CN115737562A - Polymer vesicle capable of rapidly detecting self integrity and application thereof - Google Patents
Polymer vesicle capable of rapidly detecting self integrity and application thereof Download PDFInfo
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Abstract
The invention provides a polymersome capable of rapidly detecting self integrity and application thereof. Firstly, preparing the polymersome loaded with the fluorescence donor and the fluorescence acceptor separately, then mixing the polymersome, performing freeze-drying preservation and redissolution, and detecting the fluorescence intensity generated by fluorescence energy resonance transfer (FRET) in the redissolved solution. By combining with high-flux fluorescence analysis equipment such as FRET fluorescence analysis and an enzyme labeling instrument, the integrity of the polymer vesicle can be rapidly detected in high flux without using relatively complex experimental means such as particle size test, electron microscope and the like, so that the drug, vaccine formula and freeze-drying redissolution process can be conveniently screened in high flux, and the optimization efficiency is greatly improved.
Description
Technical Field
The invention belongs to the field of high molecular materials, and particularly relates to a polymersome capable of rapidly detecting self integrity and application thereof.
Background
The key to realizing the curative effect of the medicament and the effect of the vaccine is to use the carrier to load and protect nucleic acid vaccine, nucleic acid medicament, antibody and the like with poor stability and deliver the nucleic acid vaccine, the nucleic acid medicament, the antibody and the like into specified tissues or cells in a human body.
The polymer vesicle is a macromolecular aggregate with a hollow sphere structure and is formed by orderly self-assembling amphiphilic block copolymers in a solution. Similar to liposomes, the inner water core of polymersomes can be loaded with hydrophilic molecules and the hydrophobic membrane layer can be loaded with hydrophobic molecules. Because the amphiphilic block copolymer has higher stability and modifiability compared with lipid, the polymer vesicle has certain advantages in the aspects of stability and functionality, so that the amphiphilic block copolymer becomes a new-generation carrier in the fields of drug delivery, medical diagnosis, food industry, cosmetic industry and the like.
However, despite the good stability of polymersomes, long-term storage problems remain a factor limiting their widespread use. Especially when used for loading biomacromolecules such as nucleic acid and the like, even if the biomacromolecules are wrapped in a carrier, the biomacromolecules can be degraded due to hydrolysis and the like after being stored for a certain time, so that the biological activity is lost.
The freeze-drying preparation of the medicament or the vaccine prepared by using the vacuum freeze-drying technology is one of the most effective methods for realizing the long-term storage of the medicament or the vaccine, can relieve the limitation of cold chain transportation, and is convenient for long-distance transportation at normal temperature and long-term storage at normal temperature. The vacuum freeze-drying technology is a technology for freezing a medicine water solution at a low temperature and then directly sublimating and drying water in the medicine water solution in a vacuum state. However, drug-loaded carriers face two problems during lyophilization: firstly, the medicine and the active ingredients may generate reactions such as dehydration, denaturation and the like under the conditions of low temperature, vacuum and drying; secondly, the carrier is easy to agglomerate or fuse. The two points will cause the re-dissolved medicine preparation to lose the original activity. Therefore, it is often necessary to add adjuvants to pharmaceutical formulations for protection and to optimize the conditions and procedures of lyophilization and reconstitution. The traditional method for evaluating the freeze-drying effect of the carrier-containing medicinal preparation mainly comprises the optimization of a preparation formula and the optimization of freeze-drying and redissolution processes, and then the carrier characteristic, the drug loading rate, the in vitro activity and the in vivo activity after redissolution are fed back to the formula and the process for further optimization. Because each pharmaceutical formulation may require a different protective agent, and various factors in the lyophilization conditions and procedures may influence each other, optimization of the formulation and lyophilization process is often time consuming and labor intensive.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provide a polymersome capable of rapidly detecting the self integrity.
Another object of the present invention is to provide the use of the polymersome.
The purpose of the invention is realized by adopting the following technical scheme:
a polymersome capable of rapidly detecting self-integrity, which is loaded with a fluorescence donor and a fluorescence acceptor, and can respond to a fluorescence signal when the integrity of the polymersome is destroyed.
The polymersome is composed of polymersome loaded with a fluorescence donor and polymersome loaded with a fluorescence acceptor.
In the above-mentioned polymersome, the polymersome carrying a fluorescence donor and the polymersome carrying a fluorescence acceptor may be mixed at an arbitrary ratio.
The loading method comprises the steps of modifying a fluorescence donor/acceptor on a block copolymer before preparing the vesicle, coating the fluorescence donor/acceptor on a hydrophobic layer or a hydrophilic inner cavity of the vesicle in the preparation process of the vesicle, modifying the fluorescence donor/acceptor on a former-stage copolymer after preparing the vesicle, or introducing at least one of the hydrophobic layer or the hydrophilic inner cavity of the vesicle.
The fluorescence donor and the fluorescence acceptor are molecules respectively containing different fluorescent groups, wherein the emission spectrum of the donor is obviously overlapped with the absorption spectrum of the acceptor, the excitation wavelength of the donor has no obvious influence on the acceptor, and the emission spectra of the donor and the acceptor are distinguished to a certain extent.
The polymer vesicle is assembled by amphiphilic block copolymer.
The application of the polymersome for rapidly detecting the self integrity in the evaluation of freeze-dried preparations and processes is disclosed.
A preparation method of polymersome capable of rapidly detecting self integrity comprises the following steps:
(1) Adding the block copolymer or the block copolymer and hydrophobic molecules to be loaded into an organic solvent, and dissolving to prepare a block copolymer solution;
(2) Taking a part of the block copolymer solution, and adding a fluorescence donor;
(3) Adding a fluorescent acceptor into the rest block copolymer solution;
(4) And respectively processing the block copolymer solution added with the fluorescence donor and the fluorescence acceptor to obtain polymer vesicles, and mixing the polymer vesicles and the polymer vesicles to obtain the polymer vesicles capable of rapidly detecting the self integrity.
The hydrophobic molecules in the step (1) are hydrophobic small molecule drugs.
The segmented copolymer in the step (1) is at least one of polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-polycaprolactone (PEG-PCL), PLA-PEG-PLA, PEG-PLA-PEG, PCL-PEG-PCL and PEG-PCL-PEG.
The molecular weight of the polyethylene glycol in the block copolymer is 1000-5000, and the molecular weight of the polylactic acid and the polycaprolactone is 3000-20000.
The hydrophobic small molecule drug is preferably Paclitaxel (PTX) or Doxorubicin (DOX).
The organic solvent described in step (1) is preferably Tetrahydrofuran (THF).
The ratio of the block copolymer to the organic solvent in the step (1) is preferably 2 to 15mg.
The dissolving method in the step (1) is at least one of stirring or ultrasonic oscillation.
The stirring conditions are 100-500 r/min and 15-30 min.
The fluorescence donor in the step (2) is a hydrophobic fluorescence dye donor.
The hydrophobic fluorescence donor is preferably at least one of a DiO dye or a coumarin 6 dye.
The fluorescent acceptor in the step (3) is a hydrophobic fluorescent dye acceptor.
The hydrophobic fluorescent acceptor is preferably at least one of a DiI dye or a nile red dye.
The treatment method in the step (4) is at least one of pouring into water, stirring or blow-drying to obtain a film, and dissolving the film in water and stirring.
The step (4) is mixing the polymersome added with the fluorescence donor and the polymersome added with the fluorescence acceptor according to the mass ratio of 1-3; preferably, the components are mixed according to the mass ratio of 1.
A method of evaluating lyophilized formulations and processes comprising the steps of:
(1) Freeze-drying and storing the polymersome for rapidly detecting the self integrity;
(2) And (3) redissolving the lyophilized and stored polymersome, detecting the FRET fluorescence intensity of the polymersome by using a fluorescence analysis device, and judging the integrity of the polymersome according to the FRET value.
The freeze-drying method comprises at least one of pre-freezing, sublimation drying and resolution drying.
The storage method comprises at least one of normal temperature storage, low temperature storage and ultralow temperature storage.
The redissolution method comprises at least one of oscillation, stirring and ultrasound after adding water.
The fluorescence analysis device comprises at least one of a fluorescence spectrophotometer and a high-throughput fluorescence analysis device.
The lower the FRET value, the better the integrity of the reconstituted polymersome is demonstrated.
An evaluation method is a method for obtaining the optimization effect of the freeze-drying process by the method for evaluating the freeze-drying preparation and the process.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides a polymersome capable of rapidly detecting self integrity and application thereof. Firstly, the polymer vesicles loaded with the fluorescence donor and the fluorescence acceptor are separately prepared, then are mixed, are subjected to freeze-drying preservation and redissolution, and are detected for fluorescence intensity generated by fluorescence energy resonance transfer (FRET) in a redissolved solution, so that the screening of a drug, a vaccine formula and a freeze-drying redissolution process is realized at high flux and conveniently, and the optimization efficiency is greatly improved.
Drawings
FIG. 1 is a schematic diagram of the inventive polymersome for rapidly detecting self-integrity.
Fig. 2 is a flow chart of a method for optimizing a conventional polymersome lyophilized formulation and process and a method for evaluating the lyophilized formulation and process provided by the present invention.
FIG. 3 is a transmission electron microscope result chart of the mixed polymer vesicles in example 1.
FIG. 4 is a schematic fluorescence spectrum of a mixed polymer vesicle.
FIG. 5 is a diagram showing FRET spectra showing good and poor preservation after lyophilization and reconstitution.
FIG. 6 is a transmission electron microscope result chart of vesicle destruction and fusion after freeze-drying and ultrasonic redissolution in example 6.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
In the following embodiments, if the specific test conditions are not specified, the test conditions are generally determined according to conventional test conditions or according to the test conditions recommended by the reagent company. The materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
Example 1
10mg of block copolymer PEG were weighed 2000 -PCL 5000 Then, the mixture was added to 1mL of DCM (dichloromethane), and stirred for 15 minutes to obtain a 10mg/mL block copolymer solution. Adding into20 μ L of 1mg/mL DiO dye (donor) DMSO solution. The solution was rotary evaporated to remove DCM to give a clear film. Then 10mL of deionized water is added, and the mixture is stirred for 12h at room temperature to prepare the polymer vesicle loaded with the donor.
10mg of block copolymer PEG were weighed 2000 -PCL 5000 Then, the mixture was added to 1mL of DCM, and stirred for 15 minutes to prepare a 10mg/mL block copolymer solution. Add 20. Mu.L of 1mg/mL DiI dye (acceptor) DMSO solution. The solution was rotary evaporated to remove DCM, resulting in a clear film. Then 10mL of deionized water is added, and the mixture is stirred for 12h at room temperature to prepare the polymer vesicle loaded with the receptor.
Two polymersome solutions were mixed at a ratio of 1: mixing the materials in a mass ratio of 1 to obtain the mixed polymer vesicle. The mixed vesicles prepared by the method have an average particle size of 120nm and a particle size distribution (PDI) of 0.20.
Example 2
In this example, two block copolymers with different degrees of polymerization were used to prepare polymersome, and 2mg of block copolymer PEG was weighed 2000 -PLA 15000 Added to 200. Mu.L of THF, and dissolved by ultrasonic agitation to obtain a block copolymer solution. mu.L of a 1mg/mL solution of coumarin 6 dye (donor) in THF was added. The block copolymer solution is injected into 1mL of deionized water and stirred rapidly to prepare the donor-loaded polymersome, and redundant organic solvent is removed through dialysis.
Weighing 2mg of block copolymer PEG 2000 -PCL 5000 Then, the mixture was added to 200. Mu.L of THF and dissolved by ultrasonic oscillation to obtain a block copolymer solution. mu.L of a 1mg/mL solution of Nile Red dye (acceptor) in THF was added. The block copolymer solution is injected into 1mL of deionized water and stirred rapidly to prepare the polymer vesicle loaded with the donor, and redundant organic solvent is removed through dialysis.
Two polymersome solutions were mixed at a ratio of 1:1 mass ratio was mixed. The mixed vesicles prepared by the method have an average particle size of 180nm and a particle size distribution (PDI) of 0.15.
Example 3
In this example, two block copolymers with different degrees of polymerization were used to prepare polymersome, and 2mg was weighedBlock copolymer PEG 2000 -PCL 5000 Then, the mixture was added to 200. Mu.L of THF, and dissolved by sonication to obtain a block copolymer solution. mu.L of a 1mg/mL solution of coumarin 6 dye (donor) in THF was added. The block copolymer solution is injected into 1mL of deionized water and stirred rapidly to prepare the polymer vesicle loaded with the donor, and redundant organic solvent is removed through dialysis.
Weighing 2mg of block copolymer PEG 2000 -PLA 15000 Then, the mixture was added to 200. Mu.L of THF and dissolved by ultrasonic oscillation to obtain a block copolymer solution. mu.L of a 1mg/mL solution of Nile Red dye (acceptor) in THF was added. The block copolymer solution is injected into 1mL of deionized water and stirred rapidly to prepare the polymer vesicle loaded with the donor, and redundant organic solvent is removed through dialysis.
Two polymersome solutions were mixed at 1:1, and mixing. The mixed vesicle prepared by the method has the average particle size of 150nm and the particle size distribution (PDI) of 0.20.
Example 4 validation of the stability of polymersomes at different lyophilization temperatures
Taking 3 parts of 1mL mixed polymer vesicle solution prepared in example 1, filling the solution into penicillin bottles, and respectively performing freeze-drying preservation by adopting different precooling modes: liquid nitrogen (-196 ℃), -20 ℃, 80 ℃. Placing the vial in liquid nitrogen (-196 deg.C), -20 deg.C and-80 deg.C, and taking out after the vesicle solution is completely frozen. And (3) placing the penicillin bottle in a vacuum drier, and carrying out vacuum drying for 12h at-50 ℃ to obtain a freeze-dried sample. The lyophilized samples were stored in a 4 degree refrigerator for 1 week. The samples after freeze-drying preservation treatment are taken, added with 1mL of water respectively and shaken by hand for 2 minutes for redissolution.
The FRET fluorescence intensity of the freeze-dried and redissolved polymersome is detected by a fluorescence spectrophotometer, the excitation wavelength is 483 nanometers, and the detection wavelength is 565 nanometers. According to detection, the relative fluorescence intensity of the mixed polymer vesicle solution precooled by liquid nitrogen at-20 ℃ and-80 ℃ at 565 nanometers is 1.0:2.2:1.8. meanwhile, dynamic laser scattering was used for particle size analysis, and the results showed that the average particle sizes of vesicles using the mixed polymer vesicle solutions precooled with liquid nitrogen at-20 ℃ and-80 ℃ were approximately 130nm,180nm, and 160 nm, respectivelyAnd (5) nm. It can be seen that liquid nitrogen pre-cooling is used to treat PEG 2000 -PCL 5000 The polymer vesicle is freeze-dried and stored, the number of particles which are agglomerated and collapsed after redissolution is small, the stability is superior to precooling at minus 20 ℃ and minus 80 ℃, the result of fluorescence analysis is consistent with the result of particle size analysis, the integrity of the redissolved polymer vesicle can be rapidly detected by using fluorescence detection, and complex particle size test is not needed.
Example 5 verification of the stability of polymersomes after treatment with different lyophilization stabilizers
1mL of 6 parts of the mixed polymer vesicle solution prepared in the example 2 is taken and filled in a penicillin bottle, 5% of different freeze-drying protective agents in weight ratio are added, and the influence of the freeze-drying protective agents on the freeze-drying redissolution effect is evaluated. The added lyoprotectants include: sucrose, glucose, trehalose, inulin, hydroxypropyl-beta-cyclodextrin and mannitol. And precooling by using liquid nitrogen under the freeze-drying condition uniformly, and taking out the vesicle solution after the vesicle solution is completely frozen. And (3) placing the penicillin bottle in a vacuum drier, carrying out vacuum drying for 12h at-50 ℃ to obtain a freeze-dried sample, and storing the freeze-dried sample in a 4-degree refrigerator for 1 week. The redissolution conditions were uniform and shaken up with water for 2 minutes.
The FRET fluorescence intensity of the lyophilized and redissolved polymer vesicle is detected by using an enzyme-labeling instrument, the excitation wavelength is 450 nanometers, and the detection wavelength is 620 nanometers. The results show that the freeze-dried redissolution added with sucrose, glucose, trehalose, inulin, hydroxypropyl-beta-cyclodextrin and mannitol has a relative fluorescence intensity ratio of 3.2 at 620 nm: 3.3:3.0:2.5:1.0:3.7. the particle size analysis is carried out by using dynamic laser scattering, and the result shows that the average size of the vesicles is increased by 20-100 nm and is not changed except that the particle size of the polymer vesicles protected by the hydroxypropyl-beta-cyclodextrin is almost unchanged, and the experimental result proves that the polymer vesicles treated by the hydroxypropyl-beta-cyclodextrin are less in the number of particles subjected to agglomeration and collapse and better in integrity than other freeze-drying stabilizers, so that the hydroxypropyl-beta-cyclodextrin is rapidly screened out to be the freeze-drying protective agent with better protection effect under the freeze-drying re-dissolving condition, and the particle size test result is identical with fluorescence FRET data, so that the fluorescence detection can rapidly detect the integrity of the re-dissolved polymer vesicles.
Example 6 verification of the stability of polymersomes after different reconstitution methods
1mL of 6 parts of the mixed polymer vesicle solution prepared in example 3 was filled in a vial, and 5% mannitol and 5% sucrose were added. And precooling the freeze-drying conditions by using liquid nitrogen uniformly, taking out the vesicle solution after the vesicle solution is completely frozen, carrying out low-pressure vacuum dehydration at-50 ℃ for 12h, and storing the freeze-dried sample in a 4-DEG refrigerator for 1 week. Three different reconstitution conditions were used: shake with water manually for 2 minutes, sonicate for 2 minutes, vortex for 2 minutes.
The FRET fluorescence intensity of the freeze-dried and redissolved polymer vesicle is detected by using a fluorescence spectrophotometer, the excitation wavelength is 450 nanometers, and the detection wavelength is 620 nanometers. The results show that the fluorescence intensity ratio of the freeze-dried redissolution solution after hand shaking, ultrasonic treatment and vortex treatment at 620 nm is 1.0:2.3:1.0. the vesicles after ultrasonic reconstitution were photographed using a transmission electron microscope (fig. 6), and the destruction and fusion of the vesicles were found to be significant. Experimental results prove that the ultrasonic redissolution can damage the vesicle structure, and the integrity of the redissolved polymer vesicle is poor, so that the method is not suitable for the redissolution process; the TEM result proves that the result is consistent with the FRET analysis result, and the prepared polymer vesicle capable of rapidly detecting the self integrity can rapidly detect the integrity of the polymer vesicle at high flux by combining FRET fluorescence analysis, a microplate reader and other high-flux fluorescence analysis equipment without using relatively complex experimental means such as particle size test, electron microscope and the like, so that the formula/preparation process and the freeze-drying redissolution process of the medicament and the vaccine can be conveniently screened at high flux, and the optimized efficiency is greatly improved (as shown in figures 4 and 5).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A polymersome capable of rapidly detecting self integrity, which is characterized in that the polymersome is loaded with a fluorescence donor and a fluorescence acceptor, and can respond to a fluorescence signal when the integrity of the polymersome is damaged.
2. Polymersomes according to claim 1, characterized in that:
consists of polymersome loaded with fluorescence donor and polymersome loaded with fluorescence acceptor.
3. Polymersomes according to claim 2, characterized in that:
the polymersome loaded with a fluorescence donor and the polymersome loaded with a fluorescence acceptor may be mixed in any ratio.
4. Polymersomes according to claim 1 or 2, characterized in that:
the loading method comprises the steps of modifying a fluorescence donor/acceptor on a block copolymer before preparing the vesicle, coating the fluorescence donor/acceptor on a hydrophobic layer or a hydrophilic inner cavity of the vesicle in the preparation process of the vesicle, modifying the fluorescence donor/acceptor on a former-stage copolymer after preparing the vesicle, or introducing at least one of the hydrophobic layer or the hydrophilic inner cavity of the vesicle;
the fluorescence donor and the fluorescence acceptor are molecules respectively containing different fluorescent groups, wherein the emission spectrum of the donor is obviously overlapped with the absorption spectrum of the acceptor, the excitation wavelength of the donor has no obvious influence on the acceptor, and the emission spectra of the donor and the acceptor are distinguished to a certain extent;
the polymer vesicle is assembled by amphiphilic block copolymer.
5. Use of polymersomes according to any one of claims 1 to 4 for rapid detection of their own integrity in the evaluation of lyophilized formulations and processes.
6. A preparation method of polymersome capable of rapidly detecting self integrity comprises the following steps:
(1) Adding the block copolymer or the block copolymer and hydrophobic molecules to be loaded into an organic solvent, and dissolving to prepare a block copolymer solution;
(2) Taking a part of block copolymer solution, and adding a fluorescence donor;
(3) Adding a fluorescent acceptor into the rest block copolymer solution;
(4) And respectively processing the block copolymer solution added with the fluorescence donor and the fluorescence acceptor to obtain the polymer vesicle, and mixing the polymer vesicle and the fluorescence donor and the fluorescence acceptor to obtain the polymer vesicle capable of rapidly detecting the self integrity.
7. The method of claim 6, wherein:
the hydrophobic molecules in the step (1) are hydrophobic micromolecular drugs;
the segmented copolymer in the step (1) is at least one of polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, PLA-PEG-PLA, PEG-PLA-PEG, PCL-PEG-PCL and PEG-PCL-PEG;
the molecular weight of the polyethylene glycol in the block copolymer is 1000-5000, and the molecular weight of the polylactic acid and the polycaprolactone is 3000-20000;
the hydrophobic small molecular drug is paclitaxel or adriamycin;
the organic solvent in the step (1) is tetrahydrofuran;
the proportion of the block copolymer and the organic solvent in the step (1) is 2-15mg;
the dissolving method in the step (1) is at least one of stirring or ultrasonic oscillation;
the stirring condition is 100-500 r/min and 15-30 min;
the treatment method in the step (4) is at least one of pouring the mixture into water, stirring or blow-drying the mixture to obtain a film, and dissolving the film in the water and stirring the film;
the mixing in the step (4) is carried out according to the mass ratio of 1-3 of the polymersome added with the fluorescence donor to 1-3 of the polymersome added with the fluorescence acceptor.
8. The method of claim 6, wherein:
the fluorescence donor in the step (2) is a hydrophobic fluorescence dye donor;
the fluorescent acceptor in the step (3) is a hydrophobic fluorescent dye acceptor.
9. A method of evaluating lyophilized formulations and processes, comprising the steps of:
(1) Lyophilizing and storing the polymersome for rapidly testing self-integrity according to any one of claims 1 to 4;
(2) Redissolving the lyophilized and stored polymersome, detecting the FRET fluorescence intensity of the polymersome by using fluorescence analysis equipment, and judging the integrity of the polymersome according to the FRET value;
the freeze-drying method comprises at least one of pre-freezing, sublimation drying and analytical drying;
the storage method comprises at least one of normal temperature, low temperature and ultralow temperature storage;
the redissolution method comprises at least one of oscillation, stirring and ultrasound after adding water;
the fluorescence analysis equipment comprises at least one of a fluorescence spectrophotometer and high-throughput fluorescence analysis equipment;
the lower the FRET value, the better the integrity of the reconstituted polymersome is demonstrated.
10. An evaluation method characterized in that the effect of optimization of the lyophilization process is obtained by the method for evaluating lyophilized formulations and processes according to claim 9.
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