CN103705931B - A kind of shell-droppable polymer nano carrier, its preparation method and application thereof - Google Patents

Abstract

The present invention is applicable to nanosecond medical science field, provides a kind of shell-droppable polymer nano carrier, its preparation method and application thereof.This shell-droppable polymer nano carrier, comprise TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC two kinds of bi-block copolymers, 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 outer successively toward interior, and the weight ratio of described TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC is (1-100): (1-100).This shell-droppable polymer nano carrier preparation can be biodegradable completely, possess the functions such as the corresponding and reduction response of active targeting transmission, pH, and its particle diameter and current potential can realize regulation and control simultaneously.

Description

A kind of shedding polymer nanocarrier, its preparation method and application

technical field

The invention belongs to the field of nanomedicine, and in particular relates to a shell-shedding polymer nanocarrier, a preparation method and application thereof.

Background technique

With the development of nanobiotechnology, multifunctional intelligent polymer nanoparticles have attracted more and more attention, especially in the field of cancer cell therapy. As a new type of drug carrier, multifunctional intelligent nanocarriers have been widely used in targeted delivery. Pharmaceutical field, and has good development prospects. After smart nanocarriers selectively deliver drugs to specific targets, they can release loaded drugs responsively after being stimulated by chemical signals or "triggers" such as temperature or pH. This kind of nanocarriers can be divided into active targeted delivery system and passive targeted delivery system.

The nano-passive targeted delivery system refers to the targeting of the nano-carriers to the reticuloendothelial system, so that the drug-loaded carriers gather at specific administration sites, and then release biologically active drugs. For example, long-circulating nanoparticles modified by polyethylene glycol (PEG) or polyethylene oxide (PEO) after intravenous administration, based on the permeability and retention effect (EPR effect) of solid tumors, make the carrier around the tumor tissue gather. The nano-active targeted delivery system is a specific modification of the drug carrier and then it can be targeted and transported to a specific treatment site in vivo. Due to the rapid proliferation of tumor cells, the expression of some specific receptors is enhanced, which increases the uptake of folic acid, vitamins and sugar by the cells. When the drug carrier modifies these specific receptors, it can significantly increase the uptake of the carrier by the cells and enhance the drug effect. . The active targeted delivery system has attracted more and more attention from the medical community because of its strong targeting and less toxic side effects.

Nanopolymer drug carriers have been proved to have great development prospects in the delivery of antitumor drugs, but there are still many shortcomings, such as most nanocarriers have poor stability, poor biocompatibility, and cannot be completely degraded in vivo and metabolism, it is easy to be cleared by the immune system during the circulation in the body, it is easy to accumulate in organs such as the liver, and it cannot reach the effective site of action, and it lacks functions such as active targeting to tumor sites. These shortcomings greatly limit the use of polymer nanocarriers. clinical application.

Contents of the invention

The purpose of the present invention is to provide a polymer nanocarrier with a detachable shell, which aims to solve the problem that the existing nanocarriers have poor stability, cannot be completely degraded or metabolized, and cannot simultaneously have functions such as active targeted delivery, pH response and reduction responsiveness. , and the problem that its particle size and potential cannot be adjusted.

Another object of the present invention is to provide a method for preparing a shell-shedding polymer nanocarrier that is easy to operate and controllable.

Another object of the present invention is to provide a nano-carrier drug with a detachable shell, which aims to solve the problem that the existing nano-carrier drug has poor stability, cannot be completely degraded or metabolized, and cannot have active targeted delivery, pH response and reduction response at the same time Functions such as sex, as well as the problem that its particle size and potential cannot be adjusted.

Correspondingly, the present invention also provides a preparation method of nano-carrier drug whose shell layer can be shed.

And, the application of a polymer nanocarrier whose shell can be shed in the fields of nanocarrier medicine, fluorescent dye carrier and biological probe carrier.

The embodiment of the present invention is achieved in this way, a kind of shedding polymer nanocarrier shell, including TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC two kinds of diblock polymerization The two block polymers are combined into a core-shell structure by electrostatic adsorption, and the core-shell structure is divided into four layers from outside to inside, in which the outermost layer is the polypeptide TP with active targeting, and the second layer is The outer layer is PEG that plays a protective role, and the third layer is an electrostatic adsorption layer formed by PLL (DMMA) in TP-PEG-b-PLL (DMMA)-capping agent and PLL in initiator-PLL-b-PLC , the innermost layer is a PLC hydrophobic core formed by disulfide bond crosslinking, and the weight ratio of the TP-PEG-b-PLL (DMMA)-capping agent to initiator-PLL-b-PLC is (1- 100): (1-100).

Correspondingly, a kind of preparation method of shedding polymer nanocarrier of shell layer, comprises the following steps:

Synthesis of TP-PEG-b-PLL(DMMA)-capping agent diblock polymer: PEG-b-PLL(Boc) was obtained by reacting HOOC-PEG-NH ₂ and Lys(Boc)-NCA; The amino group at the end of PLL (Boc) is blocked by a reagent to obtain PEG-b-PLL (Boc)-capping agent; the PEG carboxyl end of PEG-b-PLL (Boc)-capping agent is activated and combined with TP to obtain TP-PEG-b -PLL(Boc)-capping agent; TP-PEG-b-PLL(Boc)-capping agent removes Boc protecting group and reacts with 2,3-dimethylmaleic anhydride to obtain TP-PEG-b- PLL (DMMA) - capping agent diblock polymer;

Synthesis of Initiator-PLL-b-PLC Diblock Polymer: Initiator-PLL(Boc) is obtained by reaction of Initiator and Lys(Boc)-NCA, and Initiator-PLL is obtained by reaction with Cys(Trt)-NCA (Boc)-b-PLC (Trt) two-block polymer; remove the protecting groups Boc and Trt of lysine and cysteine side chains respectively to obtain initiator-PLL-b-PLC two-block polymer;

Shell layer can come off the formation of polymeric nano-carrier: the described TP-PEG-b-PLL (DMMA)-blocking agent diblock polymer and initiator-PLL-b-PLC diblock polymer are according to weight ratio The ratio of (1-100): (1-100) is dissolved in an organic solvent to form a homogeneous mixture solution, and the mixture solution is subjected to dialysis treatment, and freeze-dried to obtain a polymer nano-carrier with a detachable shell.

And, a nano-carrier drug with a shell that can be shed, including a hydrophobic drug and a carrier of the hydrophobic drug, the carrier is such as the above-mentioned polymer nano-carrier with a shell that can be shed, and the hydrophobic drug is loaded on the The shell can be shed from the hydrophobic core of the disulfide-crosslinked PLC of the polymeric nanocarrier.

Correspondingly, a kind of preparation method of the nano-carrier medicine that shell can fall off, comprises the following steps:

Synthesize TP-PEG-b-PLL ( DMMA)-capping agent diblock polymer and initiator-PLL-b-PLC diblock polymer;

The TP-PEG-b-PLL (DMMA)-capping agent diblock polymer, initiator-PLL-b-PLC diblock polymer and hydrophobic drug are dissolved in an organic solvent to form a mixture homogeneous solution, The mixture solution is subjected to dialysis treatment, and freeze-dried to obtain a shell-shedding polymer nanocarrier drug; wherein, TP-PEG-b-PLL (DMMA)-capping agent diblock polymer and initiator-PLL- The weight ratio of the b-PLC diblock polymer is (1-100):(1-100).

And, the application of a polymer nanocarrier whose shell can be shed in the fields of nanocarrier medicine, fluorescent dye carrier and biological probe carrier.

A kind of shedding polymer nano-carrier provided by the invention is composed of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC two kinds of diblock polymers combined by electrostatic adsorption Forming a core-shell structure with a specific structure makes the nanocarrier preparation good in stability and can be completely biodegraded and metabolized; at the same time, it has the functions of active targeted delivery, pH response and reduction responsiveness, and can easily cross the complex physiological barriers in the human body. Therefore, it can avoid being cleared by the immune system and reach the lesion smoothly; in addition, the particle size and potential of the nanocarrier preparation can be well controlled by adjusting the blending ratio of the two two-block polymers.

The present invention provides a nano-carrier drug with a shell that can be shed, because it has the above-mentioned shell-shed polymer nano-carrier, and the hydrophobic drug is loaded on the disulfide bridge of the shell-shed polymer nano-carrier. In the hydrophobic core of the connected PLC, the nano-carrier drug is stable and completely metabolized; it can simultaneously realize the functions of active targeted delivery, pH response and reduction response, and easily cross the complex physiological barriers in the human body and avoid the clearance of the immune system; and The particle size and potential of the nanocarrier drug can be well controlled by adjusting the doping ratio of the two diblock polymers.

The polymer nano-carrier with a shedding shell and the preparation method of the nano-carrier drug with a shedding shell provided by the invention are simple and controllable, and have good market prospects.

Description of drawings

Fig. 1 is a schematic diagram of the structure and assembly of the shell-shedding polymer nanocarrier and its nanocarrier drug provided by the embodiment of the present invention.

Fig. 2 is a DLS particle size diagram of a shell-shedding polymer nanocarrier provided by an embodiment of the present invention.

Fig. 3 is the cytotoxicity test of the shedable polymer nanocarrier provided by the embodiment of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

In the technical solution provided by the embodiments of the present invention, all occurrences of TP, PEG, PLL, PLC, DMMA, HPPr, b, Tutane, Boc, Trt, and DMF are explained as follows:

TP: polypeptide sequence with active targeted delivery function;

PEG: polyethylene glycol;

PLL: polylysine;

PLC: polycysteine;

DMMA: 2,3-dimethylmaleic acid;

HPPr: phenylpropionic acid;

Tutane: n-butylamine;

Boc:

Trt:

DMF: N,N-dimethylformamide;

b: Indicates that the polymer is a block polymer.

Both polyethylene glycol (PEG) and polyamino acid have good biocompatibility and biodegradability, and their degradation products are not toxic, so they are widely used in the fields of biomaterials and nanomedicine. Nanospheres modified with PEG surface have reduced cell adhesion, reduced adsorption of substances in serum, and reduced rejection and phagocytosis of macrophages. Therefore, nanomaterials modified with PEG can significantly increase their in vivo cycle time. Amino acids have a variety of side chain groups. After polymerizing amino acids, through different modifications to their side chain active groups, various polyamino acid materials with different properties can be obtained, such as hydrophilic, hydrophobic, electropositive, electropositive Negative, etc., so polyamino acid is a very potential biological material.

In view of this, the embodiment of the present invention provides a shell detachable polymer nanocarrier, including two diblocks of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC Polymers, two kinds of two-block polymers rely on electrostatic adsorption to combine into a core-shell structure, and the core-shell structure is divided into four layers from the outside to the inside, and the outermost layer is the one that can mediate active targeted delivery. Polypeptide sequence TP, the second outer layer is PEG for protection, the third layer is TP-PEG-b-PLL(DMMA)-PLL in capping agent (DMMA) and initiator-PLL-PLL in b-PLC The electrostatic adsorption layer formed, the innermost layer is a PLC hydrophobic core formed by disulfide bond crosslinking, and the weight of the TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC The ratio is (1-100):(1-100).

Specifically, in the above-mentioned shell-shedding polymer nanocarrier, by adjusting the ratio of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC two kinds of diblock polymers, To effectively control the Zeta potential size of the nanocarrier aqueous solution and the particle size of the nanocarrier drug. As a preferred embodiment, the Zeta potential of the aqueous solution of the nano-carrier whose shell can fall off is -50mv-+50mv; and/or the particle size range of the nano-carrier is 10-300nm, and Fig. 2 is the shell of the embodiment of the present invention DLS particle size plot of exfoliatable polymeric nanocarriers.

The number average molecular weight range of the PEG segment is 500KD-10000KD, the degree of polymerization of the PLL (DMMA) segment is 10-200, the degree of polymerization of the PLL segment is 10-200, and the degree of polymerization of the PLC segment is 10-200.

In the two diblock polymers of the above-mentioned TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC, in the TP-PEG-b-PLL (DMMA)-capping agent The PEG is amino-polyethylene glycol-carboxyl (HOOC-PEG-NH ₂ ), the PEG is connected to PLL (DMMA) at the amino terminal through an amide bond, and the side chain amino group of the PLL is covalently connected to DMMA through an amide bond combined. After modifying the side chain amino group of PLL by forming an amide bond with DMMA, the original positive TP-PEG-b-PLL-capping agent is transformed into a pH-sensitive electronegative material TP-PEG-b-PLL (DMMA) - capping agent. The electronegative material TP-PEG-b-PLL (DMMA)-capping agent can electrostatically adsorb a certain proportion of initiator-PLL-b-PLC, thereby adjusting the charge quantity of the shell that can shed the polymer nanocarrier as a whole, Even the charge properties of TP-PEG-b-PLL(DMMA)-capping agent changed. In the initiator-PLL-b-PLC, the N-terminal of the initiator is connected to the PLL through an amide bond.

The shell layer can shed the polymer nanocarrier, with the TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC formed by the above links as the skeleton, which can rely on electrostatic adsorption in aqueous solution Combined into a core-shell structure, automatically assembled to form a core-shell structure. The core-shell structure is divided into four layers from the outside to the inside, which are as follows: the outermost layer is a polypeptide TP with active targeting, which can be combined with specific receptors at the tumor site to achieve rapid growth of nanomicelles at the tumor site. accumulation; the second outer layer is the protective layer PEG, which can reduce the specific adsorption, thereby increasing the circulation time of the nanomicelle in the organism; the third layer is the PLL in TP-PEG-b-PLL(DMMA)-capping agent (DMMA) and PLL in Initiator-PLL-b-PLC to form an electrostatic adsorption layer through which a pH-responsive structure that the shell of the polymer nanocarrier formulation can be shed is formed. Due to the rapid proliferation of tumor tissue, the vasculature of the tumor is often unable to adequately supply nutrients and oxygen to a large number of tumor cells. Insufficient supply makes the tumor tissue hypoxic and produces lactic acid, which is hydrolyzed by ATP to generate an acidic microenvironment. Therefore, the pH of infected tissue, primary tumor and secondary tumor tissue is lower than that of normal tissue. Therefore, the electrostatic adsorption layer can realize a pH-sensitive stimulus-responsive targeted drug delivery system. The innermost layer is a PLC hydrophobic inner layer formed by disulfide bond cross-linking, which can be used to load hydrophobic drugs. With the core-shell structure of this specific structural level, the polymer nanocarrier with the shedable shell layer described in the embodiment of the present invention has multiple functions such as active targeted delivery, pH sensitivity and reduction response. Therefore, the shell layer can be shed and polymerized Bio-nanocarriers can intelligently cross the complex physiological barriers in the body, so that when the shell-shedding polymer nanocarriers are loaded with hydrophobic drugs, they can be smoothly and efficiently delivered to the target site.

In order to prevent the PLL terminal amino group of TP-PEG-b-PLL (DMMA) from bonding with the target polypeptide sequence and not increase the cytotoxicity of the carrier, a non-toxic Blocking agent, a blocking agent was introduced at the PLL end of the above-mentioned TP-PEG-b-PLL (DMMA). As a preferred embodiment, the capping agent is a carboxyl-activated alkane or an aromatic hydrocarbon. As a further preferred embodiment, the end-capping agent is HPPr, succinimide octadecanoate.

In order not to affect the function of the initiator-PLL-b-PLC, a small molecule with an amino group is introduced as an initiator in the synthesis of the above-mentioned initiator-PLL-b-PLC. As a preferred embodiment, the initiator-PLL- The initiator in b-PLC is alkanes or aromatics containing amino groups. As a further preferred embodiment, the initiator is at least one of Tutane and octadecylamine.

The shell shedable polymer nanocarrier provided by the embodiments of the present invention is combined into a core-shell structure through electrostatic adsorption between TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC; The core-shell structure has a four-layer structure, the outermost layer is a polypeptide TP with active targeting, the second outer layer is a protective PEG, the third layer is PLL (DMMA) and the electrostatic adsorption layer of PLL, and the innermost layer is two Sulfur-bonded PLC hydrophobic core. The shell layer with the specific core-shell structure can shed the polymer nanocarrier, which has the following advantages:

1. The skeleton material of the shedding polymer nanocarrier is TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC diblock polymer, which has good biocompatibility. It has low toxicity and can be completely degraded in vivo, and the degradation products are non-toxic and harmless, and can be absorbed or metabolized in vivo.

2. The shell-shedding polymer nanocarrier has polypeptide molecules for active targeted delivery, which can efficiently deliver hydrophobic drugs to the lesion site.

3. The shell-shedding polymer nanocarrier has both a shell-shedding pH-responsive structure and a reduction-responsive disulfide bond structure, which can intelligently respond to various complex physiological environments in the human body, and intelligently span various complex physiological environments in the body. Physiological barriers, therefore, can adapt to complex physiological environments in vivo and when the shell-shedding polymer nanocarriers are loaded with hydrophobic drugs, rapid release of hydrophobic small-molecule drugs at targeted locations can be achieved.

4. The shedding polymer nanocarrier has good stability, and its PEG shell can reduce non-specific adsorption and effectively protect the nanocarrier preparation from being quickly cleared by the immune system.

5. The particle size and Zeta potential of the exfoliated polymer nanocarrier can be regulated by adjusting the mixing ratio of two diblock polymers.

The shell-shedding polymer nanocarrier preparation described in the embodiment of the present invention can be prepared by the following method, of course, it can also be prepared by other methods that can obtain the shell-shedding polymer nanocarrier preparation.

Correspondingly, an embodiment of the present invention provides a method for preparing a shell-shedding polymer nanocarrier, comprising the following steps:

Synthesis of S01.TP-PEG-b-PLL (DMMA)-capping agent diblock polymer: PEG-b-PLL (Boc) is obtained by reacting HOOC-PEG-NH ₂ and Lys(Boc)-NCA; using The end-capping agent blocks the amino group at the end of PLL (Boc) to obtain PEG-b-PLL(Boc)-capping agent; activates the PEG carboxyl end of PEG-b-PLL(Boc)-capping agent and combines with TP to obtain TP-PEG -b-PLL (Boc)-blocking agent; TP-PEG-b-PLL (Boc)-blocking agent is removed from the Boc protecting group and then reacted with 2,3-dimethylmaleic anhydride to obtain TP-PEG- b-PLL(DMMA)-capper diblock polymer;

S02. Synthesis of Initiator-PLL-b-PLC Diblock Polymer: Reaction of Initiator with Lys(Boc)-NCA to Obtain Initiator-PLL(Boc), Reaction with Cys(Trt)-NCA to Obtain Initiator -PLL(Boc)-b-PLC(Trt) two-block polymer; remove the protecting group Boc and Trt of lysine and cysteine side chains to obtain initiator-PLL-b-PLC two-block polymer ;

S03. The formation of the shedding polymer nanocarrier of the shell layer: the TP-PEG-b-PLL (DMMA)-capping agent diblock polymer and initiator-PLL-b-PLC diblock polymer according to The weight ratio is (1-100):(1-100) dissolved in an organic solvent to form a homogeneous mixture solution, and the mixture solution is subjected to dialysis treatment and freeze-dried to obtain a shell-shedding polymer nanocarrier.

Specifically, in the above step S01, the synthesis of TP-PEG-b-PLL (DMMA)-capping agent diblock polymer uses HOOC-PEG _- NH as the initiator, and the amino acid anhydride ring-opening of the initiator is initiated Polymerization method to synthesize PEG-b-PLL (Boc) two-block polymer, using carboxyl-activated alkane or aromatic hydrocarbon such as HPPr as the end-capping agent to block the amino group at the end of PLL to obtain PEG-b-PLL (Boc)-capped Terminating agent diblock polymer; then the carboxyl group at the PEG end of the PEG-b-PLL (Boc)-capping agent is activated, and the PEG-b-PLL (Boc)-capping agent is linked to the targeting polypeptide TP through the activated carboxyl group Combined to obtain TP-PEG-b-PLL(Boc)-blocking agent diblock polymer; then remove the tert-butoxycarbonyl protecting group Boc of the lysine side chain to obtain TP-PEG-b-PLL-blocking Terminal agent two-block polymer; finally use the amino group of lysine side chain to react with DMMA to obtain negatively charged TP-PEG-b-PLL (DMMA)-end-capping agent two-block polymer. Wherein, the PEG is HOOC-PEG-NH ₂ , and its number average molecular weight range is 500KD-10000KD, and the number average molecular weight of the prepared TP-PEG-b-PLL (DMMA)-capping agent diblock polymer is 800KD-30000KD.

Specifically, the specific process flow of TP-PEG-b-PLL (DMMA)-capping agent diblock polymer is as follows:

S011. Dissolving HOOC-PEG-NH ₂ in DMF to form a solution with a concentration range of 1-100mg/mL, in an inert gas atmosphere-such as nitrogen protection, according to Lys(Boc)-NCA and HOOC-PEG-NH ₂ The molar ratio is (10-200):1, add Lys(Boc)-NCA monomer, and heat the reaction at a constant temperature of 30-50°C for 24-120 hours. After the reaction is completed, add 5-50 times the volume of diethyl ether, and obtain PEG-b-PLL (Boc) diblock polymer through precipitation, filtration, and drying;

S012. above-mentioned PEG-b-PLL (Boc) is dissolved in DMF, is (1-10): 1 to add end-capping agent by the mol ratio of end-capping agent and PEG-b-PLL (Boc), at room temperature (25 °C) for 12-36 hours. After the reaction is completed, add 5-50 times the volume of ether, and obtain PEG-b-PLL(Boc)-capping agent diblock polymer through precipitation, filtration and drying;

S013. According to the molar ratio of N, N-dicyclohexyl carbodiimide and PEG-b-PLL (Boc)-capping agent respectively (3-10): 1, N-hydroxysuccinimide and PEG- The molar ratio of b-PLL(Boc)-capping agent is (3-10): 1. Weigh N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide, and dissolve them in DMF to form Mix the solution, add PEG-b-PLL(Boc)-end-capping agent diblock polymer into the mixed solution, stir and react at room temperature (25°C) for 12-36 hours, then use filter membrane to filter and filter out the reaction After the by-products are produced, the molar ratio of targeting polypeptide TP and PEG-b-PLL (Boc)-capping agent is (1-3): 1. Add targeting polypeptide TP, such as breast cancer targeting The polypeptide sequence DMPGTVLP, continue to react for 24-72 hours, add 10-50 times the volume of ether, after precipitation, filtration, and drying, the TP-PEG-b-PLL(Boc)-capping agent containing the targeting peptide is obtained Diblock polymers;

S014. Dissolve the above-mentioned TP-PEG-b-PLL(Boc)-capping agent in trifluoroacetic acid, stir at room temperature (25°C) for 1-4 hours, add 5-50 times the volume of ether, and Precipitate, filter, and dry to obtain TP-PEG-b-PLL-capping agent diblock polymer;

S015. Finally, after reacting the above-mentioned TP-PEG-b-PLL-capping agent diblock polymer and DMMA in an aqueous solution of NaOH with a pH of 8.0 to 9.0 for 1-5 hours, the mixed liquid is directly loaded into Carry out dialysis treatment in the dialysis bag of 3500KD, treatment process is as follows: in the aqueous solution of NaOH of pH=8.5 dialysis 12-96 hours, change dialysis water once every 2-6 hours, then lyophilize to obtain TP-PEG-b-PLL ( DMMA) - capping agent diblock polymer.

Of course, it should be understood that in order to reduce the difficulty of the dialysis treatment in the above step S015, the product TP-PEG-b-PLL-capping agent can be predialyzed after the step of precipitation with ether in the above step S014, The treatment process is as follows: dissolve the product TP-PEG-b-PLL-capping agent filtered by ether in a polar organic solvent, and use a dialysis bag with a molecular weight cut-off of 3500KD to dialyze in water for 12-74 hours, every 2-6 The dialysis water was changed every hour, and then freeze-dried to obtain the TP-PEG-b-PLL-capping agent diblock polymer.

Taking the capping agent as HPPr as an example, the chemical equation of the synthesis reaction of TP-PEG-b-PLL(DMMA)-HPPr is as follows:

In the above step S02, the synthesis of the initiator-PLL-b-PLC two-block polymer uses an amino-containing alkane or an aromatic hydrocarbon such as Tutane as an initiator, and the amino acid anhydride ring-opening polymerization of an amino acid is initiated by the amino group on the initiator. Initiator-PLL(Boc)-b-PLC(Trt) two-block polymer; Then, initiator-PLL-b-PLC two-block polymerization is obtained by deprotecting the side chains of lysine and cysteine thing. Wherein, the number average molecular weight of the initiator-PLL-b-PLC diblock polymer is 500KD-40000KD.

The specific technological process of described initiator-PLL-b-PLC diblock polymer is as follows:

S021. The initiator is directly added to DMF to form a solution with a concentration range of 1-100mg/mL, and Lys(Boc)-NCA monomer is added under inert atmosphere conditions such as nitrogen protection, wherein Lys(Boc)-NCA monomer and The molar ratio of the initiator is (10-200): 1, and the initiator-b-PLL (Boc) diblock polymer is obtained after constant temperature reaction for 24-120 hours;

S022. Continue to add Cys(Trt)-NCA monomer to the above reaction system, the molar ratio of Cys(Trt)-NCA monomer to initiator is (10-200):1, under inert atmosphere conditions such as nitrogen protection Continue the reaction at constant temperature for 24-120 hours, add 5-50 times the volume of diethyl ether after the reaction, precipitate, filter, and dry to obtain the Tutane-PLL(Boc)-b-PLC(Trt) diblock polymer;

S023. Dissolve the above product initiator-PLL(Boc)-b-PLC(Trt) in trifluoroacetic acid containing 0.1%-10% triisopropylsilane by volume fraction, stir at room temperature (25°C) for 1-4 Hours, add 5-50 times the volume of ether, after precipitation and filtration, dissolve the phase in polar organic solvents, and use a dialysis bag with a molecular weight cut-off of 3500KD to dialyze in water for 12-96 hours, every 2-6 hours The dialysis water was changed once, and then freeze-dried to obtain the initiator-PLL-b-PLC diblock polymer.

Of course, it should be understood that in the above step S022, the initiator-b-PLL (Boc) diblock polymer obtained in S01 can also be added with 5-50 times the volume of ether, and after precipitation, filtration, and drying, Then add Cys(Trt)-NCA monomer to carry out the treatment described later.

Taking the initiator as an example, the chemical reaction equation of the synthesis reaction of Tutane-PLL-b-PLC is as follows:

In the above-mentioned step S03, the formation process of the shedding polymer nanocarrier of the shell layer is as follows: the two block polymers of the TP-PEG-b-PLL (DMMA)-capping agent and the initiator-PLL-b-PLC The block polymer is completely dissolved in the organic solvent according to the weight ratio of (1-100): (1-100) to form a uniform and transparent mixture solution, and the prepared mixture solution is placed in a dialysis bag for dialysis treatment. Freeze dried.

Specifically, the organic solvent is tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, 1,4-dioxane , at least one of dichloromethane and chloroform; the concentration of the mixture solution is 1-50 mg/mL. As another preferred embodiment, the step of the dialysis treatment is dialysis in 10-500 times the amount of water for 12-96 hours, changing the water every 2-6 hours.

The preparation method of the intelligent polymer nanocarrier with a detachable shell provided by the embodiment of the present invention has strong stability, active targeted transport, pH response and reduction responsiveness, and can load hydrophobic drugs. The preparation method is simple and reliable. control, easy to operate and promote, and has a good market prospect.

And, an embodiment of the present invention provides a nano-carrier drug with a detachable shell, including a hydrophobic drug and a carrier of the hydrophobic drug, the carrier is the above-mentioned polymer nano-carrier with a detachable shell, and the hydrophobic The drug is loaded in the hydrophobic core of the disulfide-crosslinked PLC of the shell exfoliatable polymeric nanocarriers.

Specifically, the weight ratio of the hydrophobic drug to the carrier can be set as (1-20):(2-200), and the weight ratio of the carrier to the hydrophobic drug is greater than 1. The composition and structure of the above-mentioned carrier are as described above, and will not be repeated here for the sake of saving space.

In the above-mentioned nano-carrier drug whose shell can be shed, the hydrophobic drug is loaded in the hydrophobic core of the disulfide-bonded PLC of the polymer nano-carrier that can be shed. The peptide level is about 100-1000 times that of the extracellular. When the nanocarrier enters the tumor cell, the concentration of reduced glutathione in the cell is higher than that in the extracellular. The disulfide bond is unstable in the reducing environment. The PLC hydrophobic inner layer formed by bond cross-linking is disintegrated, and the hydrophobic drug in the core can be released quickly, so as to achieve the purpose of treating tumors. Since the polymer nanocarrier with a shedable shell layer has a core-shell structure with a specific structural level, the nanocarrier drug with a shedable shell layer described in the embodiment of the present invention can simultaneously have active targeted delivery, pH sensitivity, and reduction response. Therefore, the shell-shedding nanocarrier drug can intelligently cross the complex physiological barriers in vivo, thereby efficiently delivering the loaded hydrophobic drug to the target site.

In the embodiment of the present invention, the hydrophobic drug can enter the PLC hydrophobic core layer of the nano-carrier during the formation of the carrier micelle, and automatically assemble into a shell that can shed the polymer nano-carrier drug, and its shell can shed the polymer nano-carrier drug. The structure of the carrier and the schematic diagram of the assembly of the nano-carrier drug with detachable shell are shown in Fig. 1 .

As a preferred embodiment, the hydrophobic drug is at least one selected from doxorubicin, paclitaxel, cisplatin, fluorouracil, methotrexate, and camptothecin. Of course, it should be understood that other hydrophobic drugs that can be loaded by the shell-shedding polymer nanocarrier formulations in the art can be used in the field of the embodiments of the present invention.

As a preferred embodiment, the dosage form of the nano-carrier drug whose shell layer can be shed can be made into freeze-dried powder injection or aqueous solution injection. Certainly, the polymer nano-carrier preparation with the shedding shell can be made into other dosage forms acceptable to other human bodies. When the shell-shedding polymer nanocarrier preparation is made into freeze-dried powder injection or water-soluble injection, in order to ensure that the human body can fully and effectively absorb the pharmaceutical preparation, the shell-shedding polymer nanocarrier preparation Particle size has certain requirements. As a preferred embodiment, the nanometer particle diameter of the preparation is in the range of 10-300nm. Meanwhile, as a preferred embodiment, the Zeta potential of the aqueous solution of the detachable polymer nanocarrier preparation is -50mv-+50mv. The particle size and the aqueous solution Zeta potential size of the described detachable polymer nanocarrier preparation can be controlled by controlling the TP-PEG-b-PLL (DMMA)-capping agent and initiator- with different charges in the polymer nanocarrier preparation The dosage ratio of PLL-b-PLC is controlled, so as to realize the controllability of the particle size and potential of the nanocarrier preparation.

The nano-carrier drug with a shedable shell provided by the embodiments of the present invention uses the above-mentioned shedable polymer nanocarrier as a carrier, and the hydrophobic drug is loaded on the disulfide of the shedable polymer nanocarrier. In the hydrophobic core of bonded and cross-linked PLC, the carrier drug formed is stable and completely metabolized; it can simultaneously realize functions such as active targeted delivery, pH response and reduction response, and can easily cross the complex physiological barriers in the human body and avoid clearance by the immune system; And the particle size and potential of the nano-carrier drug can be well controlled by adjusting the blending ratio of the two two-block polymers.

Correspondingly, an embodiment of the present invention provides a method for preparing a nanocarrier drug with a detachable shell, comprising the following steps:

Synthesize TP-PEG-b-PLL ( DMMA)-capping agent diblock polymer and initiator-PLL-b-PLC diblock polymer;

The TP-PEG-b-PLL (DMMA)-capping agent diblock polymer, initiator-PLL-b-PLC diblock polymer and hydrophobic drug are dissolved in an organic solvent to form a mixture homogeneous solution, The mixture solution is subjected to dialysis treatment, and freeze-dried to obtain a shell-shedding polymer nanocarrier drug; wherein, TP-PEG-b-PLL (DMMA)-capping agent diblock polymer and initiator-PLL- The weight ratio of the b-PLC diblock polymer is (1-100):(1-100).

Specifically, the synthesis steps of the above-mentioned TP-PEG-b-PLL (DMMA)-capping agent diblock polymer, and the synthesis steps of the above-mentioned initiator-PLL-b-PLC diblock polymer, in the above shell The preparation method of the detachable polymer nanocarrier has been discussed in detail, and will not be repeated here.

Specifically, the weight ratio of the hydrophobic drug to the carrier is (1-20):(1-200).

As a preferred embodiment, the hydrophobic drug is at least one selected from doxorubicin, paclitaxel, cisplatin, fluorouracil, methotrexate, and camptothecin. Of course, it should be understood that other hydrophobic drugs that can be loaded by the shell-shedding polymer nanocarrier formulations in the art can be used in the field of the embodiments of the present invention.

In the preparation step of the above-mentioned shell-shedding nanocarrier drug, in order to effectively dissolve the two-block polymer and the hydrophobic drug component in the shell-shedding polymer nanocarrier preparation, make it form a uniform, transparent For the solution, an organic solvent with excellent solubility to the above components is selected for use. As a preferred embodiment, the organic solvent is tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, 1,4-dioxo At least one of hexacyclic ring, dichloromethane, and chloroform. As another preferred embodiment, the dialysis treatment method is: adding the mixture solution into 10-500 times the volume of water for dialysis for 12-96 hours, and changing the water every 2-6 hours.

The preparation method of the nano-carrier drug with a detachable shell provided by the embodiment of the present invention is simple and controllable, and has a good application prospect.

And, the application of the intelligent polymer nano-carrier with a detachable shell in the embodiment of the present invention in the fields of nano-carrier medicine, fluorescent dye carrier, biological probe carrier and the like.

The present invention will be further described below in combination with specific implementation methods.

The synthesis of embodiment 1TP-PEG-b-PLL (DMMA)-HPPr diblock polymer

S111 Vacuumize the polymerization tube and fill it with nitrogen to protect it. Dissolve 1g of HOOC-PEG-NH ₂ with a molecular weight of _500KD in 40mL of DMF and add it to the polymerization tube. Add Lys(Boc)-NCA monomer at a ratio of 10:1, and react at a constant temperature under nitrogen protection for 24 hours. After the reaction, add 10 times of ether to precipitate, filter, and dry to obtain PEG-b-PLL(Boc) diblock polymer.

S112 Add the PEG-b-PLL (Boc) diblock polymer obtained above to 3 times the molar amount of succinimide phenylpropionate and dissolve it in 30 mL of DMF, then add it to a round-bottomed flask and stir overnight at room temperature (25°C). After the reaction, add 20 times of diethyl ether to precipitate, filter and dry to obtain PEG-b-PLL(Boc)-HPPr diblock polymer.

S113 Add the PEG-b-PLL(Boc)-HPPr diblock polymer obtained above to the dissolved N, N-dicyclohexylcarbodiimide and 3 times the molar amount of N-hydroxysuccinyl In the 20mL DMF solution of imine, after stirring the reaction at room temperature (25°C) for 24 hours, the by-products generated by the reaction were filtered through a filter membrane with a pore size of 220nm, and then added with PEG-b-PLL(Boc)-HPPr DMPGTVLP polypeptide sequence in an equimolar amount of the block polymer, continue to react for 24 hours, add 10 times of ether to precipitate, filter, and dry to obtain a TP-PEG-b-PLL(Boc)-HPPr diblock polymer containing targeting peptide.

S114 Dissolve the obtained TP-PEG-b-PLL(Boc)-HPPr diblock polymer directly in trifluoroacetic acid, stir at room temperature (25°C) for 1-4 hours, add 40 times of diethyl ether to precipitate, filter, and The obtained product was dissolved in a polar organic solvent, dialyzed in water for 72 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 4 hours, and then freeze-dried to obtain a TP-PEG-b-PLL-HPPr diblock polymer.

S115 React TP-PEG-b-PLL-HPPr diblock polymer and DMMA with 5-50 times the molar weight of lysine side chain amino group in NaOH aqueous solution with pH=8.5 for 5 hours, and then directly load the molecular weight cut-off The 3500KD dialysis bag was dialyzed in NaOH aqueous solution with pH=8.5 for 6 hours, and the dialysis water was changed every 2 hours, and then freeze-dried to obtain TP-PEG-b-PLL(DMMA)-HPPr diblock polymer.

The synthesis of embodiment 2TP-PEG-b-PLL (DMMA)-HPPr diblock polymer

S211 Vacuumize the polymerization tube and fill it with nitrogen for protection. Dissolve _2g of HOOC-PEG-NH ₂ with a number average molecular weight of 5000KD in 400mL DMF and add it to the polymerization tube. Lys(Boc)-NCA monomer was added at a molar ratio of 5:1, and reacted at a constant temperature for 12 hours under nitrogen protection. After the reaction, 25 times of diethyl ether was added to precipitate, filter, and dry to obtain PEG-b-PLL(Boc). block polymers.

S212 Add the PEG-b-PLL (Boc) diblock polymer obtained above to 5 times the molar amount of succinimide phenylpropionate and dissolve it in 50 mL of DMF, then add it to a round-bottomed flask and stir overnight at room temperature (25°C) for reaction. After the reaction, add 40 times of diethyl ether to precipitate, filter and dry to obtain PEG-b-PLL(Boc)-HPPr diblock polymer.

S213 Add the PEG-b-PLL(Boc)-HPPr diblock polymer obtained above to the N, N-dicyclohexylcarbodiimide and 5 times the molar amount of N-hydroxysuccinyl dissolved in it In 50mL of DMF solution of imine, stirred and reacted at room temperature (25°C) for 36 hours, the by-products generated by the reaction were filtered through a filter membrane with a pore size of 220nm, and then added with PEG-b-PLL(Boc)-HPPr Polypeptide sequence TP with a molar ratio of 1:2, continue to react for 36 hours, add 25 times of diethyl ether to precipitate, filter, and dry to obtain TP-PEG-b-PLL(Boc)-HPPr containing targeting peptide block polymers.

S214 Dissolve the obtained TP-PEG-b-PLL(Boc)-HPPr diblock polymer directly in trifluoroacetic acid, stir at room temperature (25°C) for 1-4 hours, add 50 times of diethyl ether to precipitate, filter, and The obtained product was dissolved in a polar organic solvent, dialyzed in water for 60 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 5 hours, and then freeze-dried to obtain a TP-PEG-b-PLL-HPPr diblock polymer.

S215 react TP-PEG-b-PLL-HPPr diblock polymer with DMMA of 5-50 times the molar weight of lysine side chain amino group in NaOH aqueous solution with pH=8.0 for 3 hours, and then directly load the molecular weight cut-off The 3500KD dialysis bag was dialyzed in NaOH aqueous solution with pH=9.0 for 8 hours, and the dialysis water was changed every 2 hours, and then freeze-dried to obtain TP-PEG-b-PLL(DMMA)-HPPr diblock polymer.

The synthesis of embodiment 3TP-PEG-b-PLL (DMMA)-HPPr diblock polymer

S311 Vacuumize the polymerization tube and fill it with nitrogen for protection. Dissolve 5 _g of HOOC-PEG-NH ₂ with a number average molecular weight of 10,000 KD in 100 mL of DMF and add it to the polymerization tube. Lys(Boc)-NCA monomer was added at a molar ratio of 10:1, and reacted at a constant temperature under nitrogen protection for 36 hours. After the reaction, 50 times of ether was added to precipitate, filtered, and dried to obtain PEG-b-PLL(Boc) two block polymers.

S312 Add the PEG-b-PLL (Boc) diblock polymer obtained above to 8 times the molar amount of succinimide phenylpropionate and dissolve it in 60 mL of DMF, then add it to a round-bottomed flask and stir at room temperature (25°C) overnight for reaction. After the reaction, add 30 times of diethyl ether to precipitate, filter and dry to obtain PEG-b-PLL(Boc)-HPPr diblock polymer.

S313 Add the PEG-b-PLL(Boc)-HPPr diblock polymer obtained above to the N, N-dicyclohexylcarbodiimide and 1 times the molar amount of N-hydroxysuccinyl dissolved in it In the 20mL DMF solution of imine, after stirring the reaction at room temperature (25°C) for 24 hours, the by-products generated by the reaction were filtered through a filter membrane with a pore size of 220nm, and then added with PEG-b-PLL(Boc)-HPPr Polypeptide sequence TP with a molar ratio of 1:3, continue to react for 48 hours, add 40 times of diethyl ether to precipitate, filter, and dry to obtain TP-PEG-b-PLL(Boc)-HPPr containing targeting peptide block polymers.

S314 Dissolve the obtained TP-PEG-b-PLL(Boc)-HPPr diblock polymer directly in trifluoroacetic acid, stir at room temperature (25°C) for 1-4 hours, add 20 times of diethyl ether to precipitate, filter, and The obtained product was dissolved in a polar organic solvent, dialyzed in water for 72 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 3 hours, and then freeze-dried to obtain a TP-PEG-b-PLL-HPPr diblock polymer.

S315 react TP-PEG-b-PLL-HPPr diblock polymer with DMMA of 5-50 times the molar weight of lysine side chain amino group in NaOH aqueous solution with pH=8.5 for 4 hours, and then directly load the molecular weight cut-off The 3500KD dialysis bag was dialyzed in NaOH aqueous solution with pH=8.5 for 5 hours, the dialysis water was changed every 2.5 hours, and then freeze-dried to obtain TP-PEG-b-PLL(DMMA)-HPPr diblock polymer.

The synthesis of embodiment 4Tutane-PLL-b-PLC diblock polymer

S421 Vacuumize the polymerization tube and fill it with nitrogen gas protection, mix 1gTutane and 20mL DMF into the polymerization tube, add Lys(Boc)-NCA single body, reacted at constant temperature for 24 hours under the protection of nitrogen, and added 10 times of diethyl ether to precipitate after the reaction, filtered, and dried to obtain the Tutane-b-PLL (Boc) diblock polymer.

S422 Add the Tutane-b-PLL (Boc) diblock polymer obtained above into the polymerization tube filled with nitrogen protection after vacuuming, and add the molar ratio of Cys(Trt)-NCA monomer to Tutane as 10:1 Cys(Trt)-NCA monomer was reacted at constant temperature (30°C) for 24 hours under the protection of nitrogen. After the reaction, 20 times of ether was added to precipitate, filtered and dried to obtain Tutane-PLL(Boc)-b-PLC(Trt) segment polymer.

S423 Dissolve the Tutane-PLL(Boc)-b-PLC(Trt) diblock polymer obtained above in trifluoroacetic acid containing 1% triisopropylsilane by volume fraction, and stir at room temperature (25°C) for 1.5 Hours, then add 20 times of diethyl ether to precipitate and filter. The resulting crude product was dissolved in DMF, dialyzed in water for 36 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 2 hours, and then freeze-dried to obtain the Tutane-PLL-b-PLC diblock polymer.

The synthesis of embodiment 5Tutane-PLL-b-PLC diblock polymer

S521 Vacuumize the polymerization tube and fill it with nitrogen for protection. Mix 1.5g Tutane and 20mL DMF into the polymerization tube and add Lys(Boc)-NCA at a molar ratio of Lys(Boc)-NCA monomer to Tutane of 100:1. The monomer was reacted at constant temperature for 72 hours under the protection of nitrogen. After the reaction, 25 times of diethyl ether was added to precipitate, filtered, and dried to obtain the Tutane-b-PLL (Boc) diblock polymer.

S522 Add the above-obtained Tutane-b-PLL (Boc) diblock polymer into the polymerization tube filled with nitrogen protection after vacuuming, and add the molar ratio of Cys(Trt)-NCA monomer to Tutane as 100:1 Cys(Trt)-NCA monomer was reacted at constant temperature (30°C) for 90 hours under nitrogen protection. After the reaction, 30 times of ether was added to precipitate, filtered and dried to obtain Tutane-PLL(Boc)-b-PLC(Trt) segment polymer.

S523 Dissolve the Tutane-PLL(Boc)-b-PLC(Trt) diblock polymer obtained above in trifluoroacetic acid containing a volume fraction of 3% triisopropylsilane, and stir at room temperature (25° C.) for 3 Hours, then add 20 times of diethyl ether to precipitate and filter. The resulting crude product was dissolved in DMF, dialyzed in water for 72 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 4 hours, and then freeze-dried to obtain the Tutane-PLL-b-PLC diblock polymer.

The synthesis of embodiment 6Tutane-PLL-b-PLC diblock polymer

S621 Vacuumize the polymerization tube and fill it with nitrogen for protection. 2gTutane and 40mL DMF are miscible and added to the polymerization tube. Lys(Boc)-NCA After the reaction, 50 times of diethyl ether was added to precipitate, filtered, and dried to obtain the Tutane-b-PLL (Boc) diblock polymer.

S622 Add the Tutane-b-PLL (Boc) diblock polymer obtained above into the polymerization tube filled with nitrogen protection after vacuuming, and add the molar ratio of Cys(Trt)-NCA monomer to Tutane as 180:1 Cys(Trt)-NCA monomer was reacted at constant temperature (30°C) for 120 hours under nitrogen protection. After the reaction, 40 times of ether was added to precipitate, filtered and dried to obtain Tutane-PLL(Boc)-b-PLC(Trt) segment polymer.

S623 Dissolve the above obtained Tutane-PLL(Boc)-b-PLC(Trt) diblock polymer in trifluoroacetic acid containing 8% triisopropylsilane by volume fraction, and stir at room temperature (25° C.) for 4 Hours, then add 20 times of diethyl ether to precipitate and filter. The resulting crude product was dissolved in DMF, dialyzed in water for 90 hours using a dialysis bag with a molecular weight cut-off of 3500KD, and the dialysis water was changed every 6 hours, and then freeze-dried to obtain the Tutane-PLL-b-PLC diblock polymer.

Example 7 Nanocarrier drug loaded with paclitaxel and its preparation

Paclitaxel-loaded nanocarrier drugs include paclitaxel and paclitaxel carrier. The paclitaxel carrier includes two diblock polymers of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC, and the two diblock polymers rely on electrostatic adsorption to form a core-shell structure, the core-shell structure is divided into four layers from the outside to the inside, in which the outermost layer is the polypeptide TP with active targeting, the second outer layer is the protective PEG, and the third layer is TP-PEG-b- The electrostatic adsorption layer formed by PLL (DMMA) in the PLL (DMMA)-capping agent and the PLL in the initiator-PLL-b-PLC, the innermost layer is the PLC hydrophobic core formed by the cross-linking of disulfide bonds, and the The weight ratio of TP-PEG-b-PLL (DMMA)-capping agent to initiator-PLL-b-PLC is 1:1. The paclitaxel is loaded in the hydrophobic inner core of the disulfide bond cross-linked PLC of the shedding polymer nanocarrier, and the weight ratio of the paclitaxel to the carrier is 1:10.

The preparation method of the nano-carrier medicine loaded with paclitaxel is as follows:

Weigh 15 mg each of TP-PEG-b-PLL(DMMA)-HPPr diblock polymer and Tutane-PLL-b-PLC diblock polymer, paclitaxel 3 mg, dissolve in 10 mL dimethyl sulfoxide, and Ultrasound for 10 minutes to fully dissolve the drug and the polymer to form a uniform, transparent organic homogeneous solution; place the prepared dimethyl sulfoxide solution in a dialysis bag with a molecular weight cut-off of 3500KD, and then dialyze in 1L of water for 48 hours. The water was changed once every 2 hours; after the dialysis, the aqueous solution of drug-loaded micelles formed in the dialysis bag was collected, and the particle size measured by DLS was 110nm, and the particle dispersion was relatively uniform.

Example 8 Nanocarrier drug loaded with doxorubicin and its preparation

Nano-carrier drug loaded with doxorubicin, including doxorubicin and doxorubicin carrier. The doxorubicin carrier includes two kinds of diblock polymers of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC, and the two kinds of diblock polymers rely on electrostatic adsorption to form Core-shell structure, the core-shell structure is divided into four layers from outside to inside, in which the outermost layer is the polypeptide TP with active targeting, the second outer layer is PEG for protection, and the third layer is TP-PEG- The electrostatic adsorption layer formed by b-PLL (DMMA)-PLL (DMMA) in the capping agent and the PLL in the initiator-PLL-b-PLC, the innermost layer is a PLC hydrophobic core formed by disulfide bond cross-linking, And the weight ratio of the TP-PEG-b-PLL(DMMA)-capping agent to the initiator-PLL-b-PLC is 1:1. The doxorubicin is loaded in the hydrophobic inner core of the disulfide bond cross-linked PLC of the shell-shedding polymer nanocarrier, and the weight ratio of the doxorubicin to the carrier is 1:5.

The preparation method of the nano-carrier drug loaded with doxorubicin is as follows:

Weigh 20 mg of TP-PEG-b-PLL(DMMA)-HPPr diblock polymer, 10 mg of Tutane-PLL-b-PLC diblock polymer, and 4 mg of doxorubicin, dissolve in 100 mL of DMF, and sonicate for 10 min at room temperature , so that the drug and the polymer are fully dissolved to form a uniform and transparent organic homogeneous solution; the prepared N,N-dimethylformyl solution is placed in a dialysis bag with a molecular weight cut-off of 2000KD, and then dialyzed in 2L of water for 60 hours , changing the water once every 4 hours; after the dialysis, the aqueous solution of drug-loaded micelles formed in the dialysis bag was collected, and then freeze-dried to obtain a powdery solid. The particle size measured by DLS is 95nm, and the particle dispersion is relatively uniform.

Example 9 Nanocarrier drug loaded with camptothecin and its preparation

Nano-carrier drugs loaded with camptothecin, including camptothecin and camptothecin carrier. The camptothecin carrier includes two kinds of diblock polymers of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC, and the two kinds of diblock polymers rely on electrostatic adsorption to form Core-shell structure, the core-shell structure is divided into four layers from outside to inside, in which the outermost layer is the polypeptide TP with active targeting, the second outer layer is PEG for protection, and the third layer is TP-PEG- The electrostatic adsorption layer formed by b-PLL (DMMA)-PLL (DMMA) in the capping agent and the PLL in the initiator-PLL-b-PLC, the innermost layer is a PLC hydrophobic core formed by disulfide bond cross-linking, And the weight ratio of the TP-PEG-b-PLL(DMMA)-capping agent to the initiator-PLL-b-PLC is 1:1. The camptothecin is loaded in the hydrophobic inner core of the disulfide bond cross-linked PLC of the shedding polymer nanocarrier, and the weight ratio of the camptothecin to the carrier is 1:2.

The preparation method of the nano-carrier drug loaded with camptothecin is as follows:

Weigh 10mg of TP-PEG-b-PLL(DMMA)-HPPr diblock polymer, 3mg of Tutane-PLL-b-PLC diblock polymer, 3mg of camptothecin, dissolve in 100mL DMF, and ultrasonicate for 10min at room temperature , so that the drug and the polymer are fully dissolved to form a uniform and transparent organic homogeneous solution; the prepared N,N-dimethylformyl solution is placed in a dialysis bag with a molecular weight cut-off of 3500KD, and then dialyzed in 2L of water for 60 hours , changing the water once every 4 hours; after the dialysis, the aqueous solution of drug-loaded micelles formed in the dialysis bag was collected, and then freeze-dried to obtain a powdery solid. The particle size measured by DLS is 78nm, and the particle dispersion is relatively uniform.

Effect Example

TP-PEG-b-PLL(DMMA)-capping agent and initiator-PLL-b-PLC two-block polymer nanocarrier cytotoxicity analysis:

Use MTT evaluation to measure the cytotoxicity of two kinds of diblock polymeric nanocarriers of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC obtained in Example 1, and the experiment comprises the following steps:

Add 100 µL of DMEM medium containing 1.2 x 104 MCF- ⁷ cells to each well of the 96-well plate. After culturing for 24 hours, a certain amount of TP-PEG-b-PLL (DMMA)-capping agent and initiator-PLL-b-PLC two kinds of diblock polymer nanocarriers, TP-PEG-b -PLL(DMMA)-capping agent and initiator-The mass ratio of PLL-b-PLC is 1:2, and the total concentration range of the polymer is 1μg/mL～200μg/mL (three parallel samples are made for each concentration) . Continue culturing for 24 hours, then discard the DMEM containing the polymer and add fresh DMEM and 100 μL MTT solution to the MCF-7 cells. After culturing for 4 hours, 100 μL DMSO was added to each plate well, and the MTT was completely dissolved by shaking at room temperature for 10 minutes. The fluorescence absorption value at 570 nm was measured with a microplate reader, and the cell survival rate at different polymer micelle concentrations was calculated.

The experimental measurement results are shown in Figure 3. It can be seen from Figure 3 that when the polymer micelle concentration was 50 μg/mL, the cell survival rate still reached over 60%, indicating that the nanocarrier has the characteristics of low toxicity.

The technical scheme provided by the present invention can be used to design polymer nanocarrier preparations with the same or similar structure but different materials. The skeleton structure of the polymer nanocarrier micelles can be replaced by a triblock polymer, wherein the PEG part cannot be replaced, and the side The PLL part of the chain-linked DMMA can be replaced by polyarginine or other amino-containing polyamino acids, PLL can be replaced by polyarginine or other cationic polyamino acids, PLC can be replaced by other hydrophobic amino acids or modified with disulfide Bonded polyamino acids are replaced, and the surface targeting polypeptide part can be replaced by targeting polypeptide sequences with amino groups.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (10)

1. a shell-droppable polymer nano carrier, comprise TP-PEG-b-PLL (DMMA)-end-capping reagent and initiator-PLL-b-PLC two kinds of bi-block copolymers, 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 the peptide T P with active targeting, the PEG of secondary skin for shielding, third layer is the static cling layer that the PLL (DMMA) in TP-PEG-b-PLL (DMMA)-end-capping reagent and the PLL in initiator-PLL-b-PLC are formed, innermost layer is the PLC hydrophobic inner core 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. shell-droppable polymer nano carrier as claimed in claim 1, is characterized in that: described end-capping reagent is alkane or the aromatic hydrocarbons of activated carboxylic; And/or

Described initiator is containing amino linear paraffin.

3. shell-droppable polymer nano carrier 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 shell-droppable polymer nano carrier, comprises the steps:

The synthesis of TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer: by HOOC-PEG-NH ₂pEG-b-PLL (Boc) is obtained by reacting with Lys (Boc)-NCA; The amino using end-capping reagent to close PLL (Boc) end obtains PEG-b-PLL (Boc)-end-capping reagent; Be combined with TP after the PEG c-terminus of activated PEG-b-PLL (Boc)-end-capping reagent and obtain TP-PEG-b-PLL (Boc)-end-capping reagent; TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer is obtained by reacting with 2,3-dimethyl maleic anhydride by after TP-PEG-b-PLL (Boc)-end-capping reagent removing Boc blocking group;

The synthesis of initiator-PLL-b-PLC bi-block copolymer: be obtained by reacting initiator-PLL (Boc) by initiator and Lys (Boc)-NCA, then be obtained by reacting initiator-PLL (Boc)-b-PLC (Trt) bi-block copolymer with Cys (Trt)-NCA; The protecting group Boc and the Trt that slough lysine and cysteine side chain respectively obtain initiator-PLL-b-PLC bi-block copolymer;

The formation of shell-droppable polymer nano carrier: 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: the ratio of (1-100) is dissolved in organic solvent and forms mixture homogeneous phase solution, described mixture solution is carried out dialysis treatment, lyophilization, obtains shell-droppable polymer nano carrier.

5. the preparation method of shell-droppable polymer nano carrier as claimed in claim 4, it is characterized in that: described organic solvent is oxolane, N, dinethylformamide, N, the 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-drug transporter that a shell is tear-away, comprise the carrier of hydrophobic drug and described hydrophobic drug, shell-droppable polymer nano carrier as described in described carrier is as arbitrary in claims 1 to 3, described hydrophobic drug load is in the hydrophobic inner core of the PLC of the disulfide bond crosslinking of described shell-droppable polymer nano carrier.

7. the nano-drug transporter that shell as claimed in claim 6 is tear-away, is characterized in that: the weight ratio of described hydrophobic drug and described carrier is (1-20): (2-200), and the weight ratio of described carrier and dewatering medicament is 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 nano-drug transporter that shell is tear-away, comprises the steps:

The method of synthesizing described TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer according to claim 4 or 5 synthesizes TP-PEG-b-PLL (DMMA)-end-capping reagent bi-block copolymer and initiator-PLL-b-PLC bi-block copolymer respectively;

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 organic solvent and form mixture homogeneous phase solution, described mixture solution is carried out dialysis treatment, lyophilization, obtains shell-droppable polymer nano carrier medicine; 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 nano-drug transporter that shell as claimed in claim 8 is tear-away, 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. one kind as arbitrary in claim 1-3 as described in the application of shell-droppable polymer nano carrier in nano-drug transporter, fluorescent dye carrier, bioprobe carrier field.