CN116236464A - Polyamino acid drug-loaded nano-particle with charge reversal function and preparation method thereof - Google Patents
Polyamino acid drug-loaded nano-particle with charge reversal function and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/40—Polyamides containing oxygen in the form of ether groups
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- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
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- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses polyamino acid drug-loaded nano particles with a charge reversal function and a preparation method thereof. Polyethylene glycol-polylysine-polyglutamic acid (mPEG-Lys) 10‑ Glu n ) As a main body, 2- (hexamethyleneimine) ethanol (NHM) is grafted on a side chain through an esterification reaction, so that a nano delivery system is constructed, and the nano delivery system has the following structural general formula:
the polyethylene glycol part in the structure is a material with good biocompatibility which is approved by the FDA and can be used for biological carriers, the glutamic acid and the lysine are respectively important raw materials for promoting the nitrogen metabolism of organisms and the synthesis of other important proteins, and the material is nontoxic and degradable and is expected to be applied to the field of medicine slow release.
Description
Technical Field
The invention relates to the field of nano-drug sustained-release carriers, in particular to a polyamino acid drug-loaded nanoparticle with a charge reversal function and a preparation method thereof.
Background
At present, medicines for oral administration or intravenous injection belong to low-molecular medicines, and the concentration of the medicines in blood is always higher than the concentration required for treatment in the early stage after the administration, which can cause anaphylactic reaction and toxic and side effects of organisms; in addition, the low molecular medicine is easy to be digested or degraded by organs such as liver and kidney, so that the phenomena of high metabolic rate, short half-life and the like are caused, and the curative effect of the medicine is affected. The appearance of the polymer drug carrier well solves the problems, wherein the polymer material is only used as a transmission system of low-molecular drugs, the drug loading is realized through chemical bonds, hydrogen bonds, ion complexation and other forms, and the polymer carrier does not have pharmacological activity or react with the drugs, so that the treatment effect of the low-molecular drugs is not affected, the polymer material has good blood and tissue compatibility, and finally can be discharged out of the body or absorbed by human bodies through degradation. Nano delivery systems rely on the small size effect and surface effect of nanoparticles to improve absorption of drugs by the human body and to achieve control over drug release. The nano-carrier can also change the distribution of the medicine in the body by means of the permeability enhancement and retention effect (Enhanced Permeability and Retention effect, EPR) of tumor blood vessels, strengthen the enrichment of the medicine in focus areas, realize the targeted release of the medicine and further reduce toxic and side effects. Nanodelivery systems have a number of advantages over direct intravenous drug injection.
The most commonly encountered problems of the nano delivery system in the process of targeted drug delivery are precipitation of nano particles and poor recognition and elimination of endothelial reticulation system, the surface of a cell membrane is electronegative, and the cationic nano particles can be combined with the cell membrane through electrostatic attraction to promote endocytosis so as to realize the uptake of the nano particles, however, the cationic nano particles are extremely easy to be adsorbed by serum proteins and accumulated in organs such as liver, lung and the like when participating in systemic circulation, so that the treatment effect of the drug-loaded nano particles is reduced, and the damage to organisms possibly caused by thrombus formation and the like; in contrast, neutral or negatively charged nanoparticles generally have longer blood circulation times, but due to their weak transmembrane transport capacity, do not facilitate penetration and uptake by cells at the tumor site, and do not readily permit drug aggregation by EPR effect even when reaching tumor tissue; because the extracellular environment of tumor tissue is weak acid, the charge inversion type drug-carrying nano particles can be constructed, so that the charge inversion type drug-carrying nano particles can be normally delivered by means of charge repulsion in normal internal circulation (pH=7.4), and the charge inversion is realized due to the triggering of an acidic environment when reaching a tumor site (pH is 5.0-6.8), so that the targeting effect of drugs is enhanced by promoting the rapid capture and uptake of cell membranes on the nano particles.
Disclosure of Invention
The invention aims to provide polyamino acid drug-loaded nano-particles with a charge reversal function and a preparation method thereof. Aiming at the defects of poor tissue compatibility, poor targeting to cancer cells and the like of the traditional drug-loaded nano particles, a novel drug-loaded nano particle capable of realizing charge reversal in tumor microenvironment (low pH) is designed.
The most commonly encountered problems of nano delivery systems in the process of targeted drug delivery are precipitation of nano particles and poor recognition and elimination by endothelial reticulation system, the surface of cell membrane is electronegative, cationic nano particles can be combined with cell membrane through electrostatic attraction to promote endocytosis so as to realize uptake of nano particles, however, the cationic nano particles are easily adsorbed by serum proteins when participating in systemic circulation and are absorbed by liver, lung and other devicesThe accumulation in the sense organ not only reduces the treatment effect of the drug-loaded nano particles, but also can cause damage to the organism due to thrombus formation and the like; the charge inversion drug-loaded nano-particles are constructed, so that the charge inversion drug-loaded nano-particles can be normally delivered by means of charge repulsion in normal internal circulation (pH=7.4), and the charge inversion is realized due to triggering of an acidic environment when reaching a tumor site (pH is 5.0-6.8), so that the targeting effect of the drug is enhanced by promoting the rapid capture and uptake of cell membranes to the nano-particles. The invention designs and prepares a series of drug-loaded nano particles with charge reversal capability, and uses polyethylene glycol-polylysine-polyglutamic acid (mPEG-Lys) 10- Glu n ) As a main body, 2- (hexamethyleneimine) ethanol (NHM) is grafted on a side chain through an esterification reaction, so that a nano delivery system is constructed; the polyethylene glycol part in the structure is a material with good biocompatibility which is approved by the FDA and can be used for biological carriers, the glutamic acid and the lysine are respectively important raw materials for promoting the nitrogen metabolism of organisms and the synthesis of other important proteins, and the material is nontoxic and degradable and is expected to be applied to the field of medicine slow release.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the polymer of the polyamino acid drug-loaded nanoparticle with the charge reversal function is a triblock copolymer, the first part is a hydrophilic macromolecular initiator polyethylene glycol chain segment, the second part is a polylysine chain segment for realizing chemical bonding drug loading, and the third part is a polyglutamic acid chain segment for grafting tertiary amine motifs, and the structural general formula is as follows:
the invention discloses a polyamino acid drug-loaded nanoparticle with a charge reversal function, which is prepared by the following steps:
(1) Synthesis of triblock copolymer [ mPEG-P (Lys-Cbz) -b-P (Glu-OBzl) ]
The raw material N-carbobenzoxy-L-lysine cyclic anhydride is dissolved by dry DMF and transferred to a reaction bottle, and is vacuumized and replaced by argon for three timesThe system is finally in inert atmosphere; macroinitiator mPEG-NH 2 Is also dissolved in DMF and slowly added into the reaction system by a syringe for reaction for 72 hours; the second raw material L-glutamic acid-5-benzyl ester is dissolved in DMF and then added into a system, and the reaction is continued for 48 hours to realize the synthesis of a second block; concentrating the mixed solution by reduced pressure distillation, taking glacial ethyl ether as a poor solvent, and obtaining yellowish powder by sedimentation, wherein the structural formula is as follows:
(2) Side chain deprotection reaction
The synthesized polyamino acid [ mPEG-P (Lys-Cbz) -b-P (Glu-OBzl) ] side chain contains benzyloxy and benzyloxycarbonyl, and needs hydrolysis reaction to realize removal of protecting groups, and the specific steps are as follows:
adding 10mL of trifluoroacetic acid into the product obtained in the previous step, stirring for a period of time until the trifluoroacetic acid is completely dissolved, dripping a hydrobromic acid/acetic acid (33%) mixed solution into a reaction bottle, keeping the process under an ice bath condition, continuing stirring at room temperature, carrying out the whole process under a closed condition, and connecting a tail gas treatment device at the upper end, wherein the solution turns red and continuously precipitates in the reaction process. After filtration, the solution was concentrated by rotary evaporation, and a pale yellow powder solid was precipitated by using glacial ethyl ether as a poor solvent, and the structural general formula was as follows:
(3) Grafting reaction of side chains
Dissolving the product of the previous step in DMF, weighing Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP), dissolving in DMF together, slowly dripping into a reaction bottle in ice bath, heating to room temperature after dripping, and continuously stirring for 5 hours to realize the activation of carboxyl; 2- (hexamethyleneimine) ethanol is diluted by DMF, slowly injected into a reaction system, continuously reacted for 48 hours, concentrated to a certain concentration, and the polymer is settled out by glacial ethyl ether to be light yellow solid, and is dried in vacuum for 24 hours for later use.
(4) Preparation of drug-loaded nanoparticles
Preparing Doxorubicin (DOX) solution with specific concentration after removing hydrochloric acid by triethylamine, dissolving the polymer prepared in the step (3) in DMF, mixing the polymer with the DOX solution, and continuously stirring the mixture at room temperature in a dark place for 24 hours, wherein chemical bonding is realized by utilizing amino groups on a polylysine side chain. After a period of time, PBS solution is added dropwise into the system and the operation is repeated for a plurality of times, so that the polymer molecules in the solution are ensured to complete the self-assembly process. The mixed solution was transferred to a dialysis bag for dialysis treatment to remove excess DOX. Finally, the solution is filtered by a water phase filter membrane to obtain the drug-loaded nano-particles, which are named DOX-Pn (NHM) -M.
Further, the proportion of the polyglutamic acid segment is 1, 2 or 3 times that of polylysine.
Further, the concentration of the prepared drug-loaded nano particles is 2 mg/mL-4 mg/mL, and the average size is 100 nm-200 nm.
The prepared drug-loaded nanoparticle has a tertiary amine structure with a charge reversal function, and can be better absorbed by cells by utilizing the protonation effect in an acidic environment.
In the invention, the in vitro release research of the model drug adopts the following method:
the polymer is made to show amphiphilicity through bonding hydrophobic DOX, and the tertiary amine structure of the side chain can generate charge interaction under acidic condition due to protonation effect to promote the release of the medicine.
Loading the concentrated drug-loaded nanoparticle solution DOX-Pn (NHM) -M into dialysis bags, respectively placing into centrifuge tubes filled with different PBS buffers, setting 37 ℃ in a constant-temperature oscillator, continuously oscillating for 90 hours at a constant rotation speed of 100rpm in the dark, taking release solution for detecting the light absorption intensity (UV-vis) of DOX within a preset time (1 h,2h,4h,7h,10h,12h,18h,24h,33h,42h,54h,66h,78h,90 h), and correspondingly supplementing the fresh buffer solution with equal volume. Each data was measured in triplicate and averaged.
The beneficial effects of the invention are as follows:
1. the three-block polyamino acid material synthesized by the invention has nontoxic and bioaffinity components.
2. Macroinitiator mPEG-NH used in the invention 2 Can act as a good hydrophilic part during self-assembly of the polymer, and plays a role in promoting the participation of the nano particles in the systemic circulation process.
3. The side chain of the triblock polyamino acid material synthesized by the invention contains a large number of carboxyl and amino groups, so that a large number of active sites are provided for chemical bonding of die-locking type medicines and grafting of other characteristic functional groups.
4. The medicine-carrying nano particle prepared by the invention has tertiary amine structure capable of responding to pH, can promote the nano particle to realize charge reversal in an acidic environment, and induce the breakage of imine bonds and the change of self-assembly structure, thereby accelerating the release of medicine.
Drawings
FIG. 1 shows the FT-IR test spectrum of the polymer in example 1 of the invention;
FIG. 2 shows the polymer of example 1 of the present invention 1 H-NMR test spectra;
FIG. 3 shows the FT-IR test spectrum of the polymer in example 2 of the invention;
FIG. 4 shows the polymer of example 2 of the present invention 1 H-NMR test spectra;
FIG. 5 shows the FT-IR test spectrum of the polymer in example 3 of the invention;
FIG. 6 shows the polymer in example 3 of the present invention 1 H-NMR test spectra;
FIG. 7 is a graph showing the particle size distribution of polymer nanoparticles in example 1 of the present invention;
FIG. 8 is a graph showing the particle size distribution of polymer nanoparticles in example 2 of the present invention;
FIG. 9 is a graph showing the particle size distribution of polymer nanoparticles in example 3 of the present invention;
FIG. 10 shows the drug release profile of drug-loaded nanoparticles of example 1 of the present invention;
FIG. 11 shows the drug release profile of drug-loaded nanoparticles in example 2 of the present invention;
fig. 12 shows a drug release profile of drug-loaded nanoparticles in example 3 of the present invention.
Detailed Description
Several examples are specifically analyzed below to describe in detail the preparation process of the novel drug-loaded nanoparticle of the present invention. The examples presented below are only for the understanding of the present invention and are not intended to limit the scope of the study and application of the present invention. The reagents and consumables used in the operation of the invention are customized by special manufacturers.
The instrument and the characterization method adopted by the invention are as follows:
(1) FT-IR was measured using a Spectrum One IR spectrometer from PE, USA. The solid sample adopts KBr tabletting, the liquid sample is coated on KBr wafer, and the wave number range of absorption spectrum scanning is 4000-500 cm -1 Scanning three times.
(2) NMR was measured at 25 ℃ using a Bruker ARX 600MHz superconducting nuclear magnetic resonance apparatus, germany. 1 H-NMR with CDCl 3 Or DMSO is used as a solvent, and TMS is used as an internal standard;
(3) the (UV-Vis) ultraviolet-visible spectrophotometer was tested using TU-1901, beijing placido, inc., with PBS solution as the background, measuring the wavelength range from 200nm to 600nm.
(4) Particle size was measured using a Malvern dynamic light scattering laser particle sizer, UK, using PBS as background, 1ml was sampled and each sample was tested three times.
Example 1
Triblock polyamino acid [ mPEG-PLys ] with charge reversal function 10 -P(Glu-NHM) 10 ]Is prepared from the nano particles with high content of active components
The chemical structural formula of the polymer is as follows:
(1) Synthesis of triblock copolymer [ mPEG-P (Lys-Cbz) 10 -b-P(Glu-OBzl) 10 ]
The raw material N-carbobenzoxy-L-lysine cyclic anhydride (0.612 g,0.002 mol) is dissolved by dry DMF and transferred to a reaction bottle, and the reaction bottle is vacuumized and replaced by argon for three times, so that the system is finally in an inert atmosphere; macroinitiator mPEG-NH 2 (1 g,0.0002 mol) was also dissolved in DMF and slowly added to the reaction system with a syringe, and the reaction was continued for 72 hours; the second raw material L-glutamic acid-5-benzyl ester (0.528 g,0.002 mol) is dissolved in 8mL of DMF and added into the system, and the reaction is continued for 48h to realize the synthesis of a second block; the mixed solution is concentrated by reduced pressure distillation, and is taken as a poor solvent, and yellowish powder named as P1-Cbz/OBzl is obtained by sedimentation, wherein the structural general formula is as follows:
(2) Side chain deprotection reaction
The triblock copolymer obtained in the previous step realizes the removal of the protecting group by utilizing hydrolysis reaction, and the specific steps are as follows:
10mL of trifluoroacetic acid is added into P1-Cbz/OBzl (1 g) and stirred for 50min until the mixture is completely dissolved, 6mL of hydrobromic acid/acetic acid (33%) mixed solution is added into the system under the ice bath condition, the mixture is cooled to room temperature after dripping, stirring is continued for 6h, the whole process is carried out under the airtight condition, a tail gas treatment device is connected at the upper end, and the reaction process solution turns red and continuously precipitates. After filtration, the solution was concentrated by rotary evaporation, and a pale yellow powder solid P1 was precipitated by using glacial ethyl ether as a poor solvent, and the solid was washed three times with ethyl ether again and dried under vacuum at 30 ℃ for 24 hours, and had the following general structural formula:
(3) Grafting reaction of side chains
Firstly, activating carboxyl, dissolving P1 (1 g) in 10mL of DMF, weighing DCC (0.470 g,0.00232 mol) and DMAP (0.05 g), dissolving in 10mL of DMF together, slowly dropwise adding the mixture into a reaction bottle in an ice bath, heating to room temperature after dropwise adding, and continuously stirring for 5 hours to realize the activation of carboxyl; 2- (Hexamethylimine) ethanol (0.332 g,0.00232 mol) was diluted with DMF, slowly injected into the reaction system, reacted at 35℃for 48h, filtered to remove by-product Dicyclohexylurea (DCU), the solution was concentrated to a certain concentration, and the polymer was precipitated from glacial ethyl ether as pale yellow solid, dried under vacuum for 24h and named P1-NHM.
(4) Preparation of drug-loaded nanoparticles
50mg of polymer P1-NHM was dissolved in 20mL of DMF, mixed with DOX solution and stirred at room temperature under continuous light-shielding for 24h, and chemical bonding was achieved by using amino groups on the polylysine side chains. Then 1mL of PBS solution with ph=7.4 was added dropwise into the system every 10min and the operation was repeated 10 times, ensuring that the polymer molecules in the solution completed the self-assembly process. The mixed solution was transferred to a dialysis bag (molecular weight cut-off 3500 Da) for dialysis treatment to remove excess DOX, the solution was replaced every 6 hours, and repeated four times. Finally, the micelle solution is filtered by a 0.45 mu M water phase filter membrane to obtain the drug-loaded nano particles, which are named DOX-P1 (NHM) -M, and the solution is required to be stored in a neutral environment all the time, so that the damage of the nano particle structure caused by the change of pH is avoided.
(5) In vitro release studies of model drugs
The polymer shows amphiphilicity and pH responsiveness by bonding hydrophobic DOX, and the tertiary amine structure of the side chain can generate charge interaction under acidic condition due to protonation effect to promote the release of the drug.
The concentrated drug-loaded nanoparticle solution DOX-P1 (NHM) -M was placed in dialysis bags, each in a centrifuge tube containing 20mL of PBS buffer with different pH values (pH=5.0; pH=6.2 and pH=7.4), the solution was continuously oscillated at a constant rotation speed of 100rpm for 90 hours under a constant temperature of 37℃in a constant temperature oscillator set at a dark place, and 3.5mL of release solution was taken for measuring the absorbance intensity (UV-vis) of DOX for a predetermined period of time (1 h,2h,4h,7h,10h,12h,18h,24h,33h,42h,54h,66h,78h,90 h) while the fresh buffer solution of equal volume was correspondingly replenished, and the absorbance of the solution at 481nm was measured to calculate the concentration of DOX. Each data was measured in triplicate and averaged.
As shown in fig. 10, the drug-loaded nanoparticle has very slow drug release rate under neutral condition, the cumulative release rate of 24 hours is 40.16%, and the cumulative release rate of tens of hours after the equilibrium is basically reached is not more than 6%, the DOX coated on the surface of the nanoparticle is mainly released, and a very small amount of drug seeps out from the core, thus proving the structural stability of the nanoparticle. The release rate at ph=6.2 is greatly improved, and the cumulative release rate at ph=5.0 for 24 hours is more up to 64.8%, which shows very significant pH sensitivity. Not only realizes chemical bonding type drug release due to the cleavage of imine bond under acidic condition, and damages self-assembly structure to release a large amount of drugs; more importantly, the tertiary amine in the acidic environment generates a protonation effect, so that a huge charge repulsive effect is caused, DOX itself also contains amino groups and other functional groups which are easy to ionize and protonate, the electrostatic effect among molecules is promoted, and the change of morphology is caused to promote the release of the medicine.
Example 2
Triblock polyamino acid [ mPEG-PLys ] with charge reversal function 10 -P(Glu-NHM) 20 ]Is prepared from the nano particles with high content of active components
The chemical structural formula of the polymer is as follows:
(1) Synthesis of triblock copolymer [ mPEG-P (Lys-Cbz) 10 -b-P(Glu-OBzl) 20 ]
The raw material N-carbobenzoxy-L-lysine cyclic anhydride (0.612 g,0.002 mol) is dissolved by dry DMF and transferred to a reaction bottle, and the reaction bottle is vacuumized and replaced by argon for three times, so that the system is finally in an inert atmosphere; macroinitiator mPEG-NH 2 (1 g,0.0002 mol) was also dissolved in DMF and slowly added to the reaction system with a syringe, and the reaction was continued for 72 hours; the second raw material L-glutamic acid-5-benzyl ester (1.052 g, 0.004mol) is dissolved in 8mL of DMF and added into the system, and the reaction is continued for 48h to realize the synthesis of a second block; concentrating the mixed solution by reduced pressure distillation, and settling with glacial ethyl ether as poor solvent to obtain micro-powderYellow powder, named P2-Cbz/OBzl, having the following structural formula:
(2) Side chain deprotection reaction
The triblock copolymer obtained in the previous step realizes the removal of the protecting group by utilizing hydrolysis reaction, and the specific steps are as follows:
10mL of trifluoroacetic acid is added into P2-Cbz/OBzl (1 g) and stirred for 50min until the mixture is completely dissolved, 6mL of hydrobromic acid/acetic acid (33%) mixed solution is added into the system under the ice bath condition, the mixture is cooled to room temperature after dripping, stirring is continued for 6h, the whole process is carried out under the airtight condition, a tail gas treatment device is connected at the upper end, and the reaction process solution turns red and continuously precipitates. After filtration, the solution was concentrated by rotary evaporation, and a pale yellow powder solid P2 was precipitated by using glacial ethyl ether as a poor solvent, and the solid was washed three times with ethyl ether again and dried under vacuum at 30 ℃ for 24 hours, and had the following general structural formula:
(3) Grafting reaction of side chains
Firstly, activating carboxyl, namely dissolving P2 (1 g) in 10mL of DMF, weighing DCC (0.956 g,0.00464 mol) and DMAP (0.1 g) to be jointly dissolved in 10mL of DMF, slowly dropwise adding the mixture into a reaction bottle under ice bath, and after dropwise adding, heating to room temperature and continuously stirring for 5 hours to realize the activation of carboxyl; 2- (Hexamethylene imine) ethanol (0.664 g,0.00464 mol) was diluted with DMF, slowly injected into the reaction system, after continuous reaction at 35℃for 48h, the by-product DCU was removed by filtration, the solution was concentrated to a certain concentration, and the polymer settled out of glacial ethyl ether as a pale yellow solid, dried under vacuum for 24h and named P2-NHM.
(4) Preparation of drug-loaded nanoparticles
50mg of polymer P1-NHM was dissolved in 20mL of DMF, mixed with DOX solution and stirred at room temperature under continuous light-shielding for 24h, and chemical bonding was achieved by using amino groups on the polylysine side chains. Then 1mL of PBS solution with ph=7.4 was added dropwise into the system every 10min and the operation was repeated 10 times, ensuring that the polymer molecules in the solution completed the self-assembly process. The mixed solution was transferred to a dialysis bag (molecular weight cut-off 3500 Da) for dialysis treatment to remove excess DOX, the solution was replaced every 6 hours, and repeated four times. Finally, the micelle solution is filtered by a 0.45 mu M water phase filter membrane to obtain the drug-loaded nano particles, which are named DOX-P2 (NHM) -M, and the solution is required to be stored in a neutral environment all the time, so that the damage of the nano particle structure caused by the change of pH is avoided.
(5) In vitro release studies of model drugs
The polymer shows amphiphilicity and pH responsiveness by bonding hydrophobic DOX, and the tertiary amine structure of the side chain can generate charge interaction under acidic condition due to protonation effect to promote the release of the drug.
The concentrated drug-loaded nanoparticle solution DOX-P2 (NHM) -M was placed in dialysis bags, each in a centrifuge tube containing 20mL of PBS buffer with different pH values (pH=5.0; pH=6.2 and pH=7.4), the solution was continuously oscillated at a constant rotation speed of 100rpm for 90 hours under a constant temperature of 37℃in a constant temperature oscillator set at a dark place, and 3.5mL of release solution was taken for measuring the absorbance intensity (UV-vis) of DOX for a predetermined period of time (1 h,2h,4h,7h,10h,12h,18h,24h,33h,42h,54h,66h,78h,90 h) while the fresh buffer solution of equal volume was correspondingly replenished, and the absorbance of the solution at 481nm was measured to calculate the concentration of DOX. Each data was measured in triplicate and averaged.
As shown in fig. 11, the drug-loaded nanoparticle has a relatively slow drug release rate under neutral conditions, a cumulative release rate of 36.69% for 24 hours, and a basic equilibrium, and the cumulative release rate of not more than 8% for several tens of hours thereafter, the DOX coated on the nanoparticle surface is mainly released, and a very small amount of drug seeps out from the core, thus proving the structural stability of the nanoparticle. The release rate at ph=6.2 was greatly improved, and the cumulative release rate at ph=5.0 for 24h was even more up to 64.28%, showing very pronounced pH sensitivity. Not only realizes chemical bonding type drug release due to the cleavage of imine bond under acidic condition, and damages self-assembly structure to release a large amount of drugs; more importantly, the tertiary amine in the acidic environment generates a protonation effect, so that a huge charge repulsive effect is caused, DOX itself also contains amino groups and other functional groups which are easy to ionize and protonate, the electrostatic effect among molecules is promoted, and the change of morphology is caused to promote the release of the medicine. The overall cumulative release rate is improved over that of example 1.
Example 3
Triblock polyamino acid [ mPEG-PLys ] with charge reversal function 10 -P(Glu-NHM) 30 ]Is prepared from the nano particles with high content of active components
The chemical structural formula of the polymer is as follows:
(1) Synthesis of triblock copolymer [ mPEG-P (Lys-Cbz) 10 -b-P(Glu-OBzl) 30 ]
The raw material N-carbobenzoxy-L-lysine cyclic anhydride (0.612 g,0.002 mol) is dissolved by dry DMF and transferred to a reaction bottle, and the reaction bottle is vacuumized and replaced by argon for three times, so that the system is finally in an inert atmosphere; macroinitiator mPEG-NH 2 (1 g,0.0002 mol) was also dissolved in DMF and slowly added to the reaction system with a syringe, and the reaction was continued for 72 hours; the second raw material L-glutamic acid-5-benzyl ester (1.578 g, 0.006mol) is dissolved in 8mL DMF and added into the system, and the reaction is continued for 48h to realize the synthesis of a second block; the mixed solution is concentrated by reduced pressure distillation, and is taken as a poor solvent, and yellowish powder named as P3-Cbz/OBzl is obtained by sedimentation, wherein the structural general formula is as follows:
(2) Side chain deprotection reaction
The triblock copolymer obtained in the previous step realizes the removal of the protecting group by utilizing hydrolysis reaction, and the specific steps are as follows:
10mL of trifluoroacetic acid is added into P3-Cbz/OBzl (1 g) and stirred for 50min until the mixture is completely dissolved, 6mL of hydrobromic acid/acetic acid (33%) mixed solution is added into the system under the ice bath condition, the mixture is cooled to room temperature after dripping, stirring is continued for 6h, the whole process is carried out under the airtight condition, a tail gas treatment device is connected at the upper end, and the reaction process solution turns red and continuously precipitates. After filtration, the solution was concentrated by rotary evaporation, and a pale yellow powder solid P3 was precipitated by using glacial ethyl ether as a poor solvent, and the solid was washed three times with ethyl ether again and dried under vacuum at 30 ℃ for 24 hours, and had the following general structural formula:
(3) Grafting reaction of side chains
Firstly, activating carboxyl, namely dissolving P3 (1 g) in 10mL of DMF, weighing DCC (1.434 g,0.00696 mol) and DMAP (0.2 g) to be jointly dissolved in 10mL of DMF, slowly dropwise adding the mixture into a reaction bottle under ice bath, and after dropwise adding, heating to room temperature and continuously stirring for 5 hours to realize the activation of carboxyl; 2- (Hexamethylimine) ethanol (0.996 g,0.00696 mol) was diluted with DMF, slowly injected into the reaction system, after continuous reaction at 35℃for 48h, the by-product DCU was removed by filtration, the solution was concentrated to a certain concentration, and the polymer settled out of glacial ethyl ether as a pale yellow solid, dried under vacuum for 24h and named P3-NHM.
(4) Preparation of drug-loaded nanoparticles
50mg of polymer P1-NHM was dissolved in 20mL of DMF, mixed with DOX solution and stirred at room temperature under continuous light-shielding for 24h, and chemical bonding was achieved by using amino groups on the polylysine side chains. Then 1mL of PBS solution with ph=7.4 was added dropwise into the system every 10min and the operation was repeated 10 times, ensuring that the polymer molecules in the solution completed the self-assembly process. The mixed solution was transferred to a dialysis bag (molecular weight cut-off 3500 Da) for dialysis treatment to remove excess DOX, the solution was replaced every 6 hours, and repeated four times. Finally, the micelle solution is filtered by a 0.45 mu M water phase filter membrane to obtain the drug-loaded nano particles, which are named DOX-P3 (NHM) -M, and the solution is required to be stored in a neutral environment all the time, so that the damage of the nano particle structure caused by the change of pH is avoided.
(5) In vitro release studies of model drugs
The polymer shows amphiphilicity and pH responsiveness by bonding hydrophobic DOX, and the tertiary amine structure of the side chain can generate charge interaction under acidic condition due to protonation effect to promote the release of the drug.
The concentrated drug-loaded nanoparticle solution DOX-P3 (NHM) -M was placed in dialysis bags, each in a centrifuge tube containing 20mL of PBS buffer with different pH values (pH=5.0; pH=6.2 and pH=7.4), the solution was continuously oscillated at a constant rotation speed of 100rpm for 90 hours under a constant temperature of 37℃in a constant temperature oscillator set at a dark place, and 3.5mL of release solution was taken for measuring the absorbance intensity (UV-vis) of DOX for a predetermined period of time (1 h,2h,4h,7h,10h,12h,18h,24h,33h,42h,54h,66h,78h,90 h) while the fresh buffer solution of equal volume was correspondingly replenished, and the absorbance of the solution at 481nm was measured to calculate the concentration of DOX. Each data was measured in triplicate and averaged.
As shown in fig. 12, the drug-loaded nanoparticle has a slow drug release rate under neutral conditions, the cumulative release rate of 24 hours is 39.59%, and the drug-loaded nanoparticle basically reaches equilibrium, and the cumulative release rate of tens of hours after the equilibrium is not more than 7%, so that DOX coated on the surface of the nanoparticle is mainly released, and a very small amount of drug seeps out from the core, thereby proving the structural stability of the nanoparticle. The release rate at ph=6.2 is greatly improved, and the cumulative release rate at ph=5.0 for 24 hours is even more up to 66.7%, which shows very significant pH sensitivity. Not only realizes chemical bonding type drug release due to the cleavage of imine bond under acidic condition, and damages self-assembly structure to release a large amount of drugs; more importantly, the tertiary amine in the acidic environment generates a protonation effect, so that a huge charge repulsive effect is caused, DOX itself also contains amino groups and other functional groups which are easy to ionize and protonate, the electrostatic effect among molecules is promoted, and the change of morphology is caused to promote the release of the medicine. The overall cumulative release rate was improved compared to examples 1 and 2, and higher concentrations of DOX could be detected during drug release.
Claims (4)
1. The polyamino acid medicine carrying nanometer particle with charge reversal function is characterized in that the structure of the block polymer is composed of three parts, the first part is hydrophilic macromolecular initiator polyethylene glycol segment, the second part is polylysine segment for doxorubicin chemical bonding, the third part is polyglutamic acid segment grafted with functional group, and the structural general formula is as follows:
2. the method for preparing the polyamino acid drug-loaded nanoparticle with charge reversal function according to claim 1, comprising the following steps:
(1) Synthesis of triblock copolymer [ mPEG-P (Lys-Cbz) -b-P (Glu-OBzl) ]
Dissolving raw material N-carbobenzoxy-L-lysine cyclic anhydride by using dry DMF (dimethyl formamide), transferring the raw material N-carbobenzoxy-L-lysine cyclic anhydride to a reaction bottle, vacuumizing, and replacing argon for a plurality of times to ensure that the system is finally in an inert atmosphere; macromolecular initiator mPEG-NH 2 Dissolving in DMF and slowly adding into a reaction system by a syringe, and continuously reacting for 72h; dissolving L-glutamic acid-5-benzyl ester in DMF, adding the solution into a system, and continuing to react for 48 hours to realize the synthesis of a second block; concentrating the mixed solution by reduced pressure distillation, taking glacial ethyl ether as a poor solvent, and obtaining powder by sedimentation, wherein the structural formula is as follows:
(2) Side chain deprotection reaction
Adding 10mL of trifluoroacetic acid into the product obtained in the previous step, stirring for a period of time until the solution is completely dissolved, dripping a hydrobromic acid/acetic acid mixed solution into a reaction bottle, keeping the process under ice bath conditions, then carrying out continuous stirring at room temperature after the process is carried out, connecting a tail gas treatment device at the upper end of the process under a closed condition, enabling the solution to turn red and continuously precipitating and separating out in the reaction process, filtering, rotationally evaporating the concentrated solution, and settling out powder solid by taking glacial ethyl ether as a poor solvent, wherein the structural formula is as follows:
(3) Grafting reaction of side chains
Dissolving the product of the previous step in DMF, weighing DCC and DMAP to be jointly dissolved in DMF, slowly dripping the mixture into a reaction bottle under ice bath, heating to room temperature after dripping, and continuously stirring for 5 hours to realize the activation of carboxyl; diluting 2- (hexamethyleneimine) ethanol with DMF, slowly injecting into a reaction system, continuously reacting for 48 hours, concentrating the solution to a certain concentration, settling out polymer with glacial ethyl ether, and drying for later use;
(4) Preparation of drug-loaded nanoparticles
Preparing DOX solution with specific concentration after triethylamine is removed from hydrochloric acid, dissolving the polymer obtained in the step (3) in DMF, mixing the solution with the DOX solution, continuously stirring the solution for 24 hours at room temperature in a dark place, utilizing amino groups on a polylysine side chain to realize chemical bonding, dropwise adding PBS solution into a system after a period of time, repeating the operation for a plurality of times to ensure that polymer molecules in the solution complete self-assembly process, transferring the mixed solution to a dialysis bag for dialysis treatment to remove redundant DOX, and finally filtering the solution through an aqueous phase filter membrane to obtain the drug-loaded nano particles.
3. The method for preparing charge-reversal drug-loaded polyamino acid nanoparticles according to claim 2, wherein the proportion of the polyglutamic acid segment is 1, 2 or 3 times that of polylysine.
4. The method for preparing the charge-reversal functional polyamino acid-loaded nanoparticle according to claim 2, wherein the concentration of the prepared drug-loaded nanoparticle is 2mg/mL to 4mg/mL, and the average size is 100nm to 200nm.
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Application publication date: 20230609 |