In Situ Liquid Crystal Gel as a New Ophthalmic Drug Delivery System for Pilocarpine Nitrate: Improving Preocular Retention and Ocular Bioavailability

: The purpose of this article is to develop an in-situ liquid crystal gel that can 34 be used as a novel ocular delivery system for pilocarpine nitrate (PN). The phytantriol 35 (PT) -based in situ liquid crystal gels were prepared by a vortex method using PT, 36 PEG400, Triglyceride (TAG) and water (in the ratio of 61.15:30:3.85:5, w/w). Firstly, 37 the internal structure of the PN-loaded liquid crystal gel was characterized by 38 polarizing microscope (PLM), small-angle X-ray scattering (SAXS), differential 39 scanning calorimetry (DSC) and rheology. In vitro drug release behavior and ex vivo 40 corneal permeation were investigated. Finally, eye irritation test ， preocular residence 41 time evaluation,were studied in vivo and compared with eye drops.Based on various 42 characterization techniques, it is proved that the internal structure of the gel is a 43 hexagonal phase.In vitro release results identified that PN could be released 44 continuously from HII gel over a period of 24 h. The in vitro obvious permeability 45 coefficient of HII gel was 3.19-fold (P < 0.01) higher than that of the eye drops. 46 Compared with eye drops,the HII gel had good bioadhesion and displayed longer 47 residence time on the eyeballs surface using fluorescent labeling technology.In 48 addition, through Corneal hydration level and eye irritation test ., it is conjectured that 49 H II gel will not cause eye irritation. In short, the formulation has the advantages of 50 high efficiency, slow release and non-toxicity, and will become a promising 51 pharmaceutical strategy to improve the efficacy of glaucoma. 52

Topical ophthalmic administration is the main method of ophthalmic treatment. 74 However, due to the high sensitivity of the eye and unique physiological barriers 75 (including corneal and conjunctival barriers, blood-water barriers and blood-retinal 76 barriers), the drug has low bioavailability and poor efficacy. Commonly used ocular 77 dosage forms are topical eye drops and ophthalmic ointments. However, most of the 78 liquid is discharged from the lacrimal duct after the eye drops are administered, and 79 may cause systemic toxicity after being absorbed through the nasopharynx. Due to its 80 greasy properties and blurred vision, eye ointment has poor patient compliance when 81 applied to eye ointment [1] . 82 Over these years, a growing number of innovative drug delivery systems have 83 been applied to the eyes. Such as liposome nanoparticles and microemulsion, to 84 extend the retention time of the eyes, thus reducing the frequency of medication and 85 increasing bioavailability. However, their drug delivery potential in ophthalmology is 86 limited by the rapid clearance of the anterior cornea due to the same rapid clearance 87 as the water-soluble eye drops. Therefore, considering the unique physiological 88 structure of the eye, effective ocular drug delivery still faces many challenges and 89 needs to find a more effective ocular delivery system. 90 In recent years, in-situ gel has become a new type of sustained release system [2,3] . 91 It is a precursor for administration and then transforms into a gel at the drug delivery 92 site. Compared with other ophthalmic preparations, the precursor preparation of liquid 93 crystal gel has a longer residence time in the eye and good curative effect. In 94 addition，in situ gel is in the form of ordinary solution, similar to eye drops, so the 95 drug is simple and good compliance [4][5][6][7][8] . Conversion from solution to gel is usually 96 caused by some physiological or external stimuli, such as variances in pH value, ionic 97 strength or temperature between the formulation outside the body and the internal 98 tissues [9] . When the formulation is exposed to artificial tears, the precursor solution is 99 transparent and can spontaneously become a gel. Since the triggering factor of this 100 phase change is due to the existence of the water environment, it can also be realized 101 directly in the physiological fluid. Compared with other methods, such as ultraviolet 102 light and temperature, this method can cause phase transition [10] more mild. According 103 to the literature, it is found that many kinds of in-situ gels have been used in eye 104 administration to improve ocular bioavailability like use of temperature-dependent 105 in-situ gelation polymer (Poloxamer) [4][5][6][7][8] , pH-dependent in-situ gelling polymers 106 (Carbopol and Hypromellose) [11][12][13] , and ionic strength-dependent in-situ gelling 107 polymer (Gellan gum). However, there are few reports on in situ liquid crystal gel for 108 ocular drug delivery.

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An amphiphilic substance containing a hydrophilic head group and a 110 hydrophobic hydrocarbon chain domain self-assembles after adding water to form a 111 long-range ordered structure called a lyotropic liquid crystal phase [8] . For example, 112 lamellar phase (Lɑ), reverse bicontinuous cubic phase (QII) and reverse bicontinuous 113 hexagonal phase (HII) have received more and more attention due to their unique 114 internal frame and broad drug capacity. Among them, the hexagonal liquid crystal as a 115 drug carrier has attracted widespread interests due to its good stability, potential drug 116 slow-release ability [14] . Phytotriol(PT) (shown in Fig.1) are generally considered safe 117 drug matrix and amphiphilic lipids with good mucosal adhesion and biocompatibility, 118 which was often utilized to form liquid crystals. The lyotropic liquid crystal delivery 119 system has many advantages, such as simple preparation, easy to use, reduced dose, 120 and sustained release effects, PT-based liquid crystal gels have been used in the 121 treatment of rheumatoid arthritis and postoperative analgesia [15] . However, there are 122 few studies on PT in ocular delivery systems.

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Glaucoma is one of the major public health problems in the world. This is a 124 chronic progressive eye disease caused by the apoptosis of retinal ganglion cells and 125 subsequent degeneration of nerve tissue. According to the survey, more than 70 126 million people in the world suffer from glaucoma, and about 10% of them are blind, 127 which makes it the main cause of irreversible blindness in the world2020 [16] . However, 128 this number may rise to 11.1 million by 2020. Pilocarpine nitrate is a drug that 129 directly acts on the parasympathetic nerve. It can directly stimulate muscarinic 130 receptor M of iris and ciliary body, cause the contraction of iris and ciliary body, open the anterior chamber angle, promote the outflow of aqueous humor through trabecular 132 meshwork structure, and produce the effect of lowering intraocular pressure [17] . It has 133 been used in the treatment of chronic open Angle glaucoma and acute closed Angle 134 glaucoma for more than 100 years [18] . At the same time, PN has been used the longest 135 as a mainstay drug for glaucoma therapy, and is one of the least-expensive and the 136 most readily available medications [19] . Applied in the eye, PN can penetrate the 137 eye wall with miosis beginning in 15-30 min which last up to 4-8 h [20] . Due to poor 138 corneal permeability, short anterior corneal retention time and poor anterior corneal 139 tear flushing, PN eye drops need to be administered frequently, generally 3-6 times a 140 day [21] . Therefore, the bioavailability is extremely low (less than 5% or even less than 141 1%). At the same time, frequent daily administration can cause a series of side effects, 142 such as pupil contraction and myopia, and even a series of gastrointestinal reactions.In 143 addition, our previous research has developed a liquid crystal gel. Based on this, we 144 made the gel into a precursor to improve compliance [22] .  According to the literature [24] and our former research (Fig.2), the formulating 171 method was as follows. Firstly, appropriate amount of PT and TAG were mixed and 172 heated to 60 ± 0.5 ℃ to prepare oil phase. Secondly, PN was placed in water at 60 ± 173 0.5 ℃ to prepare aqueous phase (PEG400 and water). Finally, the water phase in the 174 same temperature was added to the oil phase rapidly, and the mixture was 175 immediately rotated for 5 minutes to obtain a liquid crystal gel precursor. 176 Then, PT-TAG (oil phase) and PEG400-water (water phase) systems were used 177 to gradually change the weight ratio of the prescription from 10%: 90% to 90%: 10%.

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The prescription was screened by the above-mentioned preparation method, and four 179 blank formulas were prepared, namely P 0, P 1, P 2 and P 3. The optimal proportion of 180 stable self-assembled liquid crystal system was determined. Considering the solubility In the rotor method [26] , 200 mg of the formulation is accurately weighed and 192 added to a tube with a rotor (10 mm × 6 mm). The test tube was immersed in a water   Finally, a temperature scan was used to study the viscosity change with increasing 255 temperature, and the temperature setting range was 25~45 ℃. The in vitro release of PN from liquid crystal was studied by dynamic dialysis [33] .

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In short, 50 mg fresh PN liquid crystal gels were transferred into dialysis bags of the in vitro penetration study, each cornea was rinsed with normal saline to remove 315 the residual preparation on the corneal surface, and then weighed. Then, after drying 316 at 70 ± 0.5 ℃ for 12 h, the sample were weighed. In general, The HL value of 317 healthy cornea was 76~80%, corneal hydration value of more than 83% indicates 318 some degree of corneal injury [35] . The HL% value was calculated using the following  Table 1, the irritation of the samples to the eyes were judged.  The appearance before and after phase inversion was shown in Fig.3. When the 368 liquid crystal precursor was exposed to artificial tears, it undergoes a phase change It was observed that the best prescription P1 showed the "+ +" grade of gelation 377 ability, which is reported to be the most satisfactory grade [27] . The precursor 378 formulation was gelatinization immediately after exposure to PN at 37 ℃ degrees 379 can remain for a long time. showed a few crystals (as shown in Fig 4).  will cause the hydrogen bond between the PT head group and water to decrease, that 421 is, the effective size of the PT head group decreases and the α value decreases [40] . crystal gel precursor formulation [41] . When the formulation inverts, the viscosity of 435 the system increases significantly. The results showed that the liquid crystal gels had 436 pseudoplastic flow characteristics (shear thinning system) Viscosity increased at low 437 shear rate and decreased at high shear rate. One of the advantages of shear thinning 438 agents is that they have high viscosity during eye opening and stabilize tear film.

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When the blink occurs, the gel becomes thinner to prevent the irritation produced by 440 the high viscosity Newtonian fluid [42] , so that the preparation is well distributed on the 441 surface of the eye. As shown in Fig.8  As shown in Table 6, after a single administration, the in situ liquid crystal and 569 eye drops had little effect on the cornea and iris, the cornea was not turbid, and the 570 conjunctiva was free of redness, congestion, swelling and other irritation. After 571 repeated administration, the liquid crystal gel would be located in the subconjunctival 572 sac of the rabbit's eye. The conjunctiva may be slightly reddened due to blinking 573 reaction, but the score was lower than 3. The comprehensive results showed that both 574 in situ liquid crystal and PN eye drops had less irritating to rabbit eyes.   short.This showed that compared with traditional eye drops, HII gel could significantly 595 increase the contact time of the drug in front of the cornea. We speculated that the 596 good bioadhesion of PT leaded to relatively strong fluorescence intensity and slow 597 clearance. Secondly, PT may interacted with the mucin in the corneal epithelium, 598 thereby shortening the residence time of the drug on the corneal surface [49] , or PT may