dendrimer algebraically production cost and manufacture difficulty all can increase greatly and the toxicity of material also can increase, be unfavorable for that it applies.In actual applications peptide class dendrimer is made to spherical molecule, can increase to a certain extent its peripheral number of functional groups, yet, even in spherical peptide class dendrimer, because its kernel is to be also connected with peripheral amino acid by organo-functional group, and the quantity of the functional group of its kernel is extremely limited, therefore, it is also limited to increasing the effect of peripheral functional group.
Summary of the invention
For the problems referred to above, the present invention has carried out a large amount of research and has found: along with the continuous research and development of people to supramolecule self-assembly correlation technique, supramolecule self-assembly has become the important channel that builds Multifucntional nano material, yet peptide class dendrimer is carried out to supramolecule self-assembly, and particularly peptide class dendrimer and inorganic nano-particle to be carried out to the correlative study of autonomous dress also very rare.On the other hand, inorganic nano-particle, as: due to its distinctive nano functional characteristic, also increasingly extensive in the application of biomedical sector, yet, inorganic nano-particle and combination is also very rare with the research of the biomaterial of acquisition multifunction.Therefore, specialized designs of the present invention a kind of supramolecule self-assembly strategy peptide class dendrimer and inorganic nano-particle are ideally combined, obtained and there is multifunctional supramolecular hydridization peptide class dendrimer self-assembly and use the peptide class dendrimer of low algebraically can obtain the self-assembly that periphery has a large amount of functional groups by self-assembly, can meet or exceed the effect of the peptide class dendrimer of high algebraically.Meanwhile, the function of dendrimer assembly in the past that supramolecule hydridization self-assembly strategy is abundant and perfect; When realizing bioactive molecules (as gene, medicine) control release, can utilize inorganic kernel to carry out imaging and spike.
The present invention is achieved through the following technical solutions:
A peptide class dendrimer for functionalization, has core element and fan-shaped peripheral skeleton, has and can produce with inorganic nano-particle functional group or its precursor compound of coordination in its core element.
As optional mode, in the peptide class dendrimer of above-mentioned functions, described can with inorganic nano-particle produce the functional group of coordination or precursor compound be sulfydryl, disulfide linkage, phenolic hydroxyl group, a plurality of carboxyl or hydroxyl, can be corresponding with inorganic nano-particle sub-surface functional group produce the group of host-guest complexation (as the coordination of cyclodextrin-diamantane).Described sulfydryl, phenolic hydroxyl group, a plurality of carboxyl or hydroxyl (described disulfide linkage through reduction after) thus lone-pair electron can be provided and inorganic nano-particle in atom unoccupied orbital (as the unoccupied orbital of atoms metal) there is coordination, thereby realize peptide class dendrimer, be combined with the coordination of inorganic nano-particle, reach multifunction.
As optional mode, in the peptide class dendrimer of above-mentioned functions, described fan-shaped peripheral skeleton be take amino acid as repeating unit.Further, described amino acid repeating unit is at least one in L-glutamic acid, Methionin, Serine, Threonine, tyrosine, aspartic acid, arginine, Histidine.
As optional mode, in the peptide class dendrimer of above-mentioned functions, the algebraically of described peptide class dendrimer is a generation, two generations or three generations.Be preferably for two generations.
As optional mode, in the peptide class dendrimer of above-mentioned functions, described peptide class dendrimer periphery is modified with positively charged group.Make peptide class dendrimer periphery can effectively realize compression and the loading of DNA with positive charge, while electric charge repels each other and can avoid assembling, thereby guarantees dispersiveness preferably, is convenient to its application in genophore.
As optional mode, in the peptide class dendrimer of above-mentioned functions, the amino acid of described peptide class dendrimer periphery is Methionin, arginine, at least one in Histidine.
As optional mode, in the peptide class dendrimer of above-mentioned functions, the structural formula of described peptide class dendrimer is as follows:
Wherein, R1 represents the core element of fan-shaped molecule and the site part of inorganic nano-particle effect, and m represents the functionality of core element, and the functionality of core element is 1 or 2; K is peptide class dendrimer amino acid backbone, and R is positive charge amino acid for the peripheral modified with functional group of peptide class dendrimer, and K and R can be identical, also can difference, G1.0 and G2.0 represent respectively a generation and two generation peptide class dendrimer.
As optional mode, in the peptide class dendrimer of above-mentioned functions, in described core element, R1 is-S-S-or HS-.The group containing in Rm in described core element can for-OH or-COOH.R is Methionin, arginine, at least one in Histidine.
The present invention also provides a kind of supramolecule hydridization peptide class dendrimer self-assembly, comprises inorganic nano-particle kernel and peptide class dendrimer, and it is peripheral that described peptide class dendrimer is assembled in described inorganic nano-particle by coordination.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, the mode of described coordination is a kind of in the coordination of metal-sulfydryl, chelating coordination, host-guest complexation.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, described inorganic nano-particle is metal nanoparticle or inorganic non-metallic nanoparticle, is specially a kind of in quantum dot, SPIO, golden nanometer particle, rare earth nanometer particle.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, peptide class dendrimer periphery is positively charged.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, described peptide class dendrimer is low algebraically peptide class dendrimer, and its algebraically is 1 generation, 2 generations or 3 generations.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, the peptide class dendrimer that described peptide class dendrimer is any one functionalization of the present invention.
As optional mode, in above-mentioned supramolecule hydridization peptide class dendrimer self-assembly, described peptide class dendrimer is the fan-shaped molecule of core and peripheral difunctionalization.
The present invention also provides a kind of construction process of described supramolecule hydridization peptide class dendrimer self-assembly, comprises the following steps:
(1) prepare peptide class dendrimer;
(2) to the core element of peptide class dendrimer and or inorganic nano-particle carry out coordination functionalization;
(3) the peptide class dendrimer and the inorganic nano-particle that in step (2), make are fully mixed, make both self-assemblies under the driving of coordination obtain supramolecule hydridization peptide class dendrimer self-assembly.
As optional mode, the preparation of peptide class dendrimer described in step (1) can adopt the method for dispersing or convergence method or disperse-restrain the method combining.
As optional mode, described in step (1), the preparation method of peptide class dendrimer is specially:
A) amino acid is carried out to protective group: according to the difference of the core element surface functional group of peptide class dendrimer to be prepared, amino acid is protected, if core element surface functional group is that amino is protected amino acid whose amino, if core element surface functional group is that hydroxyl or carboxyl are protected amino acid whose carboxyl;
B) prepare generation peptide class dendrimer: (functionality is n to take in proportion core element, n=1 or 2), the amino acid (1.5n equivalent), condensing agent (1.5n equivalent), catalyzer (1.5n equivalent) and the organic bases (4n equivalent) that contain blocking group, at 0 ℃, under nitrogen protection condition, add anhydrous solvent to carry out dehydration condensation; Then at room temperature reaction, after reaction finishes, gained solution is through washing, and dry, concentrating under reduced pressure, obtains the first-generation peptide class dendrimer with blocking group with column chromatography for separation;
C) deprotection: accurately take first-generation peptide class dendrimer, deprotecting regent (20n equivalent), adds dissolution with solvents, and room temperature reaction is 4 hours under nitrogen protection, concentrating under reduced pressure, obtains first-generation peptide class dendrimer by extracting or precipitating;
D) prepare two generation dendrimer: take in proportion first-generation peptide class dendrimer (functionality is 2n), containing amino acid (3n equivalent), condensing agent (3n equivalent), catalyzer (3n equivalent) and the organic bases (8n equivalent) of blocking group, at 0 ℃, under nitrogen protection condition, add solvent to carry out dehydration condensation; Then at room temperature reaction, after reaction finishes, gained solution is through washing, and dry, concentrating under reduced pressure, obtains the s-generation peptide class dendrimer with blocking group by column chromatography for separation;
Repeat above deprotection and step of condensation, can obtain the 3rd, the 4th generation dendrimer.
As optional mode, described step (2) is specially, in step (1), choose and contain and can produce the functional group of coordination or the core element of its precursor compound with inorganic nano-particle, as contain sulfydryl, disulfide linkage, phenolic hydroxyl group, a plurality of carboxyls or hydroxyl, the core element of the group of functional group generation host-guest complexation that can be corresponding with inorganic nano-particle sub-surface etc., and (avoid corresponding function group to react with amino acid when preparing peptide class dendrimer) where necessary corresponding group is protected, then carry out corresponding deprotection processing making functionalization peptide class dendrimer of the present invention, or corresponding prerequisite compound is carried out to subsequent disposal make it become coordination functional group group (as to reduce acquisition sulfydryl containing the precursor compound of disulfide linkage), if select host-guest complexation mode, also need to be at the surface grafting of described inorganic nano-particle corresponding group can with the core seat of described functionalization peptide class dendrimer on corresponding group produce host-guest complexation.
As optional mode, described step (2) is specially, by contain sulfydryl, disulfide linkage, phenolic hydroxyl group, a plurality of carboxyl or hydroxyl, can be corresponding with inorganic nano-particle sub-surface the functional group core element that produces the group of host-guest complexation be connected to and take on the dendrimer that amino acid is kernel.
As optional mode, the reduction of the above-mentioned precursor compound containing disulfide linkage is specially, and the functionalization peptide class dendrimer that core element is contained to disulfide linkage, is dissolved in the mixed solvent of ethyl acetate and water, the NaBH of amount of substance such as adds
4, after stirring reaction 1 h, solution with water is diluted and is used chloroform extraction three times, merges organic phase, anhydrous MgSO
4dry, filter, revolve desolventizing, thick product uses column chromatography the peptide class dendrimer that obtains being reduced.
As optional mode, described step (3) is specially inorganic nano-particle is dispersed in organic phase, the salts solution that adds wherein the peptide class dendrimer of preparation in greatly excessive step (2), room temperature reaction 30 min, be warming up to 60 ℃ of reactions and spend the night and make to react completely, centrifugal, abandoning supernatant, repetitive scrubbing, the centrifugal supramolecule hydridization peptide class dendrimer that obtains.
As optional mode, described step (3) is specially the peptide class dendrimer part of getting preparation in step (2) and is dissolved in deionized water, add organic bases to regulate pH value weakly alkaline, add the inorganic nano-particle that is dissolved in normal hexane, add again quantitative normal hexane, be placed in ultraviolet producer and fully stir, Coordinate self-assembly reaction occurs.When inorganic nano-particle, all transfer to water, show that reaction completes substantially.Water is transferred in dialysis tubing, removed the peptide class dendrimer not reacting, after freeze-drying, obtain supramolecule hydridization peptide class dendrimer assembly.
The invention provides a kind of application of described supramolecule hydridization peptide class dendrimer self-assembly, it is characterized in that, used as genophore or pharmaceutical carrier.
As optional mode, in above-mentioned application, DNA is diluted to finite concentration with deionized water standby, supramolecule hydridization peptide class dendrimer assembly of the present invention is diluted to standby concentration with deionized water, then by above-mentioned DNA solution and supramolecule hydridization peptide class dendrimer assembly solution short mix according to a certain percentage, and hatch for some time at ambient temperature, can prepare gene-carrier complexes.
Disclosed all features in this specification sheets, or the step in disclosed all methods or process, except mutually exclusive feature and/or step, all can combine by any way.
Beneficial effect of the present invention:
1. supramolecule hydridization peptide class dendrimer self-assembly of the present invention, the introducing by coordination is assembled into inorganic nano-particle sub-surface by peptide class dendrimer.Coordination is a kind of stronger weak interaction, and the supramolecule hydridization peptide class dendrimer assembly obtaining has stable structure, dispersed preferably, has enriched dendrimer self-assembly strategy.
2, utilize supramolecule self-assembly strategy to realize the spontaneous function quick, green high-efficient of low algebraically dendrimer integrated, effectively amplify the highly branched structure of dendrimer, meet or exceed the functional effect of classical high algebraically dendrimer.And optimized classical high algebraically dendrimer characteristic, solved two hang-ups of high algebraically dendrimer toxicity and high energy consumption, low algebraically self-assembly unit is easy to synthesize, structure is accurate, and the self-assembly of gained has good biocompatibility.
3. there is accurate molecular structure, abundant surface functional group.Peptide class dendrimer has good biocompatibility, has improved the biological safety of whole system, adopts echovirus albumen clothing shell, the imitative hair style design that opens of wearing film chemistry of peptides structure, has the structure of proteinoid easily by cell endocytic.
4. supramolecule hydridization peptide class dendrimer assembly periphery has a large amount of groups with positive charge, can effectively realize compression, load and the transmission of gene.
5. except the distinctive function of dendrimer, can also embody the function of inorganic nano-particle, further realize multifunction, for example, give the function of the class dendrimer spike of supramolecule hydridization peptide and imaging.
6. supramolecule hydridization peptide class dendrimer gene vector system of the present invention, provides a kind of method of universality to build supramolecule hydridization peptide class dendrimer system, can be applied to other biological medical field.
accompanying drawing explanation:
Fig. 1 is the infrared spectrogram of the peptide of supramolecule hydridization described in the embodiment of the present invention 7 class dendrimer.
Fig. 2 is the infrared spectrogram of the peptide of supramolecule hydridization described in the embodiment of the present invention 7 class dendrimer.
Fig. 3 is the thermogravimetric curve of the dendrimer of peptide class described in the embodiment of the present invention 7.
Fig. 4 is the thermogravimetric curve of the peptide of supramolecule hydridization described in the embodiment of the present invention 7 class dendrimer.
Fig. 5 is the size distribution figure (DLS) of sample described in the embodiment of the present invention 7.
Fig. 6 is the Zeta potential figure of the peptide of supramolecule hydridization described in the embodiment of the present invention 7 class dendrimer.
Fig. 7 is the size distribution figure (DLS) of the peptide of supramolecule hydridization described in the embodiment of the present invention 8 class dendrimer/DNA mixture.
Fig. 8 is the Zeta potential figure of the size distribution figure (DLS) of the peptide of supramolecule hydridization described in the embodiment of the present invention 8 class dendrimer/DNA mixture.
Fig. 9 is the transmission electron microscope TEM photo of the size distribution figure (DLS) of the peptide of supramolecule hydridization described in the embodiment of the present invention 8 class dendrimer/DNA mixture.
Figure 10 is the cytotoxicity detection figure of sample described in the embodiment of the present invention 9 to HepG2 cell.
Figure 11 is the cytotoxicity detection figure of sample described in the embodiment of the present invention 9 to MCF-7 cell.
Figure 12 is the cytotoxicity detection figure of sample described in the embodiment of the present invention 9 to HEK293 cell.
Figure 13 is the cell cycle figure under sample described in the embodiment of the present invention 9 and HepG2 co-culture of cells, and wherein A is blank group, and B is PEI group, and C is supramolecule hydridization peptide class dendrimer/DNA mixture group.
Figure 14 is the apoptosis quantitative statistics structure iron under sample described in the embodiment of the present invention 9 and HepG2 co-culture of cells.
Figure 15 is the uciferase activity quantitative statistics result figure of take in the embodiment of the present invention 9 in the HepG2 cell of different samples after carrier transfection luciferase gene.
Figure 16 is the experimental result picture of viable cell workstation described in the embodiment of the present invention 10, and wherein A shows enrichment process in born of the same parents, and B shows core transportation, all regions of C performance core transmittance process.
Figure 17 is the TEM photo obtaining in transmission electron microscope described in the embodiment of the present invention 10 (TEM) observation experiment.
Figure 18 is the quantitative result figure of transfection experiment in the peptide of supramolecule hydridization described in the embodiment of the present invention 11 class dendrimer/DNA composite body.
Figure 19 is the quantitative result figure of bio-imaging experiment in the peptide of supramolecule hydridization described in the embodiment of the present invention 12 class dendrimer/DNA composite body.
Figure 20 is the structural representation of supramolecule hydridization peptide class dendrimer self-assembly of the present invention.
embodiment:
Embodiment is by the following examples described in further detail foregoing of the present invention again.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.Do not departing from any modification of making within the spirit and principles in the present invention, and being equal to of making according to ordinary skill knowledge and customary means replace or improve, all should be included in protection scope of the present invention.Raw materials usedly in following examples all can buy from the market.
embodiment 1:the preparation of peptide class dendrimer
A) amino acid is carried out to protective group: according to the difference of the core element surface functional group of peptide class dendrimer to be prepared, amino acid is protected, if core element surface functional group is that amino is protected amino acid whose amino, if core element surface functional group is that hydroxyl or carboxyl are protected amino acid whose carboxyl;
B) prepare generation dendrimer: (functionality is n to take in proportion core element, n=1 or 2), the amino acid (1.5n equivalent), condensing agent (1.5n equivalent), catalyzer (1.5n equivalent) and the organic bases (4n equivalent) that contain blocking group, at 0 ℃, under nitrogen protection condition, add anhydrous solvent to carry out dehydration condensation; Then at room temperature reaction, after reaction finishes, gained solution is through washing, and dry, concentrating under reduced pressure, obtains with blocking group first-generation peptide class dendrimer by column chromatography for separation;
C) deprotection: accurately take first-generation peptide class dendrimer, deprotecting regent (20n equivalent), adds dissolution with solvents, under nitrogen protection, room temperature reaction is 4 hours, deprotection, concentrating under reduced pressure, by extracting or precipitating, obtain first-generation peptide class dendrimer;
D) prepare two generation dendrimer: take in proportion first-generation peptide class dendrimer (functionality is 2n), containing amino acid (3n equivalent), condensing agent (3n equivalent), catalyzer (3n equivalent) and the organic bases (8n equivalent) of blocking group, at 0 ℃, under nitrogen protection condition, add solvent to carry out dehydration condensation; Then at room temperature reaction, after reaction finishes, gained solution is through washing, and dry, concentrating under reduced pressure, obtains with blocking group s-generation peptide class dendrimer with column chromatography for separation;
Repeat above deprotection and step of condensation, can obtain the 3rd, the 4th generation dendrimer and the amino acid modified dendrimer of positive electricity.
Condensing agent described in the present embodiment, catalyzer, organic bases, solvent, can select various condensing agents, catalyzer, organic bases, the solvent for carboxyl and amino dehydrating condensation well known in the prior art.
Can directly select in the present embodiment Dopamine HCL, tea-polyphenol or other periphery contain sulfydryl, phenolic hydroxyl group, a plurality of carboxyl or hydroxyl, can be corresponding with inorganic nano-particle sub-surface functional group produce host-guest complexation the compound of group as core element, directly prepare the dendrimer of coordination functionalization.
embodiment 2:the concrete synthesis example (synthetic route is as Fig. 1) of peptide class dendrimer
1, core element is modified.
Lipoic acid derivatives (LA-NH
2) synthetic
Take the Thioctic Acid (LA) of 5.0 g; the quadrol of single tertbutyloxycarbonyl protection of 4.6 g; the I-hydroxybenzotriazole (HOBT) of the 1-ethyl of 6.9 g-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4.9 g joins in single neck bottle of 100mL with arm; vacuumize inflated with nitrogen.With syringe, add 40 mL heavily to steam methylene dichloride (DCM), ice bath adds the DIPEA (DIPEA) of 15.7 mL under stirring, and continues to stir the deicing bath of dropping back half an hour, reacts 24 h under room temperature condition.Except after desolventizing, add chloroform to dissolve, use successively saturated NaHCO
3, NaHSO
4, NaCl washing, use anhydrous MgSO
4after dry, filter, revolve and desolventize, column chromatography (eluent is ethyl acetate (EtOAc)/DCM=1:1) separation obtains yellow powder LA-NHBoc.
Accurately take the LA-NHBoc of 2.0 g in the single neck bottle with arm, vacuumize, inflated with nitrogen, adds 4 mL heavily to steam DCM with syringe, adds the trifluoroacetic acid (TFA) of 5 mL, room temperature reaction 4 h after material dissolution.TFA is revolved in decompression, with oil pump, drains, and adds anhydrous diethyl ether to stir and obtains yellow oil, is LA-NH
2.Product, without purification, is directly used in the next step.Product, without purification, is directly used in the next step.
2, two generation peptide class dendrimer (MeO-Lys (G2)-NH
2) synthetic
Take the methyl esters protection Methionin H-Lys-OMe of 1.0 g; the Boc-Lys of 6.5 g (Boc)-OH; the HOBT of the EDCI of 3.6 g and 2.5 g is in the flask with arm of 100 mL; vacuumize, inflated with nitrogen, adds the DCM of 30 mL; ice bath stirring and dissolving; the DIPEA that adds 8 mL, continues reaction 0.5 h, changes room temperature reaction 24 h into.Except after desolventizing, add chloroform to dissolve, use successively saturated NaHCO
3, NaHSO
4, NaCl washing, use anhydrous MgSO
4after dry, filter, revolve and desolventize, column chromatography (eluent is DCM/ methyl alcohol (MeOH)=10:1) separation obtains white powder MeO-Lys (G2)-Boc.
Deprotection
Accurately take MeO-Lys (G2)-Boc of 2.0 g in the single neck bottle with arm, vacuumize, inflated with nitrogen, with syringe, add 7 mL heavily to steam DCM, the TFA that adds 5 mL after material dissolution, TFA is revolved in decompression, with oil pump, drains, add anhydrous diethyl ether to stir and obtain white precipitate, be MeO-Lys (G2)-NH
2.Product, without purification, is directly used in the next step.Product, without purification, is directly used in the next step.
The peripheral positive electricity amino acid modified (HO-Lys (G2)-Arg (the Pbf)-Boc of take is example) of 3, two generation peptide class dendrimer
Take MeO-Lys (G2)-NH of 1.5 g
2, the Boc-Arg of 11.4 g (Pbf)-OH, the EDCI of 4.1 g and 2.9 g HOBT, in the flask with arm of 100mL, vacuumize, inflated with nitrogen, adds the DMF of 30 mL, ice bath stirring and dissolving, the DIPEA that adds 9 mL, continues reaction 0.5 h, changes room temperature reaction 48h into.Except after desolventizing, add chloroform to dissolve, use successively saturated NaHCO
3, NaHSO
4, NaCl washing, use anhydrous MgSO
4after dry, filter, revolve and desolventize, column chromatography (eluent is DCM/MeOH=10:1) separation obtains white powder MeO-Lys (G2)-Arg (Pbf)-Boc.
Deprotection
Accurately take 6.0 g MeO-Lys (G2)-Arg (Pbf)-Boc in flask, add after a small amount of dissolve with methanol, add the MeOH (1 mol/L) of the NaOH of 120 mL.After question response completes, after removal of solvent under reduced pressure, add DCM, add the pH value of HCl regulator solution of 1 mol/L to 2-3, separatory, anhydrous MgSO
4dry organic phase, filters, and except desolventizing, obtaining white solid is HO-Lys (G2)-Arg (Pbf)-Boc.
4, LA-Lys (G2)-Arg-NH
2synthetic
Accurately take the LA-NH of 0.3 g
2, the HO-Lys of 4.0 g (G2)-Arg (Pbf)-Boc, the EDCI of 0.3 g and 0.2 g HOBT are in 50 mL in the flask with arm, vacuumize, inflated with nitrogen, adds the DMF of 10 mL, ice bath stirring and dissolving, the DIPEA that adds 0.9 mL, under condition of ice bath, react 0.5 h, after room temperature reaction 24 h, except desolventizing, add chloroform to dissolve, use successively saturated NaHCO
3, NaHSO
4, NaCl washing, use anhydrous MgSO
4after dry, filter, revolve and desolventize, column chromatography (eluent is DCM/MeOH=10:1) separation obtains white powder LA-Lys (G2)-Arg (Pbf)-Boc.
Deprotection
Accurately take LA-Lys (G2)-Arg (Pbf)-Boc of 2.0 g in the single neck bottle with arm, vacuumize, inflated with nitrogen, with syringe, add 4 mL heavily to steam DCM, after material dissolution, add 5 mL trifluoroacetic acids, TFA is revolved in decompression, with oil pump, drains, add anhydrous diethyl ether to stir and obtain white precipitate, be LA-Lys (G2)-Arg-NH
2.The peptide class dendrimer obtaining is dissolved in deionized water, dialysis 3 d, lyophilize is standby.
embodiment 3:the preparation of supramolecule hydridization peptide class dendrimer
The synthetic of supramolecule hydridization peptide class dendrimer can once selected arbitrarily in two kinds of methods:
Method one:
1. the reduction of peptide class dendrimer.Peptide class dendrimer is dissolved in the mixed solvent of ethyl acetate and water, the NaBH of amount of substance such as adds
4, after stirring reaction 1 h, solution with water is diluted and is used chloroform extraction three times, merges organic phase, anhydrous MgSO
4dry, filter, revolve desolventizing, thick product uses column chromatography the peptide class dendrimer that obtains being reduced.
2. coordination is assembled into supramolecule hydridization peptide class dendrimer.Inorganic nano-particle is dispersed in organic phase, the salts solution that adds wherein the greatly excessive peptide class dendrimer being reduced, room temperature reaction 30 min, being warming up to 60 ℃ of reactions spends the night and makes to react completely, centrifugal, abandoning supernatant, repetitive scrubbing, the centrifugal supramolecule hydridization peptide class dendrimer that obtains.
Method two:
Take quantitative peptide class dendrimer part (the peptide class dendrimer of coordination functionalization) and be dissolved in deionized water, add organic bases to regulate pH value weakly alkaline, add the inorganic nano-particle that is dissolved in normal hexane, add again quantitative normal hexane, be placed in ultraviolet producer and fully stir, Coordinate self-assembly reaction occurs.When inorganic nano-particle, all transfer to water, show that reaction completes substantially.Water is transferred in dialysis tubing, removed the peptide class dendrimer not reacting, after freeze-drying, obtain supramolecule hydridization peptide class dendrimer.
embodiment 4:synthesizing of supramolecule hydridization peptide class dendrimer self-assembly
Taking peptide class dendrimer ligand 1 0.0 mg is dissolved in 1 mL deionized water, add Tetramethylammonium hydroxide TMAH to regulate pH value to 7-8, add the quantum dot QDs(8 μ mol/L that is dissolved in normal hexane) 100 μ L, the normal hexane that adds again 1 mL, be placed in ultraviolet producer and fully stir, Coordinate self-assembly reaction occurs.When the fluorescence of QDs, all transfer to water, show that reaction completes substantially.Water is transferred in dialysis tubing (MWCO 1000), removed the peptide class dendrimer not reacting, after freeze-drying, obtain pink powder shape solid.
embodiment 5:the preparation of supramolecule hydridization peptide class dendrimer/DNA mixture
Supramolecule hydridization peptide class dendrimer is configured to the certain density aqueous solution, and DNA is diluted to finite concentration, standby.Then by the supramolecule hydridization peptide class dendrimer aqueous solution and DNA short mix by a certain percentage, incubated at room, to for some time, can prepare a series of mixture.
embodiment 6:the preparation of supramolecule hydridization peptide class dendrimer/DNA mixture
Supramolecule hydridization peptide class dendrimer is diluted to 2 mg/mL with deionized water, and DNA is diluted to 800 μ g/mL with deionized water.By DNA and supramolecule hydridization peptide class dendrimer and DNA short mix according to a certain percentage, under room temperature condition, give 20 min, can prepare a series of R/P than the mixture of (R/P is than being the arginine residues of dendrimer self-assembly periphery and the mol ratio of DNA phosphate group).
embodiment 7:the sign of supramolecule hydridization peptide class dendrimer
1) structural characterization
Adopt KBr pressed disc method to characterize the infrared spectra of supramolecule hydridization peptide class dendrimer, take 4 mg supramolecule hydridization peptide class dendrimers and put into mortar, add the KBr powder of 200-400 mg to mix and grind.Get powder after 200 mg grind in lozenge former, the 30s that pressurizes under 25 Mpa can obtain ingot sheet.With infrared lamp, irradiate ingot sheet with drying and waterproofing.By KBr pressed disc method, the infrared spectra of peptide class dendrimer is characterized and contrasted simultaneously.
As depicted in figs. 1 and 2, the results of FT-IR shows that supramolecule hydridization peptide class dendrimer (Fig. 1) and peptide class dendrimer (Fig. 2) are at 1660 cm to result
-1there is the stretching vibration peak of carbonyl on amido linkage (C=O) in left and right, at 1500 cm
-1there is guanidine radicals (C=N) stretching vibration peak in arginine residues in left and right, absolutely proves that peptide class dendrimer is present in supramolecule hydridization peptide class dendrimer self-assembly, has coordination between peptide class dendrimer and inorganic nano-particle.
2) thermogravimetric analysis
Accurately take the supramolecule hydridization peptide class dendrimer of 8 mg, be placed in crucible, use the thermal gravimetric analyzer of German Nai Chi company, in air atmosphere, the quality change curve with the determination of heating rate supramolecule hydridization peptide class dendrimer of 10 ℃/min from room temperature to 900 ℃.Under the same terms, peptide class dendrimer is done to thermogravimetric curve simultaneously and be used as contrast
Result as shown in Figure 3 and Figure 4, supramolecule hydridization peptide class dendrimer is comprised of two portions, center is QDs particle, periphery is peptide class dendrimer, rising along with temperature, in air atmosphere, combustion oxidation progressively of peptide class dendrimer component is also taken away by sweep gas, finally the oxide compound of the metal in remaining QDs particle.Fig. 3 is the thermogravimetric curve of peptide class dendrimer, and along with the rising of temperature, its quality declines always until completely dissolve is zero, has lost 100.00% quality.Fig. 4 is the thermogravimetric curve of supramolecule hydridization peptide class dendrimer, along with the also progressively decline of its quality of rising of temperature, but rises to after 700 ℃ in temperature, and the quality of supramolecule hydridization peptide class dendrimer substantially no longer declines, and loses altogether 40.51% quality.
3) particle diameter, current potential and nanostructure characterize
Supramolecule hydridization peptide class dendrimer is configured to finite concentration, adopts dynamic light scattering method (DLS) to measure hydration particle diameter and the Zeta potential of supramolecule hydridization peptide class dendrimer, each sample repeats 3 times.
Result as shown in Figure 5 and Figure 6, the particle diameter of supramolecule hydridization peptide class dendrimer is 7.96 ± 0.93 nm, and the particle diameter of QDs is 5 nm left and right, illustrate after self-assembly, supramolecule hydridization peptide class dendrimer surface has dendrimer to be coordinated to the surface of QDs really.The surface band positive charge of supramolecule hydridization peptide class dendrimer, current potential is 17.3 ± 0.4 mV.
Supramolecule hydridization peptide class dendrimer is carried out to pattern, diameter characterization by transmission electron microscope and atomic force microscope.The certain density supramolecule hydridization peptide class dendrimer aqueous solution is dropped in respectively on copper mesh and sheet mica, after being dried under room temperature condition, can detect.The result of transmission electron microscope and atomic force microscope is all consistent with the result of DLS.
embodiment 8:the sign of supramolecule hydridization peptide class dendrimer/DNA mixture
1) agarose gel electrophoresis analysis
Take 0.15 g agarose, the TAE damping fluid (1 *) that adds 15 mL, with microwave oven, being repeatedly heated to agarose dissolves completely, treat that temperature is cooled to 50 ℃ of left and right, coagulant liquid is poured in the groove that is inserted with template comb, shaken gently and make liquid level, after the complete cooled and solidified of isogel, extract template comb, the groove that gel is housed is put into and is marked with TAE damping fluid (1 *) electrophoresis chamber.By the different R/P of 10 μ L than (1:1,1:2.5,1:5,1:10,1:15,1:20 and 1:30) supramolecule hydridization peptide class dendrimer/DNA mixture (containing 400 ng plasmid DNA) and 2 μ L sample-loading buffers mix, then get 10 μ L mixed solution loading to 1% sepharoses and carry out electrophoresis.Condition is constant voltage mode 100 V, times 60 min.After electrophoresis, sepharose is taken out, with Goldview, dye.So gel imaging is all used the imaging of Bio-Rad ChemDoc XRS+UV gel imaging system.
Result shows, the mobility maximum of the band of naked plasmid dna; By comparison, increase along with R/P ratio, the plasmid DNA band migration rate of 1-5 road swimming band obviously reduces gradually, its strength of signal also reduces gradually until do not have completely, and the strength of signal of corresponding gel point sample hole site increases gradually, show that the negative charge of DNA is neutralized by supramolecule hydridization peptide class dendrimer.On the other hand, the fluorescence signal intensity of supramolecule hydridization peptide class dendrimer increases with the increase of R/P ratio.
2) particle diameter, current potential and morphology characterization
Supramolecule hydridization peptide class dendrimer/DNA mixture is configured to finite concentration, adopts dynamic light scattering method (DLS) to measure hydration particle diameter and the Zeta potential of supramolecule hydridization peptide class dendrimer, each sample repeats 3 times.
Particle diameter result as shown in Figure 7, the particle diameter of supramolecule hydridization peptide class dendrimer/DNA mixture is 143.33 ± 8.79 nm, the size of supramolecule hydridization peptide class dendrimer is 7.96 ± 0.93 nm, after complex plasmid DNA, the particle diameter of supramolecule hydridization peptide class dendrimer increases about 20 times, the structure of this mixture may be by a plurality of supramolecule hydridization peptide class dendrimer particles and plasmid DNA is compound forms, and in a mixture, contains a plurality of supramolecule hydridization peptide class dendrimers.
As shown in Figure 8, the Zeta potential of supramolecule hydridization peptide class dendrimer/DNA mixture is+64.5 ± 3.4 mV to Zeta potential result, far above the current potential of general dendrimer/DNA mixture.The zeta current potential of supramolecule hydridization peptide class dendrimer is+17.3 ± 0.4 mV, as can be seen here, the surface charge of the particle of supramolecule hydridization peptide class dendrimer and the compound rear formation of DNA is higher than the electric charge of supramolecule hydridization peptide class dendrimer itself, this has also reacted mixture is from the side to have a plurality of supramolecule hydridization peptide class dendrimers and DNA to be composited, and DNA is compressed by supramolecule hydridization peptide class dendrimer.
Supramolecule hydridization peptide class dendrimer/DNA mixture is carried out to pattern, diameter characterization by transmission electron microscope and atomic force microscope.Certain density supramolecule hydridization peptide class dendrimer/DNA compound water solution is dropped in respectively on copper mesh and sheet mica, after being dried under room temperature condition, can detect.
As shown in Figure 9, supramolecule hydridization peptide class dendrimer/DNA mixture presents uniform particulate state and distributes transmission electron microscope results, and size is 100-200 nm, meets with DLS result.Can clearly observe the morphological structure of supramolecule hydridization peptide class dendrimer/DNA mixture simultaneously.
Atomic force microscope result shows length and wide its height that is slightly larger than of supramolecule hydridization peptide class dendrimer/DNA mixture three-dimensional structure, and total is similar to elliposoidal.
embodiment 9:supramolecule hydridization peptide class dendrimer/DNA composite body exobiology is evaluated
1) Cytotoxic evaluation.CCK-8 method is measured the vitro cytotoxicity of supramolecule hydridization peptide class dendrimer/DNA mixture.The tumor cell inoculation of selection in the active growth phase is in 96 orifice plates, and initial cell inoculum density is 1 * 10
4individual/hole, add the corresponding culture medium culturing of 100 μ L to spend the night, prepare supramolecule hydridization peptide class dendrimer/DNA mixture of different R/P ratios and N/P than the PEI/DNA mixture that is 10, incubated at room 30 min, then with containing the corresponding substratum of 10% foetal calf serum (FBS) or without the DMEM substratum dilution of foetal calf serum for cell transfecting; Remove the substratum in 96 orifice plates, PBS(pH 7.4) N/P of the rear PEI/DNA mixture that adds respectively supramolecule hydridization peptide class dendrimer/DNA mixture containing the different R/P ratios of the substratum dilution of 10% FBS, is 10 containing the N/P of the substratum dilution of 10%FBS of twice of washing, substratum dilution is than the PEI/DNA mixture that is 10, and to make the plasmid DNA content in each hole be 200 ng, the cell being left intact as a control group; Supramolecule hydridization peptide class dendrimer/DNA mixture is hatched after 48 h together with cell, remove the mixture in hole, and with PBS(pH 7.4) washing three times, add the CCK-8 of 10 times of 100 μ L serum free mediums dilutions, hatch after 2 h, by microplate reader, measure the light absorption value at 450 nm places.4 parallel multiple holes of each R/P specific concentration design.Relative cell survival rate
Calculate as follows:
Cell?Viability?=?(OD
sample-OD
background)/(OD
control-OD
background)×100%
Result as shown in figure 10, adopts commercialization positively charged ion genophore PEI and peptide class dendrimer in contrast.For HepG2 cell, at supramolecule hydridization peptide class dendrimer/DNA mixture and HepG2 cell, hatch after 48 h, when R/P is than 2.5 to 15 time, the survival rate of cell surpasses 100%, and this explanation adds supramolecule hydridization peptide class dendrimer/DNA mixture can promote the growth of cell within the scope of this; When R/P ratio is 20, cell survival rate is 93%, and supramolecule hydridization peptide class dendrimer/DNA mixture of this concentration has started to suppress the growth of cell; When R/P increases than continuation, the survival rate of cell continues to decline, and supramolecule hydridization peptide class dendrimer/DNA mixture increases the toxicity of cell, causes the transfection efficiency of gene and the reduction of the expression amount of gene simultaneously.PEI in contrast, PEI/DNA(N/P=10) carries out transfection under serum-free condition, and the survival rate of its cell is only 50% effect, shows that PEI has high cytotoxicity.In addition people source breast cancer cell (MCF-7) and HEKC (HEK293) have been done to identical experiment, the results are shown in Figure 11 and Figure 12, consistent with HepG2 cell result.
2) apoptosis detects
Use Propidium Iodide(PT) apoptosis that Kit apoptosis test kit causes supramolecule hydridization peptide class dendrimer/DNA mixture is analyzed.Working method is as follows: the HepG2 cell after recovery is inoculated in 6 orifice plates, and the initial cell density of inoculation is 2 * 10
5individual/hole, add 2 mL containing the substratum of 10%FBS, R/P is than certain supramolecule hydridization peptide class dendrimer/DNA mixture in preparation, N/P is 10 PEI/DNA mixture and PDs/DNA mixture, incubated at room 30 min, then respectively with containing the substratum of 10%FBS with without the substratum dilution of FBS; After 24 h, remove substratum, PBS(pH 7.4) twice of washing rear add respectively 2 mL containing supramolecule hydridization peptide class dendrimer/DNA mixture of the substratum dilution of 10%FBS, the N/P of serum free medium dilution than being 10 PEI/DNA mixture and peptide class dendrimer/DNA mixture of diluting containing the substratum of 10%FBS, making the plasmid DNA content in each hole is 4 μ g, and the cell being left intact as a control group; Jointly hatch after 48 h, remove substratum, PBS(pH 7.4) clean twice; Collecting cell after trysinization, centrifugal 5 min of 1000 rpm, discard supernatant liquor; With ice-cold PBS(pH 7.4) washing twice, then according to green skies company, provide PI working fluid 500 μ L Eddy diffusion cells for experimental implementation handbook; Under 37 ℃ of conditions, lucifuge is hatched 30 min; Finally use flow cytometer to select 488 nm excitation wavelengths, 575 nm emission wavelength conditions detect sample.Each experimental group arranges 3 parallel multiple holes.
As shown in Figure 13 and Figure 14, the apoptosis rate of blank cell is 5%, and the apoptosis rate of supramolecule hydridization peptide class dendrimer/DNA mixture is that the apoptosis rate of 7%, PEI/DNA transfectional cell is 20%.Show that PEI not only can allow cell mortality, and can cause a large amount of apoptosis of survivaling cell; Supramolecule hydridization peptide class dendrimer is during as gene transfection carrier, and no matter it is all approaching with the situation of blank cell to the toxicity of cell and the apoptosis situation that causes, shows that supramolecule hydridization peptide class dendrimer has good biocompatibility.
3) cell in vitro transfection
The vivoexpression of GFP gene.HepG2 cell after recovery is inoculated in 6 orifice plates, and the initial density of inoculation is 2 * 10
5individual/hole, adds 2 mL containing the substratum of 10%FBS, overnight incubation; By method above prepare the dilution of the substratum containing 10%FBS of R/P ratio supramolecule hydridization peptide class dendrimer/DNA mixture, without the N/P of the substratum dilution of FBS than be 10 PEI/DNA mixture, containing the N/P of the substratum dilution of 10%FBS than being peptide class dendrimer/DNA mixture that 10 PEI/DNA mixture and the substratum that contains 10%FBS dilute; Remove substratum, PBS(pH 7.4) twice rear mixture that adds respectively the above-mentioned various substratum dilutions of 2 mL of washing, making the plasmid DNA content in each hole is 4 μ g, the cell being left intact is as negative control group, after 4 h, remove containing the substratum in each experimental group of PEI, and add 2 mL containing the substratum of 10%FBS, other experimental group do not process, and jointly hatch after 48 h, remove the substratum in orifice plate, and with PBS(pH 7.4) twice of washing, collecting cell after trysinization, centrifugal 5 min of 1000 rpm, discard supernatant liquor; With 500 μ L PBS(pH 7.4) Eddy diffusion cell; Finally with flow cytometer, the GFP transfection results of sample is analyzed.Each experimental group arranges 3 parallel multiple holes.
With method, investigate the impact of different incubation times on GFP efficiency gene transfection.Commercialization PEI gene transfection agent compares.
Inverted fluorescence microscope gathers the fluoroscopic image of GFP.Structure shows, blank cell is expressed without GFP, peptide class dendrimer/DNA experimental group cell does not have GFP to express yet, PEI is significantly higher than at the expression rate of its GFP of experimental group of serum-free condition transfection the experimental group that PEI has the transfection of serum condition, meets PEI as the low feature of positively charged ion transfection reagent serum tolerance.And supramolecule hydridization peptide class dendrimer/DNA experimental group is along with the increase of R/P ratio, the expression efficiency of GFP increases gradually, and the experimental group of its GFP expression rate and PEI transfection under serum-free condition maintains an equal level even slightly high.The quantitative effect of expressing for embodying GPF, utilizes flow cytometer to detect positive rate and average fluorescent strength (MFI) value of the continuous item of cell, and then detects the expression of GFP gene and the picked-up situation of supramolecule hydridization peptide class dendrimer.Result shows that engulfing all of the positive expression rate of GFP and supramolecule hydridization peptide class dendrimer progressively rise along with the increase of R/P ratio.Peptide class dendrimer/DNA group and the GFP expression rate that has serum condition PEI/DNA to organize are respectively 1% and 5%, and be 20 o'clock at R/P ratio, the GFP the positive expression rate of supramolecule hydridization peptide class dendrimer/DNA group has reached 50%, maintains an equal level with the GFP expression rate of serum-free condition PEI/DNA group.Meanwhile, the phagocytic rate of supramolecule hydridization peptide class dendrimer has surpassed 70%, and both comprehensively illustrate is 20 o'clock at R/P ratio, a large amount of cellular uptake supramolecule hydridization peptide class dendrimer/DNA mixtures, and successful expression GFP.
The vivoexpression of luciferase (luciferase).HepG2 cell after recovery be take to initial density as 1 * 10
4individual/hole is inoculated in 96 orifice plates, in cell culture incubator, cultivate after 24 h, remove substratum PBS(pH 7.4) twice rear supramolecule hydridization peptide class dendrimer/DNA mixture that adds respectively 2 mL substratum dilutions of washing, the N/P of the substratum dilution of the FBS containing 10% is than the PEI/DNA mixture that is 10, without the N/P of the substratum dilution of FBS than being 10 PEI/DNA mixture and peptide class dendrimer/DNA mixture of diluting containing the substratum of 10% FBS, making the plasmid DNA content in each hole is 200 ng, the cell being left intact is as negative control group, jointly hatch after 48 h, remove substratum, PBS(pH 7.4) washing twice, add 70 μ L lysis buffers, utilize-80 ℃ of refrigerators to carry out freeze thawing, 3 times repeatedly, the fluorescein substrate hybrid reaction of getting 20 μ L split products and 50 μ L, and by the fluorescence radiation intensity of multi-functional microplate reader detection reaction.BCA analysis of protein kit measurement for the total protein content of split product.The calculation formula of relative intensity of fluorescence RLU is as follows:
RLU=fluorescence radiation intensity/Tot Prot
Result as shown in figure 15, the transfection situation of the research supramolecule hydridization peptide class dendrimer that luciferase gene (pGL3-Luc) can be quantitative as reporter gene.Result shows, different R/P are more identical than the expression variation tendency of the activity change trend of the luciferase of experimental group and GFP, the uciferase activity of the experimental group that R/P is 20 be R/P be peptide class dendrimer experimental group uciferase activity 5 * 10
4doubly, be 30 times of serum-free PEI/DNA experimental group uciferase activity, show that the assembling of peptide class dendritic macromole forms supramolecule hydridization peptide class dendrimer and can greatly increase transfection ability.
embodiment 10:the outer intracellular transport Mechanism Study of supramolecule hydridization peptide class dendrimer/DNA composite body
1) laser co-focusing experiment.By HepG2 cell, by initial density, be 1 * 10
4individual/ware is inoculated at the bottom of glass in ware, after 24 h, adds supramolecule hydridization peptide class dendrimer/DNA mixture (every hole 300 ng plasmid DNA).Plasmid DNA in mixture is hybrid dna, and wherein 33.3% is the pGL3 plasmid of Cy5 mark, and all the other are not for having markd pGL3 plasmid.Hatch respectively 1 h, 3 h, after 8 h and 18 h, with PBS(pH 7.4) wash twice.With the substratum mark lysosome that contains Lysotracker Blue DND-22 (75 mM), then use PBS(pH 7.4) wash twice.Under laser confocal microscope (confocal laser scanning microscope, CLSM), observe and gather fluorescence picture.
Result figure provides in application documents owing to having color inconvenience, only with word, to changing plan, describe below, in figure, redness represents genophore supramolecule hydridization peptide class dendrimer, green representation DNA, blueness represents lysosome, and the nucleus sharpness of border in figure is visible.When 1 h, red fluorescence and green fluorescence overlap and show yellow, show that supramolecule hydridization peptide class dendrimer and plasmid DNA remain combined state, DNA does not depart from supramolecule hydridization peptide class dendrimer, all supramolecule hydridization peptide class dendrimer/DNA complex particles are all in cell edges, show not to be transported to immediately after supramolecule hydridization peptide class dendrimer/DNA mixture is by cellular uptake core week region.When 3 h, fluorescence (red, green) and the lysosomal fluorescence (blueness) of most of supramolecule hydridization peptide class dendrimer/DNA complex particle are overlapping, show to have entered endosome/lysosome in late period after supramolecule hydridization peptide class dendrimer/DNA mixture is by cell endocytic, but now plasmid DNA is still not separated with supramolecule hydridization peptide class dendrimer.When 8 h, red, green, blue three fluorescence has not had lap substantially, shows that supramolecule hydridization peptide class dendrimer/DNA mixture escapes from lysosome, is distributed in tenuigenin; Green fluorescence is disperse shape and is distributed in tenuigenin, and discord red fluorescence is overlapping, and this shows that now plasmid DNA is separated with supramolecule hydridization peptide class dendrimer.When 18 h, green fluorescence is mainly distributed in all regions of core and nucleus, and this shows that plasmid DNA has been transported to all regions of core and has been incorporated in cell nucleus gene.
2) viable cell workstation experiment.By HepG2 cell, by initial density, be 1 * 10
4individual/ware is inoculated at the bottom of glass in ware, after 24 h, adds supramolecule hydridization peptide class dendrimer/DNA mixture.Sample is put into viable cell workstation and cultivate, culture condition is 37 ℃, 5%CO
2air atmosphere, every 5 min carry out IMAQ one time.
Result as shown in figure 16, around little by little assembled from the film of cell by little particle, finally, having formed large aggregate near nuclear region, its fluorescence intensity strengthened greatly by red fluorescence.This shows in the endocytosis process of supramolecule hydridization peptide class dendrimer/DNA mixture, and cell is concentrated supramolecule hydridization peptide class dendrimer/DNA mixture of picked-up gradually, enters endosome, finally forms large congeries.The particle diameter of supramolecule hydridization peptide class dendrimer/DNA mixture is at 150 nm, as Figure 16 clearly shows supramolecule hydridization peptide class dendrimer/DNA mixture, there is sightless nano particle gradually by cellular uptake and be enriched in the larger particle of formation together, occur strong fluorescent signal and be observed.
3) transmission electron microscope observing experiment.Distribution for observation mixture directly perceived in bag.By HepG2 cell, by initial density, be 2 * 10
5individual/ware is inoculated in 6 orifice plates, after 24 h, adds supramolecule hydridization peptide class dendrimer/DNA mixture, jointly hatches 24 h.Peptic cell, centrifugal collecting cell, the glutaraldehyde with 0.5% is fixed cell 15 min under 4 ℃ of conditions.10
4centrifugal 15 min of rpm, abandoning supernatant, the glutaraldehyde with 3% continues fixed cell.Then use 1% perosmic anhydride to fix, then with the acetone dehydration of different concns.Cell embedding is entered in Epon812, cell is made into ultrathin section(ing), then redye with lead citrate and uranyl acetate.
As shown in figure 17, cell ultrathin section(ing) has clearly shown that SHQDs particle is in intracellular distribution to result, and has disclosed the gene transfection process of supramolecule hydridization peptide class dendrimer/DNA mixture.Supramolecule hydridization peptide class dendrimer/DNA complex particle is by cell institute endocytosis, the figure illustrates the state (white arrow indication in figure) of at least three kinds of supramolecule hydridization peptide class dendrimer/DNA mixtures, supramolecule hydridization peptide class dendrimer/DNA mixture size is in the drawings 150 nm left and right, conforms to the result of DLS.Part supramolecule hydridization peptide class dendrimer/DNA mixture is attached on cytolemma, and cytolemma depression wish is by supramolecule hydridization peptide class dendrimer/DNA mixture parcel endocytosis simultaneously; Part supramolecule hydridization peptide class dendrimer/DNA mixture is by cell endocytic and be brought together formation congeries.
embodiment 11:transfection in supramolecule hydridization peptide class dendrimer/DNA composite body
Supramolecule hydridization peptide class dendrimer/DNA mixture muscle cdna transfection experiment.The shank of the male Balb/c mouse in 4 week age is lost hair or feathers, to carry out intramuscular injection.The beta-galactosidase enzymes (β-galactosidase) of 10 μ g codings (pCMV-lacZ) or plasmid DNA (pGL3-luc) of luciferase (luciferase) and supramolecule hydridization peptide class dendrimer at PBS(pH 7.4) in carry out compound, form R/P than the supramolecule hydridization peptide class dendrimer/DNA mixture that is 20, adopt in-situ injection method direct injection to enter the tibialis anterior muscle of Balb/c mouse after hatching 30 min.After 4 d, put to death Balb/c mouse, collect its tibialis anterior muscle.PEI/DNA is as experiment contrast group.In the active testing experiment of beta-galactosidase enzymes, β-galactosidase reporter gene assay kit of the tibialis anterior muscle Yong Bi skies biotech company of Balb/c mouse is processed, with the working fluid in test kit, soak, and hatch under 37 ℃ of conditions, until there is obvious blue patch in processed muscle.In the determination of activity experiment of luciferase, the tibialis anterior muscle of Balb/c mouse is blended to homogenate with IKA homogenizer, and with 1 * the cracking of luciferase reporter gene lysis buffer, and be placed in-80 ℃ of refrigerators, until it, freeze completely afterwards and take out and thaw, so multigelation is 3 times.Then by split product with 1 * 10
4centrifugal 3 min of rotating speed of rpm, get supernatant liquor 20 μ L and 50 μ L luciferase substrates and mix, and by multi-functional microplate reader, detect its luminous intensity, and the total protein content of supernatant liquor measures and calculate its RLU value as previously mentioned with BCA.
Qualitative results shows, the muscle of supramolecule hydridization peptide class dendrimer group is after dyeing, and 5 muscle masses surfaces are obvious colors blue all, and to the eye, in injection site, most of region presents mazarine, and area is wide, scope is wide, is obvious positive reaction; Relative, the muscle tissue of PEI group only has the small portion region of contiguous injection site to be caught blueness, and area is narrow, scope is little.Contrast both dyeing situations, we can find the transfection effect that the transfection effect of supramolecule hydridization peptide class dendrimer group is organized higher than PEI far away.
Quantitative result as shown in figure 18, supramolecule hydridization peptide class dendrimer/DNA(R/P=20) uciferase activity of experimental group is 2.2 * 10
5rLU/mg albumen, PEI/DNA(R/P=10) uciferase activity of experimental group is 3.1 * 10
3rLU/mg albumen, the transfection effect of supramolecule hydridization peptide class dendrimer group than the transfection effect promoting of commercialization PEI group 70 times, two groups of data there is significant difference.Experimental result confirms that supramolecule hydridization peptide class dendrimer is as the genophore of surface band positive charge, and its transfection effect promotes than transfection effect You Liaohen great unit in the body of PEI.
Gene transfection experiment in supramolecule hydridization peptide class dendrimer/DNA mixture knurl.Cultivate a large amount of HepG2 cells, with 1 * 10
6it is subcutaneous that individual/density is only inoculated into the right back of male nude mouse in 4 week age, and under aseptic condition, raising nude mice to the size of tumour is 100mm
3time, carry out gene transfection experiment in follow-up knurl.By pCMV-p53 and supramolecule hydridization peptide class dendrimer at PBS(pH 7.4) in carry out compound, be prepared into R/P than the supramolecule hydridization peptide class dendrimer/DNA mixture that is 20, after hatching 30 min, be injected in the tumour of the right back of the body of nude mice, after 48 h, put to death nude mice and take out tumor tissues; Tumor tissues is ground and becomes powder liquid nitrogen freezing condition is lower, with RIPA lysate cracking powder, centrifugal collection supernatant liquor; BCA method is measured total protein content, and dilute sample makes total protein content same level; Supernatant liquor and protein electrophoresis sample-loading buffer are mixed, and metal bath boils 10 min; Make separation gel and concentrated glue, use sampler that protein sample is added in concentrated glue hole, under constant voltage 150 V conditions, electrophoresis 60 min; Carry out after film transfer, by pvdf membrane taking-up and primary antibodie and two anti-hatching, use ECL(enhanced chemiluminescence) test kit development, imaging in gel imaging system.
Result demonstration, the expression amount of supramolecule hydridization peptide class dendrimer group and commercialization PEI group β-actin is basically identical, illustrates that total protein content is consistent, and the p53 expressing quantity of two groups has obvious difference.The p53 protein band signal of supramolecule hydridization peptide class dendrimer group is far above the p53 protein band signal of PEI group, and this shows to compare with commercialization PEI, and supramolecule hydridization peptide class dendrimer more can activate the expression of p53 albumen in cancer cells.
embodiment 12:bio-imaging in supramolecule hydridization peptide class dendrimer/DNA composite body
1) muscle position imaging.To the shank of the Balb/c mouse in 4 week age the processing of losing hair or feathers.After 2 d, by original position intramuscular injection, supramolecule hydridization peptide class dendrimer/DNA mixture is carefully injected to the tibialis anterior muscle of mouse.Mouse, with after 4% chloral hydrate anesthesia, is used to CRi Maestro EX living imaging instrument, take 450 nm as excitation wavelength, 605 nm are emission wavelength, 1 h after injection, and 4 h, 24 h and 48 h carry out imaging research.Use Maestro measurement software to process imaging results.
Result demonstration, after intramuscular injection 48 h, supramolecule hydridization peptide class dendrimer still has strong fluorescent signal in injection site, and this is high by supramolecule hydridization peptide class dendrimer fluorescent stability, and the feature of long half time determines.
2) tumor locus imaging.The foundation of nude mice tumor model as previously mentioned, is carefully injected into tumor locus by in-situ injection by supramolecule hydridization peptide class dendrimer/DNA mixture.Mouse, with after 4% chloral hydrate anesthesia, is used to CRi Maestro EX living imaging instrument, take 450 nm as excitation wavelength, 605 nm are emission wavelength, 1 h after injection, and 4 h, 24 h and 48 h carry out imaging research.Use Maestro measurement software to process imaging results.
Result shows, during 0 h, the fluorescence signal intensity of supramolecule hydridization peptide class dendrimer is the strongest, increase along with inject time, the fluorescent signal of supramolecule hydridization peptide class dendrimer weakens gradually, during to 48 h, strength of signal during its signal intensity ratio 0 h is low, half left and right when area in the region of the signal that can detect is 0 h.After supramolecule hydridization peptide class dendrimer is injected, can longerly there is tumor locus and can not run off in this explanation, so just have higher gene delivery efficiency and expression efficiency.
3) tissue distribution research.After nude mice in-situ injection supramolecule hydridization peptide class dendrimer/DNA mixture 48 h, nude mice is put to death and dissected, get organ and the tumor tissues such as its heart, liver, spleen, lung, kidney, with PBS(pH 7.4) washing twice, be placed in CRi Maestro EX living imaging instrument, take 450 nm as excitation wavelength, 605 nm are emission wavelength, 1 h after injection, 4 h, 24 h and 48 h carry out imaging research.Use Maestro measurement software to process imaging results.
Result shows (Figure 19), the mouse main organs of supramolecule hydridization peptide class dendrimer group does not all detect the fluorescent signal of supramolecule hydridization peptide class dendrimer, strong fluorescent signal only in tumor tissues, detected, be the 20-30 of other histoorgans doubly, this shows from tumor tissues, not pass through serum loop jump to other organs of mouse in 48 h of most supramolecule hydridization peptide class dendrimer after injection, and it distributes in vivo and mainly concentrates on tumor tissues.Internal organs and the tumor tissues of blank group and PEI group do not detect fluorescent signal, the numerical value of the internal organs of its statistic data and supramolecule hydridization peptide class dendrimer group is suitable, the fluorescence that shows biological tissue itself does not have interference effect to the fluorescent signal of supramolecule hydridization peptide class dendrimer, and supramolecule hydridization peptide class dendrimer has the potentiality that become fluorescent probe in petty action object.
Claims (10)
1. a supramolecule hydridization peptide class dendrimer self-assembly, is characterized in that, comprises inorganic nano-particle kernel and peptide class dendrimer, and it is peripheral that described peptide class dendrimer is assembled in described inorganic nano-particle by coordination.
2. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, the mode of described coordination is a kind of in the coordination of metal-sulfydryl, chelating coordination, host-guest complexation.
3. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, described inorganic nano-particle is a kind of in quantum dot, ferriferrous oxide nano-particle, golden nanometer particle, rare earth nanometer particle.
4. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, peptide class dendrimer periphery is positively charged.
5. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, described peptide class dendrimer is low algebraically peptide class dendrimer, and its algebraically is 1 generation, 2 generations or 3 generations.
6. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, described peptide class dendrimer is the fan-shaped molecule of core and peripheral difunctionalization.
7. according to the supramolecule hydridization peptide class dendrimer self-assembly described in claim 1, it is characterized in that, the amino acid of described peptide class dendrimer periphery is Methionin, arginine, at least one in Histidine.
8. supramolecule hydridization peptide class dendrimer self-assembly according to claim 6, is characterized in that: the structural formula of described peptide class dendrimer is as follows:
Wherein, R1 represents the core element of fan-shaped molecule and the site part of inorganic nano-particle effect, and m represents the functionality of core element, and the functionality of core element is 1 or 2; K is peptide class dendrimer amino acid backbone, and R is positive charge amino acid for the peripheral modified with functional group of peptide class dendrimer, and K and R can be identical, also can difference, G1.0 and G2.0 represent respectively a generation and two generation peptide class dendrimer.
9. a construction process for the supramolecule hydridization peptide class dendrimer self-assembly as described in claim 1, is characterized in that, comprises the following steps:
(1) prepare peptide class dendrimer;
(2) to the core element of peptide class dendrimer and or inorganic nano-particle carry out coordination functionalization;
(3) the peptide class dendrimer and the inorganic nano-particle that in step (2), make are fully mixed, make both self-assemblies under the driving of coordination obtain supramolecule hydridization peptide class dendrimer self-assembly.
10. an application for the supramolecule hydridization peptide class dendrimer self-assembly as described in claim 1, is characterized in that, used as genophore or pharmaceutical carrier or bio-imaging and spike.
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