CN103755953B - A kind of intelligent polycation nano-carrier, its preparation method and application thereof - Google Patents
A kind of intelligent polycation nano-carrier, its preparation method and application thereof Download PDFInfo
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Abstract
The present invention is applicable to nanosecond medical science field, can mediate the intelligent polycation nano-carrier of active targeting propagation function and reduction sensitive function, its preparation method and application thereof the while of providing a kind of.This intelligent polycation nano-carrier; comprise TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; this intelligent polycation nano-carrier is divided into four layers from outside to inside successively; wherein; outermost layer is the peptide T P that can mediate active targeting transmission; the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, and innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking.This intelligent polycation nano-carrier preparation can realize the multiple loaded article of load simultaneously, and can be biodegradable completely, possess the function such as active targeting transmission and reduction responsiveness, and its particle diameter and current potential can realize regulation and control simultaneously.
Description
Technical field
The invention belongs to nanosecond medical science field, particularly relate to a kind of intelligent polycation nano-carrier, its preparation method and application thereof.
Background technology
Nano-carrier refers to can the target substance such as carrying medicament, gene and protein and have the carrier system of nanoscale.Amphipathilic block polymer nano-micelle carrier has hydrophilic segment and hydrophobic segment simultaneously, after it reaches micelle-forming concentration in water, will microphase-separated be there is in the hydrophilic segment of polymkeric substance and hydrophobic segment, spontaneously define hydrophobic segment interior, the nano-micelle of hydrophilic segment " core-shell structure copolymer " structure outside, this polymer micelle has lower micelle-forming concentration, larger solubilising space, stable kernel, can by chemistry, hydrophobic drug bag is loaded in its hydrophobic inner core by the mode such as physics and coordination, and when containing positively charged ion or anionic block in polymkeric substance simultaneously, this nano-micelle is energy load gene or protein simultaneously just, the common transmission of both realizations.
Targeting drug delivery system arrives specific physiological site, organ, tissue or cell with referring to drug selectivity, and plays pharmacological agent effect at this target site.Selectivity administration can strengthen medicine in the activity of target site and reduce its toxic side effect to non-target site, improves the therapeutic index of medicine.The method of drug targeting generally has two kinds: active targeting and passive target.Active targeting utilizes the biologic specificities such as Ag-Ab combination or ligand-receptor combination to interact to realize the targeted delivery of medicine, and passive target refers to by reducing the levels of drugs ratio increasing target/non-target site with the non-specific interaction of non-target organ, tissue and cell.
Nano-gene carrier refers to can the transfer system of load genetic stew (DNA or RNA), is mainly used in gene therapy process to target position transfer gene.Gene therapy is by importing in patient body by therapeutic gene, makes dcc gene up or express to produce medicative protein thus the effect reaching disease therapy.Gene therapy can fundamentally treat a lot of disease, is one of study hotspot of Medical and chemical field.From current present Research, the key issue of gene therapy how to use suitable carrier effectively to be imported by gene in body and makes it express, need the physiologic barrier of leap a lot of in this process, be very easy to degraded, inactivation as genetic stew or be difficult to arrive target cell interior.Cationic polymers nano-gene carrier is study hotspot nearly ten years; it utilizes reactive force adsorption of DNA between positive and negative charge or RNA; form gene/carrier complexes, this method can effectively not be degraded and easily be reached target cell interior by cytophagy by Protecting gene.Conventional cationic polymer gene vector has polymine, polylysine (PLL) etc.
Nanoprotein matter carrier refers to can the nanosystems of load protein or polypeptide, nanoprotein matter carrier be mainly used in macro-molecular protein medicine transmission or for load antibodies as vaccine adjuvant.This nanoprotein matter carrier needs with stronger positive charge, thus can stablize the protein of the macromolecule of adsorption zone negative charge.Common nanoprotein matter carrier has cationic-liposome, gel, micella etc.
Existing nano-medicament carrier existence and stability is poor, medicine easily leaks, target spot position accumulation less, biocompatibility and biodegradability poor, easily removed by immunity system, can not simultaneously a series of shortcoming such as load small-molecule drug, gene and protein, these shortcomings deposit the application that greatly limit polymer nanoparticle drug carriers.
Summary of the invention
The object of the present invention is to provide a kind of intelligent polycation nano-carrier, be intended to solve existing anti-tumor nano carrier can not the multiple loaded article of load simultaneously, poor stability, can not be completely degraded or metabolism, active targeting can not be possessed simultaneously transmit the problem that the function such as pH response and reduction responsiveness and its particle diameter can not regulate and control.
Another object of the present invention is to the preparation method of the intelligent polycation nano-carrier that a kind of easy control simple to operate is provided.
Another object of the present invention is to provide a kind of intelligent polycation nano-carrier composition, be intended to solve existing anti-tumor nano carrier can not simultaneously the multiple loaded article of load and nanometer carrier combination poor stability, can not be completely degraded or metabolism, active targeting can not be possessed simultaneously transmit the problem that the function such as pH response and reduction responsiveness and its particle diameter can not regulate and control.
Correspondingly, present invention also offers a kind of preparation method of intelligent polycation nano-carrier composition.
And a kind of intelligent polycation nano-carrier is in the application in nano-drug transporter, fluorescence dye carrier, bioprobe carrier field.
The embodiment of the present invention realizes like this; a kind of intelligent polycation nano-carrier; comprise TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; this intelligent polycation nano-carrier is divided into four layers from outside to inside successively; wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding; third layer is the positive charge layer that PLL is formed, and innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking.
Correspondingly, a kind of preparation method of intelligent polycation nano-carrier, comprises the steps:
The synthesis of TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer: by HOOC-PEG-NH
2as initiator, progressively cause amino acid N CA ring-opening polymerization synthesis PEG-b-PLL (Boc)-b-PLC (Trt); With phenylpropionic acid succinimide ester for end-capping reagent, the amino closing PLC (Trt) end obtains PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; Combine with the peptide T P that can mediate active targeting transmission after the carboxyl that activated polymer PEG holds and obtain TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is obtained after removing TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent Side chain protective group Boc group and Trt group;
The formation of intelligent polycation nano-carrier: described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent and forms homogeneous phase solution, described solution is carried out dialysis treatment, obtains intelligent polycation nano-carrier.
And, a kind of intelligent polycation nano-carrier composition, comprise nano-carrier and the loaded article of load on nano-carrier, described nano-carrier is above-mentioned intelligent polycation nano-carrier, and described loaded article load is in the hydrophobic inner core of the positive charge layer of described intelligent polycation nano-carrier and/or the PLC of disulfide bond crosslinking.
Correspondingly, a kind of preparation method of intelligent polycation nano-carrier composition, comprises the steps:
According to above-mentioned synthesis described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer method synthesis TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer;
Described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and described loaded article are carried out following process:
When loaded article is dewatering medicament, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and loaded article are dissolved in organic solvent and form homogeneous mixture solution, then carry out dialysis treatment, lyophilize, obtain the intelligent polycation nano-carrier dry powder drug being loaded with dewatering medicament.；
When loaded article be protein and/or genetic stew time, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent, then dialysis treatment is carried out, add loaded article mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with protein or genetic stew;
When loaded article is simultaneously containing dewatering medicament, protein and/or genetic stew, first described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and hydrophobic drug are dissolved in organic solvent and form homogeneous mixture solution, then dialysis treatment is carried out, add protein and/or genetic stew mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with protein or genetic stew.
And a kind of intelligent polycation nano-carrier is in the application in nano-drug transporter, fluorescence dye carrier, bioprobe carrier field.
A kind of intelligent polycation nano-carrier provided by the invention, comprise the TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer of the four-layer structure being divided into differing chemical properties from outside to inside successively, the layer of structure making this nano-carrier preparation different can the different loaded article of load, and it has good stability, can complete biodegradable and metabolism; Possess the functions such as active targeting transmission, pH response and reduction responsiveness simultaneously, easily cross over physiologic barrier complicated in human body, thus can avoid suffering immune removing and arriving focus smoothly; In addition, the particle diameter of this nano-carrier preparation can be well controlled by regulating the input amount of triblock polymer.
A kind of intelligent polycation nano-carrier composition provided by the invention, owing to having above-mentioned intelligent polycation nano-carrier, and described loaded article load is in the different structure layer of described intelligent polycation nano-carrier, make this nanometer carrier combination can meet the function of the simultaneously multiple loaded article of different nature of load, and its stable in properties, metabolism are complete; Active targeting transmission can be realized simultaneously, pH want with and function such as reduction response etc., easily cross over the physiologic barrier of complexity in human body, avoid immune removing; And the particle diameter of this nanometer carrier combination is well controlled by regulating the input amount of triblock polymer.
The preparation method of intelligent polycation nano-carrier provided by the invention and intelligent polycation nano-carrier composition thereof, method is simply controlled, has good market outlook.
Accompanying drawing explanation
Fig. 1 is structure and the assembling schematic diagram thereof of the nanometer carrier combination that the embodiment of the present invention provides.
Fig. 2 is the nano-carrier DLS grain-size graph that the embodiment of the present invention provides.
Fig. 3 is the cytotoxicity experiment figure of the nano-carrier that the embodiment of the present invention provides.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
In the technical scheme that the embodiment of the present invention provides, TP, PEG, PLL, PLC, HPPr, Boc, Trt, DMF of occurring are explained as follows:
TP: the peptide sequence that can mediate active targeting;
PEG: polyoxyethylene glycol;
PLL: polylysine;
PLC: poly-halfcystine;
HPPr: phenylpropionic acid;
Boc：
Trt：
DMF:N, dinethylformamide;
B: polymkeric substance is block polymer.
Polyoxyethylene glycol (PEG) and polyamino acid all have good biocompatibility and biodegradability, and its degraded product does not all have toxicity, are therefore widely used in biomaterial and nanosecond medical science field.Nano microsphere after PEG finishing, its cell adhesion reduces, and in serum, the adsorptivity of material reduces, and minimizing is engulfed in the rejection of scavenger cell simultaneously, and therefore, nano material, after PEG modifies, significantly can increase its time of circulating in vivo.Amino acid has multiple side-chain radical, amino acid after polymerization, by carrying out different modifications to its pendant reactive group, thus obtain the polyamino acid material of various different in kind, as hydrophilic, hydrophobic, electropositivity, electronegativity etc., so the polyamino acid very potential biomaterial that is one.
In view of this; embodiments provide a kind of intelligent polycation nano-carrier; comprise TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; this intelligent polycation nano-carrier is divided into four layers from outside to inside successively; wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding; third layer is the positive charge layer that PLL is formed, and innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking.
Particularly, in above-mentioned intelligent polycation nano-carrier, by regulating the ratio of each block of TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, effectively control the particle diameter of nano-carrier.As preferred embodiment, the DLS grain-size graph of the particle size range of described intelligent polycation nano-carrier to be 10-500nm, Fig. 2 be embodiment of the present invention intelligent polycation nano-carrier.As another preferred embodiment, the aqueous solution Zeta potential size of described intelligent polycation nano-carrier is+10mv-+50mv.
For making described intelligent polycation nano-carrier, there is acceptable cytotoxicity, to medicine, gene or albumen have efficient load, and described PEG chain segment number-average molecular weight scope is 500KD-10000KD, the described PLL segment polymerization degree is 10-200, and the polymerization degree of described PLC segment is 10-200.
Above-mentioned TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer be linear polymeric structure, wherein PEG is amino-polyoxyethylene glycol-carboxyl (HOOC-PEG-NH
2), described PEG aminoterminal is connected by amido linkage with PLL one end, and the other end of described PLL and PLC are by amido linkage covalent attachment, and end-capping reagent is connected to the tail end of PLC.
Described intelligent polycation nano-carrier, with by the above-mentioned TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer be connected to form for skeleton, in aqueous can automatic Composition formed nucleocapsid structure.Described nucleocapsid structure is divided into four layers from outside to inside, be followed successively by: outermost layer to mediate the peptide T P that active targeting transmits, due to can with some protein receptor binding of some tumour cells or endothelial cells in tumor neogenetic blood vessels specificity overexpression and realize active targeting administration object; Secondary skin is protective layer PEG, and it can reduce specific adsorption, thus increases nano-micelle cycling time in vivo; Third layer is the positive charge layer that PLL is formed, can the carrying capacity of the electronegative species such as enhancing gene and protein greatly; Innermost layer be the PLC of internal layer by disulfide bond crosslinking, form three-dimensional cross-linked reticulated hydrophobic kernel, nano-carrier stability strengthened, avoids the leakage of medicine, when arrive tumor tissues time disulfide bonds, disulfide linkage solution be cross-linked, medicine discharges fast.The hydrophobic cores that TP-PEG-b-PLL-b-PLC-HPPr triblock polymer nano-micelle has active targeting simultaneously, reduction responds and positive charge layer, therefore can simultaneously effective load hydrophobic drug, gene (DNA or RNA) and protein, be a kind of multifunctional nano carrier.
In order to the cytotoxicity preventing the terminal amino group of TP-PEG-b-PLL-b-PLC and target polypeptide sequence bonding from not increasing carrier, introduce nontoxic end-capping reagent at the end of above-mentioned TP-PEG-b-PLL-b-PLC.As preferred embodiment, described end-capping reagent is alkane or the aromatic hydrocarbons of activated carboxylic.As further preferred embodiment, described end-capping reagent is HPPr, stearic acid succinimide ester etc.
The intelligent polycation nano-carrier that the embodiment of the present invention provides, forms nucleocapsid structure by TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; This nucleocapsid structure has four-layer structure, and outermost layer is the peptide T P that can mediate active targeting transmission, and secondary skin is the PEG shielded, and third layer is the positive charge layer of PLL, and innermost layer is the PLC hydrophobic inner core of disulfide bond crosslinking.There is the intelligent polycation nano-carrier of this particular core shell structure, there is following advantage:
1, the framework material of intelligent polycation nano-carrier is TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, its good biocompatibility, toxicity is low, can be completely degraded in vivo, and degraded product is nontoxic, harmless, can be absorbed or metabolism in vivo.
2, intelligent polycation nano-carrier can mediate active targeting transmission peptide molecule TP can with some tumour cells or endothelial cells in tumor neogenetic blood vessels and specifically high expression level protein receptor binding thus can target administration be realized, reduce its toxic side effect at non-target site.
3, the innermost layer PLC of intelligent polycation nano-carrier passes through disulfide bond crosslinking, form three-dimensional cross-linked reticulated hydrophobic kernel, cation nanometer vector stabilisation is increased, avoids medicine premature leakage, when after arrival target spot, micella solution is cross-linked rapid delivery of pharmaceuticals.
4, intelligent polycation nano-carrier good stability, its PEG shell can reduce non-specific adsorption available protecting nano-carrier preparation and avoid being removed fast by immunity system.
5, intelligent polycation nano-carrier is owing to having the different structure of multilayer chemical environment, therefore, can realize simultaneously efficiently auxiliary hydrophobic drug, gene and protein even load thing arrive the function of target spot, and its particle diameter is well controlled by the structure of telomerized polymer.
Described in the embodiment of the present invention, intelligent polycation nano-carrier preparation prepares by following method, certainly, also can be obtained the method preparation of intelligent polycation nano-carrier preparation by other.
Correspondingly, embodiments provide a kind of preparation method of intelligent polycation nano-carrier, comprise the steps:
The synthesis of S01.TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer: by HOOC-PEG-NH
2as initiator, progressively cause amino acid N CA ring-opening polymerization synthesis PEG-b-PLL (Boc)-b-PLC (Trt); With phenylpropionic acid succinimide ester for end-capping reagent, the amino closing PLC (Trt) end obtains PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; Combine with the peptide T P that can mediate active targeting transmission after the carboxyl that activated polymer PEG holds and obtain TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is obtained after removing TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent Side chain protective group Boc group and Trt group;
S02. the formation of intelligent polycation nano-carrier: described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent and forms homogeneous phase solution, described solution is carried out dialysis treatment, obtains intelligent polycation nano-carrier.
Concrete, in above-mentioned steps S01, the synthesis of TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is with HOOC-PEG-NH
2for initiator, by progressively causing method synthesis PEG-b-PLL (Boc)-b-PLC (Trt) triblock polymer of amino acid N CA ring-opening polymerization, with containing the alkane of activated carboxylic or aromatic hydrocarbons if HPPr is for end-capping reagent, close the amino of PLC (Trt) end, obtain PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent triblock polymer; Then by the activated carboxylic of the PEG of PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent end, by this activated carboxyl, PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent is combined with target polypeptide TP, obtains TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent triblock polymer; Again Side chain protective group Boc and Trt of PLL (Boc) and PLC (Trt) is sloughed and obtain TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer.Wherein, described PEG is HOOC-PEG-NH
2, its number-average molecular weight scope is 500KD-10000KD, and the number-average molecular weight of prepared TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is 1500KD-60000KD.
Particularly, TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer concrete technology flow process is as described below:
S011. by HOOC-PEG-NH
2(optional number-average molecular weight scope is 500KD-10000KD) is dissolved in DMF and obtains the solution that concentration range is 1-100mg/mL, in atmosphere of inert gases-as under nitrogen protection, by Lys (Boc)-NCA and HOOC-PEG-NH
2mol ratio be (10-200): 1 adds Lys (Boc)-NCA monomer, at 30-50 DEG C thermostatically heating reaction 24-120 hour; After question response terminates, in atmosphere of inert gases-as under nitrogen protection, by Cys (Trt)-NCA monomer and HOOC-PEG-NH
2mol ratio be (10-200): 1 adds Cys (Trt)-NCA, isothermal reaction 24-120 hour at 30-50 DEG C; After question response terminates, add the ether of 5-50 times of volume, obtain PEG-b-PLL (Boc)-b-PLC (Trt) triblock polymer through precipitation, filtration, drying treatment;
S012. above-mentioned PEG-b-PLL (Boc)-b-PLC (Trt) is dissolved in DMF, be (1-10) by the mol ratio of end-capping reagent and PEG-b-PLL (Boc): 1 adds end-capping reagent, under room temperature (25 DEG C) condition, react 12-36 hour.Add the ether of 5-50 times of volume after question response terminates, obtain PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent triblock polymer through precipitation, filtration, drying treatment;
S013. N is pressed respectively, the mol ratio of N-dicyclohexylcarbodiimide and PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent is (3-10): 1, the mol ratio of N-hydroxy-succinamide and PEG-b-PLL (Boc)-end-capping reagent is (3-10): the proportional arrangement DMF mixing solutions of 1, under room temperature (25 DEG C) condition after stirring reaction 12-36 hour, use membrane filtration, after the by product that filtering reaction produces, be (1-3) in the mol ratio of target polypeptide TP and PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent: the ratio target polypeptide TP of 1, as having the peptide sequence DMPGTVLP of mammary cancer target, continue reaction adds 10-50 times of volume ether after 24-72 hour, through precipitation, filter, after drying treatment, obtain TP-PEG-b-PLL (Boc)-b-PLC (the Trt)-end-capping reagent three stage polymerization thing containing targeting peptides,
S014. being dissolved in above-mentioned TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent containing volume fraction is the trifluoroacetic acid of 0.1%-10% tri isopropyl silane, stir 1-4 hour under room temperature (25 DEG C) condition after, add the ether of 5-50 times of volume, through precipitation, filtration, drying treatment, obtain TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent three stage polymerization thing.
Certainly, be to be understood that, in order to screen out the small-molecule substance that may exist in above-mentioned TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent three stage polymerization thing, can in above-mentioned steps S014, carry out the step precipitated with ether after, product TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent is carried out pre-dialysis treatment, treatment process is as follows: product TP-PEG-b-PLL (Boc)-b-PLC (the Trt)-end-capping reagent after being filtered by ether is dissolved in polar organic solvent, use and retain dialysis tubing dialysis treatment 12-36 hour in water that number-average molecular weight is 3500KD, within every 2 hours, change water-dialyzing once, freeze-drying subsequently obtains TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent triblock polymer.
Take end-capping reagent as HPPr be example, the chemical equation of TP-PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr building-up reactions is as described below:
In above-mentioned steps S02, the forming process of intelligent polycation nano-carrier is as follows: described TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent triblock polymer is dissolved in organic solvent completely and forms homogeneous, transparent solution, obtained solution is placed in dialysis tubing and carries out dialysis treatment, lyophilize.
Particularly, described organic solvent is at least one of tetrahydrofuran (THF), DMF, N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), N-Methyl pyrrolidone, Isosorbide-5-Nitrae-dioxane, methylene dichloride, trichloromethane; The concentration of described solution is 1-50mg/mL.As another preferred embodiment, the step of described dialysis treatment is the 12-96 hour that dialyses in the water of 10-500 times amount, and every 2-6 hour changes water once.
The preparation method of the intelligent polycation nano-carrier that the embodiment of the present invention provides, the product prepared can the multiple loaded article of load, and stability is strong, possess active targeting transport, pH response and reduction responsiveness, preparation method is simply controlled, and convenient operation is promoted, and has good market outlook.
And, embodiments provide a kind of intelligent polycation nano-carrier composition, comprise nano-carrier and the loaded article of load on nano-carrier, described nano-carrier is above-mentioned intelligent polycation nano-carrier, and described loaded article load is in the hydrophobic inner core of the positive charge layer of described intelligent polycation nano-carrier and/or the PLC of disulfide bond crosslinking.
Particularly, as preferred embodiment, described loaded article is at least one in hydrophobic drug, genetic stew or protein, and as concrete preferred embodiment, described hydrophobic drug is selected from least one in Zorubicin, taxol, cis-platinum, Fluracil, Rheumatrex, camptothecine.Described genetic stew is selected from least one in cancer suppressor gene, suicide gene, siRNA, mRNA, antisense nucleic acid etc.Described protein is at least one in serum protein, pharmaceutical protein molecule, antigen, treatment peptide, somatomedin, monoclonal antibody etc.To of course it is to be understood that in this area that other can carry out the hydrophobic drug of load, genetic stew and protein with above-mentioned intelligent polycation nano-carrier preparation, all may be used in embodiment of the present invention field.
As preferred embodiment, the weight ratio of described loaded article and described carrier can be set to (1-60): (2-200), and the weight ratio of loaded article and described carrier is less than 1, when loaded article is at least one in above-mentioned hydrophobic drug, genetic stew or protein, the weight ratio of described hydrophobic drug and described carrier can be set to (1-20): (2-200); The weight ratio of described genetic stew and described carrier can be set to (1-20): (2-200); The weight ratio of described protein and described carrier can be set to (1-20): (2-200); Any two kinds in described hydrophobic drug, genetic stew or protein can be set to (1-40) with the weight ratio of described carrier: (2-200); The weight ratio of described hydrophobic drug, genetic stew or protein and described carrier can be set to (1-60): (2-200).The composition of above-mentioned carrier and structure thereof as mentioned above, in order to save length, repeat no more herein.This embodiment prepare while load hydrophobic drug, genetic stew or protein nanometer carrier combination assembling schematic diagram as shown in Figure 1.
In above-mentioned intelligent polycation nano-carrier composition, described hydrophobic drug load is in the hydrophobic inner core of the PLC of the disulfide bond crosslinking of described intelligent polycation nano-carrier; And/or described genetic stew load is at the positive charge layer of described intelligent polycation nano-carrier; And/or described protein load is at the positive charge layer of described intelligent polycation nano-carrier.Described hydrophobic drug load is in the hydrophobic inner core of the PLC of the disulfide bond crosslinking of described intelligent polycation nano-carrier, because intracellular reduced glutathion level is about extracellular 100 ~ 1000 times, after nano-carrier enters tumour cell, reduced glutathion concentration in born of the same parents is higher than outside born of the same parents, disulfide linkage is unstable in reducing environment, therefore, the PLC hydrophobic cores layer formed by disulfide bond crosslinking is disintegrated, hydrophobic drug in core is able to quick release, thus reaches the object for the treatment of tumour.Described genetic stew and/or described protein load are at the positive charge layer of described intelligent polycation nano-carrier.Because described intelligent polycation nano-carrier has the nucleocapsid structure of ad hoc structure level, make intelligent polycation nano-carrier composition described in the embodiment of the present invention can realize the function of the broad variety of load simultaneously loaded article; And can mediate the several functions such as active targeting transmission, pH sensitivity and reduction response simultaneously, therefore, loaded article to stride across physiologic barrier complicated in body intelligently, thus is delivered to target position by this intelligent polycation nano-carrier composition efficiently.
As preferred embodiment, the formulation of described intelligent polycation nano-carrier composition can be made into lyophilized injectable powder or aqueous solution injection.Certainly, intelligent polycation nano-carrier composition is made other formulations that other people body can accept.When described intelligent polycation nano-carrier composition makes lyophilized injectable powder or water-soluble injection, in order to ensure human body fully effectively absorbing this pharmaceutical preparation, there is certain requirement to the size of described intelligent polycation nano-carrier preparation.As preferred embodiment, the aqueous solution Zeta potential size of described intelligent polycation nano-carrier is+10mv-+50mv; And/or the nanometer size range of described preparation is 10-500nm.The particle diameter of described intelligent polycation nano-carrier preparation, by controlling the structure of TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer in polymer nanoparticle drug carriers, thus realizes the Modulatory character of the particle diameter of nano-carrier preparation.
The intelligent polycation nano-carrier composition that the embodiment of the present invention provides, due to above-mentioned intelligent polycation nano-carrier for carrier, therefore there are four layers of shell structure that structure is special, can realize the function of the different loads things such as the lyophobic dust of load simultaneously, protein, genetic stew, and the carrier compositions formed is stable, metabolism is complete; The functions such as active targeting transmission, pH response and reduction response can be realized simultaneously, easily cross over physiologic barrier complicated in human body, avoid immune removing; And the particle diameter of this nano-drug transporter is well controlled by regulating triblock polymer structure.
Correspondingly, embodiments provide a kind of preparation method of intelligent polycation nano-carrier composition, comprise the steps:
According to above-mentioned synthesis described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer method synthesis TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer;
Described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and described loaded article are carried out following process:
When loaded article is dewatering medicament, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and loaded article are dissolved in organic solvent and form homogeneous mixture solution, then carry out dialysis treatment, lyophilize, obtain the intelligent polycation nano-carrier dry powder drug being loaded with dewatering medicament.；
When loaded article be protein and/or genetic stew time, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent, then dialysis treatment is carried out, add loaded article mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with protein or genetic stew;
When loaded article is simultaneously containing dewatering medicament, protein and/or genetic stew, first described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and hydrophobic drug are dissolved in organic solvent and form homogeneous mixture solution, then dialysis treatment is carried out, add protein and/or genetic stew mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with protein or genetic stew.
Concrete, the synthesis step of above-mentioned TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, has done detailed discussion, has repeated no more herein in the preparation method of above-mentioned intelligent polycation nano-carrier.
Particularly, as preferred embodiment, described loaded article is at least one in hydrophobic drug, genetic stew or protein, and as concrete preferred embodiment, described hydrophobic drug is selected from least one in Zorubicin, taxol, cis-platinum, Fluracil, Rheumatrex, camptothecine.Described genetic stew is selected from least one in cancer suppressor gene, suicide gene, siRNA, mRNA, antisense nucleic acid etc.Described protein is at least one in serum protein, pharmaceutical protein molecule, antigen, treatment peptide, somatomedin, monoclonal antibody etc.To of course it is to be understood that in this area that other can carry out the hydrophobic drug of load, genetic stew and protein with above-mentioned intelligent polycation nano-carrier preparation, all may be used in embodiment of the present invention field.
As preferred embodiment, the weight ratio of described loaded article and described carrier can be set to (1-60): (1-200), when loaded article is at least one in above-mentioned hydrophobic drug, genetic stew or protein, the weight ratio of described hydrophobic drug and described carrier can be set to (1-20): (1-200); The weight ratio of described genetic stew and described carrier can be set to (1-20): (1-200); The weight ratio of described protein and described carrier can be set to (1-20): (1-200); Any two kinds in described hydrophobic drug, genetic stew or protein can be set to (1-40) with the weight ratio of described carrier: (1-200); The weight ratio of described hydrophobic drug, genetic stew or protein and described carrier can be set to (1-60): (1-200).
In the preparation process of above-mentioned intelligent polycation nano-carrier medicine, in order to triblock polymer in described intelligent polycation nano-carrier preparation effectively can be dissolved and loaded article divides, make it form homogeneous, transparent solution, select the organic solvent excellent to said components solubility property.As preferred embodiment, described organic solvent is tetrahydrofuran (THF), DMF, at least one of N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), N-Methyl pyrrolidone, Isosorbide-5-Nitrae-dioxane, methylene dichloride, trichloromethane.As another preferred embodiment, the method for described dialysis treatment is: mixture solution is joined in the water of 10-500 times of volume the 12-96 hour that dialyses, every 2-6 hour changes water once.
In the process of above-mentioned preparation intelligent polycation nano-carrier composition, due to gene and/or the easy inactivation of protein or sex change, when containing hydrophobic drug, first itself and nano-carrier are dissolved in after in organic solvent jointly, are carrying out dialysis treatment; When containing genetic stew and/or protein, first nano-carrier is dissolved in organic solvent after forming solution and carries out dialysis treatment, then add genetic stew and/or proteolytic in above-mentioned solution; When simultaneously containing hydrophobic drug and genetic stew and/or protein, first nano-carrier, hydrophobic drug are carried out dialysis treatment after being dissolved in organic solvent and forming solution, then add genetic stew and/or proteolytic in above-mentioned solution.
The preparation method of the intelligent polycation nano-carrier medicine that the embodiment of the present invention provides, method is simply controlled, has good application prospect.
And, the application of the tear-away intelligent polymer nano-carrier of shell described in the embodiment of the present invention in fields such as nano-drug transporter, fluorescence dye carrier, bioprobe carriers.
Below in conjunction with specific implementation method, the present invention is further detailed.
The synthesis of embodiment 1TP-PEG-b-PLL-b-PLC-HPPr triblock polymer
S111. polymerizing pipe being vacuumized the protection of rear inflated with nitrogen, is the HOOC-PEG-NH of 500KD by 1g number-average molecular weight
2join in polymerizing pipe, by Lys (BOC)-NCA monomer and HOOC-PEG-NH after dissolving with 20mLDMF
2mol ratio be that the ratio of 20:1 adds Lys (BOC)-NCA monomer, isothermal reaction 24 hours under nitrogen protection, then presses Cys (Trt)-NCA monomer and HOOC-PEG-NH
2mol ratio be that the ratio of 10:1 adds Cys (Trt)-NCA monomer; continue isothermal reaction under nitrogen protection 24 hours, the ether sedimentation, filtration, the drying that add 10 times after reaction terminates obtain PEG-b-PLL (BOC)-b-PLC (Trt) triblock polymer.
S112. above products therefrom PEG-b-PLL (Boc)-b-PLC (Trt) is dissolved in DMF again, add the phenylpropionic acid succinimide ester of 3 times of molar weights, room temperature (25 DEG C) reaction 24 hours, the ether sedimentation, filtration, the drying that add 15 times after reaction terminates obtained PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer.
S113. PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer is joined the N being dissolved with 3 times of molar weights, in the DMF solution of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 3 times of molar weights, at room temperature (25 DEG C) stirring reaction after 24 hours, use the by product that membrane filtration reaction produces, then the DMPGTVLP peptide sequence with polymkeric substance equimolar amount is added, continue reaction adds 20 times ether sedimentation after 24 hours, filter, drying obtains TP-PEG-b-PLL (Boc)-b-PLC (the Trt)-HPPr triblock polymer containing targeting peptides.
S114. TP-PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr is dissolved in the trifluoroacetic acid containing volume fraction 0.5% tri isopropyl silane, stir 2 hours in room temperature (25 DEG C), add the ether sedimentation of 30 times, filtration, drying obtain TP-PEG-b-PLL-b-PLC-HPPr triblock polymer crude product.Gained crude product is dissolved in DMF, use retains the dialysis tubing that number-average molecular weight is 3500KD and dialyses in water 48 hours, within every 2 hours, change water-dialyzing once, freeze-drying subsequently obtains TP-PEG-b-PLL-b-PLC-HPPr triblock polymer, and the equal number-average molecular weight of number is about 6100KD.
The synthesis of embodiment 2TP-PEG-b-PLL-b-PLC-HPPr triblock polymer
S211. polymerizing pipe being vacuumized the protection of rear inflated with nitrogen, is the HOOC-PEG-NH of 2000KD by 0.5g number-average molecular weight
2join in polymerizing pipe, by Lys (BOC)-NCA monomer and HOOC-PEG-NH after dissolving with 30mLDMF
2mol ratio be that the ratio of 40:1 adds Lys (BOC)-NCA monomer, isothermal reaction 36 hours under nitrogen protection, then presses Cys (Trt)-NCA monomer and HOOC-PEG-NH
2mol ratio be that the ratio of 20:1 adds Cys (Trt)-NCA monomer; continue isothermal reaction under nitrogen protection 72 hours, the ether sedimentation, filtration, the drying that add 30 times after reaction terminates obtain PEG-b-PLL (BOC)-b-PLC (Trt) triblock polymer.
S212. above products therefrom PEG-b-PLL (Boc)-b-PLC (Trt) is dissolved in DMF again, add the phenylpropionic acid succinimide ester of 5 times of molar weights, room temperature (25 DEG C) reaction 24 hours, the ether sedimentation, filtration, the drying that add 30 times after reaction terminates obtained PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer.
S213. PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer is joined the N being dissolved with 5 times of molar weights, in the DMF solution of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 5 times of molar weights, at room temperature (25 DEG C) stirring reaction after 36 hours, use the by product that membrane filtration reaction produces, then the DMPGTVLP peptide sequence with polymkeric substance equimolar amount is added, continue reaction adds 20 times ether sedimentation after 36 hours, filter, drying obtains TP-PEG-b-PLL (Boc)-b-PLC (the Trt)-HPPr triblock polymer containing targeting peptides.
S214. TP-PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer is dissolved in the trifluoroacetic acid containing volume fraction 1% tri isopropyl silane, stir 3 hours in room temperature (25 DEG C), add the ether sedimentation of 40 times, filtration, drying obtain TP-PEG-b-PLL-b-PLC-HPPr triblock polymer crude product.Gained crude product is dissolved in DMF, use retains the dialysis tubing that number-average molecular weight is 3500KD and dialyses in water 48 hours, within every 2 hours, change water-dialyzing once, freeze-drying subsequently obtains TP-PEG-b-PLL-b-PLC-HPPr triblock polymer, and the equal number-average molecular weight of number is about 8300KD.
The synthesis of embodiment 3TP-PEG-b-PLL-b-PLC-HPPr triblock polymer
S211. polymerizing pipe being vacuumized the protection of rear inflated with nitrogen, is the HOOC-PEG-NH of 1000KD by 1g number-average molecular weight
2join in polymerizing pipe, by Lys (BOC)-NCA monomer and HOOC-PEG-NH after dissolving with 25mLDMF
2mol ratio be that the ratio of 40:1 adds Lys (BOC)-NCA monomer, isothermal reaction 72 hours under nitrogen protection, then presses Cys (Trt)-NCA monomer and HOOC-PEG-NH
2mol ratio be that the ratio of 20:1 adds Cys (Trt)-NCA monomer; continue isothermal reaction under nitrogen protection 90 hours, the ether sedimentation, filtration, the drying that add 30 times after reaction terminates obtain PEG-b-PLL (BOC)-b-PLC (Trt) triblock polymer.
S212. above products therefrom PEG-b-PLL (Boc)-b-PLC (Trt) is dissolved in DMF again, add the phenylpropionic acid succinimide ester of 8 times of molar weights, room temperature (25 DEG C) reaction 36 hours, the ether sedimentation, filtration, the drying that add 50 times after reaction terminates obtained PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer.
S213. PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer is joined the N being dissolved with 2 times of molar weights, in the DMF solution of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 2 times of molar weights, at room temperature (25 DEG C) stirring reaction after 12 hours, use the by product that membrane filtration reaction produces, then the DMPGTVLP peptide sequence with polymkeric substance equimolar amount is added, continue reaction adds 20 times ether sedimentation after 36 hours, filter, drying obtains TP-PEG-b-PLL (Boc)-b-PLC (the Trt)-HPPr triblock polymer containing targeting peptides.
S214. TP-PEG-b-PLL (Boc)-b-PLC (Trt)-HPPr triblock polymer is dissolved in the trifluoroacetic acid containing volume fraction 0.2% tri isopropyl silane, stir 3 hours in room temperature (25 DEG C), add the ether sedimentation of 40 times, filtration, drying obtain TP-PEG-b-PLL-b-PLC-HPPr triblock polymer crude product.Gained crude product is dissolved in DMF, use retains the dialysis tubing that number-average molecular weight is 3500KD and dialyses in water 48 hours, within every 2 hours, change water-dialyzing once, freeze-drying subsequently obtains TP-PEG-b-PLL-b-PLC-HPPr triblock polymer, and the equal number-average molecular weight of number is about 7600KD.
Nanometer carrier combination that example 4 is paclitaxel loaded and preparation method thereof
Paclitaxel loaded nanometer carrier combination, comprise taxol and paclitaxel nano carrier, described paclitaxel nano carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, be divided into four layers successively from outside to inside, wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking, taxol load is in the hydrophobic inner core of the PLC of intelligent polycation nano-carrier disulfide bond crosslinking, and the weight ratio of taxol and carrier is 2:10.
The preparation method of paclitaxel loaded nanometer carrier combination is as follows:
Taking TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 10mg, taxol 2mg, use 10mL dmso solution, ultrasonic 10min under room temperature, medicine and polymkeric substance are fully dissolved, form homogeneous, transparent organic homogeneous phase solution; Being placed in by obtained dimethyl sulphoxide solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 48 hours in 1L water, within every 2 hours, changes water once; After dialysis terminates, collect the carrier micelle aqueous solution formed in dialysis tubing, it is 85nm that DLS records size, disperses more even.
Nanometer carrier combination of example 5 load Zorubicin and preparation method thereof
The nanometer carrier combination of load Zorubicin, comprise Zorubicin and adriamycin nano carrier, described adriamycin nano carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, be divided into four layers successively from outside to inside, wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking, Zorubicin load is in the hydrophobic inner core of the PLC of intelligent polycation nano-carrier disulfide bond crosslinking, and the weight ratio of Zorubicin and carrier is 1:4.
The preparation method of the nanometer carrier combination of load Zorubicin is as follows:
Take TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 80mg, Zorubicin 20mg, dissolve with 100mLDMF, ultrasonic 10min under room temperature, makes medicine and polymkeric substance fully dissolve, and forms homogeneous, transparent organic homogeneous phase solution; Being placed in by obtained DMF solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 72 hours in 2L water, within every 4 hours, changes water once; After dialysis terminates, collect the carrier micelle aqueous solution formed in dialysis tubing.It is 76nm that DLS records size, disperses more even.
Nanometer carrier combination of example 6 load P 53 gene and preparation method thereof
The nanometer carrier combination of load P 53 gene, comprise P53 gene and P53 gene nano carrier, described P53 gene nano carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, be divided into four layers successively from outside to inside, wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking, the positive charge layer that P53 gene load intelligent polycation nano-carrier PLL is formed, and the weight ratio of P53 gene and carrier is 1:10.
The preparation method of the nanometer carrier combination of load P 53 gene is as follows:
Take TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 10mg, dissolve with 4mLDMF, ultrasonic 10min under room temperature, polymkeric substance is fully dissolved, being placed in by obtained DMF solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 60 hours in 1.5L water, within every 6 hours, changes water once, after dialysis terminates, collect the polymer micelle aqueous solution formed in dialysis tubing; Measure P53 gene 0.05mg to join obtained polymer micelle aqueous solution mesoscale eddies and vibrate 2 hours, under 37 DEG C of conditions, place the nano-carrier preparation of 1 hour back loading P53 gene.DLS records size and divides 150nm, disperses more even.
Nano-composition of example 7 load siRNA gene and preparation method thereof
The nanometer carrier combination of load siRNA; comprise siRNA and siRNA nano-carrier; described siRNA nano-carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; be divided into four layers successively from outside to inside; wherein; outermost layer is the peptide T P that can mediate active targeting transmission; the PEG of secondary skin for shielding; third layer is the positive charge layer that PLL is formed; innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking; the positive charge layer that siRNA load intelligent polycation nano-carrier PLL is formed, and the weight ratio of siRNA and carrier is 1:20.
The preparation method of the nanometer carrier combination of load siRNA is as follows:
Take TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 10mg, use 5mL dmso solution, ultrasonic 10min under room temperature, polymkeric substance is fully dissolved, and being placed in by obtained dimethyl sulphoxide solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 60 hours in 1.5L water, within every 6 hours, change water once, after dialysis terminates, collect the polymer micelle aqueous solution formed in dialysis tubing, be diluted to 1mg/mL.Measure siRNA5 μ g and be placed in centrifuge tube, then enter to the obtained polymer micelle aqueous solution 100 μ L vortex oscillation 2 hours, under 37 DEG C of conditions, place the nano-carrier preparation of 1 hour back loading siRNA gene.It is about 60nm that DLS records size, disperses more even.
Nano-composition of example 8 load BSA and preparation method thereof
The nanometer carrier combination of load BSA; comprise BSA and BSA nano-carrier; described BSA nano-carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; be divided into four layers successively from outside to inside; wherein; outermost layer is the peptide T P that can mediate active targeting transmission; the PEG of secondary skin for shielding; third layer is the positive charge layer that PLL is formed; innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking; the positive charge layer that BSA load intelligent polycation nano-carrier PLL is formed, and the weight ratio of BSA and carrier is 1:20.
The preparation method of the nanometer carrier combination of load BSA is as follows:
Take TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 1mg, dissolve with 4mLDMF, ultrasonic 10min under room temperature, polymkeric substance is fully dissolved, being placed in by obtained DMF solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 60 hours in 1.5L water, within every 6 hours, changes water once, after dialysis terminates, collect the polymer micelle aqueous solution formed in dialysis tubing; Measure BSA albumen 0.05mg to join obtained polymer micelle aqueous solution mesoscale eddies and vibrate 2 hours, under 37 DEG C of conditions, place the nano-carrier preparation obtaining load BSA albumen after 1 hour.It is 95nm that DLS records size, disperses more even.
The nanometer carrier combination and preparation method thereof of example 9 simultaneously paclitaxel loaded, siRNA, BSA
Simultaneously paclitaxel loaded, siRNA, the nanometer carrier combination of BSA, comprise taxol, siRNA, BSA and nano-carrier, described nano-carrier comprises TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer, be divided into four layers successively from outside to inside, wherein, outermost layer is the peptide T P that can mediate active targeting transmission, the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking, taxol load is in the hydrophobic inner core of the PLC of intelligent polycation nano-carrier disulfide bond crosslinking, siRNA and BSA is carried on the positive charge layer that intelligent polycation nano-carrier PLL is formed, and taxol, siRNA, the total amount of BSA and the weight ratio of carrier are 3:20.
Simultaneously the preparation method of the nanometer carrier combination of paclitaxel loaded, siRNA, BSA is as follows:
Take TP-PEG-b-PLL-b-PLC-HPPr triblock polymer 20mg, taxol 2mg, use 10mL dmso solution, ultrasonic 10min under room temperature, medicine and polymkeric substance are fully dissolved, forms homogeneous, transparent organic phase solution; Being placed in by obtained dimethyl sulphoxide solution and retaining number-average molecular weight is 3500KD dialysis tubing, then dialyses 48 hours in 1L water, within every 2 hours, changes water once; After dialysis terminates, obtain the carrier micelle aqueous solution, siRNA and 0.5mgBSA of 0.5mg is joined obtained carrier micelle aqueous solution mesoscale eddies to vibrate 2 hours, under 37 DEG C of conditions, place paclitaxel loaded while of obtaining after 1 hour, siRNA, BSA nano-carrier preparation.It is 125nm that DLS records size, disperses more even.
Effect example
TP-PEG-b-PLL-b-PLC-HPPr intelligent polycation nano-carrier cytotoxicity analysis:
Use the cytotoxicity of MTT evaluating and measuring embodiment 1 gained TP-PEG-b-PLH-b-PLC-HPPr intelligence amphipathic nature polyalcohol nano-carrier, experiment comprises the steps:
In each hole of 96 orifice plates, add 100 μ L contain 1.2 × 10
4the DMEM substratum of individual MCF-7 cell.After cultivating 24h, in each hole, add the micellar solution of a certain amount of TP-PEG-b-PLL-b-PLC-HPPr, make the concentration range of polymkeric substance be 1 μ g/mL ~ 200 μ g/mL (each concentration does three Duplicate Samples).Continue to cultivate 24h, the DMEM then given up containing polymkeric substance and add fresh DMEM and 100 μ LMTT solution in MCF-7 cell.Cultivate after 4h, then join in each plate hole respectively by 100 μ LDMSO, shaken at room temperature 10min makes MTT dissolve completely, uses microplate reader to measure the fluorescent absorption value at 570nm place, calculates the survival rate of cell under different polymer micelle concentration.
Measuring result as shown in Figure 3.As shown in Figure 3, when polymer micelle concentration is 50 μ g/mL, cell survival rate still reaches more than 60%.Illustrate that this nano-carrier has the low feature of toxicity.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. an intelligent polycation nano-carrier; comprise TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer; this intelligent polycation nano-carrier is divided into four layers from outside to inside successively; wherein; outermost layer is the peptide T P that can mediate active targeting transmission; the PEG of secondary skin for shielding, third layer is the positive charge layer that PLL is formed, and innermost layer is the PLC hydrophobic inner core formed by disulfide bond crosslinking.
2. intelligent polycation nano-carrier as claimed in claim 1, is characterized in that: described end-capping reagent is alkane or the aromatic hydrocarbons of activated carboxylic.
3. a preparation method for intelligent polycation nano-carrier, comprises the steps:
The synthesis of TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer: by HOOC-PEG-NH
2as initiator, progressively cause amino acid N CA ring-opening polymerization synthesis PEG-b-PLL (Boc)-b-PLC (Trt); With phenylpropionic acid succinimide ester for end-capping reagent, the amino closing PLC (Trt) end obtains PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; Combine with the peptide T P that can mediate active targeting transmission after the carboxyl that activated polymer PEG holds and obtain TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent; TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is obtained after removing TP-PEG-b-PLL (Boc)-b-PLC (Trt)-end-capping reagent Side chain protective group Boc group and Trt group;
The formation of intelligent polycation nano-carrier: described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent and forms homogeneous phase solution, described solution is carried out dialysis treatment, obtains intelligent polycation nano-carrier.
4. an intelligent polycation nano-carrier composition, comprise nano-carrier and the loaded article of load on nano-carrier, described nano-carrier be as arbitrary in claim 1 ~ 2 as described in intelligent polycation nano-carrier, described loaded article load is in the hydrophobic inner core of the positive charge layer of described intelligent polycation nano-carrier and/or the PLC of disulfide bond crosslinking.
5. intelligent polycation nano-carrier composition as claimed in claim 4, is characterized in that: the weight ratio of described loaded article and described carrier is (1-60): (2-100), and the weight ratio of loaded article and described carrier is less than 1; And/or described loaded article is at least one in hydrophobic drug, genetic stew or protein.
6. intelligent polycation nano-carrier composition as claimed in claim 5, is characterized in that: described hydrophobic drug load is in the hydrophobic inner core of the PLC of the disulfide bond crosslinking of described intelligent polycation nano-carrier; And/or described genetic stew load is at the positive charge layer of described intelligent polycation nano-carrier; And/or described protein load is at the positive charge layer of described intelligent polycation nano-carrier.
7. the intelligent polycation nano-carrier composition as described in as arbitrary in claim 4-6, is characterized in that: described hydrophobic drug is selected from least one in Zorubicin, taxol, cis-platinum, Fluracil, Rheumatrex, camptothecine; And/or
Described genetic stew is selected from least one in cancer suppressor gene, suicide gene, siRNA, mRNA, antisense nucleic acid; And/or
Described protein is at least one in serum protein, pharmaceutical protein molecule, antigen, treatment peptide, somatomedin, monoclonal antibody.
8. the intelligent polycation nano-carrier composition as described in as arbitrary in claim 4-6, is characterized in that: the aqueous solution Zeta potential size of described intelligent polycation nano-carrier is+10mv-+50mv;
And/or the particle size range of nano-carrier is 10-500nm.
9. a preparation method for intelligent polycation nano-carrier composition, comprises the steps:
Described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer method synthesis TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is synthesized according to claim 3;
Described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and described loaded article are carried out following process:
When loaded article is dewatering medicament, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and loaded article are dissolved in organic solvent and form homogeneous mixture solution, then carry out dialysis treatment, lyophilize, obtain the intelligent polycation nano-carrier dry powder drug being loaded with dewatering medicament;
When loaded article be protein and/or genetic stew time, described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer is dissolved in organic solvent, then dialysis treatment is carried out, add loaded article mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with protein and/or genetic stew;
When loaded article is simultaneously containing dewatering medicament, protein and/or genetic stew, first described TP-PEG-b-PLL-b-PLC-end-capping reagent triblock polymer and hydrophobic drug are dissolved in organic solvent and form homogeneous mixture solution, then dialysis treatment is carried out, add protein and/or genetic stew mixing again, obtain the intelligent polycation nano-carrier pharmaceutical aqueous solution being loaded with dewatering medicament, protein and/or genetic stew.
10. one kind as arbitrary in claim 1-2 as described in the application of intelligent polycation nano-carrier in nano-drug transporter, fluorescence dye carrier, bioprobe carrier field.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029839A1 (en) * | 1997-12-12 | 1999-06-17 | Expression Genetics, Inc. | Grafted copolymers as gene carriers |
| CN101953804A (en) * | 2010-09-26 | 2011-01-26 | 苏州同科生物材料有限公司 | Shell layer dropping type nanometer medicine carrier preparation based on amphiphilic block copolymer and preparation method thereof |
| CN103272244A (en) * | 2013-01-21 | 2013-09-04 | 长春理工大学 | Medicament bonding object, preparation method and the application in tumour treatment |
-
2013
- 2013-12-12 CN CN201310687280.3A patent/CN103755953B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029839A1 (en) * | 1997-12-12 | 1999-06-17 | Expression Genetics, Inc. | Grafted copolymers as gene carriers |
| CN101953804A (en) * | 2010-09-26 | 2011-01-26 | 苏州同科生物材料有限公司 | Shell layer dropping type nanometer medicine carrier preparation based on amphiphilic block copolymer and preparation method thereof |
| CN103272244A (en) * | 2013-01-21 | 2013-09-04 | 长春理工大学 | Medicament bonding object, preparation method and the application in tumour treatment |
Non-Patent Citations (1)
| Title |
|---|
| PLL-PEG-PLL 三嵌段共聚物的制备及DNA包覆应用;杨子刚，等;《中国科学 B辑 化学》;20051220;第35卷(第6期);摘要和2.5聚离子复合物胶束形态与大小 * |
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