An ophthalmic nanoemulsion formulation for treating immune dry eye and a method of preparing the same

Abstract

The application discloses an eye nanoemulsion preparation for treating immune dry eye and a preparation method thereof, and the preparation comprises a nanoemulsion inner core loaded with tacrolimus or cyclosporine A, a simulated tear protein composite layer fixed on an oil-water interface through an amide bond, and surface modified hyaluronic acid oligosaccharide. The protein composite layer is covalently crosslinked by lysozyme and lactoferrin at an optimized molar ratio of 1:(0.8-1.2), and the hyaluronic acid is connected to the epsilon-amino group of the protein through an amide bond after EDC / NHS activation. The preparation achieves three breakthrough effects: the drug first-order release rate constant k is less than or equal to 0.25 h ‑1 in artificial tears, the drug efficacy lasts for 8-12 hours, the corneal retention time is more than 4.8 hours, the specific binding of the charge-neutral surface and mucin is relied on, the lysozyme activity retention rate is greater than or equal to 85%, and the HET-CAM irritation score is less than or equal to 0.8, thereby significantly improving the treatment safety and biocompatibility. Finally, the core advantage that the eye surface immune response and the microenvironment repair can be simultaneously inhibited through 1-2 times of daily administration is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of ophthalmic biological agents, and particularly relates to an ophthalmic nanoemulsion preparation for treating immune dry eye and a preparation method thereof. BACKGROUND

[0002] As a globally high-incidence chronic ocular surface disease, the core pathological mechanism of immune dry eye is T lymphocyte-mediated inflammatory response leading to lacrimal gland dysfunction and corneal epithelial damage. The current mainstream clinical treatment scheme has significant limitations: artificial tears can only temporarily relieve symptoms and cannot intervene in the immune process; cyclosporine A or tacrolimus eye drops (such as Restasis®) have anti-inflammatory effects, but due to the low bioavailability and strong irritation of the traditional emulsion drug delivery system, patient compliance is poor; and long-term use of glucocorticoids can easily cause intraocular pressure to rise and cataracts. In recent years, nanoemulsion technology has been tried to improve drug delivery. For example, the "cyclosporine A ophthalmic nanoemulsion" disclosed in Chinese Patent CN1140999A constructs a drug-loaded core by using medium-chain triglycerides and Poloxamer188, which improves the solubility of the drug, but does not solve the industry pain points of burst release (2h release >60%) and poor corneal retention (retention time <2h). This scheme lacks ocular surface adaptability modification, the negative charge on the surface of the nanoemulsion electrostatically repels mucin, and the absence of bioactive components cannot synergistically regulate the ocular surface microenvironment, resulting in insufficient treatment efficacy.

[0003] The deeper technical problem lies in balancing the three requirements of long-acting sustained release, ocular surface retention, and biological safety: first, the traditional nanoemulsion interface only relies on synthetic surfactants (such as phospholipids), which quickly disintegrate under the washing of tears, with a drug release half-life of less than 1.5 hours, forcing patients to administer 4-6 times a day; second, the mucin layer of the ocular surface is negatively charged, and conventional carrier formulations are difficult to effectively adhere due to charge mismatch (strong positive or strong negative); third, local application of immunosuppressants can easily cause a burning sensation, and when functional proteins (such as lysozyme) are introduced, the immobilization process often leads to conformational destruction. The existing technology has not achieved multi-dimensional adaptation of the drug delivery system to the physiological characteristics of the ocular surface, and it is urgent to break through the technical closed loop of "drug burst release - insufficient retention - loss of activity".

[0004] The present application addresses the above-mentioned defects and initiates a "bionic protein composite layer-hyaluronic acid" synergistic modification strategy: by optimizing the molar ratio of lysozyme (LYZ) and lactoferrin (LF) (1:0.8-1.2), a charge-neutral interface is covalently cross-linked by amide bonds, which simultaneously retains the antibacterial activity of LYZ (>85%) and the iron ion regulation function of LF; further anchoring EDC / NHS-activated hyaluronic acid oligosaccharides on the protein layer, and using the sugar-sugar specific binding of the ocular surface mucin, the corneal retention time is improved to more than 4.8 hours. This design overcomes the burst release of nanoemulsion (k value ≤0.25h -1), charge repulsion and biological activity damage three technical bottlenecks, the first to achieve the integration of treatment of "single administration 12 hours immunosuppression-microenvironment repair", to provide a revolutionary solution for immune dry eye. SUMMARY

[0005] In view of the above technical problems, the purpose of the present application is to provide an eye nanolipid preparation for treating immune dry eye.

[0006] The eye nanolipid preparation for treating immune dry eye comprises the following components:

[0007] (1) Nanolipid core: containing a therapeutically effective amount of immunosuppressant, selected from tacrolimus or cyclosporine A;

[0008] (2) Interfacial protein layer: a tear-like protein composite layer fixed on the oil-water interface by amide bond, composed of lysozyme (LYZ) and lactoferrin (LF) covalently cross-linked at a molar ratio of 1: (0.8-1.2).

[0009] Further preferably, the molar ratio of lysozyme to lactoferrin is 1:1, which allows lysozyme (LYZ) and lactoferrin (LF) to form a stable composite layer by electrostatic attraction and amide bond cross-linking.

[0010] The surface of the tear-like protein composite layer is also modified with hyaluronic acid oligosaccharide, and the carboxyl group is activated and connected to the epsilon-amino group of lysozyme (LYZ) or lactoferrin (LF) by amide bond.

[0011] In the pH 7.4 simulated tear fluid, the drug release rate constant k fitted by first-order kinetics is ≤0.25h -1 .

[0012] The present application also discloses a preparation method of an eye nanolipid preparation for treating immune dry eye, comprising the following steps:

[0013] S1. Preparation of drug-containing nanolipid: dissolve the above immunosuppressant in medium-chain triglyceride, add Poloxamer 188 and water phase, 50-300 bar microfluidization pre-emulsification, cycle 1-3 times, continue 500-1000 bar high pressure homogenization 2-4 times;

[0014] S2. Activation of nanolipid surface: add 1-5 mM NHS-PEG4-Maleimide to the product of S1, react at 20-37℃ for 20-40 min, and remove free reagents by ultrafiltration;

[0015] S3. Protein fixation: lysozyme (LYZ) and lactoferrin (LF) are dissolved in PBS at pH 6.5-8.0 according to a molar ratio of 1: (0.8-1.2), and the product of S2 is continuously added, and stirring is continued at 20-37℃ for 1-3h;

[0016] S4. Hyaluronic acid modification: 0.5-2% w / v hyaluronic acid oligosaccharide with pre-activated carboxyl by EDC / NHS is added, and the reaction is carried out at pH 5.5-7.0 for 0.5-2h;

[0017] S5. Purification: ultracentrifugation at 10000-18000 rpm for 20-40 min to remove free proteins.

[0018] The EDC / NHS activated carboxyl step of the hyaluronic acid oligosaccharide solution of step S4 is:

[0019] Step 1: Dissolve the hyaluronic acid oligosaccharide in 0.05-0.2M MES buffer at pH 4.8-6.2 to form a 5-20mg / mL solution;

[0020] Step 2: Continue to add EDC to the solution of step 1 to a final concentration of 5-20mM, and continue to add NHS to a final concentration of 10-50mM;

[0021] Step 3: Stir the mixed solution obtained in step 2 at 20-30℃ in the dark for 10-30min;

[0022] Step 4: Centrifuge the mixed solution after stirring in step 3 with a 5-20kDa MWCO ultrafiltration centrifuge tube at 10000-20000 rpm for 5-20min, repeat 2-4 times, and remove free reagents.

[0023] With the synergistic sustained-release effect of the protein complex layer and hyaluronic acid, daily administration of 1-2 times is achieved.

[0024] The molecular weight of the hyaluronic acid oligosaccharide is 3-5kDa.

[0025] The core particle size of the nanoemulsion is 80±10 nm, and the PDI is ≤0.25; the overall particle size after protein modification is ≤120nm, and the PDI is ≤0.3.

[0026] The thickness of the protein complex layer is 8-12 nm, and the lysozyme activity retention rate is ≥85%.

[0027] The present application needs to be explained that the core particle size (nm), the particle size after modification (nm), PDI, protein layer thickness (nm), and subsequent test items do not give test results.

[0028] Particle size and PDI were measured by dynamic light scattering (DLS) at 25℃ with three repeats. Protein layer thickness was measured by small angle X-ray scattering (SAXS) at the synchrotron radiation source with a resolution of 0.1 nm. The test procedure is not given in the following test examples.

[0029] Here, the protein complex layer thickness test method needs to be explained: after purification, the nanoemulsion sample is filled into a quartz capillary in liquid form, and SAXS test (wavelength 0.1 nm) is performed at the synchrotron radiation source, and the protein layer thickness is calculated by Guinier fitting.

[0030] The beneficial technical effects of the present application are:

[0031] 1. Long-acting sustained release and precise delivery: through the dual modification of the tear protein complex layer (LYZ / LF) and hyaluronic acid, the first-order kinetic sustained release (k≤0.25h -1 ) is realized, the drug action time is prolonged to 8-12 hours, which is greatly improved compared with ordinary nanoemulsion; secondly, the corneal retention time reaches 5.5 hours, which depends on the specific binding of the charge-neutral surface and mucin, and the retention is greatly improved.

[0032] 2. Synergistic biological activity and safety: lysozyme activity retention ≥85%: maleimide site-directed modification of inactive sulfhydryl maintains the antibacterial efficacy of LYZ. LF iron ion chelation regulates tear free iron to physiological level, inhibits oxidative stress; the irritation is significantly reduced: HET-CAM score ≤0.8, the protein layer shields the damage of surfactant.

[0033] 3. Clinical treatment advantages: the drug administration frequency is halved, 1-2 times a day, and the compliance is improved; dry eye syndrome multiple intervention: tacrolimus / cyclosporin A inhibits T cell activation, LYZ / LF repairs the ocular surface microenvironment;

[0034] Conclusion: the preparation breaks through the burst and irritation defects of traditional nanoemulsion, realizes the integration of "sustained release-retention-repair", and provides a new therapy with high efficiency and low toxicity for immunological dry eye. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The structure of an eye nanoemulsion preparation for treating immunological dry eye prepared according to an embodiment of the present application is shown in the figure.

[0036] Figure 2 The flow chart of the preparation method of an eye nanoemulsion preparation for treating immunological dry eye according to the present application is shown in the figure. DETAILED DESCRIPTION

[0037] Before particular embodiments of the present application are described, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present application, unless specifically stated otherwise.

[0038] When a numerical range is given in the embodiments, it is understood that, unless otherwise stated, each numerical range's two endpoints and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition to the specific methods, devices, materials described in the embodiments, any methods, devices, materials similar or equivalent to those described in the embodiments of the present application can be used to implement the present application according to the knowledge of the prior art possessed by those skilled in the art and the description of the present application.

[0039] Unless otherwise stated, the test methods, detection methods, preparation methods disclosed in the present application all use conventional techniques in the art.

[0040] Example 1

[0041] An ophthalmic nanoemulsion preparation for treating immune dry eye, comprising the following components:

[0042] (1) Nanoemulsion core: containing a therapeutically effective amount of an immunosuppressant selected from tacrolimus;

[0043] (2) Interfacial protein layer: a tear-like protein composite layer fixed at the oil-water interface by an amide bond, composed of lysozyme (LYZ) and lactoferrin (LF) covalently cross-linked at a molar ratio of 1:1.

[0044] The base formula is: the immunosuppressant is tacrolimus 0.05% (w / v), medium-chain triglyceride 5% (w / v), Poloxamer 188 1.5% (w / v), hyaluronic acid oligosaccharide 1% (w / v), 3 kDa, NHS-PEG4-maleimide 3 mM.

[0045] The present application also discloses a preparation method of an ophthalmic nanoemulsion preparation for treating immune dry eye, the steps are as follows:

[0046] S1. Preparation of drug-containing nanoemulsion: dissolve the above-mentioned immunosuppressant in medium-chain triglyceride, add Poloxamer 188 and aqueous phase, 200 bar microfluidic pre-emulsification, cycle 2 times, continue 800 bar high pressure homogenization 3 times;

[0047] S2. Activate the surface of nanoemulsion: add 2 mM NHS-PEG4-Maleimide to the product of S1, react at 25°C for 30 min, and remove free reagent by ultrafiltration;

[0048] S3. Protein fixation: dissolve lysozyme (LYZ) and lactoferrin (LF) in PBS at pH 7.4 according to a molar ratio of 1:1, continue to add the product of S2, and stir at 25°C for 2 h;

[0049] S4. Hyaluronic acid modification: add 1% w / v hyaluronic acid oligosaccharide with pre-activated carboxyl by EDC / NHS, and react at pH 6.5 for 1 h; the molecular weight of the hyaluronic acid oligosaccharide is 3 kDa;

[0050] S5. Purification: remove free protein by ultracentrifugation at a speed of 12,000 rpm for 30 min.

[0051] The EDC / NHS activated carboxyl step of the hyaluronic acid oligosaccharide solution of S4 is as follows:

[0052] Step 1: dissolve the hyaluronic acid oligosaccharide in 0.1 M MES buffer at pH 5.5 to form a 10 mg / mL solution;

[0053] Step 2: continue to add EDC to the solution of step 1 to a final concentration of 10 mM, and continue to add NHS to a final concentration of 25 mM;

[0054] Step 3: stir the mixed solution obtained in step 2 at 25°C in the dark for 20 min;

[0055] Step 4: centrifuge the mixed solution after stirring in step 3 using a 10 kDa MWCO ultrafiltration centrifuge tube at a speed of 15,000 rpm for 10 min, repeat for 3 times, and remove free reagent.

[0056] Figure 1 A schematic structural diagram of an eye nanoemulsion preparation for treating immune dry eye syndrome prepared in an embodiment of the present application. Among them, the yellow part 1 represents the drug-loaded core, the core is medium-chain triglyceride (MCT), and the drug molecules (represented by purple dots 2) are uniformly distributed inside; the light blue short line or ring band 5 represents the interfacial activation layer, which is composed of NHS-PEG4-Maleimide and located at the oil-water interface; the green ring structure 3 represents lysozyme (LYZ), and the red ring structure 4 represents lactoferrin (LF), which together constitute the tear-like protein composite layer; the deep blue long chain 6 in the outermost layer shows the hyaluronic acid modification layer, which is anchored on the ε-amino group of the protein through an amide bond.

[0057] Figure 2 A flow chart of a preparation method of an eye nanoemulsion preparation for treating immune dry eye syndrome in an embodiment of the present application.

[0058] Example 2

[0059] An ophthalmic nanoemulsion preparation for treating immune dry eye, comprising the following components:

[0060] (1) Nanoemulsion core: containing a therapeutically effective amount of an immunosuppressant selected from tacrolimus;

[0061] (2) Interfacial protein layer: a composite layer of tear-like proteins fixed at the oil-water interface by amide bond, composed of lysozyme (LYZ) and lactoferrin (LF) covalently cross-linked at a molar ratio of 1:0.8.

[0062] The base formula is: the immunosuppressant is tacrolimus 0.05% (w / v), medium-chain triglyceride 5% (w / v), Poloxamer 188 1.5% (w / v), hyaluronic acid oligosaccharide 1% (w / v), 3 kDa, NHS-PEG4-Maleimide 5 mM.

[0063] The application also discloses a preparation method of an ophthalmic nanoemulsion preparation for treating immune dry eye, comprising the following steps:

[0064] S1. Preparation of drug-containing nanoemulsion: dissolve the above-mentioned immunosuppressant in medium-chain triglyceride, add Poloxamer 188 and an aqueous phase, and perform microfluidization pre-emulsification at 200 bar, circulate twice, and continue high-pressure homogenization at 800 bar for 3 times;

[0065] S2. Activation of the surface of the nanoemulsion: add 2 mM NHS-PEG4-Maleimide to the product of S1, react at 25°C for 30 min, and remove free reagents by ultrafiltration;

[0066] S3. Protein fixation: dissolve lysozyme (LYZ) and lactoferrin (LF) in PBS at pH 7.4 according to a molar ratio of 1:1, continue to add the product of S2, and stir at 25°C for 2 h;

[0067] S4. Hyaluronic acid modification: add 1% w / v hyaluronic acid oligosaccharide with a pre-activated carboxyl group by EDC / NHS, and react at pH 6.5 for 1 h; the molecular weight of the hyaluronic acid oligosaccharide is 3 kDa;

[0068] S5. Purification: remove free proteins by ultracentrifugation at a centrifugal speed of 12000 rpm for 30 min.

[0069] The step of activating the carboxyl group of the hyaluronic acid oligosaccharide solution in S4 is:

[0070] Step 1: Dissolve hyaluronic acid oligosaccharide in 0.1 M MES buffer at pH 5.5 to make a 10 mg / mL solution;

[0071] Step 2: Continue to add EDC to the solution of Step 1 to a final concentration of 10 mM, and continue to add NHS to a final concentration of 25 mM;

[0072] Step 3: Stir the mixed solution obtained in Step 2 at 25°C in the dark for 20 min;

[0073] Step 4: Centrifuge the mixed solution after stirring in Step 3 using a 10 kDa MWCO ultrafiltration centrifuge tube at 15000 rpm for 10 min, repeat 3 times, and remove the free reagents.

[0074] Example 3

[0075] An ophthalmic nanoemulsion preparation for treating immune dry eye, comprising the following components:

[0076] (1) Nanoemulsion core: containing a therapeutically effective amount of an immunosuppressant selected from tacrolimus;

[0077] (2) Interfacial protein layer: a composite layer of tear-like proteins fixed at the oil-water interface by amide bonds, composed of lysozyme (LYZ) and lactoferrin (LF) covalently cross-linked at a molar ratio of 1:1.2.

[0078] The base formula is: the immunosuppressant is tacrolimus 0.05% (w / v), medium-chain triglyceride 5% (w / v), Poloxamer 188 1.5% (w / v), hyaluronic acid oligosaccharide 1% (w / v), 3 kDa, NHS-PEG4-maleimide 1 mM.

[0079] The present application also discloses a preparation method of an ophthalmic nanoemulsion preparation for treating immune dry eye, comprising the following steps:

[0080] S1. Preparation of drug-containing nanoemulsion: dissolve the above-mentioned immunosuppressant in medium-chain triglyceride, add Poloxamer 188 and aqueous phase, pre-emulsify by microfluidization at 200 bar, circulate 2 times, and continue to homogenize at high pressure of 800 bar for 3 times;

[0081] S2. Activation of nanoemulsion surface: add 2 mM NHS-PEG4-maleimide to the product of S1, react at 25°C for 30 min, and remove the free reagents by ultrafiltration;

[0082] S3. Protein fixation: dissolve lysozyme (LYZ) and lactoferrin (LF) in PBS at pH 7.4 according to a molar ratio of 1:1, continue to add the product of S2, and stir at 25°C for 2 h;

[0083] S4. Hyaluronic acid modification: 1% w / v hyaluronic acid oligosaccharide pre-EDC / NHS activated carboxyl group was added and reacted for 1 h at pH 6.5; the hyaluronic acid oligosaccharide had a molecular weight of 3 kDa;

[0084] S5. Purification: ultracentrifugation at 12000 rpm for 30 min to remove free proteins.

[0085] The EDC / NHS activated carboxyl group step of the hyaluronic acid oligosaccharide solution of step S4 was as follows:

[0086] Step 1: Dissolve the hyaluronic acid oligosaccharide in 0.1 M MES buffer at pH 5.5 to make a 10 mg / mL solution;

[0087] Step 2: Continue to add EDC to the solution of step 1 to a final concentration of 10 mM, and continue to add NHS to a final concentration of 25 mM;

[0088] Step 3: Stir the mixed solution obtained in step 2 at 25°C in the dark for 20 min;

[0089] Step 4: Centrifuge purify the mixed solution after stirring in step 3 using a 10 kDa MWCO ultrafiltration centrifuge tube at 15000 rpm for 10 min, repeated 3 times, to remove free reagents.

[0090] Comparative Example 1

[0091] The difference between Comparative Example 1 and Example 1 was that there was no protein layer. That is, there was no artificial tear protein complex layer fixed on the oil-water interface by amide bonds.

[0092] Comparative Example 2

[0093] Chinese Patent CN1140999A discloses "Cyclosporin A-loaded eye nanolipid".

[0094] Test Example

[0095] Test Example 1: Lysozyme activity retention rate (%), release rate constant k (h -1 ), corneal retention time (h), HET-CAM score test, goblet cell density test, IL-1β level detection.

[0096] Test Standards:

[0097] Lysozyme activity: T. lyticus method, 410 nm absorbance change rate (ΔA / min);

[0098] "Lysozyme activity retention rate (%) = (modified LYZ activity / free LYZ activity) x 100%", wherein the free LYZ activity is defined as the value of ΔA / min of unmodified LYZ in PBS.

[0099] Drug release: Dialysis bag method (artificial tears, pH 7.4) HPLC detection, first-order kinetic fitting k value;

[0100] Ocular surface retention: Fluorescently labeled rabbit cornea imaging in vivo confocal microscopy, quantitative fluorescence;

[0101] Irritation: HET-CAM villous membrane allantoic test vascular injury score (0-21 points);

[0102] Goblet cell density test: conjunctival imprint cytology + Periodic Acid-Schiff (PAS) staining quantification

[0103] Operation procedure: (1) Animal model: New Zealand white rabbits (2.5-3.0 kg) phenol red cotton thread test to screen for dry eye (tear secretion <5 mm / 5 min), n=10 / group. (2) Dosing regimen: Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 preparation eye drops, twice a day x 28 days. (3) Sampling: After anesthesia, 0.22 μm cellulose ester membrane was applied to the inferior fornix conjunctiva for 5 seconds; immediately fixed in 10% neutral formalin. (4) PAS staining: immerse in 1% periodic acid (5 min) continue with Schiff reagent (15 min) continue with hematoxylin restain; observe cells under a microscope. (5) Quantitative analysis: count 5 non-overlapping areas (400x) per sample; density (number / mm 2 ) = goblet cell number / field area x 10 6 .

[0104] IL-1β level detection: Test standard: enzyme-linked immunosorbent assay (ELISA)

[0105] Operation procedure: (1) Tear collection: before and after 28 days of administration, capillary collection of non-irritating tears (5 μL / eye); add protease inhibitors (Complete TM, Roche) and stored at appropriate temperature below zero. (2) Sample processing: tear fluid was centrifuged (12000 rpm, 10 min, 4°C) and the supernatant was taken; total protein of conjunctival tissue was extracted with RIPA lysis buffer (BCA quantification standardization). (3) ELISA detection (human IL-1 beta kit for rabbit source): tear fluid supernatant / tissue lysis buffer was added to pre-coated plate (100 μL / well); incubated at 4°C overnight, continued to wash the plate, continued to add biotinylated antibody (1:1000, 37°C, 1 h); continued to add HRP-streptavidin (1:2000, 37°C, 30 min), then TMB color development, then drop 2M H2SO4 to stop, finally read value at 450 nm by microplate reader.

[0106] Calculation: IL-1 beta (pg / mg) = (sample OD value-blank OD) / (standard curve slope) x dilution factor / total protein concentration.

[0107] Table 1 Lysozyme activity retention rate (%), release rate constant k (h -1 ), corneal retention time (h), HET-CAM score test results

[0108] Group Lysozyme activity retention rate (%) release rate constant k (h -1 )]]> Corneal retention time (h) HET-CAM score test results Example 1 92 0.18 5.5±0.4 0.5 Example 2 86 0.22 4.8±0.3 0.8 Example 3 88 0.20 5.0±0.3 0.7 Comparative Example 1 -- 0.45* 1.5±0.3* 4.2*

[0109] Note that * means not up to standard (activity≥85%, k≤0.25 h -1 , retention≥4 h, score≤2).

[0110] (1) The difference mechanism of lysozyme activity retention rate (possible reason analysis): Example 1, LYZ:LF molar ratio is 1:1, the activity retention rate reaches 92%, because the charge shielding is best. Because at this time LF and LYZ form a neutral complex in the pH 7.4 environment, completely wrapping the LYZ active center; Example 2, LYZ:LF molar ratio is 1:0.8, the activity retention rate is 86%, because the lack of LF leads to the exposure of LYC, and the LF coverage is not complete, so that the LYZ active center is partially exposed to the tear esterase, and reversible inactivation occurs; Example 3, LYZ:LF molar ratio is 1:1.2, the activity retention rate is 88%, because of the steric hindrance effect, the collision frequency of excess LF and LYZ increases, inducing the conformational micro-change of LYZ. (2) Possible reason analysis of release rate constant (k value) change: Comparing examples 1-3, the LYZ:LF molar ratio of example 1 is 1:1, the protein layer has high density, low porosity, high diffusion energy barrier, and large hydration layer thickness. The possible reason is that at this time the electrostatic attraction is large, which leads to the close arrangement of proteins, so the pore size is small, resulting in the largest drug diffusion resistance; LF contains a hydrophilic domain, and when the LYZ:LF molar ratio is 1:1, a continuous hydration layer is formed, delaying the precipitation of lipid-soluble drugs. (3) Factors affecting corneal retention time: For the LYZ:LF molar ratio of example 1, due to the charge matching effect, the complex near-electrically neutral leads to the smallest electrostatic repulsion, so that the mucin binding force is the strongest; secondly, the synergistic effect of hyaluronic acid. Because the electrically neutral surface makes the hyaluronic acid oligosaccharide (negatively charged) directionally stretched, so that more sugar chains are combined with the mucin glycosyl group. (4) Analysis of safety differences in HET-CAM scores: Example 1 has no significant irritation because the protein layer completely shields the nanolipid inner core; Example 2 causes mild inflammation due to residual positive charge; for example 3, excess LF leads to imbalance of iron ion chelation.

[0111] In summary: the essence of LYZ:LF=1:1 optimization, this ratio realizes four balances: (1) Electrostatic balance: charge neutralization eliminates ocular surface irritation; (2) Spatial balance: double-protein dense packing forms diffusion pores smaller than 2 nm; (3) Functional balance: LYZ antibacterial activity and LF anti-inflammatory activity synergize; (4) Interface balance: continuous distribution of hydrophilic / hydrophobic domains supports the directional anchoring of hyaluronic acid.

[0112] Comparative Example 1 and Comparative Example 1, (1) Lysozyme activity retention rate: first, the root cause is that there is no lysozyme in the unmodified protein layer of Comparative Example 1. The deep impact is the lack of LYZ antibacterial barrier, and the risk of secondary infection of dry eye syndrome increases. (2) Release rate constant k: in the drug-carrier interaction, Example 1 is LF hydrophobic domain anchoring tacrolimus, and Comparative Example 1 is simply physically wrapped and easy to leak. (3) Corneal retention time: triple inactivation mechanism, charge repulsion: the negative charge on the surface of Comparative Example 1 repels mucin; lack of adhesion protein: no mucin binding domain of LF; hyaluronic acid failure: the lack of fixed hyaluronic acid leads to being washed away by tears. (4) HET-CAM score: in terms of surface activity damage, for Example 1, the protein layer shields Poloxamer 188, and for Comparative Example 1, Poloxamer 188 directly dissolves the corneal phospholipid; in terms of osmotic pressure imbalance, the LF of Example 1 regulates the osmotic pressure of tears, and the osmotic pressure of the nanoemulsion of Comparative Example 1 leads to corneal dehydration; in terms of oxidative stress, for Example 1, LF chelates free iron, but for Comparative Example 1, no protection leads to a 5-fold increase in tear MDA level.

[0113] Summary: irreplaceability of the protein layer.

[0114] Possible reasons for the overall performance loss of Comparative Example 1: physical barrier loss leading to drug burst release; biological interface dysfunction leading to corneal retention; protection function zero leading to irritation; Example 1 achieves functional structuring through the basic protein layer, while Comparative Example 1 is only a simple drug delivery system, which cannot meet the needs of dry eye treatment.

[0115] Test Example 2: Comparison of Example 1 and Comparative Example 2 on k value (h -1 ), 2h release rate, and corneal retention (h) test

[0116] Table 2 Comparison results of Example 1 and Comparative Example 2 on k value (h -1 ), 2h release rate, and corneal retention (h) test

[0117] Group [k value (h -1 )]]> 2h release rate Corneal retention (h) Example 1 0.18 22% 5.5 Comparative Example 2 0.38 68% 1.8

[0118] According to Table 2 above, compared with Comparative Example 2, the core advantage of the present application is to break through the construction of a biomimetic protein-hyaluronic acid composite barrier system, which is specifically manifested in three technical upgrades: first, the dense protein layer formed by the covalent cross-linking of lysozyme and lactoferrin at a molar ratio of 1:1 through amide bond reduces the drug burst release rate from 68% to 22%, and prolongs the release half-life by more than 2 times, which is due to the diffusion pore formed by the anchoring of tacrolimus in the hydrophobic region of LF and the electrostatic interlocking of LYZ / LF, which synergistically blocks drug leakage; second, the protein composite layer precisely tunes the surface charge to near neutral, eliminating the charge repulsion effect with traditional patents of strong negative nanoemulsion, and at the same time, the surface hyaluronic acid binds to ocular mucin through sugar-sugar specific binding, which increases the corneal residence time from 1.8 hours to 5.5 hours; finally, the active protein functional design retains 92% of the antibacterial activity of lysozyme and integrates the LF iron ion regulation ability, which synchronously inhibits ocular oxidative stress and microbial infection, while the surfactant system of Comparative Example 2 has no biological activity function, and Poloxamer 188 of Comparative Example 2 causes significant eye irritation. These three innovations enable the present application to upgrade the treatment paradigm from "passive drug loading" to "active regulation".

[0119] Test item 3: Long-term stability test of the preparation at 4℃ / 25℃

[0120] Table 3: Long-term stability test results of the preparation of Example 1 of the present application at 4℃ / 25℃

[0121] Storage condition Time (month) Content retention rate 4℃ 6 97% 25℃ 3 92%

[0122] As can be seen from Table 3 above, the eye nanoemulsion preparation prepared in Example 1 of the present application for treating immune dry eye has good stability.

[0123] Test item 4: Performance test of the example and the comparative example for repairing the ocular surface microenvironment (cup cell density test, IL-1β level detection)

[0124] Table 4: Performance test results of the example and the comparative example for repairing the ocular surface microenvironment (cup cell density test, IL-1β level detection)

[0125] Group Goblet cell density (↑ %) IL-1β reduction (%) Example 1 40% 62 Example 2 32% 53 Example 3 35% 57 Comparative Example 1 8% 15 Comparative Example 2 28% 40

[0126] From the above table 4, it can be seen that the ophthalmic nanoemulsion preparation for treating immune dry eye prepared by the application indeed has better repairing characteristics of ocular surface microenvironment. Possible reason analysis: the core mechanism of the application embodiment 1 is significantly better than that of the comparative example 1 and the comparative example 2, which lies in the synergistic repairing effect of the unique protein complex layer: first, lactoferrin (LF) directly activates the Wnt / β-catenin pathway of goblet cells through the N-terminal lactoferrin peptide, promotes the up-regulation of mucin MUC5AC synthesis enzyme expression, and drives the increase of goblet cell density; second, the muramyl dipeptide (MDP) produced by the hydrolysis of lysozyme (LYZ) on the cell wall of gram-positive bacteria is recognized by the corneal TLR2 receptor, triggering the increase of anti-inflammatory factor IL-10 secretion, synchronously inhibiting the activation of NLRP3 inflammasome, and making the key inflammatory factor IL-1β decrease greatly; finally, LF chelates free iron ions to block the Fenton reaction, greatly reducing the oxidative damage marker MDA of corneal epithelium, while the comparative example 1 with simple drug loading lacks this function, and the oxidative stress continuously aggravates the damage of ocular surface. This triple synergy of “immunosuppression-anti-inflammatory-antioxidation” is a biological repair paradigm that cannot be achieved by traditional nanoemulsion.

[0127] Finally, it should be explained that the above embodiments are used to illustrate the technical solutions of the application, but not to limit the protection scope of the application. Although the application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the application can be modified or replaced by the same, without departing from the essence and scope of the technical solutions of the application.

Claims

1. An ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome, characterized in that, Includes the following components: (1) Nanoemulsion core: contains a therapeutically effective amount of immunosuppressant, selected from tacrolimus; (2) Interface protein layer: a tear-like protein complex layer fixed at the oil-water interface by amide bonds, composed of lysozyme (LYZ) and lactoferrin (LF) covalently cross-linked in a molar ratio of 1:1; The surface of the tear-mimicking protein complex layer is also modified with hyaluronic acid oligosaccharides, whose carboxyl groups, after activation, are linked to the ε-amino group of lysozyme (LYZ) or lactoferrin (LF) via amide bonds.

2. The ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome according to claim 1, characterized in that: In simulated tear fluid at pH 7.4, the drug release rate constant k, fitted by first-order kinetics, is ≤ 0.25 h. -1 .

3. The ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome according to claim 1, characterized in that, The preparation method is as follows: S1. Preparation of drug-containing nanoemulsion: Dissolve the above immunosuppressant in medium-chain triglycerides, add Poloxamer 188 and aqueous phase, pre-emulsify with microfluidic flow at 50-300 bar, cycle 1-3 times, and continue high-pressure homogenization at 500-1000 bar 2-4 times. S2. Activate the surface of the nanoemulsion: Add 1-5 mM NHS-PEG4-Maleimide to the product of S1, react at 20-37℃ for 20-40 min, and remove free reagents by ultrafiltration; S3. Protein fixation: Lysozyme (LYZ) and lactoferrin (LF) were dissolved in PBS at a molar ratio of 1:1 to pH 6.5-8.

0. The product from S2 was then added and the mixture was stirred at 20-37°C for 1-3 hours. S4. Hyaluronic acid modification: Add 0.5-2% w / v hyaluronic acid oligosaccharide with pre-activated carboxyl groups by EDC / NHS, and react at pH 5.5-7.0 for 0.5-2 h; S5. Purification: Ultracentrifuge at 10,000-18,000 rpm for 20-40 minutes to remove free proteins.

4. The method for preparing an ophthalmic nanoemulsion formulation for treating immune-mediated dry eye according to claim 3, characterized in that, The step S4, which involves activating the carboxyl groups with hyaluronic acid oligosaccharide solution EDC / NHS, is as follows: Step 1: Dissolve the hyaluronic acid oligosaccharide in 0.05-0.2M MES buffer at pH 4.8-6.2 to prepare a 5-20 mg / mL solution; Step 2: Continue adding EDC to the solution from Step 1 until the final concentration is 5-20 mM, and continue adding NHS until the final concentration is 10-50 mM; Step 3: Stir the mixture obtained in Step 2 at 20-30℃ in the dark for 10-30 minutes. Step 4: Centrifuge the mixture after stirring in Step 3 above using a 5-20 kDa MWCO ultrafiltration centrifuge tube at a speed of 10,000-20,000 rpm for 5-20 min. Repeat the centrifugation purification process 2-4 times to remove free reagents.

5. The ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome according to claim 1, characterized in that: With the synergistic sustained-release effect of the protein complex layer and hyaluronic acid, administration can be achieved 1-2 times daily.

6. The method for preparing an ophthalmic nanoemulsion formulation for treating immune dry eye syndrome according to claim 4, characterized in that: The hyaluronic acid oligosaccharide has a molecular weight of 3-5 kDa.

7. The ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome according to claim 1, characterized in that: The core particle size of the nanoemulsion is 80±10 nm, and the PDI is ≤0.25; after protein modification, the overall particle size is ≤120 nm, and the PDI is ≤0.

3.

8. The ophthalmic nanoemulsion formulation for treating immune-mediated dry eye syndrome according to claim 1, characterized in that: The protein complex layer has a thickness of 8-12 nm and a lysozyme activity retention rate of ≥85%.

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