WO2016155051A1

WO2016155051A1 - Reducible and degradable polyzwitterion nanomicelle and preparation method thereof

Info

Publication number: WO2016155051A1
Application number: PCT/CN2015/077150
Authority: WO
Inventors: 倪才华; 吴鲁艳; 张丽萍; 石刚
Original assignee: 江南大学
Priority date: 2015-03-31
Filing date: 2015-04-22
Publication date: 2016-10-06
Also published as: AU2015377981A1; CN104788689A; AU2015377981B2; CN104788689B

Abstract

The present invention relates to the technical field of biomedical materials, and in particular relates to a reducible and degradable polyzwitterion nanomicelle and a preparation method thereof. Firstly, copolycondensation is performed with taurine, N,N-Bis(acryloyl)cystamine and dodecylamine by Michael addition mechanism. The synthesis obtains a terpolymer of poly(taurine-co-N,N-bis(acrylyl)cystamine-co-dodecylamine). Then, a nanomicelle is formed by self-assembling in an aqueous solution. Since the terpolymer chain segment contains an amino, a disulfide bond and zwitterion and other structural units simultaneously, it has a sensitive responsiveness to pH and reduction. At the same time, the zwitterion gives the nanomicelle excellent anti-protein non-specific adsorption properties. The nanomicelle of the present invention has no cytotoxicity, can be completely degraded in vivo, and has a high prospect as an anticancer drug carrier.

Description

Reductively degradable poly zwitterionic nanomicelle and preparation method thereof

Technical field

The invention relates to the technical field of biomedical materials, in particular to a reductively degradable poly zwitterionic nanomicelle and a preparation method thereof.

Background technique

Polymer nanomicelles as a chemotherapeutic drug carrier highlight a bright future in the field of malignant tumor treatment. Injectable nano-micelles, along with blood circulation, can pass through certain barriers of body tissues, reach the tumor site, release the drug at the lesion site, greatly improve the bioavailability of the drug and reduce toxic side effects. The ideal nanomicelle as a drug carrier needs to meet the following basic conditions: suitable micelle size, generally within 200 nm; stable in the long-term circulation in vivo, no non-specific protein adsorption; with a certain drug loading rate and targeting Controlled release characteristics; good biocompatibility and degradability.

In order to ensure the stability of the nanomicelles in an aqueous medium, the outer layer of the micelle is usually modified with a hydrophilic material. Currently, the most commonly used hydrophilic material is polyethylene glycol (PEG). However, in recent years, studies have shown that PEG has certain hydrophobicity in addition to hydrophilicity, and is oxidized in the presence of oxygen and transition metal ions (present in most biochemical-related solutions); possible immunization of PEG-modified protein drugs The reaction was also observed; in addition, the PEG shell shielding effect of the nanomicelles was not conducive to the uptake in the nanomicelle cells.

In recent years, some researchers have conducted more research on non-fouling materials and found that poly-sex ions have unique anti-pollution properties. Common zwitterionic polyelectrolytes are: poly(sulfobetaine 2-methyl methacrylate) (PSBMA), poly(carboxybetaine 2-methyl methacrylate) (PCBMA), poly(2-methylpropene) Acyloxyethylphosphorylcholine (PMPC) and the like, as shown in Figure 1, have been identified as effective non-fouling materials which maintain the stability of micelles in complex media such as serum. Therefore, poly-zitter materials may be an excellent substitute for PEG.

However, the polyazonia ions reported in the literature are usually obtained by polymerization of an ethylenic monomer containing positive and negatively charged groups. They are non-degradable, difficult to excrete in the body, and do not meet the requirements for use in human body. Application bottlenecks.

Summary of the invention

In order to solve the above technical problems, an object of the present invention is to provide a reductively degradable poly zwitterionic nanomicelle having good biocompatibility, which can be completely degraded in the body without residue, and a preparation method thereof.

The present invention provides a reductively degradable poly zwitterionic nanomicelle comprising a terpolymer synthesized from three monomers of N,N-bis(acryloyl)cystamine, a fatty amine and taurine, wherein the fatty amine The carbon chain length ranges from C8 to C16. If the carbon chain length is less than C8, the terpolymer is less hydrophobic and it is difficult to form micelles. If the carbon chain length is greater than C16, the solubility of the terpolymer will change. Poor, it is also difficult to form micelles; in order to prevent the molecular weight of the terpolymer from becoming smaller, the sum of the number of moles of N,N-bis(acryloyl)cystamine in the terpolymer and the number of moles of fatty amine and taurine Equally, and the molar ratio of N,N-bis(acryloyl)cystamine, taurine and fatty amine in the terpolymer is from 1:0.2 to 0.9:0.8 to 0.1. The three monomer units have their respective functional roles in the nanomicelle: 1) N,N-bis(acryloyl)cystamine contains disulfide bonds, which are stable in a non-reducing environment, but in a reducing environment Breaking occurs, so that the micelles have reduction responsiveness; 2) fatty amines, non-toxic and harmless, soluble in solvents such as methanol and ethanol, have certain hydrophobicity, are often used in the synthesis of surfactants, and their hydrophobicity is high. The self-assembly and drug-loading properties of the material lay the foundation; 3) Taurine has many physiological functions and is an essential nutrient for human health. The taurine molecule contains both amino and sulfonic acid groups, and thus has zwitterionic properties. It also gives nanomicelles excellent anti-protein non-specific adsorption properties.

In order to effectively balance the hydrophobicity and solubility of the terpolymer, the fatty amine used in the present invention is preferably one selected from the group consisting of dodecylamine having a carbon chain length of C12 as a monomer of the terpolymer, dodecylamine. It is a linear aliphatic primary amine with a relatively balanced hydrophobicity and solubility.

The invention also provides a preparation method of the reductively degradable poly zwitterionic nanomicelle, which comprises the following steps in sequence:

1) N, N-bis (acryloyl) cystamine, fatty amine and taurine three monomers by a copolycondensation reaction to obtain a terpolymer, the fatty amine carbon chain length range of C8-C16;

2) The terpolymer is purified and dried, and the solvent is selected from tetrahydrofuran, or N,N-dimethylformamide, or dioxane, or isopropanol or absolute ethanol to obtain a terpolymer solution. These solvents are all excellent solvents in the organic synthesis reaction. More preferably, the solvent is tetrahydrofuran, and tetrahydrofuran is a heterocyclic organic compound. It is one of the strong polar ethers and is used as a chemical reaction. a medium-polar solvent, a colorless, volatile liquid;

3) adding ultrapure water to the terpolymer solution under continuous stirring;

4) The ternary copolymer solution obtained in the step 3) is subjected to dialysis treatment to obtain nano micelles.

Specifically, in the step 1), the reaction system of the copolycondensation reaction is a mixed solvent containing deionized water, methanol and ethanol. Specifically, the volume ratio of the deionized water, methanol and ethanol is from 7:2:1 to 1.5, and the ratio of each component in the mixed solvent can be appropriately adjusted according to the reaction conditions.

Specifically, in the step 1), the reaction condition of the copolycondensation reaction is heating in a 50 ° C oil bath under nitrogen protection for not less than 3 days.

Specifically, in the step 2), the terpolymer is purified by dialysis, dried, and dissolved in tetrahydrofuran to obtain a terpolymer solution.

Specifically, in the step 4), a dialysis bag having a molecular weight cut off of 3,500 is used, and dialysis treatment is performed for not less than 48 hours to obtain nano micelles. The purpose of the dialysis bag is to remove unreacted small molecules and oligomers, because polymers with too low molecular weight are not easy to form micelles, and dialysis bags have various specifications, and 3500 can be selected.

Michael addition is the addition reaction of a nucleophile to an α,β-unsaturated carbonyl compound. The N,N-bis(acryloyl)cystamine molecule contains a double bond, while the taurine and dodecylamine molecules contain a primary amino group, and a double bond with a primary amino group can undergo a typical Michael addition to form a polycondensation reaction product. According to the principle of the polycondensation reaction, a high molecular weight polymer can be obtained when the equimolar ratio of the two types of groups participating in the polycondensation reaction is obtained. Therefore, in the design of the synthetic formulation, the present invention adopts the molar ratio of N,N-bis(acryloyl)cystamine to the sum of the moles of taurine and dodecylamine, and N,N-bis(acryloyl)cystamine. The ratio of the moles of taurine to the fatty amine is from 1:0.2 to 0.9:0.8 to 0.1, whereby a high molecular weight copolymer is efficiently obtained. The copolymer segment contains structural units such as an amino group, a disulfide bond, and a zwitterion.

The invention also provides a reductively degradable poly zwitterionic nanomicelle, which is used in the preparation of a chemotherapeutic drug carrier. Glutathione is a reducing tripeptide that is 100 to 1000 times more concentrated in tumor cells than in body fluids. The drug-loaded nanomicelle enters into the tumor cell. Under the reduction of glutathione, the disulfide bond in the polymer is broken, the polymer is degraded, and the drug is released; at the same time, the tumor cell has a weak acidic environment, the nanometer The micelle contains an amino group, so the nanomicelle has both pH and reducing sensitivity, and the micelle structure changes under the stimulation of the tumor cell environment, prompting drug release.

With the above solution, the present invention has at least the following advantages:

1. Since the copolymer segment contains structural units such as an amino group and a disulfide bond, it has sensitive pH and reduction responsiveness, and changes in micelle structure in a weakly acidic and reducing environment inside the tumor cell promotes the drug freed;

2. The poly-zinc ions in the nano-micelles give the nano-micelles excellent anti-protein non-specific adsorption properties, so the nano-micelles have special anti-pollution properties;

3. The disulfide bond is located in the polymer backbone, and after the nanomicelle enters the tumor cell, it is reduced under the stimulation of high concentration of glutathione in the cell, and the disulfide bond is broken, so the nanomicelle is completely degraded in the body, no Residue, as a carrier of anticancer drugs, has practical application value;

4. Nanomicelles are non-cytotoxic and meet the safety standards for human use;

5. The addition of Michael to the synthesis of high polymer, the method is simple, the conditions are mild, no catalyst and other additives are needed, no by-products are produced, the reaction is complete, and the product is pure.

The above description is only an overview of the technical solutions of the present invention, and the technical means of the present invention can be more clearly understood and can be implemented in accordance with the contents of the specification. Hereinafter, the preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

DRAWINGS

Figure 1 is a zwitterionic polyelectrolyte in the prior art;

Figure 2 is a schematic view showing the copolycondensation reaction of N,N-bis(acryloyl)cystamine, taurine and dodecylamine in the present invention;

3 is a transmission electron micrograph of a reductively degradable poly zwitterionic nanomicelle in the present invention;

Figure 4 is a particle size distribution of the reductively degradable poly zwitterionic nanomicelles at pH 7.4 in the present invention,

In the figure, a, b, c, d, and e represent N, N-bis(acryloyl)cystamine, taurine and dodecylamine in a molar ratio of 1:0.2:0.8, 1:0.5:0.5, respectively. Reducible degradable poly zwitterionic nanomicelle obtained by reaction at 1:0.6:0.4, 1:0.8:0.2, 1:0.9:0.1;

Figure 5 shows the zeta potential of poly- zwitterionic nanomicelles as a function of pH,

The molar ratio of N,N-bis(acryloyl)cystamine, taurine and dodecylamine is 1:0.2:0.8;

6 is a particle size change of a reductively degradable poly zwitterionic nanomicelle in a 10 mM glutathione solution at different times in the present invention,

In the figure, PBS and GSH represent a phosphate buffer solution and a glutathione solution, respectively;

7 is a particle size change of a reductively degradable poly zwitterionic nanomicelle in different protein solutions in the present invention,

In the figure, BSA stands for bovine serum albumin and FBS stands for fetal bovine serum;

Figure 8 is a graph showing the cytotoxicity of reductively degradable poly zwitterionic nanomicelles in the present invention.

detailed description

The specific embodiments of the present invention are further described in detail below with reference to the drawings and embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

1) Synthesis of N,N-bis(acryloyl)cystamine:

11.6 g of cystamine dihydrochloride was added to a 250 mL single-necked flask, and then cystamine dihydrochloride was stirred and dissolved by adding 50 mL of distilled water. The flask was placed in an ice-water mixture at 0 ° C; 8 g of sodium hydroxide solid was weighed and dissolved in 20 mL of distilled water, and the dissolved sodium oxygen oxide solution was added to a single-mouth flask at a time, and the previously purified 19 mL was prepared. The acryloyl chloride was mixed with 3 mL of dichloromethane to form a solution, and the mixture was dropped into a one-necked flask through a constant pressure dropping funnel. After the dropwise addition was completed in 40 minutes, the reaction was controlled to react at 25 ° C for 16 hours. The product was filtered, washed with deionized water three times and then recrystallised from ethyl acetate.

2) Synthesis of poly(taurine-co-N,N-bis(acryloyl)cystamine-co-dodecylamine) terpolymer:

In a 50 mL three-necked flask, 3.5 mL of distilled water, 1 mL of methanol, and 0.5 mL of ethanol were mixed to form a solution, and N,N-bis(acryloyl)cystamine, taurine, and dodecylamine were dissolved in the above solution in nitrogen. Under the protection, the reaction solution was heated to 50 ° C in an oil bath and reacted for 3 days. The product was dissolved in 10 mL of ultrapure water, transferred to a dialysis bag with a molecular weight cutoff of 3500-7000 D, dialyzed for 4 days, and then lyophilized to obtain poly(taurine-co-N,N-bis(acryloyl)cystamine. -co-dodecylamine) terpolymer. The copolycondensation reaction process of N,N-bis(acryloyl)cystamine, taurine and dodecylamine is shown in FIG. The corresponding dissolution formula ratio of N,N-bis(acryloyl)cystamine, taurine and dodecylamine in the mixed solution, as shown in Table 1, the present invention only provides five kinds of terpolymer synthesis formula, specifically used In the process, according to the application requirements of different sizes of nanomicelles, the ratio of the three monomers is adjusted.

Table 1 List of ternary copolymer synthesis formula

3) Preparation of reductively degradable poly zwitterionic nanomicelles:

10 mg of poly(taurine-co-N,N-bis(acryloyl)cystamine-co-dodecylamine) terpolymer was dissolved in 1 mL of tetrahydrofuran solvent, and dropped into the solution during continuous stirring. Add 10 mL of ultrapure water. After stirring for 3 h, the solution was transferred into a dialysis bag with a molecular weight cutoff of 3,500, and dialysis for 48 h to obtain a reductively degradable poly zwitterionic nanomicelle. The molecular weight cut-off of the dialysis bag can be adjusted according to the specific use process, and is usually not less than 3,500. As shown in Fig. 3, it is a scanning electron micrograph of the reductively degradable poly zwitterionic nanomicelle. In the figure, ae corresponds to the ternary copolymer of five kinds of ae in Table 1, and it can be seen that the nanomicelle has a substantially spherical shape. The particle size distribution is relatively uniform, but when the proportion of taurine in the component is large, the micelle morphology changes to a columnar shape.

According to the application requirements of different size of the injected nano-micelle, the ratio of the three monomers can be adjusted to obtain nano-micelles of different particle sizes. As shown in Fig. 4, with the increase of taurine content, the nano-micelle The particle size is increased. Due to the molar ratio of N,N-bis(acryloyl)cystamine:taurine:dodecylamine=1:0.8:0.2, the particle size of the nanomicelle is relatively moderate and representative. The chemical properties of the nanomicelles at different particle sizes are relatively uniform. Therefore, the nanomicelles mentioned in the subsequent embodiments of the present invention refer to the nanomicelles at this ratio unless otherwise specified.

Example 2

The pH sensitivity of the reductively degradable poly zwitterionic nanomicelles:

The reductively degradable poly zwitterionic nanomicelles obtained in Example 1 were respectively placed in a buffer solution of pH=5.0, 6.5, 7.4, 10.0, and the zeta potential was observed by a zeta potential meter. The results are shown in Fig. 5, wherein N, N-bis(acryloyl)cystamine: taurine: dodecylamine molar ratio = 1:0.8:0.2; in Figure 5, when the pH is 7.4, the solution is weakly alkaline, and the amino group in the micelle is difficult The proton is ingested, and the positive charge is artificially prepared. The negatively charged ions in the micelles are dominant, so they have a negative zeta potential value. At acidic conditions of pH 5.0 and 6.5, the amino groups in the micelles take up protons to form ammonium-based positive ions, and as time increases, the degree of protonation increases, so the zeta potential is converted to a positively charged value.

Example 3

Reduction sensitivity of reductively degradable poly- zwitterionic nanomicelles:

The reductively degradable poly zwitterionic nanomicelle obtained in Example 1 was placed in a glutathione solution having a concentration of 10 mM, wherein N,N-bis(acryloyl)cystamine in the nanomicelle: taurine: ten The molar ratio of diamine was 1:0.8:0.2, and the particle size change of the nanomicelle was tested by laser light scattering instrument at different times to observe the reduction responsiveness. As shown in Fig. 6, the nanomicelle was free of glutathione. In the PBS buffer solution of peptide (GSH), the particle size did not change after 24 hours; however, the nanomicelles in the 10 mM glutathione (GSH) solution showed a small particle size after 12 hours, indicating that there were some double When the sulfur bond is broken, the molecular weight of the copolymer decreases, and the copolymer re-self-assembles to form nano-micelles with smaller particle size; as the nano-micelles increase in time in the GSH solution, more disulfide bonds are broken, and the copolymer Turning into a small molecule, the micelles tend to disintegrate, so the process of increasing the particle size occurs.

Example 4

Non-specific adsorption properties of anti-bovine serum albumin that can reduce polyphosphorus nano-micelles:

The reductively degradable poly zwitterionic nanomicelles obtained in Example 1 (in which the molar ratio of N,N-bis(acryloyl)cystamine:taurine:dodecylamine = 1:0.8:0.2) were respectively contained. Concentration of 45g / L Bovine serum albumin and PBS buffer solution with pH=7.4 were incubated for 24 hours. The particle size of the nanoparticles was monitored by laser light scattering instrument to observe the non-specific adsorption performance of anti-bovine serum albumin. The results are shown in Figure 7. The particle size of the nanomicelles after contact with different proteins is compared with the particle size before contact with the protein. In the figure, BSA stands for bovine serum albumin and FBS stands for fetal bovine serum; as can be seen from Figure 7: After the five nanomicelle samples synthesized were contacted with bovine serum albumin and fetal bovine serum solution for a certain period of time, the particle size remained unchanged, as in the protein-free buffer solution. This result indicates that the nanomicelle and protein are There was no mutual adsorption, and the non-specific adsorption performance of anti-bovine serum albumin of nanomicelles was verified.

Example 5

Biocompatibility of reductively degradable poly zwitterionic nanomicelles:

In a water bath at 37 ° C, 3T3 cells frozen at -80 ° C were quickly thawed, transferred to a centrifuge tube containing 7 mL of RPMI-1640 medium, centrifuged at 800 rpm, and containing 10% calf serum. The RPMI-1640 medium was pipetted into a single cell suspension, which was transferred to a 50 mL culture flask and incubated at 37 ° C in a 5% CO 2 incubator.

The cytotoxicity of the nanoparticles formed by the reductive degradation of poly-sex ion nano-micelles was tested by MTT method, and the mouse fibroblasts were inoculated into 96-well plates at about 1.2×105/mL. 100 μL of wells, cultured for 24 h, aspirate the original culture solution in each well, and add 100 μL of negative control solution (pH=7.4 and 10% calf serum RPMI-1640 medium with pH=6.5) and positive control solution (0.64) per well. % phenol medium), sample group (sample group containing pH=7.4 and pH=6.5 10% calf serum RPMI-1640 medium), continue to be placed in 37 ° C, 5% CO2 incubator, culture separately 24h, 48h. Each set has 4 parallel holes. After the culture plate was taken out, the cell growth was observed by an inverted microscope. After adding MTT 20 μL, and continuing to culture for 4 hours, the liquid in the wells in the culture plate was exhausted, dimethyl sulfoxide was added, and the absorbance value (A) was measured at 570 nm with a microplate reader to calculate the cell survival rate. . As shown in Figure 8, the survival rate of 3T3 and Hela cells in different concentrations of nanomicelle solution is between 94% and 100%. Under the same conditions, the survival data of the two cells are close to each other; The increase in micelle concentration, although the cell survival rate decreased, but overall was greater than 80%, in line with biocompatibility standards.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that It will be apparent to those skilled in the art that various modifications and changes can be made without departing from the spirit of the invention.

Claims

A method for preparing a reductively degradable poly zwitterionic nanomicelle, which comprises the following steps in sequence:

1) N, N-bis (acryloyl) cystamine, fatty amine and taurine three monomers by a copolycondensation reaction to obtain a terpolymer, the fatty amine carbon chain length range of C8-C16;

2) the terpolymer is purified and dried and dissolved to obtain a terpolymer solution;

3) adding ultrapure water to the terpolymer solution under continuous stirring;

4) The ternary copolymer solution obtained in the step 3) is subjected to dialysis treatment to obtain nano micelles.
The method for preparing a reductively degradable poly zwitterionic nanomicelle according to claim 1, wherein in the step 1), the reaction system of the copolycondensation reaction is a mixed solvent comprising deionized water, methanol and ethanol.
The method according to claim 1, wherein the fatty amine is dodecylamine.
The method for preparing a reductively degradable poly zwitterionic nanomicelle according to claim 1, wherein in the step 1), the reaction condition of the copolycondensation reaction is 50 ° C, and the oil bath is heated under nitrogen protection. 3 days.
The method for preparing a reductively degradable poly zwitterionic nanomicelle according to claim 1, wherein in the step 2), the terpolymer is purified by dialysis, dried, and dissolved in tetrahydrofuran to obtain three Metapolymer solution.
The method for preparing a reductively degradable poly zwitterionic nanomicelle according to claim 1, wherein in the step 4), a dialysis bag having a molecular weight cut off of 3,500 is used, and the dialysis treatment is not less than 48 hours to obtain a nanometer. Micellar.
The method for preparing a reductively degradable poly zwitterionic nanomicelle according to claim 1, wherein the molar amount of N,N-bis(acryloyl)cystamine and the fatty amine and taurine in the three monomers The sum of the mole numbers is equal, and the ratio of the molar ratio of N,N-bis(acryloyl)cystamine, taurine and fatty amine is 1:0.2 to 0.9:0.8 to 0.1.
A reductively degradable poly zwitterionic nanomicelle characterized by comprising a terpolymer synthesized from three monomers of N,N-bis(acryloyl)cystamine, fatty amine and taurine, wherein the fat The amine has a carbon chain length ranging from C8 to C16.
The reductively degradable poly zwitterionic nanomicelle of claim 8 wherein: The molar ratio of N,N-bis(acryloyl)cystamine in the terpolymer is equal to the sum of the moles of the fatty amine and taurine, and the N,N-bis(acryloyl group) in the terpolymer The ratio of moles of cystamine, taurine and fatty amine is from 1:0.2 to 0.9:0.8 to 0.1.
The use of the reductively degradable poly zwitterionic nanomicelle according to any one of claims 8 or 9 for the preparation of a chemotherapeutic drug carrier.