CN115192578A - Preparation of mixed micelle carrying quercetin and nintedanib - Google Patents
Preparation of mixed micelle carrying quercetin and nintedanib Download PDFInfo
- Publication number
- CN115192578A CN115192578A CN202210699802.0A CN202210699802A CN115192578A CN 115192578 A CN115192578 A CN 115192578A CN 202210699802 A CN202210699802 A CN 202210699802A CN 115192578 A CN115192578 A CN 115192578A
- Authority
- CN
- China
- Prior art keywords
- quercetin
- nintedanib
- chitosan oligosaccharide
- mixed micelle
- 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.)
- Granted
Links
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- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 48
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 title claims abstract description 48
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 48
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- XZXHXSATPCNXJR-ZIADKAODSA-N nintedanib Chemical compound O=C1NC2=CC(C(=O)OC)=CC=C2\C1=C(C=1C=CC=CC=1)\NC(C=C1)=CC=C1N(C)C(=O)CN1CCN(C)CC1 XZXHXSATPCNXJR-ZIADKAODSA-N 0.000 title claims abstract description 27
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Images
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- A61P27/02—Ophthalmic agents
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Abstract
The invention belongs to the field of biological medicine, and relates to a preparation method of a mixed micelle carrying quercetin and nintedanib, which comprises the following steps: the peptide-modified targeted esterase sensitivity nano-carrier is constructed by taking quercetin as a hydrophobic block material and natural chitosan oligosaccharide as a hydrophilic block material. The invention constructs the multifunctional active targeting nano-carrier by means of pharmaceutical preparation, and provides an effective solution for improving the bioavailability of the drug in eyes. Quercetin is used as a hydrophobic block material, and the positive charge adsorbability and the adhesiveness of natural chitosan oligosaccharide are used as hydrophilic block materials to construct the peptide modified targeted esterase sensitivity nano-carrier. The carrier can realize long retention and high compatibility of the preparation on the ocular surface and the synergistic delivery of the quercetin and the nintedanib.
Description
Technical Field
The invention belongs to the field of biological medicine, and mainly relates to preparation of a mixed micelle carrying quercetin and nintedanib.
Background
The information disclosed in this background section is only for enhancement of 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 that is already known to a person 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 focus corneal disease in preclinical and clinical studies is mostly dependent on corneal stroma or subconjunctival injection, however, the fear and pain of patients caused by frequent injection and the adverse consequences caused by irreversible mechanical injury caused by frequent vigilance are brought. The local eye drops have the advantages of no wound, less adverse reaction and the like, and are the first choice for treating anterior segment diseases. However, the traditional eye-dropping 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 focal sites of corneal neovascularization reside in the corneal stroma, which for drug delivery first requires penetration of both the dynamic, hydrophilic tear film and the static, lipophilic corneal epithelium, requiring formulations that have both solubility in the aqueous phase and penetration of the lipophilic epithelial layer. Indeed, limited by the denser tight junctions in the corneal epithelium, the pharmacokinetic results of some research efforts show that penetration of the nanoparticies through the anterior segment delivery pathway of the corneal epithelium is even more difficult than the posterior segment delivery of the conjunctiva-sclera-choroid in ocular non-invasive topical administration. The polymer nano micelle has a hydrophobic block and a hydrophilic block, can realize self-assembly in water to form a stable structure to encapsulate a hydrophobic drug, and is applied as eye drops by using a clear aqueous solution, so that no interference is caused to vision.
Disclosure of Invention
In order to solve the problems, the multifunctional active targeting nano-carrier is constructed by a pharmaceutical preparation means, so that an effective solution is provided for improving the bioavailability of the drug in the eyes. Quercetin is used as a hydrophobic block material, and the positive charge adsorbability and the adhesiveness of natural chitosan oligosaccharide are used as hydrophilic block materials to construct the peptide modified targeted esterase sensitivity nano-carrier. The carrier can realize long retention and high compatibility of the preparation on the ocular surface and the cooperative delivery of quercetin and nintedanib.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for preparing a mixed micelle carrying quercetin and nintedanib, comprising:
and (2) constructing the peptide-modified targeted esterase sensitivity nano-carrier by using the quercetin as a hydrophobic block material and the natural chitosan oligosaccharide as a hydrophilic block material.
In a second aspect of the present invention, there is provided a quercetin and nintedanib loaded mixed micelle prepared by the above method.
In a third aspect of the present invention, there is provided an application of the above-mentioned quercetin and nintedanib-loaded mixed micelle in the preparation of a drug carrier or a drug delivery system.
In a fourth aspect of the present invention, there is provided an application of the above-mentioned quercetin and nintedanib-loaded mixed micelle in the preparation of eye drops.
In a fifth aspect of the invention, the application of the quercetin and nintedanib carrying mixed micelle in preparing a medicament for treating anterior segment ocular segment pathological changes is provided.
In a sixth aspect of the present invention, there is provided a blank pharmaceutical carrier comprising: the mixed micelle carrying quercetin and nintedanib.
In a seventh aspect of the invention, there is provided a pharmaceutical formulation or drug delivery system comprising: the mixed micelle carrying quercetin and nintedanib.
The invention has the beneficial effects that:
(1) Corneal Neovascularization (CNV) is one of the major causes of severe visual impairment. The formation of CNV can impair visual sensitivity and even lead to vision loss. Statistically, over 150 ten thousand cases of corneal blindness are reported each year. The eye acts as a human ocular organ and there are many specific physiological barriers and clearance mechanisms. These barriers result in short retention time after administration of conventional eye drops, poor intraocular tissue penetration, and extremely low bioavailability, which imposes limitations on drug therapy. The invention constructs the multifunctional nano micelle by means of a pharmaceutical preparation so as to realize the synergistic attenuation effect of the corneal neovascularization treatment.
(2) The operation method is simple, universal and easy for large-scale production.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the present application;
FIG. 2 is a synthesis scheme of modified chitooligosaccharide-quercetin polymer (GSCQ) of dipeptide;
FIG. 3 is a graph of particle size and Zeta potential of GSCQ and GSCQ @ NTB;
FIG. 4 is a representation of the microscopic morphology of the nanomicelles;
FIG. 5 is a graph showing changes in particle size distribution measured by the DLS method;
FIG. 6 is a graph of cumulative release rates at various time points;
FIG. 7 is a graph of fluorescent tissue distribution and imaging of a sample;
FIG. 8 is a drug concentration-time curve;
FIG. 9 is a graph of corneal epithelium integrity as observed by slit-lamp cobalt blue light;
FIG. 10 is a diagram showing histological changes of cornea observed by Hematoxylin-Eosin (H & E) staining;
FIG. 11 is a representative plot of disease progression for each treatment group after induction of neovasculature;
fig. 12 is a disease score heatmap;
FIG. 13 is a graph showing the results of quantifying the corneal neovascularization length;
fig. 14 is a diagram for quantifying the area of corneal neovascularization.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 mixed micelle carrying quercetin and nintedanib comprises the following steps:
performing carboxylation modification on quercetin by adopting 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;
reacting glycyl sarcosine with Fmoc protected N-terminal amino with the chitosan oligosaccharide-quercetin conjugate CSQT in the presence of a catalyst, and removing an Fmoc protecting group to obtain a dipeptide modified chitosan oligosaccharide-quercetin polymer GSCQ.
In some embodiments, the mass ratio of quercetin to succinic anhydride is 3-5: 1.
in some embodiments, the carboxylation modification of quercetin 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 under inert gas atmosphere, in the absence of light, at room temperature.
In some embodiments, the mass ratio of quercetin to Fmoc-GS is 0.8 to 1.2:1.
in some embodiments, the amide ester bond of the Fmoc protecting group is selectively cleaved in piperidine/dichloromethane solution.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
EXAMPLE 1 preparation of dipeptide-modified Chitosan oligosaccharide-Quercetin Polymer
The amidosarcosine-modified chitosan oligosaccharide-quercetin polymer was synthesized by the following reaction. Firstly, performing carboxylation modification on quercetin by using succinic anhydride in a DMSO anhydrous system, and connecting the carboxylated quercetin with amino of chitosan oligosaccharide under the catalysis of EDC/NHS to synthesize a chitosan oligosaccharide-quercetin Conjugate (CSQT). Secondly, connecting the active carboxyl of glycinyl sarcosine with Fmoc protection N-terminal amino with the unreacted amino of chitosan oligosaccharide (Fmoc-GSCQ) by using amide reaction in an EDC/NHS system; finally, removing Fmoc protecting group to obtain dipeptide modified chitooligosaccharide-quercetin polymer (GSCQ), and the synthetic route is shown in figure 2.
Firstly, 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 stirred for 24 hours at 600rpm in a constant-temperature water bath at 50 ℃ under the conditions of nitrogen protection and light shielding to carry out carboxylation (carboxy-QRT). Dissolving 0.2875g EDC & HCl and 0.1725g NHS in 2mL DMSO respectively, adding into the carboxy-QRT reaction solution respectively, and stirring for 30min to activate carboxyl;
0.12g of chitosan oligosaccharide was weighed and dissolved in 15mL of deionized water, stirred to be completely dissolved, and placed in an ice-water bath. The carbon-QRT reaction solution activated in the previous step was slowly dropped into the chitosan oligosaccharide solution, and the ice bath was removed after 10 min. Stirring and reacting at 600rpm for 24h under the conditions of nitrogen protection, light protection and room temperature to obtain a crude product. The crude product was put into dialysis bags (MWCO =2000 Da), dialyzed against DMSO: H2O =1 for 2 days, and then dialyzed against deionized water for 2 days, thereby removing insoluble quercetin and other unreacted small molecules. Filtering the dialysate by using a Buchner funnel, pre-freezing the filtrate in a refrigerator at-80 ℃ for 12h, and freeze-drying for 36h by using a freeze dryer to obtain a light yellow solid, namely the product CSQT.
Secondly, 0.3g of Fmoc-GS is weighed and dissolved in 30mL of deionized water to be uniformly stirred and dispersed. 0.46g of EDC & HCl and 0.15g of NHS were dissolved in 10mL of deionized water, and the solutions were poured into the Fmoc-GS reaction solution, and stirred for 30min to activate the exposed carboxyl group of Fmoc-GS.
Weighing 0.15g of CSQT, dissolving in 30mL of deionized water, slowly dropwise adding the solution into the Fmoc-GS reaction solution with activated carboxyl, and stirring at 600rpm for 24 hours under the conditions of nitrogen protection, light protection and room temperature to connect Fmoc-GS to unreacted chitosan oligosaccharide amino. 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. And pre-freezing the liquid obtained after dialysis at-80 ℃ for 12h, and freeze-drying for 36h to obtain a solid product Fmoc-GSCQ.
In the third step, the amide ester bond of Fmoc protecting group was cleaved selectively in 20% piperidine/dichloromethane (v/v) solution. 50mg of Fmoc-GSCQ was dissolved in a solvent prepared from 2mL of piperidine and 8mL of dichloromethane. Stirring the obtained solution under the protection of nitrogen for 2h to remove the Fmoc protecting group, performing rotary evaporation to remove dichloromethane, redissolving with 10mL of deionized water, dropwise adding 1M HCl to adjust the pH of the solution to 7.0, dialyzing the deionized water for 2 days by a dialysis bag (MWCO =2000 Da), removing residual organic reagents and micromolecules, and performing freeze drying to obtain the final product GSCQ polymer.
Example 2 preparation of blank Polymer nanoparticles
The blank nano micelle is prepared by a film dispersion method. 5mg of GSCQ polymer was weighed out and dispersed in 3mL of methanol, and stirred for 4 hours for uniform dispersion. The sample was placed in a rotary evaporator and methanol was evaporated at 40 ℃ and 80rpm to form a uniform film on the wall of the eggplant-shaped bottle. Placing the eggplant-shaped bottle in a normal-temperature vacuum drying oven overnight, adding 10mL of deionized water after the overnight, placing the bottle in a water-bath ultrasonic instrument for ultrasonic hydration for 40min at 37 ℃, and filtering an ultrasonic sample through a 0.8-micron water-based microporous filter membrane to obtain the GSCQ polymer nano micelle solution.
Example 3 preparation of Nydanib-loaded Polymer nanomicelles
The drug-loaded nano micelle (GSCQ @ NTB) is prepared by a film dispersion method. 5mg of GSCQ polymer is weighed and dispersed in 2mL of methanol, and is stirred for 0.5h to be uniformly dispersed, and then 1mL of NTB methanol solution with different concentrations of NTB is dripped, and the stirring is continued for more than 4 h. The sample is placed in a rotary evaporator, and methanol is evaporated at 40 ℃ and 80rpm, so that the polymer forms a uniform coating on the wall of the eggplant-shaped bottle. Placing the eggplant-shaped bottle in a vacuum drying oven at normal temperature overnight, adding 10mL of deionized water after the overnight, and placing the bottle in a water bath ultrasonic instrument for ultrasonic hydration for 40min at 37 ℃. Centrifuging the sample at 6000rpm for 10min to remove the non-entrapped solid drug, and filtering with 0.8 μm water system microporous membrane to obtain GSCQ @ NTB polymer nano micelle solution.
Placing the GSCQ @ NTB polymer micelle solution at-80 deg.C for pre-freezing for 12h, and lyophilizing for 36h to obtain GSCQ @ NTB solid powder.
In order to improve the drug loading of the nano-micelle, the variation trend of the drug loading and the encapsulation efficiency of the nano-micelle is evaluated by screening different charge ratios of NTB and GSCQ polymers, and the result is shown in Table 1. Along with the increase of the NTB feeding amount, the medicine loading amount shows a rising trend. When the ratio of NTB to GSCQ is increased from 0.5 to 10 to 1.5, the drug loading is increased from (4.43 +/-0.16)% to (8.67 +/-0.20)%, which indicates that the nano drug-loading system has good drug loading capacity. However, when the ratio of NTB to GSCQ is increased from 1 to 10 to 1.5, the encapsulation efficiency of the system is significantly reduced from (89.0 ± 2.09)% to (63.3 ± 1.60)%, which may result from the amount of NTB charged exceeding the loading capacity of the polymer. Therefore, the optimal feeding proportion is selected when the ratio is 1.
TABLE 1 Effect of different Nintedanib dosages on GSCQ nanomicelle drug loading and encapsulation efficiency (n = 3)
Particle size and potential: the nano micelle solution obtained by preparation is diluted to a proper multiple, the particle size and Zeta potential of GSCQ and GSCQ @ NTB are respectively inspected, and figure 3 shows representative nano micelle particle size distribution and Zeta potential, and the result shows that the micelle particle size distribution is uniform and the stability is better.
Microscopic morphology: taking the prepared blank micelle and the drug-loaded micelle, diluting the blank micelle and the drug-loaded micelle to a proper concentration by deionized water, dropwise adding the diluted blank micelle and the drug-loaded micelle to a copper net coated with a carbon film, dropwise adding 2% (w/v) of phosphotungstic acid solution for sample negative dyeing, airing, and then placing under a transmission electron microscope to characterize the microscopic morphology of the nano micelle, wherein the microscopic morphology of the nano micelle is shown in figure 4 (a scale bar =0.3 μm). The result shows that the polymer self-assembles to form the nano micelle with uniform spherical shape, uniform particle size, good dispersibility and no obvious aggregation.
Examination of esterase sensitivity: in order to examine the change of the particle size distribution of the nano-micelle in the presence of esterase, the sensitivity of the esterase is verified. Taking a certain amount of blank GSCQ nano micelle solution, respectively adding 20U/mL pig liver esterase, placing in a constant temperature oscillator at 37 ℃ and 100rpm for incubation for 1h and 4h, and determining 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 examination of Nanomicelle carrying Nintedanib Polymer
The release behavior of nintedanib from the nanocolloid was investigated using dynamic membrane dialysis. The study was divided into three groups, the release medium containing 0.2% tween 80 in PBS buffer pH = 7.4. Adding 1mL of methanol solution dissolved with 40 mu g of NTB free medicine into the first group of dialysis bags; adding 1mL of GSCQ @ NTB micelle solution loaded with 40 μ g of NTB into the second group of dialysis bags; to examine esterase sensitivity, 1mL of GSCQ @ NTB micelle solution loaded with 40. Mu.g of NTB and containing pig liver esterase at a concentration of 20U/mL was added to the third group of dialysis bags. The solutions were placed in dialysis bags with molecular weight cut-off of 2kDa, and the dialysis bag openings were tightened and placed in centrifuge tubes containing 8mL release medium. The centrifuge tubes were incubated with shaking in a constant temperature water bath shaker at 37 ℃ and 100rpm, 0.5mL of release medium was sampled at the corresponding time points, and an equal volume of fresh medium was added to the centrifuge tubes. The medium sample solution is filtered through a 0.22 μm microporous filter membrane, NTB content is measured by HPLC, and the cumulative release rate at different time points is calculated as shown in figure 6.
Experimental example 2 visualization experiment of distribution of fluorescence in eyes
In order to more intuitively observe and contrast the transport pathway of nanomicelles in ocular tissues, the nanomicelles were labeled using C6 as a fluorescent probe. The method is divided into a free C6 group and a GSCQ @ C6 group, and the intraocular transport metabolism condition of the medicament and the preparation is researched. Before the experiment, the New Zealand rabbit is balanced for three days in a constant light environment around the clock, 8 rabbits are fixed, 50 muL of free C6 with the concentration of 250 mug/mL is dripped into the right conjunctival sac of the rabbit, 50 muL of GSCQ @ C6 eye drops with the loading equal dosage of C6 is dripped into the left conjunctival sac of the rabbit, eyelids are pulled slightly up and down to ensure that the eye drops are uniformly dispersed on the ocular surface, and the fixation is released after 15min to allow the rabbit to freely move.
Rabbits were sacrificed at 15, 30, 60, 120, 240, 360, 480, and 720min after administration, and the eyeballs were dissected and washed with 20mL of physiological saline. Tissue specimens were frozen at-80 ℃, cut into 16 frozen sections and stained for nuclei with DAPI. The fluorescence tissue distribution of the sample was observed under confocal microscope and imaged as shown in fig. 7.
The 12h internal corneal fluorescence distributions for the free C6 group and the GSCQ @ C6 group are shown in FIG. 6. A stronger fluorescence distribution of the nanopreparation group than that of the free drug group was observed. The GSCQ group started to be efficiently taken up by corneal epithelial cells at 30min and started to penetrate the corneal epithelium into the corneal stroma, was uniformly distributed in the stroma at 1h and partially reached the corneal endothelial layer, and then, until 8h, fluorescence remained in the stroma until 12h disappeared. Whereas little fluorescence is retained in the epithelial and stromal layers of the free C6 group, and little fluorescence appears in the stromal layers within 2-4 h.
Experimental example 3 measurement of corneal drug Retention concentration
The new zealand rabbits were equilibrated for three days in a day-night constant light environment before the experiment, which was measured in parallel with 3 groups. Each group fixes 8 rabbits, 50 muL of free NTB with the concentration of 250 mug/mL is dripped into the right conjunctival sac of the rabbit, 50 muL of GSCQ @ NTB eye drops carrying medicines with equal dosage is dripped into the left conjunctival sac, eyelids are pulled slightly up and down to keep the eye drops evenly dispersed on the ocular surface, and after 15min, the fixation is released to allow the rabbits to freely move.
Rabbits were sacrificed at 15, 30, 60, 120, 240, 360, 480, and 720min after administration, and the ocular surface was rinsed with 10mL of physiological saline and dehydrated with a sterile dry cotton ball. Rabbit eyeballs are removed, corneal tissue is dissected under a stereoscopic microscope and placed in an EP tube. Precisely weighing, cutting into pieces with side length of about 1-2mm, and soaking in 400 μ L methanol. Sealing, soaking at 4 deg.C for 24 hr to obtain leachate, centrifuging at 10000rpm for 10min, collecting leachate supernatant, detecting by HPLC, and calculating NTB concentration in corneal leachate.
And (3) evaluating the bioavailability degree and bioavailability speed of the test preparation GSCQ @ NTB micelle in the cornea by drawing a drug concentration-time curve by taking free NTB as a reference preparation, and calculating the area under the drug concentration-time curve by GraphPad Prism 9 to obtain the relative bioavailability of the GSCQ @ NTB micelle.
Relative bioavailability (F) R ) Calculated as follows:
formula (I) neutralized AUC NTB And AUC GSCQ@ Area under the drug concentration-time curves, D, for the reference and test formulations, respectively NTB And D GSCQ@ The reference and test formulations are administered at the same dose.
The trend of NTB drug-time concentration changes in corneal leachate after rabbit corneal drug administration was determined by HPLC, and the drug concentration-time curve is shown in fig. 8. Within 12h of local administration, the AUC of free NTB is 446.7 +/-21.56, the AUC of the GSCQ @ NTB preparation is 3431 +/-120.6, and the relative bioavailability of the GSCQ @ NTB is 768.1 percent which is calculated by the area under the drug-time concentration curve and is far higher than that of the free NTB group. Wherein the drug concentration of free NTB and GSCQ @ NTB reached Cmax at 1h, cmax of GSCQ @ NTB was 3.66 times that of NTB group, and the concentration of GSCQ @ NTB group was 13.5 times higher than that of free group at 4h as the free drug was rapidly metabolized in the eye, which is consistent with the results of intraocular fluorescence distribution.
Experimental example 4 rabbit ocular surface compatibility test
The stimulation and biocompatibility of gscq @ ntb drug loaded micelles in rabbit eye (n = 3) was evaluated by a modified Draize test: mu.L of the drug-loaded micelle solution (250. Mu.g/mL) was dripped into the conjunctival sac of the right eye of the rabbit, and 50. Mu.L of 0.9% physiological saline was dripped into the left eye of the rabbit as a control. The medicine is administered twice a day in the morning and evening, and is administered for 21 days. After the beginning of administration, 1d,7d,14d and 21d, the ophthalmic sodium fluorescein staining test paper is placed on the lower eyelid of the rabbit for 3min and then taken out, and the integrity of the corneal epithelium is observed by slit lamp cobalt blue light, as shown in fig. 9; each group of corneas was dissected at 21d, and histological changes of the corneas were observed by Hematoxylin-Eosin (H & E) staining after tissue sectioning as shown in FIG. 10.
The irritancy of the rabbit ocular surface by the gscq @ ntb micelle formulation was verified by continuous administration for 21 days. The result of fluorescein sodium staining proves that no obvious injury is seen on the rabbit cornea after 21 days of continuous administration of the GSCQ @ NTB micelle preparation. H & E staining results show that after the administration of the GSCQ @ NTB group is finished, the rabbit corneal epithelial cells are arranged tightly and uniformly, the thickness of the rabbit corneal epithelial cells is not changed obviously compared with that of a normal saline group, the matrix layer structure is intact, and the phenomena of fibroplasia and inflammatory cell infiltration are avoided. In addition, under 21-day slit lamp observation, no obvious complication such as edema and inflammation appears in eyes, which indicates that the stimulation of the GSCQ @ NTB micelle is low and meets the use standard of the ophthalmic preparation.
Experimental example 5 pharmacodynamic study of synergistic delivery of Quercetin and Nintedanib
The rabbit cornea neovascular disease model is constructed as follows: general anesthesia was induced by intravenous injection of 10% chloral hydrate into the ear margin at a dose of 2mL/kg. Fixing on operating table after successful general anesthesia, opening left eyelid with eye speculum, dripping 5% povidone iodine into conjunctival sac for disinfection, gently pulling up and down eyelid to make eye drops stay on eye surface for 1min, and washing conjunctival sac with 50mL physiological saline. The excess water in the conjunctival sac is wiped off by a sterile cotton stick, and then 50 mu L of oxybuprocaine hydrochloride eye drops are dripped into the conjunctival sac of the operation eye to relieve local anesthesia and pain. Soaking sterilized blank drug sensitive paper with diameter of 8.0mm in sterile forceps at concentration of 1 mol.L -1 Taking out the filter paper from the sodium hydroxide solution for 5 seconds, slightly throwing off excessive alkali liquor, placing the filter paper at the tangent position of the left eye of the rabbit and the corneal limbus, taking down the filter paper after placing for 40 seconds, immediately flushing the operative eye with 300mL of physiological saline for 60 seconds, and taking down the eyelid retractor.
Taking the observation of the invasion of new blood vessels into the corneal limbus for growth and no obvious complications such as infection, perforation and the like as the standard for successful modeling, 25 experimental New Zealand rabbits are randomly divided into a normal saline group, a GSCQ blank micelle group, an NTB free drug group, a dexamethasone sodium phosphate group and a GSCQ @ NTB drug-loaded micelle group, each group is divided into 5, marked and raised in cages. The medicine is administrated twice a day in the morning and evening, each time is 50 mu L, the administration concentration is 250 mu g/mL, blank micelles are administrated in the concentrations of medicine-carrying micelles, and levofloxacin eye drops are used for eye drop instillation to prevent surgical eye infection.
On days 4, 9 and 14, the rabbit eyes were examined for scores using a hand-held slit lamp, with the following criteria:
corneal haze: 0= completely transparent; 1= slightly blurred and easy to see iris and pupil; 2= slightly opaque, but the iris and pupil can be observed; 3= opaque, hardly visible pupil; 4= completely opaque, no pupil was observed.
Length of new blood vessel: 0= no new blood vessels; 1= neovascularization at the limbus; 2= neovascularization across the limbus and near the central location; 3= neovasculature crosses the corneal center.
Evaluation of vascular lumen: 0= no new blood vessels; 1= a hand-held slit lamp is required to observe the new blood vessels; 2= new blood vessels are easily seen with a hand-held slit lamp; 3= neovascularisation was observed without slit lamp.
The quantification of the corneal neovascularization is calculated by Image-Pro Plus software, and after the range of a proportional scale and a CNV is defined, the software can automatically calculate the length of the blood vessel; the area of the new blood vessel is processed in an Image-Pro Plus software auxiliary mode, and the area of the sector of the blood vessel growth is calculated to be processed in a quantification mode, wherein the formula is as follows:
where S is defined as the area of CNV, C is defined as the sector proportion covered by neovasculature after 12 equal portions of corneal circumference are equally divided, L is defined as the length of the longest blood vessel from the corneal 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 heatmap is shown in fig. 12, quantification of corneal neovascularization length is shown in fig. 13, and quantification of corneal neovascularization area is shown in fig. 14.
As shown in fig. 12, 5 rabbits per group were labeled a-E, and total scores for corneal neovascular disease progression were recorded at 4, 9, and 14 days, with different colors corresponding to the rightmost legend. As can be seen from fig. 13 and 14, the neovascular length and area were significantly reduced in the NTB group, DEX group, and gscq @ NTB group, as compared to the saline group; compared with the DEX group, the area of the corneal neovascularization area of the GSCQ @ NTB group is further reduced, the length of the blood vessels is further reduced remarkably, and the total clinical disease score is reduced remarkably, which indicates that the GSCQ @ NTB group can remarkably inhibit the growth of the neovascularization. The reason is that the GSCQ @ NTB group enhances the retention of the ocular surface and the uptake of corneal tissues, so that the preparation has deeper and more uniform distribution on a corneal stroma layer, and further proves that the combined application of quercetin and nintedanib has better inhibition effect on the disease progression of corneal neovascularization and has certain pharmacodynamic advantages.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a mixed micelle carrying quercetin and nintedanib is characterized by comprising the following steps:
performing carboxylation modification on quercetin by adopting 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;
reacting glycyl sarcosine with Fmoc protected N-terminal amino with the chitosan oligosaccharide-quercetin conjugate CSQT in the presence of a catalyst, and removing an Fmoc protecting group to obtain a dipeptide modified chitosan oligosaccharide-quercetin polymer GSCQ.
2. The method for preparing the quercetin and nintedanib-loaded mixed micelle according to claim 1, wherein the mass ratio of quercetin to succinic anhydride is 3-5: 1.
3. the method of claim 1, wherein the quercetin and nintedanib-loaded mixed micelle is carboxylated and modified by succinic anhydride in a DMSO anhydrous system.
4. The method of claim 1, wherein the catalyst comprises: EDC & HCl, NHS.
5. The method for preparing the quercetin and nintedanib-loaded mixed micelle according to claim 1, wherein the mass ratio of the quercetin to the chitosan oligosaccharide is 2-5: 1.
6. the method for preparing the quercetin and nintedanib-loaded mixed micelle according to claim 1, wherein the reaction between the carboxylated quercetin and the chitosan oligosaccharide is performed under inert gas atmosphere, in the absence of light, and at room temperature.
7. The method of claim 1, wherein the mass ratio of quercetin to Fmoc-GS is 0.8-1.2: 1.
8. the method for preparing quercetin and nintedanib-loaded mixed micelles according to claim 1, wherein the amide ester bond of the Fmoc protecting group is selectively cleaved in piperidine/dichloromethane solution.
9. The quercetin and nintedanib loaded mixed micelle prepared by the method according to any one of claims 1-8.
10. The quercetin and nintedanib mixed micelle of claim 9 for use in the preparation of a pharmaceutical carrier, a drug delivery system, an eye drop or a medicament for the treatment of anterior segment ocular lesions.
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| US20220000838A1 (en) * | 2020-07-04 | 2022-01-06 | Lebrun Labs LLC | Formulations and methods related to eye irritation |
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