Encapsulation of azithromycin ion pair in liposome for enhancing

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Encapsulation of azithromycin ion pair in liposome for enhancing ocular delivery and therapeutic efficacy on dry eye Tianyang Ren, Xiaoyang Lin, Qianying Zhang, Dongmei You, Xiaoyu Liu, Xiaoguang Tao, Jingxin Gou, Yu Zhang, Tian Yin, Haibing He, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00516 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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Molecular Pharmaceutics

Encapsulation of azithromycin ion pair in liposome for enhancing ocular delivery and therapeutic efficacy on dry eye Tianyang Ren1, Xiaoyang Lin1, Qianying Zhang1, Dongmei You1, Xiaoyu Liu1, Xiaoguang Tao1, Jingxin Gou1, Yu Zhang1, Tian Yin2, Haibing He1, Xing Tang1, *

1

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical

University, Shenyang 110016, Liaoning, PR China 2

School of Functional Food and Wine, Shenyang Pharmaceutical University,

Shenyang 110016, Liaoning, PR China

*Corresponding author: Professor Xing Tang, Department of Pharmaceutics Science, Shenyang Pharmaceutical University. E-mail: [email protected] 103 Wenhua Road, Shenyang 110016, Liaoning, PR of China Tel: +8602423986343; Fax: +8602423911736;

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The aim of this work was to design a novel ocular delivery carrier based on liposomes loaded with azithromycin (AZM) for the treatment of dry eye (DE) disease. To improve the drug loading efficiency, an AZM-cholesteryl hemisuccinate (CHEMS) ion pair (ACIP) was first prepared, and the successful formation of the ACIP was characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD), which demonstrated a stable interaction between CHEMS and AZM. The ACIP-loaded liposome (ACIP-Lip) appeared as spherical particles under TEM, with a uniform particle size of 60±2 nm and zeta potential of -20.3±4.6 mV. The entrapment efficiency (EE) and drug loading (DL) of ACIP-Lip were greatly improved to 95.6±2.0 % and 9.2±0.7 % respectively, which was attributed to the enhanced loading capacity of the liposomes through use of the ion pair and addition of MCT. The ACIP-Lip also exhibited a high stability during a three-month storage period at both 4 °C and 25 °C. In vitro release of AZM from ACIP-Lip was pH-dependent, with a more rapid release at pH 6.0 than at pH 7.4, which is beneficial for ocular therapy. Furthermore, the corneal permeation of AZM was enhanced by ACIP-Lip, demonstrating an apparent permeability coefficient (Papp×106) of 8.92±0.56 cm/s, which was approximately 2-fold that of the AZM solution. Finally, an in vivo pharmacodynamical study showed that the essential symptoms of DE rats were significantly improved by ACIP-Lip with a high efficiency and superior to hyaluronic acid sodium eye drops in the market. Hence, ACIP-Lip is a promising formulation for DE treatment.

Keywords: azithromycin, ion pair, liposome, corneal permeation, dry eye


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1. Introduction Dry eye (DE) is multifactorial disease characterized by corneal epithelium damage, inflammation of ocular surface, eye discomfort and visual disturbance, and it can have a large impact on quality of life1-3. DE is suffered by millions of people worldwide, and it has become an important health problem of ophthalmology. Meibomian gland dysfunction (MGD) is a prevalent cause of DE 4, and is a chronic, diffuse abnormality of the meibomian glands, normally characterized by terminal duct obstruction and/or changes in the glandular secretion5, 6. Meibomian glands normally produce abundant lipids in lysosomes, and secrete them into lateral ducts and onto the ocular surface. These secretions could strengthen the stability and decrease evaporation of the tear film, and therefore play a critical role in the protection of the eye. MGD directly leads to instability of the tear film, which has been recognized as one of the major mechanism of DE7. As well, inflammation of the ocular surface is commonly involved in DE-induced corneal lesions, and has been shown as a main contributor to the severity of DE. Thus, anti-inflammatory treatments are also an important option for the treatment of DE symptoms8, 9. Azithromycin (AZM) (Figure 1A) is a long-acting derivative of erythromycin, and has long been used as an antibiotic due to its good effect against chlamydia and bacteria, including for treatment of eye infections caused by bacteria10. AZM eye drops (AzaSite ®), approved by the FDA, have been marketed in the United States since 2007 and primarily used to treat bacterial conjunctivitis

11

. More importantly,

recent clinical trials have revealed that AZM can be used as a potentially effective and well tolerated therapy for MGD12, 13. AZM efficiently suppresses MGD associated conjunctival inflammation and growth of lid bacteria

13, 14

. As well, AZM accelerated

the accumulation of free cholesterol, neutral lipids and lysosomes in human meibomian gland epithelial cells

15, 16

, which restores the lipid properties of the

meibomian gland secretions12. Based on this, it is suggested that AZM may be beneficial for the treatment of DE. Unfortunately, AZM is almost completely insoluble in water and has a low solubility in oil, and thus it is of importance to develop a new ocular delivery system with high drug loading of AZM and excellent ACS Paragon Plus Environment


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physicochemical stability to improve the ocular bioavailability.

Figure 1. Chemical structure of azithromycin (A) and cholesteryl hemisuccinate (B).

Recently, ion pairing has been used as a novel chemical strategy to increase the drug loading and skin penetration of various drug delivery systems, as a result of increasing the lipophilicity or solubility of drugs17, 18. Unlike covalent modification, the ion pair approach reversibly changes the physicochemical properties of the ionized drug by means of Coulomb attraction, and therefore there is no change in the structure or pharmacological actions of the conjugates

19

. On the basis of

Brønsted-Lowry acid-base theory, hydrogen-bonded ion pair is easily formed by strong acids and bases through proton transfer, particularly in solvents with a low dielectric constant20.


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Where

is a conventional hydrogen bonded compound;

is a hydrogen-bonded ion pair

17

. Conventional electrostatic

effects determine the formation of weak hydrogen bonds (Equilibrium I). However, when the hydrogen bonds are stronger, proton transfer will play an important factor in the formation of hydrogen-bonded ion pairs (Equilibrium II)

19

. In our study,

cholesteryl hemisuccinate (CHEMS) (Figure 1B) was selected as the lipophilic counter-ion to increase the lipophilicity of AZM, as recent studies have demonstrated that CHEMS can significantly increase the lipid solubility of azithromycin21, 22. The formation of the ion pair was expected to enhance trans-cornea delivery and increase corneal permeability. Ocular diseases are customarily treated with topical eye drops, and thus the drugs are delivered locally in the region of the eye23. However, it is hard to increase the ocular bioavailability of drugs from topical solutions, and only about 5 % of the drug in eye drops reaches the inner ocular tissues24. Liposomes are a type of microsphere carrier formed by lipid bilayers, and are commonly used for nano drug delivery systems. Particularly, in recent years, liposome systems have obtained great popularity as ocular drug delivery platforms25. Drugs can be both encapsulated in the internal aqueous phase, as well as dispersed in lipid bilayers, which leads to an improvement in solubility and permeability through the cornea. Additionally, liposomal delivery of drugs is also a prolonged and controlled delivery26, and thus the efficacy of the drugs can be extended. Finally, liposomes can be delivered with the convenience of an ophthalmic drop, and can help confine the site of administration. In this work, an AZM-CHEMS ion pair (ACIP) was prepared and loaded into the phospholipid bilayers of liposomes. The physicochemical properties of ACIP-loaded liposomes (ACIP-Lip) were systematically characterized to verify the improvements in drug loading, encapsulation efficiency and stability during storage by use of the ion pairing method. In vitro drug release and corneal permeation of ACIP-Lip were investigated, and finally the in vivo effect of ACIP-Lip on DE was evaluated in DE rat ACS Paragon Plus Environment


models.

2. Materials and methods 2.1 Materials and animals AZM (Shanghai Sunve Pharmaceutical Co., Ltd., Shanghai, China, purity 94.5%), CHEMS (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), Egg yolk lecithin PC-98T and DSPE-PEG 2000 (Shanghai A.V.T. Pharmaceutical L.T.D., Shanghai, China), Long chain triglyceride (LCT) and Medium chain triglyceride (MCT) (Lipoid KG, Ludwigshafen, Germany). Double distilled water (DDW) was used and the other reagents were analytical or HPLC grade. Male SD rats (200~220 g) and New Zealand white rabbits (2.0~2.5 kg) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). All animal experiments were conducted according to the Guiding Principles in Shenyang Pharmaceutical University. The experiments were approved by the ethical committee of Shenyang Pharmaceutical University.

2.2 Preparation and characterization of ion pair ACIP was prepared by the solvent evaporation method. Equimolar amounts of AZM and CHEMS were dissolved in cold chloroform, stirred for 20 min in an ice bath, and then the chloroform was evaporated under nitrogen. The remaining chloroform was evaporated in a vacuum drying chamber at 40 °C for 5 days, and the resultant white powder was stored in a bottle at 4 °C. The physical mixture (PhM) was prepared by blending AZM and CHEMS in a porcelain mortar for 10 min. AZM, CHEMS, ACIP and PhM were analysed by DSC, XRD and FTIR as follows. For DSC, samples (2~3 mg) were sealed in aluminum pans fitted with perforated lids, and an empty pan was used as a control. The samples were heated from 25 °C to 250 °C at a rate of 10 °C/min in a nitrogen atmosphere (DSC-1, Mettler-Toledo, Switzerland). XRD was obtained by a D/Max-2400 diffractometer (Rigaku Instrument, Japan) at room temperature under Cu Kα radiation over the range of 5-60°(2θ) in increments of 0.5 °/min at 56 kV and 182 mA. FTIR spectra were recorded on a BRUKER IFS 55 FTIR system between 4000 and 400 cm−1 through the KBr disk method. ACS Paragon Plus Environment

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2.3 Preparation of ACIP-Lip ACIP-Lip with an AZM concentration of 10.0 mg/mL was prepared by a thin film dispersion and homogenization method. 1.65 % (w/v) ACIP, 1.85 % (w/v) CHEMS, 10.0 % (w/v) PC-98T, 0.5 % (w/v) DSPE-PEG 2000, 2.0 % (w/v) MCT and 0.05 % (w/v) α-tocopherol were dissolved in an appropriate amount of anhydrous ethanol at 60 °C and then transferred to a flask. The organic solvent was removed in a rotary evaporator. For the aqueous phase, phosphate buffer (pH 8.0) was prepared and heated to 60 °C. Following removal of the solvent, 0.05 % (w/v) EDTA-2Na, 0.003 % (w/v) benzalkonium bromide and the aqueous phase was transferred to the flask, which was then placed in an ultrasonic bath (KQ-300DE, Kunshan, China) at 60 °C for 30 min. The coarse liposomes were then further homogenized in a micro jet homogenizer (Noozle Fluid Technology, Co., Ltd) with cooling water circulating. The pH of the final liposomes was adjusted to 8.0 with 1M NaOH, and the product was passed through a liposome extruder (ATS Engineering Limited, EX1, Shanghai, China) with a 100 nm polycarbonate membrane. Finally, the ACIP-Lip formulation was transferred to vials with the addition of nitrogen gas. AZM solution (AZM-Sol) was prepared by dissolving AZM in a citric acid and sodium citrate buffer solution at pH 4.0, and then the solution was adjusted to pH 7.0.

2.4 Characterization of ACIP-Lip 2.4.1. Particle size distribution (PSD) and zeta potential The PSD and zeta potential of ACIP-Lip were measured with a NicompTM 380 Particle Sizing system (Santa Barbara, California, USA). Samples were diluted properly with DDW to obtain an optimum scattering intensity before measurements at 25°C. 2.4.2. Encapsulation efficiency (EE) and drug loading (DL) The EE and DL of ACIP-Lip were measured after separation of ACIP-Lip by the ultrafiltration method. Ultrafiltration was carried out using VIVASPIN 4 filters (VIVASCIENCE Ltd. Co., Germany) at 810 g for 25 min, with a molecular weight cut-off of 10,000 Da. The quantity of free AZM in the aqueous phase was measured by HPLC. ACS Paragon Plus Environment


The EE and DL of ACIP-Lip were then calculated according to the following equations: Wi − Wf × 100 Wi Wi − Wf × 100 DL (%) = Wl

EE (%) =

Where Wi is the total drug content, Wf is the amount of free drug in the aqueous phase, and Wl is the weight of liposomes containing drug. 2.4.3. Morphology The morphology of ACIP-Lip was observed under TEM (JEOL, Tokyo, Japan). ACIP-Lip suspension was diluted with a staining agent (3 % phosphotungstic acid, pH 6.5) at a 3:1 volume ratio. Then, the diluted ACIP-Lip was dropped onto a copper grid and the excess was removed using filter paper. The sample was dried in air for 6 h at room temperature before observation. 2.4.4. Stability study A fresh batch of ACIP-Lip was prepared and stored at 4 ± 2 °C and 25 ± 2 °C. The physical appearance, PSD, pH value, drug content and EE were measured at set time intervals over three months in order to evaluate the physical and chemical stability.

2.5 In vitro release of AZM from liposomes The ACIP-Lip suspension (1 mL, donor solution) was placed in a dialysis membrane (MD34, AMERICA) with a molecular weight cut-off of 8,000~14,000 Da, and was immersed in 30 mL of pH 6.0 or 7.4 PBS. The medium was kept at 37 °C under continuous magnetic stirring at 100 rpm. At predetermined time intervals, 2 mL of release medium was withdrawn and the same volume of fresh medium was replaced. The AZM-Sol was used as the control. The content of drug released was determined by HPLC, and the mean release percentages were calculated.

2.6 In vitro corneal permeation An in vitro corneal permeation study was carried out using modified Franz-type diffusion cells with a diffusion area of 0.64 cm2 27, 28. Rabbits of an average weight of 2.0~2.5 kg were sacrificed, and then corneas with a 2 mm ring of sclera were excised


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and installed above diffusion cells. The epithelial side faced the donor cell. The receptor cell was filled with 3 mL of simulated tear fluid, and maintained at 34±1 °C under stirring at 250 rpm. The test samples (3 mL, 1mg/mL AZM) were placed in the donor cells and then sealed with cling film to prevent evaporation. At appropriate time intervals for a total period of 480 min, 0.3 mL of permeated sample was withdrawn and replaced with an equal volume of fresh simulated tear fluid into the receptor cell. The removed samples were filtered through a 0.45µm membrane and measured by the HPLC as above. AZM-Sol was used as a control. The amount of drug permeated through the cornea was plotted versus time, and the apparent corneal permeability coefficient (cm/s) was determined according to the following equation28: Papp =

∆ܳ ∆t ∗ ‫ܥ‬଴ ∗ ‫ ∗ ܣ‬60

Where △Q/△t is the slope of the linear portion (µg per minute), C0 is the initial drug concentration (µg/cm3), A is the corneal surface area (cm2), and 60 is the conversion constant from minutes to seconds.

2.7 In vivo effect of ACIP-Lip on DE disease A DE rat model was used to evaluate the effect of ACIP-Lip on DE. The DE rat model was induced with topical administration of benzalkonium chloride as reported by Lin et al.29. Briefly, the eyes of the rats were instilled with 20µL of 0.2% benzalkonium chloride three times daily (9 AM, 3 PM and 9 PM) for 6 days. The rat models were evaluated by break-up time of tear film (BUT) and slit lamp after fluorescein sodium staining (FS)8, 9. Eighteen DE rats were randomly divided into six groups (3 rats with 6 eyes per group): NS (control group), Levofloxacin hydrochloride eye drops (LH, 5mL: 15mg, United Laboratories Co., Ltd.) group, Hyaluronic acid sodium eye drops (HAS, 5mL: 5mg, United Laboratories Co., Ltd.) group, AZM-Sol (10mg/mL) group, ACIP-Lip (10mg/mL) group and LH+HAS group. The eyes of model rats in different groups were instilled with 20µL of different eye drops three times daily (9 AM, 3 PM and 9 PM) for 7 days. On days 1, 2, 4 and 7, BUT and FS



were performed, and on day 7, the rats were sacrificed and the ocular global tissues were carefully dissected and harvested for histological analysis. H&E staining was performed on the histologic sections of ocular global tissues for histological analysis30. To determine the BUT, the rats were anesthetized with an intraperitoneal injection of 40 mg/kg pentobarbital sodium. 2% sodium fluorescein was instilled in the conjunctival sac, and then the eyelids were closed manually 3 times. The ocular surface was observed under a slit lamp microscope. When an indicator of DE was first observed on the ocular surface, the BUT was recorded in seconds. After recording the BUT, corneal epithelial damage was graded with a cobalt blue filter under a slit lamp microscope. The cornea was divided into 4 areas that were individually scored from 0 to 3, and the final score was obtained by adding the 4 scores together with a total maximal score of 12 and analyzed as described in reference.

2.8 Statistical analysis Data is presented as the mean ± standard deviation (SD). A Student’s t-test was used to analyze the statistical significance of differences between two groups. Statistical significant difference was set at P < 0.05, and very significant difference was defined as P < 0.01.

3. Results and discussion 3.1 Verification of ACIP formation 3.1.1. Characterization of ACIP Figure 2A shows the DSC curves of AZM, CHEMS, PhM and the equivalent molar ACIP. Pure AZM and CHEMS samples showed specific endothermic peaks at approximately 125 °C and 186 °C, respectively, which corresponds to their respective melting temperatures. The analysis of the PhM of AZM and CHEMS showed a subdued endothermic signal at approximately 132 °C, which was attributed to the solubilization of AZM into CHEMS or a solid state interaction induced by heating and dilution31. In the DSC curve of ACIP, a new endothermic peak appeared at 152 °C, and the characteristic peaks of AZM and CHEMS completely disappeared, indicating the formation of a new chemical substance. ACS Paragon Plus Environment

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Figure 2. The DSC thermograms (A), XRD spectra (B) and FTIR spectra (C) of AZM, CHEMS, physical mixture (PhM) and AZM-CHEMS ion pairs (ACIPs), (D) The formation process of ACIPs and its interaction with the phospholipid bilayer. Figure 2B shows the XRD spectra of AZM, CHEMS, PhM and ACIP. The XRD spectra demonstrated that AZM and CHEMS were in a crystalline state, which was apparent due to the numerous distinct peaks. The XRD spectra of PhM depicted superimposed signals of the two compounds. However, the characteristic peaks of azithromycin and CHEMS were completely removed in the XRD of ACIP, indicating that ACIP was a new existing form in an amorphous state. The FTIR spectra are shown in Figure 2C. The spectrum of AZM displays the characteristic peaks of the O–C=O stretching vibration in the lactone ring at 1721.2 cm−1 and OH bands at 3494.3 cm−1. CHEMS exhibits distinct bands at 3424.9 cm−1 relating to the OH stretching vibration and 1709.6 cm−1 for the C=O stretching vibration. As a control, the spectrum of PhM showed a clear superimposition of the signals of the two pure components32. However, the signals in the FTIR spectra of



ACIP exhibited some differences compared with the other compounds, likely due to the formation of a new complex. Specifically, the C=O signal of CHEMS shifted to 1732.1 cm−1, and the new signals at 1572.3 cm−1 were related to the bending vibration of an ammonium ion, indicating the happening of proton translocation between the carboxyl and amino groups. Additionally, a characteristic peak of an N-H bending vibration from the dimethylamino group at 757.0 cm−1 region was observed, confirming the formation of a new coordinate bond between the proton from CHEMS and the ion-pair electron from the N atom of AZM. 3.1.2 Formation mechanism of ACIP by analysis of Coulomb force Ion pairs are formed by the binding of oppositely charged ions, which depends on Coulomb force. The interaction between ions can be expressed by the Coulomb force formula as follows: F=

q1 q2 εr2

Where F is the force of attraction, q1 and q2 are the magnitudes of the electrical charges, ε is the dielectric constant of the medium, and r is the distance between the ions. As seen from the formula, the smaller the value of ε, the larger the value of F will result, and thus an ion pair will be more easily formed in solvents with lower dielectric constants33. Conversely, in solvents with high dielectric constants, the ion pairs can more easily form hydrogen bonds with the solvent molecules, and so the interaction between ions may be weakened. Therefore, in this work, chloroform, which has a low dielectric constant, was used as the solvent to increase the interaction between the ion pairs, ensuring their final stability19. In addition, the distance r between AZM and CHEMS decreases gradually in the preparation of ion pair complexes, and so the interaction F increases gradually34. The principles and mechanisms of the preparation process are shown in Figure 2D. When AZM and CHEMS are dissolved in chloroform, the two ions are solvated, and as the solvent evaporates, the distance between AZM and CHEMS decreases and they eventually share the same solvent molecule. After the solvent is evaporated to the point where


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the saturation concentration is reached, the AZM and CHEMS molecules accumulate in the fixed space order. Finally, AZM and CHEMS interact with each other and form hydrogen bonding ion pairs. The ion pairs insert in the molecular chain of phospholipids and can be firmly loaded between phospholipid bilayer membranes. The planar chemical structure of ACIP were shown in Figure 3.

Figure 3. The chemical structure of ACIP.

3.2 Preparation and characterization of ACIP-Lip Ion pairing is a useful strategy to increase the loading of AZM in liposomes. A low EE (73.5 %) and DL (1.5 %) were obtained when AZM was loaded in liposomes without CHEMS, likely due to its low solubility in water and oil. After the formation of ACIP, the EE and DL of liposomes were greatly improved to 82.6% and 5.2%, respectively. This is attributed to both the enhanced solubility of AZM in oil and compatibility with the fatty acid chains of phospholipid molecules. As well, the hydrophobic region of the CHEMS chain further enhanced the lipophilicity of ACIP. To further improve the drug loading, a small amount of oil (2.0%, w/v) was added in the formulation of ACIP-Lip. The commonly used liquid lipids, MCT and LCT, were selected to assess the effect of the oil on the physicochemical properties of the ACIP-Lip. It was observed that the addition of MCT in ACIP-Lip maintained a good physical appearance, a small and uniform particle size (60 nm), a high EE (95.6 %) and DL (9.2 %), and that all properties were improved over the comparative addition of LCT (data not provided). Compared with LCT, MCT has a smaller molecular ACS Paragon Plus Environment


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weight and larger water solubility, as well as a lower pKa value. MCT is known to improve the solubilization ability of drugs, and therefore, MCT was selected as the oil component of the liposomes. The MCT increased the solubility and stability of ACIP in the phospholipid bilayer, which resulted in a decrease in the leakage rate of the liposomal azithromycin and an increase in encapsulation efficiency. Further, the composition of the oil has a large influence on the physicochemical stability of liposomes35, 36. The PSD, physical appearance, EE and DL were chosen as the major parameters for evaluation, as summarized in Table 1. The schematic diagram of the structure of ACIP-Lip is illustrated in Figure 4A. It is reported that the PSD is a key factor that affects the permeability of liposomes for intraocular drug delivery. Particles with a size of less than 100 nm generally show improved cell uptake

34, 37

. The mean particle size of ACIP-Lip was

60±2 nm, with a poly-dispersity index below 0.2, suggesting a uniform monodisperse phase. The zeta potential can create an electrical barrier on the surface of the particles and acts as a ‘repulsive factor’ that prevents aggregation of the spheres38. The zeta potential of the ACIP-Lips was found to be -20.3±4.6 mV, which indicates that this suspension has excellent physical stability. The TEM micrograph of ACIP-Lip (Figure 4B) confirms that the liposomes were spherical in appearance and the particle size ranged from 50 to 100 nm in accordance with the PSD results. Thus, by use of the ion pairing technique and addition of MCT, AMZ was successfully encapsulated between phospholipid bilayers of liposomes through their hydrophobic interactions.

Table 1. Characterization of AZM-Lip and ACIP-Lip with or without MCT addition. (n=3) Parameters

AZM-Lip

ACIP-Lip (without MCT)

ACIP-Lip (with MCT)

Physical appearance

Turbid

Transparent and uniform

Transparent and uniform

Mean size (nm)

183±5

77±6

60±2

P.I.

0.41±0.18

0.24±0.02

0.18±0.04


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Zeta Potential (mV)

-1.3±0.7

-8.4±2.3

-20.3±4.6

EE (%)

73.5±2.6

82.6±4.2

95.6 ± 2.0

DL (%)

1.5±0.3

5.2±1.3

9.2 ± 0.7

Figure 4. The schematic diagram of the structure of ACIP-Lip (A) and the TEM photograph of ACIP-Lip (B). 3.3 Stability study The storage stability of ACIP-Lip was studied at 4 ± 2 °C and 25 ± 2 °C over a period of three months. The physical appearance and PSD were measured to assess the physical stability, while pH value and drug content were used to evaluate the chemical stability. As shown in Table 2, ACIP-Lip was stable over a three-month storage period. After storage, the physical appearance of ACIP-Lip was transparent and uniform with no significant changes. At 4 °C, the particle size and P.I. value of



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ACIP-Lip did not greatly change, while there was a slight increase at 25 °C. This indicated that the small particles had partially aggregated during storage at 25 °C, potentially due to the slow oxidation of the phospholipids which leads to an increase of the viscosity of the eye drops. The pH value of ACIP-Lip was mildly reduced at 25 °C compared with that of the fresh samples, which was likely attributed to the production of fatty acids by hydrolysis of the phospholipid molecules. Additionally, the EE and content of ACIP-Lip did not decrease significantly at both 4 °C and 25 °C after three months, indicating that no obvious drug leakage or precipitation was occurring during storage. All the parameters suggested a high physicochemical stability of ACIP-Lip. This can be attributed to both the ion pair formation stabilizing ACIP-Lip by increasing the solubility and compatibility of AZM in phospholipid bilayers, as well as the addition of MCT also playing an important role in stabilizing the structure of ACIP-Lip38, 39.

Table 2. Stability of ACIP-Lip stored at 4 and 25 °C for three months. (n=3) Parameters

0

3 months (4°C)

Transparent and Transparent

3 months (25°C) and Transparent

and

Physical appearance uniform

uniform

uniform

Mean size (nm)

60±2

63±2

68±3

P.I.

0.18±0.04

0.19±0.04

0.25±0.08

pH value

7.96 ± 0.02

7.93± 0.02

7.74± 0.06

EE (%)

95.6 ± 2.0

95.3 ± 2.5

94.5 ± 3.7

Content (%)

100.3±0.5

100.1±0.7

99.8±0.3

3.4 In vitro drug release study The release profiles of ACIP-Lip and AZM-Sol in medium of different pH values are shown in Figure 5. AZM-Sol demonstrated fast drug release at either pH 6.0 or pH 7.4, with a completed release within 4h. Compared with AZM-Sol, ACIP-Lip showed a much slower drug release at both pH 6.0 and pH 7.4, indicating that loading the


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AZM ion pair form into the liposomes significantly changed the drug release profile. As shown in Figure 5, less than 70 % of AZM was released from the liposomes within 72h. However, there were some differences between the release rate at pH 6.0 and pH 7.4 when AZM was released from ACIP-Lip. The accumulated release percentage of ACIP-Lip reached 59.3 % at 12 h at pH 6.0, but only 45.7 % of AZM was released at pH 7.4 during the same period. Thus, the drug release was faster at pH 6.0 than pH 7.4 during the initial period. The quick release of AZM from liposomes in an acidic environment could be attributed to the pH responsiveness of liposomes based on the protonation of the CHEMS groups

40

. In addition, according to previous studies, CHEMS exhibits

pH-sensitive polymorphic phase behavior

21, 41

. CHEMS is a compound that changes

the shape of the molecule as the pH value changes38. CHEMS is an inverted cone at neutral pH, showing a lamellar structure, however, when the pH is around 5.8, the degree of protonation will increase, the charge will be lost, and the hydration will be reduced42. At the same time, its headgroup becomes smaller, changing from obconic for cylindrical or conical, the lamellar structure will disappear43, and so the encapsulated material will be released. The pH-responsive release behavior is of benefit for application of liposomes in the eyes. When the AZM-loaded liposomes enter into the eyes, a slow release of AZM could be achieved when the drug was transported in the eyes (pH 6.4~7.7), however, a further quick release of AZM could be stimulated by the acidic environment where the bacteria reside.



Figure 5. In vitro release of AZM in pH 6.0 and 7.4 medium. (n=3)

3.5 In vitro corneal permeation The corneal epithelium forms the outer layer of the eye, and is the pivotal barrier to ocular drug delivery

44

. Approximately 90 % resistance to hydrophilic drugs and

10 % to hydrophobic drugs in ocular delivery were attributed to the epithelium lipophilic nature45. AZM is neither a hydrophilic or hydrophobic drug, and therefore has a low corneal permeability. The formation of ACIP efficiently improved the lipophilicity of AZM, and the transcorneal permeability could be further enhanced by loading ACIP into the nano-sized liposomes. Hence, the in vitro corneal permeation of ACIP-Lip was evaluated to verify its ability to enhance the ocular bioavailability of AZM. Figure 6 shows the curves of the accumulated content of AZM permeated across the cornea as a function with time when administrated as ACIP-Lip or AZM-Sol. The transcorneal penetration parameters of ACIP-Lip and AZM-Sol are summarized in Table 3. A linear relationship between the quantity of AZM permeated through the cornea and time could be obtained within 0~480 min, which indicates that good


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corneal integrity was maintained throughout the experiment. The apparent permeability coefficient (Papp×106) of AZM-Sol was 4.43±0.27 cm/s. As expected, ACIP-Lip exhibited a much higher Papp×106 of 8.92±0.56 cm/s, which was about 2-fold higher than that of AZM-Sol. This result demonstrated that the corneal permeation of AZM was significantly improved by ACIP-Lip. For intraocular drug delivery, the particle size is a pivotal factor that affects the tissue penetration, and nano-sized particles demonstrate enhanced cell uptake46. The ACIP-Lip has a size distribution smaller than 100nm, which is considered an important role in increasing ocular drug delivery. As well, the increased lipophilicity of AZM by the formation of ion pair and the high compatibility of ACIP-Lip with the corneal epithelial cells is another key factor, and so ACIP-Lip was useful for increasing the ocular bioavailability of AZM.

Figure 6. In vitro corneal permeation profiles of ACIP-Lip and AZM-Sol. (n=3)

Table 3. Transcorneal penetration parameters of ACIP-Lip and AZM-Sol. (n=3) Samples

Linear

R2


Papp×106 (cm·s-1)


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ACIP-Lip

Y=0.3426x+12.442

0.9935

8.92±0.56

AZM-Sol

Y=0.1702x+4.0045

0.9927

4.43±0.27

3.6 In vivo effect of ACIP-Lip on DE A DE rat model was induced with topical administration of benzalkonium chloride. The BUT and FS score of the DE models were 5.26±0.62 s and 12.0, respectively, significantly different from those of normal rats (20.12±0.45 s and 0, respectively). After 7 days treatment, all treated groups showed a great improvement in the BUT (Figure 7A) and FS score (Figure 7B) when compared to the NS group. ACIP-Lip, LH+HAS and HAS treatments steadily increased the mean BUT over time, and by the end of the treatment period, the mean BUTs were almost at the normal level. Compared with the HAS treatment, ACIP-Lip and LH+HAS treatments improved the mean BUT to normal levels in a shorter time (by Day 4), indicating that ACIP-Lip and LH+HAS had a higher efficiency in treatment for DE. AZM-Sol and LH treatments also increased the mean BUT in the treatment period, but to a lesser degree compared to ACIP-Lip and LH+HAS groups. As shown in Figure 7B and Figure 8, the results in FS of the treated groups were in line with the BUT. The FS scores were significantly reduced in ACIP-Lip, LH+HAS and HAS groups when compared to NS group. On day 2 and day 4, the FS scores in ACIP-Lip and LH+HAS treatments were significantly lower than that in the HAS treatment, indicating that ACIP-Lip and LH+HAS treatments had a more rapid effect. AZM-Sol treatment also caused an obvious reduction in FS scores compared with the NS group, but they were still obviously higher than ACIP-Lip and LH+HAS treatments, suggesting that ACIP-Lip and LH+HAS are superior to AZM-Sol in the treatment of DE. The histology of the cornea and conjunctiva of rats after treatment were assessed with H&E staining as shown in Figure 9. Figure 9A-a depicts a normal cornea of a healthy rat with smooth epithelium and no inflammatory infiltrations, while the cornea of the DE model was damaged with irregular epithelial surface, thinning of the epithelial layer and severe inflammatory cell infiltration (Figure 9A-b). In NS group,


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no obvious change was observed on the cornea compared with DE group. The LH treatment (Figure 9A-d) ameliorated the inflammatory infiltrations, however the cornea was still irregular on the surface and had vascular congestion in the stroma. After treatment with HAS or AZM-Sol, the damaged corneal epithelium was significantly improved, but there was serious congestion in the AZM-Sol group and a moderate inflammatory reaction in the HAS group. For ACIP-Lip and LH+HAS groups, the corneas of rats were recovered to normal levels, indicating that both ACIP-Lip and LH+HAS have an excellent therapeutic effect on DE disease. In the histological analysis of conjunctiva (Figure 9B), inflammatory infiltrations and conjunctival congestions could be clearly observed in the DE group. Similarly, the ACIP-Lip and LH+HAS treatments could also cure the conjunctival damage. DE is a multifactorial disease on the ocular surface, and tear film instability and inflammation are the significant symptoms in the development of this disease8, 9. In this experiment, HAS eye drops were used as a positive control to treat DE, and showed a great improvement in signs of DE syndrome. It is clear that HAS could promote water retention properties and enhance tear film stability in DEs due to its viscoelasticity7, 47. Compared with HAS, ACIP-Lip showed a faster and improved therapeutic effect for the DE rats. This is due to AZM being able to enhance the stability and prevent evaporation of the tear film by increasing the accumulation and secretion of lipids in human meibomian gland epithelial cells16. As well, AZM is quite effective in suppressing the MGD associated conjunctival inflammation and growth of lid bacteria, which is also considered a key factor for treatment of DE9. To further study the role of the antibiotic and anti-inflammatory effect in the recovery of the ocular surface damage, LH alone or in combination with HAS (LH+HAS) was used in the DE treatment. As a result, the LH treatment showed a mild therapeutic effect that was not significantly better than the NS treatment, however the LH+HAS treatment showed a great improvement in DE symptoms and was similar to the ACIP-Lip group. These results indicated that the antibiotic and anti-inflammatory effect of AZM played an adjuvant effect in accelerating the recovery of DE disease, and was a contributor to ACIP-Lip being superior to HAS in the treatment of DE ACS Paragon Plus Environment


syndrome. Further, ACIP-Lip was able to efficiently deliver AZM to the ocular acting sites and increase the ocular bioavailability, which is attributed to the high drug loading by ion pair and superiority of the nano-sized liposome. Thus, ACIP-Lip was improved over AZM-Sol for DE treatment. In addition, the lipids contained in ACIP-Lip also contribute in the restoration of a normal precorneal tear film by replenishing deficient tear film lipids and strengthening their stability8. In all, ACIP-Lip provides a new efficient therapy for DE disease by tear film stabilization and inflammation suppression.

Figure 7. The change of the BUT (A) and FS score (B) on day 1, day 2, day 4 and day 7 after treatment in each group. ⁎ Significantly different compared with NS


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group (P < 0.05), ⁎⁎ Very significantly different (P < 0.01), # significantly different compared with HAS group (P < 0.05), && significantly different compared with AZM-Sol group (P < 0.01).

Figure 8. Representative photographs of fluorescein sodium staining for each group on day 1, day 2, day 4 and day 7. A: NS group, B: LH group, C: HAS group, D: AZM-Sol group, E: ACIP-Lip group, F: HAS+LH group.



Figure 9. H&E stained histological sections of cornea (A) and conjunctiva (B) of rats after treatment. a: Normal group, b: DE group, c: NS group, d: LH group, e: HAS group, f: AZM-Sol group, g: ACIP-Lip group, h: HAS+LH group. 4. Conclusions This work describes a novel formulation of AZM liposomes prepared through an ion pair method, and the formation of ACIP was verified by DSC, XRD and FTIR. Owing to the use of ion pair, the EE and DL of ACIP-Lip were greatly improved, and the obtained ACIP-Lip also exhibited excellent stability during storage. This was attributed to the improved lipophilicity and compatibility with phospholipid molecules and the enhanced loading capacity of the liposome by the addition of MCT. Compared with AZM solution, the ACIP-Lip showed a sustained and pH sensitive in vitro release of AZM, with a more rapid release rate at pH 6.0 than at pH 7.4, which is beneficial for ocular therapy. The corneal permeation of AZM was also enhanced by ACIP-Lip by approximately 2-fold to AZM-Sol. An in vivo pharmacodynamical study confirmed that the symptoms of DE rats were significantly improved by ACIP-Lip with a high efficiency superior to HAS eye drops. The efficient therapy of ACIP-Lip


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for DE disease was realized by tear film stabilization and inflammation suppression. These results indicate that ACIP-Lip is a promising ophthalmic drug for DE therapy.

Notes The authors declare no competing financial interest. Acknowledgements This work is supported by the National Natural Science Foundation of China (No. 81673378). The authors appreciated Dr. Amanda Pearce for her correction of the manuscript.

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Graphical Abstract


Encapsulation of azithromycin ion pair in liposome for enhancing

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