CN115192578B - Preparation of quercetin and nilamide mixed micelle - Google Patents
Preparation of quercetin and nilamide mixed micelle Download PDFInfo
- Publication number
- CN115192578B CN115192578B CN202210699802.0A CN202210699802A CN115192578B CN 115192578 B CN115192578 B CN 115192578B CN 202210699802 A CN202210699802 A CN 202210699802A CN 115192578 B CN115192578 B CN 115192578B
- Authority
- CN
- China
- Prior art keywords
- quercetin
- chitosan oligosaccharide
- gscq
- ntb
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- 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
- A61K9/08—Solutions
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to the field of biological medicine, and relates to a preparation method of a quercetin and nilamide mixed micelle, which comprises the following steps: the quercetin is used as a hydrophobic block material, the natural chitosan oligosaccharide is used as a hydrophilic block material, and the peptide-modified targeted esterase-sensitive nano-carrier is constructed. The invention constructs the multifunctional active targeting nano-carrier by means of a pharmaceutical preparation, and provides an effective solution for improving the bioavailability of the drug in eyes. The peptide-modified targeted esterase-sensitive nano-carrier is constructed by taking quercetin as a hydrophobic block material and taking positive charge adsorption and adhesiveness of natural chitosan oligosaccharide as a hydrophilic block material. The carrier can realize long retention and high compatibility of the preparation on the ocular surface, and synergistic delivery of quercetin and nidanib.
Description
Technical Field
The invention belongs to the field of biological medicine, and mainly relates to a preparation method of a quercetin and nilamide mixed micelle.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Efficient delivery of drugs to ocular targets is a very challenging task. In recent years, the delivery of deep foci of cornea in preclinical and clinical studies is mostly dependent on corneal stroma or subconjunctival injection, however, frequent injection is performed to give fear and pain to patients, and the adverse effects caused by irreversible mechanical injury are warned at any time. The local eye drops have the advantages of no wound, less adverse reaction and the like, and are the preferred way for treating the anterior segment lesion. However, the traditional eye drop strategy cannot change the pharmacokinetic characteristics of the monoclonal antibody and the small molecule tyrosine inhibitor, such as short residence time in eyes, poor tissue penetrability and low bioavailability. The focus of corneal neovascularization is located in the corneal stroma, and for drug delivery, it is first necessary to penetrate the dynamic hydrophilic tear film and the static lipophilic corneal epithelium, which requires that the formulation have both dissolution in the aqueous phase and penetration into the lipophilic epithelium. In fact, limited by the denser tight junction in the corneal epithelium, the pharmacokinetic results of some studies show that in non-invasive topical administration of the eye, the anterior segment delivery pathway of the nanoformulation through the corneal epithelium is even more difficult than posterior segment delivery of the conjunctiva-sclera-choroid. The polymer nano micelle has a hydrophobic block and a hydrophilic block, can realize self-assembly in water to form a stable structure for encapsulating the hydrophobic drug, and can be applied as eye drops by using clear aqueous solution, so that no interference is caused to vision.
Disclosure of Invention
In order to solve the problems, the invention constructs the multifunctional active targeting nano-carrier by means of a pharmaceutical preparation, and provides an effective solution for improving the bioavailability of the drug in eyes. The peptide-modified targeted esterase-sensitive nano-carrier is constructed by taking quercetin as a hydrophobic block material and taking positive charge adsorption and adhesiveness of natural chitosan oligosaccharide as a hydrophilic block material. The carrier can realize long retention and high compatibility of the preparation on the ocular surface, and synergistic delivery of quercetin and nidanib.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a quercetin and nilamide cloth loaded mixed micelle, which comprises the following steps:
the method is characterized in that quercetin is used as a hydrophobic block material, natural chitosan oligosaccharide is used as a hydrophilic block material, and a peptide-modified targeting esterase-sensitive nano-carrier is constructed, so that the peptide-modified targeting esterase-sensitive nano-carrier is obtained.
In a second aspect of the invention, a quercetin and nilamide cloth-loaded mixed micelle prepared by the method is provided.
In a third aspect, the invention provides an application of the quercetin-carried and nintedanib mixed micelle in preparation of a drug carrier or a drug delivery system.
In a fourth aspect, the invention provides an application of the quercetin and nilamide mixed micelle in preparing eye drops.
In a fifth aspect, the invention provides an application of the quercetin and nilamide mixed micelle in preparing a medicament for treating anterior ocular segment lesions.
In a sixth aspect of the invention, there is provided a blank drug carrier comprising: the quercetin and nilanib loaded mixed micelle.
In a seventh aspect of the invention, there is provided a pharmaceutical formulation or drug delivery system comprising: the quercetin and nilanib loaded mixed micelle.
The invention has the beneficial effects that:
(1) Corneal Neovascularization (CNV) is one of the main causes of serious impairment of vision. The formation of CNV can impair visual acuity and even lead to vision loss. Statistically, more than 150 ten thousand cases of corneal blindness are reported each year. The eye serves as a human visual organ and there are many specific physiological barriers and clearance mechanisms. These barriers result in short residence times after administration of conventional eye drops, poor penetration of intraocular tissue, and extremely low bioavailability, which presents a number of limitations for drug therapy. The invention constructs the multifunctional nano micelle by means of a pharmaceutical preparation so as to realize the synergistic and toxicity-reducing effects of cornea neovascularization treatment.
(2) The operation method is simple, has universality and is easy for large-scale production.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of the present application;
FIG. 2 is a synthetic scheme for a dipeptide modified chitosan oligosaccharide-quercetin polymer (GSCQ);
FIG. 3 is a graph of particle size and Zeta potential of GSCQ and GSCQ@NTB;
FIG. 4 is a diagram depicting the microscopic morphology of nanomicelle;
FIG. 5 is a graph showing the change in particle size distribution measured by the DLS method;
FIG. 6 is a graph showing cumulative release rates at various time points;
FIG. 7 is a graph of fluorescence tissue distribution and imaging of a sample;
FIG. 8 is a graph of drug concentration versus time;
FIG. 9 is an integrity chart of a slit lamp cobalt blue observation of the corneal epithelium;
FIG. 10 is a graph of histological changes of the cornea observed with Hematoxylin-Eosin (H & E) staining;
FIG. 11 is a representative graph of disease progression for each treatment group after induction of new blood vessels;
FIG. 12 is a disease scoring heatmap;
FIG. 13 is a graph showing the result of quantification of corneal neovascularization length;
fig. 14 is a graph showing quantification of corneal neovascularization area.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A preparation method of a quercetin and nilamide cloth loaded mixed micelle comprises the following steps:
carboxylating and modifying quercetin by succinic anhydride to obtain carboxylated quercetin;
reacting the carboxylated quercetin with chitosan oligosaccharide in the presence of a catalyst to obtain a chitosan oligosaccharide-quercetin conjugate CSQT;
under the existence of a catalyst, glycyl sarcosine of Fmoc-protected N-terminal amino is reacted with the chitosan oligosaccharide-quercetin conjugate CSQT, and Fmoc protecting groups are removed, so that the dipeptide modified chitosan oligosaccharide-quercetin polymer GSCQ is obtained.
In some embodiments, the mass ratio of quercetin to succinic anhydride is 3-5: 1.
in some embodiments, quercetin is carboxylated with succinic anhydride in a DMSO anhydrous system
In some embodiments, the catalyst comprises: edc·hcl, NHS.
In some embodiments, the mass ratio of quercetin to chitosan oligosaccharide is 2-5: 1.
in some embodiments, the reaction of carboxylated quercetin with chitosan oligosaccharide is performed in an inert gas atmosphere, protected from light, at room temperature.
In some embodiments, the mass ratio of quercetin to Fmoc-GS is from 0.8 to 1.2:1.
in some embodiments, the amide linkage of the Fmoc protecting group is selectively cleaved in a piperidine/dichloromethane solution.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
EXAMPLE 1 preparation of dipeptide modified Chitosan oligosaccharide-Quercetin Polymer
The aminoacyl sarcosine modified chitosan-quercetin polymer was synthesized by the following reaction. In the first step, carboxylic acid modification is carried out on quercetin by utilizing succinic anhydride under a DMSO anhydrous system, and the carboxylated quercetin is connected with amino groups of chitosan oligosaccharide under the catalysis of EDC/NHS to synthesize a chitosan oligosaccharide-quercetin Conjugate (CSQT). Secondly, utilizing amide reaction, and connecting the active carboxyl of glycyl sarcosine of Fmoc-protected N-terminal amino group with unreacted amino group of chitosan oligosaccharide (Fmoc-GSCQ) under EDC/NHS system; finally removing Fmoc protecting group to obtain the dipeptide modified chitosan oligosaccharide-quercetin polymer (GSCQ), and the synthetic route is shown in figure 2.
In the first step, 0.303g of quercetin and 0.131g of succinic anhydride which are dried to constant weight are precisely weighed and dissolved in 10mL of DMSO, and the quercetin is carboxylated (carboxy-QRT) by stirring for 24 hours at 600rpm in a constant temperature water bath at 50 ℃ under the protection of nitrogen and in the dark. 0.2875g of EDC. HCl and 0.1725g of NHS are respectively dissolved in 2mL of DMSO, and are respectively put into carboxy-QRT reaction solutions to be stirred for 30min so as to activate carboxyl groups;
0.12g of chitosan oligosaccharide was weighed into 15mL of deionized water, stirred to dissolve completely, and placed in an ice-water bath. Slowly dripping the carboxy-QRT reaction solution activated in the previous step into the chitosan oligosaccharide solution, and removing the ice bath after 10 min. The reaction was stirred at 600rpm for 24h under nitrogen protection, light protection and room temperature to give the crude product. The crude product was placed in a dialysis bag (mwco=2000 Da), dialyzed against DMSO: h2o=1:1 as dialysate for 2 days, and dialyzed against deionized water for 2 days to remove insoluble quercetin and other unreacted small molecules. Filtering the dialyzate by using a Buchner funnel, pre-freezing the filtrate in a refrigerator at the temperature of-80 ℃ for 12 hours, and freeze-drying the filtrate for 36 hours by using a freeze dryer to obtain a pale yellow solid, namely the product CSQT.
And secondly, weighing 0.3g of Fmoc-GS, dissolving in 30mL of deionized water, and stirring and dispersing uniformly. 0.46g EDC. HCl and 0.15g NHS were dissolved in 10mL deionized water, and added to Fmoc-GS reaction solution, respectively, and stirred for 30min to activate exposed carboxyl groups of Fmoc-GS.
0.15g of CSQT is weighed and dissolved in 30mL of deionized water, slowly added dropwise to Fmoc-GS reaction solution of activated carboxyl groups, and stirred at 600rpm for 24 hours under the conditions of nitrogen protection, light shielding and room temperature so as to enable Fmoc-GS to be connected to unreacted chitosan oligosaccharide amino groups. The resulting reaction solution was put into a dialysis bag (mwco=2000 Da) and dialyzed against deionized water for 2 days to remove unreacted small molecules. The liquid obtained after dialysis is placed at-80 ℃ for pre-freezing for 12 hours, and the solid product Fmoc-GSCQ is obtained after 36 hours of freeze-drying.
Third, the amide linkage of the Fmoc protecting group was selectively cleaved in a 20% piperidine/dichloromethane (v/v) solution. 50mgFmoc-GSCQ was weighed out and dissolved in a solvent prepared from 2mL of piperidine and 8mL of methylene chloride. The obtained solution is stirred for 2 hours under the protection of nitrogen to remove Fmoc protecting groups, after dichloromethane is removed by rotary evaporation, 10mL of deionized water is used for redissolution, 1M HCl is added dropwise to adjust the pH of the solution to 7.0, dialysis bags (MWCO=2000 Da) are used for dialysis on the deionized water for 2 days, residual organic reagents and small molecules are removed, and the final product GSCQ polymer is obtained after freeze drying.
EXAMPLE 2 preparation of blank Polymer nanoparticles
The blank nano micelle is prepared by adopting a film dispersion method. 5mg of GSCQ polymer was weighed and dispersed in 3mL of methanol, and stirred for 4 hours to uniformly disperse. The sample was placed in a rotary evaporator and methanol was distilled off at 40℃and 80rpm to form a uniform film of polymer on the walls of the eggplant-shaped bottle. And placing the eggplant-shaped bottle in a normal temperature vacuum drying oven overnight, adding 10mL of deionized water after overnight, placing in a water bath ultrasonic instrument, carrying out ultrasonic hydration at 37 ℃ for 40min, and filtering the ultrasonic sample through a 0.8 mu m water system microporous filter membrane to obtain the GSCQ polymer nano micelle solution.
Example 3 preparation of Nidani-cloth-loaded Polymer nanomicelle
The drug-loaded nano micelle (GSCQ@NTB) is prepared by adopting a film dispersion method. 5mg of GSCQ polymer is weighed and dispersed in 2mL of methanol, stirred for 0.5h to be uniformly dispersed, then 1mL of NTB methanol solution with different concentrations of NTB is dripped, and stirring is continued for more than 4 h. The sample was placed in a rotary evaporator and methanol was distilled off at 40℃and 80rpm to form a uniform film of polymer on the walls of the eggplant-shaped bottle. The eggplant-shaped bottle is placed in a normal temperature vacuum drying oven overnight, 10mL of deionized water is added after overnight, and the eggplant-shaped bottle is placed in a water bath ultrasonic instrument for ultrasonic hydration at 37 ℃ for 40min. Centrifuging the ultrasonic sample at 6000rpm for 10min to remove the non-entrapped solid medicine, and filtering the sample through a 0.8 mu m water system microporous filter membrane to obtain GSCQ@NTB polymer nano micelle solution.
And (3) placing the GSCQ@NTB polymer micelle solution in a pre-frozen state at the temperature of-80 ℃ for 12 hours, and freeze-drying for 36 hours to obtain GSCQ@NTB solid powder.
In order to increase the drug loading of the nano-micelle, the change trend of the drug loading and encapsulation efficiency of the nano-micelle was evaluated by screening different feeding ratios of NTB and GSCQ polymers, and the results are shown in Table 1. With the increase of the NTB feeding amount, the drug loading amount tends to rise. When the ratio of NTB to GSCQ is increased from 0.5:10 to 1.5:10, the drug loading rate is increased from (4.43+/-0.16)% to (8.67+/-0.20)%, which shows that the nano drug loading system has good drug loading capacity. However, when the ratio of NTB to GSCQ is increased from 1:10 to 1.5:10, the encapsulation efficiency is obviously reduced from (89.0+/-2.09)% to (63.3+/-1.60)%, and the obvious reduction of the encapsulation efficiency is probably caused by that the feeding amount of the NTB exceeds the loading capacity of the polymer. Therefore, the optimum ratio of 1:10 was selected, and the drug loading was (8.17.+ -. 0.18)%, and the encapsulation efficiency was (89.0.+ -. 2.09)%.
Table 1 effect of different amounts of nidanib on GSCQ nanomicelle drug loading and encapsulation efficiency (n=3)
Particle size and potential: the prepared nano micelle solution is diluted to proper multiples, the particle sizes and Zeta potentials of GSCQ and GSCQ@NTB are respectively examined, and the particle size distribution and Zeta potential of the representative nano micelle are shown in figure 3, so that the result shows that the micelle particle size distribution is uniform and the stability is good.
Microcosmic morphology: the prepared blank micelle and drug-loaded micelle are diluted to proper concentration by deionized water, dripped on a copper mesh coated with a carbon film, and 2% (w/v) of phosphotungstic acid solution is dripped for sample negative dyeing, and the mixture is dried and placed under a transmission electron microscope to characterize the microscopic morphology of the nano micelle, as shown in fig. 4 (scale bar=0.3 μm). The result shows that the polymer self-assembles to form nano-micelle with uniform sphere, uniform particle size, good dispersibility and no obvious aggregation.
Esterase sensitivity examination: to examine the change in the particle size distribution of the nano-micelle in the presence of esterase to verify its esterase sensitivity. Taking a certain amount of blank GSCQ nano micelle solution, respectively adding 20U/mL pig liver esterase, placing in a constant temperature oscillator for incubation for 1h and 4h under the condition of 37 ℃ and 100rpm, and measuring the change of the particle size distribution by a DLS method, wherein the change is shown in figure 5.
Experimental example 1 in vitro release behavior investigation of Nidamanib Polymer-loaded nanomicelle
The release behavior of nilanib from nanomicelle was examined using dynamic membrane dialysis. The study was divided into three groups, release medium containing 0.2% tween 80 in PBS buffer at ph=7.4. 1mL of methanol solution in which 40 μg of NTB free drug is dissolved is added into a first group of dialysis bags; 1mL of GSCQ@NTB micelle solution containing 40 mug NTB is added into a second group of dialysis bags; to investigate esterase sensitivity, 1mL of gscq@ntb micelle solution, loaded with 40 μg NTB, was added to the third set of dialysis bags, and it contained pig liver esterase at a concentration of 20U/mL. The solutions were placed in dialysis bags with a molecular weight cut-off of 2kDa, respectively, and the dialysis bags were fastened to the opening and placed in centrifuge tubes containing 8mL of release medium. The centrifuge tube was incubated at 37℃with shaking in a thermostatic water bath shaker at 100rpm, 0.5mL of release medium was sampled at the corresponding time points, and the centrifuge tube was replenished with an equal volume of fresh medium. The medium sample was removed and passed through a 0.22 μm microporous filter membrane, the NTB content was measured by HPLC, and the cumulative release rate at various time points was calculated as shown in FIG. 6.
Experimental example 2 intraocular distribution fluorescence visualization experiment
In order to more intuitively observe and compare the transport pathway of the nano-micelle in the eye tissue, the nano-micelle was labeled with C6 as a fluorescent probe. The drugs and formulations were studied for their in-ocular transport metabolism, divided into free C6 and gscq@c6 groups. Before the experiment, the New Zealand rabbits are balanced for three days in a constant light environment around the clock, 8 rabbits are fixed, 50 mu L of free C6 with the concentration of 250 mu g/mL is dripped into the right conjunctival sac of the rabbits, 50 mu L of GSCQ@C6 eye drops with equal dose of C6 are dripped into the left conjunctival sac, eyelids are slightly pulled up and down to ensure that the eye drops are uniformly dispersed on the ocular surface, and the rabbits are allowed to freely move after the fixation is released for 15 min.
Rabbits were sacrificed 15, 30, 60, 120, 240, 360, 480, 720min after administration, and eyeballs were dissected and rinsed with 20mL of physiological saline. Tissue specimens were frozen at-80 ℃, cut into 16 frozen sections and nuclei stained with DAPI. The fluorescent tissue distribution of the sample was observed under a confocal microscope and imaged as shown in fig. 7.
The 12h internal corner-fluorescence distribution of free C6 and gscq@c6 groups is shown in fig. 6. A stronger fluorescence profile can be observed for the nano-formulation group than for the free drug group. The GSCQ group started to be efficiently taken up by the corneal epithelial cells at 30min and started to penetrate the corneal epithelium into the corneal stroma, uniformly distributed in the stroma and partially reaching the corneal endothelial layer at 1h, followed by a period of up to 8h, where fluorescence remained until 12h disappeared. Whereas little fluorescence resides in the epithelial and matrix layers of the free C6 group, only a few fluorescence occurs in the matrix layer within 2-4 hours.
Experimental example 3 determination of the residual concentration of corneal drug
New Zealand rabbits were equilibrated in a constant light environment around the clock for three days prior to the experiment, which was performed in parallel with 3 groups. 8 rabbits are fixed in each group, 50 mu L of free NTB with the concentration of 250 mu g/mL is dripped into right conjunctival sac of the rabbits, 50 mu L of GSCQ@NTB eye drops with the same dose of medicine are dripped into left conjunctival sac, eyelids are gently pulled up and down to enable the eye drops to be uniformly dispersed on the eye surface, and the fixation is released after 15min to allow the rabbits to freely move.
Rabbits were sacrificed 15, 30, 60, 120, 240, 360, 480, 720min after administration, the ocular surface was rinsed with 10mL of physiological saline, and water was removed by dipping with a sterile dry cotton ball. The rabbit eyeballs were removed and corneal tissue was dissected under a stereoscopic microscope and placed in an EP tube. Precisely weighing, cutting to 1-2mm, and soaking in 400 μl methanol. Sealing, soaking at 4deg.C for 24 hr to obtain extract, centrifuging at 10000rpm for 10min, collecting supernatant, detecting by HPLC, and calculating NTB concentration in cornea extract.
The bioavailability degree and the bioavailability speed of the GSCQ@NTB micelle of the tested preparation in the cornea are evaluated by drawing a drug concentration-time curve by taking the free NTB as a reference preparation, and the area under the drug concentration-time curve is calculated by using GraphPad Prism 9 to obtain the relative bioavailability of the GSCQ@NTB micelle.
Relative bioavailability (F) R ) The calculation is performed as follows:
neutralization AUC NTB And AUC GSCQ@ The areas under the drug concentration-time curves of the reference preparation and the test preparation, respectively, D NTB And D GSCQ@ Respectively reference preparationsDosage of the test formulation.
HPLC measured the trend of NTB drug-time concentration change in the corneal leachate after rabbit cornea administration, and the drug concentration-time curve is shown in fig. 8. The AUC of the free NTB is 446.7 ±21.56, the AUC of the gscq@ntb preparation is 3431±120.6, and the relative bioavailability of gscq@ntb is 768.1% by the area under the drug-time concentration curve, which is far higher than that of the free NTB group. Wherein, the drug concentration of free NTB and GSCQ@NTB reaches Cmax at 1h, cmax of GSCQ@NTB is 3.66 times of that of NTB group, and the concentration of GSCQ@NTB group is 13.5 times higher than that of free group at 4h along with rapid metabolism of free drug in eyes, which is consistent with the result of fluorescence distribution in eyes.
Experimental example 4 eye surface compatibility test of Rabbit
Irritation and biocompatibility of gscq@ntb drug-loaded micelles in rabbit eyes (n=3) were evaluated by modified Draize test: mu.L of the drug-loaded micelle solution (250. Mu.g/mL) was instilled into conjunctival sac of right eye of rabbit, and 50. Mu.L of 0.9% physiological saline was instilled into left eye of rabbit as a control. The administration was fixed twice daily in the morning and evening for 21 days. 1d,7d,14d and 21d after the start of administration, the ophthalmic sodium fluorescein staining test paper was placed on the lower eyelid of the rabbit for 3min and then taken out, and the integrity of the corneal epithelium was observed by cobalt blue light of a slit lamp, as shown in fig. 9; the histological changes of the cornea were observed by taking each group of cornea at 21d and staining with Hematoxylin-Eosin (H & E) after tissue section as shown in fig. 10.
Irritation of the gscq@ntb micelle formulation to the rabbit ocular surface was verified by 21 days of continuous dosing. The result of fluorescein sodium staining proves that the rabbit cornea is not obviously damaged after the GSCQ@NTB micelle preparation is continuously administered for 21 days. H & E staining results show that after the GSCQ@NTB group is dosed, rabbit cornea epithelial cells are closely and uniformly arranged, compared with a normal saline group, no obvious thickness change exists, the matrix layer structure is complete, and fibrous tissue hyperplasia and inflammatory cell infiltration phenomena are avoided. In addition, no obvious complications such as edema and inflammation appear in eyes after 21 days of slit lamp observation, which shows that GSCQ@NTB micelle has low irritation and meets the use standard of the ophthalmic preparation.
Experimental example 5 QuercusSynergistic delivery pharmacodynamic studies of Succinum and Nidamib
The rabbit cornea neovascular disease model is constructed as follows: general anesthesia was induced by the marginal intravenous injection of 10% chloral hydrate at an anesthetic dose of 2mL/kg. After the general anesthesia is successful, the eye speculum is fixed on an operating table, the left eyelid is opened by an eyelid speculum, 5% povidone iodine is dripped into a conjunctival sac for sterilization, and after the eyelid is slightly pulled up and down to enable eye drops to stay on the ocular surface for 1min, the conjunctival sac is flushed with 50mL of physiological saline. Excess water in conjunctival sac was wiped off with sterile cotton swab, and then 50 μl of obukaine hydrochloride eye drops were instilled in the conjunctival sac of the operation eye to relieve pain with local anesthesia. Soaking sterilized blank medicated paper sheet with diameter of 8.0mm in 1 mol.L concentration with sterile camera -1 After taking out, slightly throwing away the redundant alkali liquor, placing the filter paper sheet at the tangential position of the left eye and the limbus of the rabbit, taking down after placing for 40s, immediately flushing the operation eye with 300mL of physiological saline for 60s, and taking down the eyelid speculum.
Taking the obvious complications such as observed invasion of new blood vessels into limbus growth and no infection, perforation and the like as the standard of successful modeling, 25 experimental New Zealand rabbits are randomly divided into a physiological saline group, a GSCQ blank micelle group, an NTB free drug group, a dexamethasone sodium phosphate group and a GSCQ@NTB drug carrying micelle group, wherein 5 rabbits are marked and fed in separate cages. The medicine is administrated twice a day in the morning and at the evening, 50 mu L of the medicine is administrated each time, the administration concentration is 250 mu g/mL, blank micelle is administrated in the concentration of medicine carrying micelle and the like, and the levofloxacin eye drops are conventionally used for eye dropping to prevent surgical eye infection.
On days 4, 9 and 14, the rabbit surgical eyes were scored with a hand-held slit lamp, with the following scoring criteria:
corneal haze: 0 = completely transparent; 1 = slightly blurred and easy to see iris and pupil; 2 = slightly opaque, but iris and pupil can be observed; 3 = opaque, almost no pupil visible; 4 = completely opaque, no pupil was observed.
Length of the new blood vessel: 0 = no new blood vessel; 1 = there is a new blood vessel at the limbus; 2 = new blood vessel crossing the limbus and approaching the central location; 3 = neovascularization across the center of the cornea.
Vascular lumen assessment: 0 = no new blood vessel; 1 = new blood vessels need to be observed with a hand-held slit lamp; 2 = new blood vessel is easily seen with a hand held slit lamp; new blood vessels can be observed in the case of 3=no slit lamp.
Quantification of the corneal neovascularization is calculated through Image-Pro Plus software, and after a scale and a CNV range are defined, the software can automatically calculate the length of the blood vessel; the area of the new blood vessel is treated in an auxiliary way through Image-Pro Plus software, and the quantitative treatment is carried out by calculating the fan-shaped area of the blood vessel growth, wherein the formula is as follows:
where S is defined as the CNV area, C is defined as the proportion of the sector covered by the neovascularization after 12 aliquots of corneal circumference are averaged, L is defined as the length of the longest vessel from the limbus, and R is defined as the corneal radius.
Representative images of disease progression for each treatment group after induction of neovascularization are shown in fig. 11, disease scoring heatmaps are shown in fig. 12, quantification of corneal neovascularization length results are shown in fig. 13, and quantification of corneal neovascularization area is shown in fig. 14.
As shown in fig. 12, 5 rabbits in each group were labeled a-E, and total scores of corneal neovascularization disease progression were recorded for 4, 9, and 14 days, with different colors corresponding to the right-most legend. As can be seen from fig. 13 and 14, the length and area of the neovasculature of each of the NTB group, DEX group and gscq@ntb group were significantly reduced as compared to the normal saline group; compared with the DEX group, the cornea neovascularization area of the gscq@ntb group is further reduced, the blood vessel length is further significantly reduced, and the clinical disease total score is significantly reduced, which indicates that the gscq@ntb group can significantly inhibit the growth of the neovascularization. This is due to the fact that the GSCQ@NTB group enhances the retention of the ocular surface and the uptake of cornea tissues, so that the preparation has deeper and more uniform distribution in the cornea stroma layer, and further proves that the combined application of quercetin and Nidamib has better inhibition effect on the disease progression of cornea neovascularization and has certain pharmacodynamics advantage.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for preparing a dipeptide modified chitosan oligosaccharide-quercetin polymer, which is characterized by comprising the following steps:
carboxylating and modifying quercetin by succinic anhydride to obtain carboxylated quercetin;
reacting the carboxylated quercetin with chitosan oligosaccharide in the presence of a catalyst to obtain a chitosan oligosaccharide-quercetin conjugate CSQT;
under the condition of the existence of a catalyst, fmoc-GS and the chitosan oligosaccharide-quercetin conjugate CSQT are reacted, fmoc protecting groups are removed, and the dipeptide modified chitosan oligosaccharide-quercetin polymer GSCQ is obtained;
the mass ratio of the quercetin to the succinic anhydride is 3-5: 1, a step of;
the mass ratio of the quercetin to the chitosan oligosaccharide is 2-5: 1, a step of;
the mass ratio of the quercetin to the Fmoc-GS is 0.8-1.2: 1.
2. the method for preparing the dipeptide modified chitosan oligosaccharide-quercetin polymer according to claim 1, wherein the quercetin is subjected to carboxylation modification by using succinic anhydride in a DMSO anhydrous system.
3. The method for preparing a dipeptide modified chitosan oligosaccharide-quercetin polymer according to claim 1, wherein the catalyst comprises: edc·hcl, NHS.
4. The method for preparing a dipeptide modified chitosan oligosaccharide-quercetin polymer according to claim 1, wherein the reaction of carboxylated quercetin and chitosan oligosaccharide is carried out in inert gas atmosphere under the conditions of light shielding and room temperature.
5. The method for preparing a dipeptide modified chitosan oligosaccharide-quercetin polymer according to claim 1, wherein the amide ester bond of the Fmoc protecting group is cleaved in a piperidine/dichloromethane solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699802.0A CN115192578B (en) | 2022-06-20 | 2022-06-20 | Preparation of quercetin and nilamide mixed micelle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699802.0A CN115192578B (en) | 2022-06-20 | 2022-06-20 | Preparation of quercetin and nilamide mixed micelle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115192578A CN115192578A (en) | 2022-10-18 |
| CN115192578B true CN115192578B (en) | 2023-05-30 |
Family
ID=83575627
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210699802.0A Active CN115192578B (en) | 2022-06-20 | 2022-06-20 | Preparation of quercetin and nilamide mixed micelle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115192578B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982168A (en) * | 2010-11-02 | 2011-03-02 | 山东大学 | Quercetin nano-micelle preparation and preparation method thereof |
| CN104324384A (en) * | 2014-10-24 | 2015-02-04 | 山东大学 | Hyaluronic acid-quercetin conjugate self-assembly micelle preparation and preparation method thereof |
| CN106038485A (en) * | 2016-06-30 | 2016-10-26 | 中国海洋大学 | Preparation method of nano-micelles with mucous layer permeation and P-gp inhibition dual effects |
| CN106727309A (en) * | 2016-12-20 | 2017-05-31 | 广州中大南沙科技创新产业园有限公司 | Polymer micelle solution containing Quercetin and its preparation method and application |
| CN110302391A (en) * | 2019-07-01 | 2019-10-08 | 大连民族大学 | A kind of glucan-Quercetin polymer medicament carrying micelle preparation and preparation method thereof |
| CN112138001A (en) * | 2020-09-25 | 2020-12-29 | 山东大学 | Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220000838A1 (en) * | 2020-07-04 | 2022-01-06 | Lebrun Labs LLC | Formulations and methods related to eye irritation |
-
2022
- 2022-06-20 CN CN202210699802.0A patent/CN115192578B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982168A (en) * | 2010-11-02 | 2011-03-02 | 山东大学 | Quercetin nano-micelle preparation and preparation method thereof |
| CN104324384A (en) * | 2014-10-24 | 2015-02-04 | 山东大学 | Hyaluronic acid-quercetin conjugate self-assembly micelle preparation and preparation method thereof |
| CN106038485A (en) * | 2016-06-30 | 2016-10-26 | 中国海洋大学 | Preparation method of nano-micelles with mucous layer permeation and P-gp inhibition dual effects |
| CN106727309A (en) * | 2016-12-20 | 2017-05-31 | 广州中大南沙科技创新产业园有限公司 | Polymer micelle solution containing Quercetin and its preparation method and application |
| CN110302391A (en) * | 2019-07-01 | 2019-10-08 | 大连民族大学 | A kind of glucan-Quercetin polymer medicament carrying micelle preparation and preparation method thereof |
| CN112138001A (en) * | 2020-09-25 | 2020-12-29 | 山东大学 | Quercetin-low molecular weight heparin-paclitaxel conjugate, preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115192578A (en) | 2022-10-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200390692A1 (en) | Apparatus and formulations for suprachoroidal drug delivery | |
| Zhang et al. | Sustained intravitreal delivery of dexamethasone using an injectable and biodegradable thermogel | |
| CN1318093C (en) | Injections for eye tissue containing drug bound to polyethylene glycol | |
| CN108883207A (en) | Tree-shaped body-biological adhesive polymer hydrogel nanometer glue and its purposes | |
| WO2020103958A1 (en) | Nanocrystalline eye drop, preparation method and use thereof | |
| JP2014129408A (en) | Novel biomaterials for ocular drug delivery and method for making and using the same | |
| EA030318B1 (en) | Non-linear multiblock copolymer-drug conjugates for the delivery of active agents | |
| JP2014510724A (en) | Thermoresponsive hydrogel composition | |
| JP2010519183A (en) | Polymerization using protein precipitation for elution of physiological solutions | |
| CN106794152A (en) | For preventing the nano particle for being loaded with glucocorticoid that corneal allograft repels and new vessels is formed | |
| Wu et al. | Cell penetrating peptide TAT-functionalized liposomes for efficient ophthalmic delivery of flurbiprofen: Penetration and its underlying mechanism, retention, anti-inflammation and biocompatibility | |
| CN110693831B (en) | Preparation method of long-ocular-surface retention and high-corneal-permeability drug-loaded nano-micelle | |
| JPH04364855A (en) | Coated lens in eye and coating composition therefor | |
| Li et al. | Sustaining intravitreal residence with L-arginine peptide-conjugated nanocarriers | |
| CN115192578B (en) | Preparation of quercetin and nilamide mixed micelle | |
| WO2020050779A1 (en) | Hydrogels with tunable electrostatic properties | |
| CN113797162B (en) | An ophthalmic preparation for treating macular edema, optic neuritis and non-infectious endophthalmitis by eye drop administration | |
| Schaefer et al. | Evaluation of Intracameral Pentablock copolymer Thermosensitive gel for sustained drug delivery to the anterior chamber of the eye | |
| US20230081539A1 (en) | Suprachoroidal delivery of drug particles to reduce toxicity | |
| WO2022156373A1 (en) | Ophthalmic preparation for treating macular edema, optic neuritis and non-infectious endophthalmitis through eye drop administration | |
| CN114831930B (en) | Collagen assembled eye drops, preparation method and application | |
| Ramesh | Ocular barriers and ocular drug delivery: Bridging the gap using nanomicelles as drug carriers | |
| Junnuthula et al. | Intravitreal Polymeric Nanocarriers with Long Ocular Retention and Targeted Delivery to the Retina and Optic Nerve Head Region. Pharmaceutics 2021, 13, 445 | |
| CN117752617A (en) | Application of amphiphilic cationic substance modified mPEG-PCL nanoparticle in preparation of medicament for treating retinal diseases | |
| Wang et al. | An active transport dual adaptive nanocarrier designed to overcome the corneal microenvironment for neovascularization therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |