Ion-responsive plumbagin nanoparticles, preparation method and application thereof

Abstract

The application provides an ion-responsive linarin nanoparticle and a preparation method and application thereof, and comprises the following steps: preparing GC-PLGA nanoparticles by nanometer precipitation method through glycol chitosan and polylactic acid-glycolic acid copolymer, incubating the GC-PLGA nanoparticles with linarin to obtain GC-PLGA-LIN nanoparticles; reacting gellan gum with methacrylic anhydride to obtain methylated gellan gum; reacting the methylated gellan gum with arginine-glycine-aspartic acid peptide to obtain MeGG-RGD; reacting the MeGG-RGD with hyaluronic acid to obtain MeGG-RGD-HA; mixing and resolubilizing the MeGG-RGD-HA and the GC-PLGA-LIN nanoparticles in water, and self-assembling to obtain the ion-responsive linarin nanoparticle MRH-GPLNP. The nanoparticle can exhibit a better protection effect on linarin, and can improve the bioavailability and stability of the linarin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to an ion-responsive plumbagin nanoparticle for protecting corneal epithelial cells and relieving high-osmotic stress of corneal epithelial cells, and a preparation method and application thereof. BACKGROUND

[0002] A series of related eye diseases are induced by the long-term exposure of corneal epithelial cells and the like to an environment induced by a high-osmotic medium, such as dry eye (DE), Sjogren's syndrome (SS), Sjogren's syndrome-related keratoconjunctivitis sicca (SS-KCS), and laser refractive surgery-laser-assisted in situ keratomileusis (LASIK)-induced neurotrophic epitheliopathy (LNE), which also constitutes a disease related to high-osmotic stress. Among them, DE is an ocular surface disease that affects millions of people worldwide and is a serious health problem worldwide. According to the International TFOS Tear Film and Ocular Surface Society Deliberations II (TFOS DEWS II), DE is defined as "an ocular surface multifactorial disease characterized by a loss of tear film homeostasis with associated ocular symptoms in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles". Common symptoms of DE, such as foreign body sensation, burning and stinging, can affect the productivity of patients and reduce the quality of life; if not treated in time through appropriate treatment methods, the chronic inflammatory state involved in DE can lead to decreased vision or blindness.

[0003] An important mechanism of DE is the link between tear hyperosmolarity and the severity of inflammation. The core mechanism of the disease is evaporation-induced tear hyperosmolarity, which triggers countless signal events and release of proinflammatory mediators in the surface epithelial cells. The combined effects of high-osmotic stress and inflammation are believed to cause epithelial cell damage and loss of barrier function in the cornea and conjunctiva. DE increases with age, and women are more affected. In fact, DE recurs in tear evaporation, which subsequently leads to an increase in tear film osmolarity, and the high-osmotic pressure of the surface epithelial cells leads to water and electrolyte imbalance between the intracellular and extracellular compartments, thereby reducing the cell volume. The dramatic shrinkage of the cells can lead to changes in cell survival, cell membrane and cytoskeleton integrity, and denaturation of cytoplasmic proteins.

[0004] The tear environment with high-osmoticity can produce a large amount of proinflammatory markers such as tumor necrosis factor and some matrix metalloproteinases such as MMP-9 or MMP-7. Therefore, long-term exposure to tear film hyperosmolarity can be the basis of the disease. This in vivo increase is accompanied by a decrease in the barrier function of the corneal epithelium, increasing the likelihood of pathogenic infiltration and worsening of inflammatory conditions. The lack of correlation between DE symptoms and tear film osmolarity can be due to the time-dependent variability of tear film osmolarity concentration or technical limitations in obtaining reproducible tear film osmolarity values.

[0005] Effective osmoprotectants need to be found to manage DE. Osmoprotectants are biocompatible compounds with osmolytic activity that can change the water-absorbing capacity of cells. They are usually small, neutral and hydrophilic substances that do not interfere with cell function. Each osmoprotectant works in a different way, with a wide variety of kinetics and intracellular internalization. When a hyperosmotic environment occurs or hyperosmotic stress can cause cell damage, the cell can be given the uptake of osmoprotectant substances. In addition, some of them can even be internalized in the cell by specific mediated transporters before the hyperosmotic environment. The amount and time of substances that cells can retain play a crucial role in their effectiveness.

[0006] Linarin (LIN), belonging to flavonoids, is a representative component of the traditional Chinese medicine Flos Genkwa, which is commonly used to treat eye diseases. Studies have shown that Flos Genkwa can be used for red and swollen eyes, eye nebula, dark eyes due to liver deficiency, and blurred vision. Linarin has various biological activities such as antibacterial, anti-aging, anti-hyperosmotic, anti-irritation, bidirectional regulation of the body, anti-fatigue, etc.

[0007] However, linarin is a heat-sensitive compound, which is easily degraded by heat, insoluble in water, and has poor lipid solubility, resulting in its application being limited. SUMMARY

[0008] The purpose of the present application is to provide an ion-responsive linarin nanoparticle and a preparation method and application thereof, which can exhibit better protection to linarin and improve the bioavailability and stability of linarin.

[0009] The present application is realized by the following technical solutions:

[0010] A preparation method of an ion-responsive linarin nanoparticle, comprising:

[0011] GC-PLGA nanoparticles are prepared by nanoprecipitation method through glycol chitosan and polylactic acid-glycolic acid copolymer, the GC-PLGA nanoparticles are incubated with linarin, and GC-PLGA-LIN nanoparticles are obtained;

[0012] Methylated gellan gum is obtained by reacting gellan gum with methacrylic anhydride; MeGG-RGD is obtained by reacting the methylated gellan gum with arginine-glycine-aspartic acid peptide; and MeGG-RGD-HA is obtained by reacting MeGG-RGD with hyaluronic acid;

[0013] MeGG-RGD-HA and GC-PLGA-LIN nanoparticles are mixed and dissolved in water, self-assembled, and ion-responsive linarin nanoparticles MRH-GPLNP are obtained.

[0014] Preferably, the glycol chitosan and polylactic acid-glycolic acid are prepared into GC-PLGA nanoparticles by nano-precipitation method, specifically:

[0015] The glycol chitosan and polylactic acid-glycolic acid are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is added to water to obtain a suspension, and the organic solvent in the suspension is evaporated, followed by solid-liquid separation to obtain the GC-PLGA nanoparticles.

[0016] Preferably, the GC-PLGA nanoparticles are incubated with linarin to obtain GC-PLGA-LIN nanoparticles, specifically: the GC-PLGA nanoparticles are resuspended in a 0.5-2.0 mg / mL linarin aqueous solution, and incubated at room temperature for 3-5 h, and then the obtained product is freeze-dried to obtain the GC-PLGA-LIN nanoparticles.

[0017] Preferably, the mass ratio of the polylactic acid-glycolic acid copolymer and the glycol chitosan is (50-100) mg:(15-50) mg.

[0018] Preferably, the gellan gum is reacted with methacrylic anhydride to obtain methylated gellan gum, specifically:

[0019] The gellan gum is dissolved in water, and methacrylic anhydride is added to the obtained solution, and the pH is adjusted to 8.0-8.2, and the reaction is carried out for 6-10 h to obtain a MeGG solution; the MeGG solution is dialyzed, and freeze-dried to obtain the methylated gellan gum, wherein the mass ratio of the gellan gum and the methacrylic anhydride is (1-1.5) g:(2-8) mL.

[0020] Preferably, the methylated gellan gum is reacted with arginine-glycine-aspartic acid peptide to obtain MeGG-RGD, specifically: the methylated gellan gum is dissolved in 2-(N-morpholino) ethanesulfonic acid buffer, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and the reaction is carried out for 2-4 h; then, arginine-glycine-aspartic acid peptide solution is added, and the reaction is carried out, and the obtained reaction solution is dialyzed and freeze-dried to obtain MeGG-RGD; wherein the ratio of the methylated gellan gum, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and arginine-glycine-aspartic acid peptide is (100-150) mg:(88-100) mg:(0.2-0.3) g:(35-75) μg.

[0021] Preferably, the MeGG-RGD is reacted with hyaluronic acid to obtain MeGG-RGD-HA, specifically:

[0022] Dissolve hyaluronic acid in water to obtain a HA solution; dissolve 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in water to obtain an EDC / NHS solution; add the EDC / NHS solution to the HA solution, stir and react for 1-2 hours, then add a MeGG-RGD NaHCO3 solution to undergo an amide reaction, dialyze, and freeze-dry to obtain MeGG-RGD-HA.

[0023] Preferably, the mass ratio of hyaluronic acid and MeGG-RGD is (100-150) mg:(15-30) mg.

[0024] The ion-responsive geniposide nanoparticles obtained by the preparation method.

[0025] The ion-responsive geniposide nanoparticles are used in the preparation of a drug for relieving hypertonic stress of corneal epithelial cells.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The present application encapsulates flavonoid geniposide by a combination of nano-precipitation, methyl modification and self-assembly, which shows better protection for geniposide and improves the utilization rate of geniposide. The physical cross-linked hydrogel of gellan gum (GG) often loses stability in the body after implantation due to the exchange of divalent cations with monovalent cations existing in the physiological environment at a high concentration. In order to overcome this shortcoming, the gellan gum backbone is methyl-modified. Due to the unique physiology and anatomy of the eye, the first obstacle of traditional drug delivery is that the anterior surface area of the cornea is limited, and the volume of eye drops available for the ocular surface is limited (about 30 muL). The methyl-modified gellan gum can form better ionic bonds with the cations of the ocular surface (such as corneal epithelial cells) without losing stability, and has the ionic response characteristics of sol-gel. It relieves the problem that most of the volume of the drug solution is eliminated due to the sudden increase in the volume of tears during the first reflex blinking process of traditional eye drops.

[0028] The biological and therapeutic properties of chitosan (beta-(1-4)-2-amino-2-deoxy-D-glucan) include antibacterial and anticancer effects, as well as biodegradability, non-toxicity and low immunogenicity. However, its disadvantage is that it is only soluble in acidic solutions with a pH value lower than 6. In order to overcome this solubility limitation, the present application introduces glycol chitosan (GC), which has the properties of chitosan and good solubility in aqueous solutions.

[0029] In order to protect the drug from the degradation agent during the drug delivery process and promote its absorption, the present application introduces polylactic acid-glycolic acid copolymer (PLGA) as a drug nano-carrier, which is degraded into lactic acid and glycolic acid two monomer units when hydrolyzed in vivo, is a natural metabolite, is easy to be discharged out of the body in the form of carbon dioxide and water, and is non-toxic.

[0030] The present application targets the nanoparticles containing the arginine-glycine-aspartic acid peptide Arg-Gly-Asp (RGD) to the eye, which can be connected with the fibronectin accumulated in the corneal epithelium after injury, and the RGD motif on the fibronectin binds to the surface alpha v Beta 1 integrin of the corneal epithelial cells, promotes the release of epidermal growth factor (EGF), stimulates cell spreading and migration, and plays an important role in treating corneal epithelial defects.

[0031] On the ocular surface, the viscosity of the tear film containing hyaluronic acid (HA) decreases between blinks, thereby evenly distributing the tear film. Once stationary, it regains a higher viscosity, thereby prolonging its residence time on the ocular surface. Hyaluronic acid is rich in hydroxyl groups that attract water molecules, thereby thickening and stabilizing the tear film and reducing mechanical trauma to the ocular surface through lubrication and aiding re-epithelialization.

[0032] In summary, the present application designs nanoparticles containing the above multifunctional properties by a. preparing methylated modification of gellan gum, covalently binding with RGD, and amidating with HA; b. nanoprecipitating ethylene glycol chitosan and polylactic acid-glycolic acid, then co-incubating with geniposide, and finally mixing and resolubilizing a and b nanoparticles for self-assembly, so that the nanoparticles have protective effects such as promoting proliferation and growth, restoring migration, adjusting cell size, regulating the intrinsic mode of cell growth, and other cell pathways of human SV40 immortalized corneal epithelial cells under hypertonic stress conditions. Due to its small size effect, the carrier has a larger specific surface area. Therefore, the geniposide nanoparticles have higher stability, biocompatibility, higher penetration protection, and have health care and therapeutic effects such as protecting ocular corneal epithelial cells and relieving hypertonic stress response. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is an atomic force microscope (AFM) two-dimensional planar image of MRH-GPLNP prepared in Example 1 in an aqueous solution;

[0034] Figure 2 is an atomic force microscope (AFM) 3-D stereoscopic image of MRH-GPLNP prepared in Example 1 in an aqueous solution;

[0035] Figure 3 is a scanning electron microscope (SEM) image (x100K) of MRH-GPLNP prepared in Example 1 in an aqueous solution;

[0036] Figure 4 is the size size and skewness distribution image of MRH-GPLNP prepared in Example 1;

[0037] Figure 5 is the Fourier transform infrared spectrogram of GG and MeGG in Example 1;

[0038] Figure 6 is the nuclear magnetic resonance hydrogen spectrum (1H NMR) graph of GG in Example 1;

[0039] Figure 7 is the nuclear magnetic resonance hydrogen spectrum (1H NMR) graph of MeGG in Example 1;

[0040] Figure 8 is the X-ray photoelectron spectroscopy (XPS) spectrum analysis graph of MeGG in Example 1;

[0041] Figure 9 is the X-ray photoelectron spectroscopy (XPS) spectrum analysis graph of MeGG-RGD in Example 1;

[0042] Figure 10 is the zeta potential graph of PLGA, GC-PLGA, GC-PLGA-LIN, MRH-GPLNP in Example 1;

[0043] Figure 11 is the sol diagram of MRH-GPLNP / ultrapure water group (A), free morin / ultrapure water group (B), MRH-GPLNP / artificial tear fluid group (C), gellan gum / artificial tear fluid group (D);

[0044] Figure 12 is the sol-gel phase inversion diagram of MRH-GPLNP / ultrapure water group (A), free morin / ultrapure water group (B), MRH-GPLNP / artificial tear fluid group (C), gellan gum / artificial tear fluid group (D);

[0045] Figure 13 is the effect of MRH-GPLNP prepared in Example 1 on the proliferation of HCEC cells under hyperosmotic stress conditions;

[0046] Figure 14 is the effect of MRH-GPLNP prepared in Example 1 on the cell size of HCEC cells under hyperosmotic stress conditions;

[0047] Figure 15 is the effect of MRH-GPLNP prepared in Example 1 on the cell migration of HCEC cells under hyperosmotic stress conditions. DETAILED DESCRIPTION

[0048] For a further understanding of the present application, the application will be described in conjunction with the embodiments, which are intended to further explain the features and advantages of the present application, and are not intended to limit the claims of the present application.

[0049] The preparation method of the ion-responsive geniposide nanoparticles of the present application comprises the following steps:

[0050] Step 1, preparation of ethylene glycol chitosan-poly(lactic-co-glycolic acid)-geniposide (GC-PLGA-LIN) nanoparticles

[0051] The GC-PLGA nanoparticles are prepared by the nano-precipitation method: poly(lactic-co-glycolic acid) and ethylene glycol chitosan are dissolved in a mixed solvent of acetone and anhydrous ethanol to obtain a mixed solution; in the mixed solvent, the volume ratio of acetone to anhydrous ethanol is (5-10):1. Then the mixed solution is added to deionized water under stirring at a rate of 500-750 rpm to obtain a suspension. Next, the suspension is continuously stirred at a rate of 500-750 rpm overnight to evaporate the organic solvents in the suspension. Then the GC-PLGA nanoparticles are collected by centrifugation at 20000-30000 x g for 15-20 min and washed several times in ultrapure water. Then the GC-PLGA nanoparticles are resuspended in a LIN aqueous solution and incubated at room temperature for 3-5 h, and then the resulting product is freeze-dried to obtain GC-PLGA-LIN nanoparticles.

[0052] wherein the ratio of PLGA, GC, mixed solvent and deionized water is (50-100) mg:(15-50) mg:(6-20) mL:(45-90) mL.

[0053] Step 2, preparation of methylated gellan gum-arginine-glycine-aspartic acid peptide-hyaluronic acid (MeGG-RGD-HA) nanoparticles

[0054] Methylated gellan gum (MeGG) is synthesized by reacting gellan gum (Mw1 / 4 10000000) with methacrylic anhydride (MA): gellan gum is added to water and dissolved at 90-95℃, 50-60℃ MA is added to the resulting solution, the reaction lasts for 6-10 h, the pH is adjusted to 8.0-8.2 periodically with 5.0-5.5 M NaOH solution to obtain a MeGG solution; the MeGG solution is purified by dialysis (membrane molecular weight cut-off is 11-14 kD) for 3-5 d to remove excess MA, and the purified MeGG is obtained by freeze-drying and stored in a dry and dark place. Wherein the ratio of GG, MA and deionized water is (1-1.5) g:(2-8) mL:(100-150) mL.

[0055] MeGG was added to 2-(N-morpholino)ethanesulfonic acid (MES) buffer (to keep the pH of the solution constant in the following chemical reaction steps, closer to the physiological pH 7.4), dissolved at 50-55℃, adjusted to pH 6.0-6.2, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the resulting solution and reacted at 37℃ for 2-4h; then, the refined glycine aspartic acid peptide solution was added and continuously reacted at room temperature overnight; the resulting reaction solution was dialyzed for 2-5d to obtain MeGG-RGD, which was freeze-dried and kept at 4-5℃. Among them, the ratio of MeGG, EDC, NHS and RGD is (100-150) mg:(88-100) mg:(0.2-0.3) g:(35-75) μg.

[0056] Hyaluronic acid was dissolved in water at a concentration of 5-7mg / mL to obtain a HA solution. EDC and NHS were dissolved in water at a concentration of 38-50mg / mL (mass ratio of EDC:NHS=(19-20):21) to obtain an EDC / NHS solution. The EDC / NHS solution was added to the HA solution and reacted at room temperature for 1-2h under stirring to activate the carboxyl functional group of HA, then 0.1-0.2M NaHCO3 solution of MeGG-RGD was added, the carboxyl group of HA reacted with the amino group of RGD in MeGG-RGD to form an amide, and was kept at 4℃ for 72-96h under stirring. The mixture was purified by dialysis (MWCO: 3kDa) and deionized (DI) water for 72-96h, freeze-dried to obtain MeGG-RGD-HA. Among them, the mass ratio of HA and MeGG-RGD is (100-150) mg:(15-30) mg.

[0057] Step 3, MeGG-RGD-HA and GC-PLGA-LIN nanoparticles were mixed and reconstituted in water, and the final MeGG-RGD-HA-GC-PLGA-LIN nanoparticles, MRH-GPLNP, were self-assembled.

[0058] Example 1: Preparation of MRH-GPLNP nanoparticles

[0059] Preparation of GC-PLGA-LIN nanoparticles

[0060] PLGA polymer (50 mg) and GC (15 mg) were dissolved in 6 mL of a mixed solvent of acetone and absolute ethanol to obtain a mixed solution, with a volume ratio of acetone to absolute ethanol of 5:1. The mixed solution was then added to 45 mL of deionized water under stirring at a rate of 500 rpm. Next, the resulting suspension was continuously stirred at a rate of 500 rpm overnight to evaporate the organic solvents in the solution. The GC-PLGA nanoparticles were then collected by centrifugation at 20,000 x g for 15 min and washed three times in ultrapure water. The GC-PLGA nanoparticles were then resuspended in a LIN aqueous solution (LIN concentration of 1 mg / mL) and incubated at room temperature for 3 h, after which the product was lyophilized to obtain GC-PLGA-LIN nanoparticles.

[0061] Preparation of MeGG-RGD-HA nanoparticles

[0062] GG (1 g) was added to water and dissolved at 90°C, 8 mL of MA at 50°C was added to the resulting solution, and the reaction was continued for 6 h, with the pH being adjusted to 8.0 at regular intervals with a 5.0 M NaOH solution, and the resulting MeGG solution was purified by dialysis (membrane molecular weight cut-off of 11-14 kD) against distilled water for at least 3 d to remove excess MA. The purified MeGG was obtained by freeze-drying and stored in a dry, light-protected place.

[0063] MeGG (100 mg) was added to a 2-(N-morpholino)ethanesulfonic acid buffer and dissolved at 50-55°C, and the pH was adjusted to 6.0. EDC (88 mg) and NHS (0.2 g) were added to the resulting solution and allowed to react at 37°C for 2 h. Then, an RGD solution (RGD 35 μg) was added and continuously reacted overnight at room temperature, and the solution was dialyzed for 2 d to obtain MeGG-RGD, which was kept at 4°C after freeze-drying, and RGD functionalization was confirmed by X-ray photoelectron spectroscopy elemental analysis.

[0064] HA (100 mg) was dissolved in water at a concentration of 5 mg / mL to obtain an HA solution. EDC and NHS were dissolved in water at a concentration of 38 mg / mL (mass ratio of EDC:NHS = 19:21) to obtain an EDC / NHS solution. The EDC / NHS solution was added to the HA solution and reacted at room temperature for 1 h under stirring, then a 0.1 M NaHCO3 solution of MeGG-RGD (15 mg) was added to conjugate with the RGD peptide and kept at 4°C for 72 h under stirring. The mixture was purified by dialysis (MWCO: 3 kDa) and deionized water for 72 h and lyophilized to obtain MeGG-RGD-HA.

[0065] Preparation of MRH-GPLNP nanoparticles

[0066] The MeGG-RGD-HA and GC-PLGA-LIN nanoparticles were mixed and reconstituted to self-assemble the final MeGG-RGD-HA-GC-PLGA-LIN nanoparticles, namely MRH-GPLNP.

[0067] As shown in Figure 5 , the methyl characteristic peak of MeGG appeared at 1700 cm -1 around, indicating that the methyl modification was successful. Figure 6 As shown in Figure 7 , the H NMR spectrum of MeGG showed the presence of four characteristic peaks corresponding to the -CH of rhamnose (δ 5.29 ppm), the -CH of glucuronic acid (δ 5.11 ppm), the -CH of glucose (δ 4.88 ppm) and the -CH3 of rhamnose (δ 1.45 ppm). 1 The H NMR spectrum showed obvious peaks in the double bond region (δ 5.50-7.00 ppm) and a sharp peak corresponding to the -CH3 of the methyl methacrylate group (δ 2.09 ppm) on the spectrum of the modified GG, confirming the methacrylation of GG. Figure 8 and Figure 9 As shown in

[0068] Referring to the method of Example 1, the sample addition amount was optimized, and the experimental conditions of each group are shown in Table 1 below.

[0069] Table 1 Conditions of different experimental groups

[0070]

[0071]

[0072] Example 2: Morphological characterization of MRH-GPLNP prepared in Example 1

[0073] The apparent morphology of MRH-GPLNP (sample 2) prepared in Example 1 was characterized by atomic force microscopy. The scale bar can show the average particle size. 100 μL of MRH-GPLNP under freeze-drying conditions at a concentration of 0.1 mg / mL was taken to characterize and evaluate the overall morphology.

[0074] As shown in Figure 1 , the AFM two-dimensional planar image of MRH-GPLNP in aqueous solution shows that the size is about 150 nm. Figure 2 is the AFM 3-D stereoscopic image of MRH-GPLNP; Figure 3 is the SEM image (×100K) of MRH-GPLNP, Figure 4 was analyzedFigure 3 The size of MRH-GPLNP was about 150 nm, which was similar to the AFM result, and tended to be a normal distribution. Figure 10 The surface zeta potential showed that the change of positive and negative potential of PLGA, GC-PLGA, GC-PLGA-LIN and MRH-GPLNP showed that MRH-GPLNP was successfully self-assembled and synthesized.

[0075] Example 3: Determination of entrapment efficiency and drug loading

[0076] The percentage of encapsulation efficiency (EE) and drug loading (DL) of MRH-GPLNP was determined. MRH-GPLNP nanoparticles were separated from the solution at 15000 rpm at 4°C using a microcentrifuge (TOMY, MX-305, high-speed refrigerated microcentrifuge), and the obtained clear supernatant was analyzed after diluting the unencapsulated oleanolic acid LIN drug (triplicate for each). The total percentage of drug, i.e. entrapment (EE) and loading (DL), was calculated by the following equations: EE = (total amount of drug added - total amount of free drug added) / total amount of drug added x 100 (1), DL = (total amount of drug added - total amount of free drug added) / weight of nanoparticles recovered (mg) x 100 (2).

[0077] The determination of entrapment efficiency and drug loading was carried out for samples 1-16, respectively, and the results showed that the addition amount condition in example 1 (sample 2) was the optimal addition amount. Through calculation, the encapsulation efficiency of MRH-GPLNP in sample 2 was 91% and the drug loading was 66.67%, which was the highest encapsulation efficiency and drug loading.

[0078] Example 4: Solubilization and ion response characteristics of MRH-GPLNP of example 1

[0079] In order to evaluate the solubilization and ion response characteristics of MRH-GPLNP, the effect of retention in the pre-corneal area was simulated. 15 mL of artificial tears was left to volatilize at 37°C for 24 h to accelerate the concentration of inorganic salts in the concentrated artificial tears. The experimental groups were: MRH-GPLNP / ultrapure water group (A), free oleanolic acid / ultrapure water group (B), MRH-GPLNP / artificial tears group (C), gellan gum / artificial tears group (D). 0.1 mg of gellan gum, free oleanolic acid and MRH-GPLNP of example 1 were dissolved in 0.25 mL of ultrapure water, respectively, and at the same time, 60°C water bath for 5 min, then the temperature was adjusted to 37°C water bath for 5 min. The MRH-GPLNP / artificial tears group and the gellan gum / artificial tears group continued to be added with concentrated artificial tears at a speed of every 10 μL / 2 s for 2 min and then cooled for 2 min. The MRH-GPLNP / ultrapure water group and the free oleanolic acid / ultrapure water group were added with the same amount of ultrapure water under the same conditions.

[0080] AsFigure 11 Compared with A, the sample tube of B showed obvious precipitation phenomenon, which indicated the necessity of packaging of geniposide and improved the solubility of geniposide. Since the viscosity of the gellan gum solution is thermoreversible. With the increase of temperature, the viscosity decreases sharply, but it returns to the original state after cooling. When the solution is cooled, a double helix structure is produced. Ionic gelation requires monovalent or divalent cations, and the consistency of the gel can be changed by changing the concentration and type of ions present in the gel. The salt particles contained in the artificial tears, such as sodium ions or potassium ions, can form a cation-induced phase transition structure, thereby producing a stronger gel channel. MRH-GPLNP showed a liquid flow state before being combined with artificial tears, and after interacting with the positive cations in the artificial tears, it formed a transparent gel solid state, and when the sample tube was inverted, it did not flow down, Figure 12 The phase transition of liquid-solid ionic response characteristics was shown, which indicated that the MRH-GPLNP / artificial tear group was endowed with ionic response characteristics due to the introduction of gellan gum.

[0081] Example 5: Culture of human SV40 immortalized corneal epithelial cell line

[0082] The cells used in the experiment were human corneal epithelial cells (HCEC) derived from human SV40 immortalized corneal epithelial cell line (CRL-11135, HCE-2; ATCC), and the culture medium used was DMEM / F12, with additional 10% fetal bovine serum and 10 ng / mL human epidermal growth factor, and the culture medium was replaced every day. The culture condition was 37℃, 5% CO2 in the incubator.

[0083] Example 6: Effect of MRH-GPLNP of Example 1 on the proliferation of HCEC cells under hyperosmotic stress

[0084] Select HCEC cells in the logarithmic growth phase, add 0.25% trypsin for digestion, resuspend and blow into single cells after adding culture solution, count, and adjust the final concentration of cells to 1.0×10 cHCEC cells were seeded in 96-well plates at a density of 5 x 104cells / mL with 150 μL cell suspension per well and incubated in an incubator for 24 h. The culture medium was aspirated and samples were added for 6 h incubation, with a sample concentration of 10 μg / mL, and an equal amount of PBS buffer was added to the control group. The cells were treated as follows: control group (normal concentration condition), hyperosmotic group (osmotic pressure range of 550 mOsm / L, 120 mM sodium chloride (NaCl) was added), hyperosmotic + experimental free geniposide group, hyperosmotic + experimental MRH-GPLNP group. The osmotic pressure was added 2 h before the addition of NaCl. Then 25 μL of 5 mg / mL MTT solution was added per well, and the wells were incubated in a 37 °C incubator with a CO2concentration of 5% and a saturated humidity condition for 4 h. The culture medium in each well was gently aspirated. 200 μL of DMSO was added to each well. After the crystals were fully dissolved, the absorbance value A of each well was measured at 570 nm using a microplate reader, and the cell proliferation rate was calculated. Cell proliferation rate = (absorbance value of experimental group - absorbance value of control group) / absorbance value of control group.

[0085] Figure 13 The MTT experiment results of the effect of MRH-GPLNP on the proliferation of HCEC cells under hyperosmotic stress conditions showed that the cell proliferation rates were different for different samples: control group (1.67 ± 0.06), hyperosmotic group (1.13 ± 0.06), hyperosmotic + free geniposide group (1.37 ± 0.06), and hyperosmotic + MRH-GPLNP group (1.57 ± 0.06). There were significant differences between the groups (P < 0.05). Compared with the control group, the cell proliferation rate of the hyperosmotic group was significantly down-regulated. Compared with the hyperosmotic group, the cell proliferation rate of the hyperosmotic + free geniposide group had a significant upward trend, and the upward trend of the hyperosmotic + MRH-GPLNP group was more obvious. Therefore, under hyperosmotic culture conditions, the growth of HCEC cell viability was reduced, and the cell proliferation was inhibited. Free geniposide significantly promoted cell proliferation under hyperosmotic conditions, and MRH-GPLNP had a more intense promoting effect on cell proliferation.

[0086] Example 7: Cell size analysis of MRH-GPLNP of Example 1 under hyperosmotic stress conditions of HCEC cells

[0087] HCEC cells in the logarithmic growth phase were selected, and the cell size of HCEC after exposure to a hypertonic environment (500 mOsm / L) for 16 hours was analyzed. Under isotonic conditions, comparisons were made between each concentration and the base value. Analysis of previously measured samples was performed at a medium flow rate of <10000 events / s over a time range of 300 s. After selecting hypertonic conditions for the model, fluctuations in cell size were analyzed. Different osmoprotective substances (free geniposide and MRH-GPLNP, with a final concentration of 10 μg / mL of geniposide) were tested for their regulation of cell size by pre-incubating the cells with them for 8 h before adding the hypertonic solution.

[0088] Figure 14 Figure 8 is a graph showing the effect of MRH-GPLNP on the cell size of HCEC cells under hypertonic stress conditions; the cell size calculation experiment results show that different sample groups have different effects on the size of the cells. Compared with the control group, the cell size of the hypertonic group was reduced to 62.3%. Compared with the hypertonic group, the cell size of the hypertonic + free geniposide group was restored by 17.7%, and the cell size of the hypertonic + MRH-GPLNP group was restored by 28.7%. There were significant differences between the groups (P<0.05). Compared with the control group, the cell size of the hypertonic group was significantly reduced. Compared with the hypertonic group, the cell size of the hypertonic + free geniposide group had a significant upregulation trend, and the hypertonic + MRH-GPLNP group had a more obvious upregulation trend. Thus, under hypertonic culture conditions, the size of HCEC cells is reduced, which has the effect of reducing cell size. Free geniposide has a significant protective effect on cell size under hypertonic conditions, and MRH-GPLNP has a more intense inhibitory effect on cell size after encapsulation.

[0089] Example 8: Scratch test

[0090] To detect HCEC cell migration, HCEC cells were seeded in a 6-well plate at 5x10 5 cells per well, and after 24 h of culture, the cells were evenly spread on the bottom of the well. A 1 mL sterile pipette tip was vertically scratched in the center of the well, the floating cells on the bottom of the well were aspirated, and the well was washed with PBS. Then, 1% fetal bovine serum was added to the culture medium, and the cells were treated according to the different groupings described above. ImageJ software was used to calculate the width of the scratch. The relative migration distance = 12 h migration distance - 0 h migration distance.

[0091] Figure 15The observation and comparison of the migration ability of HCEC cells under the hyperosmotic stress condition by the MRH-GPLNPs of Example 1 can analyze that different samples in different groups have different degrees of cell migration: the control group 515.00±5.00, the hyperosmotic group (346.67±15.27), the hyperosmotic+free morin group (431.33±19.76), and the hyperosmotic+MRH-GPLNP group (488±24.88). The migration degree of cells in each group has a significant difference (P<0.05). Compared with the control group, the migration distance of cells in the hyperosmotic group is significantly reduced. Compared with the hyperosmotic group, the migration distance of cells in the hyperosmotic+free morin group is significantly increased, and compared with the hyperosmotic group, the migration distance of cells in the hyperosmotic+MRH-GPLNP group is more significantly increased. Therefore, the migration ability of HCEC cells under the hyperosmotic culture condition is weakened, free morin has a promoting effect on the cell migration under the hyperosmotic condition, and the MRH-GPLNPs after encapsulation have a significant promoting effect on the migration distance of cells.

Claims

1. A method for preparing ion-responsive strychnosine nanoparticles, characterized in that, include: GC-PLGA nanoparticles were prepared by a nanoprecipitation method using ethylene glycol chitosan and polylactic acid-glycolic acid copolymer. GC-PLGA nanoparticles were then incubated with strychnine to obtain GC-PLGA-LIN nanoparticles. Gellan gum was reacted with methacrylic anhydride to obtain methylated gellan gum. The methylated gellan gum was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The reaction was carried out for 2-4 h. Then, arginine aspartic acid peptide solution was added, and the reaction was carried out. The resulting reaction solution was dialyzed and freeze-dried to obtain MeGG-RGD. Hyaluronic acid was dissolved in water to obtain HA solution. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in water to obtain EDC / NHS solution. EDC / NHS solution was added to HA solution, and the reaction was stirred for 1-2 h. Then, MeGG-RGD NaHCO3 solution was added to carry out amide reaction. The reaction was dialyzed and freeze-dried to obtain MeGG-RGD-HA. MeGG-RGD-HA and GC-PLGA-LIN nanoparticles were mixed and dissolved in water, and then self-assembled to obtain ion-responsive monaxioline nanoparticles MRH-GPLNP.

2. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The preparation of GC-PLGA nanoparticles by reacting ethylene glycol chitosan and polylactic acid-glycolic acid via a nanoprecipitation method is specifically as follows: Ethylene glycol chitosan and polylactic acid-glycolic acid were dissolved in an organic solvent to obtain a mixed solution. The mixed solution was added to water to obtain a suspension. The organic solvent in the suspension was evaporated, and then the solid and liquid were separated to obtain GC-PLGA nanoparticles.

3. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The process of incubating GC-PLGA nanoparticles with strychnine to obtain GC-PLGA-LIN nanoparticles specifically involves: resuspending GC-PLGA nanoparticles in a 0.5-2.0 mg / mL aqueous solution of strychnine and incubating at room temperature for 3-5 hours, then lyophilizing the resulting product to obtain GC-PLGA-LIN nanoparticles.

4. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The mass ratio of the polylactic acid-hydroxyacetic acid copolymer to ethylene glycol chitosan is (50-100) mg: (15-50) mg.

5. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The gellan gum reacts with methacrylic anhydride to obtain methylated gellan gum, specifically: Gellan gum was dissolved in water, and methacrylic anhydride was added to the resulting solution to adjust the pH to 8.0-8.

2. The reaction was carried out for 6-10 hours to obtain a MeGG solution. The MeGG solution was dialyzed and freeze-dried to obtain methylated gellan gum, wherein the mass ratio of gellan gum to methacrylic anhydride was (1-1.5) g: (2-8) mL.

6. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The ratio of the methylated gellan gum, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and arginine aspartic peptide is (100-150) mg: (88-100) mg: (0.2-0.3) g: (35-75) µg.

7. The method for preparing ion-responsive strychnosine nanoparticles according to claim 1, characterized in that, The mass ratio of hyaluronic acid to MeGG-RGD is (100-150) mg: (15-30) mg.

8. Ion-responsive strychnoside nanoparticles obtained by the preparation method according to any one of claims 1-7.

9. The use of the ion-responsive strychnine nanoparticles according to claim 8 in the preparation of a drug to relieve hyperosmolar stress in corneal epithelial cells.

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