CN110694057A

CN110694057A - Preparation method of nano composite micelle cancer vaccine capable of controllably capturing/releasing antigen

Info

Publication number: CN110694057A
Application number: CN201911087188.7A
Authority: CN
Inventors: 史林启; 李雪; 黄帆; 刘阳; 马如江; 安英丽
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-01-17

Abstract

A method for preparing a nano composite micelle cancer vaccine capable of controllably capturing/releasing antigen. The vaccine is constructed based on a pH-responsive PCL-b-PEG/PCL-b-PAE composite micelle, a hydrophobic micro-region formed by a pH-responsive chain segment (PAE) of the PCL-b-PAE when the pH is greater than 6.5 can capture antigen through hydrophobic effect, and the PAE is protonated when the pH is less than 6.5, changes from a hydrophobic state to a hydrophilic state and is electropositive, so that the antigen can be rapidly released. The preparation method is simple and nontoxic, realizes controllable capture and release of the antigen through the ingenious design of the PAE, and has wide application prospect in the aspect of cancer vaccines.

Description

Preparation method of nano composite micelle cancer vaccine capable of controllably capturing/releasing antigen

Technical Field

The invention belongs to the field of nano biological medicine materials, and discloses a simple nano vaccine for activating the immune response of the antigen specificity of an organism, which is developed by capturing and loading antigen protein by utilizing a specific hydrophobic micro-region on the surface of a composite micelle and releasing the antigen protein in response in a tumor subacid environment.

Background

Since the first demonstration of vaccination by the 18 th century uk doctor edward janna, vaccines have been considered as one of the first line of treatment for the control of cancer and infectious diseases. Vaccination aims to elicit the immune response of the host by antigen administration, and is the only most effective pharmaceutical intervention in modern medicine. Traditionally, the injection of attenuated live or killed whole pathogens into the body triggers a protective immune response without causing disease. When the same pathogen invades again, the body can rapidly generate corresponding immune response to eliminate the pathogen. After decades of research, various vaccines have been developed for tumor therapy, including cell vaccines, DNA/RNA vaccines, protein/polypeptide vaccines, etc. Among them, protein/polypeptide vaccines have gained widespread attention due to their high specificity and relatively low cost. However, the clinical effectiveness of protein/polypeptide vaccines is still further improved. For example: the existing antigen load is mainly to bond antigen protein on a carrier or to wrap the antigen protein in a hydrophobic core through chemical bond, which faces the problems that the protein structure is easy to degrade, inactivate, release slowly and can not be completely released.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a preparation method of a cancer vaccine which captures an antigen through a hydrophobic acting force and loads the antigen on a PAE hydrophobic micro-region of a nano composite micelle; in addition, the proton sponge effect of the PAE can realize the complete release of the antigen under the acidic condition, and the effectiveness of the vaccine is improved.

Technical scheme of the invention

A nanometer composite micelle cancer vaccine capable of controllably capturing/releasing antigen is based on PCL-b-PEG/PCL-b-PAE nanometer composite micelle, a hydrophobic micro-region formed by a pH response chain segment (PAE) when the pH is more than 6.5 can be used for capturing the antigen, and the antigen can be rapidly released when the pH is less than 6.5.

A method for preparing a nano composite micelle cancer vaccine capable of controllably capturing/releasing antigen comprises the following steps:

1) synthesis of PCL-b-PEG

Polyethylene glycol (PEG)_n-OH) and epsilon-CL in a mass ratio of 1:50 in an appropriate amount of redistilled toluene and are subjected to a liquid nitrogen freeze-evacuation-thaw procedure, which is cycled three times. Reacting for 12h at 110 ℃ to obtain PCL-b-PEG_nThe amphiphilic block polymer is precipitated by ethyl acetate and dried in vacuum to obtain white powder;

2) synthesis of PCL-b-PAE

Dissolving 2-hydroxyethyl acrylate and epsilon-CL in a proper amount of redistilled toluene according to the mass ratio of 1:50, and circulating for three times through liquid nitrogen freezing-vacuumizing-unfreezing processes. Reacting for 12h at 110 ℃ to obtain PCL monoacrylate; then PCL monoacrylate, hexane-1, 6-diol-diacrylate (HDD),4, 4' -trimethylene-dipiperidine (TDP) are subjected to Michael addition polymerization reaction in the mass ratio of 1:30:31 to prepare PCL-b-PAE;

3) preparation of composite micelles

Dissolving PCL-b-PEG prepared in the step 1) and PCL-b-PAE prepared in the step 2) in DMF according to the mass ratio of 1: 1. The polymer solution was dropwise added to acidic water of pH 6 under ultrasonic conditions, and ten minutes later, the micelle solution was transferred to a dialysis bag having a molecular weight cutoff of 3500 for dialysis for 3 days. The micelle is concentrated to constant volume by ultrafiltration before use;

4) preparation of vaccines

Taking 1ml of the composite micelle solution with the concentration of 1mg/ml prepared in the step 3), adding 400ug/ml of chicken ovalbumin, and performing shake incubation at 37 ℃ for 1h to obtain the nano composite micelle cancer vaccine;

the invention has the advantages and beneficial effects that:

the invention provides a simple and effective nano cancer vaccine for activating antigen-specific immune response. The method can controllably capture/release the antigen through hydrophobic interaction, can protect the antigen from being degraded and efficiently release, has simple preparation, lower cost and convenient operation, and has great value for the development of vaccines.

Drawings

FIG. 1 shows the preparation process of the nano vaccine of the present invention.

FIG. 2 is the distribution of MSPM @ OVA nanoparticles.

FIG. 3 is the fluorescence spectra of nanoparticles and OVA protein, used to study binding sites of OVA and nanoparticles.

Fig. 4 shows the release behavior of the nano-vaccine under different pH environments.

Figure 5 is an in vitro safety assessment of nanoparticles.

Detailed Description

Example 1:

a method for preparing a nano composite micelle cancer vaccine capable of controllably capturing/releasing antigen comprises the following steps and related evaluation:

1) synthesis of PCL-b-PEG

Drying the treated 0.1mmol PEG_nAdding the-OH and 5mmol of epsilon-CL treated by vacuum distillation into a 50mL dry eggplant-shaped bottle, mixing, dissolving with 10mL redistilled anhydrous toluene, adding one drop of stannous octoate (Sn (Oct))₂). Then, after three cycles of freezing by liquid nitrogen, vacuum pumping, nitrogen filling and thawing, the reaction was carried out for 12 hours in an oil bath at 110 ℃ under the protection of nitrogen. After the reaction, the reaction mixture was diluted with an appropriate amount of dichloromethane and then precipitated in ten times the volume of ethyl acetate. After the precipitation is completed, the white powdery solid obtained by suction filtration, washing and vacuum drying is the PCL-b-PEGn.

2) Synthesis of PCL-b-PAE

0.8mmol of 2-hydroxyethyl acrylate and 40mmol of ε -CL are dissolved in 10ml of redistilled toluene and 1 drop of stannous octoate is added. Freezing by liquid nitrogen, vacuumizing, filling nitrogen and unfreezing, circulating for three times, and reacting for 12 hours in an oil bath at 110 ℃ under the protection of nitrogen. Then precipitating in ethyl glacial ether with ten times of volume, and drying the crude product in a vacuum oven to obtain the PCL monoacrylate. Then 0.1mmol PCL monoacrylate, 3mmol HDD and 3.1mmol TDP were dissolved in 10ml chloroform. Stirring at 55 deg.C for 3 days, precipitating all the solution into excessive diethyl ether, and vacuum filtering to obtain PCL-b-PAE.

3) Preparing a composite micelle;

according to the mass ratio of 1:1, PCL-b-PEG and PCL-b-PAE are dissolved in DMF. The polymer solution was dropwise added to acidic water of pH 6 under ultrasonic conditions, and ten minutes later, the micelle solution was transferred to a dialysis bag having a molecular weight cutoff of 3500 for dialysis for 3 days.

4) Preparing a vaccine;

taking 1ml of the composite micelle solution with the concentration of 1mg/ml prepared in the step 3), adding 400ug/ml of chicken ovalbumin, and performing shake incubation at 37 ℃ for 1h to obtain the nano composite micelle cancer vaccine. As shown in fig. 1, the preparation process of MSPM @ OVA; FIG. 2 is an evaluation of micelle particle size by Dynamic Light Scattering (DLS), and the average particle size was 150 nm.

5) Studying the binding positions of the nanoparticles and the OVA protein;

the binding sites of OVA and nanoparticles were studied by fluorescence spectroscopy of Cy 5-labeled nanoparticles and Cy 3-labeled OVA; excitation at the excitation wavelength of Cy3 (515nm) was the maximum emission wavelength of Cy3 at 570nm, and 670nm was the maximum emission wavelength of Cy 5. Fluorescence resonance energy transfer occurs only when the distance between the energy donor and acceptor is less than 10 nm. As shown in FIG. 3, after MSPM-PAE-Cy5 was incubated with Cy3-OVA, a distinct peak appeared in the fluorescence spectrum at 670nm, indicating that OVA protein was successfully captured in PAE hydrophobic domain.

6) Release of OVA;

the release of OVA was assayed by placing MSPM @ OVA in buffer solution at pH7.4 and pH 5.0. As shown in FIG. 4, OVA is released more rapidly in a buffered solution at pH 5.0; whereas in a ph7.4 environment, OVA release is slower.

7) Safety assessment of nanoparticles

This was done by in vitro cytotoxicity experiments. Five nanoparticle solutions (5, 10, 50, 100, 200ug/mL) were prepared at different concentrations, and DC 2.4 cells were selected for cytotoxicity experiments (MTT). The results are shown in fig. 5, and the survival rate of the cells is above 90% at each concentration, which indicates that the nanoparticles prepared by the method have no obvious cytotoxicity in vitro.

Claims

1. A nanocomposite micelle cancer vaccine for controlled capture/release of antigen, comprising: the nano composite micelle is PCL-b-PAE/PCL-b-PEG, a hydrophobic micro-region formed by a pH responsive chain segment (PAE) of the vaccine when the pH is more than 6.5 can be used for capturing an antigen, and the antigen can be quickly released when the pH is less than 6.5.

2. The controlled capture/release nanocomposite micelle cancer vaccine according to claim 1, wherein PAE is in a hydrophobic state under neutral conditions; under acidic condition, proton sponge effect is generated, and the product is changed into hydrophilic state.

3. A method for preparing the antigen controllable trapping/releasing nano-composite micelle cancer vaccine of claim 1, which is characterized by comprising the following steps:

1) synthesizing PCL-b-PEG;

polyethylene glycol (PEG)_n-OH) and epsilon-caprolactone (epsilon-caprolactone, which is abbreviated as epsilon-CL hereinafter) are dissolved in redistilled toluene according to the mass ratio of 1:50, and the solution is subjected to liquid nitrogen freezing-vacuumizing-unfreezing process for three times; reacting at 110 ℃ for 12h, precipitating by using ethyl acetate and drying in vacuum to obtain PCL-b-PEG_nA white powder;

2) synthesizing PCL-b-PAE;

dissolving 2-hydroxyethyl acrylate and epsilon-CL in redistilled toluene according to the mass ratio of 1:50, and circulating for three times through the processes of liquid nitrogen freezing, vacuumizing and unfreezing; reacting for 12h at 110 ℃ to obtain PCL monoacrylate; then, PCL monoacrylate, hexane-1, 6-diol-diacrylate (HDD) and 4, 4' -trimethylene-dipiperidine (TDP) are fed in the mass ratio of 1:30:31 to carry out Michael addition polymerization reaction to prepare PCL-b-PAE;

3) preparing a composite micelle;

dissolving the PCL-b-PEG prepared in the step 1) and the PCL-b-PAE prepared in the step 2) in DMF according to the mass ratio of 1:1, dropwise adding the polymer solution into acidic water with pH value of 6 under the ultrasonic condition, after ten minutes,

transferring the micelle solution into a dialysis bag with the molecular weight cutoff of 3500 for dialysis for 3 days; the micelle is concentrated to constant volume by ultrafiltration before use;

4) preparing a vaccine;

taking 1ml of the composite micelle solution with the concentration of 1mg/ml prepared in the step 3), adding 400ug/ml of chicken ovalbumin, and performing shake incubation at 37 ℃ for 1h to obtain the nano composite micelle cancer vaccine.