CN103705931A - Shell-droppable polymer nano carrier as well as preparation method and application thereof - Google Patents
Shell-droppable polymer nano carrier as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention is applicable to the field of nano medicine, and provides a shell-droppable polymer nano carrier as well as a preparation method and an application thereof. The shell-droppable polymer nano carrier comprises two diblock polymers: a TP-PEG-b-PLL (DMMA)-end capping agent and an initiator-PLL-b-PLC; the two diblock polymers are combined into a core-shell structure through electrostatic adsorption; the core-shell structure is divided into four layers sequentially from outside to inside; and the weight ratio of the TP-PEG-b-PLL (DMMA)-end capping agent to the initiator-PLL-b-PLC is (1-100):(1-100). The shell-droppable polymer nano carrier preparation can be completely biodegraded and also has the functions of active targeting transfer, pH response and reduction response; and the particle size and electric potential can be controlled.
Description
Technical field
The invention belongs to nanosecond medical science field, relate in particular to the tear-away polymer nanoparticle drug carriers of a kind of shell, its preparation method and application thereof.
Background technology
Development along with nanometer biotechnology, multifunctional intelligent polymer nano granules is more and more subject to people's attention, particularly in cancerous cell treatment field, multifunctional intellectual nano-carrier has been widely applied to target drug-carrying field as a kind of new drug carrier, and has good development prospect.Intelligent nano carrier is delivered to drug selectivity after specific target spot, after being excited by chemical signal or temperature or pH etc. " trigger ", can discharge responsively by carrying medicament, this nano-carrier can be divided into initiatively targeted delivery system and passive target transmission system.
Nanometer passive target transmission system refers to that nano-carrier has targeting to reticuloendothelial system, and medicine carrying carrier is assembled at specific medicine-feeding part, then discharges bioactive medicine.For example, the long-circulating nanoparticles that Polyethylene Glycol (PEG) or polyethylene glycol oxide (PEO) are modified is after intravenous administration, and the permeability based on solid tumor and retention effect (EPR effect), make carrier assemble at tumor tissues around.Nanometer initiatively targeted delivery system is pharmaceutical carrier to be carried out to specific modification then can be transported to specific therapentic part by directed targeting in vivo.Because tumor cell proliferation is rapid, some specific expression of receptor strengthen, and cell is increased the picked-up of folic acid, vitamin and sugar, when pharmaceutical carrier, have modified after these specific receptors, can significantly improve the picked-up of cell to carrier, strengthen drug effect.Initiatively targeted delivery system, because of advantages such as its targeting are strong, toxic and side effects is little, is more and more subject to the attention of the world of medicine.
Nanometer polymer pharmaceutical carrier has been proved to be has great development prospect aspect transmission antitumor drug, but still there is a lot of deficiencies at present, as most nano-carrier less stable, biocompatibility is bad, degradable and metabolism in vivo, in cyclic process, easily by immune system, removed in vivo, thereby easily occur to assemble can not arrive useful effect position and lack at organs such as livers, be initiatively targeted to the functions such as tumor locus, these shortcomings have limited polymer nanoparticle drug carriers application clinically greatly.
Summary of the invention
The object of the present invention is to provide the tear-away polymer nanoparticle drug carriers of a kind of shell, be intended to solve existing nano-carrier poor stability, can not be completely degraded or metabolism, can not possess simultaneously the functions such as initiatively targeted delivery, pH response and reduction response, with and particle diameter and the current potential problem that can not regulate and control.
Another object of the present invention is to provide a kind of preparation method of the tear-away polymer nanoparticle drug carriers of shell of easy control simple to operate.
The nano-carrier medicine that provides a kind of shell tear-away is provided a further object of the present invention, be intended to solve existing nano-carrier medicine stability poor, can not be completely degraded or metabolism, can not possess simultaneously the functions such as initiatively targeted delivery, pH response and reduction response, with and particle diameter and the current potential problem that can not regulate and control.
Correspondingly, the present invention also provides the preparation method of the tear-away nano-carrier medicine of a kind of shell.
And the tear-away polymer nanoparticle drug carriers of a kind of shell is in the application in nano-carrier medicine, fluorescent dye carrier, bioprobe carrier field.
The embodiment of the present invention is to realize like this, the tear-away polymer nanoparticle drug carriers of a kind of shell, comprise two kinds of bi-block copolymers of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC, two kinds of described bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, described nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is to have the initiatively peptide T P of targeting, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is (1-100): (1-100).
Correspondingly, the preparation method of the tear-away polymer nanoparticle drug carriers of a kind of shell, comprises the steps:
Synthesizing of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer: by HOOC-PEG-NH
2and Lys (Boc)-NCA reaction obtains PEG-b-PLL (Boc); Use the amino of end-capping reagent sealing PLL (Boc) end to obtain PEG-b-PLL (Boc)-end-capping reagent; After the PEG c-terminus of activated PEG-b-PLL (Boc)-end-capping reagent, be combined with TP and obtain TP-PEG-b-PLL (Boc)-end-capping reagent; After being removed to Boc blocking group, TP-PEG-b-PLL (Boc)-end-capping reagent obtains TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer with the reaction of 2,3-dimethyl maleic anhydride;
Initiator-PLL-b-PLC bi-block copolymer synthetic: by initiator and Lys (Boc)-NCA reaction, obtain initiator-PLL (Boc), then and Cys (Trt)-NCA reaction obtain initiator-PLL (Boc)-b-PLC (Trt) bi-block copolymer; The protecting group Boc and the Trt that slough respectively lysine and cysteine side chain obtain initiator-PLL-b-PLC bi-block copolymer;
The formation of the tear-away polymer nanoparticle drug carriers of shell: be (1-100) according to weight ratio by described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer: ratio (1-100) is dissolved in organic solvent and forms mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers of shell.
And, the nano-carrier medicine that a kind of shell is tear-away, the carrier that comprises hydrophobic drug and described hydrophobic drug, the tear-away polymer nanoparticle drug carriers of described carrier shell described above, described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell.
Correspondingly, the preparation method of the nano-carrier medicine that a kind of shell is tear-away, comprises the steps:
Method according to above-mentioned synthetic described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer is synthesized respectively TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer;
Described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer, initiator-PLL-b-PLC bi-block copolymer and hydrophobic drug are dissolved in to organic solvent and form mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers medicine of shell; Wherein, the photograph weight ratio of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer is (1-100): (1-100).
And the tear-away polymer nanoparticle drug carriers of a kind of shell is in the application in nano-carrier medicine, fluorescent dye carrier, bioprobe carrier field.
The tear-away polymer nanoparticle drug carriers of a kind of shell provided by the invention, by TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymers of initiator-PLL-b-PLC, rely on Electrostatic Absorption to be combined into the nucleocapsid structure with ad hoc structure, make this nano-carrier preparation stability good, can complete biodegradable and metabolism; Possess initiatively the functions such as targeted delivery, pH response and reduction response simultaneously, easily cross over physiologic barrier complicated in human body, thereby can avoid suffering immune removing and arrive smoothly focus; In addition, the particle diameter of this nano-carrier preparation and current potential can be by regulating the ratio of two kinds of bi-block copolymer fusion to be well controlled.
The nano-carrier medicine that a kind of shell provided by the invention is tear-away, owing to thering is the tear-away polymer nanoparticle drug carriers of above-mentioned shell, and described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, make that this nano-carrier medicine is stable, metabolism is complete; Can realize the functions such as initiatively targeted delivery, pH response and reduction response simultaneously, easily cross over physiologic barrier complicated in human body, avoid immune removing; And the particle diameter of this nano-carrier medicine and current potential can be by regulating the doping ratio of two kinds of bi-block copolymers to be well controlled.
The preparation method of the nano-carrier medicine that the tear-away polymer nanoparticle drug carriers of shell provided by the invention and shell thereof are tear-away, method is simply controlled, has good market prospect.
Accompanying drawing explanation
Fig. 1 is structure and the assembling schematic diagram thereof of the tear-away polymer nanoparticle drug carriers of shell that provides of the embodiment of the present invention and nano-carrier medicine thereof.
Fig. 2 is the DLS grain-size graph of the tear-away polymer nanoparticle drug carriers of shell that provides of the embodiment of the present invention.
Fig. 3 is the cytotoxicity test of the tear-away polymer nanoparticle drug carriers of shell that provides of the embodiment of the present invention.
The specific embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, 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, is not intended to limit the present invention.
In the technical scheme that the embodiment of the present invention provides, the TP occurring, PEG, PLL, PLC, DMMA, HPPr, b, Tutane, Boc, Trt, DMF are explained as follows:
TP: there is the initiatively peptide sequence of targeted delivery function;
PEG: Polyethylene Glycol;
PLL: polylysine;
PLC: poly-cysteine;
DMMA:2,3-dimethyl maleic acid;
HPPr: benzenpropanoic acid;
Tutane: n-butylamine;
Boc：
Trt：
DMF:N, dinethylformamide;
B: expression polymer is block polymer.
Polyethylene Glycol (PEG) and polyamino acid all have good biocompatibility and biodegradability, and its catabolite all do not 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 macrophage simultaneously, and therefore, nano material, after PEG modifies, can significantly increase the time that it circulates in vivo.Aminoacid has multiple side-chain radical, aminoacid after polymerization, by its side chain active group is carried out to different modifications, thereby obtain the polyamino acid material of various different in kinds, as hydrophilic, hydrophobic, electropositivity, electronegativity etc., so polyamino acid is a kind of very potential biomaterial.
In view of this, the embodiment of the present invention provides a kind of shell tear-away polymer nanoparticle drug carriers, comprise two kinds of bi-block copolymers of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC, two kinds of described bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, described nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is for mediating the initiatively peptide sequence TP of targeted delivery, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is (1-100): (1-100).
Particularly, in the tear-away polymer nanoparticle drug carriers of above-mentioned shell, can, by regulating the ratio of TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymers of initiator-PLL-b-PLC, effectively control the Zeta potential size of nano-carrier aqueous solution and the particle diameter of nano-carrier medicine.As preferred embodiment, the aqueous solution Zeta potential of the nano-carrier that described shell is tear-away size is-50mv-+50mv; And/or the particle size range of nano-carrier is 10-300nm, the DLS grain-size graph of the tear-away polymer nanoparticle drug carriers of shell that Fig. 2 is the embodiment of the present invention.
Described PEG segment number-average molecular weight scope is 500KD-10000KD, and described PLL (DMMA) the segment degree of polymerization is 10-200, and the degree of polymerization of described PLL segment is 10-200, and the degree of polymerization of described PLC segment is 10-200.
In two kinds of bi-block copolymers of above-mentioned TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC, the PEG in described TP-PEG-b-PLL (DMMA)-end-capping reagent is amino-Polyethylene Glycol-carboxyl (HOOC-PEG-NH
2), described PEG is connected by amido link with PLL (DMMA) at aminoterminal, and the side chain amino of described PLL and DMMA are by amido link covalent bond.After side chain amino by the mode modification PLL with DMMA formation amido link, the TP-PEG-b-PLL-end-capping reagent being originally positive is changed into electronegativity material TP-PEG-b-PLL (the DMMA)-end-capping reagent of pH responsive type.This electronegativity material TP-PEG-b-PLL (DMMA)-end-capping reagent can pass through a certain proportion of initiator-PLL-b-PLC of Electrostatic Absorption, thereby the amount of charge of adjusting the tear-away polymer nanoparticle drug carriers integral body of shell, even makes TP-PEG-b-PLL (DMMA)-end-capping reagent charge property change.In described initiator-PLL-b-PLC, the N of described initiator end is connected by amido link with PLL.
The tear-away polymer nanoparticle drug carriers of described shell, TP-PEG-b-PLL (DMMA)-end-capping reagent and the initiator-PLL-b-PLC forming by above-mentioned link of take is skeleton, in aqueous solution, can rely on Electrostatic Absorption to be combined into nucleocapsid structure, automatic Composition forms nucleocapsid structure.Described nucleocapsid structure is divided into four layers from outside to inside, is followed successively by: outermost layer is to have the initiatively peptide T P of targeting, and it can, by the special receptors bind with tumor locus, realize nano-micelle at the Rapid Accumulation of tumor locus; Inferior skin is protective layer PEG, and it can reduce specific adsorption, thereby increases nano-micelle circulation time in vivo; The 3rd layer is the PLL(DMMA in TP-PEG-b-PLL (DMMA)-end-capping reagent) and initiator-PLL-b-PLC in the Electrostatic Absorption layer that forms of PLL, by this Electrostatic Absorption layer, form the tear-away pH response structure of shell of polymer nanoparticle drug carriers preparation.Because tumor tissues is bred rapidly, the vascular system of tumor often can not be supplied with nutrient substance and oxygen fully to a large amount of tumor cells, inadequate keeping makes tumor tissues anoxia, produce lactic acid, through ATP hydrolysis, produce an acid microenvironment, so the pH of infected tissue, primary tumo(u)r and secondary tumor tissue is low than normal structure.Therefore, this Electrostatic Absorption layer can be realized the stimuli responsive type targeted drug delivery system of pH responsive type.Innermost layer is formed, be can be used for the PLC hydrophobic core layer of load hydrophobic drug by disulfide bond crosslinking.The nucleocapsid structure with this ad hoc structure level, make described in the embodiment of the present invention that the tear-away polymer nanoparticle drug carriers of shell has initiatively targeted delivery simultaneously, pH is responsive and the several functions such as reduction response, therefore, the tear-away polymer nanoparticle drug carriers of this shell can stride across physiologic barrier complicated in body intelligently, thereby when the tear-away polymer nanoparticle drug carriers load of shell has hydrophobic drug, can successfully it be delivered to target position efficiently.
In order to prevent the PLL terminal amino group of TP-PEG-b-PLL (DMMA) and the cytotoxicity that target polypeptide sequence bonding does not increase carrier, at the end of above-mentioned TP-PEG-b-PLL-b-PLC, introduce nontoxic end-capping reagent, at the PLL end of above-mentioned TP-PEG-b-PLL (DMMA), introduced end-capping reagent.As preferred embodiment, the alkane that described end-capping reagent is activated carboxylic or aromatic hydrocarbons.As further preferred embodiment, described end-capping reagent is HPPr, stearic acid succinimide ester.
In order not affect the function of initiator-PLL-b-PLC, in above-mentioned initiator-PLL-b-PLC synthetic, introduced with amino micromolecule as initiator, as preferred embodiment, in described initiator-PLL-b-PLC, initiator is for containing amino alkane or aromatic hydrocarbons.As further preferred embodiment, described initiator is at least one in Tutane, 18-amine..
The tear-away polymer nanoparticle drug carriers of shell that the embodiment of the present invention provides is combined into nucleocapsid structure by the Electrostatic Absorption between TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC; This nucleocapsid structure has four-layer structure, and outermost layer is to have the initiatively peptide T P of targeting, and inferior skin is the PEG shielding, and the 3rd layer is the Electrostatic Absorption layer of PLL (DMMA) and PLL, and innermost layer is the PLC hydrophobic inner core of disulfide bond crosslinking.The tear-away polymer nanoparticle drug carriers of shell with this particular core shell structure, has following advantage:
1, the framework material of the tear-away polymer nanoparticle drug carriers of shell is TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC bi-block copolymer, its good biocompatibility, toxicity is low, can be completely degraded in vivo, and catabolite is nontoxic, harmless, can be absorbed in vivo or metabolism.
2, the tear-away polymer nanoparticle drug carriers of shell has the initiatively peptide molecule of targeted delivery, can efficiently transmit hydrophobic drug and arrive lesions position.
3, the tear-away polymer nanoparticle drug carriers of shell has the disulfide bond structure of the tear-away pH response structure of shell and reduction response simultaneously, the physiological environment of various complexity in can intelligent response human body is, the physiologic barrier that strides across various complexity in body of intelligence, therefore, physiological environment complicated in body can be adapted to and when the tear-away polymer nanoparticle drug carriers load of described shell has dewatering medicament, hydrophobic small-molecule drug can be realized in the quick release of target location.
4, the tear-away polymer nanoparticle drug carriers good stability of shell, its PEG shell can reduce non-specific adsorption and effectively protect nano-carrier preparation to avoid being removed fast by immune system.
5, the particle diameter of the tear-away polymer nanoparticle drug carriers of shell and Zeta potential can regulate and control by the ratio that regulates two kinds of bi-block copolymers to mix.
Described in the embodiment of the present invention, the tear-away polymer nanoparticle drug carriers preparation of shell can prepare by following method, and certainly, prepared by the method that also can obtain the tear-away polymer nanoparticle drug carriers preparation of shell by other.
Correspondingly, the embodiment of the present invention provides the preparation method of the tear-away polymer nanoparticle drug carriers of a kind of shell, comprises the steps:
Synthesizing of S01.TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer: by HOOC-PEG-NH
2and Lys (Boc)-NCA reaction obtains PEG-b-PLL (Boc); Use the amino of end-capping reagent sealing PLL (Boc) end to obtain PEG-b-PLL (Boc)-end-capping reagent; After the PEG c-terminus of activated PEG-b-PLL (Boc)-end-capping reagent, be combined with TP and obtain TP-PEG-b-PLL (Boc)-end-capping reagent; After being removed to Boc blocking group, TP-PEG-b-PLL (Boc)-end-capping reagent obtains TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer with the reaction of 2,3-dimethyl maleic anhydride;
S02. initiator-PLL-b-PLC bi-block copolymer is synthetic: by initiator and Lys (Boc)-NCA reaction, obtain initiator-PLL (Boc), then and Cys (Trt)-NCA reaction obtain initiator-PLL (Boc)-b-PLC (Trt) bi-block copolymer; The protecting group Boc and the Trt that slough lysine and cysteine side chain obtain initiator-PLL-b-PLC bi-block copolymer;
S03. the formation of the tear-away polymer nanoparticle drug carriers of shell: be (1-100) according to weight ratio by described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer: ratio (1-100) is dissolved in organic solvent and forms mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers of shell.
Concrete, in above-mentioned steps S01, TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer synthetic with HOOC-PEG-NH
2for initiator, by the amino of initiator, cause synthetic PEG-b-PLL (Boc) bi-block copolymer of method of aminoacid anhydride ring-opening polymerisation, take containing the alkane of activated carboxylic or aromatic hydrocarbons as HPPr be end-capping reagent, the amino of sealing PLL end, obtains PEG-b-PLL (Boc)-end-capping reagent bi-block copolymer; Then by the activated carboxylic of the PEG end of PEG-b-PLL (Boc)-end-capping reagent, by this activated carboxyl, PEG-b-PLL (Boc)-end-capping reagent is combined with target polypeptide TP, obtains TP-PEG-b-PLL (Boc)-end-capping reagent bi-block copolymer; Again the tertbutyloxycarbonyl protecting group Boc of lysine side-chain is sloughed and obtains TP-PEG-b-PLL-end-capping reagent bi-block copolymer; Finally utilize amino and the DMMA reaction of lysine side-chain to obtain electronegative TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer.Wherein, described PEG is HOOC-PEG-NH
2, its number-average molecular weight scope is 500KD-10000KD, the number-average molecular weight of prepared TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer is 800KD-30000KD.
Particularly, TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer concrete technology flow process is as described below:
S011. by HOOC-PEG-NH
2be dissolved in and in DMF, form the solution that concentration range is 1-100mg/mL, atmosphere of inert gases-as under nitrogen protection, press Lys (Boc)-NCA and HOOC-PEG-NH
2mol ratio be (10-200): 1 adds Lys (Boc)-NCA monomer, heated at constant temperature reaction 24-120 hour at 30-50 ℃.The ether that adds 5-50 times of volume after question response finishes, obtains PEG-b-PLL (Boc) bi-block copolymer through precipitation, filtration, dried;
S012. above-mentioned PEG-b-PLL (Boc) being dissolved in DMF, is (1-10) by the mol ratio of end-capping reagent and PEG-b-PLL (Boc): 1 adds end-capping reagent, under room temperature (25 ℃) condition, reacts 12-36 hour.The ether that adds 5-50 times of volume after question response finishes, obtains PEG-b-PLL (Boc)-end-capping reagent bi-block copolymer through precipitation, filtration, dried;
S013. press respectively N, the mol ratio of N-dicyclohexylcarbodiimide and PEG-b-PLL (Boc)-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): 1 ratio takes N, N-dicyclohexylcarbodiimide and N-hydroxy-succinamide, and dissolve and be configured to mixed solution with DMF, PEG-b-PLL (Boc)-end-capping reagent bi-block copolymer is added in mixed solution, under room temperature (25 ℃) condition after stirring reaction 12-36 hour, use membrane filtration, after the by-product that filtering reaction produces, mol ratio in target polypeptide TP and PEG-b-PLL (Boc)-end-capping reagent is (1-3): 1 ratio adds target polypeptide TP, as there is the peptide sequence DMPGTVLP of breast carcinoma targeting, continue reaction and after 24-72 hour, add the ether of 10-50 times of volume, through precipitation, filter, after dried, obtain TP-PEG-b-PLL (the Boc)-end-capping reagent bi-block copolymer containing targeting peptides,
S014. above-mentioned TP-PEG-b-PLL (Boc)-end-capping reagent is dissolved in to trifluoroacetic acid, under room temperature (25 ℃) condition, stir after 1-4 hour, the ether that adds 5-50 times of volume, through precipitation, filtration, dried, obtains TP-PEG-b-PLL-end-capping reagent bi-block copolymer;
S015. finally above-mentioned TP-PEG-b-PLL-end-capping reagent bi-block copolymer and DMMA are reacted after 1-5 hour in the aqueous solution of the NaOH of pH=8.0～9.0, mixing material is directly packed in the bag filter that molecular cut off is 3500KD and carries out dialysis treatment, treatment process is as follows: 12-96 hour dialyses in the aqueous solution of the NaOH of pH=8.5, every 2-6 hour changes water-dialyzing once, and lyophilizing subsequently obtains TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer.
Certainly, be to be understood that, in order to reduce the difficulty of dialysis treatment in above-mentioned steps S015, after the step of can be in above-mentioned steps S014, precipitating with ether, product TP-PEG-b-PLL-end-capping reagent is carried out to pre-dialysis treatment, treatment process is as follows: the product TP-PEG-b-PLL-end-capping reagent after ether is filtered is dissolved in polar organic solvent, use bag filter dialysis treatment 12-74 hour in water that molecular cut off is 3500KD, every 2-6 hour changes water-dialyzing once, and lyophilizing subsequently obtains TP-PEG-b-PLL-end-capping reagent bi-block copolymer.
Take end-capping reagent as HPPr be example, the chemical equation of TP-PEG-b-PLL (DMMA)-HPPr synthetic reaction is as described below:
In above-mentioned steps S02, described initiator-PLL-b-PLC bi-block copolymer synthetic be take containing amino alkane or aromatic hydrocarbons if Tutane is initiator, causes synthetic initiator-PLL (Boc)-b-PLC (Trt) bi-block copolymer of method of aminoacid anhydride ring-opening polymerisation by the amino on initiator; Then by sloughing the protecting group of lysine and cysteine side chain, obtain initiator-PLL-b-PLC bi-block copolymer.Wherein, the number-average molecular weight of described initiator-PLL-b-PLC bi-block copolymer is 500KD-40000KD.
Described initiator-PLL-b-PLC bi-block copolymer concrete technology flow process is as described below:
S021. initiator is directly joined and in DMF, form the solution that concentration range is 1-100mg/mL, in inert atmosphere conditions as added Lys (Boc)-NCA monomer under nitrogen protection, wherein the mol ratio of Lys (Boc)-NCA monomer and initiator is (10-200): 1, after isothermal reaction 24-120 hour, obtain initiator-b-PLL (Boc) bi-block copolymer;
S022. in above-mentioned reaction system, continue to add Cys (Trt)-NCA monomer, the mol ratio of described Cys (Trt)-NCA monomer and initiator is (10-200): 1, in inert atmosphere conditions as continued isothermal reaction 24-120 hour under nitrogen protection, the ether that adds 5-50 times of volume after reaction finishes, obtains Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer through precipitation, filtration, dried;
S023. above product initiator-PLL (Boc)-b-PLC (Trt) is dissolved in the trifluoroacetic acid that contains volume fraction 0.1%-10% tri isopropyl silane, in room temperature (25 ℃), stir 1-4 hour, add 5-50 times of volume ether, after precipitation, filtration treatment, to the phase be dissolved in polar organic solvent, use bag filter that molecular cut off the is 3500KD 12-96 hour that dialyses in water, every 2-6 hour changes water-dialyzing once, and lyophilizing subsequently obtains initiator-PLL-b-PLC bi-block copolymer.
Certainly, should be appreciated that in above-mentioned steps S022, also initiator-the b-PLL obtaining in S01 (Boc) bi-block copolymer can be added to the ether of 5-50 times of volume, after precipitation, filtration, dried, then add Cys (Trt)-NCA monomer to carry out aftermentioned processing.
Take initiator for being example, and the chemical equation of Tutane-PLL-b-PLC synthetic reaction is as follows:
In above-mentioned steps S03, the forming process of the tear-away polymer nanoparticle drug carriers of shell is as follows: by described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer, according to weight ratio, be (1-100): ratio (1-100), be dissolved in organic solvent completely and form homogeneous, transparent mixture solution, the mixture solution making is placed in to bag filter and carries out dialysis treatment, lyophilization.
Particularly, described organic solvent is at least one of oxolane, DMF, N,N-dimethylacetamide, dimethyl sulfoxide, N-Methyl pyrrolidone, Isosorbide-5-Nitrae-dioxane, dichloromethane, chloroform; The concentration of described mixture 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 doubly measuring at 10-500, and every 2-6 hour changes water once.
The preparation method of the tear-away intelligent polymer nano-carrier of shell that the embodiment of the present invention provides, the product stability preparing is strong, possess initiatively targeting transportation, pH response and reduction response, can load hydrophobic drug, preparation method is simply controlled, convenient operation is promoted, and has good market prospect.
And, the embodiment of the present invention provides a kind of shell tear-away nano-carrier medicine, the carrier that comprises hydrophobic drug and described hydrophobic drug, described carrier is the tear-away polymer nanoparticle drug carriers of above-mentioned shell, and described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell.
Particularly, the weight ratio of described hydrophobic drug and described carrier can be set to (1-20): (2-200),, and the weight ratio of described carrier and dewatering medicament is greater than 1.The composition of above-mentioned carrier and structure thereof as mentioned above, in order to save length, repeat no more herein.
In the tear-away nano-carrier medicine of above-mentioned shell, described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, because intracellular reduced glutathion level is approximately extracellular 100～1000 times, when nano-carrier enters after tumor cell, reduced glutathion concentration in born of the same parents is higher than outside born of the same parents, disulfide bond is unstable in reducing environment, therefore, the PLC hydrophobic cores layer forming by disulfide bond crosslinking disintegrates, hydrophobic drug in core is able to quick release, thereby reach the object for the treatment of tumor.Because the tear-away polymer nanoparticle drug carriers of described shell has the nucleocapsid structure of ad hoc structure level, make described in the embodiment of the present invention that the tear-away nano-carrier medicine of shell can have initiatively targeted delivery simultaneously, pH is responsive and the several functions such as reduction response, therefore, the tear-away nano-carrier medicine of this shell can stride across physiologic barrier complicated in body intelligently, thereby the hydrophobic drug of load is delivered to target position efficiently.
In the embodiment of the present invention, described hydrophobic drug can enter the PLC hydrophobic core layer of nano-carrier in the micelle formation process of carrier, automatic Composition becomes the tear-away polymer nanoparticle drug carriers medicine of shell, and the assembling schematic diagram of the nano-carrier medicine that the structure of the tear-away polymer nanoparticle drug carriers of its shell and shell thereof are tear-away as shown in Figure 1.
As preferred embodiment, described hydrophobic drug is selected from least one in amycin, paclitaxel, cisplatin, fluorouracil, methotrexate, camptothecine.Of course it is to be understood that other hydrophobic drugs that can carry out load with the tear-away polymer nanoparticle drug carriers preparation of above-mentioned shell in this area, all can be in embodiment of the present invention field.
As preferred embodiment, the dosage form of the nano-carrier medicine that described shell is tear-away can be made into lyophilized injectable powder or aqueous solution injection.Certainly, the tear-away polymer nanoparticle drug carriers preparation of shell is made to other dosage forms that other people body can be accepted.When the tear-away polymer nanoparticle drug carriers preparation of described shell is made lyophilized injectable powder or water solublity injection, in order to guarantee the fully effectively absorption of human body to this pharmaceutical preparation, the size of the tear-away polymer nanoparticle drug carriers preparation of described shell is had to certain requirement.As preferred embodiment, the nanometer particle size scope of described preparation is 10-300nm.Meanwhile, for as preferred embodiment, described tear-away polymer nanoparticle drug carriers preparation aqueous solution Zeta potential size is-50mv-+50mv.The particle diameter of described tear-away polymer nanoparticle drug carriers preparation and aqueous solution Zeta potential size, can with TP-PEG-b-PLL (the DMMA)-end-capping reagent of different electric charges and the input ratio of initiator-PLL-b-PLC, control by controlling in polymer nanoparticle drug carriers preparation, thereby realize the Modulatory character of particle diameter and the current potential of nano-carrier preparation.
The tear-away nano-carrier medicine of shell that the embodiment of the present invention provides, owing to take the tear-away polymer nanoparticle drug carriers of above-mentioned shell, it is carrier, and described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, and the carrier medicament of formation is stable, metabolism is complete; Can realize the functions such as initiatively targeted delivery, pH response and reduction response simultaneously, easily cross over physiologic barrier complicated in human body, avoid immune removing; And the particle diameter of this nano-carrier medicine and current potential can be by regulating the mix proportions of two kinds of bi-block copolymers to be well controlled.
Correspondingly, the embodiment of the present invention provides the preparation method of the tear-away nano-carrier medicine of a kind of shell, comprises the steps:
Method according to above-mentioned synthetic described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer is synthesized respectively TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer;
Described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer, initiator-PLL-b-PLC bi-block copolymer and hydrophobic drug are dissolved in to organic solvent and form mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers medicine of shell; Wherein, the photograph weight ratio of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer is (1-100): (1-100).
Concrete, the synthesis step of above-mentioned TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer, and the synthesis step of above-mentioned initiator-PLL-b-PLC bi-block copolymer, in the preparation method of the tear-away polymer nanoparticle drug carriers of above-mentioned shell, done detailed discussion, repeated no more herein.
Particularly, the weight ratio of described hydrophobic drug and described carrier is (1-20): (1-200).
As preferred embodiment, described hydrophobic drug is selected from least one in amycin, paclitaxel, cisplatin, fluorouracil, methotrexate, camptothecine.Of course it is to be understood that other hydrophobic drugs that can carry out load with the tear-away polymer nanoparticle drug carriers preparation of above-mentioned shell in this area, all can be in embodiment of the present invention field.
In the preparation process of the nano-carrier medicine that above-mentioned shell is tear-away, in order effectively to dissolve bi-block copolymer and the hydrophobic drug component in the tear-away polymer nanoparticle drug carriers preparation of described shell, make it form homogeneous, transparent solution, select the organic solvent good to said components solubility property.As preferred embodiment, described organic solvent is oxolane, DMF, at least one of N,N-dimethylacetamide, dimethyl sulfoxide, N-Methyl pyrrolidone, Isosorbide-5-Nitrae-dioxane, dichloromethane, chloroform.As another preferred embodiment, the method for described dialysis treatment is: mixture solution is joined to the 12-96 hour that dialyses in the water of 10-500 times of volume, every 2-6 hour changes water once.
The preparation method of the nano-carrier medicine that shell that the embodiment of the present invention provides is tear-away, method is simply controlled, has good application prospect.
And the tear-away intelligent polymer nano-carrier of shell is in the application in the fields such as nano-carrier medicine, fluorescent dye carrier, bioprobe carrier described in the embodiment of the present invention.
Below in conjunction with specific implementation method, the present invention is further detailed.
Synthesizing of embodiment 1TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer
S111 is by inflated with nitrogen protection after polymerization pipe evacuation, the HOOC-PEG-NH that is 500KD by 1g molecular weight
2after dissolving with 40mL DMF, join in polymerization pipe, by Lys (Boc)-NCA monomer and HOOC-PEG-NH
2mol ratio be 10:1 ratio adds Lys (Boc)-NCA monomer, under nitrogen protection, isothermal reaction is 24 hours, adds ether sedimentation, filtration, dry PEG-b-PLL (Boc) bi-block copolymer that obtains of 10 times after reaction finishes.
S112 joins in round-bottomed flask and stirs reaction overnight in room temperature (25 ℃) after adding 3 times of mole benzenpropanoic acid succinimide esters to dissolve with 30mL DMF above gained PEG-b-PLL (Boc) bi-block copolymer, adds ether sedimentation, filtration, dry PEG-b-PLL (the Boc)-HPPr bi-block copolymer that obtains of 20 times after reaction finishes.
S113 joins by above gained PEG-b-PLL (Boc)-HPPr bi-block copolymer the N that is dissolved with 3 times of moles, in the DMF solution of the 20mL of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 3 times of moles, at room temperature (25 ℃) stirring reaction after 24 hours, use the by-product of the membrane filtration reaction generation that aperture is 220nm, then add and the DMPGTVLP peptide sequence of PEG-b-PLL (Boc)-HPPr bi-block copolymer equimolar amounts, continue reaction and after 24 hours, add the ether sedimentation of 10 times, filter, dry TP-PEG-b-PLL (the Boc)-HPPr bi-block copolymer obtaining containing targeting peptides.
S114 is directly dissolved in trifluoroacetic acid by gained TP-PEG-b-PLL (Boc)-HPPr bi-block copolymer, in room temperature (25 ℃), stir 1-4 hour, the ether sedimentation, the filtration that add 40 times, products therefrom is dissolved in to polar organic solvent, use the bag filter that molecular cut off is 3500KD in water, to dialyse 72 hours, within every 4 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL-HPPr bi-block copolymer.
S115 reacts the DMMA of TP-PEG-b-PLL-HPPr bi-block copolymer and the 5-50 times of amino mole of lysine side-chain 5 hours in the aqueous solution of the NaOH of pH=8.5, then the bag filter that directly packs molecular cut off into and be 3500KD is dialysed 6 hours in the aqueous solution of the NaOH of pH=8.5, within every 2 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer.
Synthesizing of embodiment 2TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer
S211 is by inflated with nitrogen protection after polymerization pipe evacuation, the HOOC-PEG-NH that is 5000KD by 2g number-average molecular weight
2after dissolving with 400mL DMF, join in polymerization pipe, by Lys (Boc)-NCA monomer and HOOC-PEG-NH
2mol ratio be 5:1 ratio adds Lys (Boc)-NCA monomer, under nitrogen protection, isothermal reaction is 12 hours, adds ether sedimentation, filtration, dry PEG-b-PLL (Boc) bi-block copolymer that obtains of 25 times after reaction finishes.
S212 joins in round-bottomed flask and stirs reaction overnight in room temperature (25 ℃) after adding 5 times of mole benzenpropanoic acid succinimide esters to dissolve with 50mL DMF above gained PEG-b-PLL (Boc) bi-block copolymer, adds ether sedimentation, filtration, dry PEG-b-PLL (the Boc)-HPPr bi-block copolymer that obtains of 40 times after reaction finishes.
S213 joins by above gained PEG-b-PLL (Boc)-HPPr bi-block copolymer the N that is dissolved with 5 times of moles, in the DMF solution of the 50mL of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 5 times of moles, at room temperature (25 ℃) stirring reaction after 36 hours, use the by-product of the membrane filtration reaction generation that aperture is 220nm, then add and the mole of PEG-b-PLL (Boc)-HPPr bi-block copolymer than being the peptide sequence TP of 1:2, continue reaction and after 36 hours, add the ether sedimentation of 25 times, filter, dry TP-PEG-b-PLL (the Boc)-HPPr bi-block copolymer obtaining containing targeting peptides.
S214 is directly dissolved in trifluoroacetic acid by gained TP-PEG-b-PLL (Boc)-HPPr bi-block copolymer, in room temperature (25 ℃), stir 1-4 hour, the ether sedimentation, the filtration that add 50 times, products therefrom is dissolved in to polar organic solvent, use the bag filter that molecular cut off is 3500KD in water, to dialyse 60 hours, within every 5 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL-HPPr bi-block copolymer.
S215 reacts the DMMA of TP-PEG-b-PLL-HPPr bi-block copolymer and the 5-50 times of amino mole of lysine side-chain 3 hours in the aqueous solution of the NaOH of pH=8.0, then the bag filter that directly packs molecular cut off into and be 3500KD is dialysed 8 hours in the aqueous solution of the NaOH of pH=9.0, within every 2 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer.
Synthesizing of embodiment 3TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer
S311 is by inflated with nitrogen protection after polymerization pipe evacuation, the HOOC-PEG-NH that is 10000KD by 5g number-average molecular weight
2after dissolving with 100mL DMF, join in polymerization pipe, by Lys (Boc)-NCA monomer and HOOC-PEG-NH
2mol ratio be 10:1 ratio adds Lys (Boc)-NCA monomer, under nitrogen protection, isothermal reaction is 36 hours, adds ether sedimentation, filtration, dry PEG-b-PLL (Boc) bi-block copolymer that obtains of 50 times after reaction finishes.
S312 joins in round-bottomed flask and stirs reaction overnight in room temperature (25 ℃) after adding 8 times of mole benzenpropanoic acid succinimide esters to dissolve with 60mL DMF above gained PEG-b-PLL (Boc) bi-block copolymer, adds ether sedimentation, filtration, dry PEG-b-PLL (the Boc)-HPPr bi-block copolymer that obtains of 30 times after reaction finishes.
S313 joins by above gained PEG-b-PLL (Boc)-HPPr bi-block copolymer the N that is dissolved with 1 times of mole, in the DMF solution of the 20mL of the N-hydroxy-succinamide of N-dicyclohexylcarbodiimide and 1 times of mole, at room temperature (25 ℃) stirring reaction after 24 hours, use the by-product of the membrane filtration reaction generation that aperture is 220nm, then add and the mole of PEG-b-PLL (Boc)-HPPr bi-block copolymer than being the peptide sequence TP of 1:3, continue reaction and after 48 hours, add the ether sedimentation of 40 times, filter, dry TP-PEG-b-PLL (the Boc)-HPPr bi-block copolymer obtaining containing targeting peptides.
S314 is directly dissolved in trifluoroacetic acid by gained TP-PEG-b-PLL (Boc)-HPPr bi-block copolymer, in room temperature (25 ℃), stir 1-4 hour, the ether sedimentation, the filtration that add 20 times, products therefrom is dissolved in to polar organic solvent, use the bag filter that molecular cut off is 3500KD in water, to dialyse 72 hours, within every 3 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL-HPPr bi-block copolymer.
S315 reacts the DMMA of TP-PEG-b-PLL-HPPr bi-block copolymer and the 5-50 times of amino mole of lysine side-chain 4 hours in the aqueous solution of the NaOH of pH=8.5, then the bag filter that directly packs molecular cut off into and be 3500KD is dialysed 5 hours in the aqueous solution of the NaOH of pH=8.5, within every 2.5 hours, change water-dialyzing once, lyophilizing subsequently obtains TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer.
Synthesizing of embodiment 4Tutane-PLL-b-PLC bi-block copolymer
S421 is by inflated with nitrogen protection after polymerization pipe evacuation; after being dissolved each other, 1g Tutane and 20mL DMF join in polymerization pipe; the ratio that is 20:1 in the mol ratio of Lys (Boc)-NCA monomer and Tutane adds Lys (Boc)-NCA monomer; under nitrogen protection, isothermal reaction is 24 hours, adds ether sedimentation, filtration, dry Tutane-b-PLL (Boc) bi-block copolymer that obtains of 10 times after reaction finishes.
S422 adds above gained Tutane-b-PLL (Boc) bi-block copolymer in the polymerization pipe of inflated with nitrogen protection after evacuation; the ratio that is 10:1 in the mol ratio of Cys (Trt)-NCA monomer and Tutane adds Cys (Trt)-NCA monomer; constant temperature under nitrogen protection (30 ℃) reacts 24 hours, adds ether sedimentation, filtration, dry Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer that obtains of 20 times after reaction finishes.
S423 is dissolved in above gained Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer to contain in the trifluoroacetic acid that volume fraction is 1% tri isopropyl silane, in room temperature (25 ℃), stir 1.5 hours, then add ether sedimentation, the filtration of 20 times.Gained crude product is dissolved in to DMF, uses the bag filter that molecular cut off is 3500KD in water, to dialyse 36 hours, within every 2 hours, change water-dialyzing once, lyophilizing subsequently obtains Tutane-PLL-b-PLC bi-block copolymer.
Synthesizing of embodiment 5Tutane-PLL-b-PLC bi-block copolymer
S521 is by inflated with nitrogen protection after polymerization pipe evacuation; after being dissolved each other, 1.5g Tutane and 20mL DMF join in polymerization pipe; the ratio that is 100:1 in the mol ratio of Lys (Boc)-NCA monomer and Tutane adds Lys (Boc)-NCA monomer; under nitrogen protection, isothermal reaction is 72 hours, adds ether sedimentation, filtration, dry Tutane-b-PLL (Boc) bi-block copolymer that obtains of 25 times after reaction finishes.
S522 adds above gained Tutane-b-PLL (Boc) bi-block copolymer in the polymerization pipe of inflated with nitrogen protection after evacuation; the ratio that is 100:1 in the mol ratio of Cys (Trt)-NCA monomer and Tutane adds Cys (Trt)-NCA monomer; constant temperature under nitrogen protection (30 ℃) reacts 90 hours, adds ether sedimentation, filtration, dry Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer that obtains of 30 times after reaction finishes.
S523 is dissolved in above gained Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer to contain in the trifluoroacetic acid that volume fraction is 3% tri isopropyl silane, in room temperature (25 ℃), stir 3 hours, then add ether sedimentation, the filtration of 20 times.Gained crude product is dissolved in to DMF, uses the bag filter that molecular cut off is 3500KD in water, to dialyse 72 hours, within every 4 hours, change water-dialyzing once, lyophilizing subsequently obtains Tutane-PLL-b-PLC bi-block copolymer.
Synthesizing of embodiment 6Tutane-PLL-b-PLC bi-block copolymer
S621 is by inflated with nitrogen protection after polymerization pipe evacuation; after being dissolved each other, 2g Tutane and 40mL DMF join in polymerization pipe; the ratio that is 180:1 in the mol ratio of Lys (Boc)-NCA monomer and Tutane adds Lys (Boc)-NCA monomer; under nitrogen protection, isothermal reaction is 120 hours, adds ether sedimentation, filtration, dry Tutane-b-PLL (Boc) bi-block copolymer that obtains of 50 times after reaction finishes.
S622 adds above gained Tutane-b-PLL (Boc) bi-block copolymer in the polymerization pipe of inflated with nitrogen protection after evacuation; the ratio that is 180:1 in the mol ratio of Cys (Trt)-NCA monomer and Tutane adds Cys (Trt)-NCA monomer; constant temperature under nitrogen protection (30 ℃) reacts 120 hours, adds ether sedimentation, filtration, dry Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer that obtains of 40 times after reaction finishes.
S623 is dissolved in above gained Tutane-PLL (Boc)-b-PLC (Trt) bi-block copolymer to contain in the trifluoroacetic acid that volume fraction is 8% tri isopropyl silane, in room temperature (25 ℃), stir 4 hours, then add ether sedimentation, the filtration of 20 times.Gained crude product is dissolved in to DMF, uses the bag filter that molecular cut off is 3500KD in water, to dialyse 90 hours, within every 6 hours, change water-dialyzing once, lyophilizing subsequently obtains Tutane-PLL-b-PLC bi-block copolymer.
Nano-carrier medicine and preparation thereof that embodiment 7 is paclitaxel loaded
Paclitaxel loaded nano-carrier medicine, comprises paclitaxel and paclitaxel carrier.Described paclitaxel carrier comprises TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymers of initiator-PLL-b-PLC, two kinds of bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, this nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is to have the initiatively peptide T P of targeting, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is 1:1.Described paclitaxel loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, and the weight ratio of described paclitaxel and described carrier is 1:10.
The preparation method of paclitaxel loaded nano-carrier medicine is as follows:
Take respectively TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer and each 15mg of Tutane-PLL-b-PLC bi-block copolymer, paclitaxel 3mg, use 10mL dmso solution, ultrasonic 10min under room temperature, medicine and polymer are fully dissolved, form homogeneous, transparent organic homogeneous phase solution; It is 3500KD bag filter that the dimethyl sulphoxide solution making is placed in to molecular cut off, then in 1L water, dialyses 48 hours, within every 2 hours, changes water once; After dialysis finishes, collect the carrier micelle aqueous solution forming in bag filter, it is 110nm that DLS records size, and granule disperses relatively homogeneous.
Nano-carrier medicine and the preparation thereof of embodiment 8 load amycin
The nano-carrier medicine of load amycin, comprises amycin and amycin carrier.Described amycin carrier comprises TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymers of initiator-PLL-b-PLC, two kinds of bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, this nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is to have the initiatively peptide T P of targeting, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is 1:1.Described amycin loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, and the weight ratio of described amycin and described carrier is 1:5.
The preparation method of the nano-carrier medicine of load amycin is as follows:
Take respectively TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer 20mg and each 10mg of Tutane-PLL-b-PLC bi-block copolymer, amycin 4mg, with 100mL DMF, dissolve, ultrasonic 10min under room temperature, medicine and polymer are fully dissolved, form homogeneous, transparent organic homogeneous phase solution; By the N making, it is 2000KD bag filter that N-dimethyl formyl solution is placed in molecular cut off, then in 2L water, dialyses 60 hours, within every 4 hours, changes water once; After dialysis finishes, collect the carrier micelle aqueous solution forming in bag filter, then lyophilization obtains pulverulent solids.It is 95nm that DLS records size, and granule disperses relatively homogeneous.
Nano-carrier medicine and the preparation thereof of embodiment 9 load camptothecines
The nano-carrier medicine of load camptothecine, comprises camptothecine and camptothecine carrier.Described camptothecine carrier comprises TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymers of initiator-PLL-b-PLC, two kinds of bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, this nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is to have the initiatively peptide T P of targeting, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is 1:1.Described camptothecine loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell, and the weight ratio of described camptothecine and described carrier is 1:2.
The preparation method of the nano-carrier medicine of load camptothecine is as follows:
Take respectively TP-PEG-b-PLL (DMMA)-HPPr bi-block copolymer 10mg and each 3mg of Tutane-PLL-b-PLC bi-block copolymer, camptothecine 3mg, with 100mL DMF, dissolve, ultrasonic 10min under room temperature, medicine and polymer are fully dissolved, form homogeneous, transparent organic homogeneous phase solution; By the N making, it is 3500KD bag filter that N-dimethyl formyl solution is placed in molecular cut off, then in 2L water, dialyses 60 hours, within every 4 hours, changes water once; After dialysis finishes, collect the carrier micelle aqueous solution forming in bag filter, then lyophilization obtains pulverulent solids.It is 78nm that DLS records size, and granule disperses relatively homogeneous.
Effect embodiment
Two kinds of bi-block copolymer nano-carrier cytotoxicity analysis of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC:
The cytotoxicity that uses MTT evaluating and measuring embodiment 1 gained TP-PEG-b-PLL (DMMA)-end-capping reagent and two kinds of bi-block copolymer nano-carriers of initiator-PLL-b-PLC, experiment comprises the steps:
In each hole of 96 orifice plates, add 100 μ L to contain 1.2 * 10
4the DMEM culture medium of individual MCF-7 cell.Cultivate after 24h, in each hole, add two kinds of bi-block copolymer nano-carriers of a certain amount of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC, the mass ratio of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is 1:2, and the total concentration scope of polymer is 1 μ g/mL～200 μ g/mL (each concentration is done three Duplicate Samples).Continue to cultivate 24h, then give up the DMEM that contains polymer and add fresh DMEM and 100 μ L MTT solution in MCF-7 cell.Cultivate after 4h, then 100 μ L DMSO are joined respectively in each plate hole, room temperature vibration 10min dissolves MTT 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%, illustrates that this nano-carrier has the advantages that toxicity is low.
Technical scheme provided by the invention can be used for identical or the similar but polymer nanoparticle drug carriers preparation that material is different of project organization, the framing structure of its polymer nanoparticle drug carriers micelle can be replaced by triblock polymer, wherein peg moiety can not replace, the PLL part that side chain connects DMMA can replace containing amino polyamino acid with poly arginine or other, PLL can be used the polyamino acid of poly arginine or other cationizations to replace, PLC can replace with other hydrophobic aminoacid or the polyamino acid that is modified with disulfide bond, surface targets can be used with amino target polypeptide sequence and replace to polypeptide portion.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. the tear-away polymer nanoparticle drug carriers of shell, comprise two kinds of bi-block copolymers of TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC, two kinds of described bi-block copolymers rely on Electrostatic Absorption to be combined into nucleocapsid structure, described nucleocapsid structure is divided into four layers from outside to inside successively, wherein, outermost layer is to have the initiatively peptide T P of targeting, inferior skin is the PEG shielding, the 3rd layer is the Electrostatic Absorption layer that PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC form, the PLC hydrophobic inner core of innermost layer for being formed by disulfide bond crosslinking, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is (1-100): (1-100).
2. the tear-away polymer nanoparticle drug carriers of shell as claimed in claim 1, is characterized in that: the alkane that described end-capping reagent is activated carboxylic or aromatic hydrocarbons; And/or
Described initiator is for containing amino linear paraffin.
3. the tear-away polymer nanoparticle drug carriers of shell as claimed in claim 1 or 2, is characterized in that: the aqueous solution Zeta potential size of the nano-carrier that described shell is tear-away is-50mv-+50mv;
And/or the particle size range of nano-carrier is 10-300nm.
4. a preparation method for the tear-away polymer nanoparticle drug carriers of shell, comprises the steps:
Synthesizing of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer: by HOOC-PEG-NH
2and Lys (Boc)-NCA reaction obtains PEG-b-PLL (Boc); Use the amino of end-capping reagent sealing PLL (Boc) end to obtain PEG-b-PLL (Boc)-end-capping reagent; After the PEG c-terminus of activated PEG-b-PLL (Boc)-end-capping reagent, be combined with TP and obtain TP-PEG-b-PLL (Boc)-end-capping reagent; After being removed to Boc blocking group, TP-PEG-b-PLL (Boc)-end-capping reagent obtains TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer with the reaction of 2,3-dimethyl maleic anhydride;
Initiator-PLL-b-PLC bi-block copolymer synthetic: by initiator and Lys (Boc)-NCA reaction, obtain initiator-PLL (Boc), then and Cys (Trt)-NCA reaction obtain initiator-PLL (Boc)-b-PLC (Trt) bi-block copolymer; The protecting group Boc and the Trt that slough respectively lysine and cysteine side chain obtain initiator-PLL-b-PLC bi-block copolymer;
The formation of the tear-away polymer nanoparticle drug carriers of shell: be (1-100) according to weight ratio by described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer: ratio (1-100) is dissolved in organic solvent and forms mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers of shell.
5. the preparation method of the tear-away polymer nanoparticle drug carriers of shell as claimed in claim 4, it is characterized in that: described organic solvent is oxolane, N, dinethylformamide, N, at least one of N-dimethyl acetylamide, dimethyl sulfoxide, N-Methyl pyrrolidone, Isosorbide-5-Nitrae-dioxane, dichloromethane, chloroform;
And/or the concentration of mixture solution is 1-50mg/mL.
6. the nano-carrier medicine that shell is tear-away, the carrier that comprises hydrophobic drug and described hydrophobic drug, the tear-away polymer nanoparticle drug carriers of shell as described in described carrier is as arbitrary in claim 1～3, described hydrophobic drug loads in the hydrophobic inner core of PLC of disulfide bond crosslinking of the tear-away polymer nanoparticle drug carriers of described shell.
7. the tear-away nano-carrier medicine of shell as claimed in claim 6, is characterized in that: the weight ratio of described hydrophobic drug and described carrier is (1-20): (2-200), and described carrier and dewatering medicament weight ratio be greater than 1; And/or
Described hydrophobic drug is selected from least one in amycin, paclitaxel, cisplatin, fluorouracil, methotrexate, camptothecine.
8. a preparation method for the tear-away nano-carrier medicine of shell, comprises the steps:
Method according to claim 5 or 6 synthetic described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymers and initiator-PLL-b-PLC bi-block copolymer is synthesized respectively TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer;
Described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer, initiator-PLL-b-PLC bi-block copolymer and hydrophobic drug are dissolved in to organic solvent and form mixture homogeneous phase solution, described mixture solution is carried out to dialysis treatment, lyophilization, obtain the tear-away polymer nanoparticle drug carriers medicine of shell; Wherein, the weight ratio of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer is (1-100): (1-100).
9. the preparation method of the tear-away nano-carrier medicine of shell as claimed in claim 8, is characterized in that: the weight ratio of described hydrophobic drug and described carrier is (1-20): (1-200);
And/or described hydrophobic drug is selected from least one in amycin, paclitaxel, cisplatin, fluorouracil, methotrexate, camptothecine.
10. the tear-away polymer nanoparticle drug carriers of shell is in the application in nano-carrier medicine, fluorescent dye carrier, bioprobe carrier field.
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