CN109134849B

CN109134849B - Polylipopeptide vesicle with negative inner membrane as well as preparation method and application thereof

Info

Publication number: CN109134849B
Application number: CN201810826941.9A
Authority: CN
Inventors: 邓超; 邱敏; 钟志远
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-07-25
Filing date: 2018-07-25
Publication date: 2022-02-25
Anticipated expiration: 2038-07-25
Also published as: CN109134849A

Abstract

The invention discloses a polyester peptide vesicle with a negative inner membrane as well as a preparation method and application thereof, belonging to the technical field of high polymer materials and applied chemistry. Firstly, simply preparing a PEG-b-PAPA-b-PASp triblock copolymer through NCA ring-opening polymerization and deprotection; the prepared cRGD-CLP has the characteristics of small size, good stability, high protein entrapment capacity and the like; the cRGD-CLP (SAP-cRGD-CLP) loaded with saporin protein can be specifically targeted to alpha_vβ₃Integrin over-expressed tumor cells and strong cytotoxicity to A549 lung cancer cells; the cRGD-CLP can be circulated in vivo, thereby promoting the enrichment of protein drugs in tumor sites and completely inhibiting the growth of in-situ A549 lung cancer. Therefore, the vesicle is simple to prepare, high in protein loading, good in tumor targeting and has great potential in protein treatment of tumors.

Description

Polylipopeptide vesicle with negative inner membrane as well as preparation method and application thereof

Technical Field

The invention relates to a polyester peptide vesicle with negative inner membrane as well as a preparation method and application thereof, belonging to the technical field of high polymer materials and applied chemistry.

Background

Protein drugs have shown unique advantages in cancer therapy. Compared with the traditional micromolecule chemotherapy drugs, the therapeutic protein drugs such as monoclonal antibodies, enzymes, hormones and the like have the characteristics of strong specificity, high activity and the like. However, the clinical transformation of protein drugs is greatly hindered by the defects of instability of the internal structure, easy degradation and inactivation in the in vivo circulation process, poor cell penetration, immunogenicity and the like. Therefore, how to deliver therapeutic proteins that are required to function inside cells safely and efficiently into cancer cells is a great challenge in the field of protein therapy. The polymersome has a huge hydrophilic inner cavity and can be used for loading protein, and the firm wall membrane can isolate the protein from the outside, thereby playing the role of protecting the protein. However, the conventional polymersome has a low efficiency of entrapping proteins.

Disclosure of Invention

In order to solve the problems, the invention designs and synthesizes an asymmetric triblock lipopeptide and a lipopeptide vesicle which is prepared from the asymmetric lipopeptide and has an asymmetric membrane structure, wherein the inner membrane of the lipopeptide vesicle is negative electricity, and the lipopeptide vesicle is used for efficient entrapment and targeted delivery of protein drugs.

The first purpose of the invention is to provide a poly-lipopeptide material, wherein the poly-lipopeptide material is polyethylene glycol-b-poly (2-aminocaproic acid) -b-polyaspartic acid ((PEG-b-PAPA-b-PAsp)), and the structural formula of the poly-lipopeptide material is shown as follows:

wherein, the value range of x is 40-2300, the value range of y is 30-800, and the value range of z is 1-500.

In one embodiment of the present invention, the peptide material further includes a targeting peptide material obtained by modifying a specific targeting molecule at the end of the hydrophilic segment of polyethylene glycol.

In one embodiment of the present invention, the specific targeting molecule is a short peptide, a small molecule targeting molecule, an antibody, a polysaccharide or a monosaccharide.

In one embodiment of the invention, the antibody further comprises an antibody fragment.

In one embodiment of the invention, the short peptide is cRGD with the sequence of cRGDfC, cNGQ with the sequence of cNGQGEQc, CC-9 with the sequence of CSNIDARAC or CPP33 with the sequence of RLWMRWYSPRTRAYGC.

In one embodiment of the invention, the small molecule targeting molecule is folic acid or anisamide.

The second purpose of the invention is to provide a preparation method of the above-mentioned lipopeptide material, comprising the following steps: with polyethylene glycol-amino (PEG-NH)₂) Sequentially initiating 2-aminocaproic acid N-carboxyl internal anhydride (APA-NCA) and beta-benzyl-L-aspartic acid-N-carboxyl internal anhydride monomer (BLA-NCA) to prepare polyethylene glycol-b-poly (2-aminocaproic acid) -b-poly (beta-benzyl-L-aspartic acid) (PEG-b-PAPA-b-PBLA) triblock copolymer as a macromolecular initiator, and then sequentially carrying out ring-opening polymerization on the triblock copolymerAnd removing benzyl by deprotection to obtain the polyester peptide material.

In one embodiment of the invention, the molecular weight of PEG is preferably 5kDa, the molecular weight of PAPA is preferably 11kDa and the molecular weight of PASp is preferably 2 kDa.

In one embodiment of the present invention, the method specifically comprises the following steps:

(1) adding a DMF solution of a 2-aminocaproic acid-N-carboxyl internal anhydride monomer into a DMF solution of polyethylene glycol-amino to react under the condition of nitrogen;

(2) adding a beta-benzyl-L-aspartic acid-N-carboxyl internal anhydride monomer into the reaction solution obtained in the step (1) to continue reacting, then adopting ethyl glacial ether to precipitate, and drying to obtain polyethylene glycol-b-poly (2-aminocaproic acid) -b-poly (beta-benzyl-L-aspartic acid);

(3) removing benzyl from polyethylene glycol-b-poly (2-aminocaproic acid) -b-poly (beta-benzyl-L-aspartic acid) by deprotection method, precipitating with ethyl acetate, and drying to obtain polyethylene glycol-b-poly (2-aminocaproic acid) -b-polyaspartic acid.

In one embodiment of the present invention, the above preparation method can be represented as follows:

in one embodiment of the present invention, in the step (1), the reaction is carried out under the conditions of 30 to 40 ℃ for 60 to 80 hours.

In one embodiment of the present invention, in the step (2), the reaction is carried out under the conditions of 30 to 40 ℃ for 60 to 80 hours.

The third purpose of the invention is to provide the application of the polypeptide material in protein drug delivery.

In one embodiment of the invention, the application is that the polyester peptide material is prepared into a polyester peptide vesicle for protein drug delivery.

The fourth purpose of the invention is to provide a polyester peptide vesicle, which is prepared from the polyester peptide material, and comprises that the polyester peptide material is prepared from polyester peptide material without targeting molecules, or the polyester peptide vesicle is prepared from polyester peptide material without targeting molecules, and the polyester peptide vesicle is surface-modified by targeting molecules.

The fifth purpose of the invention is to provide the application of the lipopeptide vesicle in protein drug delivery.

The invention has the beneficial effects that:

1. the polyethylene glycol-b-poly (2-aminocaproic acid) -b-polyaspartic acid prepared by the invention can be obtained by gradual ring-opening polymerization and subsequent deprotection reaction. The whole preparation process is simple and direct, and has good controllability.

2. The polyester peptide vesicle disclosed by the invention has the characteristics of small size, good stability, high protein entrapment capacity and the like.

3. The preparation method is simple, the used raw materials are wide in source and good in repeatability, and the application prospect is good.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of PEG-b-PAPA-b-PBLA in example 1;

FIG. 2 is a nuclear magnetic hydrogen spectrum of PEG-b-PAPA-b-PASp in example 2;

FIG. 3 is a graph of the particle size and profile of cRGD-CLP (A), transmission electron microscopy image of cRGD-CLP (B), serum stability (C), in vitro release of FITC-CC-cRGD-CLP (D), and cytotoxicity results of cRGD-CLP and CLP in examples 3, 4, and 5 (E);

FIG. 4 is a graph showing the results of the cell flow of cRGD-CLP loaded with protein in example 5 in A549 lung cancer cells (A) and its cytotoxicity on A549 cells (B);

FIG. 5 is a graph showing the results of the blood circulation studies of Cy 5-labeled cRGD-CLP in healthy mice in example 6 (A) and its biodistribution in A549-Luc lung carcinoma in situ mice (B);

FIG. 6 is a graph showing the results of the anti-tumor activity of SAP-cRGD-CLP-loaded mice of example 7 in the A549-Luc orthotopic lung cancer, wherein A is a tumor growth curve, B is a weight change curve of the mice, and C is a survival curve of the mice.

Detailed Description

In order to better understand the essence of the invention, the technical contents of the invention are explained in detail below by way of examples.

Example 1: synthesis of polyethylene glycol-b-poly (2-aminocaproic acid) -b-poly (beta-benzyl-L-aspartic acid) (PEG-b-PAPA-b-PBLA) triblock copolymer

The PEG-b-PAPA-b-PBLA triblock copolymer is prepared by dissolving PEG-NH in DMF under nitrogen₂The compound is prepared by sequentially initiating 2-aminocaproic acid-N-carboxyl internal anhydride monomer (APA-NCA) and beta-benzyl-L-aspartic acid-N-carboxyl internal anhydride monomer (BLA-NCA) to ring-opening polymerization by using a macroinitiator. The specific synthesis steps are as follows: under nitrogen, 10mL of APA-NCA (0.52g, 1.76mmol) in DMF was added rapidly to PEG-NH₂After reacting at 35 ℃ for 72 hours in DMF (2mL), the second monomer BLA-NCA was added, and the reaction was continued for 72 hours. After the reaction is finished, the reaction solution is precipitated for many times in excess of 20 times of glacial ethyl ether, and finally, the white solid product is obtained after vacuum-pumping and drying. Yield: 83 percent.

The nuclear magnetic spectrum of PEG-b-PAPA-b-PBLA is shown in figure 1,¹H NMR(600MHz,CDCl₃/CF₃COOH (9/1, v/v), FIG. 1, delta) 7.19(5H, -C₆H₅),5.02(2H,C₆H₅CH₂-),4.45(1H,-COCHNH-),3.73(4H,-OCH₂CH₂O-),3.47(3H,-OCH₃),2.92(2H,-COCH₂-),1.80(2H,-CH(NH)CH₂CH₂-),1.24(24H,-CH₂(CH₂)₁₂CH₃),0.86(3H,-CH₂CH₃)。

Example 2: synthesis of PEG-b-PAPA-b-PASp copolymer

The PEG-b-PAPA-b-PASp triblock copolymer is obtained by deprotecting PEG-b-PAPA-b-PBLA in HBr. The method comprises the following specific steps: HBr (33 wt.% HOAc,0.3mL, 1.66mmol) was added to a solution of PEG-b-PAPA-b-PBLA (0.3g, 0.015mmol) in trifluoroacetic acid (3mL) and reacted at 0 ℃ for 2 hours. After the reaction is finished, excessive ethyl glacial ether is used for repeated precipitation for many times, and finally the product is dried in vacuum to obtain a white solid product. Yield: 80 percent.

The nuclear magnetic spectrum of PEG-b-PAPA-b-PASp is shown in figure 2,¹H NMR(600MHz,CDCl₃/CF₃COOH (9/1, v/v), FIG. 2, delta) 4.50(1H, -COCHNH-),3.72(4H, -OCH)₂CH₂O-),3.47(3H,-OCH₃),2.99(2H,-COCH₂-),1.82(2H,-CH(NH)CH₂CH₂-),1.24(24H,-CH₂(CH₂)₁₂CH₃),0.85(3H,-CH₂CH₃)。

TABLE 1 characterization of PEG-b-PAPA-b-PBLA triblock copolymers

^aBy¹H NMR was calculated.^bMeasured by GPC (mobile phase: CHCl)₃(ii) a Flow rate: 0.8 mL/min; temperature: 40 ℃; standard sample preparation: polystyrene).

Example 3: preparation of lipopeptide vesicles with asymmetric membrane structure and determination of Critical Aggregation Concentration (CAC) of the same

The cRGD modified poly-peptide vesicle (cRGD-CLP) is prepared by a solvent replacement method. The brief steps are as follows: 0.2mL of a solution containing 20 mol.% cRGD-PEG-b-PAPA (M) at a concentration of 2mg/mL_n6.0-11.3kg/mol) and 80 mol.% mPEG-b-PAPA-b-PAsp (M)_n5.0-10.1-1.4kg/mol) was added dropwise to 0.8mL of PB buffer medium (10mM, pH 7.4) followed by dialysis in PB for 8 hours, changing the dialysis medium every 2 hours. The Critical Aggregation Concentration (CAC) of the lipopeptide vesicles was measured by fluorescence using pyrene as a fluorescent probe. The concentration range of cRGD-CLP and CLP is 1.2X 10^-5To 0.1mg/mL and the concentration of pyrene in each sample was 0.6. mu.M. The fluorescence intensities at 372nm and 383nm were recorded by a fluorescence spectrophotometer at 330nm excitation. The ratio of the vesicle concentration to the fluorescence intensity at 372nm and 383nm is plotted, and the inflection point of the obtained curve is the CAC value of the curve. Dynamic laser light scattering (DLS) indicated that the cRGD-CLP particle size was about 80 nm and the particle size distribution was narrow (fig. 3A). From the transmission electron microscope images, it can be found that the cRGD-CLP has a spherical vesicle structure (FIG. 3B). cRGD-CLP surface electrodeThe potential was-9.46 mV (Table 2). We found that the Critical Aggregation Concentration (CAC) of cRGD-CLP was 2.46mg/L (table 2) and remained stable for a long time in PB with 10% FBS (fig. 3C), showing good stability, which is related to the lipid-lipid accumulation effect present in its wall membrane. The properties of the non-targeting vesicle CLP, such as particle size, distribution, surface potential and the like, are similar to those of the cRGD-CLP.

TABLE 2 characterization of empty vesicles

^aThe particle size and particle size distribution of the vesicles were determined by dynamic laser light scattering.

^bThe surface potential was measured by electrophoresis in PB at pH 7.4.

^cMeasured by a fluorescence spectrophotometer.

Example 4: loading of proteins and in vitro Release behavior Studies

The cRGD-CLP loaded with protein is prepared as described above by adding the polymer solution dropwise to PB buffer medium containing Cytochrome C (CC), FITC-labeled CC, Cy 5-labeled CC or sapogenotoxin protein (SAP) dissolved therein, and dialyzing the unloaded protein in PB medium using a dialysis bag having a cut-off molecular weight of 35 ten thousand. The Protein Loading (PLC) and loading efficiency (PLE) can be measured by UV spectrophotometer or fluorescence spectrometer. PLC and PLE can be calculated by the following equations:

PLC (wt.%) x 100 (sum of mass of protein loaded in vesicles/polymer and mass of protein)

PLE (%) × 100 (mass of protein loaded in vesicle/mass of protein initially added) × 100

We investigated the in vitro release behavior of proteins by dialysis in PB medium (pH 7.4,10mM) at 37 ℃. The brief steps are as follows: 0.5mL of FITC-CC-cRGD-CLP (0.2mg/mL) was placed in a dialysis bag with a cut-off of 35 ten thousand, placed in 25mL of PB medium and then placed in a constant temperature shaker at 37 ℃. At a predetermined time point, 5mL of the released solution was removed and then supplemented with a corresponding volume of fresh medium. The amount of FITC-CC in each sample was then determined by fluorescence spectroscopy. From table 3, it can be known that the cRGD-CLP can achieve high encapsulation efficiency of FITC-CC through electrostatic interaction between PAsp and FITC-CC, and when the theoretical drug loading (PLC) is 2-10 wt.%, the encapsulation efficiency of protein is 93-100%, which is significantly higher than that of vesicles assembled by diblock copolymers. Notably, the in vitro release results of FITC-CC showed that the release of FITC-CC did not exceed 25% within 24 hours under conditions mimicking the normal physiological environment of humans (pH 7.4, 37 ℃) (FIG. 3D), further demonstrating the excellent stability of the protein-loaded vesicles.

TABLE 3 characterization of FITC-CC-cRGD-CLP

^aMeasured by an ultraviolet spectrophotometer.

^bThe particle size and particle size distribution of FITC-CC-cRGD-CLP were determined by DLS.

^cMeasured by electrophoresis.

Example 5: endocytosis and cytotoxicity assays in cells

We used the MTT method to evaluate the antitumor activity of SAP-loaded cRGD-CLP and CLP (SAP-cRGD-CLP, SAP-CLP) against A549 cells. A549 cells are paved into a 96-well plate, after 24 hours of culture, SAP-cRGD-CLP, SAP-CLP and SAP with set concentrations are added, and after 4 hours of incubation, the culture medium is replaced by fresh culture medium to remove the drugs which are not endocytosed by the cells. After 68 hours, MTT solution was added. After continuously culturing for 4 hours, adding 150 mu L DMSO into each hole to dissolve the generated crystallin, measuring the absorbance value at 570nm by using an enzyme-labeling instrument, adjusting to zero by using a blank hole of the culture medium, and calculating the cell survival rate. Toxicity testing of empty vectors to a549 cells was similar to that described above. A549 cells were plated in 96-well plates, cultured for 24 hours, and then cRGD-CLP and CLP at set concentrations were added, followed by continuous incubation for 48 hours without medium change. The experimental results showed that the empty vesicles were almost non-toxic to a549 cells even at high concentrations of 1mg/mL, indicating good biocompatibility (fig. 3E). First, we investigated the endocytosis and intracellular release behavior of FTITC-CC-cRGD-CLP in a549 cells by flow cytometry. The brief steps are as follows: after A549 cells were cultured in a 6-well plate for 24 hours, FITC-CC-cRGD-CLP and FITC-CC-CLP (FITC-CC concentration: 40. mu.g/mL) were added, and after incubation for 4 hours, the cells were digested, washed, centrifuged, and finally dispersed in 0.5mL of PBS and tested by flow cytometry. The results of the experiment showed that FITC-CC-cRGD-CLP-treated A549 cells exhibited twice the fluorescence intensity as the FITC-CC-CLP group (FIG. 4A).

Notably, from FIG. 4B, it can be seen that SAP-loaded cRGD-CLP (SAP-cRGD-CLP) exhibited a strong killing effect on A549 cells at a semi-lethal concentration (IC)₅₀) 16.3nM, the toxicity of SAP-CLP to cells in the non-targeting group, although not as good as the targeting group, was also better at inhibiting tumor cell growth, its IC for A549 cells₅₀It was 29.2 nM. Free saporin, however, does not substantially inhibit the proliferation of cancer cells because of its very poor ability to enter cells. Thus, the prepared cRGD-CLP can efficiently deliver therapeutic proteins to alpha_vβ₃Tumor cells overexpressing integrin.

Example 6: blood circulation and biodistribution experiments

All animal experimental procedures were in compliance with the relevant provisions of the experimental animal center at the university of suzhou. To study the in vivo circulation time of CLP and cRGD-CLP, we injected Cy 5-labeled CLP and Cy 5-labeled cRGD-CLP into healthy Balb/c mice via tail vein (amount of Cy 5: 6 nmol). Mouse blood samples were collected at 0.05, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours post-injection, and the amount of Cy5 in each sample was measured by fluorescence spectroscopy. The results show that both vectors are capable of long circulation in vivo, with an elimination half-life (t)_1/2,β) 3.5 and 3.2 hours, respectively (fig. 5A).

We use in situ A549 lung cancer as model to determine the enrichment amount of nano-drug in tumor and other normal organs. Cy5-CC-cRGD-CLP, Cy5-CC-CLP and Cy5-CC (0.4. mu. mol Cy5-CC equiv./kg) were injected into tumor-bearing mice via tail vein. After 6 hours, the mice were sacrificed, the tumors and the major organs were collected, the tumors were weighed and ground after washing with PBS, finally Cy5-CC was extracted with DMSO, and the amount of Cy5-CC in each sample was detected with a fluorescence spectrometer. Experimental results show that Cy5-CC-cRGD-CLP and Cy5-CC-CLP have higher enrichment in mouse lung, while Cy5-CC-cRGD-CLP group ((7.73% ID/g) has significantly more enrichment in lung than Cy5-CC-CLP group (4.90% ID/g) (FIG. 5B), which is mainly because vesicle surface modified cRGD can help to enhance the targeting and enrichment of tumor, in contrast, free Cy5-CC has very little enrichment at tumor site (1.94% ID/g), which is mainly because free protein is easily degraded, adsorbed and eliminated in vivo.

Example 7: evaluation of in vivo antitumor Activity of SAP-cRGD-CLP

We evaluated the in vivo anti-tumor effect of SAP-cRGD-CLP using in situ A549-Luc lung cancer as a model. Tumor-bearing mice were randomly divided into 3 groups of 6 mice each (of which 1 was used for histological analysis and the remaining 5 were used to observe survival and weight changes). SAP-cRGD-CLP and SAP-CLP were injected into mice via tail vein (administration dose: 16.7nmol SAP equiv./kg), and were administered once every 4 days for a total of 4 times. We used the IVIS II small animal in vivo imaging system (Caliper Life Sciences) to measure the bioluminescence intensity in the mouse lung to indirectly calculate the tumor size. Mice were injected intraperitoneally with 100 μ L of D-fluorescein potassium salt (15mg/mL in PBS) prior to imaging. The body weight of the mice was measured every two days, and the relative body weight change of the mice was measured by m/m₀(m₀Calculated as mouse body weight on day 0). We found that lung fluorescence intensity was lower in SAP-cRGD-CLP treated mice than at day 0 on day 16 of treatment (FIG. 6A), indicating that it was effective in inhibiting tumor growth. Although the SAP-CLP in the non-targeting group can also inhibit the growth of the tumor to a certain extent, the effect is obviously inferior to that in the targeting group, and the fact that the cRGD is modified on the surface of the vesicle can enhance the enrichment of the nano-drug at the tumor part and promote the uptake of the nano-drug by the tumor cells is proved, thereby achieving the purpose of enhancing the curative effect. From FIG. 6B we found the treatment groupThe body weight of the mouse does not change obviously, and the nano-drug on the surface has small toxic and side effects. Mouse survival experiments showed that SAP-cRGD-CLP significantly prolonged survival of mice, with median survival of 50 days in the treated group (fig. 6C), and 41 and 28 days in the SAP-CLP and PBS groups, respectively.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. The polyester peptide vesicle is characterized by being prepared from polyester peptide materials, and comprises polyester peptide materials without targeting molecules, polyester peptide materials with the targeting molecules, or polyester peptide vesicle surfaces prepared from polyester peptide materials without the targeting molecules are modified with the targeting molecules;

the polyester peptide material is polyethylene glycol-bPoly (2-aminocaproic acid) -b-polyaspartic acid having the formula:

2. The lipopeptide vesicle of claim 1, wherein the lipopeptide material is modified with a specific targeting molecule at the end of a hydrophilic segment of polyethylene glycol.

3. The depsipeptide vesicle of claim 2, wherein the specific targeting molecule is a short peptide, a small molecule targeting molecule, an antibody, a polysaccharide, or a monosaccharide.

4. The depsipeptide vesicle of claim 3, wherein the short peptide is cRGD having the sequence cRGDfC, cNGQ having the sequence cNGQGEQc, CC-9 having the sequence CSNIDARAC, or CPP33 having the sequence RLWMRWYSPRTRAYGC.

5. The depsipeptide vesicle of claim 3, wherein the small molecule targeting molecule is folic acid or anisamide.

6. The use of the depsipeptide vesicle of claim 1 in the preparation of a protein drug carrier, wherein the protein drug is saporin.