CN115089725B - Nanometer targeting drug delivery system for targeting outer hair cells and loading forskolin, and preparation method and application thereof - Google Patents

Nanometer targeting drug delivery system for targeting outer hair cells and loading forskolin, and preparation method and application thereof Download PDF

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CN115089725B
CN115089725B CN202210429290.6A CN202210429290A CN115089725B CN 115089725 B CN115089725 B CN 115089725B CN 202210429290 A CN202210429290 A CN 202210429290A CN 115089725 B CN115089725 B CN 115089725B
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forskolin
targeting
liposome
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CN115089725A (en
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查定军
安晓刚
陈俊
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Air Force Medical University of PLA
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Abstract

The invention discloses a targeting external hair cell and forskolin-loaded nano targeting drug delivery system and a preparation method and application thereof, and belongs to the technical field of biological medicines. The targeting drug delivery system takes liposome with a phospholipid bilayer structure as a carrier, carries a drug forskolin and a fluorescent marker, enables carboxyl groups on the surface of the liposome to react with amino groups of targeting polypeptide LS19 to form amide bonds under the action of carbodiimide compounds, and couples LS19 to the surface of the liposome, so that the inner ear targeting drug delivery system is prepared. The drug delivery system can overcome the inner ear blood-labyrinth barrier, target OHCS, and realize fluorescent imaging visualization, safety and long-term stable sustained release. The system can be locally administered in the inner ear of a mouse and can be targeted to be delivered to cochlear hair cells, and the therapeutic drug forskolin is stabilized and slowly released, so that noise or cisplatin-induced hearing loss is prevented. The targeted drug delivery system provided by the invention has the potential to solve the problem of inner ear drug delivery and carry out clinical transformation application.

Description

Nanometer targeting drug delivery system for targeting outer hair cells and loading forskolin, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a targeting external hair cell and forskolin-loaded nano targeting drug delivery system, and a preparation method and application thereof.
Background
Sensorineural hearing loss (sensorineural hearing loss, SNHL) caused by ototoxic drugs, noise exposure, genetics, trauma, etc. has become the most common sensorineural disease in the world, and clinically lacks effective targeted drugs, therapeutic regimens and protective measures. Inner ear drug delivery therapy SNHL is one of the hot spots of ear science research, however the biggest challenge is inner ear drug delivery drug. The accessibility of the inner ear and the blood-labyrinthine barrier (BLB, fig. 1) greatly limit inner ear drug delivery and release, most drugs cannot reach the entire cochlea quickly across BLB, or the amount of target cells is too small, the efficacy is very low, and conventional modes of administration are clearly unsuitable for inner ear drug delivery and treatment. In addition, many drugs have short plasma half-lives, and there are large variations among individuals with changes in body metabolism, all of which affect the effective delivery and absorption of the drug. Therefore, how to overcome BLB, achieving targeted delivery of inner ear is a leading edge and important scientific issue for inner ear drug delivery treatment of sensorineural hearing loss.
Based on the latest literature and our earlier experimental results, a potential targeted therapeutic drug Forskolin for preventing cisplatin ototoxicity and noise deafness is discovered. Forskolin (FSK for short) is a diterpenoid compound extracted from plant Coleus forskohlii, which was first described in 1981 as a cardiac active drug, and is an activator and antioxidant of Adenylate Cyclase (AC), significantly increasing intracellular cAMP levels. FSK has been shown to be a specific drug for the treatment of heart disease, hypertension, diabetes and asthma, but it has little research and application in the otology field, nor has it been applied to the treatment of clinical otology diseases. One of the currently accepted hearing loss mechanisms caused by noise, cisplatin and the like is oxidative stress injury, a great deal of research proves that FSK has antioxidant and anti-inflammatory effects in various tissues and cell types, and the latest literature reports that FSK can reduce ROS level, reduce cisplatin-noise-induced hearing loss and possibly be a potential targeted therapeutic drug for otology prevention of cisplatin ototoxicity and noise deafness. However, FSK also has BLB problems that make it ineffective, so it is urgent to construct a novel targeted drug delivery system for FSK that overcomes BLB and is suitable for the inner ear, to achieve targeted delivery and release of FSK in the inner ear, and to confirm targeting, bio-application safety and effectiveness of the drug delivery system.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a nano targeting drug delivery system for targeting peripheral hair cells and loading forskolin, a preparation method and application thereof, and aims to solve the problem that the forskolin can not cross a blood-labyrinth barrier and treat sensorineural deafness.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses a targeting external hair cell and a nano targeting drug delivery system for loading forskolin, which comprises a liposome, wherein a fluorescent marker is loaded in a water phase in the liposome, the forskolin is loaded in an oil phase, and a targeting polypeptide LS19 is modified on the surface of the liposome;
wherein the amino acid sequence of the targeting polypeptide LS19 is shown as SEQ ID No. 1.
Preferably, the targeting polypeptide LS19 is coupled to the surface of the liposome through an amide bond formed by the carbodiimide compound and DSPE-mPEG-COOH.
Preferably, the liposome carrier comprises lecithin and cholesterol.
Further preferably, the fluorescent label is rhodamine-B.
The invention also discloses a preparation method of the nano targeting drug delivery system for targeting the outer hair cells and loading forskolin, which comprises the following steps:
1) Preparation of Forskolin and fluorescent marker-rhodamine B-loaded Liposome
Weighing lecithin, DSPE-mPEG-COOH, cholesterol and Forskolin dissolved in DMSO, and adding chloroform for full dissolution; evaporating the dissolved reaction liquid until chloroform is completely volatilized; adding a rhodamine B-containing purified water solution for hydration, ultrasonic treatment, washing, filtering and sizing to prepare an internal water phase loaded rhodamine B and an oil phase loaded Forskolin liposome;
2) Targeting polypeptide LS-19 for carrying out functional modification on liposome
Washing the liposome prepared in the step 1) by using MES, fixing the volume, adding the targeting polypeptide LS-19 and the carbodiimide compound, and reacting overnight; and centrifuging the reaction solution, collecting the supernatant, ultrafiltering and washing, wherein the supernatant is the Lipid-Forskolin-rhodamine B-LS19 liposome.
Preferably, the mass ratio of lecithin, cholesterol, forskolin, rhodamine B and targeting polypeptide LS-19 is 130:20:10:3:6.
preferably, in step 1), the dissolution is ultrasonic dissolution; the washing adopts purified water ultrafiltration washing; filtering with 0.22 μm filter membrane; in step 2), the ultrafiltration is performed using purified water.
The invention also discloses application of the nano targeting drug delivery system for targeting the outer hair cells and loading forskolin in preparation of otology pharmaceutical preparations.
Preferably, the otology drug is a drug for treating sensorineural hearing loss.
Preferably, the otic drug formulation is delivered by injection or topical administration to the inner ear.
Compared with the prior art, the invention has the following beneficial effects:
according to the targeting delivery system for targeting the outer hair cells and loading forskolin, provided by the invention, the delivery system can specifically aim at OHCS, liposome with a phospholipid bilayer structure is taken as a carrier, potential therapeutic drugs Forskolin (FSK) of the otology and fluorescent markers are loaded, the targeting polypeptide LS19 is coupled to the surface of the liposome by utilizing the function of specifically recognizing actin prestin specifically expressed in the inner ear hair cells by the targeting polypeptide LS19, so that the inner ear targeting delivery system (LS 19-FSK-NP) is prepared, and a stable inner ear targeting nanomaterial is assembled and formed. The liposome selected by the drug delivery system is a long-circulating ligand targeted drug-carrying liposome, is more intelligent, has lower toxicity, is easy to commercialize, has targeting property, slow release property, cell affinity and histocompatibility, can drive drug-carrying nano particles to be actively targeted and delivered to cochlear target cells through receptor-ligand interaction, and stabilizes and slowly releases therapeutic drugs. The targeted drug delivery in the inner ear is realized through the fluorescence liposome mediated forskolin, and the advantages are that the inner ear blood-labyrinth barrier can be overcome, the targeted effect on OHCs is realized, the fluorescence imaging visualization is realized, and the drug is safely and stably released continuously for a long time, so that better drug delivery effect and prevention effect can be achieved, and the defects and side effects of conventional drug administration are greatly reduced. The system can be locally administrated in the inner ear of a mouse, can be targeted to be delivered to cochlear hair cells, and can stably and slowly release a therapeutic drug FSK, so that noise or cisplatin-induced hearing loss can be effectively prevented. The targeted drug delivery system is an effective strategy for solving the problem of inner ear drug delivery, is also an effective tool in the field of ear scientific research, and has great clinical transformation application potential.
Further, under the action of the carbodiimide compound, the carboxyl on the surface of the liposome reacts with the amino of the targeting polypeptide LS19 to form an amide bond, so that the targeting polypeptide LS19 can be coupled to the surface of the drug-loaded fluorescent liposome.
The preparation method of the nano targeted drug delivery system for targeting the outer hair cells and loading the Forskolin is simple in preparation process, raw materials are easy to obtain, the encapsulation rate of the Forskolin can reach 35.7%, the drug loading rate of the Forskolin can reach 2.68%, the coupling rate of the targeting polypeptide LS19 can reach 84.919292%, the Forskolin can be loaded successfully, and the targeted delivery of the Forskolin can be realized across an inner ear blood-labyrinth barrier.
Drawings
FIG. 1 is a schematic diagram of the structure of a blood-labyrinth barrier;
FIG. 2 is a diagram of a targeting peripheral hair cell and forskolin loaded nano-targeting drug delivery system according to the present invention;
FIG. 3 is a diagram of the Forskolin HPLC detection result (200. Mu.g/mL) of the present invention;
FIG. 4 is a Forskolin standard graph of the present invention;
FIG. 5 is a graph of the DLS detection results of the present invention; wherein, the graph A is the DLS detection result of Lipid-Forskolin-rhodamine B-LS1, the graph B is the DLS detection result of Lipid-rhodamine B-LS19, and the graph C is the DLS detection result of Lipid-Forskolin-rhodamine B;
FIG. 6 is a graph showing the Zeta potential detection result of the present invention; wherein, the graph a is the Zeta potential detection result of the Lipid-Forskolin-rhodamine B-LS19, the graph B is the Zeta potential detection result of the Lipid-rhodamine B-LS19, and the graph c is the Zeta potential detection result of the Lipid-Forskolin-rhodamine B;
FIG. 7 is a graph showing the result of transmission electron microscopy according to the present invention; wherein, the graph A is the transmission electron microscope detection result of Lipid-Forskolin-rhodamine B-LS19, the graph B is the transmission electron microscope detection result of Lipid-rhodamine B-LS19, and the graph C is the transmission electron microscope detection result of Lipid-Forskolin-rhodamine B;
FIG. 8 is a graph of the detection result of fluorescence emission spectrum according to the present invention;
FIG. 9 is a LS19 standard graph of the present invention;
FIG. 10 is a diagram of a mouse cochlear hair cell targeting validation of the present invention; wherein, figure a is cochlea DAPI immunostaining (blue), figure b is Myosin7a immunostaining (OHCs) (green), figure c is LS19-FSK-NP immunostaining (red), figure d is trichromatic immunofluorescence combination, and figure e is a partial magnified view of figure d.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in other sequences than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
according to the targeting external hair cell and forskolin loaded nano targeting drug delivery system provided by the invention, liposome with a phospholipid bilayer structure is taken as a carrier, a fluorescent marker is loaded on an internal water phase (hydrophilic) of the carrier, a treatment drug Forskolin (FSK) is loaded on an oil phase (hydrophobic), carboxyl on the surface of the liposome reacts with amino of a targeting polypeptide LS19 to form an amide bond under the action of a carbodiimide compound, and the targeting polypeptide LS19 (targeting prestin specifically expressed in the identified external hair cell) is coupled to the surface of the liposome to form a co-assembly, so that the novel targeting external hair cell and forskolin loaded nano targeting drug delivery system is constructed;
wherein the liposome carrier comprises lecithin and cholesterol, and the fluorescent marker is selected from rhodamine-B.
The preparation method of the nano targeting drug delivery system for targeting the outer hair cells and loading forskolin provided by the invention comprises the following steps:
1. preparation of Forskolin and fluorescent marker-rhodamine B-loaded Liposome
130mg lecithin (purchased from Shanghai Ala Biotechnology Co., ltd.), 20mg DSPE-mPEG-COOH (purchased from Guangzhou Carbonic technologies Co., ltd.), 20mg cholesterol (purchased from Shanghai Ala Biotechnology Co., ltd.), and 10mg Forskolin (dissolved in 200. Mu.L DMSO; purchased from APEXBIO Co.) were accurately weighed into a centrifuge tube, and 8mL chloroform was added and the mixture was thoroughly dissolved by sonication; transferring the dissolved reaction solution into a 100mL eggplant-shaped bottle, placing the bottle on a rotary evaporator, and performing rotary evaporation at 70 ℃ and 30rpm until the chloroform is completely volatilized; the eggplant-shaped bottle was removed, and 8mL of a purified aqueous solution containing 3mg of rhodamine B (available from Haiaa Ding Shenghua technologies Co., ltd.) was added thereto, and hydrated at 40℃and 17rpm for 40 minutes; taking down the eggplant-shaped bottle, performing water bath ultrasonic treatment for 1min, taking out the solution to a 10mL centrifuge tube, and performing probe ultrasonic treatment (20% power/2 s stop for 3 s/ice bath) for 9min; the solution was transferred to a 100kDa ultrafiltration tube and washed 5 times (3777 g,20 min/time) with purified water (pH 7-8) by ultrafiltration; filtering with 0.22 μm filter membrane, and fixing volume with purified water to 6 mL to obtain liposome (namely Lipid-Forskolin-rhodamine B, FSK-NP for short) loaded with rhodamine B in inner water phase and Forskolin loaded with oil phase.
2. Targeting peptide LS19 prepared by carrying out functional modification on Forskolin and fluorescent marker-rhodamine B loaded liposome
The experimental procedure was as follows: 60mg of the liposome prepared in the step (1) was washed with 15mM MES (sodium fatty acid methyl sulfonate; pH 5.5; available from Shanghai Ala Biotechnology Co., ltd.) and fixed to a volume of 9.251mL, and 6mg of the targeting polypeptide LS-19 (0.0404 mg/mL, 148.5. Mu.L; purified water was dissolved, and 1% NaHCO was added thereto) was vortexed 3 Adjusting the pH value to be neutral; targeting polypeptide LS-19 is made by Shanghai blaze Biotechnology Co., ltd; the amino acid sequence is shown in Table 1), 6mg EDC (10 mg/mL, 600. Mu.L; purchased from Sigma company), and reacted overnight (more than 20 hours) at 37℃on a shaker at 150 rpm; after the reaction is finished, transferring the reaction solution into an ultrafiltration tube of 100kDa, centrifuging, collecting a supernatant (used for detecting the coupling rate of the polypeptide), ultrafiltering by adopting purified water, and washing for 5 times, wherein the supernatant is a liposome coupled with the targeting peptide LS19 (namely a Lipid-Forskolin-rhodamine B-LS19 liposome, LS19-FSK-NP for short).
Table 1 amino acid sequence listing of targeting polypeptide LS-19
Name of the name Amino acid sequence Sequence number
Targeting polypeptide LS19 LSTHTTESRSMVGGSCGGS SEQ.ID.NO.1
The nano targeting drug delivery system for targeting the outer hair cells and loading forskolin is detected and characterized
1. Sample preparation
1. Preparation of sample 1 (Lipid-Forskolin-rhodamine B-LS19 Liposome, abbreviated as LS 19-FSK-NP)
The preparation method is the same as the preparation method step 1 and step 2 of the nano targeting drug delivery system targeting the outer hair cells and loading forskolin.
2. Preparation of sample 2 (Lipid-rhodamine B-LS19 liposomes, abbreviated as LS 19-NP)
Step 1: accurately weighing 65mg of lecithin, 10mg of DSPE-mPEG-COOH and 10mg of cholesterol in a centrifuge tube, adding 4mL of chloroform, and fully dissolving by ultrasound; transferring the dissolved reaction solution into a 100mL eggplant-shaped bottle, placing the bottle on a rotary evaporator, and performing rotary evaporation at 70 ℃ and 30rpm until chloroform is completely volatilized; taking down the eggplant-shaped bottle, adding 4mL of purified water solution containing 3mg of rhodamine B, and hydrating at 40 ℃ and 17rpm for 40min; taking down the eggplant-shaped bottle, taking out the solution to a 10mL centrifuge tube after ultrasonic treatment in water bath for 1min, and performing ultrasonic treatment (20% power/2 s stop for 3 s/ice bath) on the probe for 9min; the solution was transferred to a 100KD ultrafiltration tube and washed 5 times with purified water (3777 g,20 min/time); filtering with 0.22 μm filter membrane, and fixing volume with purified water to 3mL to obtain liposome of rhodamine B (Lipid-rhodamine B, NP for short).
Step 2: step 2 of the preparation method of the nano targeting drug delivery system targeting the outer hair cells and loading forskolin.
3. Preparation of sample 3 (Lipid-Forskolin-rhodamine B liposome, abbreviated as FSK-NP)
The preparation method is the same as the preparation method step 1 of the nano targeting drug delivery system targeting the outer hair cells and loading forskolin.
Samples 1, 2 and 3 can be prepared according to the preparation methods provided by the present invention, or can be custom made by company according to the methods described above.
2. Detection characterization
Characterization one: detection of Forskolin drug loading and encapsulation efficiency by HPLC
1. Detection of Forskolin standard curve samples by HPLC
(1) 25mg of Forskolin was dissolved in 500. Mu.L of DMSO at a concentration of 50mg/mL and diluted with acetonitrile to 12.5, 25, 50, 100, 150 and 200. Mu.g/mL.
(2) HPLC detection conditions:
column type: c18; column temperature: 30 ℃; flow rate: 1mL/min; mobile phase: acetonitrile: water = 50:50;
injection volume: 20. Mu.L; detection wavelength: 210nm; retention time: about 6.4.
The detection result of 200 mug/mL Forskolin HPLC is shown in FIG. 3, the prepared Forskolin standard curve is shown in FIG. 4, and the standard curve is y=5590.7x+9528.2 (R 2 ＝0.9992)。
2. HPLC (high Performance liquid chromatography) detection for Forskolin drug-loading and encapsulation efficiency
1mg of Lipid-Forskolin-rhodamine B-LS19 liposome prepared by the method is taken, and Lipid-Forskolin-rhodamine B-LS19 liposome is taken: demulsifier = 1:9 mass ratio acetonitrile solution containing 10% Triton X-100 (total volume 727. Mu.L) was added, sonicated for 20min, centrifuged to remove precipitate, and filtered through 0.22 μm and detected by HPLC.
The peak area values detected were calculated from the standard curve of FIG. 4, and found that 1mg of liposome contained 26.8. Mu.g of Forskolin, the total solid content of liposome was 133.2mg, and the amount of Forskolin dosed was 10mg.
The formula for the Forskolin encapsulation rate is as follows:
carrying the coating capacity of 26.8 mug and the feeding amount of 10mg/133.2mg into the formula, and calculating to obtain the Forskolin encapsulation rate of 35.7%;
the formula for calculating the Forskolin drug loading is as follows:
the total solid content of the liposome with the inclusion capacity of 26.8 mug is brought into the formula, and the calculated Forskolin drug loading rate is 2.68%.
Characterization two: DLS detection
The DLS data for samples 1, 2 and 3 are shown in FIG. 5, with hydrodynamic sizes (Mean by Number) of 116.6nm, 119.6nm and 97.4nm for the three samples, respectively. The hydrodynamic size increases after coupling of polypeptide LS-19 compared to sample 1 and sample 3, further indicating that the polypeptide has been successfully coupled to the surface of sample 1.
And (3) characterization: zeta potential detection
The Zeta potential of samples 1, 2 and 3 is shown in FIG. 6, with Zeta potentials of-18.58 mV, -18.07mV and-44.23 mV for the three samples, respectively. The absolute value of Zeta potential decreases after coupling of polypeptide LS-19 compared to sample 1 and sample 3, further indicating that the polypeptide has been successfully coupled to the surface of sample 1.
Characterization four: transmission electron microscope detection result
The sizes and the morphologies of the samples 1, 2 and 3 are shown in fig. 7, and the particle sizes of the samples are about 100nm, which indicates that the particle sizes of the liposome are not obviously influenced by the drug loading forskolin and the modified targeting polypeptide LS-19, and the requirements that the average particle size of the liposome is about 100nm are met, so that the drug loading rate is relatively high and the liposome is easier to enter cells.
Characterization five: fluorescence spectrum detection result
Taking 3 groups of samples, and respectively adding purified water to dilute to 0.5mg/mL, wherein the total amount is 2mL; another 20. Mu.L of 1mg/mL rhodamine B aqueous solution was diluted to 2mL with purified water. And detecting fluorescence emission spectrums of rhodamine B and samples 1-3 by adopting a fluorescence spectrum.
The fluorescence emission spectra of rhodamine B aqueous solution and samples 1, 2 and 3 are shown in FIG. 8, and the fluorescence emission spectra of samples 1-3 have no obvious difference in excitation wavelength position compared with the rhodamine B aqueous solution, which indicates that rhodamine B is successfully loaded in samples 1, 2 and 3.
Characterization five: target protein coupling rate
Sample to be measured: after sample 1 was coupled to the targeting polypeptide LS-19, the supernatant was ultrafiltered and centrifuged.
And detecting the content of the target polypeptide LS19 in the supernatant by adopting the BCA, and calculating the coupling rate of the target polypeptide LS19 according to the feeding amount.
Standard curves were made using targeting polypeptide LS-19: 20.2mg of the targeting polypeptide LS19 was dissolved in 500. Mu.L of aqueous solution and diluted with MES to 125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.625. Mu.g/mL and 7.8125. Mu.g/mL.
The prepared LS19 standard curve is shown in FIG. 9, the coupling rate of the target polypeptide LS19 is calculated according to the BCA detection result of the sample to be detected, and the calculation result is shown in Table 2:
TABLE 2 BCA assay for targeting polypeptide LS19
Concentration mg/mL calculated from standard curve Actual concentration mg/mL Concentration of reaction mg/mL Coupling ratio%
0.015080708 0.150807084 1 84.919292
The results in Table 2 show that the coupling rate of the targeting polypeptide LS-19 reaches 84.9%, which indicates that the coupling effect is good, and inner ear targeted delivery and release of forskolin can be achieved through receptor-ligand interaction.
Characterization six: mouse cochlear hair cell targeting verification experiment
After adult male C57BL/6J mice were anesthetized with 5% chloral hydrate, the mice were placed on a constant temperature hot plate to maintain their body temperature constant, the post-aural hairs of the mice were removed, 75% alcohol was sterilized, small scissors were used to cut behind the ears, the round window niche was exposed under a microscope, inner ear drug delivery was performed by the administration mode of the round window membrane route, 10uL of the constructed drug delivery system nanoparticles were slowly injected under a microscope with a drawn glass needle, and then the wound was sutured and sterilized.
Mice are sacrificed after 2-3 hours of administration, and cochlea is taken for paraformaldehyde fixation, EDTA decalcification treatment and other operations, and frozen sections and immunofluorescence staining are carried out. In immunofluorescence experiments, myosin7a was used to label hair cells, DAPI was used to line nuclei, and laser confocal photographing was performed to verify that the drug delivery system was targeted to cochlear hair cells.
The results in fig. 10 demonstrate that the drug delivery system accumulates in large amounts at coti, confirming its specific delivery to hair cells with high targeting.
In summary, according to the nano targeting drug delivery system for targeting the outer hair cells and loading the Forskolin, provided by the invention, the liposome can successfully load fluorescent markers rhodamine B and Forskolin, and the targeting polypeptide LS-19 can be successfully coupled to the surface of the Lipid-Forskolin-rhodamine B-LS19 liposome, so that targeted delivery and release of the Forskolin in the inner ear are realized.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<110> Chinese people's air force medical university
<120> nanometer targeting drug delivery system for targeting outer hair cells and loading forskolin, preparation method and application
<141> 2022-04-12
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 1
Leu Ser Thr His Thr Thr Glu Ser Arg Ser Met Val Gly Gly Ser Cys
1 5 10 15
Gly Gly Ser

Claims (8)

1. The nanometer targeting drug delivery system for targeting the outer hair cells and loading the forskolin is characterized by comprising a liposome, wherein a fluorescent marker is loaded on the water phase in the liposome, the forskolin is loaded on the oil phase, and a targeting polypeptide LS19 is modified on the surface of the liposome;
wherein the amino acid sequence of the targeting polypeptide LS19 is shown as SEQ.ID.NO. 1; the targeting polypeptide LS19 is coupled on the surface of the liposome through an amide bond formed by a carbodiimide compound and DSPE-mPEG-COOH; the liposome carrier comprises lecithin and cholesterol.
2. A nano-targeted drug delivery system for targeting peripheral hair cells and carrying forskolin according to claim 1, wherein the fluorescent label is rhodamine-B.
3. A method for preparing a forskolin-loaded nano-targeted drug delivery system targeting peripheral hair cells according to claim 2, which is characterized by comprising the following steps:
1) Preparation of Forskolin and fluorescent marker-rhodamine B-loaded Liposome
Weighing lecithin, DSPE-mPEG-COOH, cholesterol and Forskolin dissolved in DMSO, and adding chloroform for full dissolution; evaporating the dissolved reaction liquid until chloroform is completely volatilized; adding a rhodamine B-containing purified water solution for hydration, ultrasonic treatment, washing, filtering and sizing to prepare an internal water phase loaded rhodamine B and an oil phase loaded Forskolin liposome;
2) Targeting polypeptide LS-19 for carrying out functional modification on liposome
Washing the liposome prepared in the step 1) by using MES, fixing the volume, adding the targeting polypeptide LS-19 and the carbodiimide compound, and reacting overnight; and centrifuging the reaction solution, collecting the supernatant, ultrafiltering and washing, wherein the supernatant is the Lipid-Forskolin-rhodamine B-LS19 liposome.
4. A method of preparing a Forskolin-loaded nanotargeting delivery system for targeting peripheral hair cells according to claim 3, wherein the mass ratio of lecithin, cholesterol, forskolin, rhodamine B and targeting polypeptide LS-19 is 130:20:10:3:6.
5. a method of preparing a forskolin-loaded nanotargeting delivery system for targeting peripheral hair cells according to claim 3, wherein in step 1), the dissolution is performed by ultrasonic dissolution; the washing adopts purified water ultrafiltration washing; filtering with 0.22 μm filter membrane; in step 2), the ultrafiltration is performed using purified water.
6. Use of a forskolin-loaded nano-targeted delivery system according to claim 1 or 2 for the preparation of an otologic pharmaceutical formulation.
7. The use according to claim 6, wherein the otologic drug is a drug for the treatment of sensorineural hearing loss.
8. The use according to claim 6, wherein the otic pharmaceutical formulation is delivered by injection or by topical administration to the inner ear.
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