Carrier-free nanoparticles with synergistic effect of Chinese and western medicines as well as preparation method and application of carrier-free nanoparticles

Abstract

The invention relates to the field of biological medicine, in particular to a carrier-free nanoparticle which is formed by self-assembly of cyclosporine A and costunolide through intermolecular weak interaction. The invention also provides a preparation method of the traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of medicines for treating xerophthalmia. The carrier-free nano eye drops based on the synergistic effect of Chinese and western medicines are used for anti-inflammatory treatment of the xerophthalmia, a new strategy is provided for precise diagnosis and treatment of the xerophthalmia, the problems of single target spot, limited curative effect and the like in traditional treatment are solved, and more efficient inflammation control is expected to be achieved. While the curative effect is improved, the tolerance risk caused by long-term use is reduced, double optimization of the curative effect and safety is realized, and the limitation of traditional single drug treatment is broken through. By adopting the carrier-free nano preparation, the delivery efficiency of the medicine on the ocular surface is effectively improved, the bioavailability is enhanced, powerful support is provided for improving the curative effect of the medicine, and the carrier limitation bottleneck is broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a carrier-free nanoparticle with synergistic effect of traditional Chinese medicine and western medicine, and a preparation method and application thereof. BACKGROUND

[0002] Dry eye disease (DED) is a high-incidence ocular surface disease worldwide, and is showing a trend of younger age, seriously affecting the quality of life of patients. The existing treatment mainly uses artificial tears, CsA and other local administration, although the curative effect is significant, but there are tolerance problems. For example:

[0003] (1) Traditional single drug CsA eye drops:

[0004] Technical features: classic formula 0.05% CsA + castor oil / polysorbate 80, etc., forming micron-nano emulsion droplets (100-200 nm). Its mechanism of action is to inhibit T cell activation, down-regulate IL-2 / TNF-α, restore tear secretion and goblet cell density. Approved scheme: BID, treatment course ≥3-6 months. Main disadvantages: (1) Corneal penetration rate ≈5%, low bioavailability. (2) Contains castor oil, preservatives, 15-20% of patients have burning / stinging, conjunctival hyperemia. (3) Slow onset (≥4-6 weeks), long-term high-frequency administration is required, and the compliance is poor. (4) Long-term retention of oily carrier, risk of meibomian gland blockage and corneal epithelial microvilli damage.

[0005] (2) Carrier-containing nano eye drops (already on the market or in clinical research stage)

[0006] Technical features (take representative products as an example): Restasis® (0.05% CsA nanoemulsion) - castor oil emulsion system; Cequa® (0.09% CsA nanomicelles) - oil-free amphiphilic block copolymer; Cycloome® (0.05% CsA microemulsion) - smaller particle size 20-50 nm. Through "carrier-drug" complex, the corneal retention and transmembrane transport are improved, and the theoretical bioavailability is increased by 2-5 times. Some preparations can reduce irritation and reduce administration frequency. Main disadvantages: The carrier itself may still cause irritation: castor oil, ethanol, high concentration of surfactants may cause conjunctival hyperemia and blurred vision. Carrier stability problems: emulsion aggregation, lipid oxidation, particle size increases during long-term storage, affecting the uniformity of curative effect. Carrier degradation products / auxiliaries (such as polysorbate, benzalkonium chloride) can induce chronic inflammation or corneal toxicity. The process is complex, the cost is high, and the sterile filling is more difficult than traditional emulsion in large-scale production.

[0007] (3) Common bottlenecks of the two

[0008] CsA itself is extremely difficult to dissolve in water, has a large molecular weight (1202 Da), and corneal penetration resistance still exists. Whether traditional emulsions or nano systems with carriers, as long as they contain oil or high concentrations of surfactants, it is difficult to completely eliminate the "burning / painful" complaints. In the patient experience, multiple daily dosing + long-term treatment, compliance is still a real problem.

[0009] The development of nanomedicine opens up a new path for the treatment of dry eye syndrome, and the carrier-free nano preparation can improve the efficiency of drug delivery. However, there is no report on a carrier-free nanoparticle for the treatment of dry eye syndrome, its preparation method and application. SUMMARY

[0010] The purpose of the present application is to provide a carrier-free nanoparticle for the synergistic effect of traditional Chinese medicine and Western medicine mainly for the anti-inflammatory treatment of dry eye syndrome (DED) to play a role in tear film repair, inflammatory factor regulation and ocular surface microenvironment improvement. The present application also provides a preparation method of the nanoparticle and its application in the treatment of dry eye syndrome.

[0011] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0012] The construction idea of "carrier-free nanoparticle of traditional Chinese medicine and Western medicine co-assembly" is adopted: cyclosporine A (CsA, Western medicine) and costunolide (COS, traditional Chinese medicine) are self-assembled through intermolecular weak interaction (hydrogen bond, hydrophobic bond, etc.), without traditional carriers. The technical features of the present application can be summarized as "three no three self one synergy", as follows:

[0013] 1) Three no

[0014] No carrier: completely get rid of any additional aids such as surfactants, lipids, polymers, etc.

[0015] No organic solvent: one-step self-assembly in aqueous phase throughout, green process.

[0016] No chemical modification: CsA and COS maintain the original drug structure without covalent coupling.

[0017] 2) Three self

[0018] Self-identification: the cyclic hydrophobic skeleton of CsA and the sesquiterpene lactone hydrophobic surface of COS are complementary and embedded through hydrophobic bonds.

[0019] Self-assembly: hydrogen bonds (N-methyl of CsA and conjugated double bond / aromatic ring of COS) provide directionality to drive the formation of spherical nanoparticles with a particle size of <200 nm.

[0020] Self-stabilization: the "core-shell" structure of hydrophobic core + hydrophilic shell has steric hindrance, and the particle size changes <10% at room temperature.

[0021] 3) a synergy

[0022] CsA (immunosuppression) + COS (anti-inflammatory) are synergistically co-delivered in the same nanoparticle, which can shorten the onset time and reduce the frequency of administration.

[0023] Based on the above technical scheme, the first aspect of the present application provides a carrier-free nanoparticle (CsA@COS) formed by self-assembly of cyclosporine A (CsA) and costunolide (COS) through intermolecular weak interaction.

[0024] Further, the intermolecular weak interaction is a carbon-hydrogen bond, a hydrophobic bond, etc.

[0025] Further, in the carrier-free nanoparticle, the molar ratio of cyclosporine A to costunolide is 2:1.

[0026] Further, the preparation method of the carrier-free nanoparticle is to dissolve cyclosporine A and costunolide in ethanol at a molar ratio of 2:1, then slowly add the cyclosporine A solution to distilled water under stirring, slowly add the costunolide solution, mix the two solutions thoroughly, and further self-assemble the complex in the solution to form the carrier-free nanoparticle CsA@COS.

[0027] The second aspect of the present application provides a preparation method of the carrier-free nanoparticle (CsA@COS) as described above, which is to dissolve cyclosporine A and costunolide in ethanol at a molar ratio of 2:1, then slowly add the cyclosporine A solution to distilled water under stirring, slowly add the costunolide solution, mix the two solutions thoroughly, and further self-assemble the complex in the solution to form the carrier-free nanoparticle CsA@COS.

[0028] Further, the stirring speed is 500-800 rpm.

[0029] Further, after the two solutions are added, continue to stir for a period of time, and the stirring time is 10-15 min.

[0030] Further, the concentration of the cyclosporine A solution is 2 mM; and the concentration of the costunolide solution is 1 mM.

[0031] Further, the preparation method of the carrier-free nanoparticle: on a magnetic stirrer, 100 μL of 2 mM cyclosporine A solution is slowly added to 1 mL of distilled water, the dropping speed is controlled, then 100 μL of 1 mM costunolide solution is added, and the two solutions are fully mixed at a uniform and slow speed. The stirring speed is 500-800 rpm, no heating is needed, and the operation can be carried out at room temperature. After the dropping is completed, continue to stir for 10-15 min. The compound is further self-assembled in the solution to form CsA@COS nanoparticles. During the stirring process, CsA and costunolide gradually combine to form a compound through intermolecular interaction.

[0032] In a third aspect of the present application, the carrier-free nanoparticle (CsA@COS) is applied to the preparation of a drug for treating dry eye.

[0033] Further, the drug for treating dry eye is eye drops.

[0034] In a fourth aspect of the present application, a carrier-free nanoparticle eye drop is provided. The carrier-free nanoparticle (CsA@COS) is purified and concentrated, and the concentrated solution is diluted with physiological saline to obtain a CsA@COS nanoparticle solution. The pH value of the solution is adjusted to be between 6.5 and 7.5.

[0035] Further, the following purification methods can be used according to the organic solvent, such as dialysis method. The formed nanoparticle solution is transferred to a dialysis bag, and deionized water is used for dialysis to remove residual organic solvents and uncoated CsA. After dialysis, the nanoparticle solution is centrifuged or ultrafiltered to concentrate the nanoparticles to obtain a CsA@COS nanoparticle dispersion liquid for subsequent preparation of eye drops.

[0036] Further, the preparation of the eye drops: the concentrated solution is diluted with an appropriate amount of physiological saline to obtain a CsA@COS nanoparticle solution. A buffer (such as a phosphate buffer) is added to adjust the pH value of the solution to be between 6.5 and 7.5 to meet the physiological environment of the eye.

[0037] Further, the optimal use concentration of the CsA@COS eye drops is 15 μM, based on the concentration of CsA.

[0038] In a fifth aspect of the present application, the carrier-free nanoparticle eye drop is applied to the preparation of a drug for treating dry eye.

[0039] Based on current research progress and clinical needs, the use of CsA (cyclosporine A) and COS (costunolide) in the treatment of dry eye syndrome with western medicine synergistic effect of carrier-free nano eye drops mainly reflects the following aspects:

[0040] 1) Anti-inflammatory and immune regulation

[0041] CsA: By inhibiting T cell activation and the release of inflammatory cytokines such as IL-2 and TNF-α, it can reduce the core inflammatory response of dry eye syndrome.

[0042] COS: As a natural anti-inflammatory ingredient, it can synergistically enhance the anti-inflammatory effect of CsA by blocking the NF-κB pathway, reducing immune damage to the cornea and conjunctiva.

[0043] 2) Promote tear and mucin secretion

[0044] CsA can directly stimulate lacrimal glands and conjunctival goblet cells, increase tear secretion and mucin production, and improve tear film stability.

[0045] 3) Advantages of carrier-free nanotechnology

[0046] High bioavailability: Carrier-free nano systems such as lipid nanoparticles or self-assembled nanomicelles can overcome the poor water solubility of CsA, significantly improve drug retention time and penetration rate on the ocular surface, and reduce the frequency of administration.

[0047] Reduce side effects: Avoid the burning sensation or blurred vision caused by traditional carriers of CsA such as castor oil, and improve patient compliance.

[0048] 4) Expand indications

[0049] Dry eye combined with autoimmune diseases: such as Sjogren's syndrome, thyroid-related eye disease, etc., CsA@COS nano preparations can take into account systemic immune abnormalities and local ocular inflammation.

[0050] Postoperative or drug-induced dry eye: Reduce the need for long-term hormone eye drops, and reduce the risk of hormone-induced glaucoma.

[0051] 5) Clinical potential and conversion prospects

[0052] Similar CsA nano preparations (such as Cyclasol®) have shown faster onset of action (2 weeks) and better tolerance than traditional emulsions, and the addition of COS can further shorten the course of treatment or reduce the concentration of CsA, reducing the potential toxicity of long-term medication.

[0053] In summary, this nano eye drop will bring a broader application prospect for the treatment of dry eye syndrome through the innovation of Chinese and western medicine synergy and drug delivery technology, and provide new ideas for expanding treatment strategies.

[0054] Compared with the prior art, the application has the advantages that:

[0055] In the CsA@COS carrier-free nanodrop eye drops of the application, CsA is used as an immunomodulator, and is safer than a corticosteroid in long-term use; COS is the main active ingredient of traditional Chinese medicine Saussurea, and has anti-inflammatory activity. The CsA@COS carrier-free nanodrop eye drops of the application combine CsA and COS for the first time, utilize the multi-target synergistic anti-inflammatory effect, and block the inflammatory cascade reaction through the complementary action of the synergistic mechanism of traditional Chinese medicine and western medicine. The core advantages are that:

[0056] 1. Carrier-free: The irritability is minimized by removing castor oil, surfactants and preservatives, the side effects caused by the oily carrier are eliminated, and the corneal affinity is improved;

[0057] 2. Dual-target anti-inflammatory: CsA inhibits T cells + COS blocks NF-κB, synergistically enhances the anti-inflammatory efficiency, and shortens the onset time;

[0058] 3. High permeability: The problem of hydrophobic drug penetration is solved, self-assembly <200 nm hydrophilic nanoparticles are used to prolong the corneal retention time and improve the bioavailability;

[0059] 4. Low frequency: Once a day can maintain an effective concentration, and the compliance is significantly better than the traditional BID scheme;

[0060] 5. Simple process: The carrier-free self-assembly system saves the complex emulsification-freeze drying process, and the cost is lower.

[0061] The application provides a carrier-free nanodrop eye drops based on the synergistic action of traditional Chinese medicine and western medicine for anti-inflammatory treatment of dry eye, provides a new strategy for precise diagnosis and treatment of dry eye, solves the problems of single target and limited efficacy in traditional treatment, and is expected to realize more efficient inflammation control. While improving the efficacy, the risk of resistance caused by long-term use is reduced, the dual optimization of efficacy and safety is realized, and the limitation of traditional single drug treatment is broken through. The carrier-free nanodrug of the application effectively improves the delivery efficiency of the drug on the ocular surface, enhances the bioavailability, provides strong support for improving the efficacy of the drug, and breaks through the bottleneck of carrier limitation. BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is a particle size diagram of the nanoparticle CsA@COS eye drop in Example 2;

[0063] FIG. 2 is a scanning electron microscope diagram of the nanoparticle CsA@COS eye drop in Example 3;

[0064] FIG. 3 is a stability diagram of the nanoparticle CsA@COS eye drop in Example 4;

[0065] FIG. 4 is a molecular docking diagram of the nanoparticle CsA@COS eye drop in Example 5;

[0066] Figure 5 is a cell viability graph of HCE-T cells treated with different concentrations in Example 6, including CsA, COS, and CsA@COS eye drops;

[0067] Figure 6 is a confocal laser scanning microscope (CLSM) image of HCE-T cells treated with nanoparticle CsA@COS eye drops for 6 hours in Example 7. DETAILED DESCRIPTION

[0068] The specific embodiments provided by the present application are described in detail below with reference to the accompanying examples.

[0069] Example 1: Preparation of nanoparticle CsA@COS eye drops

[0070] 1) Solution preparation: 11.616 mg of cyclosporine A (molecular weight 232.32) was accurately weighed and added to 500 μL of organic solvent ethanol, and stirred on a magnetic stirrer until completely dissolved, to prepare a 100 mM cyclosporine A stock solution, which was then diluted with ethanol to 2 mM for use. 12.0261 mg of costunolide (molecular weight 1202.61) was weighed and dissolved in 100 μL of ethanol to prepare a 100 mM costunolide stock solution, which was then diluted with ethanol to 1 mM for use.

[0071] 2) Mixing and self-assembly: 100 μL of 2 mM cyclosporine A solution was slowly added to 1 mL of distilled water on a magnetic stirrer, controlling the dropwise speed, and then 100 μL of 1 mM costunolide solution was added, ensuring uniform and slow dropwise speed to allow the two solutions to mix thoroughly. The stirring speed and stirring time have an important influence on the formation of the complex. The stirring speed is generally 500-800 rpm, without heating, and the complex can be formed at room temperature. After the dropwise addition is completed, continue to stir for a period of 10-15 min. The complex is further self-assembled in the solution to form CsA@COS nanoparticles. During the stirring process, CsA and costunolide gradually combine to form a complex through intermolecular interaction forces.

[0072] 3) Purification and concentration: the formed nanoparticle solution can be purified according to the following purification methods using organic solvents, such as dialysis method. The nanoparticle solution is transferred to a dialysis bag and dialyzed with deionized water to remove residual organic solvents and uncoated CsA. After dialysis, the nanoparticle solution is centrifuged or ultrafiltered to concentrate the nanoparticles, obtaining a CsA@COS nanoparticle dispersion solution for subsequent preparation of eye drops.

[0073] 4) Preparation of eye drops: The concentrated solution is diluted with an appropriate amount of normal saline to obtain a CsA@COS nanoparticle solution. Add a buffer (such as phosphate buffer) to adjust the pH of the solution to between 6.5 and 7.5 to meet the physiological environment of the eye.

[0074] Example 2: Particle size diagram of nanoparticle CsA@COS

[0075] Using a particle size analyzer, 1 mL of nanoparticle solution is added to a cuvette and placed in the particle size analyzer for measurement. Each sample is measured 3-5 times, and the average value is taken as the final result. The measurement temperature is 25°C, and the temperature needs to be equilibrated for each measurement. As shown in Figure 1 , the average particle size of CsA@COS nanoparticles is mainly distributed in the range of 170-190 nm, which meets the screening standard of particle size <200 nm.

[0076] Example 3: Scanning electron micrograph of nanoparticle CsA@COS

[0077] 1 mL of nanoparticle dispersion is added dropwise to a silicon wafer, naturally dried, and then gold spraying is performed to increase the conductivity of the sample. Then the sample is placed in a scanning electron microscope to observe the morphology, size and dispersion of the nanoparticles at different magnifications. In this experiment, a Zeiss Sigma 300 field emission scanning electron microscope and an Oxford Xplore 30 energy spectrometer are used, as shown in Figure 2 , the SEM image can intuitively show the actual morphology of the nanoparticles, providing important information for further understanding the properties of the nanoparticles.

[0078] Example 4: Stability diagram of nanoparticle CsA@COS

[0079] In order to evaluate the stability of CsA@COS nanoparticles under different conditions, they are placed in different time (1d, 3d, 5d, 7d, 9d) respectively, and the changes of particle size and PDI are measured regularly. Through the stability study, the shelf life of the nanoparticles can be determined, as shown in Figure 3 , the particle size of the nanoparticles does not change significantly during storage, reflecting the good uniformity and stability of the size distribution of CsA@COS nanoparticles.

[0080] Example 5: Molecular docking diagram of nanoparticle CsA@COS

[0081] Molecular docking technology was used for simulation analysis. First, the molecular structure information of CsA and COS was obtained from the relevant chemical database. Then, molecular docking was realized by using AutoDock 4.2.6 software package, the coordinates of the docking box center were set as the center of the small molecule to completely cover the entire small molecule structure, the grid number in XYZ direction was set to 30x30x30, the docking number was set to 100, and the rest of the parameters used the default value. Energy optimization used Amber14 force field, and the optimization process was divided into two steps: first, 1000 steps of steepest descent method optimization, and then 5000 steps of conjugate gradient method for further optimization of the structure, and the final result was used as the model for subsequent analysis.

[0082] The complex conformation obtained by molecular docking of CsA and COS is shown in FIG. 1, and the predicted binding energy between CsA and COS molecules is -4.200 kcal / mol. The two molecules are mainly combined by hydrogen bond interaction, and a total of 4 carbon hydrogen bonds are formed. In addition, there are 3 hydrophobic interactions (alkyl-alkyl interaction). The docking results show that CsA can stably combine with COS to form a complex. Figure 4

[0083] FIG. 1 shows the complex conformation obtained by molecular docking of CsA and COS.

[0084] Human corneal epithelial cells HCE-T were selected for cell experiments. First, the toxicity of the nanoparticles to HCE-T cells was evaluated, and the MTT method was used to detect the proliferation inhibition effect of different concentrations of nanoparticles on HCE-T cells. HCE-T cells (1x10 5 HCE-T cells (1x10 Figure 5 ​As shown, the cell viability of HCE-T cells treated by different concentrations of CsA, COS and CsA@COS eye drops, by the method of concentration gradient step by step, to screen the best use concentration of CsA@COS eye drops is 15 μM, using this concentration on HCE-T cells almost no toxicity.

[0085] Example 7: Confocal laser scanning microscope (CLSM) images of HCE-T cells treated by nanoparticle CsA@COS eye drops for 6 hours.

[0086] HCE-T cells were seeded in 24-well plates at a coverage of about 20%, and placed in a 5% CO2, 37°C incubator for overnight culture. Remove the old culture medium, add the following drug-containing but serum-free culture medium: Dil fluorescent probe labeled CsA@COS eye drops, the concentration of CsA@COS eye drops is 15 μM, return to the incubator for continue to avoid light culture for 6 hours. After incubation, the cells were fixed and DAPI staining. After mounting, confocal laser scanning microscope (CLSM) was used to evaluate the uptake of nanoparticles by cells. The results are shown in Figure 6 As shown, the orange-red fluorescence is mostly distributed around the nucleus, indicating that the nanoparticles CsA@COS eye drops prepared by the patent have higher cell uptake efficiency.

[0087] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A carrier-free nanoparticle, characterized in that, The cyclosporine A and the costunolide are self-assembled by intermolecular weak interaction to form the carrier-free nanoparticles.

2. The carrier-free nanoparticle of claim 1, wherein, The intermolecular weak interaction is a carbon-hydrogen bond and a hydrophobic bond.

3. The carrier-free nanoparticle of claim 1, wherein, The molar ratio of the cyclosporine A to the costunolide in the carrier-free nanoparticles is 2:

1.

4. A method of preparing a carrier-free nanoparticle as claimed in claim 1, wherein, The cyclosporine A and the costunolide are dissolved in ethanol respectively with a molar ratio of 2:1, then the cyclosporine A solution is slowly added into distilled water under stirring, and then the costunolide solution is slowly added into the cyclosporine A solution, and the two solutions are mixed thoroughly, and the complex is further self-assembled in the solution to form the carrier-free nanoparticles.

5. The production method according to claim 4, characterized by, The stirring speed is 500-800 rpm.

6. The preparation method according to claim 4, characterized in that, After the two solutions are added completely, the stirring is continued for 10-15 min.

7. The preparation method according to claim 4, characterized in that, The concentration of the cyclosporine A solution is 2 mM, and the concentration of the costunolide solution is 1 mM.

8. The use of the carrier-free nanoparticles as claimed in any one of claims 1-3 in the preparation of a medicament for treating dry eye.

9. A carrier-free nanodrop eye drop solution, characterized in that, The carrier-free nanoparticles as claimed in any one of claims 1-3 are purified and concentrated, the concentrated solution is diluted with physiological saline to obtain a nanoparticle solution, and the pH value of the solution is adjusted to be between 6.5 and 7.

5.

10. The use of the carrier-free nanoparticle eye drop as claimed in claim 9 in the preparation of a medicament for treating dry eye.

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