CN110652593A - Nucleus-targeted nano-drug carrier and preparation method and application thereof - Google Patents

Nucleus-targeted nano-drug carrier and preparation method and application thereof Download PDF

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CN110652593A
CN110652593A CN201910808571.0A CN201910808571A CN110652593A CN 110652593 A CN110652593 A CN 110652593A CN 201910808571 A CN201910808571 A CN 201910808571A CN 110652593 A CN110652593 A CN 110652593A
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pamam
nls
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韩世松
帅心涛
万文清
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Sun Yat Sen University
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Abstract

The invention discloses a nucleus targeting nano-drug carrier and a preparation method and application thereof. The cell nucleus targeting nano-drug carrier is prepared by grafting cell nucleus targeting peptide NLS on the surface of PAMAM to obtain PAMAM-NLS; wherein part of amino groups on the periphery of the PAMAM are respectively connected with a cell nucleus targeting peptide NLS, and the molar ratio of the amino groups on the periphery of the PAMAM to the cell nucleus targeting peptide NLS is 128: 20-128: 60. The invention takes the dendrimer PAMAM with small particle size as the basis, a certain amount of nuclear localization signal peptide NLS is modified on the surface of the dendrimer PAMAM, the size of the dendrimer PAMAM is controlled within 25nm, the recognition effect of the nuclear localization signal peptide NLS and a nuclear pore complex on a nuclear membrane is utilized, and simultaneously, the dendrimer PAMAM with small size can efficiently and smoothly pass through a cell nucleus and promote a nano carrier to be transferred into the cell nucleus, namely, a chemical drug or a gene drug with a target spot in the cell nucleus can be loaded to enter the cell nucleus, the drug effect is improved, and the use amount and the side effect of the drug are reduced; meanwhile, the nano-carrier can be degraded in vivo, and the problem of in vivo residue can not be caused.

Description

Nucleus-targeted nano-drug carrier and preparation method and application thereof
Technical Field
The invention relates to the technical field of nano-drugs, in particular to a cell nucleus targeting nano-drug carrier and a preparation method and application thereof.
Background
The biological body has a very complex physiological environment, the medicines for treating diseases need to pass through multiple obstacles from the intake to the exertion of the functions of human beings, only a small part of medicines which can exert the drug effect are always exerted finally, the treatment effect is seriously influenced, and meanwhile, strong toxic and side effects are brought. The nano-drug targeting delivery anti-tumor agent can promote the drug to pass through multiple biological barriers, enrich and release more tumor tissue cells, achieve the tumor targeting of drug delivery, weaken toxic and side effects and improve the effect of inhibiting tumors. The targeted nano-drug delivery becomes one of the most promising methods and research centers for tumor treatment, and has important significance for drug research of other diseases.
The delivery of drugs in the human body needs to overcome a plurality of obstacles including blood circulation, tumor enrichment, endocytosis, endosomal escape and the like, and a great deal of research is carried out on the several links of drug delivery at present. However, the true target of many drugs is in the nucleus, including some chemotherapy drugs such as adriamycin and camptothecin which destroy DNA, and gene therapy which needs to input up-regulated expressed genes. The research on the cell nucleus targeted transfer is weak at present, on one hand, the research is less, the efficiency of entering the cell nucleus is low, on the other hand, only a few nano materials can carry medicines to efficiently target and enter the cell nucleus, and most of the nano materials are inorganic nano particles which are not biodegradable. This is because the targeted delivery of the cell nucleus has strict requirements, and the foreign drug into the cell nucleus can only pass through the nuclear pore, so that the size is very limited. On the other hand, by virtue of the recognition of the nuclear targeting peptide and nuclear pore complex, the size limit of nanoparticles that can pass through the nuclear pore can be significantly increased to several tens of nanometers. Therefore, it is of great significance to develop and research drug carriers capable of targeting cell nuclei to pass through nuclear pores, and modification of cell nucleus localization signal peptides and application of small-sized nanoparticles can help to achieve the purpose.
Disclosure of Invention
The invention aims to provide a cell nucleus targeting nano-drug carrier aiming at the problems of small quantity, low efficiency, inorganic nano-particles as main materials and the like of the cell nucleus targeting transfer carrier. The cell nucleus targeting nano-drug carrier takes the dendrimer polyamide-amine PAMAM as a main carrier, and grafts the cell nucleus targeting peptide NLS, so that the drug carrier PAMAM-NLS which can efficiently target to enter the cell nucleus of a tumor is obtained, the size of the carrier is within 25nm, the drug carrier can efficiently enter the cell nucleus, and chemotherapeutic drugs such as DOX or plasmid DNA can be carried into the cell nucleus. Further, a tumor targeting group folic acid FA and PEG for promoting stability can be introduced, so that the nano-drug is multifunctional, and long blood circulation, tumor targeting endocytosis and cell nucleus targeting transportation are realized.
The invention also aims to provide a preparation method of the cell nucleus targeting nano-drug carrier.
The invention further aims to provide application of the cell nucleus targeting nano-drug carrier.
The above object of the present invention is achieved by the following scheme:
a nucleus-targeted nano-drug carrier is prepared by connecting amino on the surface of PAMAM with multiple nucleus-targeted peptides NLS.
The cell nucleus targeting nano carrier is based on dendrimer PAMAM, the dendrimer PAMAM is a highly branched polyamide-amine structure and is spherical nano particles, the size of 5-generation PAMAM is about 5nm, and 128 amino groups are arranged on the surface of a single molecule. The natural small size and the surface amino group are utilized, after a certain amount of nuclear localization signal peptide NLS is modified on the surface of the nano carrier, the size of the nano carrier is controlled within 25nm, and therefore the recognition effect of the nuclear localization signal peptide and a nuclear pore complex on a nuclear membrane is utilized to promote the nano carrier to be transported into a cell nucleus.
The cell nucleus targeting nano-carrier can wrap chemotherapeutic drugs in hydrophobic gaps through hydrophobic effect, or compound DNA through electrostatic effect, so that the drugs are carried and transferred into cell nucleus.
When all amino groups on the periphery of the PAMAM are grafted with NLS, the residual amino groups are insufficient, so that groups such as PEG and FA can not be continuously modified, and the surface electropositivity of the nano-carrier is influenced; however, when the amount of NLS grafted is small, the recognition ability of the complex with nuclear pore is insufficient, and the nuclear targeting ability is limited.
Preferably, the molar ratio of the peripheral amino groups of the PAMAM to the cell nucleus targeting peptide NLS is 128: 20-60. The nucleus targeting nano-drug carrier with the ratio within the range can realize good nucleus targeting function.
Preferably, the PAMAM is a 5 th generation polyamidoamine dendrimer, a single molecule of which has 128 amino groups; the cell nucleus targeting peptide NLS is derived from SV40, and the functional sequence of the cell nucleus targeting peptide NLS is PKKKRKV. The size of the 5 th generation polyamide-amine dendrimer is about 5nm, and 128 amino groups are arranged on the surface of a single molecule.
Preferably, in the cell nucleus targeting nano-drug carrier, the PAMAM can also be grafted with one or two of FA or PEG. Furthermore, a plurality of functions are introduced by modifying a tumor targeting group Folic Acid (FA) and/or a group PEG for promoting stability, and the like, so that tumor targeting endocytosis and further cell nucleus targeting drug transfer are realized.
The invention also protects the preparation method of the cell nucleus targeting nano-drug carrier, firstly, the amino on the periphery of the PAMAM reacts with Traut's reagent to be converted into sulfydryl; then, the sulfhydrylation PAMAM and NLS are mixed and dissolved in water, tri (2-carboxyethyl) phosphine and triethylamine are added for reaction, and after the reaction is finished, the cell nucleus targeting nano drug carrier PAMAM-NLS can be obtained by dialysis and freeze-drying.
Preferably, the preparation process of the thiolated PAMAM is as follows: dissolving PAMAM in a polar solvent, adding Traut's reagent, triethylamine and tri (2-carboxyethyl) phosphine, uniformly mixing, and reacting in an inert gas atmosphere to obtain the sulfhydrylated PAMAM.
Preferably, the polar solvent is methanol, ethanol or water; more preferably methanol.
The inert gas is preferably nitrogen.
Preferably, in the preparation process of the sulfhydrylated PAMAM, the reaction molar ratio of PAMAM, Traut's reagent, triethylamine and tris (2-carboxyethyl) phosphine is as follows: 1:60:120: 120; the reaction temperature is room temperature, and the reaction time is 12-24 h;
in the reaction process of the sulfhydrylated PAMAM and NLS, the reaction molar ratio of the sulfhydrylated PAMAM, the NLS, triethylamine and tri (2-carboxyethyl) phosphine is as follows: 1: 20-60: 120: 120; the reaction temperature is room temperature, and the reaction time is 24-48 h.
Preferably, the nuclear localization signal peptide NLS is synthesized by Fmoc protection strategy of polypeptide solid-state synthesis technology, and is connected with maleimide propionic acid (Mal) group at the nitrogen terminal.
More preferably, the NLS is synthesized by: rink Amide AM resin is adopted, dry DMF is taken as a solvent, amino acid protected by Fmoc is taken as a raw material, HoBt and HBTU are taken as condensation reagents, and DIEA is taken as an alkali catalyst; boiling with 10mg/mL ninhydrin solution in methanol, and maintaining for 1 min to observe whether the resin is discolored or not to check whether the condensation reaction is complete; fmoc protection group removal was performed with 20% piperidine/DMF (v/v) for 5+15 min. After all amino acids are grafted, introducing maleimide group into the N end of the polypeptide, wherein the reagent is maleimide propionic acid, and the coupling method is the same as that of the amino acids.
The cracking agent formula adopts 95% trifluoroacetic acid, 2.5% trimethylsilane and 2.5% deionized water to react for 2 h. The structure of the polypeptide was confirmed by ESI-MS mass spectral characterization.
Preferably, the PAMAM and the activated PEG and/or FA are mixed uniformly and react to obtain the PAMAM grafted with the PEG and/or FA; then, the PAMAM is reacted with Traut's reagent to convert the residual amino on the surface of the PAMAM into sulfydryl; and finally, mixing the cells with NLS in water, adding tri (2-carboxyethyl) phosphine and triethylamine for reaction, dialyzing and freeze-drying after the reaction is finished to obtain the FA and/or PEG grafted cell nucleus targeted nano drug carrier PAMAM-NLS.
The application of the cell nucleus targeting nano-drug carrier is also in the protection scope of the invention, and the cell nucleus targeting nano-drug carrier can load chemical drugs or gene drugs.
Preferably, the target of the chemical or genetic drug is in the nucleus. For example, the chemical drug may be doxorubicin DOX, which enters the pores of PAMAM by hydrophobic interaction after alkaline desalting, i.e. successfully loaded on the carrier; the gene medicine is plasmid pDNA which is negative charge, the nucleus targeting nano-medicine carrier is positive charge, and the pDNA can be loaded on the nucleus targeting nano-medicine carrier through electrostatic interaction.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the dendrimer PAMAM with small particle size as the base, after a certain amount of nuclear localization signal peptide NLS is modified on the surface of the dendrimer PAMAM, the size of the dendrimer PAMAM is controlled within 25nm, the recognition effect of the nuclear localization signal peptide NLS and a nuclear pore complex on a nuclear membrane can be realized, simultaneously, the dendrimer PAMAM with small size can efficiently and smoothly pass through a cell nucleus, the nano-carrier is promoted to be transferred into the cell nucleus, and simultaneously, the chemical drug or the gene drug with a target point in the cell nucleus can be loaded to enter the cell nucleus, so that the drug effect is improved, and the use amount and the side effect of the drug are.
Furthermore, a tumor targeting group folic acid FA and PEG for promoting stability can be introduced into the cell nucleus targeting nano-drug carrier, so that the nano-drug is multifunctional, has good stability of aqueous solution and serum, can target more enrichment of tumor cells in tumor tissues, realizes long blood circulation, tumor targeting endocytosis and cell nucleus targeting transportation, and improves the drug treatment effect.
In addition, the PAMAM and the nuclear localization signal peptide are high molecular materials rich in amide structures, can be degraded in vivo, and do not have the problems of in vivo residue and non-degradability of inorganic nanoparticles.
Drawings
FIG. 1 is the chemical structure of NLS prepared in example 1 and its ESI-MS mass spectrum.
FIG. 2 is the nuclear magnetic spectrum of the PAMAM in examples 1, 2 and 3 labeled with FITC fluorescence and modified with-SH, NLS, FA and PEG.
FIG. 3 is a transmission electron micrograph of PAMAM-NLS/FA/PEG prepared in example 2.
FIG. 4 is a confocal laser photograph of PAMAM-NLS prepared in example 3 cultured in C6 cells for 6 h.
FIG. 5 is a confocal laser photograph of PAMAM-NLS/FA/PEG prepared in example 3 cultured in C6 cells for 6 h.
FIG. 6 is a confocal laser photograph labeled with YOYO-1 after culturing the PAMAM-NLS/FA/PEG-loaded p53 plasmid in 7402 cells for 6h in example 4.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of nucleus-targeted dendrimer nanocarriers
The preparation process of the cell nucleus targeting peptide modified PAMAM-NLS comprises the following specific steps:
1. synthesis of Nuclear localization Signal peptide NLS (Mal-PKKKRKV): the Fmoc protection strategy is manually synthesized by adopting a solid polypeptide synthesis method, and the amino acid-containing peptide is prepared on Rink Amide AM resin in a mode of gradually condensing amino acid. Specifically, freshly distilled dry DMF was used as a reaction solvent, 3 equivalents of Fmoc amino acid, HoBt, HBTU and 8 equivalents of DIEA were added to each step of the condensation of amino acids to react for 2 hours, and the reaction was checked for completion by boiling with 10mg/mL of ninhydrin in methanol and maintaining for 1 minute to observe whether the resin was discolored or not.
Fmoc protecting group removal in each step was performed with 20% piperidine/DMF (v/v) for 5+15 min. After all amino acids are grafted, introducing maleimide group into the N end of the polypeptide, wherein the reagent is maleimide propionic acid, and the coupling method is the same as that of the amino acids. Washing with DMF, anhydrous methanol and dichloromethane for three times, vacuum drying, adding cracking agent (95% trifluoroacetic acid, 2.5% trimethylsilane and 2.5% deionized water) for reaction for 2h, cracking the polypeptide from the resin, suction filtering, collecting filtrate, rotary evaporating for concentration, adding anhydrous ether to generate precipitate, centrifuging, vacuum drying to obtain NLS Peptide (Mal-PKKKRKV-NH2), and determining the polypeptide structure by ESI-MS mass spectrum characterization.
The chemical structural formula of NLS and ESI-MS mass spectrum thereof are shown in FIG. 1. As can be seen from FIG. 1, the theoretical molecular weight of NLS is identical to the measured molecular weight of the product, which is 1035, and it is confirmed that the polypeptide has been successfully synthesized.
2. Sulfhydrylation modification of dendrimer PAMAM: 1.0G of PAMAM (generation 5, G5) in methanol (20% content) was taken, 95mg of Traut's reagent (100eq) was added, dissolved in 10mL of methanol +2mL of deionized water, and 400uL (4eq) of triethylamine and 200mg (1eq) of tris (2-carboxyethyl) phosphine (TCEP) were added and dissolved completely by sonication. Vacuumizing, introducing nitrogen, and reacting overnight under stirring. Transferring into a dialysis bag with MWCO of 3500, dialyzing with methanol, adding ether for precipitation, and vacuum drying to obtain PAMAM-SH. The synthesis was confirmed by hydrogen nuclear magnetic resonance spectroscopy.
3. Preparing a cell nucleus targeting nano-drug carrier PAMAM-NLS: 30mg of PAMAM-SH is taken out and dissolved in water, 40 times of Mal-PKKKRKV peptide is added, 6mg of TCEP and 12uL of triethylamine are added, and the mixture is magnetically stirred for reaction for 2 days. Then dialyzing with water, and freeze-drying to obtain the cell nucleus targeting nano-drug carrier PAMAM-NLS.
Example 2 preparation of multifunctional Nanocarriers with Nuclear targeting, tumor targeting, and Long blood circulation
Meanwhile, the cell nucleus targeting nano-drug carrier PAMAM-NLS grafted with FA and PEG is prepared by the following specific process:
1. preparation of PAMAM-FA/PEG: 30mg of G5 PAMAM was reacted with 30eq of PEG2000 and 30eq of FA; wherein, DCC/NHS is used for activating PEG and FA, PAMAM is added, and the mixture is stirred and reacted for 2 days by magnetic force; transferring into dialysis bag, dialyzing with methanol, dialyzing with water, and lyophilizing to obtain PAMAM-FA/PEG product.
2. Synthesis of PAMAM-FA/PEG/SH: the PAMAM-FA/PEG was added to Traut's reagent to react, and the remaining part of amino groups on the surface of PAMAM were converted into thiol groups, in the same manner as in step 2 of example 1.
3. Preparation of PAMAM-FA/PEG/NLS: PAMAM-FA/PEG/SH is taken to react with NLS peptide prepared in the step 1 in the example 1; the specific reaction process is the same as that of the step 3 of the example 1, so that a PAMAM-FA/PEG/NLS solid product is obtained, and the chemical structure of the PAMAM-FA/PEG/NLS solid product is shown as the following figure:
the dendritic core is a 5-generation PAMAM dendrimer, the black mark part is PEG, the blue mark part is FA, and the red mark part is NLS; as can be seen from the above structural schematic diagram, PEG, FA and NLS are grafted on the surface of PAMAM.
The transmission electron micrograph of PAMAM-NLS/FA/PEG is shown in FIG. 3, from which it can be seen that the nano-carrier is relatively uniform particles, and most of the size is between 10-20 nm.
Example 3 fluorescent labeling
PAMAM, PAMAM-NLS prepared in example 1, and PAMAM-NLS/FA/PEG 3 carriers prepared in example 2 were modified with FITC for green fluorescent labeling.
The specific method for modifying the fluorescent dye FIT comprises the following steps: respectively dissolving the carrier materials in deionized water, adding 5eq of FITC and 5eq of triethylamine, stirring for one day in a dark place, and dialyzing water to obtain various FITC fluorescence labeled nano carriers.
The nuclear magnetic resonance spectrogram of FITC-labeled PAMAM, PAMAM-NLS and PAMAM-NLS/FA/PEG is shown in figure 2. As can be seen from FIG. 2, the characteristic peaks of each polymer can be clearly detected, thus proving that 3 carriers have been successfully synthesized.
Example 4 Nuclear targeting Nanoparticulate Carrier PAMAM-NLS Loading drug
1. Loaded chemical drugs
Taking doxorubicin DOX as an example, the PAMAM-NLS loaded with DOX is prepared by the following specific preparation process: dissolving 20mg of PAMAM-NLS material in methanol, adding DOX 5mg, adding triethylamine 2mg, magnetically stirring for one day in a dark place, and dialyzing water to obtain DOX @ PAMAM-NLS.
By referring to the method, PAMAM-NLS is replaced by PAMAM-NLS/FA/PEG, and DOX @ PAMAM-NLS/FA/PEG can be prepared.
2. Gene-loaded medicine
Taking the composite plasmid pDNA as an example, the PAMAM-NLS loaded with the pDNA is prepared by the following specific preparation process: dissolving 10mg of PAMAM-NLS material in deionized water, adding pDNA (taking pDNA of EGFP gene as an example) with different masses, slightly swirling, and mixing for more than 0.5 h to obtain the PAMAM-NLS @ pDNA.
The compounding condition of the vector and the DNA is detected through agarose gel electrophoresis experiments, and multiple experiments show that when the mass ratio of the PAMAM-NLS to the DNA is more than or equal to 3:1, the vector can completely compound the DNA (all plasmid DNA is compounded with the vector through electrostatic interaction).
By referring to the method, PAMAM-NLS is replaced by PAMAM-NLS/FA/PEG, and then PAMAM-NLS/FA/PEG @ pDNA can be prepared.
Example 5 Nuclear Targeted delivery Effect of Nanoparticulate Carrier
The sizes of the empty nano-carriers (PAMAM, PAMAM-NLS and PAMAM-NLS/FA/PEG) or the nano-carriers loaded with drugs (DOX @ PAMAM-NLS, DOX @ PAMAM-NLS/FA/PEG, PAMAM-NLS @ pDNA, PAMAM-NLS/FA/PEG @ pDNA) are determined by dynamic light scattering, and the sizes and the distribution of the appearances are observed by a transmission electron microscope to ensure that the sizes are within 25 nm.
The cell nucleus targeting effect is observed by a confocal fluorescence microscope after cell co-culture, and the PAMAM-NLS/FA/PEG are respectively grafted with green fluorescence FITC or chemotherapeutic drug DOX encapsulating red fluorescence or composite plasmid pDNA to prepare FITC-PAMAM-NLS, FITC-PAMAM-NLS/FA/PEG, DOX @ PAMAM-NLS/FA/PEG and PAMAM-NLS/FA/PEG @ pDNA; the specific detection method comprises the following steps:
mixing C6 cell or 7402 cell at 5 × 104The density of each hole is inoculated in a confocal cell culture dish, materials to be researched (FITC-PAMAM-NLS, FITC-PAMAM-NLS/FA/PEG, DOX @ PAMAM-NLS/FA/PEG, PAMAM-NLS/FA/PEG @ pDNA) are added after 24 hours, the culture medium is sucked after incubation for certain time (1 hour, 3 hours, 6 hours and 12 hours), PBS is used for washing for 3 times, Hoechest33342 is used for staining cell nucleus for 15min, the culture medium is sucked again, PBS is used for washing for 3 times, and a normal culture medium is added; observing under a laser confocal fluorescence microscope, wherein the cell nucleus is blue fluorescence, FITC is green fluorescence, and the pDNA is marked by YOYO-1 before being added and is also green fluorescence.
The fluorescence intensity and distribution of the nano-drug in the cells were observed, and the results are shown in FIGS. 4 to 6.
FIG. 4 is a confocal laser photograph of PAMAM-NLS labeled FITC prepared in example 1 (example 3) cultured in C6 cells for 6 hours. The fluorescence and distribution of FITC labeled nano-carrier FITC-PAMAM-NLS in cells are shown in (1), (2) cell nuclei stained with DAPI (DAPI), (3) an overlay image of carrier green fluorescence and cell nucleus blue fluorescence, and (4) an intensity linear graph of green fluorescence and yellow fluorescence in a streaked passing range of the image (3). As can be seen from FIG. 4, the nano-carrier PAMAM-NLS enters the cell nucleus in a large amount, and the distribution of the nano-carrier PAMAM-NLS in the cell nucleus is overlapped with the position of the cell nucleus, so that an obvious cell nucleus targeting effect is realized.
FIG. 5 is a confocal laser photograph of PAMAM-NLS/FA/PEG-labeled FITC prepared in example 2 (example 3) cultured in C6 cells for 6 h. The fluorescence intensity of the cell nucleus is shown in the graph (3). As can be seen from FIG. 5, the nano-carrier PAMAM-NLS/FA/PEG enters the cell nucleus in a large amount, and an obvious cell nucleus targeting effect is realized.
FIG. 6 is a confocal laser photograph of PAMAM-NLS/FA/PEG-loaded p53 plasmid labeled with YOYO-1 and cultured in 7402 cells for 6h in example 4. Wherein (1) is a photograph of DAPI-stained nuclei, (2) is the fluorescence and distribution of YOYO-1-labeled p53 plasmid, (3) a photograph of bright field of 7402 cells, and (4) is an overlay of the graphs (1-3). As can be seen from FIG. 4, the plasmid DNA p53 entered the interior of the cell in large quantities, and the distribution in the cell approximately overlapped with the nucleus, achieving good target delivery effect of the nucleus.
The experiment shows that the nano-carrier can enter the cell nucleus and can also carry pDNA to enter the cell nucleus, thereby achieving good cell nucleus targeting transfer effect.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A cell nucleus targeting nano-drug carrier is characterized in that amino on the surface of PAMAM is connected with a plurality of cell nucleus targeting peptides NLS to obtain the PAMAM-NLS.
2. The cell nucleus-targeted nano-drug carrier of claim 1, wherein the molar ratio of the peripheral amino groups of the PAMAM to the cell nucleus-targeted peptide NLS is 128: 20-60.
3. The nuclear-targeted nanomedicine carrier of claim 1, wherein the PAMAM is a generation 5 polyamidoamine dendrimer having 128 amino groups per molecule; the cell nucleus targeting peptide NLS is derived from SV40, has a functional sequence of PKKKRKV, and is connected with a maleimidopropionic acid group at the nitrogen end.
4. The nuclear-targeted nano-drug carrier of claim 1, wherein in the nuclear-targeted nano-drug carrier, the PAMAM can be grafted with one or both of FA and PEG.
5. The method for preparing the cell nucleus targeting nano-drug carrier as claimed in any one of claims 1 to 3, wherein the amino group at the periphery of PAMAM reacts with Traut's reagent to be converted into sulfhydryl group; and then, dissolving sulfhydrylation PAMAM and NLS in water, adding tri (2-carboxyethyl) phosphine and triethylamine for reaction, and dialyzing and freeze-drying after the reaction is finished to obtain the cell nucleus targeting nano-drug carrier PAMAM-NLS.
6. The method for preparing the cell nucleus targeting nano-drug carrier according to claim 5, wherein the preparation process of the thiolated PAMAM is as follows: dissolving PAMAM in a polar solvent, adding Traut's reagent, triethylamine and tri (2-carboxyethyl) phosphine, uniformly mixing, and reacting in an inert gas atmosphere to obtain the sulfhydrylated PAMAM.
7. The method for preparing the cell nucleus targeting nano-drug carrier according to claim 6, wherein the polar solvent is methanol, ethanol or water; in the preparation process of the sulfhydrylation PAMAM, the reaction molar ratio of the PAMAM, Traut's reagent, triethylamine and tri (2-carboxyethyl) phosphine is as follows: 1:60:120: 120; the reaction temperature is room temperature, and the reaction time is 12-24 h;
in the reaction process of the sulfhydrylated PAMAM and NLS, the reaction molar ratio of the sulfhydrylated PAMAM, the NLS, triethylamine and tri (2-carboxyethyl) phosphine is as follows: 1: 20-60: 120: 120; the reaction temperature is room temperature, and the reaction time is 24-48 h.
8. The preparation method of the cell nucleus targeting nano-drug carrier of claim 4 is characterized in that the PAMAM and the activated PEG and/or FA are mixed uniformly and react to obtain the PAMAM grafted with PEG and/or FA; then, the PAMAM is reacted with Traut's reagent to convert the residual amino on the surface of the PAMAM into sulfydryl; and finally, mixing the cells with NLS in water, adding tri (2-carboxyethyl) phosphine and triethylamine for reaction, dialyzing and freeze-drying after the reaction is finished to obtain the FA and/or PEG grafted cell nucleus targeted nano drug carrier PAMAM-NLS.
9. The use of the nuclear-targeted nano-drug carrier as claimed in any one of claims 1 to 4, wherein the nuclear-targeted nano-drug carrier can be loaded with chemical drugs or gene drugs.
10. The use of the nuclear-targeted nanomedicine carrier of claim 9, wherein the target of the chemical or genetic agent is within the nucleus.
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