CN102516395A - Polypeptide carrier used for improving targeting ability and transfection efficiency of medicine/gene, and purpose thereof - Google Patents
Polypeptide carrier used for improving targeting ability and transfection efficiency of medicine/gene, and purpose thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide carrier used for improving targeting ability and transfection efficiency of a medicine/gene. The polypeptide carrier has a structure represented by a formula (I): TATp-Cys-LHRHp (I), wherein TATp is a TAT fragment of HIV, Cys is Cysteine, LHRHp is an analogue of luteinizing hormone releasing hormone LHRH. The polypeptide carrier provided by the invention carries a large amount of positive charges. The polypeptide carrier has good water-solubility and high targeting ability aiming at sexual hormone dependent tumor and liver cancer. The carrier can directly carry negatively charged nucleic acid or medicine, and mediate the nucleic acid or medicine into cells. Also, the carrier can be used for modifying other gene or medicine carriers, such that tumor targeting abilities and transfection efficiency of the gene or medicine carriers can be improved. Therefore, a novel carrier is provided for gene/medicine delivering.
Description
Technical field
The invention belongs to technical field of biological materials, be specifically related to a kind of target property of medicine/gene and the peptide carrier and purposes of transfection efficiency of improving.
Background technology
Tumour is one of current modal morbidity and cause of death, and there is more than 1,000 ten thousand new cases and 6,200,000 deaths every year in the whole world.Updated statistics shows that recent two decades comes, and it is nearly 30 percent that Chinese cancer mortality has risen, and just has one to die from cancer among per four, five dead persons, occupies first of the cause of death.But cancer chemotherapeutic drug also causes normal cell dead in kill cancer cell owing to lack target property at present, causes patient's whole body toxic side effect bigger.Therefore gene/the pharmaceutical carrier that develops efficient target plays crucial effects in oncotherapy.
Cell-penetrating peptides (Cell penetrating peptides) is one type and can directly passes cytolemma fast and get into intracellular polypeptide; And the biologically active substance that can carry multiple different size and character gets into cell; Comprise small-molecule substance, polypeptide, polypeptide-nucleic acid (peptidenucleo acid; PNA), protein, DNA, siRNA, nano particle and micella etc., this character makes it possibly become the excellent drug carrier.Wherein, The TAT polypeptide is a present more cell-penetrating peptides of research, derive from HIV virus tat protein (Transcriptional activator of transcription, TAT); Sequence is YGRKKRRQRRR; Mainly form, have a large amount of positive charges, and have good hydrophilicity by basic amino acids arginine and Methionin.Existing experiment confirm TATp modified liposome can obviously improve its inside and outside transfection efficiency, and external transfection efficiency can reach 30%-50%, and has obviously reduced the toxicity when the simple lipid body mediates transfection.But get into cell but not rely on acceptor because TATp directly wears film, inorganizable cell targeted.
The main policies that improves the tumor-targeting treatment is to tumour cell related antigen (tumor-associated antigens; TAAs) solid support material is modified, made it have higher selectivity and improve the absorption of tumour cell whereby gene to tumour cell.Discover; Luteinizing hormone-releasing hormone (LRH) acceptor (luteinizing hormone-releasing hormone receptor; LHRHR) rely on overexpression on tumour such as the cytolemma such as mammary cancer, ovarian cancer and prostate cancer in liver cancer and plurality hormone; Apparently higher than the expression on the respective organization human cell membrane, and on the cytolemma of normal liver,kidney,spleen, the heart, lung, brain, thymus gland and skeletal muscle tissue, do not have and detectedly to express.Small peptide (the sequence: EHWSYGLRPG) that luteinizing hormone-releasing hormone (LRH) (LHRH) is made up of 10 amino acid.Existing cell in vitro experiment showed, that the LHRH modification can obviously improve the target property of contained medicine to mammary cancer, ovarian cancer cell.
In view of problem discussed above, it is a kind of to the strong target property of tumour and carrier efficiently that expectation provides.
Summary of the invention
The objective of the invention is to overcome a gene/pharmaceutical carrier target property difference and the low difficult problem of transfection efficiency that prior art exists, a kind of target property of medicine/gene and peptide carrier of transfection efficiency of improving is provided.
Second purpose of the present invention provides the purposes of the peptide carrier of a kind of target property that improves medicine/gene and transfection efficiency.
The 3rd purpose of the present invention provides the multipolymer that the peptide carrier beautify chitosan of a kind of target property that improves medicine/gene and transfection efficiency forms.
The 4th purpose of the present invention provides the purposes of the multipolymer that the peptide carrier beautify chitosan of a kind of target property that improves medicine/gene and transfection efficiency forms.
Technical scheme of the present invention is summarized as follows:
A kind of target property of medicine/gene and peptide carrier of transfection efficiency of improving, said peptide carrier has the structure of formula (I):
TATp-Cys-LHRHp(I)
Wherein: TATp is the TAT fragment of HIV;
Cys is a halfcystine;
LHRHp is the analogue of r-hLH LHRH.
The aminoacid sequence of said TATp is: N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg; Or its reverse sequence is: N end-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg;
The aminoacid sequence of said LHRHp is: N end-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu; Or its reverse sequence:
N end-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly.
Said TATp-Cys-LHRHp sequence is shown in the SEQ ID NO.1:
N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
Or shown in the SEQ ID NO.2: the N end
-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly;
Or shown in the SEQ ID NO.3: the N end
-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
Or the end of N shown in the SEQ ID NO.4
-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly.
The purposes of aforementioned polypeptides carrier.
The multipolymer that the peptide carrier beautify chitosan forms.
The purposes of above-mentioned multipolymer.
Compared with prior art, advantage of the present invention is following:
What the present invention relates to a kind ofly improves the target property of medicine/gene and the peptide carrier of transfection efficiency has a large amount of positive charges; Have good water-solubility and also have the very strong specific aim hormone-dependent tumor and the target property of liver cancer in addition; Can directly carry electronegative nucleic acid or medicine and mediate its entering cell, also can modify other gene or pharmaceutical carrier and improve its tumor-targeting and transfection efficiency.For gene/delivery of drug provides a kind of novel carriers.
Two peptide T ATp that the present invention relates to are connected through halfcystine with LHRHp; Can two peptide molecules be linked to other carrier with Y shape form orientation; Can give full play to the function of two peptide molecules; And can reduce the non-specific spinoff of TATp, for the efficient targeting modification of gene/pharmaceutical carrier provides a kind of novel method.
A kind of target property that improves medicine/gene that the present invention relates to and the peptide carrier of transfection efficiency do not have special requirement to other carrier surface chemical group during to other carrier modification, therefore use wider.
The multipolymer that the peptide T ATp-Cys-LHRHp that the present invention relates to and chitosan form can be applicable to transgene carrier and pharmaceutical prepn (for example drug-carrying nanometer particle, gel) etc.
Description of drawings
Fig. 1 peptide carrier mass-spectrogram.
Fig. 2 peptide carrier performance liquid collection of illustrative plates.
Fig. 3. peptide modified chitin copolymer synthetic route chart.
Fig. 4. peptide modified chitin copolymer infrared spectrogram.Wherein, solid line is a chitosan, and dotted line is peptide modified chitin copolymer.
Fig. 5. peptide modified chitin copolymer isoelectric point determination result.
Fig. 6. peptide modified chitin copolymer carries gene nanoparticle gel blocking result.
Fig. 7. peptide modified chitin copolymer carries gene nanoparticle AFM.
Fig. 8. peptide modified chitin copolymer carries gene nanoparticle stability result.
Wherein: the peptide modified chitin copolymer of Fig. 8-1 carries gene nanoparticle Zeta potential changing conditions.
The peptide modified chitin copolymer of Fig. 8-2 carries gene nano particle diameter changing conditions.
Fig. 9. peptide modified chitin copolymer carries the gene nanoparticle and carries the transfection results of luciferase gene pGL3-control nanoparticle to people's normal liver cell LO2 and liver cancer cell HepG2.
Figure 10. cell transfecting 24 back inverted fluorescence microscope observationss
The peptide modified chitin copolymer of A carries the gene nanoparticle and carried gene nanoparticle transfection people normal liver cell LO2 24 hours;
The peptide modified chitin copolymer of B carried gene nanoparticle transfection human liver cancer cell HepG2 24 hours;
C is Lipofectamine2000 transfection people normal liver cell LO2 24 hours;
D is Lipofectamine2000 transfection human liver cancer cell HepG2 24 hours.
Figure 11 cell transfecting is the flow cytometry result after 24 hours.
A: peptide modified chitin copolymer carried pEGFP gene nanoparticle transfection people normal liver cell LO2 after 24 hours, and streaming result shows 0.06% cell expressing GFP;
B: peptide modified chitin copolymer carried pEGFP gene nanoparticle transfection human liver cancer cell HepG2 after 24 hours, and streaming result shows 7.77% cell expressing GFP;
2000 years pEGFP genes of C:Lipofectamine nanoparticle transfection people normal liver cell LO2 is after 24 hours, and streaming result shows 2.7% cell expressing GFP;
2000 years pEGFP genes of D:Lipofectamine nanoparticle transfection human liver cancer cell HepG2 is after 24 hours, and streaming result shows 9.26% cell expressing GFP.
Embodiment
Through specific embodiment the present invention is further described below:
Peptide T ATp-Cys-LHRHp of the present invention is owing to be the difunctional peptide that is connected to form through the halfcystine two peptide species molecules (TATp and LHRHp) that length is suitable; Can this difunctional peptide (TATp-Cys-LHRHp) be linked to other gene/pharmaceutical carriers with Y shape form orientation through 3-(2-pyridine dimercapto) propionic acid n-hydroxysuccinimide eater sulfydryl linking agents such as (SPDP); Like chitosan; To give full play to two polypeptide (TATp and LHRHp) function separately; Tomour specific target recognition reaction and TATp like LHRHp carry the function that other material gets into cell, and reduce the non-specific cell absorption that TATp causes.
Below in conjunction with specific embodiment the present invention is further described.
Synthetic and the sign of peptide T ATp-Cys-LHRHp
Use Peptide synthesizer, adopt the solid-phase polypeptide synthetic technology to carry out polypeptide and synthesize, the gained polypeptide shows that through mass spectrometry results its molecular ion peak is 2657 (see figure 1)s, and is consistent with the polypeptide molecular weight of expection, analyzes through HPLC, and its purity is greater than 99% (see figure 2)
TATp-Cys-LHRHp carries the preparation of gene nanoparticle
With the peptide carrier (TATp-Cys-LHRHp) and the DNA aqueous solution that makes soluble in water respectively, then respectively with two aqueous solution in 37 ℃ of water-baths 10 minutes; Is by N/P (mol ratio?) than be 10: 1 with the quick mixing of two solution and whirlpool concussion 50 seconds, promptly get TATp-Cys-LHRHp and carry the gene nanoparticle.The median size of nanoparticle is about 82nm, and the zeta current potential is 26mV.
N/P can select in 2: 1～20: 1, and the concussion time was selected in 40～60 seconds, and the TATp-Cys-LHRHp that obtains carries the gene nanoparticle, and its median size is about 70～90nm, and the zeta current potential is at 20-40mY.
TATp-Cys-LHRHp carries the cell transfecting experiment of gene nanoparticle
Carry the gene nanometer particle process method according to embodiment 2TATp-Cys-LHRHp; The TATp-Cys-LHRHp of preparation N/P than 10: 1 carries the gene nanoparticle; Then with this nanoparticle difference transfection human liver cancer cell BEL-7402 and people's normal liver cell LO2; The amount of transfection nanoparticle is calculated as 5 μ g DNA/ml with DNA, and transfection is after 24 hours, and high intension viable cell imaging results shows that this nanoparticle has stronger target property to liver cancer cell BEL-7402; Higher to its transfection efficiency, then very low to normal liver cell LO2 transfection efficiency.
The preparation of TATp-Cys-LHRHp beautify chitosan multipolymer
Concrete grammar comprises the steps:
(1) it is soluble in water to take by weighing the chitosan that molecular weight is 5000-8000 (CS), processes mass concentration and be 1% the aqueous solution; (2) 3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester (SPDP) is dissolved in the DMSO 99.8MIN. (DMSO), processing concentration is 20mM solution;
(3) be that 1: 3 ratio the solution that said step (1) is obtained and the solution of step (2) acquisition mix room temperature reaction 60 minutes in the mol ratio of the free amine group on SPDP molecule and the CS molecule;
(4) contain the desalting column of the phosphate buffered saline buffer balance molecular weight cut-off 5000 of 1mM YD 30 with pH=7.5, the reaction solution that obtains with the cf-centrifugation step (3) of 4000xg then 25 minutes, and centrifugal 3 times to remove free SPDP molecule;
(5) the TATp-Cys-LHRHp polypeptide is dissolved in the deoxidation distilled water, processes mass concentration and be 1% the aqueous solution; Said TATp-Cys-LHRHp amino acid sequence of polypeptide is:
N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
(6) by a certain percentage (mol ratio of the free amine group on TATp-Cys-LHRHp peptide molecule and the CS molecule is 1: 3) mixes the product that step (4) obtains with the solution that step (5) obtains, and reacts 12 hours in stirring at room;
(7) contain the desalting column of second molecular weight cut-off 5000 of phosphate buffered saline buffer balance of 1mM YD 30 with pH=7.5; Be 4000xg centrifugation step (6) gained reaction solution 25 minutes then with cf-, and centrifugal 3 times promptly obtain TATp-Cys-LHRHp beautify chitosan multipolymer with purified product; With seal after the lyophilize of said TATp-Cys-LHRHp beautify chitosan multipolymer place-20 ℃ subsequent use.
Shown in Figure 3 is the synthetic synoptic diagram of TATp-Cys-LHRHp beautify chitosan multipolymer.
Annotate: in the building-up process of TATp-Cys-LHRHp beautify chitosan multipolymer, add the SPDP and the TATp-Cys-LHRHp molecule of respective amount in the reaction respectively, with the TATp-Cys-LHRHp beautify chitosan multipolymer of preparation different degree of substitution.
Fig. 4 is the infrared spectrogram of TATp-Cys-LHRHp beautify chitosan multipolymer, and the amino peak of CS is at 1658.07cm
-1The place, and amino peak obviously reduces in TATp-Cys-LHRHp beautify chitosan multipolymer, while imido base peak 1555.56cm
-1The place, peak obviously strengthens.By TATp-Cys-LHRHp beautify chitosan multipolymer shown in Figure 3 promptly is through amino reaction on sulfydryl on the polypeptide and the chitosan molecule, makes the TATp-Cys-LHRHp polypeptide through the hydrogen on the substituted-amino polypeptide is linked on the chitosan molecule.Therefore synthesize successfully according to this results of IR explanation TATp-Cys-LHRHp beautify chitosan multipolymer.
Embodiment 5
The titration of TATp-Cys-LHRHp beautify chitosan multipolymer iso-electric point
TATp-Cys-LHRHp beautify chitosan multipolymer is made into the aqueous solution that concentration is 0.5mg/ml; With the 0.5N NaOH aqueous solution and the 0.05N NaOH aqueous solution as titrating solution; Adopt laser particle analyzer through the soda acid autotitrating method; Begin titration to pH value=12 from initial pH; Every interval pH 0.5 measures a zeta current potential, and the iso-electric point that finally records TATp-Cys-LHRHp beautify chitosan multipolymer is 11.3 (as shown in Figure 5), infers thus; TATp-Cys-LHRHp beautify chitosan multipolymer is should be positively charged in about 7 the water in the pH value, and theoretical this multipolymer can interact through positive and negative charge with electronegative DNA or medicine and form nano complex.
The preparation that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nano complex
TATp-Cys-LHRHp beautify chitosan multipolymer is prepared different nanoparticles with the EGFP-C1 DNA according to different N/P ratio.At first that TATp-Cys-LHRHp beautify chitosan multipolymer and DNA is soluble in water respectively, the concentration of aqueous dna is 0.2mg/ml, and TATp-Cys-LHRHp beautify chitosan copolymer solution concentration compares and difference according to different N/P.Two solution are placed 37 ℃ of water-baths 10 minutes respectively, then two solution equal-volumes are mixed, whirlpool shakes 45-55 second immediately, promptly gets required nanoparticle.The gained nanoparticle is carried out gel retardation assasy, show that by Fig. 6 the N/P ratio is that 1: 1 o'clock TATp-Cys-LHRHp beautify chitosan multipolymer can wrap up DNA fully.Adopt laser particle analyzer to carry out particle diameter and Zeta potential mensuration the gained nanoparticle; Result such as following table and shown in Figure 7; The particle diameter of most nanoparticles is between 70-85nm, and current potential is about 30mV, and the AFM imaging results shows that each nanoparticle is than regular circular.Each nanoparticle is placed room temperature, respectively at the 1st, 2,3,7,14 day nanoparticle is carried out particle diameter and Zeta potential mensuration, with the stability of observation nanoparticle.The particle diameter and the Zeta potential that remove the nanoparticle of N/P than 1: 4 shown in Fig. 8-1 and Fig. 8-2 change greatly, and promptly outside the less stable, other selected proportioning nanoparticle is all relatively stable.
Table. the peptide modified chitin copolymer of different N/P carries the particle diameter and the Zeta potential of gene nanoparticle
TATp-Cys-LHRHp beautify chitosan multipolymer carries gene nanoparticle cell in vitro transfection results
Is 10: 1 preparation nanoparticle with luciferase gene pGL3-control by the N/P ratio with TATp-Cys-LHRHp beautify chitosan multipolymer; Then with nanoparticle difference transfection human liver cancer cell HepG2 and people's normal liver cell LO2; And with commercialization transfection reagent Lipofectamine 2000 as positive control, the amount of transfection nanoparticle is calculated as 5 μ g DNA/ml with DNA.Transfection is cell employing inverted fluorescence microscope observation and flow cytometry analysis after 24 hours; Fig. 9 and Figure 10 show that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nanoparticle liver cancer cell is had stronger target property, and normal liver cell is not almost had transfection.Figure 11 carries the flow cytometry result of gene nanoparticle cell in vitro transfection after 24 hours for TATp-Cys-LHRHp beautify chitosan multipolymer; It is 7.77% to the transfection efficiency of liver cancer cell HepG2 that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nanoparticle; And normal liver cell is merely 0.06%, be about 130 times; And TATp-Cys-LHRHp beautify chitosan multipolymer carry the gene nanoparticle to the transfection efficiency of HepG2 near commercialization transfection reagent Lipofectamine 2000, its selectivity to liver cancer cell then is higher than Lipofectamine 2000 far away.
Embodiment 8
The preparation of TATp-Cys-LHRHp beautify chitosan multipolymer (II)
Concrete grammar is following:
(1) it is soluble in water to take by weighing the chitosan of molecular weight 5000-8000, processes mass concentration and be 5% solution;
(2) SPDP is dissolved among the DMSO, processing concentration is 20mM solution;
(3) be that 1: 10 ratio the solution that said step (1) is obtained and the solution of step (2) acquisition mix room temperature reaction 30 minutes in the mol ratio of the free amine group on SPDP molecule and the CS molecule;
(4) contain the desalting column of the phosphate buffered saline buffer balance molecular weight cut-off 5000 of 1mM YD 30 with pH=7.5, the reaction solution that obtains with the cf-centrifugation step (3) of 4000xg then 25 minutes, and centrifugal 3 times to remove free SPDP molecule;
(5) the TATp-Cys-LHRHp polypeptide is dissolved in the deoxidation distilled water, processes mass concentration and be 1% the aqueous solution; Said TATp-Cys-LHRHp amino acid sequence of polypeptide is:
N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
(6) be that 1: 10 ratio is mixed the product that step (4) obtains with the solution that step (5) obtains in the mol ratio of the free amine group on TATp-Cys-LHRHp peptide molecule and the CS molecule, reacted 10 hours in stirring at room;
(7) contain the desalting column of second molecular weight cut-off 5000 of phosphate buffered saline buffer balance of 1mM YD 30 with pH=7.5; Be 4000xg centrifugation step (6) gained reaction solution 25 minutes then with cf-, and centrifugal 4 times promptly obtain TATp-Cys-LHRHp beautify chitosan multipolymer with purified product; With seal after said TATp-Cys-LHRHp beautify chitosan multipolymer (II) lyophilize place-20 ℃ subsequent use.
The preparation of TATp-Cys-LHRHp beautify chitosan multipolymer (III)
Preparation process is following:
(1) it is soluble in water to take by weighing the chitosan of molecular weight 5000-8000, processes mass concentration and be 1% solution;
(2) SPDP is dissolved among the DMSO, processing concentration is 20mM solution;
(3) be that 9: 10 ratio the solution that said step (1) is obtained and the solution of step (2) acquisition mix room temperature reaction 45 minutes in the mol ratio of the free amine group on SPDP molecule and the CS molecule;
(4) contain the desalting column of the phosphate buffered saline buffer balance molecular weight cut-off 5000 of 1mM YD 30 with pH=7.5, the reaction solution that obtains with the cf-centrifugation step (3) of 4000xg then 30 minutes, and centrifugal 4 times to remove free SPDP molecule;
(5) the TATp-Cys-LHRHp polypeptide is dissolved in the deoxidation distilled water, processes mass concentration and be 2% the aqueous solution; Said TATp-Cys-LHRHp amino acid sequence of polypeptide is:
N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly;
(6) be that 9: 10 ratio is mixed the product that step (4) obtains with the solution that step (5) obtains in the mol ratio of the free amine group on TATp-Cys-LHRHp peptide molecule and the CS molecule, reacted 14 hours in stirring at room;
(7) contain the desalting column of second molecular weight cut-off 5000 of phosphate buffered saline buffer balance of 1mM YD 30 with pH=7.5; Be 4000xg centrifugation step (6) gained reaction solution 30 minutes then with cf-, and centrifugal 5 times promptly obtain TATp-Cys-LHRHp beautify chitosan multipolymer with purified product; With seal after said TATp-Cys-LHRHp beautify chitosan multipolymer (III) lyophilize place-20 ℃ subsequent use.
The preparation of TATp-Cys-LHRHp beautify chitosan multipolymer (IV)
Concrete grammar comprises the steps:
(1) it is soluble in water to take by weighing molecular weight 190000-310000 chitosan, processes mass concentration and be 0.5% solution;
(2) SPDP is dissolved among the DMSO, processing concentration is 20mM solution;
(3) be that 1: 10 ratio the solution that said step (1) is obtained and the solution of step (2) acquisition mix room temperature reaction 45 minutes in the mol ratio of the free amine group on SPDP molecule and the CS molecule;
(4) contain the desalting column of the phosphate buffered saline buffer balance molecular weight cut-off 5000 of 1mM YD 30 with pH=7.5, the reaction solution that obtains with the cf-centrifugation step (3) of 4000xg then 40 minutes, and centrifugal 5 times to remove free SPDP molecule;
(5) the TATp-Cys-LHRHp polypeptide is dissolved in the deoxidation distilled water, processes mass concentration and be 3% the aqueous solution; Said TATp-Cys-LHRHp amino acid sequence of polypeptide is:
N end-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
(6) be that 1: 10 ratio is mixed the product that step (4) obtains with the solution that step (5) obtains in the mol ratio of the free amine group on TATp-Cys-LHRHp peptide molecule and the CS molecule, reacted 12 hours in stirring at room;
(7) contain the desalting column of second molecular weight cut-off 5000 of phosphate buffered saline buffer balance of 1mM YD 30 with pH=7.5; Be 4000xg centrifugation step (6) gained reaction solution 40 minutes then with cf-, and centrifugal 3 times promptly obtain TATp-Cys-LHRHp beautify chitosan multipolymer with purified product; With seal after said TATp-Cys-LHRHp beautify chitosan multipolymer (IV) lyophilize place-20 ℃ subsequent use.
The preparation of TATp-Cys-LHRHp beautify chitosan multipolymer (V)
Concrete grammar comprises the steps:
(1) it is soluble in water to take by weighing molecular weight 190000-310000 chitosan, processes mass concentration and be 0.5% solution;
(2) SPDP is dissolved among the DMSO, processing concentration is 20mM solution;
(3) be that 1: 1 ratio the solution that said step (1) is obtained and the solution of step (2) acquisition mix room temperature reaction 60 minutes in the mol ratio of the free amine group on SPDP molecule and the CS molecule;
(4) contain the desalting column of the phosphate buffered saline buffer balance molecular weight cut-off 5000 of 1mM YD 30 with pH=7.5, the reaction solution that obtains with the cf-centrifugation step (3) of 4000xg then 30 minutes, and centrifugal 4 times to remove free SPDP molecule;
(5) the TATp-Cys-LHRHp polypeptide is dissolved in the deoxidation distilled water, processes mass concentration and be 5% the aqueous solution; Said TATp-Cys-LHRHp amino acid sequence of polypeptide is:
N end-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly;
(6) be that 1: 1 ratio is mixed the product that step (4) obtains with the solution that step (5) obtains in the mol ratio of the free amine group on TATp-Cys-LHRHp peptide molecule and the CS molecule, reacted 15 hours in stirring at room;
(7) contain the desalting column of second molecular weight cut-off 5000 of phosphate buffered saline buffer balance of 1mM YD 30 with pH=7.5; Be 4000xg centrifugation step (6) gained reaction solution 40 minutes then with cf-, and centrifugal 4 times promptly obtain TATp-Cys-LHRHp beautify chitosan multipolymer with purified product; With seal after said TATp-Cys-LHRHp beautify chitosan multipolymer (V) lyophilize place-20 ℃ subsequent use.
The experiment proof; The method that adopts embodiment 6 with the TATp-Cys-LHRHp beautify chitosan multipolymer of the method for embodiment 8-embodiment 11 preparation respectively with DNA according to different N/P than the different nanoparticles of preparation; The result is except that the nanoparticle less stable of N/P than 1: 4, and other selected proportioning nanoparticle is all relatively stable.
It is 4: 1 preparation nanoparticle with luciferase gene pGL3-control by the N/P ratio with TATp-Cys-LHRHp beautify chitosan multipolymer that the TATp-Cys-LHRHp beautify chitosan multipolymer of the method preparation of use embodiment 8-embodiment 11 adopts the method for embodiment 6, then nanoparticle is carried out transfection (final concentration is 5 μ g DNA/ml) to human liver cancer cell HepG2 and people's normal liver cell LO2 respectively.Behind the transfection 24h; Transfectional cell is carried out cracking; Getting 20 μ l cracking supernatants is added in the 100 μ l luciferase detection reagent; Detect through Chemiluminescence Apparatus, the result shows that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nanoparticle liver cancer cell is had obvious selectivity, and its transfection efficiency is about 80-110 times of normal liver cell.
It is 10: 1 preparation nanoparticle with luciferase gene pGL3-control by the N/P ratio with TATp-Cys-LHRHp beautify chitosan multipolymer that the TATp-Cys-LHRHp beautify chitosan multipolymer of the method preparation of use embodiment 8-embodiment 11 adopts the method for embodiment 6; Then with nanoparticle difference transfection human liver cancer cell HepG2 and people's normal liver cell LO2; And with commercialization transfection reagent Lipofectamine 2000 as positive control, the amount of transfection nanoparticle is calculated as 5 μ gDNA/ml with DNA.Transfection is cell employing inverted fluorescence microscope observation and flow cytometry analysis after 24 hours; The result shows that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nanoparticle liver cancer cell is had stronger target property, and normal liver cell is not almost had transfection.TATp-Cys-LHRHp beautify chitosan multipolymer carries the flow cytometry result of gene nanoparticle; It is 2.3-6.1% to the transfection efficiency of liver cancer cell HepG2 that TATp-Cys-LHRHp beautify chitosan multipolymer carries the gene nanoparticle; And normal liver cell is merely 0.02-0.08%, be about 75-108 doubly; And TATp-Cys-LHRHp beautify chitosan multipolymer carry the gene nanoparticle to the transfection efficiency of HepG2 near commercialization transfection reagent Lipofectamine 2000, its selectivity to liver cancer cell then is higher than Lipofectamine 2000 far away.
Claims (7)
1. one kind is improved the target property of medicine/gene and the peptide carrier of transfection efficiency, it is characterized in that said peptide carrier has the structure of formula (I):
TATp-Cys-LHRHp(I)
Wherein: TATp is the TAT fragment of HIV;
Cys is a halfcystine;
LHRHp is the analogue of r-hLH LHRH.
2. peptide carrier according to claim 1 is characterized in that the aminoacid sequence of said TATp is: N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg; Or its reverse sequence is: N end-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg;
3. peptide carrier according to claim 1 is characterized in that the aminoacid sequence of said LHRHp is: N end-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu; Or its reverse sequence:
N end-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly.
4. peptide carrier according to claim 1 is characterized in that said TATp-Cys-LHRHp sequence is shown in the SEQ ID NO.1:
N end-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
Or shown in the SEQ ID NO.2: the N end
-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly;
Or shown in the SEQ ID NO.3: the N end
-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Gly-Arg-Leu-Trp (D type)-Tyr-Ser-Trp-His-Glu;
Or the end of N shown in the SEQ ID NO.4
-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg-Cys-Glu-His-Trp-Ser-Tyr-Trp (D type)-Leu-Arg-Gly.
5. the purposes of the peptide carrier of one of claim 1-4.
6. the multipolymer that forms of the peptide carrier beautify chitosan of one of claim 1-4.
7. the purposes of the multipolymer of claim 6.
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| CN103031337A (en) * | 2012-09-28 | 2013-04-10 | 北京吉利奥生物科技发展有限公司 | Small nucleic acid molecule delivery technology |
| CN104815333A (en) * | 2015-04-05 | 2015-08-05 | 北京工业大学 | Preparation method and applications of poly ion micelle nano-particles |
| CN113058042A (en) * | 2021-04-01 | 2021-07-02 | 江苏中慧元通生物科技有限公司 | Preparation method of nasal-spray lipid nanoparticles capable of stably delivering RNA molecules |
| CN113069416A (en) * | 2021-03-31 | 2021-07-06 | 华中科技大学 | Active targeting amphiphilic polypeptide composite nano micelle prodrug and preparation and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031337A (en) * | 2012-09-28 | 2013-04-10 | 北京吉利奥生物科技发展有限公司 | Small nucleic acid molecule delivery technology |
| CN104815333A (en) * | 2015-04-05 | 2015-08-05 | 北京工业大学 | Preparation method and applications of poly ion micelle nano-particles |
| CN104815333B (en) * | 2015-04-05 | 2017-10-31 | 北京工业大学 | A kind of preparation method and applications of polyion micelle nano-particle |
| CN113069416A (en) * | 2021-03-31 | 2021-07-06 | 华中科技大学 | Active targeting amphiphilic polypeptide composite nano micelle prodrug and preparation and application thereof |
| CN113069416B (en) * | 2021-03-31 | 2022-05-17 | 华中科技大学 | Active targeting amphiphilic polypeptide composite nano micelle prodrug and preparation and application thereof |
| CN113058042A (en) * | 2021-04-01 | 2021-07-02 | 江苏中慧元通生物科技有限公司 | Preparation method of nasal-spray lipid nanoparticles capable of stably delivering RNA molecules |
| CN113058042B (en) * | 2021-04-01 | 2023-06-30 | 易慧生物技术(上海)有限公司 | Preparation method of lipid nanoparticle capable of being subjected to nasal spraying and used for stably delivering RNA molecules |
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