CN113577030A - Preparation method of micro-fluidic technology-based drug microcarrier for acquired deafness - Google Patents
Preparation method of micro-fluidic technology-based drug microcarrier for acquired deafness Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
- A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0046—Ear
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1658—Proteins, e.g. albumin, gelatin
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- 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
- A61P27/16—Otologicals
Abstract
The invention relates to a preparation method of a micro-carrier for acquired deafness based on a micro-fluidic technology, wherein the micro-fluidic technology is utilized to control the generation of an internal and external two-phase fluid, and the generation process of the internal and external two-phase fluid comprises the following steps: s1: preparing micron-scale liquid drops for tympanum injection by utilizing shearing force and interfacial tension between two immiscible phase fluids, and carrying hearing protective drugs and auxiliary agents for increasing the permeability of a round window membrane in situ; s2: the droplets are solidified by polymerization to form the drug microcarriers. Compared with the prior art, the drug microcarrier prepared in the invention can be used for treating acquired deafness by tympanogram injection, wherein the drug microcarrier is filled in the middle ear cavity by tympanogram injection, and the drug is slowly released by natural degradation of the microcarrier and enters the inner ear through the round window membrane, so that the effect of treating acquired deafness is achieved, and the drug microcarrier has the advantages of small side effect and damage, lasting effect, obvious effect, simplicity and easiness in operation and the like.
Description
Technical Field
The invention relates to the field of biological materials and conversion medicine, in particular to a preparation method of a micro-carrier of a medicine aiming at acquired deafness based on a micro-fluidic technology.
Background
Tympanogram injection is one of the commonly used treatments for acquired deafness. The tympanum injection can improve the local drug concentration of the inner ear, improve the drug absorption rate, reduce the side effect of protective drugs such as dexamethasone and the like on the whole body, and reduce the influence of the drugs on the anti-tumor property of cisplatin. Has unique advantages and is a safe and effective treatment mode.
Traditional tympanogram injections commonly used drugs such as dexamethasone, triamcinolone acetonide, etc. are injected into the middle ear cavity, however, the drugs are easily lost through the eustachian tube. Due to the short residence time of the drug in the middle ear cavity, it is difficult to cross the round window membrane into the inner ear resulting in low drug benefit. In addition, drugs lost through the eustachian tube are often absorbed by the human body through the digestive tract, thereby causing major side effects. To solve the problem of drug loss, the head is kept in a special position for a long time, and the method has poor patient compliance. In addition, the risk of perforation of the tympanic membrane and infection of the middle ear is increased by repeated tympanometry injections.
Thus, the traditional method of directly injecting the drug through the tympanic cavity limits the efficacy of the drug to treat hearing loss.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for preparing a micro-carrier of a drug aiming at acquired deafness based on a micro-fluidic technology, the micro-carrier of the drug which is coated in situ is prepared by controlling a two-phase fluid by the micro-fluidic technology, the micro-carrier can be applied to tympanum injection, the micro-carrier of the drug is filled in a middle ear cavity, the drug is slowly released through the natural degradation of the micro-carrier and enters the inner ear through a round window membrane, and the effect of treating the acquired deafness is achieved.
The purpose of the invention can be realized by the following technical scheme:
the technical scheme aims to protect a preparation method of a micro-carrier of a medicine aiming at acquired deafness based on a micro-fluidic technology, the preparation method is to control the generation of an internal and external two-phase fluid by utilizing the micro-fluidic technology, and the generation process of the internal and external two-phase fluid comprises the following steps:
s1: preparing micron-scale liquid drops by utilizing shearing force and interfacial tension between two immiscible phases, and carrying hearing protective drugs and auxiliary agents for increasing the permeability of the round window membrane in situ;
s2: the droplets are solidified by polymerization to form the drug microcarriers.
Further, the step of S1 includes:
s1-1: preparation of emulsion template
Constructing a coaxial co-flow type micro-fluidic chip, preparing a corresponding polymerizable high-molecular prepolymer solution according to the hydrophilic and hydrophobic properties of the encapsulated hearing protection drug and the adjuvant, dissolving the drug and the adjuvant in the polymerizable high-molecular prepolymer solution, injecting immiscible internal and external phase solutions through internal and external phase channels of the micro-fluidic chip, and forming monodisperse emulsion droplets by utilizing the shear force and surface tension between the two phase solutions;
s1-2: droplet parameter regulation
Emulsion droplets with different sizes are obtained through the size and the shape of channels of all parts of the microfluidic chip, the flow velocity, the viscosity and the like of a continuous phase and a disperse phase.
Further, the step of S2 includes:
based on the physicochemical properties of the polymerizable high-molecular prepolymer solution in step S1-1, ultraviolet light or heat is used to induce a high-molecular crosslinking reaction, so that emulsion droplets are cured to obtain a drug microcarrier containing a protective drug and an auxiliary agent, the residual solvent is removed, and the drug microcarrier is dried and stored in a container.
Furthermore, the micro-fluidic chip is a three-dimensional coaxial internal and external co-flow micro-channel made of glass capillary tubes.
Furthermore, the pipe diameter of the tip of an inner phase glass tube in the microfluidic chip is less than 50 μm, and the pipe diameter of a collecting tube is less than 300 μm;
the flow velocity range of the inner phase of the micro-fluidic chip is 0.1-0.2ml/h, the flow velocity range of the outer phase is 5-10ml/h, and the flow velocity ratio of the inner phase to the outer phase is controlled to be 1: 500-1: within 1000, the inner phase fluid is broken into micro-droplets by means of high-speed narrow-jet (narrow-jet) jet.
Further, the main body material of the inner phase fluid in S1 is a prepolymer solution of one or more materials of polylactic-co-glycolic acid (PLGA), methacrylate gelatin (Gel-MA), sodium alginate, etc.
Further, in S1, the external phase fluid is a component that is biologically safe and immiscible with the internal phase fluid.
When the inner phase fluid is aqueous solution, the outer phase fluid is one or more natural oils selected from corn oil, sunflower oil, castor oil, etc., and is mixed with surfactant such as SPAN 80.
Further, the hearing protective drug in S1 is one or more of dexamethasone, triamcinolone acetonide, alpha lipoic acid and N-acetyl-L-cysteine.
Further, the adjuvant for increasing the permeability of the round window membrane in the S1 is one or more of benzyl alcohol, histamine, saponin and caprate.
Further, the drug microcarrier prepared in S2 has a diameter of 1-50 μm, and the protective drug and adjuvant can be slowly released by slow degradation of the material, see fig. 2.
Compared with the prior art, the invention has the following technical advantages:
(1) the invention designs a micro-fluidic chip, which enables inner-phase fluid to be broken into micro-droplets in a high-speed narrow-jet (narrow jetting) jet mode under the action of high viscosity force by changing the sizes of an inner-phase channel and a collecting pipe and the flow rates of inner-phase fluid and outer-phase fluid, so as to form drug-loaded microspheres with the particle size of less than 50 mu m for tympanogram injection.
(2) Compared with the traditional preparation method, the method for preparing the drug microcarrier for tympanogram injection in a microfluidic mode is controllable in size, high in encapsulation efficiency and drug loading, high in biological safety and wide in selectable range of material carriers.
(3) The drug microcarrier prepared by microfluidics can be applied to drugs for treating acquired deafness by tympanogram injection, a degradable material with high biological safety is used as a main body of the microcarrier, and a protective drug and an auxiliary agent are slowly released along with natural degradation of the material, so that long-term administration and drug slow release are realized, the times of tympanum puncture are reduced, the effect of one-time injection long-term protection is realized, and the risks of permanent perforation and infection of the tympanum can be reduced.
(4) The drug microcarrier prepared by microfluidics can be applied to the process of treating acquired deafness by tympanogram injection, and in the application process, the hearing protective drug is slowly released, the eustachian tube loss is less, and the side effect of the whole body is small; in the specific application process, the permeability of the round window membrane is increased, the inner ear part can maintain higher drug concentration, and the drug treatment effect is obviously improved.
(5) The micro-fluidic drug carrier can be applied to the process of treating acquired deafness by tympanogram injection, so as to treat deafness and sudden deafness caused by gentamicin, cisplatin, noise and the like.
(6) The drug microcarrier prepared by microfluidics can be applied to the process of treating acquired deafness by tympanogram injection, wherein the microcarrier has less influence on the inner ear caused by sound wave transmitted by the middle ear compared with the traditional hydrogel.
(7) The micro-fluidic prepared drug micro-carrier can be applied to a method for treating acquired deafness by tympanogram injection, the main material of the drug micro-carrier does not enter the inner ear, the material can be naturally degraded, the degraded product is slowly discharged through the eustachian tube, and no obvious toxic or side effect is caused to the inner ear.
Drawings
FIG. 1 is a schematic flow diagram of the fabrication of drug microcarriers by microfluidic chips;
FIG. 2 is a schematic diagram showing the degradation of dexamethasone sodium phosphate-gelatin methacrylate (Dexsp-GelMA) drug microcarriers;
FIG. 3 is a schematic representation of the injection of a drug microcarrier-sodium chloride solution into the middle ear cavity of a guinea pig by tympanogram injection, where the protective effect of the drug microcarrier on hearing was verified by the guinea pig;
FIG. 4 is a structure and particle size distribution diagram of dexamethasone sodium phosphate-gelatin methacrylate (Dexsp-GelMA) after curing under an optical microscope.
Detailed Description
In consideration of research and development conception, different from direct injection, the micro-carrier material is used for encapsulating the drug, so that the directional delivery and the sustained release of the drug can be realized, the rapid loss of the drug is avoided, and the utilization rate of the drug is improved. The traditional methods for preparing the drug microcarrier mainly comprise a mechanical stirring method, an emulsion solvent volatilization method, a spray drying method and the like, but the microcarrier prepared by the traditional method has uneven size distribution, uneven particle size, poor monodispersity and poor encapsulation rate; and the traditional microcapsule causes the burst release of the drug after the shell is degraded, and the slow release effect is not good.
The micro-fluidic technology adopted by the invention forms liquid drops through the mutual shearing action and the surface tension of mutually insoluble solutions of various phases, can wrap one or more medicines with different properties, and has the advantages of wide material and medicine selection range, high medicine encapsulation rate and good slow release performance. More importantly, the microcarrier prepared by the micro-fluidic technology has controllable size, controllable structure and good monodispersity. The invention can prepare a degradable microcarrier loaded with hearing protective drugs and auxiliary agents with good biocompatibility by utilizing a microfluidic technology, and protects hearing loss by injecting the drug microcarrier into the middle ear cavity through the tympanic cavity. The advantages that the material is naturally degraded, the permeability of the round window membrane is improved, and the hearing protective drug can be continuously released in the middle ear cavity, so that the purpose of continuously treating hearing loss can be achieved by single injection. In specific application, the treatment time window and treatment indications of the drug microcarriers can be changed by changing the types of the microcarrier materials and the protective drugs, so that the treatment effect is optimized.
When the drug microcarrier is specifically applied, the hearing protective drug is slowly released through natural degradation of the microcarrier, and meanwhile, the auxiliary agent is used to increase the permeability of the round window membrane and improve the effect of the hearing protective drug entering the inner ear.
When the drug microcarrier is used specifically, a 2ml syringe and a No. 6 flat-mouth needle with the length of 60mm are used for carrying out tympanum puncture in the upper front quadrant of the tympanum and injecting the drug microcarrier into the middle ear cavity.
When the drug microcarrier is specifically applied, the indications of the drug microcarrier mainly comprise hearing loss and sudden deafness caused by gentamicin, cisplatin and noise.
When the drug microcarrier is specifically applied, the defects that the traditional drug is directly injected into the middle ear cavity through the tympanic cavity, the retention time is short, the traditional drug cannot easily and rapidly cross the round window membrane barrier, the drug is easy to flow to the digestive tract through the eustachian tube, and the side effect is large can be solved, and the defects that the drug microcarriers prepared by other methods are poor in uniformity, poor in slow release performance, difficult to inject through the tympanic cavity through an injector and the like can be overcome.
Specifically, the drug microcarrier is characterized in that the particle size of the drug microcarrier is 1-50 μm, and the drug microcarrier is loaded with hearing protective drugs and auxiliary agents, so that the permeability of the round window membrane is improved, the concentration of the hearing protective drugs in the inner ear is increased, the treatment effect of the hearing protective drugs is improved, and the acquired deafness is treated continuously.
In specific application, the method for treating hearing loss by tympanogram injection of the drug microcarrier comprises the following steps:
(1) the preparation steps of microfluidic loading are as follows:
drawing a glass capillary tube by using a tube drawing instrument, grinding the tube opening to be less than 50 mu m by using abrasive paper, soaking in alcohol, and ultrasonically cleaning. The glass capillary microfluidic chip is assembled by a drawn glass capillary, glass capillaries with the outer diameters of 1000 mu m, 300 mu m and 100 mu m, a glass slide, a sample application needle and quick-drying glue.
(2) The preparation method of the drug microcarrier comprises the following steps:
preparing a polymerizable high-molecular prepolymer solution from degradable materials with the same hydrophilicity and hydrophobicity and high biological safety according to the hydrophilicity and hydrophobicity of the hearing protective drug and the auxiliary agent, mixing the hearing protective drug and the auxiliary agent in a certain proportion, and adding an ultraviolet initiator HMPP in a proper proportion into the prepolymer solution; selecting one or more of oleum Arachidis Hypogaeae, oleum Helianthi, and oleum ricini with good biological safety and immiscible with inner phase, and mixing with SPAN80 at a certain ratio to obtain outer phase solution; respectively injecting the internal phase solution and the external phase solution into an internal phase channel and an external phase channel of the microfluidic chip through peristaltic pumps; the diameter of the liquid drop is changed by adjusting the flow rate of the solution of the inner phase and the solution of the outer phase; ultraviolet light curing or heating curing emulsion liquid drops; the residual solvent was removed by rinsing with absolute ethanol and ultrapure water several times, dried and stored in a container.
(3) The step of the injection of the drug microcarrier through the tympanic cavity:
mixing the dried drug microcarrier and normal saline according to a proportion, uniformly mixing by ultrasonic oscillation, injecting into an injector, puncturing the tympanic membrane in the front upper quadrant of the tympanic membrane under the guidance of an otoscope or a microscope, slightly enlarging the puncture hole by using a needle to facilitate the balance of the air pressure of the middle ear cavity and the external air pressure, and slowly injecting the drug microcarrier-normal saline mixed solution into the middle ear cavity.
Wherein, the inner phase solution is selected from one or more of materials with good biocompatibility and degradability, such as gelatin methacrylate (GelMA), polylactic acid-glycolic acid copolymer (PLGA), sodium alginate, etc.; in-situ loading hearing protective medicine (one or more of dexamethasone sodium phosphate, triamcinolone acetonide, N-acetyl-L-cysteine and alpha-lipoic acid) and adjuvant (one or more of methanol, histamine, saponin and caprate) for increasing permeability of round window membrane; and a certain proportion of HMPP (phenyl acetone) was mixed.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
Preparation of dexamethasone sodium phosphate-saponin-loaded methyl methacrylate gelatin (Dexsp-GelMA) microcarrier and antagonism of hair cell damage caused by cisplatin:
1. preparation of dexamethasone sodium phosphate-saponin-loaded methyl methacrylate gelatin (Dexsp-GelMA) microcarrier
(1) Manufacturing a single-emulsion microfluidic chip: drawing a glass capillary tube by using a tube drawing instrument, grinding the tube opening to be less than 50 mu m by using abrasive paper, soaking in alcohol, and ultrasonically cleaning. The glass capillary microfluidic chip is assembled by a drawn glass capillary, glass capillaries with the outer diameters of 1000 mu m, 300 mu m and 100 mu m, a glass slide, a sample application needle and quick-drying glue.
(2) Preparing a single emulsion template: referring to fig. 1, various solutions are prepared, 50mg of dexamethasone sodium phosphate and 130ug of saponin are dissolved in 1ml of mixed aqueous solution of 8% (w/v) GelMA and 6% (v/v) HMPP (phenyl acetone) to serve as aqueous phases, the aqueous phases are uniformly mixed by ultrasonic oscillation, 500 μ l of SPAN80 is dissolved in 10ml of castor oil to serve as oil phases, the two phases are respectively connected with teflon tubes through injectors and are led into two-phase inlets of a microfluidic chip, the flow rate of a peristaltic pump is controlled, and two-phase fluid is sheared into monodisperse single emulsion droplets with the particle size of less than 50 μm in the mobile phase.
(3) Curing the single emulsion template: and (3) vertically irradiating the generated emulsion liquid drop for 10min by ultraviolet light to solidify the emulsion liquid drop into balls, rinsing the emulsion liquid drop for multiple times by absolute ethyl alcohol and ultrapure water to remove residual solvent, and storing the emulsion liquid drop in a container after blowing and drying the emulsion liquid drop in a blowing dryer at 30 ℃. FIG. 4 is a structure and particle size distribution diagram of dexamethasone sodium phosphate-gelatin methacrylate (Dexsp-GelMA) under an optical microscope after ultraviolet light curing.
(4) Characterization of drug carrier microspheres: the surface structure of the microsphere is observed under an optical microscope, the drug microcarrier is photographed by software, and the particle size of the microcarrier is measured, so that the diameter of the microcarrier is about 33 mu m, the size is uniform, and the sphericity and the monodispersity are good.
(5) Weighing 1mg of dried Dexsp-GelMA microcarrier, dispersing in 1ml of PBS solution, placing in a constant-temperature shaking table with the rotation speed of 100rpm and the temperature of 37 ℃, centrifuging for 24h each hour, taking 200 mu l of supernatant, supplementing 200 mu l of fresh PBS, continuing shaking, sampling once a day, and adding fresh PBS for continuing shaking. Collecting the supernatant at different time points, and determining the concentration of dexamethasone sodium phosphate by an ultraviolet spectrophotometer.
(6) The dexamethasone sodium phosphate was determined to have a measurement wavelength of 242nm by full wavelength scanning. And (3) calculating the drug release amount of the supernatant by detecting the drug content of the supernatant, and drawing an in-vitro release curve of the drug microcarrier dexamethasone sodium phosphate.
Dexsp-GelMA microcarriers antagonize cisplatin damage to hair cells:
(1) cell inoculation: the hair cells used in the experiment are a mouse-derived HEI-OC1 cell line, and the passaged HEI-OC1 cells are inoculated into a ninety-six-well plate and cultured for 24 h.
(2) Drug carriers act on cells: the pure dexamethasone sodium phosphate cell culture medium containing 0ug/ml, 2.5ug/ml, 25ug/ml, 50ug/ml and 75ug/ml Dexsp and the dexamethasone sodium phosphate-GelMA drug-loaded microsphere culture medium are arranged in 8 groups, the pure culture medium is arranged as a control group, and each group has 3 duplicate wells. The corresponding culture medium and 30 mu M cisplatin are respectively added into each group, the cisplatin is not added into a control group, 10 mu l CCK8 is added into each hole after 24h of culture, the incubation is carried out for 1h at 37 ℃, and the cell activity is detected by an enzyme-labeling instrument.
Dexsp-GelMA microcarriers antagonize cisplatin-induced hearing loss and cochlear hair cell damage in guinea pigs:
(1) selecting guinea pigs: 16 adult guinea pigs with normal hearing were selected and their hearing thresholds at 4, 8, 16, 24, and 32kHz were measured by ABR.
(2) Tympanum injection of drug microcarriers: 16 guinea pigs that underwent ABR detection were randomly divided into 4 groups: Dexsp-GelMA, Dexsp, GelMA, physiological saline. Referring to fig. 3, about 116mg of Dexsp-GelMA drug microcarrier, 86mg GelMA and 30mg Dexsp are respectively weighed and dissolved in 1ml of physiological saline, injected into a 1ml syringe, and slowly injected into the middle ear cavity of the guinea pig after puncturing the tympanic membrane in the front upper quadrant of the guinea pig tympanic membrane through the external auditory canal by a plastic needle with the length of about 20mm under a microscope. The control group was injected with 100. mu.l of physiological saline in the tympanic cavity.
(3) Cisplatin-impaired guinea pig hearing: after feeding guinea pigs for 1 day, cisplatin-physiological saline solution at a concentration of 1mg/kg was prepared, and intraperitoneal injection was performed to the guinea pigs at a dose of 12 mg/kg.
(4) Evaluation of drug microcarriers for guinea pig hearing protection: ABR testing of guinea pigs on day 6 to assess threshold 4, 8, 16, 24, 32kHz demonstrated that the drug microcarriers antagonize cisplatin-induced hearing loss in guinea pigs.
(5) Evaluation of outer hair cell loss: and (3) killing the guinea pigs after ABR detection, taking out the auditory follicles, puncturing the bone at the tops of the snails by using a steel needle, then placing the guinea pigs into 4% paraformaldehyde for fixation overnight, peeling off the basement membrane of the guinea pigs, then using 1% Triton to break the membrane for 30min, respectively dyeing by using DAPI and Phalloidin for 10min, washing by using PBS for three times, sealing the slices, and observing the hair cell loss condition under a fluorescence microscope.
Example 2
The preparation of polylactic-co-glycolic acid (PLGA) microcarriers carrying N-acetyl-L cysteine and the antagonism of the damage of cisplatin to hair cells are carried out by a microfluidic technology:
1. preparation of a poly (lactic-co-glycolic acid) (PLGA) microcarrier for dexamethasone.
(1) Manufacturing a single-emulsion microfluidic chip: the procedure of making the single emulsion microfluidic chip satisfying the requirements was the same as in example 1.
(2) Preparing a single emulsion template: preparing various phase solutions, dissolving dexamethasone into a polylactic acid-glycolic acid copolymer (PLGA) methanol and dichloromethane solution as an internal phase, wherein the external phase is a 2 wt% PVA solution, connecting Teflon tubes through an injector respectively, leading the external phase into two-phase inlets of a microfluidic chip, and controlling the flow rate of a peristaltic pump to enable two-phase fluid to be sheared into monodisperse single emulsion droplets with the particle size of less than 50 mu m in a flowing phase.
(3) Curing the single emulsion template: and (3) placing the generated monodisperse single emulsion droplets in a rotary evaporator (39 degrees, 80rpm), vacuumizing and decompressing to completely volatilize the methylene dichloride serving as the PLGA solvent, filtering and taking out after the droplets are determined and solidified, and washing with ethanol for multiple times to remove redundant impurities on the surface.
(4) Characterization of drug microcarriers: and observing the surface structure of the microcarrier under an optical microscope, and measuring the particle size of the microcarrier to obtain the microcarrier with uniform diameter and size and good sphericity and monodispersity.
Polylactic-co-glycolic acid (PLGA) microcarriers of N-acetyl-L cysteine antagonize cisplatin damage to hair cells:
(1) cell inoculation: the procedure was as in example 1, 2- (1)
(2) Drug carriers act on cells: pure N-acetyl-L cysteine cell culture medium and poly (lactic-co-glycolic acid) (PLGA) microcarrier culture medium containing 0mM, 2.5mM, 5mM, 10mM and 15mM of N-acetyl-L cysteine were set for 10 groups, and the pure culture medium was set as a control group, and each group had 3 duplicate wells. The corresponding culture and 30 mu M cisplatin were added to each group, the control group was not added with cisplatin, 10 mu l of CCK8 was added to each well after 24h of culture, incubation was carried out at 37 ℃ for 1h, and cell activity was detected by a microplate reader.
Polylactic-co-glycolic acid (PLGA) microcarriers of N-acetyl-L cysteine antagonize cisplatin-induced hearing loss and cochlear hair cell damage in guinea pigs:
(1) selecting guinea pigs: the procedure was the same as in 3- (1) of example 1.
(2) Tympanum injection of drug microcarriers: 16 guinea pigs that underwent ABR detection were randomly divided into 4 groups: polylactic-co-glycolic acid (PLGA) microcarriers of N-acetyl-L-cysteine, polylactic-co-glycolic acid (PLGA) microcarriers and normal saline. Respectively taking polylactic acid-glycolic acid copolymer (PLGA) microcarrier, polylactic acid-glycolic acid copolymer (PLGA) microcarrier and N-acetyl-L-cysteine/normal saline solution of the same N-acetyl-L-cysteine concentration, injecting the mixture into a 1ml syringe, puncturing the tympanic membrane in the front upper quadrant of the guinea pig tympanic membrane through an external auditory canal by a plastic needle with the length of about 20mm under a microscope, and then slowly injecting the solution into the middle ear cavity of the guinea pig. The control group was injected with 100. mu.l of physiological saline in the tympanic cavity.
(3) Cisplatin-impaired guinea pig hearing: the procedure was the same as in 3- (3) of example 1.
(4) Evaluation of drug microcarriers for guinea pig hearing protection: the procedure was the same as in 3- (4) of example 1.
(5) Evaluation of outer hair cell loss: the procedure was the same as in 3- (5) of example 1.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A preparation method of a micro-carrier of a medicine aiming at acquired deafness based on a micro-fluidic technology is characterized in that the preparation method is to control the generation of an internal and external two-phase fluid by utilizing the micro-fluidic technology, and the generation process of the internal and external two-phase fluid comprises the following steps:
s1: preparing micron-scale liquid drops by utilizing shearing force and interfacial tension between two immiscible phases, and carrying hearing protective drugs and auxiliary agents for increasing the permeability of the round window membrane in situ;
s2: the droplets are solidified by polymerization to form the drug microcarriers.
2. The method for preparing a micro-carrier of a drug for acquired deafness based on microfluidic technology as claimed in claim 1, wherein the step of S1 comprises:
s1-1: preparation of emulsion template
Constructing a coaxial co-flow type micro-fluidic chip, preparing a corresponding polymerizable high-molecular prepolymer solution according to the hydrophilic and hydrophobic properties of the encapsulated hearing protection drug and the adjuvant, dissolving the drug and the adjuvant in the polymerizable high-molecular prepolymer solution, injecting immiscible internal and external phase solutions through internal and external phase channels of the micro-fluidic chip, and forming monodisperse emulsion droplets by utilizing the shear force and surface tension between the two phase solutions;
s1-2: droplet parameter regulation
Emulsion droplets with different sizes are obtained through the size and the shape of channels of all parts of the microfluidic chip, the flow velocity, the viscosity and the like of a continuous phase and a disperse phase.
3. The method for preparing a micro-carrier of a drug for acquired deafness based on microfluidic technology as claimed in claim 1, wherein the step of S2 comprises:
based on the physicochemical properties of the polymerizable high-molecular prepolymer solution in step S1-1, ultraviolet light or heat is used to induce a high-molecular crosslinking reaction, so that emulsion droplets are cured to obtain a drug microcarrier containing a protective drug and an auxiliary agent, the residual solvent is removed, and the drug microcarrier is dried and stored in a container.
4. The method for preparing a micro-fluidic-technology-based drug carrier for acquired deafness according to claim 1, wherein the micro-fluidic chip is a three-dimensional coaxial inner-outer co-flow micro-channel made of glass capillary.
5. The method for preparing a micro-carrier of a drug for acquired deafness based on microfluidic technology of claim 4, wherein the tube diameter of the tip of the inner phase glass tube in the microfluidic chip is less than 50 μm, and the tube diameter of the collecting tube is less than 300 μm;
the flow velocity range of the inner phase of the micro-fluidic chip is 0.1-0.2ml/h, the flow velocity range of the outer phase is 5-10ml/h, and the flow velocity ratio of the inner phase to the outer phase is controlled to be 1: 500-1: within 1000 f, the internal phase fluid is broken up into microdroplets in a high-speed narrow jet.
6. The preparation method of the micro-carrier for the acquired deafness based on the microfluidic technology as claimed in claim 1, wherein the main material of the internal phase fluid in S1 is a prepolymer solution of one or more materials selected from polylactic acid-glycolic acid copolymer, methacrylate gelatin, sodium alginate, etc.
7. The method for preparing a micro-carrier of a drug for acquired deafness based on microfluidic technology of claim 1, wherein the fluid of the outer phase in S1 is a component that is biologically safe and immiscible with the fluid of the inner phase.
8. The preparation method of the micro-fluidic technology-based drug microcarrier for acquired deafness according to claim 1, wherein the hearing protective drug in S1 is one or more of dexamethasone, triamcinolone acetonide, alpha lipoic acid, and N-acetyl-L-cysteine.
9. The method for preparing a micro-carrier of a drug for acquired deafness based on microfluidic technology of claim 1, wherein the adjuvant for increasing permeability of round window membrane in S1 is one or more of benzyl alcohol, histamine, saponin, and caprate.
10. The method for preparing a drug microcarrier for acquired deafness based on microfluidic technology of claim 1, wherein the diameter of the drug microcarrier prepared in S2 is 1-50 μm.
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