CN113577011B - Tacrolimus ophthalmic preparation, preparation method and application thereof - Google Patents

Abstract

The invention discloses a tacrolimus ophthalmic preparation, a preparation method and application thereof. The invention polymerizes the monofunctional POSS, PEG and PPG to obtain the copolymer MPEP, the copolymer MPEP shows sol-gel transition behavior sensitive to temperature, and the thermally responsive hydrogel improves the water solubility of the drug after carrying the dry eye drug tacrolimus, improves the adhesiveness on the ocular surface mucosa, prolongs the drug retention time and forms a long-acting ocular drug delivery system. The tacrolimus ophthalmic formulation of the present invention shows a better dry eye treatment effect compared to other tacrolimus formulations.

Description

Tacrolimus ophthalmic preparation, preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a preparation of tacrolimus.

Background

Dry eye is a multifactorial ocular surface disorder with tear film instability and hyperpermeability. Inflammation caused by dry eye can lead to damage to the corneal epithelium and abnormal nerve sensations. According to clinical epidemiological surveys of multiple research centers worldwide, the prevalence of dry eye accounts for 4.1% to 23.7% of the general population. Dry eye has a great impact on the quality of life and economic burden on patients.

Currently, eye drops containing the hydrophobic macrolide immunosuppressant tacrolimus (FK 506) can alleviate dry eye by blocking T-cell dependent immune responses. However, FK506 is a highly hydrophobic substance, is hardly soluble in water, and is poorly soluble in hydrophilic formulations, which reduces the efficacy of the drug, even causing burns and stings in some patients. In addition, eye movements (e.g., blinking, tear secretion, and nasolacrimal drainage) can rapidly clear FK506 from the ocular surface, resulting in low bioavailability of existing commercial eye drops. In order to ensure therapeutic effects, it is often necessary to increase the frequency of drug administration to four to five times per day, but this in turn increases the risk of side effects.

Therefore, further studies have been required for improvement of the water solubility of the drug, extension of the residence time of the drug on the ocular surface, and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a tacrolimus ophthalmic preparation, a preparation method and application thereof.

One of the technical schemes adopted for solving the technical problems is as follows:

an ophthalmic tacrolimus preparation wherein tacrolimus is carried on MPEP (poly (MPOSS-PEG-PPG carbamate) copolymer), which is a copolymer of MPOSS, PEG (polyethylene glycol) and PPG (polypropylene glycol).

The MPEP can self-assemble to form micelle and wrap the tacrolimus in the micelle to form drug-loaded micelle, namely the tacrolimus ophthalmic preparation.

Further, the tacrolimus ophthalmic formulation may be converted to a gel on the ocular surface. At ocular surface temperatures (about 34.5 ℃), the Critical Gel Temperature (CGT) of MPEP is about 5wt%, i.e., the concentration of MPEP in tacrolimus ophthalmic formulations is about 5wt% and above, which converts to gel on the ocular surface.

In one embodiment: in the tacrolimus ophthalmic preparation, the weight ratio of tacrolimus to MPEP is 1: 15-25.

In one embodiment: in the raw materials for copolymerizing to form the MPEP, the feeding weight ratio of PEG to PPG is 1-3: 0.5 to 1.5 percent of MPOSS, and the weight percentage of the batch is 0.3 to 3 percent.

Preferably, the MPOSS is added in a weight percentage of 1.5-2.5%, and the obtained MPEP has the best effect.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

a method of preparing an ophthalmic tacrolimus formulation comprising: dissolving the MPEP and the tacrolimus in a volatile organic phase, and dropwise adding the MPEP and the tacrolimus into an aqueous phase under stirring; removing the volatile organic phase to obtain the tacrolimus ophthalmic preparation, wherein the tacrolimus ophthalmic preparation is a drug-loaded micelle formed by self-assembling MPEP to form a micelle and wrapping the tacrolimus in the micelle.

In one embodiment: the volatile organic phase comprises acetone and the aqueous phase comprises phosphate buffer.

In one embodiment: the preparation method of the MPEP comprises the following steps: under the protective atmosphere, PEG, PPG and MPOSS react in anhydrous toluene at 105-115 ℃ for 20-30 h by taking HDI (1, 6-hexamethylene diisocyanate) as a chain extender and DBT (dibutyl tin dilaurate) as a catalyst to obtain the MPEP.

The third technical scheme adopted by the invention for solving the technical problems is as follows:

use of an ophthalmic tacrolimus formulation in the manufacture of a medicament for the treatment of dry eye.

Preferably, the dosage form of the medicament comprises eye drops; eye ointment, eye gel, etc. may also be used.

The fourth technical scheme adopted for solving the technical problems is as follows:

the application of the polymer MPEP as a pharmaceutical excipient. For example, MPEP can be used as an auxiliary material for preparing ophthalmic medicines (such as eye drops, eye pastes, eye gels and the like), and can also be used as an auxiliary material for preparing other types of medicines (such as injections, external medicines and the like). The Critical Gel Temperature (CGT) of MPEP varies from body to body, but basically MPEP concentrations of about 5wt% and above can be converted to gel in situ.

Preferably, the dosage form of the medicament comprises eye drops; eye ointment, eye gel, etc. may also be used.

According to the invention, a series of copolymer MPEP is synthesized by MPOSS, PEG and PPG, the introduction of hydrophobic POSS groups promotes the self-assembly of the copolymer MPEP, and influences the micelle and gelation characteristics, so that the copolymer MPEP has sol-gel transition behavior sensitive to temperature, thereby further increasing the application possibility in biomedicine. The MPEP improves the water solubility of FK506, the MPEP hydrogel MPEP-FK506 loaded with FK506 has strong adhesive force with the ocular surface, prolongs the retention time of the medicine, improves the symptoms of xerophthalmia, and has good biocompatibility and safety. Compared with the existing commercial FK506 and F127-FK506 preparations, the MPEP-FK506 hydrogel has better efficacy in xerophthalmia.

The equipment, reagents, processes, parameters, etc. according to the present invention are conventional equipment, reagents, processes, parameters, etc. unless otherwise specified, and are not exemplified.

All ranges recited herein are inclusive of all point values within the range.

The terms "about," "about," or "about" and the like as used herein refer to a range of + -20% of the stated range or value.

In the present invention,% is weight percent and ratio is weight percent unless otherwise specified.

In the present invention, the "room temperature" is a conventional ambient temperature, and may be 10 to 30 ℃.

Compared with the background technology, the technical proposal has the following advantages:

the invention provides an eye adhesive thermal response hydrogel MPEP, which is PEG-PPG copolymer modified by POSS groups. MPOSS-PEG-PPG has the ability to self-assemble at the ocular surface, with micellizing and gelling properties. In addition, the hydrogel can carry FK506 with poor water solubility, remarkably improve the retention force of FK506 on the ocular surface, effectively relieve the xerophthalmia of mice, inhibit the xerophthalmia more effectively than the drugs currently available in a mouse xerophthalmia model, and show excellent biocompatibility and low toxicity in vitro and in vivo. The thermal response hydrogel MPEP containing POSS has great application prospect in developing long-acting eye drops and effectively treating eye diseases.

Drawings

Fig. 1 is used to illustrate the synthesis and self-assembly of MPEP, wherein: (a) For illustration MPOSS, PEG and PPG were synthesized into MPEP by polyaddition; (b) To illustrate the self-assembly of MPEP loaded with FK506 and the process of forming hydrogels in water.

FIG. 2 is a schematic view of ¹ H NMR and FT-IR spectra, wherein: (a) is MPOSS (upper) and 2MPEP (lower); (b) FT-IR spectra for MPOSS, PEG, PPG and three MPEP copolymers; (c) 1H NMR spectrum of 0.5 MPEP; (d) 1H NMR spectrum of 1 MPEP. ¹ The solvent of H NMR was CDCl ₃ 。

Fig. 3 is a thermal performance test result, wherein: (a) is the TGA profile of the MPOSS and three MPEP copolymers; (b) a DSC cooling curve for the MPOSS, PEG, and MPEP copolymers; (c) is a DSC heating profile of MPOSS, PEG and MPEP copolymers; and (d) is an enlarged view of the dotted line box in (c).

Fig. 4 is a graph illustrating the performance test results of MPEP sol, wherein: (a) UV-vis spectra of DPH-1MPEP aqueous solutions with different concentrations at room temperature; (b) CMC value of 1MPEP determined by extrapolation; (c) To illustrate the change in transmittance at 500nm of three aqueous MPEP solutions (2 wt%) with increasing temperature; (d) For illustration of the size distribution of micelles in a 1MPEP aqueous solution (0.5 wt%) at three temperatures; (e) The method is used for explaining the change of micelle size with temperature and concentration in the 1MPEP water solution; (f) To illustrate that the size of micelles in an aqueous MPEP solution at 70 ℃ varies with the type and concentration of MPEP; (g) CMC value of 0.5MPEP determined by extrapolation; (h) is the CMC value of 2MPEP determined by extrapolation.

Fig. 5 is a phase diagram, wherein: (a) 1MPEP; (b) Pluronic F127; (c) 0.5MPEP; (d) 2MPEP.

Fig. 6 is used to illustrate rheological properties, wherein: (a) a temperature sweep profile of 1MPEP (6 wt%); (b) Is a rheological characteristic curve of 1MPEP (6 wt%) during temperature rise; (c) a temperature sweep profile of 0.5MPEP (8 wt%); (d) a temperature sweep profile of 2MPEP (5 wt%); (e) A rheological profile of 0.5MPEP (8 wt%) during temperature rise; (f) Is a rheological characteristic curve of 2MPEP (5 wt%) during the temperature rise.

Fig. 7 is used to illustrate the morphological structure of MPEP micelles and MPEP hydrogels, wherein: (a) a particle size distribution of 1MPEP and 2 MPEP; (b) TEM photographs of 1MPEP micelle and 2MPEP micelle; (c) SEM pictures of 1MPEP, 2MPEP and F127 hydrogels.

Fig. 8 is used to illustrate the properties of hydrogels, wherein: (a) Light transmittance for 1MPEP, 2MPEP, F127 and commercial carbomer hydrogels; (b) In vitro degradation curves for 1MPEP, 2MPEP and F127 hydrogels; (c) In vitro drug release behavior of FK506 in 1MPEP, 2MPEP and F127 hydrogels; (d) is in vitro cytotoxicity of hydrogels to HCE cells; (e) Transparency photographs of hydrogels for commercial carbomers, commercial FK506, and F127-FK506, 1MPEP-FK506, 2MPEP-FK506 loaded with FK 506.

Fig. 9 is a surface plasmon resonance test result for evaluating binding affinity of a polymer to mucin, wherein: (a) 1MPEP; (b) 2MPEP; (c) F127; 3.906. Mu.M, 7.813. Mu.M, 15.63. Mu.M, 31.25. Mu.M, 62.5. Mu.M are sequentially arranged from top to bottom in each graph.

Fig. 10 is an in vivo evaluation experiment result of hydrogel, wherein: (a) Is a slit lamp photograph (arrows indicate the position of the hydrogel on the cornea and indicate the separation of the hydrogel from the cornea); (b) OCT images of hydrogels on the ocular surface after blinking (arrows indicate the position of the hydrogel on the cornea and indicate the separation of the hydrogel from the cornea); (c) OGD staining results for each group; (d) To measure tear production results using the phenol red line test; (e) is an image of OGD staining intensity statistics. Data are expressed as mean ± standard error (s.e.m.), P <0.05, P <0.01, P <0.001.

Fig. 11 is a histological image of the cornea and conjunctiva, wherein: (a) Representative images of immunofluorescent staining for MMP-3 (rectangular indicates areas of corneal epithelial MMP-3 immunofluorescent staining); (b) Representative images of MMP-9 immunofluorescent staining of conjunctiva.

Fig. 12 is a staining result of PAS and CD4, wherein: (a) Goblet cells in each group of conjunctiva are shown for PAS staining results (arrows indicate goblet cells); (b) CD4 staining results, showing the number and location of cd4+ T cells infiltrating each group of conjunctiva (arrows indicate cd4+ T cells); (c) is a quantitative analysis of goblet cell counts; (d) is a quantitative analysis of the expression level of CD4 protein. Data are expressed as mean ± standard error (s.e.m.), P <0.05, P <0.01, P <0.001.

Fig. 13 is H & E staining results of corneal and conjunctival tissue sections, wherein: (a) is the cornea; (b) is conjunctiva.

FIG. 14 is a graph illustrating the ocular surface pharmacokinetic results of MPEP hydrogels with commercial FK506, F127-FK 506.

Fig. 15 is a safety test result of a hydrogel, wherein: (a) is a fundus image; (b) fluorescent fundus angiography; (c) OCT images showing retinal structure and morphology (dashed lines represent scanned portions of the murine retina, rectangular represents a partial view of the retina).

Detailed Description

The invention is further described below with reference to the drawings and examples.

Example 1 Synthesis of Poly (MPOSS-PEG-PPG urethane) copolymers (MPEP)

The MPEP of this example is a copolymer of MPOSS (available from Hybrid Plastics (MS, USA)), PEG (polyethylene glycol) and PPG (polypropylene glycol). The synthesis process uses HDI (1, 6-hexamethylene diisocyanate) as a chain extender and DBT (dibutyl tin dilaurate) as a catalyst. The weight ratio of PEG to PPG is fixed as 2:1. the weight percent of MPOSS fed was set to 0.5, 1 or 2wt%, respectively, and the resulting copolymer was represented as nMPP according to the different weight percent MPOSS fed, where n represents the weight percent MPOSS fed, MP represents MPOSS, E represents PEG, and P represents PPG.

Take the 0.5MPEP formulation as an example: 10g of starting material (comprising 6.63g of PEG (3.3 mmol), 3.32g of PPG (1.6 mmol) and 0.05g of MPOSS (5.3X10) ^-5 mol)) and 100mL of anhydrous toluene were added to a 250mL round bottom flask and traces of water and most of toluene were removed by azeotropic distillation in a rotary evaporator until only 10mL of toluene remained. This procedure was repeated twice. Subsequently, stirring was carried out at 110℃under an argon atmosphere, and 0.84mL of HDI (5.2 mmol) and two drops of DBT were added for reaction; 10mL of anhydrous toluene was added in addition whenever the reaction became too viscous to allow the magnetic stirrer to rotate. After 24 hours of reaction, the precipitate was precipitated with hexane-diethyl ether (3:7 v/v) mixture, redissolved in IPA (isopropanol) and dialyzed against deionized water for 3 days for purification. Finally, 0.5MPEP was obtained by freeze-drying for 3 days, with a copolymer yield of 70%.

1MPEP and 2MPEP are obtained with reference to the above method.

Example 2 molecular characterization of MPEP

Gel Permeation Chromatography (GPC): measured at 40℃using a Waters GPC system (Shimadzu) equipped with two Phenogel columns (103 and) (size: 300 x 7.80 mm) and differential refractive detector. The eluent was HPLC grade THF (tetrahydrofuran) and the eluent flow rate was 1mL/min. Polymethyl methacrylate standards were used to generate calibration curves.

¹ H Nuclear Magnetic Resonance (NMR) spectroscopy: measured at room temperature using a 500MHz NMR spectrometer (JEOL, japan). Chemical shift reference solvent CDCl ₃ A solvent peak at 7.3 ppm.

Fourier transform infrared (FT-IR) spectrum: measured using a Spectrum 2000FT-IR spectrophotometer (Perkin Elmer, USA) at room temperature. Mixing the copolymer MPEP with KBr and tabletting at 4,000-400 cm ^--1 In the wave number range of 4cm ^-1 Is scanned 32 times at the resolution of (c).

The results were as follows:

different fromThe MPOSS content random multiblock copolymer MPEP is synthesized from MPOSS, PEG and PPG by addition polymerization, with DBT as catalyst and HDI as chain extender (FIG. 1 (a)). From the GPC results in Table 1, it can be seen that the MPEP copolymers have similar molecular weights and dispersitiesAfter 24 hours of reaction, there are an average of 15 to 18 blocks per polymer chain.

TABLE 1 molecular characterization and thermal Properties of copolymer MPEP

Table 1 (subsequent table)

a is composed of ¹ H NMR spectrum was calculated. The degradation temperature was determined by TGA method. c DSC method to measure crystallization temperature. d DSC method to measure the melting temperature. The glass transition temperature was measured by e DSC method.

The chemical structure of MPEP copolymer is determined by ¹ H NMR spectrum verification (fig. 2 (a) (c) (d)). Typically, the PEG block has CH at 3.6ppm ₂ Characteristic peaks are generated. The PPG blocks have CH at 1.1ppm,3.4ppm and 3.5 ppm, respectively ₃ 、CH ₂ And characteristic peaks of CH. HDI bonds show CH at 1.3ppm, 1.5ppm and 3.1ppm ₂ Characteristic peaks of the groups. Reference to MPOSS ¹ H NMR spectrum through seven CH's attributed to isobutyl ₃ And CH (CH) ₂ The 0.9ppm and 0.6ppm peaks confirm the presence of MPOSS blocks in MPEP copolymers. The integral ratio of all these peaks is consistent with the theoretical value. The composition of the MPEP copolymer was calculated from the integral ratio, and the result was close to the feed ratio (Table 1).

The success of the synthesis was further demonstrated by comparing the FT-IR spectrum of the MPEP copolymer with that of MPOSS, PEG, PPG (FIG. 2 (b)). At 1705cm ^-1 And 1535cm ^-1 A new absorption band appears nearby, representing a new absorption band composed of hydroxyl groups/isocyanidesC=o and N-H in the urethane linkage formed by the acid ester reaction. At about 1106cm ^-1 Overlapping absorption bands of C-O-C in PEG and PPG blocks and Si-O-Si in MPOSS blocks were observed. The Si-C of the small amount of MPOSS will be at about 1253cm ^-1 Where a medium absorption band is created. In addition, at 3000-2800 cm ^–1 The absorption bands in the range correspond to the saturated C-H present in all three blocks.

Example 3 thermal Properties of MPEP

Thermogravimetric analysis (TGA): performed on a Q500 TGA analyzer (TA Instruments in the united states) under a nitrogen atmosphere. At 20 ℃ for min ^-1 The rate of (2) increases from room temperature to 800 ℃.

Differential Scanning Calorimetry (DSC): calibration was performed on a Q100 photo DSC analyzer (TA Instruments, USA) using indium. Heating the sample from-80deg.C to 200deg.C, and then heating at 20deg.C for 20 min ^-1 Is cooled at a rate of (2). The heating/cooling cycle was repeated twice and the data from the second cycle was taken for analysis.

The results were as follows:

the thermal properties of MPEP copolymers were investigated by TGA and DSC and the relevant parameters are shown in table 1. The TGA results show that the thermal stability of all three copolymers is good, and the 5% weight loss degradation temperature of all three copolymers is higher than 300 ℃. At the same time, the MPOSS undergoes two-step degradation after 266℃and 392℃due to the decomposition of the vertex groups and the transformation of the Si-O-Si cage structure into ceramic, respectively (FIG. 3 (a)). This change is reflected in the TGA profile of the MPEP copolymer, where the first step overlaps with the degradation of the PEG and PPG blocks, and the second step is performed after about 570 ℃.

DSC results showed that a single Tg of the MPEP copolymer occurred during heating (fig. 3 (d)) indicating that the MPOSS had good miscibility with the polymer and no microphase separation. The heating profile of the MPOSS shows two endothermic peaks at 0 ℃ and 162 ℃, possibly corresponding to two crystal types (fig. 3 (c)). When incorporated into polymer systems, bulk aggregation of MPOSS is limited due to low levels. Thus, for MPEP copolymers, only one endothermic peak due to the PEG block was observed around 38℃which is lower than the Tm of pure PEG due to copolymerization. In contrast, the cooling profile of MPEP copolymer shows an exothermic peak around-19℃and a weaker shoulder on the left (FIG. 3 (b)), which may be due to a broad distribution of molecular weights.

Example 4 self-Assembly Property of MPEP

Determination of Critical Micelle Concentration (CMC): aqueous MPEP copolymer solutions of different concentrations starting from 1wt% were prepared using a gradient dilution method and then mixed with DPH methanol solution (0.6 mM) at 50:1 by volume. After reaching equilibrium overnight at 4 ℃, UV-vis spectra were obtained on a UV-2501PC spectrophotometer (shimadzu) at room temperature in the range of 320-460 nm.

Determination of minimum critical solution temperature (LCST): the LCST value of the aqueous MPEP copolymer solution (2 wt%) was measured by UV-visible spectrometry on a UV-2501PC spectrophotometer (Shimadzu, japan) equipped with a temperature control module, with a temperature gradient of 1℃and an equilibration time of 5 minutes/step, with a temperature rise from 25℃to 75 ℃.

Particle size analysis: the particle size and distribution of the aqueous MPEP copolymer solutions at different concentrations (0.1, 0.5 and 1 wt.%) and at different temperatures (25, 37 and 70 ℃) were measured on a Nano ZetaSizer system by Dynamic Light Scattering (DLS) method, with a laser wavelength of 633nm and a scattering angle of 173 ℃.

The results were as follows:

in aqueous solution, amphiphilic MPEP copolymers tend to self-assemble into micelles from monomers driven by hydrophobic interactions. Such micelles have a hydrophobic core formed by the MPOSS and PPG blocks, and a hydrophilic moiety formed by the PEG blocks, which can encapsulate hydrophobic molecules in aqueous solution, thereby producing a stabilizing and solubilising effect. Based on this, CMC values of MPEP copolymers were determined from UV-vis absorption spectra using hydrophobic DPH as fluorescent probe. As the concentration increases from 0.0005wt% to 1wt%, absorption peaks around 344, 358, and 378nm become stronger (fig. 4 (a)). The absorbance at 378nm and 400nm (A378-A400) plotted against the logarithmic concentration gave CMC values of 0.082wt%,0.075wt% and 0.071wt%, respectively, of 0.5MPEP,1MPEP and 2MPEP, which decreased slightly with increasing MPOSS content (FIG. 4 (b) (g) (h)).

Since the hydrophilic/hydrophobic transition of PPG is temperature dependent, the self-assembly behaviour will change, thus achieving a delicate balance between hydrophilicity and hydrophobicity. As shown in (fig. 1 (b)), the MPEP-formed micelles further aggregate reversibly at higher temperatures or concentrations. Macroscopic appearance is seen as cloudiness of the MPEP aqueous solution. The LCST value of a copolymer is generally defined as the temperature at which a 50% reduction in transmittance occurs at 500nm at a given concentration. As can be seen from (fig. 4 (c)), the LCST value of the MPEP copolymer changed with a change in the content of the MPOSS, and at a concentration of 2wt%, the LCST value of 0.5MPEP was 53.6 ℃, the LCST value of 1MPEP was 60..0 ℃, and the LCST value of 2MPEP was 66.0 ℃.

This micelle conversion at low concentrations was also confirmed by the change in particle size. Table 2 shows the average particle size of particles in MPEP aqueous solutions at different temperatures and concentrations as measured by DLS method. By comparison, the particle size showed a slight decrease when the temperature was increased from 25 ℃ to 37 ℃. The particle size increases to different extents were observed after heating the sample to 70 c, the higher the concentration the more pronounced the particle size increase due to the greater proportion of micellar aggregates (fig. 4 (d) (e)). Moreover, the MPOSS content of the copolymer also affects the particle size, especially at relatively high temperatures. As shown in fig. 4 (f), the particle size at 70 ℃ is positively correlated with the content of MPOSS, and the difference between the different concentrations increases because the strong hydrophobicity of MPOSS can promote self-assembly and aggregation of micelles.

TABLE 2 particle size of micelles formed by MPEP copolymer in aqueous solution

Measurement of average particle size at different concentrations by aDLS method

EXAMPLE 5 Sol-gel transition behavior of MPEP

Determination of sol-gel transition behavior: an aqueous MPEP copolymer solution was prepared in 4-mL vials at a concentration ranging from 2wt% to 20 wt%. After 1 day at 4 ℃ to ensure complete dissolution, the sample was gradually heated in a water bath from 4 ℃ to 80 ℃, with a temperature gradient of 2 ℃, and equilibration time of 5 minutes/step. Gelation temperature is defined as the critical temperature at which a firm gel is observed in an inverted vial.

Rheological analysis: this was done on a Discovery DHR-3 parallel plate mixed rheometer. The results were measured from low temperature to body temperature and from 4 ℃ to 80 ℃ at a heating rate of 5 ℃/min, with strain fixed at 1% and frequency fixed at 1Hz.

The results were as follows:

as the concentration of the MPEP aqueous solution increases, the micelles will accumulate in a network and gradually become a non-flowable gel (fig. 1 (b)). This example investigated the sol-gel transition behavior of MPEP copolymers. As shown in fig. 5, the MPEP aqueous solution may undergo four phases as the temperature increases from 4 ℃ to 80 ℃): transparent sols, transparent gels, cloudy gels and dehydrated gels. Among them, the transition points of transparent gels and turbid gels are difficult to determine, and they are classified together as gel phases. In contrast, commercially available Pluronic F127 underwent three stages, including transparent sol, transparent gel and transparent sol. The opacity of MPEP thermogels may be due to the particular mesh size of their micellar network approaching the wavelength of visible light. The different final states may be due to excessive hydrophobic interactions between the multiple MPOSS and PPG blocks, collapsing the micelle network and repelling water at high temperatures. In addition, the Critical Gel Temperature (CGT) of MPEP copolymers decreases with increasing concentration and is affected by the content of MPOSS. At the same concentration, the CGT of MPEP copolymers with higher content of MPOSS is lower. The CGC of 0.5MPEP,1MPEP and 2MPEP are 7wt%,5wt% and 5wt%, respectively, which is much lower than the CGC of Pluronic F127, which is advantageous for biomedical applications.

This example investigated the temperature responsiveness of MPEP copolymers by rheology. The concentration of 0.5MPEP of the test sample was chosen to be 8wt%,1MPEP was 6wt%, and 2MPEP was 5wt%, with the corresponding CGT in the phase diagrams being about 32 ℃,24 ℃ and 26 ℃, respectively. According to the temperature scan in the range of 4 to 80 c (fig. 6), both the storage modulus G' and the loss modulus g″ increase at different rates with increasing temperature and exhibit sol-gel transition. CGTs for 0.5MPEP,1MPEP and 2MPEP were about 32.0 ℃,16.3 ℃ and 17.8 ℃, respectively. The CGT values of 0.5MPEP obtained by both methods agree, but the CGT values of 1MPEP and 2MPEP obtained from the rheological measurements are lower than those obtained from the phase diagram, probably because the temperature at which G' equals G "is virtually semi-solid and not sufficiently hard to be seen as a gel in the tube-inversion method. Starting at a temperature of about 70 ℃, G' and G "began to drop slightly and then again approached again, indicating gel collapse and consistent with the tube-inversion method results. In addition, the temperature was raised from a lower temperature (e.g., 15 ℃) to body temperature (about 37 ℃) to evaluate the material's response rate to temperature changes. As can be seen from fig. 6, a fast sol-gel transition is achieved almost in synchronization with the temperature change, which means that MPEP is used as an injectable material that can form hydrogels in situ in biomedical applications.

Example 6 MPEP micelle transmission electron microscopy images and MPEP hydrogel scanning electron microscopy images

And (3) obtaining an electron microscope image: the particle size of the micelles was measured using a Malvern nano ZetaSizer particle sizer (U.S.), and the morphology of the micelles was photographed using a Transmission Electron Microscope (TEM) (Hitachi HT-7800). Hydrogels were prepared with 1MPEP, 2MPEP and F127 polymers, respectively, and rapidly placed in a-80 ℃ freezer prior to photography with a Scanning Electron Microscope (SEM). Subsequently, the sample was freed of moisture using a freeze dryer and gold sprayed (40 seconds) onto the small pieces of dried sample. The morphology of the hydrogels was observed using a field emission scanning electron microscope (FE-SEM) (zeiss supra 55). The detection voltage is 5kV, and the amplification factor is 500 times.

The results were as follows:

the morphology of the polymer micelles was examined by TEM and the hydrogel morphology by SEM. Particle sizes of 1MPEP and 2MPEP micelles were measured at room temperature. The particle size distribution is shown in FIG. 7 (a). The morphology of the 1MPEP and 2MPEP polymer micelles was approximately circular, and the particle size was similar to that of the particle sizer, as shown in fig. 7 (b). Further, morphological structures of 1MPEP, 2MPEP and F127 hydrogels were observed using SEM (fig. 7 (c)). The prepared F127 dry product is in a powder shape, has no obvious space structure, and 1MPEP retains a clear pore structure, which shows that the product has better swelling performance. 2MPEP has a pronounced lamellar structure, probably due to the higher POSS content, which may alter the structural properties of the hydrogels.

EXAMPLE 7 transmittance of MPEP blank hydrogel

Determination of light transmittance of blank hydrogel: the temperature sensitive copolymers 1MPEP and 2MPEP can form hydrogels at 5wt% concentration at the ocular surface temperature (34.5 ℃) while F127 concentration needs to reach 20wt% to form gels at the ocular surface temperature. PBS solutions containing 5wt%1MPEP,5wt%2MPEP and 20wt% F127 were prepared, respectively. 100 μl of each set of samples was added to a 96-well plate and incubated at 34.5 ℃ to form hydrogels; commercial liquid carbomers were used as controls. Full wavelength scanning was performed using a multifunctional microplate reader (Multiskan FC, sammer feichi technology, usa) to determine absorbance values.

The transmittance of the hydrogel was calculated according to the formula a = -log T%, where a is absorbance and T% is transmittance. Hydrogels with a light transmittance of greater than 90% are considered transparent and translucent between 10% and 90% in the visible wavelength range of 390-780 nm; light transmittance below 10% is opaque.

The results were as follows:

hydrogels having a light transmittance of greater than 90% in the visible wavelength range of 390 to 780nm are considered transparent. The hydrogel applied to the eye needs to have high transparency, the present example tests absorbance of 1mpep,2mpep and F127 after conversion into gel, and calculates light transmittance according to formula a = -log T% (fig. 8 (a)). The transmittance of the hydrogel was greater than 90% in the visible wavelength range, indicating that the transparency of 1mpep,2mpep and F127 was very good without affecting vision (fig. 8 (e)). There is no significant difference in light transmittance from commercial liquid carbomers.

Example 8 degradation of MPEP hydrogels and in vitro drug Release of FK506

Degradation analysis of blank hydrogels: samples of 500 μl of 1mpep,2mpep and F127 solutions were added to the microcentrifuge tube, respectively. After the hydrogel was formed at 34.5 ℃, 500 μl of PBS was slowly added to the top of the hydrogel. The microcentrifuge tube was placed vertically in a thermostatted shaker and degradation was performed at 34.5℃and 50 rpm. At a specific time point, the supernatant liquid was removed and weighed, after subtracting the weight of the blank microcentrifuge tube, the weight of the remaining hydrogel was recorded, and fresh PBS was then added to continue degradation.

High Performance Liquid Chromatography (HPLC) determines the concentration of FK 506: chromatographic column: ZORBAX SB-C18 (5 μm, 4.6X100 mm); mobile phase: acetonitrile: 0.25% phosphoric acid in water (60:40, v/v); ultraviolet detection wavelength: 215nm; flow rate: 1mL/min; column temperature: 50 ℃; sample injection amount: 10 mu L. FK506 powder was precisely weighed, dissolved in acetonitrile to a concentration of 1mg/mL, and used as a stock solution. FK506 stock solutions were diluted in mobile phase to a series of concentration gradient standard solutions of 1, 2, 4, 12, 24, 32, 64 and 128 μg/mL. The standard solutions of the series of concentration gradients were subjected to HPLC chromatography and concentration-peak area standard curves were created. The fitted curve is y=19.059x+13.581, r ² ＝0.9999。

Preparation of FK506 hydrogel: FK506 is first entrapped in amphiphilic micelles due to its poor water solubility. 20:1 material/drug weight ratio, 1mpep,2mpep, f127 were dissolved in acetone, equal amounts of FK506 were added, respectively, and the mixture of FK506 and each material was added dropwise to PBS with stirring. Volatile acetone was removed by nitrogen purging to produce 1MPEP-FK506 (1M-FK 506), 2MPEP-FK506 (2M-FK 506) and F127-FK506 drug loaded micelle solutions. The precipitate was removed by centrifugation at 2000rpm and the concentration of FK506 in the supernatant was measured by HPLC. The sample concentrations were calculated and adjusted and a weight of material was added to ensure that the final concentrations of 1mpep,2mpep and F127 were approximately 5%,5% and 20%, respectively. The final concentration of FK506 was set to 0.1% according to the usual clinical concentration and stored for use.

In vitro drug release experiments: mu.L of a sample of 1M-FK506, 2M-FK506 or F127-FK506 solution was added to a microcentrifuge tube and a hydrogel was formed in a thermostatic water bath at 34.5 ℃. Subsequently, 500 μl of 1×pbs (containing 0.2% tween) was slowly added to the top of the hydrogel as a release medium. The microcentrifuge tube was placed vertically in a thermostatted shaker and drug release experiments were performed at 50rpm and 34.5 ℃. At a specific time point, the upper release medium was removed and replaced with 500 μl fresh PBS until the hydrogel was completely degraded. HPLC was performed on the collected release medium to measure the concentration of FK506, and then a drug release profile was constructed.

The results were as follows:

in ocular applications, the hydrogel has a slow release effect, so that the copolymer can exert better therapeutic effect when being prepared into hydrogel. 1MPEP,2MPEP and F127 hydrogels were prepared and in vitro degradation experiments were performed to compare degradation rates. As shown in fig. 8 (b), the degradation rate of F127 was very fast, and it was almost completely degraded on day 1. However, the degradation rate of 1MPEP hydrogel was slower and the 2MPEP hydrogel was completely degraded at day 6, whereas the degradation rate was slower and the complete degradation was only nearly complete at day 14.

This example prepares 1MPEP,2MPEP and F127 hydrogels containing FK 506. FK506 encapsulation rates of 1MPEP and 2MPEP can reach 93.4% and 92.8%, respectively, and drug loading rates of FK506 are 4.46% and 4.43%, respectively. Subsequently, a blank copolymer is added to achieve the proper hydrogel concentration and the temperature is raised to convert the solution to a gel state. As shown in fig. 8 (e), the transparency of FK506 solutions in F127, 1MPEP and 2MPEP polymers was significantly improved compared to Commercial-FK506, indicating that the polymer micelles significantly improved the solubility of FK506 in aqueous solutions. In order to examine the release rate of FK506 from the hydrogel, the present example also conducted an in vitro drug release experiment (fig. 8 (c)). The results show that FK506 is released most quickly from F127 hydrogel, released slowly from 1MPEP hydrogel and released most slowly from 2MPEP hydrogel, which shows that the hydrogel containing POSS group has higher strength and better slow release effect.

Example 9 in vitro cytotoxicity of MPEP hydrogels

Cytotoxicity of hydrogels to Human Corneal Epithelial (HCE) cells: 1M-FK506, 2M-FK506 and F127-FK506 solutions were prepared by the method of example 8. 1MPEP,2MPEP, F127, 1M-FK506, 2M-FK506 and F127-FK506 solutions were stored at-80℃for three days, and 500. Mu.L of each solution was then added to the cell culture plate to form a hydrogel at 37 ℃. Subsequently, 1mL of medium was added and incubated at 37℃for 48 hours. The supernatant was collected as hydrogel-drug extract, diluted with medium to FK506 concentrations of 0.5, 0.25, 0.125, 0.0625 and 0.03125mg/mL, respectively, with 0mg/mL FK506 group as control group.

Cell viability was assessed using the MTT assay: immortalized Human Corneal Epithelium (HCE) cell lines were purchased from American Type Culture Collection (ATCC) and cultured in DMEM/F12 medium supplemented with hEGF, insulin, FBS and penicillin-streptomycin. HCE cells were seeded onto 96-well plates and incubated for 24 hours. Dilute hydrogel-drug extracts were added, incubated for 48 hours, then the medium was removed, replaced with MTT solution, and incubated for another 4 hours. The supernatant was removed and DMSO was substituted to dissolve formazan crystals. Finally use An absorbance reader (Molecular Devices) measures absorbance at 492 nm; cell survival curves were then constructed.

The results were as follows:

to test the cytocompatibility of hydrogels, this example uses the MTT method to evaluate the toxicity of hydrogel extracts to HCE cells (fig. 8 (d)). The results indicate that, irrespective of the presence of FK506, the F127 hydrogel extract showed the highest cytotoxicity, the 1MPEP extract showed low cytotoxicity, and the 2MPEP extract had the lowest cytotoxicity. This is probably because F127 degrades at the fastest rate, resulting in more material entering the hydrogel-drug extract. The degradation rate of 1MPEP and 2MPEP hydrogels is slow, and thus cytotoxicity is reduced.

Example 10 interaction between mucin and POSS Polymer

Surface plasmon resonance: the interaction between mucin and polymer was analyzed using a BIAcore-T200 biomolecular interaction analyzer with surface plasmon resonance. The purchased mucin was dialyzed in ice-cold PBS for 48 hours to replace the salts in the original sample, followed by freeze-drying of the mucin. Acetate buffers of different pH values were formulated to screen the appropriate pH for protein coupling. Mucin was immobilized on CM5 sensor chips using an amino coupling method, with a final immobilization level of mucin of about 10,000RU. 1MPEP,2MPEP and F127 were diluted sequentially to 62.5. Mu.M, 31.25. Mu.M, 15.625. Mu.M, 7.8125. Mu.M, 3.90625. Mu.M and 0. Mu.M, respectively, using PBS as working buffer. Various polymer solutions of various concentrations were flowed through the chip at a contact time of 120s, a flow rate of 30. Mu.L/min and a separation time of 450 s. The chip was then washed with pH 2.5 glycine buffer and PBS buffer at an operating temperature of 2 ℃. The interaction between mucin and polymer was analyzed using Biacore T200 evaluation software (version 2.0) and with 1:1 in combination with model fitting curves to calculate the kinetic constant KD values.

The results were as follows:

in general, mucus can interact with foreign substances through electrostatic interactions, van der Waals forces, hydrophobic forces, hydrogen bonding, and the like. The mucous membrane surface of eyeball is covered with a layer of mucous, and the main component of mucous is mucin. The adhesion of the drug can be enhanced by enhancing the interaction of the drug with the mucin on the surface of the eyeball. POSS-containing polymers can enhance adhesion to mucins, as the POSS group can promote protein anchoring. To explore the potential mechanism by which POSS-containing polymers enhance mucoadhesion, the present example uses surface plasmon resonance (Biacore T200 system) to verify the binding affinity between the polymer and mucin.

Binding affinity is expressed in terms of equilibrium dissociation constant (KD), with smaller KD values resulting in greater binding affinity of the ligand to its target. As shown in FIG. 9, the binding force between polymer F127 and mucin was relatively weak, and the KD value was about 11.380. Mu.M. The polymers 1MPEP and 2MPEP containing POSS groups have significantly improved affinity for mucin, with KD values of 0.508. Mu.M and 0.409. Mu.M, respectively. The results indicate that the addition of POSS groups to the polymer chains helps to improve binding affinity to mucins. Presumably, this is because the strongly hydrophobic POSS groups can interact with the exposed hydrophobic domains around mucins. Thus, POSS-containing polymers can be used to improve the adhesion of drug delivery systems to ocular surfaces, extending drug retention times.

Example 11 clinical evaluation of MPEP hydrogels in a mouse Dry eye model

Animal model: female C57BL/6 mice (weight 25-35 g; age 8-12 weeks) were purchased from the university of Xiamen animal center (Xiamen Fujian, china). Animal protocols conform to the declaration of the institute of vision and ophthalmic research (ARVO) regarding the use of animals in ophthalmic and vision research. The study plan was approved by the ethical committee of experimental animals at the university of Xiamen. The mice were kept in a well ventilated environment with a humidity of 30.+ -. 3% and a temperature of 23-25 ℃. Five consecutive days, mice were subcutaneously injected with scopolamine hydrobromide (DS 5) (0.25 g/100 mL) under the armpit daily to establish a dry eye model.

Topical drug treatment and slit lamp imaging: in the mouse dry eye model, 5. Mu.L of eye drops (PBS, 1MPEP,2MPEP,F127,Commercial-FK506, F127-FK506, 1M-FK506, 2M-FK 506) were administered four times daily for 5 days for topical drug treatment. After the administration of the eye drops, the mice were manually blinked, the cornea was examined and the hydrogels on the ocular surface were observed by a slit lamp.

Tear secretion test: tear secretion tests were performed at the end of eye drop treatment. The phenol red cotton wire was placed into the subconjunctival vault and the mice were forced to remain open for 15 seconds. The phenol red cotton line became red after contact with tear, and the length of the red portion was measured in millimeters.

Corneal fluorescein staining: the mice were manually blinked several times by dropping 5% green dextran fluorescent dye (OGD) onto the surface of the mice eyes for 1 minute. Mice were then euthanized and the cornea was rinsed 5 times with 1mL saline. The cornea was photographed under 470nm fluorescence excitation using a fluorescence dissecting microscope (Leica, wetzlar, germany). Fluorescence intensity of the corneal OGD staining was quantitatively analyzed using NIS Elements software.

Histological staining: the mice were picked up for at least 24H with 4% formaldehyde, embedded in paraffin, sectioned (5 μm thick), mounted on glass slides and stained using hematoxylin and eosin (H & E) and PAS (Periodic Acid-Schiff stand) staining kit. Observations were made using an optical microscope (Eclipse E400 with DS-Fi1, nikon, mellville, N.Y.). For PAS staining, goblet cells of the upper and lower conjunctiva were quantitatively counted using NIS Elements software.

Immunofluorescent staining: after sampling, the tissues were OCT embedded and stored at-80 ℃. Frozen sections of samples (5 μm thick) were mounted on slides, acetone fixed, blocked with 2% Bovine Serum Albumin (BSA), and the frozen sections incubated with anti-matrix metalloproteinase 3 (MMP-3), anti-matrix metalloproteinase 9 (MMP-9) and anti-CD 4 antibodies. The following day, sections were washed 3 times with PBS and incubated with Alexa Fluor 488 conjugated donkey anti-sheep IgG or Alexa Fluor 488 conjugated donkey anti-rabbit IgG for 1 hour in the dark at room temperature. Finally, the sections were fixed with DAPI and visualized and imaged using a fluorescence microscope (leica dm 2500).

Western blotting: mouse conjunctival tissue was surgically isolated and dissolved in cold RIPA buffer containing a protease and phosphatase inhibitor cocktail. The purified protein was extracted and then quantified using BCA assay kit. An equal amount of protein was separated on a 12% Tricine gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% bsa for 2h at room temperature and then incubated overnight with anti-CD 4 antibodies. The next day, membranes were washed 3 times with Tris Buffered Saline (TBST) containing 0.05% tween 20, and then incubated with HRP-conjugated goat anti-rabbit IgG for 1 hour. Protein bands were visualized using a superchemiluminescent kit and images were captured by a ChemiiDoc XRS system (BioRad Laboratories, inc.) from philadelphia, pa. Densitometry was performed using ImageJ software.

Optical Coherence Tomography (OCT) examination, fundus imaging, and fluorescent fundus angiography: OCT examination, fundus imaging and fluorescence fundus angiography were performed using an optrocbe system (china). After 7 days of administration of various FK 506-containing eye drops (Commercial-FK 506, F127-FK506, 1M-FK506, 2M-FK 506), the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.0075 mL/mL), and pupils were dilated using the compound topiramate eye drops. Retinal OCT and fundus images of each eye were taken centered on the optic nerve head. After dosing and manual blinking, corneal OCT image shots were taken. Prior to performing fluorescent fundus angiography, mice were intraperitoneally injected with 1% fluorescein sodium (0.0075 mL/g) and fluorescent fundus angiography images were taken in the same manner as the fundus imaging procedure.

Statistical analysis: all data are expressed as mean ± standard error of mean (s.e.m.). Statistical significance was assessed using GraphPad Prism 8.0 software for one-way ANOVA, tukey post-hoc test, according to the normalization of data distribution. P <0.05 was significantly different.

The results were as follows:

hydrogels can remain on the ocular surface for a longer period of time than conventional eye drops, thereby providing a sustained effect. Blinking and tear washout can lead to drug loss, while the adhesive properties of hydrogels can prevent them from being cleared from the ocular surface. The in situ thermoreversible crosslinkability of the hydrogels on the ocular surface was evaluated by slit lamp and OCT. As shown in the slit lamp image in FIG. 10 (a), topical application of Commercial-FK506 did not produce residue on the cornea. In contrast, these formulations gel on the cornea after topical administration of F127-FK506, 1M-FK506, and 2M-FK 506. However, F127-FK506 and 1M-FK506 hydrogels showed separation from the cornea after blinking. Whereas 2M-FK506 hydrogel adhered well to the cornea and its integrity was not compromised by blinking, indicating that 2M-FK506 hydrogel provided greater adhesion. The surface of the adhesion between the hydrogel and the cornea is then visualized by Optical Coherence Tomography (OCT). Gelation of the F127-FK506 hydrogel resulted in the coating on the cornea breaking after blinking (arrow in the F127-FK506 group of FIG. 10 (b)). The 1M-FK506 hydrogel appeared as a non-uniform gel on the cornea after blinking (arrow in the 1M-FK506 group of FIG. 10 (b)). In contrast, 2M-FK506 hydrogels formed smooth gels on the cornea, blinking did not cause loss or damage to the hydrogel (arrows in the 2M-FK506 group of FIG. 10 (b)), indicating that 2M-FK506 hydrogels effectively prolonged the duration of drug action on the ocular surface.

Different eye drops were used to evaluate the efficacy of FK 506-loaded hydrogels in a mouse dry eye model. After staining the cornea with OGD dye, the corneal epithelial loss appears green under fluorescence. As can be seen from fig. 10 (c), OGD staining was severe in the control group, the DS5 group, the PBS treatment, the F127 treatment, the 1MPEP treatment, and the 2MPEP treatment groups. However, the Commercial-FK506, F127-FK506, 1M-FK506 and 2M-FK506 groups showed lower OGD staining, indicating that these four formulations had good repair of dry stress induced corneal epithelial defects. Topical application of 2M-FK506 significantly reduced the intensity of the corneal OGD staining (fig. 10 (e)). Due to its greater adhesion to the cornea, the 2M-FK506 hydrogel protects the corneal epithelium from the dry environment for a long period of time by sustained release of FK 506. Tear production is a very important clinical parameter in assessing dry eye progression because tear can protect corneal epithelial cells. The topical administration of 2M-FK506 significantly increased tear production compared to the other groups, but did not differ significantly from the F127-FK506 group (fig. 10 (d)). Based on these results, 2M-FK506 is an effective formulation to reverse the insufficient tear production due to dry eye.

Example 12 effects of MPEP hydrogels on cornea and conjunctiva in a mouse Dry eye model

The results were as follows:

MMP-3 and MMP-9 are key indicators of dry eye-induced damage to corneal barrier function; thus, levels of MMP-3 and MMP-9 in the corneal epithelium were studied by immunofluorescent staining (FIG. 11). Commercial FK506, F127-FK506, 1M-FK506 and 2M-FK506 topical application can reduce MMP-3 and MMP-9 expression in the corneal epithelium. Among these hydrogels, topical application of 2M-FK506 can minimize MMP-3 and MMP-9 levels, indicating improved corneal barrier function at dry pressure.

Dry eye can not only impair the corneal barrier function, but can also lead to corneal epithelial roughness. From the results of the corneal H & E staining, 2M-FK506 was shown to contribute more to the morphological restoration of the corneal epithelium than the other groups. Furthermore, goblet cells are simple columnar epithelial cells that secrete mucin to form a tear film that resists changes in the ocular surface environment, but inflammation caused by dry eye can result in goblet cell loss. The 2M-FK506 hydrogel can significantly save loss of goblet cells under dry pressure (FIG. 12 (a) (c) and FIG. 13 (b)), indicating that 2M-FK506 inhibits inflammation for a long period of time and plays a positive role in promoting recovery of ocular surface damage.

Dry eye is a chronic inflammatory immune disease characterized by infiltration of cd4+ T cells in the conjunctiva. FK506 (tacrolimus) binds to immunophilins cyclophilin and FKBP12, which inhibit calcineurin activity and direct metabolic reprogramming of cd4+ T cells to enhance their immunosuppressive effects. The results indicate that topical application of 2M-FK506 is effective in inhibiting scopolamine hydrobromide-induced conjunctival cd4+ T cell infiltration (fig. 12 (b)). Considering that cd4+ T cells play a key role in FK506 treatment of dry eye, further validating CD4 expression, 2M-FK506 was found to be effective in inhibiting CD4 expression (fig. 12 (d)), suggesting that 2M-PEP works better in concert with FK 506.

Example 13 eye surface pharmacokinetic study of MPEP hydrogels

Animal model: new Zealand white rabbits (male, weight 2.0-3.0 kg) were purchased from the university of Xiamen animal center (Xiamen Fujian, china). Each group of drugs (commercial FK506, F127-FK506, 1M-FK506, 2M-FK 506) was administered on the ocular surface at 50. Mu.L. The eyelid was manually closed for about 1 minute and the formulation was allowed to contact the cornea. A second administration is then performed. Subsequently, at different predetermined time points (1-, 2-, 4-and 8-hours, cornea n=4 was taken for each time point of each group), the anesthetized animals were euthanized. The cornea was dissected, washed with 0.9% physiological saline and dried on filter paper, and then the cornea was weighed. All corneas obtained were collected and stored at-80 ℃. A series of triple quadrupole LC/MS/M (AB SCIEX QTRAP6500+, USA) analyses were performed.

FK506 was extracted by protein precipitation, ascomycin was used as an Internal Standard (IS), and a 100ng/mL stock solution of IS was prepared with methanol at the time of use. The cornea was homogenized in 500. Mu.L of Deuterium Depleted Water (DDW), IS (100 ng/mL, 100. Mu.L) and methanol (800. Mu.L) were added to the cornea homogenate and vortexed for 5 minutes. The mixture was then centrifuged (12000 rpm. Times.4℃,10 minutes). Aliquots of the supernatant of the centrifuged homogenate were dried by nitrogen flow for at least 2 hours. The residue was vortexed in methanol (100 μl). The sample was centrifuged again (12000 rpm. Times.10 min, 4 ℃) and the resulting supernatant (80. Mu.L) was transferred for analysis.

The sample (5. Mu.L) was chromatographed on a liquid chromatograph (acquisition UPLC I-class, waters, USA), column (Acquity UPLC BEH C; 1.7 μm, 2.1X100 mm; waters, USA), gradient elution of mobile phase A (containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid): the initial mobile phase was 40% b and held for 1 minute; then rise to 95% b over 4 minutes and hold for 2.5 minutes; the mobile phase was returned to the original mobile phase after 0.1 min and equilibrated for 2.4 min, eluting at 50 ℃ at a flow rate of 0.4 mL/min. The procedure for mass spectrometry (AB SCIEX QTRAP6500+, usa) is as follows: CUR:40psi, temperature: 550 ℃, ion spray: 5500V, gas 1:55psi, gas 2:60psi. The detector employs a multiple reaction monitoring technique (MRM), conversion parameters: FK506:826.4/616.4, 826.4/415.3; ascomycin: 814.4/604.4.

The results were as follows:

in ocular pharmacokinetic studies, a significant increase in drug concentration in rabbit cornea at 1, 2, 4 and 8 hours post-dose was observed for 2M-FK506 compared to F127-FK506 and commercial FK506 (fig. 14). Compared to F127-FK506, MPEP-FK506 hydrogels are effective against ocular clearance by adhering to the ocular surface, which significantly increases the drug concentration of FK506 in the cornea. Also, the thermally responsive hydrogel system effectively increased the concentration of FK506 in the cornea compared to commercial FK 506. FK506 is released slowly and continuously in the hydrogel system. Thus, MPEP-FK506 hydrogels with enhanced ocular surface adhesion and thermal response properties can enhance ocular surface administration effects.

EXAMPLE 14 evaluation of safety of MPEP hydrogels on mice

The results were as follows:

studies have shown that nanoparticle-induced chronic immune responses can lead to damage to retinal vascular layers, retinal cell degeneration and neovascularization. The above examples have demonstrated the therapeutic efficacy of MPEP-FK506 formulations in a mouse dry eye model; the safety of such hydrogels still requires verification. Thus, fundus imaging, fluorescence fundus angiography, and OCT examination were performed 7 days after topical administration of the different formulations. Fundus imaging is a common technique for observing the morphology of the retina, optic disc, macular area, and retinal blood vessels, and for examining whether there is bleeding, exudation, hemangioma, retinal degeneration, retinal holes, neovascular, atrophic spots, pigment disorders, retinal changes, and the like. Fundus angiography is mainly used to further observe microscopic structural changes of fundus blood vessels. From fundus images and fluorescent fundus angiography, no apparent fundus vascular leakage, enlargement of blood vessels, or abnormal pathways were observed after MPEP-FK506 formulation treatment (fig. 15 (a) (b)). OCT is an ophthalmic diagnostic technique with the advantages of non-invasiveness, high resolution and rapid imaging speed at the cellular level, and solving the problem of in vivo investigation of the retina. After OCT examination of the retina on day 7, it was found that the structure of each retinal layer was aligned, no significant retinal detachment or degeneration was observed, and no difference in retinal thickness was observed between groups (fig. 15 (c)).

The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.

Claims (6)

1. A tacrolimus ophthalmic formulation characterized in that: in the tacrolimus ophthalmic preparation, tacrolimus is carried on MPEP, wherein the MPEP is a copolymer of MPOSS, PEG and PPG; the tacrolimus ophthalmic formulation turns into a gel on the ocular surface; in the raw materials for copolymerizing to form the MPEP, the feeding weight ratio of PEG to PPG is 1-3: 0.5 to 1.5 weight percent of MPOSS, and 0.3 to 3 weight percent of MPOSS; in the tacrolimus ophthalmic preparation, the weight ratio of tacrolimus to MPEP is 1: 15-25.

2. The tacrolimus ophthalmic formulation according to claim 1, characterized in that: the feeding weight percentage of the MPOSS is 1.5-2.5%.

3. A process for the preparation of an ophthalmic tacrolimus formulation according to any one of claims 1 or 2, characterized in that: comprising the following steps: dissolving the MPEP and the tacrolimus in a volatile organic phase, and dropwise adding the MPEP and the tacrolimus into an aqueous phase under stirring; removing the volatile organic phase to obtain the tacrolimus ophthalmic preparation.

4. A process for the preparation of an ophthalmic tacrolimus formulation according to claim 3, characterized in that: the preparation method of the MPEP comprises the following steps: under the protective atmosphere, PEG, PPG and MPOSS react in anhydrous toluene at 105-115 ℃ by taking HDI as a chain extender and DBT as a catalyst to obtain the MPEP.

5. Use of the tacrolimus ophthalmic formulation of any one of claims 1 or 2 in the manufacture of a medicament for the treatment of dry eye.

6. Use according to claim 5, characterized in that: the dosage form of the medicine comprises eye drops.