CN113069552A - Nano particles and application thereof - Google Patents

Nano particles and application thereof Download PDF

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CN113069552A
CN113069552A CN202110233466.6A CN202110233466A CN113069552A CN 113069552 A CN113069552 A CN 113069552A CN 202110233466 A CN202110233466 A CN 202110233466A CN 113069552 A CN113069552 A CN 113069552A
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peg
dspe
chitosan
emodin
mbi
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邹川
陈蕾
吉春兰
卢钊宇
林启展
刘旭生
卢富华
郭瑞
冯龙宝
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Guangdong Hospital of Traditional Chinese Medicine
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    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
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Abstract

The invention relates to the technical field of drug carriers, in particular to a nanoparticle and application thereof. According to the invention, DSPE-PEG-CS polymer is formed by distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH) and chitosan, and then 5-amino-2-Mercaptobenzimidazole (MBI) is used for thiolating the polymer to form a sulfhydryl polymer DSPE-PEG-CS-MBI drug carrier, wherein the drug carrier has amphipathy, and in a water phase, a hydrophobic end of the drug carrier can wrap a hydrophobic drug emodin to form an oil-in-water type nano micelle, so that the solubility of the emodin is improved; meanwhile, sulfhydryl on DSPE-PEG-CS-MBI can be oxidized to form disulfide bond to adhere to the surface of mucous membrane, so as to prolong the detention time of the disulfide bond on the mucous membrane and facilitate the slow release of emodin.

Description

Nano particles and application thereof
Technical Field
The invention relates to the technical field of drug carriers, in particular to a nanoparticle and application thereof.
Background
Chronic Kidney Disease (CKD) is caused by a number of disease pathways that irreversibly alter kidney function and structure. Recent national prevalence surveys have shown that Chronic Kidney Disease (CKD) has become an important public health problem in china. In developed countries, the incidence of CKD is high, with over 14% of adults suffering from CKD. Uremic toxins can contribute to CKD complications, and there is increasing evidence that uremic retention solutes originate from the metabolism of colonic microorganisms, such as indoleamines derived from bacterial metabolites, which can contribute to uremic toxins through the gut-renal axis, including indoxyl sulfate. Indoxyl sulfate, derived from intestinal flora fermentation, is an independent risk factor for CKD and cardiovascular disease. On the other hand, studies have shown that the intestinal flora is altered in CKD status, and this malnutrition in turn promotes the development of CKD. Studies have shown that by targeting the colonic environment CKD can be effectively treated by altering uremic toxins and intestinal flora.
Herbs have historically been used to treat chronic kidney disease and their related natural compounds are considered important sources of drug discovery. Emodin (1,3, 8-trihydroxy-6-methylanthraquinone, Emodin) is a naturally occurring anthraquinone, found in the roots and bark of many plants, especially the important active ingredient of Chinese herbal medicine rhubarb, which has laxative, antibacterial, immunosuppressive and diuretic effects. Rhubarb has been a traditional Chinese medicine for relaxing bowels since ancient times, and is widely used for reducing uremic toxins to treat CKD in China. But since the early 1950 s it was used more frequently by colonic administration than by oral administration. Studies have shown that rheum based soup enemas can reduce uremic toxins, including urea, creatinine, and indoxyl sulfate, preserve kidney function and help patients avoid dialysis. Treatment of rhubarb via the colonic route can modulate multiple targets of gut microbiota and metabolic toxins through the gut renal axis, thereby improving kidney function. However, the single enema treatment method is easy to cause symptoms of abdominal pain and diarrhea due to the violent property of the emodin, and the emodin has extremely poor water solubility, so that the emodin has low utilization rate in the administration route, and meanwhile, the retention time of the enema is short, frequent rectal administration is needed, and the frequent administration easily causes anal or rectal mucosa injury to patients.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a nano particle, wherein a liposome is used as a drug carrier to load emodin, so that the defects that simple emodin is poor in water solubility and the anus or rectal mucosa is easily damaged by frequent drug administration for a patient are overcome.
In order to achieve the purpose, the invention adopts the technical scheme that: providing a nanoparticle comprising a drug carrier on the outside of the particle and emodin on the inside of the particle; the drug carrier is liposome.
The application of the drug carrier in improving the utilization rate of the insoluble drug is common, wherein the liposome is a good drug carrier. The invention adopts liposome as a drug carrier to load emodin, and overcomes the defects that simple emodin has poor water solubility and is easy to cause anal or rectal mucosa injury to patients after frequent administration.
As a preferable embodiment of the nanoparticle of the invention, the liposome is DSPE-PEG-CS, and the DSPE-PEG-CS is obtained by connecting DSPE-PEG-COOH and chitosan.
Polyethylene glycol (PEG) is a high molecular material with good water solubility and dispersibility, various functional groups can be endowed by grafting modification, and a polymer with one hydrophilic end and one hydrophobic end can be formed after modification of the PEG by a hydrophobic compound Distearoylphosphatidylethanolamine (DSPE), so that the PEG becomes an excellent hydrophobic drug carrier.
Chitosan (CS), also known as 2-amino 2-deoxy- β -D-glucose, is a deacetylated product of chitin. Because the chitosan is nontoxic, has the characteristics of excellent biocompatibility, easy degradation in organisms and the like, and is widely used in the field of medical dressings. The invention links distearoyl phosphatidyl ethanolamine-polyethylene glycol DSPE-PEG-COOH with chitosan to obtain liposome DSPE-PEG-CS, and the loaded emodin has good biocompatibility, and the nano particle loaded with emodin has adhesive property, thus prolonging the detention time of the drug on mucosa, and avoiding the defect of easy anal or rectal mucosa injury to patients caused by frequent administration.
As a preferable embodiment of the nanoparticle of the invention, the DSPE-PEG-CS is obtained by condensation reaction of carboxyl of DSPE-PEG-COOH and amino on chitosan.
As a preferred embodiment of the nanoparticle of the present invention, the condensation reaction comprises the steps of:
s11, activating carboxyl on DSPE-PEG-COOH by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS;
s12, adding chitosan for reaction.
As a preferred embodiment of the nanoparticle of the present invention, the liposome thiolates chitosan on the basis of DSPE-PEG-CS.
Chitosan can form hydrogen bond and electrostatic action with mucoprotein, and has good biological adhesion performance, but the adhesion effect based on non-covalent bond can not ensure the sustained release of the drug at the designated position, and the application of chitosan is limited, and the adhesion performance of chitosan is obviously improved after sulfhydrylation, because sulfhydrylation polymer can form disulfide bond with the mucoprotein layer and is specifically combined with the abundant subregion of cysteine in mucoprotein.
In a preferred embodiment of the nanoparticle of the present invention, the thiolation is carried out by thiolating a hydroxyl group on the chitosan using 5-amino-2-mercaptobenzimidazole.
5-amino-2-Mercaptobenzimidazole (MBI) is an intermediate of ilaprazole serving as a medicine for treating the intestinal ulcer, so that the MBI can not only make chitosan sulfhydrylated, but also can play a certain treatment effect on the intestinal ulcer.
As a preferred embodiment of the nanoparticle of the present invention, the thiolation comprises the steps of:
s21, adding an oxidant to aldehyde the hydroxyl on the chitosan in the DSPE-PEG-CS, and then adding a terminator to terminate the reaction to obtain aldehyde distearoylphosphatidylethanolamine-polyethylene glycol-chitosan DSPE-PEG-CS-CHO;
s22, adding 5-amino-2-mercaptobenzimidazole MBI into DSPE-PEG-CS-CHO for incubation, and then adding sodium cyanoborohydride for reaction to obtain sulfhydrylated distearoylphosphatidylethanolamine-polyethylene glycol-chitosan DSPE-PEG-CS-MBI.
In a preferred embodiment of the nanoparticle of the present invention, the oxidizing agent is sodium periodate solution, and the terminating agent is ethylene glycol.
The chitosan structural formula contains a large number of hydroxyl groups, the hydroxyl groups generate aldehyde groups under the oxidation of sodium periodate serving as an oxidant, and when the hydroxyl groups are excessively oxidized, the hydroxyl groups are firstly changed into the aldehyde groups from the hydroxyl groups, and then the aldehyde groups are changed into carboxyl groups from the aldehyde groups, so that ethylene glycol is required to be added to stop the reaction so as to prevent the hydroxyl groups from being excessively oxidized.
The invention provides a preparation method of the nano-particle, which comprises the following steps:
s31, mixing the drug carrier and the emodin, dissolving in DMSO, performing ultrasonic treatment, dropwise adding the solution into deionized water while performing ultrasonic treatment by using a cell crushing instrument, performing ultrasonic treatment for 10-30 min after the dropwise adding is finished, and centrifuging to remove insoluble precipitates;
and S32, dialyzing the mixed solution by using deionized water, and freeze-drying the solution to obtain the nano particles.
The invention also provides application of the nano-particles in preparation of a medicine for treating chronic kidney diseases through enema.
The invention has the beneficial effects that:
(1) the DSPE-PEG-CS polymer is formed by distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH) and chitosan, and the polymer is sulfhydrylated by 5-amino-2-Mercaptobenzimidazole (MBI) on the basis to form sulfydryl polymer DSPE-PEG-CS-MBI, wherein the sulfydryl polymer can load hydrophobic drug emodin to be used as a slow release drug;
(2) the sulfhydrylation DSPE-PEG-CS-MBI drug carrier prepared by the invention has amphipathy, in a water phase, a hydrophobic end of the drug carrier can wrap a hydrophobic drug emodin to form an oil-in-water type nano micelle, and the solubility of the emodin is improved; meanwhile, sulfhydryl on DSPE-PEG-CS-MBI can be oxidized to form disulfide bond to adhere to the surface of mucous membrane, so as to prolong the detention time of the disulfide bond on the mucous membrane and facilitate the slow release of emodin.
Drawings
FIG. 1: the invention relates to a DSPE-PEG-CS-MBI infrared spectrogram.
FIG. 2: the invention relates to a DSPE-PEG-CS-MBI nuclear magnetic hydrogen spectrogram.
FIG. 3: an electron transmission electron microscope image of DSPE-PEG-CS-MBI/Emodin.
FIG. 4: the drug release curve of DSPE-PEG-CS-MBI/Emodin is disclosed.
FIG. 5: the result of the cytotoxicity test of Emodin and DSPE-PEG-CS-MBI/Emodin is shown in the figure.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
EXAMPLE 1 preparation of distearoylphosphatidylethanolamine-polyethylene glycol-chitosan (DSPE-PEG-CS)
Taking 100mg of DSPE-PEG-COOH (molecular weight 2000Da, purchased from Aladdin, product number D163587-100mg), dissolving the DSPE-PEG-COOH in 20ml of dimethyl sulfoxide (DMSO), weighing 8mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), adding the weighed solution into the solution, stirring for 15min, then weighing 12mg of N-hydroxysuccinimide, adding the weighed N-hydroxysuccinimide into the solution, stirring for 15min, slowly and dropwise adding the mixed solution into 60ml of DMSO containing 0.5g of chitosan, reacting for 6h, dialyzing the mixed solution for three days by using deionized water after the reaction is finished, freezing the solution at-80 ℃, and freeze-drying to obtain the DSPE-PEG-CS.
EXAMPLE 2 preparation of formylated distearoylphosphatidylethanolamine-polyethylene glycol-chitosan (DSPE-PEG-CS-CHO)
Weighing 50mg of DSPE-PEG-CS prepared in example 1, dissolving in 15ml of water, slowly dropwise adding a solution containing 30mg of sodium periodate under the condition of keeping out of the sun, reacting for 6 hours, dropwise adding 300 mu l of ethylene glycol to terminate the reaction, dialyzing the mixed solution for three days by using deionized water, freezing the solution at-80 ℃ after dialysis is finished, and freeze-drying to obtain DSPE-PEG-CS-CHO.
EXAMPLE 3 preparation of thiolated Distearoylphosphatidylethanolamine-polyethylene glycol-chitosan (DSPE-PEG-CS-MBI)
0.05g of 5-amino-2-Mercaptobenzimidazole (MBI) and 0.02g of DSPE-PEG-CS-CHO prepared in example 2 are weighed, dissolved in 40ml of DMSO, incubated at room temperature for 2h, then 0.02g of sodium cyanoborohydride is added, reaction is carried out at room temperature for 48h, the mixed solution is dialyzed by deionized water for three days, after dialysis is finished, the solution is frozen at-80 ℃, freeze-dried to obtain DSPE-PEG-CS-MBI, and the product is stored at 4 ℃.
Example 4 preparation of thiolated Distearoylphosphatidylethanolamine-polyethylene glycol-chitosan/Emodin (DSPE-PEG-CS-MBI/Emodin)
Weighing 0.02g of DSPE-PEG-CS-MBI and 0.01g of Emodin in the embodiment 3, mixing the two, dissolving in 5ml of DMSO, performing ultrasound for 15min, slowly dripping the solution into 40ml of deionized water, performing ultrasound while dripping the solution by using a cell crusher in the process, performing ultrasound for 15min after dripping is finished, centrifuging at 10000rpm to remove insoluble precipitates, dialyzing the mixed solution for three days by using the deionized water, performing dialysis, freezing the solution at-80 ℃, and performing freeze-drying to obtain the DSPE-PEG-CS-MBI/Emodin, wherein the product is stored at 4 ℃.
Example 5 DSPE-PEG-CS-MBI Performance assay
(1) Infrared testing
The DSPE-PEG-CS-MBI prepared in the example 3 is measured by a Fourier infrared spectrometer at 4000-399cm by a potassium bromide tabletting method-1Infrared spectrum in the range.
The results are shown in FIG. 1, in which 2881cm in the infrared spectrum-1Is PEG upper-CH21759cm, stretching vibration peak of C-H bond of (2)-11653cm of a stretching vibration peak at an amido bond C ═ O bond-1The peak is the stretching vibration absorption peak of C ═ N double bond formed by aldehyde group and amino group on DSPE-PEG-CS-CHO, 754cm-1And 700cm-1The compound is a bending vibration absorption peak outside a C-H bond surface on a benzene ring in a 5-amino-2-Mercaptobenzimidazole (MBI) structure, and the success of modification of the nano system can be proved.
(2) Nuclear magnetism
3-5 mg of DSPE-PEG-CS-MBI is weighed and dissolved in a proper amount of deuterated reagent (heavy water D)2O), then loading into a clean nuclear magnetic tube, and performing nuclear magnetic structure measurement by using a nuclear magnetic resonance spectrometer at room temperature.
The results are shown in FIG. 2, in which the nuclear magnetic hydrogen spectrum corresponds to absorption peaks of hydrogen on benzene ring in the structure of 5-amino-2-Mercaptobenzimidazole (MBI) at chemical shifts 7.08 and 7.5, and corresponds to absorption peaks of hydrogen on polyethylene glycol (PEG) at 3.65.
Example 6 DSPE-PEG-CS-MBI/Emodin Performance determination
(1) Transmission Electron Microscopy (TEM) test
The DSPE-PEG-CS-MBI/Emodin solution prepared in the embodiment 4 is filtered by a 0.22um filter membrane, the filtrate is dripped on a carbon supporting membrane of a copper net, and after the filtrate is naturally dried in the air, the carbon supporting membrane is placed in a high-resolution transmission electron microscope to observe the overall morphology and the particle size distribution of the nanoparticles.
As shown in FIG. 3, it can be seen from the transmission electron micrograph that the poorly soluble drug Emodin (black dots in the figure) is encapsulated by the liposome nanoparticle DSPE-PEG-CS-MBI, and the prepared DSPE-PEG-CS-MBI/Emodin has better dispersibility.
(2) Drug loading and nanoparticle encapsulation efficiency determination
The concentration of the emodin in the nanoparticle compound is measured by an ultraviolet spectrophotometer to calculate the drug loading rate and the nanoparticle encapsulation rate, and the calculation formula is as follows: the drug loading is the weight of the drug in the compound/the total weight of the compound; the encapsulation rate is the weight of the drug loaded in the nanomaterial/total drug dose.
The DSPE-PEG-CS-MBI/Emodin nanoparticle composite prepared in example 4 was measured to have an encapsulation rate of 86.3% and a drug loading of 4.11%.
(3) Sustained drug release
An appropriate amount of the DSPE-PEG-CS-MBI/Emodin nanoparticle complex prepared in example 4 was placed in a dialysis bag with a cut-off molecular weight of 10000, and the dialysis bag was placed in a 25mL test tube, added to a pH of 7.4, 15mL of pbs buffer, and ultrasonically dispersed to form a uniform nanoparticle suspension. The nanoparticle suspension was placed in a closed test tube, maintained at 37 ℃ in a constant temperature shaking incubator, shaken at 100rpm, sampled periodically, and supplemented with an equal amount of fresh buffer in the test tube, keeping the volume constant. And measuring absorbance at 436nm of the changed filtrate by using an ultraviolet spectrophotometer, calculating release amount, and drawing a cumulative release curve.
The result is shown in fig. 4, and it can be seen from the drug sustained release curve that in the first 12 hours, the release rate of the drug is relatively fast, the cumulative release amount is about 40%, in the later time, the emodin can still be slowly released from the nano system, and the final release amount can reach about 60%, indicating that the system has the sustained release function.
(4) In vitro cytotoxicity
The cytotoxicity of DSPE-PEG-CS-MBI/Emodin and Emodin alone on mouse fibroblasts (L929) was evaluated by a method for detecting cell activity by CCK-8. The specific operation steps are as follows: l929 cells were first seeded at a density of 5000 cells/well in 96-well plates and then placed in a carbon dioxide incubator overnight for adherence. Subsequently, the original medium was aspirated, and replaced with fresh complete medium containing DSPE-PEG-CS-MBI/Emodin and Emodin at different concentrations, selected in the range of 10-100. mu.g/ml, 5 replicates per concentration. The cells were then incubated in an incubator for 24h, after which the cells were washed once with PBS and 100. mu.l of fresh medium (containing 10% CCK-8) was added to each well. Placing the mixture in an incubator for incubation for a period of time, finally detecting and recording the absorbance at the wavelength of 450nm by using a microplate reader, and calculating the cell survival rate by the following formula:
cell survival (%) × (experimental absorbance-blank absorbance)/(negative control absorbance-blank absorbance) × 100%.
As shown in FIG. 5, it can be seen that the cell survival rate of the unencapsulated Emodin was about 80% at a concentration of 50. mu.g/ml, and that the cell survival rate was only about 55% at a concentration of 70. mu.g/ml, in which the Emodin exhibited a high cytotoxicity; the result that the cells still have about 80% survival rate when the concentration of the Emodin in the nano system is 70 mu g/ml for the nano system DSPE-PEG-CS-MBI/Emodin containing the same content of the Emodin shows that the Emodin has lower toxicity after being coated by the DSPE-PEG-CS-MBI compared with the Emodin not coated by the carrier.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A nanoparticle comprising a drug carrier on the outside of the particle and emodin on the inside of the particle; the drug carrier is liposome.
2. Nanoparticle according to claim 1, wherein said liposome is DSPE-PEG-CS, obtained by attaching DSPE-PEG-COOH to chitosan.
3. The nanoparticle as claimed in claim 2, wherein the DSPE-PEG-CS is obtained by condensation reaction of carboxyl group of DSPE-PEG-COOH and amino group of chitosan.
4. A nanoparticle according to claim 3, wherein the condensation reaction comprises the steps of:
s11, activating carboxyl on DSPE-PEG-COOH by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS;
s12, adding chitosan for reaction.
5. Nanoparticle according to claim 2, wherein said liposome comprises a DSPE-PEG-CS based thiolation of chitosan.
6. A nanoparticle according to claim 5, wherein said thiolation is achieved by thiolating a hydroxyl group on the chitosan using 5-amino-2-mercaptobenzimidazole.
7. A nanoparticle according to claim 6, wherein the thiolation comprises the steps of:
s21, adding an oxidant to aldehyde the hydroxyl on the chitosan in the DSPE-PEG-CS, and then adding a terminator to terminate the reaction to obtain aldehyde distearoylphosphatidylethanolamine-polyethylene glycol-chitosan DSPE-PEG-CS-CHO;
s22, adding 5-amino-2-mercaptobenzimidazole MBI into DSPE-PEG-CS-CHO for incubation, and then adding sodium cyanoborohydride for reaction to obtain sulfhydrylated distearoylphosphatidylethanolamine-polyethylene glycol-chitosan DSPE-PEG-CS-MBI.
8. A nanoparticle according to claim 7, wherein the oxidising agent is a sodium periodate solution and the terminating agent is ethylene glycol.
9. A method for preparing nanoparticles according to claim 1, characterized in that it comprises the following steps:
s31, mixing the drug carrier and the emodin, dissolving in DMSO, performing ultrasonic treatment, dropwise adding the solution into deionized water while performing ultrasonic treatment by using a cell crushing instrument, performing ultrasonic treatment for 10-30 min after the dropwise adding is finished, and centrifuging to remove insoluble precipitates;
and S32, dialyzing the mixed solution by using deionized water, and freeze-drying the solution to obtain the nano particles.
10. Use of a nanoparticle according to any one of claims 1 to 9 in the manufacture of a medicament for use in enema of chronic kidney disease.
CN202110233466.6A 2021-03-03 2021-03-03 Nano particles and application thereof Pending CN113069552A (en)

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