Ex vivo and in vivo evaluation of the effect of coating a coumarin-6

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Ex vivo and in vivo evaluation of the effect of coating a coumarin-6-labeled nanostructured lipid carrier with chitosan-N-acetylcysteine on rabbit ocular distribution Dandan Liu, Jinyu Li, Bingchao Cheng, Qingyin Wu, and Hao Pan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00069 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017

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Molecular Pharmaceutics

Ex vivo and in vivo evaluation of the effect of coating a coumarin-6-labeled nanostructured lipid carrier with chitosan-N-acetylcysteine on rabbit ocular distribution Dandan Liu1,2, Jinyu Li2, Bingchao Cheng2, Qingyin Wu1, Hao Pan3,*

1

School of Biomedical & Chemical Engineering, Liaoning Institute of Science and

Technology, Benxi 117004, PR China; 2

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, PR

China; 3

College of Pharmacy, Liaoning University, Shenyang 110036, PR China

1

ACS Paragon Plus Environment


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ABSTRACT This study is focused on further understanding the characteristics of chitosan-N-acetylcysteine

surface-modified

nanostructured

lipid

carriers

(CS-NAC-NLCs) in their interaction with ocular mucosa. Coumarin-6 (C6)-labeled NLCs, including uncoated NLCs, chitosan hydrochloride (CH)- and CS-NAC-coated NLCs, were developed using a melt-emulsification technique and subsequently decorated with different types or portions of chitosan derivatives. Mucoadhesion was evaluated ex vivo using a flow-through process with fluorescence detection. The results demonstrated that the presence of CS-NAC on the C6-NLC surface provided the most obvious enhancement in adhesion due to the formation of both noncovalent (ionic) and covalent (disulfide bridges) interactions with mucus chains. Meanwhile, the concentration of CS-NAC in the formulation positively influenced the viscosity of the nanoparticles and hence prolonged their retention in the ocular tissue. Transcorneal penetration studies revealed that CS-NAC-NLC particles were able to penetrate through the entire corneal epithelium primarily via a transcellular route. The transport depth and velocity were strongly rely on the modification material and the particle size. Ex vivo fluorescence imaging and in vivo ocular distribution investigations showed that C6 was broadly distributed in rabbit eye tissues and absorbed by aqueous humor after CS-NAC-NLC instillation. In relation to C6 eye drops, CS-NAC-NLCs achieved considerably higher Cmax (4.01-fold), MRT0-∞ (1.87-fold) and AUC0-∞ (16.29-fold) in the aqueous humor. Moreover, the increase in drug absorption was greater in the cornea than in the conjunctiva. Thereby, it is possible to draw a conclusion that CS-NAC-NLCs presented great potential for drug application to the front portion of the eye.

Key words: Coumarin-6, chitosan-N-acetylcysteine, mucoadhesion, nanostructured lipid carriers, transcorneal mechanism, ocular distribution.

2


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1. INTRODUCTION Topical instillation of an active compound into the eye is usually considered as the most efficient and widely accepted way to treat all sorts of ophthalmic diseases. No less than 90% of these conventional preparations are eye drops, particularly to solve front-of-the-eye problems 1. In the front half of the eyeball, corneal and conjunctival epithelium, together with the overlying tear film, act as physiological barriers to keep the eye from infection and desiccation

2, 3

. The cornea is a

non-vascularized barrier consisting of three main layers: namely the outermost epithelium, the middle stroma layer, and the inner endothelial layer, which has tight junctions and showing strong obstruction to passive diffusion of drug molecules

4, 5

.

In addition, despite drug delivery through conjunctival route is one hundred times greater than that of the cornea, it owns the identical type of tight junctions as well and further retard passive movement of hydrophilic molecules. Conjunctival drug absorption is usually ineffective because the limbal area is full of blood capillaries and lymphatics, thereby dissolved drug can be dissipated into blood and lymphatic flow and further reducing the ophthalmic bioavailability 3, 6. The tear film is produced by tears, which has a multifunctional role, including entrapment of debris, microorganisms, and even drugs. Furthermore, the tear membrane is a dynamic solution that go through rapid restoration and consequently prevents the residence of the drugs on the surface of the cornea, creating an additional barrier for the intraocular contents. In addition to the anatomical barriers, the defense mechanisms of the eye, including the blinking reflex, lachrymal secretion and nasolacrimal drainage, will lead to short precorneal retention time and a further reduction in ophthalmic bioavailability 7

. Due to the above reasons, no more than 5% of the total administered drug reaches

the intraocular tissues 8, 9. In recent years, strategies to enhance the transcorneal bioavailability have been extensively studied. Numerous nanomedicines such as polymeric micelles10, liposomes11, dendrimers12, and nanoparticles13 have been formulated and evaluated for ocular drug administration. Improving corneal penetration and prolonging precorneal residence time are the main methods applied to overcome precorneal restraints

14-16

. Among the vehicles reported thus far, nanostructured lipid carriers

(NLCs) is currently considered an efficient vector to enhance the ophthalmic bioavailability, because of their superiorities such as controllable drug release, high drug loading and easy to scale-up.

17-19

. Recent works have proved that the surface

modification of NLCs may influence its ocular mucosa interaction. The positive charge carried by the NLCs was observed to beneficial in prolong the pre-corneal 3



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retention time of the nanoparticles20. As a new type of mucoadhesive materials, thiolated chitosan (TCS) exhibits reinforced mucoadhesion compared to the undecorated parent polymer, namely chitosan (CS), thanks to the formation of covalently disulfide bridges between free thiol groups of the TCS and cysteine-rich domains of mucus glycoproteins

21

. In our previous study, a new type of TCS was

developed by chemically decoration of CS with N-acetyl-L-cysteine (NAC). With the idea of synergistically improving ophthalmic bioavailability, we designed a combination system of chitosan-N-acetylcysteine (CS-NAC)-coated NLCs for curcumin ocular delivery22. The results indicated that the system was able to significantly enhance the in vitro transcorneal penetration, improve the precorneal residence and eventually increase the ocular absorption of curcumin compared with the uncoated NLCs and chitosan hydrochloride (CH)-modified NLCs. In addition, the effects were proportional to the thiolation level of CS-NAC. Therefore, the CS-NAC-based colloidal carrier showed a number of distinct advantages for ophthalmic administration. Despite these positive results, the mechanism of action of the novel system has not been illuminated. Meanwhile, it is worth noting that the therapeutic target for the surface ophthalmic diseases, such as conjunctivitis or keratitis, is the ocular surface. But when treating intraocular diseases, such as glaucoma or uveitis, an improvement in corneal permeability would be desired. Accordingly, distribution of a drug in ophthalmic tissues following topical application would be of great importance for the treatment of different ocular problems. Based on these considerations, the main objective of the present research was to quantify and illustrate the mechanism of interaction between CS-NAC-coated NLCs and the rabbit cornea. In the present work, we developed a series of colloidal drug carriers: uncoated NLCs, CH-coated NLCs, and surface-modified NLCs with CS-NAC polymer solution of various concentrations; all of them were loaded with the fluorescent marker coumarin-6 (C6). The mucoadhesion, ex vivo/in vivo transcorneal mechanism, and ocular distribution of these nanoparticles were all investigated. The results were analyzed to elucidate the influence of the different factors (polymer type, particle size, thiol surface concentration, etc.) possibly participated in the interaction between these colloidal systems and the ocular mucosa. 2. EXPERIMENTAL SECTION 2.1. Reagents and Animals. Coumarin-6 (C6) was acquired from Shanghai Aladdin Co., Ltd. (China). Miglyol 812N was supplied by Sasol (Germany). Glyceryl monostearate (GMS) was supplied by Tianjin Bodi Chemical Holding Co., Ltd. (China). Polyoxyl 15 4


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hydroxystearate (Solutol® HS15) was from BASF (Germany). Gelucire 44/14 was kindly donated by Gattefosse (France). CS-NAC (the amount of free sulfhydryl group equals 496.7±17.1 µmol/g) was synthesized through covalent modification of CS with NAC in our previous study 22. Chitosan hydrochloride (CH), as the control polymer, was synthesized using the same method without addition of NAC to the coupling reaction22. Male New Zealand albino rabbits of 2.0~2.5 kg were acquired from Shenyang Pharmaceutical University Lab Animal Center. The experiments were agreed by the Animal Ethical Committee of Shenyang Pharmaceutical University (approval number: SYPU-IACUC-2016-0921-401), and all rabbits were managed according to the Laboratory Animal Care Principles. The animals were fed with standard pellet diet, and allowed free access to water. 2.2. Preparation of the Formulations. The melt-emulsification method as described in our previous report22 was applied to prepare the NLCs labeled with C6. Firstly, the mixture of C6 (5 mg), GMS (112 mg), Miglyol 812 N (69 mg) and Solutol® HS15 (89 mg) was melted at 75 °C to form the oil phase. Meanwhile, 5 mL of the aqueous phase containing 2 mg/mL Gelucire 44/14 was heated up to 75 °C and mixed with the melted lipid phase under magnetic stirring at 600 rpm for 5 min. Then, the obtained emulsion was immediately cooled in an ice bath (0 °C) to form the NLCs. To obtain CS-NAC or CH surface-modified NLCs, an aliquot of the above NLC suspension and the same volume of CS-NAC solution (0.1%, 0.5%, or 1%, w/v) or CH solution (0.1%, w/v) were mixed together under agitation, followed by incubation at room temperature with constant stirring for 30 min. For the preparation of C6 eye drops, 2.5 mg of C6 was dissolved in 5 mL of 15% propylene glycol solution. All preparation processes were performed in an aseptic room under aseptic conditions, and the preparations were all sterilized by filtration through a 0.22 µm filter 23. C6-NLCs surface-modified with different concentrations of CS-NAC were designated as CS-NAC-NLC-1(0.1%), CS-NAC-NLC-2 (0.5%), and CS-NAC-NLC-3 (1%), respectively. 2.3. Characterization of the NLCs. Nano ZS90 instrument was applied to determine the mean particle size (PS), polydispersity index (PDI) and zeta potential (ZP) of the NLCs (Malvern Instruments, UK) at room temperature. The encapsulation efficiency (EE, %) of the C6 labeled NLCs was determined using an ultrafiltration centrifugation method 22. Briefly, 0.5 mL of the NLC sample was added to the upper chamber of an Amicon® centrifugal filter (MWCO 10 kDa, Millipore Co., USA) and centrifuged at 1933×g for 15 min. The unentrapped C6 in the filtrate was analyzed from a standard curve of C6 in 5



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acetonitrile using a Multimode Microplate Reader (Excitation/Emission: 464 nm/504 nm, Varioskan Flash, Thermo Fisher Scientific Corporation, USA). The following equation was used to calculate EE: EE (%) =

(W Tatal − W Free ) × 100 W Tatal

where WTotal and WFree were the total weight of C6 in the NLCs and the non-encapsulated C6 in the filtrate, respectively. 2.4. Mucoadhesion Study. Mucoadhesive studies were performed using a fluorescence microscope (Olympus BX50, Tokyo, Japan) attached with an AxioCam MRm camera (Zeiss, Germay). The rabbit eyes were removed immediately after the animals were slaughtered. To make the cornea flatten, an eyeball was pierced to discharge the vitreous humor prior to the experiment. Before sample administration, background images were captured. Then 40 µL of the labeled preparation was placed onto the corneal surface, and fluorescence microscopy images were again taken, followed by washing cycles each with 20 mL of artificial tear fluid (ATF) irrigated onto the corneal surface at 4 mL/min using a syringe pump24. ATF was prepared by dissolving 3.35 g NaCl, 0.0305 g CaCl2, and 1 g NaHCO3 in 500 mL of deionized water. The microscopy images were also analyzed by ImageJ software in 8-bit grayscale after each wash. To normalize the mean fluorescence values, the background signals captured for the samples before contact with the C6 preparations were deducted. The experiments were conducted in triplicate each with different regions on the rabbit ocular. The tests were also carried out directly on glass slides without corneal tissue. 2.5. Ex vivo Transcorneal Study The rabbit corneas were obtained promptly after the animals were sacrificed, and were used within 30 min of enucleation. Standard eye bank techniques were applied to dissect corneoscleral buttons. The isolated corneal tissue (exposed area 0.5 cm2) was mounted in the Franz diffusion cell, separating the donor from the receptor compartment. The endothelial side was filled with 4 mL of glutathione bicarbonate Ringer’s (GBR) buffer containing 1.2% Tween 80, and the epithelial side was filled with 200 µL of the preparation (equivalent to 100 µg of C6) supplemented with GBR-1.2% Tween 80 solution to 1 mL. After 15 min or 120 min of the transcorneal experiments at 34±0.5 °C, the tissues were inserted into a Coverwell™ image chamber (Invitrogen), and the pictures were obtained within the x-y vertical mode (sequential scanning from 0 to 40 µm in a plane parallel to the ocular surface) using a 40× immersion objective. Optical images were captured using the z-stack mode, with the ocular surface fixed as the original position 25. 6


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2.6. In vivo Corneal Permeation Experiment To investigate the in vivo drug transcorneal behavior within the rabbit eye, an inverted fluorescence microscope (Olympus BX50, Tokyo, Japan) was employed. First, 200 µL of the preparation was dripped into the left eye of an animal every 2 min a total of 5 times. The contralateral eye was administrated with normal saline (control). After a given time (15 min, 120 min or 240 min), the rabbits were euthanized by an air embolism, and the eyes were instantly removed and sliced vertically along the sagittal plane using a CM 1900 cryostat microtome (Leica, Germany). The cornea slices were both observed under normal light and a fluorescence microscope. 2.7. Ex vivo fluorescence images of ocular tissues C6 distribution in different ocular tissues was investigated using an in vivo imaging device. Briefly, 200 µL of the labeled preparations (containing 100 µg of C6) was dripped into the lower conjunctival sac of each rabbit eye. The blank tissues administered with an equal volume of normal saline were used as the control. After euthanasia at 240 min, the cornea, conjunctiva-sclera, iris-ciliary body and crystalline lens were isolated and repeatedly washed with saline. After sucking the liquid with filter paper, the tissues were immediately examined using an in vivo imaging system (Carestream FX Pro, USA). 2.8. In vivo ocular distribution study The animals were divided into six groups randomly, each comprising six rabbits. In brief, 200 µL of the preparations containing 100 µg of C6 were administrated in each eye of the animals. The rabbits were euthanized by air embolism after 5, 30, 60, 120 and 240 min of administration. The aqueous humor sample was immediately obtained from the anterior chamber using a 30-gauge needle attached to a syringe. Cornea and conjunctiva-sclera were subsequently carefully dissected under a microscope, rinsed with normal saline, blotted dry, and placed in pre-weighed vials. The vials were re-weighed to calculate the weight of the tissues. Afterward, the tissue samples were homogenized using a High-Speed Dispersator (XHF-D, Scientz, China) in an ice bath for 3~5 min, and then centrifuged at 13,600 ×g for 30 min. Tissue samples and aqueous humor were directly diluted with acetonitrile to 500 µL, followed by vortexing for 2 min, and then filtered using a 0.22 µm syringe filter. Finally, extracted tissue homogenates were analyzed using a Multimode Microplate Reader (Excitation/Emission wavelength: 464 nm/504 nm). Extracted tissue from the untreated contralateral eyes were used as the control. The concentration of C6 in various tissues was determined using a standard curve. 2.9. Statistical analysis The data were analyzed using independent sample t-tests, and p< 0.05 was considered significantly different. All experiments were performed at least in 7



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triplicate. 3. RESULTS AND DISCUSSION 3.1 Characterization of the NLCs The aim of our study was to further illuminate the mechanism of the CS-NAC coating on the transcorneal behavior of the NLCs by comparing the CH-modified NLCs with naked NLCs. As listed in Table 1, the properties of the NLCs were evaluated. The PS of unmodified C6-NLCs was 50.71±0.45 nm, with a negative zeta potential value of -15.36±0.28 mV. Coating with cationic polymers significantly increased the PS values and altered the surficial charge distribution. Both the CH and CS-NAC coating decreased the electron density encircling the surface of NLCs, and making zeta potential shift from negative to positive. Meanwhile, with an increase in the CS-NAC concentration, the observed increase in PS and ZP values of the surface-modified NLCs were obtained. The results demonstrated that CH or CS-NAC was absorbed onto the surface of the nanoparticles, and the higher CS-NAC copolymer concentrations resulted in more CS-NAC anchored onto the nanoparticles. With respect to size distribution, all the PDI values were less than 0.3, which indicated that the NLCs were homogeneously distributed. Additionally, the EE values of all NLCs were larger than 90%, this may due to the lipophilicity of C6. Compared with the pure NLCs and the CH-modified NLCs, the EE values of the CS-NAC-NLCs were all increased. This phenomenon could be attributed to the generation of disulphide linkage between thiolated polymers, thus efficiently preventing C6 escape from the particles. Table 1. Characterization of the NLCs (mean ± SD, n=3). Responses Polymer Concentration

Uncoated NLCs

CH-NLCs

CS-NAC-NLC-1

CS-NAC-NLC-2

CS-NAC-NLC-3

0

0.1

0.1

0.5

1

(%, w/v) PS (nm)

50.71±0.45

83.04 ± 1.87

86.74± 1.35

149.52±1.43

270.25 ±1.81

PDI

0.12±0.01

0.26 ± 0.05

0.15 ±0.01

0.19 ±0.02

0.26 ±0.03

ZP (mV)

-15.36± 0.28

30.23 ± 0.25

20.37±0.24

34.62±0.64

53.23±0.19

EE (%)

90.06 ± 1.22

90.89 ± 2.34

95.82 ±1.28

97.63 ±2.25

98.05 ±1.44

3.2. Mucoadhesion Study. In our study, the retention experiments were conducted in vitro using a flow-through method with fluorescence detection, respectively15, 16, 26-28. The glass slide was applied as a non-mucosal reference to clarify whether the mucoadhesion behavior observed could be simply ascribed to the viscosity of the preparations or particular mucoadhesive effect. Figure 1 shows representative fluorescence pictures

8


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and the retention curves for the preparations detained on the glass slide and the surface of the eye. According to Figure 1(a) and (c), C6 eye drops and C6-NLCs were washed off from the glass slide very quickly and disappeared completely under the first or second wash. After surface modification with CH or CS-NAC, the retention of C6-NLCs on the glass was significantly improved. But CH-NLC nanoparticles were washed from the glass surface more rapidly than the thiolated particles. C6-CH-NLCs could only stay after 3 washes. Its thiolated counterpart (CS-NAC-NLC-1) was removed from the glass slides after 4 wash cycles. For CS-NAC-NLC-2 and CS-NAC-NLC-3, fluorescence signals could be detected even after 5 washes. The results observed on the glass slide depended largely on the viscosity of the preparations. As depicted in Figure 1(b) and (d), all the preparations exhibited better adhesion and spreading property on the corneal surface in comparison with the glass group, especially for C6-CS-NAC-NLCs. Both C6 eye drops and C6-NLCs demonstrated a very sharp drop in fluorescence intensity after the first two washes. Retention of the CH-NLC particles was a little bit higher than that of the solution and pure NLCs. This may be due to the polymeric nature of CH, whose positively charged macromolecules can permeate through the mucosal layer of the corneal epithelium and form hydrogen bonding or electrostatic interactions (non-covalent bonds) with mucins to generate an interpenetration layer 29. As expected, the strongest mucoadhesive performance was observed for thiolated nanoparticles, which were detained on the eye surfaces even after 5 washes. This may be attribute to the formation of disulfide bridges (covalent bonds) together with the electrostatic attraction between thiol groups of the CS-NAC and cysteine-rich regions of mucus glycoproteins on the cornea. Moreover, mucoadhesion studies were conducted for CS-NAC-NLCs of different sizes and thiol content. After coating with increased amount of CS-NAC ranging from 0.1% to 1% (w/v), the PS and thiol content of the particles were all clearly increased. The mucoadhesive ability in this series was found to follow the following trend: CS-NAC-NLC-1 (86.74±1.35 nm)CS-NAC-NLC-3. The results further confirmed that it would be difficult for large particles to pass through the intercellular spaces or be internalized into the cytoplasm.

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Figure 2. Confocal laser fluorescence microscopy observation of the corneal cross-sections up to the depth of 40 µm after 15 min and 120 min of ex vivo cornea permeation study. (A) eye drops; (B) NLCs; (C) CH-NLCs; (D) CS-NAC-NLC-1; (E) CS-NAC-NLC-2; (F) CS-NAC-NLC-3

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Figure 3. Confocal laser fluorescence microscopy observation of the corneal surface after 120 min of ex vivo cornea permeation study. The small pictures inserted are the sections parallel to the corneal surface captured as deep as the fluorescence can be observed for different preparations. 3.4. In vivo Corneal Permeation Experiment Although the in vitro and ex vivo results obtained for CS-NAC-NLCs are promising, only in vivo experiments can truly evaluate the influence of physiological mechanisms such as the pressure originate from eyelid wiping and tear flow on the preparations. To evaluate the capacity of different types of NLCs to improve the in vivo corneal permeation of a drug, an inverted fluorescence microscope was employed to investigate the transcorneal penetration behavior of labeled preparations after administration. According to Figure 4, the green fluorescent signals of C6 was primarily focused on the corneal epithelium for all preparations 15 min after application. For C6-CS-NAC-NLCs, weak signals could even be detected in the stroma and the endothelium. After 120 and 240 min of exposure, the signal shifted progressively into the interior of the cornea for all preparations except the C6 eye drops and the naked NLC groups. For CH-NLCs, although the green fluorescence could be observed in the stroma at 240 min, the signals were quite weak. Furthermore, CS-NAC-coated NLCs exhibited a stronger fluorescent signals and deeper transmission intensity into the entire cornea than the other preparations. At 240 min, the corneal endothelium was also well stained, especially for C6-CS-NAC-1. The results sustained the hypothesis that drugs could penetrate through the cornea more 14


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efficiency after being loaded into CS-NAC surface-modified NLCs and were consistent with the results obtained in the ex vivo transcorneal experiment.

Figure 4. Inverted fluorescence micrographs after in vivo corneal permeation of labeled NLCs and eye drops. (A) eye drops; (B) NLCs; (C) CH-NLCs; (D) CS-NAC-NLC-1; (E) CS-NAC-NLC-2; (F) CS-NAC-NLC-3. Scale bar: 150µm 3.5 Ex vivo fluorescence imaging of ocular tissues Ex vivo imaging device was applied to further investigate the influence of the CS-NAC coating on the transcorneal migration behavior of the different NLCs. As depicted in Figure 5, the fluorescence strength of all the eye tissues progressively decayed from the outer to the inner of the eyes. The fluorescent signals from the eye drops were observed to be the weakest (group A). Stronger fluorescence intensity was observed for the tissues treated with naked NLCs (group B), but the signals detected in the conjunctiva-sclera and cornea were quite weak. This indicated that C6 was not able to penetrate through the cornea effortlessly due to the physical barriers of the cornea and conjunctiva-sclera. For CH- or CS-NAC-coated NLCs, fluorescent signals 15



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could be detected in all tissues, and the signal intensity in this series was found to follow this trend: CS-NAC-NLC-1>CS-NAC-NLC-2>CS-NAC-NLC-3>CH-NLC. The CS-NAC-NLC-1 group got the most significant enhancement in transcorneal migration ability, which was consistent with the results of the ex vivo and in vivo corneal transmission tests.

Figure 5. Ex vivo fluorescence picture of ocular tissues from rabbits administrated with labeled preparations. (A) eye drops; (B) NLCs; (C) CH-NLCs; (D) CS-NAC-NLC-1; (E) CS-NAC-NLC-2; (F) CS-NAC-NLC-3. 3.6 Distribution of C6 in ocular tissues After topical instillation of the labeled solution or the NLCs, the distribution of C6 in various rabbit eye tissues was examined and the results are illustrated in Figure 6. C6 was widely distributed in eye tissues and absorbed by the aqueous humor. For all formulations, C6 achieved maximum peak concentration (Cmax) at 30 min in the cornea and conjunctiva-sclera and at 60 min in the aqueous humor. A short transcorneal absorption delay time (lag time) of approximately 30 min was observed in the aqueous humor for all preparations. This can be explained by the fact that in the first 15 min, C6 was delivered from the cornea epithelium into the hydrophilic stroma progressively (see section 3.3 and section 3.4), and a drug reservoir was formed in the stroma, which hindered the delivery of hydrophobic drugs because of the hydrophilic property of the barrier33. The results were identical to those obtained in our previous in vitro corneal permeation studies 22. The pharmacokinetic parameters of C6 are shown in Table 2. Cmax, MRT0-∞, and AUC0-∞ of the CS-NAC-NLC groups in all ocular tissues were significantly higher than those of the other groups, including the CH-NLC group (p

Ex vivo and in vivo evaluation of the effect of coating a coumarin-6

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