Meniere syndrome targeted drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to a Meniere syndrome targeted drug delivery system as well as a preparation method and application thereof. The system is composed of a liposome carrier, a VEGFR-2 antibody, dexamethasone and betahistine, wherein the liposome carrier is composed of distearoyl phosphatidylcholine (DSPC) and cholesterol in a molar ratio of 4: 1; the VEGFR-2 antibody is modified on the surface of the liposome; and the dexamethasone and the betahistine are loaded in the liposome carrier in a molar ratio of 1: 3. The antibody is modified on the surface of the liposome through an EDC / NHS covalent coupling technology, so that the drug penetrates through the inner ear blood lost barrier and is accurately delivered to inner ear vascular vein cells. And the particle size of the liposome is 120.9 nm. Animal experiments show that the inner ear drug concentration of a targeted group reaches 12.5 mu g / g and is 3.3 times that of a non-targeted group; after treatment, the vertigo attack frequency is reduced by 74.4%, the hearing threshold is improved by 25.3 dB, and no significant systemic toxicity exists. The preparation process is simple, and an efficient and safe new strategy is provided for targeted therapy of Meniere syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a targeted drug delivery system for Meniere's syndrome, its preparation method and application. In particular, the present invention realizes the penetration of the inner ear blood-labyrinth barrier by modifying the surface of liposomes with vascular endothelial growth factor receptor-2 (VEGFR-2) antibody, enabling precise delivery of drugs to the stria vascularis cells of the inner ear, significantly improving the bioavailability of dexamethasone and betahistine, and reducing systemic side effects. Background Art

[0002] Meniere's syndrome is an inner ear disease, and its main symptoms include recurrent vertigo, fluctuating hearing loss, tinnitus and a feeling of ear fullness. The pathological mechanism of this disease has not been fully clarified, but it is generally believed to be related to endolymphatic hydrops in the inner ear. Endolymphatic hydrops in the inner ear leads to increased inner ear pressure, which in turn affects auditory and vestibular functions. Currently, the treatment methods for Meniere's syndrome mainly include drug treatment, surgical treatment and lifestyle adjustment, but these methods often have problems such as limited efficacy, large side effects or strong invasiveness.

[0003] Drug treatment is a common means for Meniere's syndrome. Commonly used drugs include diuretics, glucocorticoids (such as dexamethasone) and vestibular inhibitors (such as betahistine). However, traditional systemic drug delivery methods have problems such as uneven drug distribution, poor targeting and large systemic side effects, and it is difficult to effectively deliver drugs to the diseased sites in the inner ear. The existence of the inner ear blood-labyrinth barrier further limits the penetration of drugs into the inner ear, resulting in unsatisfactory treatment effects.

[0004] In recent years, nano-drug delivery systems have shown great potential in the field of drug delivery. In particular, liposomes, as a kind of nano-carrier with good biocompatibility, can effectively encapsulate drugs and achieve targeted delivery through surface modification. Liposomes have the advantages of controllable particle size, high drug loading capacity, good stability, etc., and can achieve precise drug delivery by modifying specific antibodies or ligands on the surface. For example, vascular endothelial growth factor receptor-2 (VEGFR-2) is highly expressed in the stria vascularis cells of the inner ear. By modifying the surface of liposomes with VEGFR-2 antibody, targeted delivery of drugs to the stria vascularis cells of the inner ear can be achieved, thereby increasing the local concentration of drugs in the inner ear and reducing systemic side effects. Summary of the Invention

[0005] The present invention aims to solve the technical problems of poor drug targeting, large systemic side effects and insufficient drug synergistic effect in the existing treatment of Meniere's syndrome. For this purpose, the present invention provides a targeted drug delivery system for Meniere's syndrome, which is characterized in that it includes a liposome carrier composed of distearoyl phosphatidylcholine (DSPC) and cholesterol, a VEGFR-2 antibody modified on the surface of the liposome by the EDC / NHS cross-linking method, and a combined drug of dexamethasone and betahistine encapsulated in the liposome.

[0006] Through the specific binding of the VEGFR-2 antibody, this drug delivery system precisely delivers drugs to the stria vascularis cells of the inner ear, significantly increasing the drug concentration at the inner ear target site. Dexamethasone and betahistine are encapsulated in a molar ratio of 1:3 to exert a synergistic therapeutic effect, effectively controlling the onset of vertigo and hearing loss. The liposome carrier can reduce the distribution of drugs in non-target tissues and lower systemic side effects. In addition, this drug delivery system has high stability, with a liposome particle size of approximately 120.9 nm, an encapsulation efficiency of 92.1%, and the particle size growth ≤ 10% after storage at 4°C for 30 days. Description of the Drawings

[0007] Figure 1 It is a schematic structural diagram of the targeted drug delivery system of the present invention. The figure shows the structural composition of the liposome carrier, the encapsulated drugs (dexamethasone and betahistine), and the VEGFR-2 antibody modified on the surface of the liposome.

[0008] Figure 2 It is a liposome particle size distribution diagram. Detected by dynamic light scattering (DLS), the results show that the average particle size of the liposome is 120.9 nm.

[0009] Figure 3 It is a transmission electron micrograph of the liposome. It conforms to Figure 2 the measured particle size, with good morphology and no damage.

[0010] Figure 4 It is the detection result of the inner ear drug concentration in the animal model. Detected by HPLC method, the inner ear drug concentrations (dexamethasone and betahistine) in the targeted drug delivery group are 12.5 μg / g and 37.2 μg / g respectively, significantly higher than those in the non-targeted drug delivery group and the blank control group (not detected).

[0011] Figure 5 It is the curve of the change in the frequency of vertigo attacks in the Meniere's syndrome mouse model. After 4 weeks of treatment, the frequency of vertigo attacks in the targeted drug delivery group decreased by 75.5%, that in the non-targeted drug delivery group decreased by 30.5%, and there was no obvious change in the blank control group.

[0012] Figure 6 It is the detection result of biosafety. By detecting the blood biochemistry (ALT, AST, BUN) of the mice injected with the prepared materials on days 0, 1, 3, and 5. The results show that there are no statistical differences in the indicators of each group (P>0.05). Detailed Description of the Invention

[0013] Example 1

[0014] The preparation of the targeted drug delivery system is carried out as follows:

[0015] In this example, the preparation process of the targeted drug delivery system is as follows: Dissolve distearoyl phosphatidylcholine (DSPC, purchased from Sigma-Aldrich, 40 μmol) and cholesterol (purchased from Avanti Polar Lipids, 10 μmol) in a chloroform-methanol mixed solvent (volume ratio 2:1, the solvent is purchased from the National Pharmaceutical Group), transfer it to a 50 mL round-bottom flask, connect a rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), with a vacuum degree of 0.09 MPa, a water bath temperature of 40 °C, a rotation speed of 60 rpm, and continuously evaporate for 30 minutes to form a uniform lipid film. Subsequently, place the lipid film in a 55 °C constant temperature water bath, add phosphate buffer solution (PBS, pH 7.4, purchased from Gibco) containing dexamethasone (3 mM, purchased from MCE) and betahistine (9 mM, purchased from TargetMol), and hydrate for 30 minutes. During this period, manually shake the flask every 5 minutes to ensure complete hydration. After hydration is completed, transfer the liposome solution to a probe sonicator (Ningbo Xinzhi Biotechnology), set the power to 100 W and the frequency to 20 kHz, and sonicate for 10 minutes (each time for 2 minutes, with a 1-minute interval of ice bath cooling to avoid overheating) to obtain drug-loaded liposomes.

[0016] Dissolve the VEGFR-2 monoclonal antibody (ab237634, purchased from Abcam) in PBS buffer (pH 6.0), add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 10 mM, purchased from Sigma-Aldrich) and N-hydroxysuccinimide (NHS, 20 mM, purchased from Sigma-Aldrich), and activate at room temperature for 30 minutes. Mix the activated antibody solution with the drug-loaded liposomes at a molar ratio of 1:50, and react with shaking in a 25 °C constant temperature shaker (Shanghai Zhicheng Analytical Instrument) for 2 hours. After the reaction is completed, transfer the mixture to an ultrafiltration centrifugal tube (cut-off molecular weight 100 kDa, purchased from Millipore), and centrifuge at 8000×g for 15 minutes (Beckman Coulter) to remove unbound antibodies and free drugs, and obtain the purified targeted drug delivery system. Finally, mix the targeted drug delivery system with 5% mannitol (w / v, purchased from the National Pharmaceutical Group), aliquot it into vials, pre-freeze it (store it in an -80 °C refrigerator for 4 hours), and then transfer it to a freeze dryer (Labconco). Set the program: pre-freeze at -40 °C for 4 hours, primary dry at -20 °C for 12 hours, and secondary dry at 25 °C for 6 hours to finally obtain a freeze-dried preparation. When reconstituting, add 1 mL of normal saline (0.9% NaCl, purchased from Beyotime Biotechnology) and vortex for 30 seconds until completely dissolved. Figure 1 The structural schematic diagram of the targeted drug delivery system is shown. The figure shows the structural composition of the liposome carrier, the encapsulated drugs (dexamethasone and betahistine), and the VEGFR-2 antibody modified on the surface of the liposome, and it can achieve the function of targeting to the inner ear.

[0017] Example 2

[0018] Characterization and stability verification of the drug-loaded liposomes were carried out as follows:

[0019] Particle size and Zeta potential detection: The particle size distribution and surface potential of the liposomes were detected by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90). The liposome solution was diluted to 0.1 mg / mL with PBS buffer (pH 7.4), transferred to a quartz sample cell, and the detection temperature was set at 25 °C, the scattering angle was 173°, the laser wavelength was 633 nm, the detection time was 60 seconds, and each sample was measured 3 times and the average value was taken. As Figure 2 shown, the average particle size of the liposomes was 120.9 nm, the particle size distribution of the liposomes was uniform, meeting the requirements of the drug delivery system. The Zeta potential was -15.2 mV, indicating that the liposome surface was negatively charged, which was beneficial to improving the stability of the preparation. The morphology of the liposomes was further confirmed by transmission electron microscopy (TEM), showing a typical spherical structure, and the particle size distribution was consistent with the DLS results. The TEM image showed that the liposome membrane structure was intact, without obvious aggregation or rupture, as Figure 3 shown.

[0020] Liposome stability assessment: The liposomes were stored at 4 °C, 25 °C and 37 °C respectively, and samples were taken on the 1st, 7th, 14th and 30th days to detect the particle size and PDI. The results showed that there was no obvious change in the particle size and PDI of the liposomes at 4 °C (particle size: 121 ± 4 nm); the particle size increased slightly at 25 °C (125 ± 5 nm); the particle size increased to 130 ± 6 nm at 37 °C, indicating that the liposomes had good stability at room temperature, but needed to be further optimized under high temperature conditions. The results are shown in Table 1.

[0021] Table 1 Liposome stability assessment

[0022] Storage conditions Day 1 Day 7 Day 14 Day 30 4℃ 120.35 ± 0.52 nm 121.12 ± 0.48 nm 121.45 ± 0.50 nm 122.03 ± 0.55 nm 25℃ 120.35 ± 0.52 nm 123.27 ± 0.49 nm 124.56 ± 0.53 nm 125.18 ± 0.57 nm 37℃ 120.35 ± 0.52 nm 128.42 ± 0.58 nm 129.67 ± 0.60 nm 130.25 ± 0.62 nm

[0023] Example 3

[0024] Construction and verification of an animal model of Meniere's syndrome were carried out as follows:

[0025] Animal model construction: Female BALB / c mice aged 6 - 8 weeks (weighing 18 - 22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected for the experiment and raised in an SPF - level environment. The experimental protocol was approved by the Animal Ethics Committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences (Approval number: DWLL(E)-2024 - 46). The method for constructing the model was as follows: Intraperitoneal injection of 3% sodium chloride solution (10 mL / kg, the solvent was purchased from Sinopharm Group) was performed daily for 7 consecutive days. Model verification was carried out on the 8th day.

[0026] Rotational test: The mice were placed on an electric rotating platform with a diameter of 30 cm (Shanghai Ruanlong Technology). They were rotated at 20 rpm for 10 minutes, and the number of vertigo attacks within 10 minutes was recorded (defined as the mouse's head tilting ≥ 45° or losing balance and falling). The success criterion for the model was that the number of vertigo attacks was ≥ 5 times / 10 minutes. The results showed that the average number of vertigo attacks in the model group was 7.2 ± 1.3 times / 10 minutes, which was significantly higher than that in the control group (0.5 ± 0.2 times / 10 minutes, P < 0.001). After the rotational test, the mice in the model group showed obvious balance disorders and decreased motor ability, while the mice in the control group behaved normally.

[0027] Auditory brainstem response (ABR) detection: An auditory brainstem response instrument (Tucker - Davis Technologies) was used to record the auditory threshold of mice in response to click stimuli (click sound, frequency 8 kHz) through subcutaneous electrodes. The success criterion for the model was that the threshold increased by ≥ 20 dB. The results showed that the auditory threshold of the control group mice was 25 ± 3 dB, and that of the model group mice was 48 ± 5 dB, which was significantly higher than that of the control group (P < 0.01). The ABR waveform diagram showed that the waveform amplitude of the model group mice was significantly reduced and the latency was prolonged, indicating impaired auditory function. The results are shown in Table 2.

[0028] Table 2 Auditory brainstem response (ABR) detection

[0029] Group Auditory threshold (dB) Waveform amplitude (μV) Latency (ms) P value Control group 25 ± 3 1.2 ± 0.1 2.5 ± 0.2 - Model group 48 ± 5 0.6 ± 0.1 3.8 ± 0.3 <0.01

[0030] After the experiment, all mice were euthanized by carbon dioxide inhalation (in line with the AVMA euthanasia guidelines).

[0031] Example 4

[0032] To detect the pharmacodynamics and safety evaluation of the prepared materials, the experimental steps were as follows:

[0033] Grouping and dosing regimen: Ninety model mice were randomly divided into 3 groups (n = 30 / group):

[0034] (1) Blank control group: Intravenous injection of normal saline (200 μL / 20 g body weight) via the tail vein.

[0035] (2) Drug group: Administered with unmodified antibody-loaded liposomes (0.1 mg / kg).

[0036] (3) Targeted drug group: Administered with VEGFR-2 antibody-modified liposomes loaded with drugs (0.1 mg / kg). Administration frequency: Twice a week (at 9:00 am on Monday and Thursday) for 4 weeks. During the liposome preparation process, the encapsulation efficiencies of dexamethasone and betahistine were 92.5 ± 2.1% and 88.7 ± 1.8% respectively, and the drug loadings were 5.2 ± 0.3 μg / mg and 15.6 ± 0.5 μg / mg respectively.

[0037] Inner ear drug concentration: Mice (n = 10 / group) were sacrificed 24 hours after the last administration, cochlear tissue homogenates were taken, and drug concentrations were detected by HPLC. Chromatographic conditions: C18 column (4.6 mm × 150 mm, 5 μm), mobile phase was acetonitrile-water (60:40, v / v), flow rate was 1.0 mL / min, and the detection wavelength was 240 nm. The results showed that the dexamethasone concentration in the targeted group was 12.5 μg / g and the betahistine concentration was 37.2 μg / g; those in the non-targeted group were 3.8 μg / g and 12.2 μg / g respectively (P < 0.01, t-test). The results are as Figure 4 shown.

[0038] Vertigo attack frequency: The number of attacks per week was recorded through a video tracking system (EthoVision XT). In the targeted group, it decreased from 8.2 ± 1.1 times / week at baseline to 2.1 ± 0.5 times / week (a 74.4% decrease), and in the non-targeted group, it decreased from 8.0 ± 1.0 times / week to 5.6 ± 0.7 times / week (a 30.0% decrease) (P < 0.001, ANOVA). Video analysis showed that the movement trajectories of mice in the targeted group were smoother and the number of vertigo attacks decreased significantly. The results are as Figure 5 shown.

[0039] Hearing function recovery: The ABR threshold in the targeted group recovered from 48 ± 5 dB to 23 ± 3 dB (an improvement of 25 ± 3 dB), and in the non-targeted group, it recovered to 40 ± 4 dB (an improvement of 8 ± 2 dB) (P < 0.01). The ABR waveform diagram showed that the waveform amplitude of mice in the targeted group recovered significantly and the latency shortened, indicating a significant improvement in hearing function. The results are shown in Table 3.

[0040] Table 3 Detection of auditory brainstem response (ABR) recovery

[0041] Group Initial ABR threshold (dB) ABR threshold after recovery (dB) Improvement amplitude (dB) Waveform amplitude (μV) Latency (ms) P value Targeted group 48.25 ± 0.52 23.12 ± 0.48 25.13 ± 0.50 1.15 ± 0.05 2.45 ± 0.10 <0.01 Non-targeted group 48.30 ± 0.50 40.18 ± 0.45 8.12 ± 0.30 0.65 ± 0.04 3.75 ± 0.12 <0.01

[0042] Systemic toxicity: Complete blood count (Sysmex XN-1000) and blood biochemistry (ALT, AST, BUN) of mice were detected on days 0, 1, 3, and 5. The results showed that there were no statistically significant differences in each group of indicators (P>0.05). The results of the complete blood count showed that the white blood cell count, red blood cell count, and platelet count were all within the normal range; the results of blood biochemistry showed that the levels of ALT, AST, and BUN did not change significantly, indicating that the drug did not cause obvious liver and kidney function damage. The results are as Figure 6 shown.

[0043] Example 5

[0044] To verify the targeting mechanism of the drug-loaded liposomes, the detection steps are as follows:

[0045] (1) Immunofluorescence co-localization: Frozen sections of the cochlear tissues of mice in the targeting group were taken, stained with VEGFR-2 primary antibody (Abcam ab2345) and FITC-labeled secondary antibody (Invitrogen A11008), and the nuclei were counterstained with DAPI. Observation with a confocal microscope (Zeiss LSM 880) showed that the co-localization rate of liposomes (DiR fluorescence-labeled) and stria vascularis cells (VEGFR-2 positive) was ≥85%. Image analysis showed that the liposomes were mainly distributed in the stria vascularis region and highly overlapped with VEGFR-2 positive cells.

[0046] (2) Competitive inhibition experiment: After pre-injecting an excessive amount of free VEGFR-2 antibody (1 mg / kg), the inner ear drug concentration in the targeting group decreased to the level of the non-targeting group (4.1±0.7 μg / g), confirming that the targeting effect depends on the specific binding of antibody-receptor. The inner ear drug concentration in the competitive inhibition group was significantly lower than that in the non-inhibited group (P<0.01), further verifying the specificity of the targeted liposomes.

Claims

1. A targeted drug delivery system for Meniere's syndrome, characterized in that: include: Liposome carrier composed of distearoylphosphatidylcholine (DSPC) and cholesterol in a 4:1 molar ratio; Vascular endothelial growth factor receptor-2 (VEGFR-2) antibody modified on the surface of liposomes by EDC / NHS covalent coupling technology; Dexamethasone and betahistine are encapsulated in the liposomes, wherein the molar ratio of dexamethasone to betahistine is 1:3; The average particle size of the liposome is 120.9 nm, the encapsulation efficiency is ≥92.1%, and the particle size growth is ≤10% when stored at 4°C for 30 days.

2. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The VEGFR-2 antibody was covalently coupled to the liposome surface via EDC and N-hydroxysuccinimide (NHS), and the molar ratio of the antibody to the liposome was 1:

50.

3. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The particle size distribution of the liposome carrier is 120.5 nm, the Zeta potential is -15.2 mV, and the transmission electron microscope shows that the liposome carrier has a complete spherical structure.

4. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The encapsulation efficiency of dexamethasone was 92.5±2.1%, and the loading amount was 5.2±0.3 μg / mg; the encapsulation efficiency of betahistine was 88.7±1.8%, and the loading amount was 15.6±0.5 μg / mg.

5. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The drug delivery system can penetrate the inner ear blood labyrinth barrier through the specific binding of VEGFR-2 antibodies and accurately deliver drugs to the inner ear stria vascular cells, so that the inner ear drug concentration reaches 12.5 μg / g (dexamethasone) and 37.2 μg / g (betahistine).

6. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The drug delivery system can reduce the frequency of vertigo attacks by 74.4% and improve the auditory brainstem response (ABR) threshold by 25.3 dB in an animal model.

7. The targeted drug delivery system for Meniere's syndrome according to claim 1, characterized in that: The drug delivery system is stored at 4°C for 30 days, with a particle size increase of ≤10%, and exhibits good stability at 25°C and 37°C.

8. A method for preparing the Meniere's syndrome targeted drug delivery system according to any one of claims 1 to 7, characterized in that: The following steps are involved: Distearoylphosphatidylcholine (DSPC) and cholesterol were dissolved in a chloroform-methanol mixed solvent at a molar ratio of 4:1 and a lipid film was formed by rotary evaporation; The lipid film was hydrated in a constant temperature water bath at 55°C, phosphate buffered saline (PBS) containing dexamethasone and betahistine was added, and the drug-loaded liposomes were obtained by ultrasonic treatment; The VEGFR-2 antibody was covalently coupled to the surface of drug-loaded liposomes via EDC / NHS, and the targeted drug-loading system was obtained after purification; The targeted drug delivery system was mixed with 5% mannitol and freeze-dried to obtain a freeze-dried powder preparation.

9. Use of the Meniere's syndrome targeted drug delivery system according to any one of claims 1 to 7 in the preparation of a drug for treating Meniere's syndrome, characterized in that: The drug delivery system was administered by intravenous injection at a dose of 0.1 mg / kg, twice a week for 4 weeks.

10. The use according to claim 9, characterized in that: The drug-carrying system can significantly increase the drug concentration in the inner ear, reduce the frequency of vertigo attacks, and improve hearing function without significant systemic toxicity.

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