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TPGS modified nanoliposomes as an effective ocular delivery system to treat glaucoma
International Journal of Pharmaceutics 553 (2018) 21–28
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
TPGS modified nanoliposomes as an effective ocular delivery system to treat glaucoma
T
Quansheng Jina,1, Huili Lia,1, Zhaohui Jina,1, Lingjing Huanga, Fazhan Wanga, Yang Zhoua, ⁎ ⁎ Yongmei Liua, Chunling Jianga, James Oswaldb, Jinhui Wua, , Xiangrong Songa, a
State Key Laboratory of Biotherapy/Geriatrics and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy, Sichuan University, Chengdu 610041, China b School of Nanotechnology Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanoliposomes TPGS Brinzolamide Ocular drug delivery system Pharmacodynamics
The aim of this study is to investigate the potential of D-alpha-tocopheryl poly (ethylene glycol 1000) succinate (TPGS) modified nanoliposomes as an ophthalmic delivery system of brinzolamide (Brz) for glaucoma treatment. The Brz loaded nanoliposomes containing TPGS (T-LPs/Brz) were firstly developed by a thin-film dispersion method. The average particle size was 96.87 ± 4.43 nm. The entrapment efficiency of the Brz was 95.41 ± 3.03% and the drug loading was 4.00 ± 0.13%. T-LPs/Brz exhibited obvious sustained release of Brz; in stark contrast to the normal liposomes of Brz (LPs/Brz) and the commercial formulation AZOPT® (Brz ophthalmic suspension, Brz-Sus). Enhanced trans-corneal transport of Brz was achieved with T-LPs/Brz. Compared with both Brz-Sus and LPs/Brz, the apparent permeability coefficient (Papp) of T-LPs/Brz was 10.2 folds and 1.38 folds higher, respectively. Moreover, T-LPs/Brz extended the cornea residence of Brz. White New Zealand rabbits treated with T-LPs/Brz had 3.18 folds and 1.57 folds Brz concentration 2 h after treatment than Brz-Sus and LPs/ Brz, respectively. Further pharmacodynamic studies showed that T-LPs/Brz maintained an effective intraocular pressure (IOP) reduction from 3 h to 11 h after administration, while Brz-Sus and LPs/Brz presented effective IOP decreases from 3 h to 6 h and 3 h to 8 h respectively. The preliminary safety evaluation demonstrated that T-LPs/ Brz had no significant side effects; specifically, no cornea damage and eye irritation. All the results indicated that TPGS modified nanoliposomes were a promising ocular delivery carriers for Brz to treat glaucoma. As such, TLPs/Brz might be worthy of further translational study.
1. Introduction Human eyes are well protected from the environment by various barriers, among which the cornea is the most protective outer layer. Eye drops for topical instillation, a conventional eye treatment route; often have low topical ophthalmic bioavailability (< 5%). This low ophthalmic bioavailability is mainly due to poor drug permeation across the cornea. Moreover, rapid washout by the tear and lachrymal drainage systems also leads to the low bioavailability of conventional eye drops. Despite this, eye drops are still one of the preferred dosage forms by the patients, accounting for over 90% of the market in 2014 (Cholkar et al., 2014b). Therefore, the development of novel eye drops with enhanced corneal permeation and extended cornea retention is needed to improve eye drop therapeutic efficacy. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), one of
FDA-approved safe pharmaceutical adjuvants, is a water-soluble derivative of natural vitamin E created via esterification with polyethylene glycol 1000 (Guo et al., 2013). As a novel nonionic surfactant, it has been widely used as an absorption enhancer, permeation enhancer, emulsifier, solubilizer, stabilizer and P-glycoprotein inhibitor (Guo et al., 2013; Tan et al., 2017). Moreover, many TPGS-based drug delivery systems (DDS) have also been developed for cancer therapy. Recently, it has been reported that TPGS can be used to solubilize Coenzyme Q10 or riboflavin in eye drops without any side effect in clinical trials. TPGS can also enhance the corneal permeation of riboflavin-5′-phosphate when combinationally used (Ostacolo et al., 2013). Possible reason is that it interacts with P-glycoprotein and, perhaps, with other drug transporter proteins (Collnot et al., 2006). However, there are limited reports about TPGS-based DDS for eye drops (Caruso et al., 2016; Cholkar et al., 2015; Cholkar et al., 2014a;
⁎
Corresponding authors. E-mail addresses: [email protected] (J. Wu), [email protected] (X. Song). 1 These authors contributed equally to this article. https://doi.org/10.1016/j.ijpharm.2018.10.033 Received 8 June 2018; Received in revised form 8 October 2018; Accepted 10 October 2018 Available online 11 October 2018 0378-5173/ © 2018 Published by Elsevier B.V.
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S100. The organic solvents were subsequently removed by a rotary evaporator at 37 °C to form a thin lipid film. Phosphate buffer solution (pH 7.4) was used to hydrate the thin lipid film for 1 h at 60 °C to obtain a colloidal dispersion. T-LPs/Brz were finally obtained after sonicating the colloidal dispersion for 3 min at 100 W in an ice bath and filtering with a 0.22-μm syringe filter for purpose of sterilization. The preparation of the normal liposomes of Brz (LPs/Brz) was performed as described in the previous study (Li et al., 2016a,b). The blank liposomes (LPs and T-LPs) were prepared following the same method without the addition of Brz.
Duan et al., 2015; Fogagnolo et al., 2013; Vadlapudi et al., 2014; Warsi et al., 2014). To our knowledge, it’s still unclear if TPGS can be used to promote the ocular penetration of drug-loaded nanoliposomes. Conventional liposomes, consisting of an outer bilayer of lipids enclosing a central aqueous core, vary in size from 10 nm to 1 mm or greater. They are of sufficient flexibility to be formulated as eye drops (Agarwal et al., 2016). Liposomes can easily solubilize hydrophobic drugs by embedding them in the lipid bilayer (Honda et al., 2013) and enhance the corneal permeability of lipophilic drugs through the close contact with cornea and conjunctiva and prolonged corneal contact time (Agarwal et al., 2016; Rathod and Deshpande, 2010). Our group previously used liposomes to deliver the therapeutic glaucoma drug brinzolamide (Brz) with poor water solubility (Li et al., 2016a; Wang et al., 2018). The topical application presented enhanced intraocular pressure lowering effect mainly because of the increased permeation. However, clinical application using conventional liposomes for topical ophthalmic drug delivery of Brz is hindered by instability and aggregation (Agarwal et al., 2016). Thus, it’s urgent to explore novel liposomes for eye drops. Brz is a highly specific, non-competitive, reversible, and effective inhibitor of carbonic anhydrase II (CA-II), which is able to suppress formation of aqueous humor in the eye and thus decrease IOP. Brz is an effective drug for the treatment of glaucoma (Siesky et al., 2008). Its commercially available formulation AZOPT® (Brz-Sus) is a sterile aqueous suspension due to its high lipophilicity (Siesky et al., 2008). Patients often subsequently feel uncomfortable due to the obvious granular sensation of the suspension, leading to tears and rapid clearance of the administered Brz-Sus. Although we have used an inclusion complex or liposomes to increase patient compliance, further improvement of Brz bioavailability still needs to be explored. In this study, Brz loaded nanoliposomes containing TPGS (T-LPs/Brz) were developed as a novel ocular delivery system for topical administration. We showed, for the first time, that TPGS modified nanoliposomes can improve the cornea penetration and retention time of Brz, thereby enhancing in vivo efficacy for glaucoma treatment.
3. Characterization of T-LPs/Brz 3.1. Particle size and zeta potential The particle size, polydispersity index, and zeta potential of T-LPs/ Brz was determined using a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., UK). The samples were diluted with distilled water 5 times before measurement. All measurements were carried out in triplicate. 3.2. Morphology The morphology of T-LPs/Brz was determined by transmission electron microscopy (TEM, (H-600; Hitachi, Tokyo, Japan). Briefly, a drop of the liposome solution was placed onto on copper grids, and then they were stained with 2% phosphotungstic acid (PTA) for 30 s. Samples were air-dried for 10 min at room temperature and then observed at an acceleration voltage of 100 kV. 3.3. Entrapment efficiency and drug loading efficiency The unencapsulated Brz was removed from the nanoliposomes solution by centrifugation at 50,000 rpm for 1 h at 4 °C, followed by removal of the supernatant. Then, one milliliter of acetonitrile was added into the sediment of the liposomes and sonicated for 20 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was diluted with acetonitrile. The Brz content was quantified by reverse-phase high performance liquid chromatography (HPLC) at 254 nm with a C18 column (4.6 mm × 150 mm–5 μm, Atlantis column) using a combination of two solvent systems of acetonitrile/water (40/60, v/v) as a mobile phase. Entrapment efficiency (EE%) and drug loading (DL%) was calculated by the following calculation Eq. (1) and Eq. (2):
2. Materials and methods 2.1. Materials Brz (purity > 99%) was purchased from Dalian Meilun Biology Technology Company (Dalian, China). TPGS was bought from Sigma Co. (St Louis, MO, USA). Soybean phosphatidylcholine (S100) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol was obtained from Shanghai Yuanju Biology Technology Company (Shanghai, China). AZOPT® was received from Alcon Laboratories (Puurs, Belgium). All other reagents were of analytical grade.
EE% = amount of the Brz in nanoliposomes/initial Brz amount ∗ 100% (1)
DL% = amount of the Brz in nanoliposomes/total amount of the liposome∗ 100%
(2)
2.2. Cell culture and animals 3.4. In vitro Brz release Murine fibroblasts L929 cells were obtained from the American Type Culture Collection (ATCC) and were cultured at 37 °C, 5% CO2 incubator in a phenol-red free RPMI 1640 with L-glutamine, 10% fetal bovine serum and 1% penicillin–streptomycin (Lonza, Germany). New Zealand rabbits (2.0–2.5 kg) were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, Sichuan, China). All animal procedures were approved and supervised by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, Sichuan, China).
The release rate of Brz from T-LPs/Brz was investigated by dynamic dialysis techniques. In short, 2 mL of T-LPs/Brz, LPs/Brz and Brz solutions were placed in different dialysis bags (MWCO = 144,000 Da). Each dialysis bag was completely immersed in 40 mL of simulated tear fluid (STF) at 37 °C under mild stirring. At pre-determined time intervals, 0.5 mL of the samples were taken from the solution for drug content analysis and replaced with 0.5 mL of the fresh release STF. The amount of Brz released into the STF was quantified by the HPLC method described above. All the data was presented as the mean of the results of experiments run in triplicate.
2.3. Preparation of T-LPs/Brz
3.5. Corneal permeability study and corneal hydration level
T-LPs/Brz were prepared using a modified thin-film hydration method (Li et al., 2016a,b). Briefly, S100 and cholesterol were dissolved in chloroform/methanol (4:1, v/v) solution. Then, Brz and TPGS, dissolved in acetone, were added to the mixture solution of cholesterol and
The transcorneal transport of Brz was studied using a Franz diffusion chamber (Gupta et al., 2013) which consisted of a donor and a 22
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were done thrice, and the mean values were taken. The mean percentage decrease in IOP was calculated by the following Eq. (6)
receiver compartment. Glutathione Bicarbonate Ringer (GBR) buffer was used as the medium and the temperature was maintained at 37 ± 1 °C under mixing conditions by using a magnetic stirrer. The cornea and 2 mm ring sclera were cut and stored in GBR buffer within 20 min after the rabbits were sacrificed. The obtained cornea was first mounted between the donor and receptor compartments through the sclera while the epithelium of the cornea was kept facing the donor compartment. After that, 1 mL of the GBR solution was added to the receiver compartment, while 0.5 mL of T-LPs/Brz or LPs/Brz was added to the donor compartment. Next, 0.3 mL of the samples were taken out at 15, 30, 60, 90, 120, 180, 240, 300, and 360 min from the receiver compartment and replaced with fresh GBR buffer. The total content of Brz permeating across the cornea was quantified by the HPLC method as described above. The experiment was done in triplicate. The apparent permeation coefficient (Papp, cm/s) of each formulation was calculated by Eq. (3):
Papp = (ΔQ/Δt )/(C0 ∗A∗60)
ΔIOP% = (IOPcontroleye−IOPtreatmenteye)/IOPcontroleye × 100% 4. Preliminary safety evaluation 4.1. In vitro cytotoxicity
L929 cells were seeded in a 96-well plate (2 × 104 cells per well). After 24 h of culture, the cells were washed with PBS and incubated with increasing concentrations of Brz, LPs/Brz, T-LPs and T-LPs/Brz (0.1–1000 μg/mL) for 24 h. The medium was then removed, and cell viability was determined by MTT assay (Oka et al., 1992; Vijayakumar and Ganesan, 2012). Untreated cells were used as reference for cell viability determination. The experiment was conducted in triplicate and results were expressed as mean ± standard deviation.
(3)
4.2. Eye irritation study of T-LPs/Brz
where C0 is the initial concentration of Brz in the donor chamber (μg/ mL) and A is the corneal surface area for diffusion (cm2), ΔQ/Δt is the permeation rate (μg/min) of Brz across the cornea as calculated from the slope of the straight line relating the cumulative infiltration of Brz to time. Corneal hydration levels (HL, %) were investigated by measuring total water content. Each corneal sample was carefully removed from the scleral ring and weighed (WW). The dry corneal weight (Wd) was then obtained after the cornea was dessicated at 100 °C for 6 h and weighed to determine HL% was determined by Eq. (4):
HL% = (Ww−Wd)/Ww ∗100
18 male healthy New Zealand rabbits were used as testing animals and randomly divided into three groups. The self-contrast method was used. The right eye was directly given 50 μL of the drug solution (1 mg/ mL Brz in LPs/Brz and T-LPs/Brz, 10 mg/mL Brz in AZOPT®) and the left eye was given the same amount of isotonic sodium chloride solution as a control. The rabbit eyes were passively closed for 30 s after administration. Once a day for 2 weeks, the topical state of the eyes was observed before each administration and 1, 2, 24, 48 and 72 h after the last administration. According to scoring criterion (Alany et al., 2006), the cornea, iris, edema, secretions and conjunctival scores were assessed respectively and the irritation score of each eye was calculated as the sum of the five parts. The final irritation score was calculated as the average of the sum of the scores between the 6 rabbits in each respective group. The relationship between response scores and irritation potential was shown in Table 1.
(4)
3.6. Study on the precorneal residence time According to the methods previously described (Ding et al., 1992), the study was performed by using Schimer Test with some modifications. Briefly, 50 μL of each prepared formulation or a control commercial formulation were added into the conjunctiva of the eye of New Zealand rabbits using a micro sampler. Pre-weighed filter paper with a diameter of 8 mm was then inserted into the lower eyelid position at 0.5, 1, 2 and 3 h after the eye drop administration. The filter paper was removed immediately after tear absorption and weighed again. The weight difference of filter paper was recorded and the density of tear fluid was regarded as 1.005 g/mL. The filter paper containing tear fluid was placed into a 1.5 mL eppendorf tube with 200 μL methanol, and Brz was extracted by vortexing for 30 min at room temperature. After centrifugation at 13000 rpm for 10 min, the supernatant containing Brz was quantified by HPLC. The concentration of Brz was calculated by the following equation:
C0 =
C×V×ρ Δm
(6)
4.3. H&E staining For the histopathological analysis, the excised cornea were immersed in a 4% paraformaldehyde solution, embedded in paraffin, sectioned, and processed for hematoxylin and eosin (H&E) staining. Images were acquired on a light microscope (Olympus, Tokyo, Japan). 4.4. Statistical analysis Statistical analysis was performed by one-way ANOVA. p values < 0.05 were considered statistically significant, and extreme significance levels were set at p < 0.01 and p < 0.001. 5. Results
(5)
where C0 is the initial concentration of Brz, C is the concentration of Brz diluted by methanol, V is the volume of methanol, ρ is the density of tear fluid, nearly 1.005 g/mL, Δm is the weight difference of filter paper between the twice weighing.
5.1. Preparation of T-LPs/Brz The processing parameters of T-LPs/Brz were optimized according to our preliminary screening studies. This included the lipid-to-cholesterol (L/C) molar ratio, lipid-to-drug (L/D) molar ratio, and the
3.7. In vivo efficacy investigation Table 1 The relationship between response scores and irritation potential.
Three rabbits were used for each formulation. Each formulation (50 µL, 1 mg/mL Brz in LPs/Brz and T-LPs/Brz, 10 mg/mL Brz in AZOPT®) was administered topically into the upper quadrant of the right eye, which was then immediately followed by manual blinking for three times. The left eye was left to serve as a control. The IOP was measured at different time intervals after dosing (viz. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h post-dosing) by using a Schiotz Tonometer (Rudolf Riester GmbH and Co., KG, Germany). All the measurements 23
Cumulative score
Assessment
0–3 4–8 9–12 13–16
Nonirritant Slightly irritant Moderately irritant Seriously irritant
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Fig. 1. Effects of various processing parameters on the mean diameter and drug entrapment efficiency of T-LPs/Brz. A, the lipid-to-cholesterol (L/C) molar ratio; B, lipid-to-drug (L/D) molar ratio; C, concentrations of TPGS. All data is expressed as mean ± SD for n = 3. *P < 0.05, **P < 0.01.
but a dramatic distinction appeared at 120–360 min. As presented in Table 2, the apparent permeation coefficients (Papp) of the T-LPs/Brz was 1.38 folds that of the LPs /Brz (p < 0.05) and 10.2 folds that of AZOPT® (p < 0.001). Precorneal residence time was also studied and displayed in Fig. 3B. At 60 min, there was no significant difference in Brz concentration in tear fluid among the three groups. However, TLPs/Brz achieved the highest drug concentration at 120 min. Compared to AZOPT®, the drug concentration of T-LPs/Brz was 3.18 folds higher (p < 0.05) at 180 min, which indicated that the novel nanoliposomes dramatically prolonged the corneal residence time of Brz and thus improved the drug absorption.
percentage of TPGS. As the molar percent of cholesterol increased, EE% increased at first and then reached a peak (at about 100%) when the L/ D was held constant at a molar ratio of 7:7 (Fig. 1A). The L/D molar ratio was closely related to the encapsulation rate of Brz. As shown in Fig. 1B, 95% EE was achieved when the L/D molar ratio was 10:1. However, EE% did not change significantly with a further increase of the L/D molar ratio to 20:1. In order to get optimal T-LPs/Brz with high EE% and small size, the L/C and L/D molar ratios were set at 7:7 and 10:1, respectively. Furthermore, various amounts of TPGS were introduced to assess the penetration profiles of TPGS modified liposomes. Fig. 1C displayed that 1% TPGS (w/w) in T-LPs/Brz was enough to achieve the strongest permeation of Brz. As such, the optimal process for preparing T-LPs/Brz was determined to be a L/C molar ratio of 7:7 and a L/D molar ratio of 10:1 with a concentration of 1% TPGS (w/w).
5.4. Intraocular pressure lowering effect It was reported that 15% or higher average reduction in IOP was considered as effective in IOP control (Akman et al., 2005). IOP in both eyes of rabbits was monitored for 12 h. The maximal IOP reduction (up to 35.17%) was achieved 4 h after instillation of T-LPs/Brz, while LPs/ Brz showed the maximal IOP reduction of 28.97% 5 h after administration. As seem in Fig. 4, T-LPs/Brz caused 26.38% reduction of IOP 2 h after administration. Whereas, only a 21.19% and 24.87% IOP reduction was in AZOPT® and LPs/Brz groups, respectively. T-LPs/Brz, similar to LPs/Brz, displayed an effective IOP reduction for about 11 h. In contrast, AZOPT® maintained an effective IOP reduction only over a period of 6 h. In summary, T-LPs/Brz showed significantly lower IOP than the control groups after treatment for 4–10 h. These results clearly demonstrated that T-LPs/Brz were the most effective prepared treatment for IOP reduction.
5.2. Pharmaceutical properties The optimum T-LPs/Brz presented an EE% of 95.41 ± 3.03% (n = 3), DL% of 4.00 ± 0.13% (n = 3) and a mean particle size of 96.87 ± 4.43 nm (n = 3) (Fig. 2A) with a narrow size distribution (the polydispersity index was 0.188 ± 0.011, n = 3). The zeta potential value was about neutral, with the value of -1.17 ± 1.91 mV (n = 3). No significant difference in EE, size, and zeta potential among the three batches of T-LPs/Brz was observed, indicating a reproducible preparation process. As shown in Fig. 2B, T-LPs/Brz had slightly blue opalescence and evident Tyndall effect. The morphology of the T-LPs/Brz was observed by TEM. Most of particles were nearly spherical in shape with an average diameter of approximate 90 nm (Fig. 2C). This was in accordance with the results obtained by photon correlation spectroscopy using a Zetasizer Nano ZS90. Moreover, despite the small absolute value of the zeta potential, our preliminary stability study proved that the T-LPs/Brz were stable for at least 10 days at 4 °C and 25 °C (Fig. 2D). A significant improvement over LPs/Brz which are only physically stable for 3 days at 4 °C (Li et al., 2016a). The in vitro release profile of T-LPs/Brz was illustrated in Fig. 2E, which differed from Brz or LPs/Brz. T-LPs/Brz displayed no burst release in the first 1 h. Furthermore, compared to Brz and the LPs/Brz, TLPs/Brz delayed the drug release to a large extent. Interestingly, AZOPT® also exhibited the same slow release properties as T-LPs/Brz for the first 4 h. The release rate of T-LPs/Brz was significantly lower than that of AZOPT®, Brz and LPs/Brz after 4 h, which demonstrated the better sustained-release properties of T-LPs/Brz.
5.5. Preliminary safety evaluation The development of an effective and non-toxic treatment strategy is still a challenging task for ocular drug delivery. In this study, an MTT assay was employed to test the influence of different formulations on the metabolic activity of L929 cells (Bauer et al., 2012; Luo et al., 2016). As a standard assay of cell viability, this method was based on the colorimetric analyses of living cells. As shown in Fig. 5A, T-LPs/Brz, LPs/Brz, Brz, T-LPs and LPs, had no apparent influence on cell viability (near 100%) of L929 cells even with extremely high concentrations of Brz of up to 1000 μg/mL. This study leads to the conclusion that the nanoliposomes could be non-toxic as eye drops. HL (Corneal hydration levels) is a parameter frequently used to evaluate cornea damage. Normal HL of the cornea is between 75% and 80%. Change in this level demonstrates damage to the epithelium and/ or endothelium (Vega et al., 2008). As seen in Table 3, the HL of the TLPs/Brz was 78.56%, indicating that the T-LPs/Brz are safe for corneal tissue. To examine the ocular tolerability of T-LPs/Brz, an eye irritation experiment was performed by using the Draize test in rabbit eyes (Li et al., 2013). The in vivo results were summarized in Table 4. AZOPT®
5.3. Ex vivo corneal permeation A corneal penetration study was carried out to evaluate the effect of T-LPs/Brz on the drug transcorneal transportation. As shown in Fig. 3A, the transcorneal drug permeation of the T-LPs/Brz was 2 times and 5 times higher than LPs/Brz and AZOPT®, respectively. Initially, there was no significant difference among AZOPT®, LPs/Brz and T-LPs/Brz, 24
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Fig. 2. Characterization of T-LPs/Brz. A, the size and Zeta Potential of T-LPs/Brz; B, the appearance of T-LPs/Brz demonstrating obvious Tyndall effect; C, Transmission electron microscopy (TEM) image of T-LPs/Brz. Scale bar, 100 nm; D, the stability of T-LPs/Brz in 4 °C (left) and 25 °C (right); E, Release profile of TLPs/Brz at 37 °C in STF. All data is expressed as mean ± SD for n = 3.
nanoliposomes encapsulating Brz for glaucoma therapy. The therapeutic effects and preliminary safety profile of the T-LPs/Brz was investigated to assess the potential application of this new preparation of Brz as eye drops. T-LPs/Brz with good EE and particle size were successfully prepared after optimization of processing factors including L/C molar ratio, L/D molar ratio and TPGS content. As presented in Fig. 1, the mean size and EE were influenced significantly by the content of lipid and cholesterol. It is well known that cholesterol reduces the flexibility of membranes in the preparation process (Hathout et al., 2007; Kirby et al., 1980; Senior and Gregoriadis, 1982). Excessive cholesterol content hinders Brz uptake into the phospholipid bilayer space, resulting in reduced encapsulation efficiency. Moreover, a large amount of cholesterol might also compete with Brz for the hydrophobic space inside the
caused relatively severe eye irritation including conjunctival congestion, corneal opacification and secretions. While T-LPs/Brz, similar to LPs/Brz, only resulted in small amounts of conjunctival inflammation and no negative clinical signs such as corneal opacity and conjunctival redness or abnormality. This demonstrates that the T-LPs/Brz have little short or long term irritant effects in rabbits. The results of a histological analysis using a light microscope were shown in Fig. 5B. The corneal epithelium of the rabbits treated by TLPs/Brz retained an intact structure, providing further proof that T-LPs/ Brz caused no eye irritation.
6. Discussion The goal of this study was to construct TPGS modified 25
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Fig. 3. Corneal permeation of T-LPs/Brz in vitro. A, drug permeation of T-LPs/Brz at different times, B. Brz concentration in tear fluid at different times. All data is expressed as mean ± SD for n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. Table 2 The apparent permeation coefficients (Papp). Papp(*10−6cm/s) T-LPs/Brz LPs/Brz AZOPT®
3.55 ± 0.12*** 2.58 ± 0.04*** 0.35 ± 0.01
&
Date are mean ± SD. *** P < 0.001 versus AZOPT® group. & P < 0.05 versus LPs/Brz group.
nanoliposomes. Therefore, further increasing the cholesterol content (when the L/C molar ratio was set at 7:14) did not enhance Brz entrapment. Fig. 1A showed that more lipids resulted in a bigger size. This is probably due to incomplete hydration of the lipid film caused by the high viscosity of lipids (Mourtas et al., 2009). The amount of TPGS was also optimized to enhance the permeation of Brz. T-LPs/Brz exhibited the strongest corneal penetration when the concentration of TPGS was 1% (w/w) and it was observed that, more TPGS cannot further improve drug permeation. Furthermore, it has been reported that P-gp expression in rabbit corneas inhibits drug penetration (Dey et al., 2003). A 1% (w/w) concentration of TPGS is enough to inhibit P-gp expression, and as a result, leading to improved permeation of Brz. It is known that high concentrations of nonionic surfactants are not advised for ophthalmic formulations (Mu et al., 2005). Therefore, the optimal amount of TPGS in T-LPs/Brz was set at 1% (w/w) in this study. The size of the optimized T-LPs/Brz was < 100 nm with a slightly negative surface charge, which probably resulted from the phosphatidylcholine head group on the outer nanoliposome surface (Ascenso et al., 2013). The introduction of TPGS might contribute to improved stability of the T-LPs/Brz when compared with LPs/Brz at 4 °C and 25 °C. Vitamin E succinate and PEG, two parts of TPGS, each provide large steric hindrance and a hydrophilic chain on the surface of the TLPs/Brz, respectively. This can prevent aggregation of the nanoliposomes (Guo et al., 2013). Despite the relative improvement in stability, the T-LPs/Brz are still only stable for < 20 days. Thus, freezing the preparation should be further investigated for longer-term storage of TLPs/Brz. T-LPs/Brz displayed a better sustained release profile when compared with LPs/Brz. There was no burst release of Brz in T-LPs/Brz in the first 1 h. Which is indicative of complete entrapment of Brz in TLPs/Brz without any drug free in solution or adherent on the liposome surface. It has been reported that TPGS affects the release behavior of nanoparticle drug delivery systems (Li et al., 2016b). This makes the Brz slowly diffuse from the liposome vehicle which contains TPGS and phospholipids. Further slowing the release rate of Brz is the hydrophilic PEG chains in the TPGS on the surface of the T-LPs/Brz. These chains retard the release of the hydrophobic Brz into the diffusion medium (Raju et al., 2013). As such, T-LPs/Brz released < 30% of their Brz after
Fig. 4. The IOP reduction of white New Zealand rabbits treated by T-LPs/Brz, LPs/Brz and AZOPT®. All data is expressed as mean ± SD for n = 6. * P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 5. Preliminary Safety Evaluation of the T-LPs/Brz. A, cell viability at different concentrations treated with different formulations, namely LPs, TPGS-LPs, Brz, LPs/Brz and T-LPs/Brz; B, representative H&E images (200X) of the cornea of New Zealand rabbits treated with PBS(Control), AZOPT®, LPs/Brz and Brz-TPGS-LPs.
biomechanical and biochemical features, and conjunctiva cavity volume (York and Steiling, 1998). As expected, T-LPs/Brz exhibited enhanced and sustained IOP reduction effect when compared with LPs/ Brz and AZOPT® as shown in Fig. 4. The promising efficacy of T-LPs/Brz on glaucoma treatment may be mainly attributed to the improved precorneal retention and transcorneal transportation. The effective inhibition of P-gp by TPGS can avoid clearance in the cornea and promote quick drug penetration and cornea absorption (Ostacolo et al., 2013; Wempe et al., 2009). Moreover, the sustained-release profile of the TLPs/Brz is important for glaucoma treatment due to the maintenance of a constant Brz concentration in the cornea (Feng et al., 2007). Ocular tolerability is vital to the potential application for novel eye preparations. In this study, several experiments were employed to investigate the preliminary safety of T-LPs/Brz. The in vitro cytotoxicity test showed that T-LPs/Brz were non-toxic on L929 cells, because both the vector T-LPs and drug Brz were safe. Brz can be absorbed systemically after topical administration. Excessive concentration of Brz in the circulatory system may cause side effects of sulfonamides. But due to the exist of the blood-ocular barrier (Maurice, 1984), the amount of the Brz into the circulatory system through local administration is even less. In view of this, we speculate that its biodistribution may not affect the safety of the formulation. Eye irritation experiments and histological analysis demonstrated that T-LPs/Brz had no long or short-term irritant effects. Consistent with literature (Khalil et al., 2017; Lajunen et al., 2016), T-LPs/Brz with nearly neutral surface charge and small size do not induce eye inflammation. Generally, rabbit eyes are more susceptible to extraneous substances than human’s, it is therefore reasonable to believe that T-LPs/Brz possess excellent ocular tolerability.
Table 3 The HLs of AZOPT®, LPs/Brz and T-LPs/Brz. HL (%) AZOPT® LPs/Brz T-LPs/Brz
80.23 ± 2.20 78.35 ± 2.35 78.56 ± 1.10
Table 4 The results of the eye irritation score. The degree of stimulation
Cornea Iris Conjunctival Edema Secretions Sum score
Physiological saline
AZOPT®
LPs/Brz
T-LPs/Brz
0 0 0 0 0 0
2 0 1 0 2 5
0 0 1 1 0 2
0 0 1 1 0 2
1 h. This sustained release performance might be conducive to the retention in the cornea and thus provide potent and prolonged therapeutic efficacy. Although it looks like the plateau phase appeared when Brz was released around 80% out of TLPs/Brz, a slight increase of the percentage of Brz release can be observed. So it can be inferred that 100% release of Brz out of TLPs/Brz might be achieved if the time was prolonged. Drug trans-corneal transportation and precorneal retention are widely used to evaluate ophthalmic drug delivery systems, providing vital information which help to predict the bioactivity of eye drops. Both human and rabbit corneas express P-gp (Katragadda et al., 2006), which is an ATP-dependent efflux pump and can actively remove previously absorbed drugs (Warsi et al., 2014). TPGS was reported to interact with P-gp and inhibit its efflux function of exogenous drugs (Ostacolo et al., 2013; Wempe et al., 2009). In this study, T-LPs/ Brz enhanced Brz permeation and improved Brz concentration in tear fluid (as shown in Fig. 3 and Table 2) in contrast to LPs/Brz. This effect is probably due to the TPGS in T-LPs/Brz efficiently inhibiting the efflux of Brz mediated by P-gp. Moreover, T-LPs/Brz can release Brz slowly and protect Brz against drainage through tears, hence enhancing the precorneal retention and trans-corneal transportation. New Zealand rabbits were selected as model animals to evaluate the efficacy of IOP reduction and safety of T-LPs/Brz in this study, because they have relatively large eyes, sharing many anatomical features with humans such as eyeball size, internal structure, optical system,
7. Conclusions We successfully developed an innovative ocular delivery system (TLPs) which can efficiently incorporate hydrophobic molecules (Brz) and improve their therapeutic efficacy. Brz is entrapped by T-LPs to form novel nanoliposomes (T-LPs/Brz) with high efficiency by a simple preparation method. T-LPs/Brz with a nearly-neutral surface charge displayed a better sustained release performance and storage stability when compared with LPs/Brz. Ex vivo corneal transport experiments demonstrated that T-LPs/Brz improve the penetration of Brz across the cornea to a large extent. In vivo experiments found that T-LPs/Brz achieved extended pre-corneal retention of Brz and prolonged reduction of IOP with high efficacy. Moreover, no eye irritation or lesions were observed in healthy white rabbits. Cumulatively, these results support the conclusion that TPGS modified nanoliposomes are an 27
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effective delivery system for Brz to treat glaucoma.
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8. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 9. Author contributions Xiangrong Song conceived and designed the study. Quansheng Jin, Huili Li, Zhaohui Jin, Fazhan Wang, Lingjing Huang and Yang Zhou performed experiments. Quansheng Jin, Chunling Jiang and Yongmei Liu analyzed data. Quansheng Jin wrote the paper. Jinhui Wu and James Oswald reviewed and edited the manuscript. All authors read and approved the manuscript. 10. Funding This work was financially supported by Sichuan Province Science and Technology Support Program (2015SZ0234 and 2017GZ0413) and the National Natural Science Foundation of China (No. 81600006). References Agarwal, R., Iezhitsa, I., Agarwal, P., Abdul Nasir, N.A., Razali, N., Alyautdin, R., Ismail, N.M., 2016. Liposomes in topical ophthalmic drug delivery: an update. Drug Delivery 23, 1075–1091. Akman, A., Cetinkaya, A., Akova, Y.A., Ertan, A., 2005. Comparison of additional intraocular pressure-lowering effects of latanoprost vs brimonidine in primary openangle glaucoma patients with intraocular pressure uncontrolled by timolol-dorzolamide combination. Eye 19, 145–151. Alany, R.G., Rades, T., Nicoll, J., Tucker, I.G., Davies, N.M., 2006. W/O microemulsions for ocular delivery: evaluation of ocular irritation and precorneal retention. J. Control. Rel.: Off. J. Control. Rel. Soc. 111, 145–152. Ascenso, A., Cruz, M., Euleterio, C., Carvalho, F.A., Santos, N.C., Marques, H.C., Simoes, S., 2013. Novel tretinoin formulations: a drug-in-cyclodextrin-in-liposome approach. J. Lipos. Res. 23, 211–219. Bauer, M., Lautenschlaeger, C., Kempe, K., Tauhardt, L., Schubert, U.S., Fischer, D., 2012. Poly(2-ethyl-2-oxazoline) as alternative for the stealth polymer poly(ethylene glycol): comparison of in vitro cytotoxicity and hemocompatibility. Macromol. Biosci. 12, 986–998. Caruso, C., Ostacolo, C., Epstein, R.L., Barbaro, G., Troisi, S., Capobianco, D., 2016. Transepithelial corneal cross-linking with vitamin E-enhanced riboflavin solution and abbreviated, low-dose UV-A: 24-month clinical outcomes. Cornea 35, 145–150. Cholkar, K., Gunda, S., Earla, R., Pal, D., Mitra, A.K., 2015. Nanomicellar topical aqueous drop formulation of rapamycin for back-of-the-eye delivery. AAPS PharmSciTech 16, 610–622. Cholkar, K., Hariharan, S., Gunda, S., Mitra, A.K., 2014a. Optimization of dexamethasone mixed nanomicellar formulation. AAPS PharmSciTech 15, 1454–1467. Cholkar, K., Vadlapudi, A., Dasari, S.R., Mitra, A.K., 2014b. Ocular Drug Delivery. Collnot, E.M., Baldes, C., Wempe, M.F., Hyatt, J., Navarro, L., Edgar, K.J., Schaefer, U.F., Lehr, C.M., 2006. Influence of vitamin E TPGS poly(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell monolayers. J. Control. Rel. Off. J. Control. Rel. Soc. 111, 35–40. Maurice, D., 1984. Ocular Pharmacokinetics, Anonymous Pharmacology of the Eye. Springer, pp. 19–116. Dey, S., Patel, J., Anand, B.S., Jain-Vakkalagadda, B., Kaliki, P., Pal, D., Ganapathy, V., Mitra, A.K., 2003. Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines. Investig. Ophthalmol. Visual Sci. 44, 2909–2918. Ding, S., Chen, C.C., Salome-Kesslak, R., Tang-Liu, D.D., Himmelstein, K.J., 1992. Precorneal sampling techniques for ophthalmic gels. J. Ocular Pharmacol. 8, 151–159. Duan, Y., Cai, X., Du, H., Zhai, G., 2015. Novel in situ gel systems based on P123/TPGS mixed micelles and gellan gum for ophthalmic delivery of curcumin. Colloid. Surf. B Biointerf. 128, 322–330. Feng, S.S., Zhao, L., Zhang, Z., Bhakta, G., Win, K.Y., Dong, Y., Shu, C., 2007. Chemotherapeutic engineering: vitamin E TPGS-emulsified nanoparticles of biodegradable polymers realized sustainable paclitaxel chemotherapy for 168h in vivo. Chem. Eng. Sci. 62, 6641–6648. Fogagnolo, P., Sacchi, M., Ceresara, G., Paderni, R., Lapadula, P., Orzalesi, N., Rossetti, L., 2013. The effects of topical coenzyme Q10 and vitamin E D-alpha-tocopheryl polyethylene glycol 1000 succinate after cataract surgery: a clinical and in vivo confocal study. Ophthalmologica. J. int. d'ophtalmol. Int. J. Ophthalmol. Zeitsch. Augen. 229, 26–31. Guo, Y., Luo, J., Tan, S., Otieno, B.O., Zhang, Z., 2013. The applications of Vitamin E
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
TPGS modified nanoliposomes as an effective ocular delivery system to treat glaucoma
T
Quansheng Jina,1, Huili Lia,1, Zhaohui Jina,1, Lingjing Huanga, Fazhan Wanga, Yang Zhoua, ⁎ ⁎ Yongmei Liua, Chunling Jianga, James Oswaldb, Jinhui Wua, , Xiangrong Songa, a
State Key Laboratory of Biotherapy/Geriatrics and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy, Sichuan University, Chengdu 610041, China b School of Nanotechnology Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanoliposomes TPGS Brinzolamide Ocular drug delivery system Pharmacodynamics
The aim of this study is to investigate the potential of D-alpha-tocopheryl poly (ethylene glycol 1000) succinate (TPGS) modified nanoliposomes as an ophthalmic delivery system of brinzolamide (Brz) for glaucoma treatment. The Brz loaded nanoliposomes containing TPGS (T-LPs/Brz) were firstly developed by a thin-film dispersion method. The average particle size was 96.87 ± 4.43 nm. The entrapment efficiency of the Brz was 95.41 ± 3.03% and the drug loading was 4.00 ± 0.13%. T-LPs/Brz exhibited obvious sustained release of Brz; in stark contrast to the normal liposomes of Brz (LPs/Brz) and the commercial formulation AZOPT® (Brz ophthalmic suspension, Brz-Sus). Enhanced trans-corneal transport of Brz was achieved with T-LPs/Brz. Compared with both Brz-Sus and LPs/Brz, the apparent permeability coefficient (Papp) of T-LPs/Brz was 10.2 folds and 1.38 folds higher, respectively. Moreover, T-LPs/Brz extended the cornea residence of Brz. White New Zealand rabbits treated with T-LPs/Brz had 3.18 folds and 1.57 folds Brz concentration 2 h after treatment than Brz-Sus and LPs/ Brz, respectively. Further pharmacodynamic studies showed that T-LPs/Brz maintained an effective intraocular pressure (IOP) reduction from 3 h to 11 h after administration, while Brz-Sus and LPs/Brz presented effective IOP decreases from 3 h to 6 h and 3 h to 8 h respectively. The preliminary safety evaluation demonstrated that T-LPs/ Brz had no significant side effects; specifically, no cornea damage and eye irritation. All the results indicated that TPGS modified nanoliposomes were a promising ocular delivery carriers for Brz to treat glaucoma. As such, TLPs/Brz might be worthy of further translational study.
1. Introduction Human eyes are well protected from the environment by various barriers, among which the cornea is the most protective outer layer. Eye drops for topical instillation, a conventional eye treatment route; often have low topical ophthalmic bioavailability (< 5%). This low ophthalmic bioavailability is mainly due to poor drug permeation across the cornea. Moreover, rapid washout by the tear and lachrymal drainage systems also leads to the low bioavailability of conventional eye drops. Despite this, eye drops are still one of the preferred dosage forms by the patients, accounting for over 90% of the market in 2014 (Cholkar et al., 2014b). Therefore, the development of novel eye drops with enhanced corneal permeation and extended cornea retention is needed to improve eye drop therapeutic efficacy. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), one of
FDA-approved safe pharmaceutical adjuvants, is a water-soluble derivative of natural vitamin E created via esterification with polyethylene glycol 1000 (Guo et al., 2013). As a novel nonionic surfactant, it has been widely used as an absorption enhancer, permeation enhancer, emulsifier, solubilizer, stabilizer and P-glycoprotein inhibitor (Guo et al., 2013; Tan et al., 2017). Moreover, many TPGS-based drug delivery systems (DDS) have also been developed for cancer therapy. Recently, it has been reported that TPGS can be used to solubilize Coenzyme Q10 or riboflavin in eye drops without any side effect in clinical trials. TPGS can also enhance the corneal permeation of riboflavin-5′-phosphate when combinationally used (Ostacolo et al., 2013). Possible reason is that it interacts with P-glycoprotein and, perhaps, with other drug transporter proteins (Collnot et al., 2006). However, there are limited reports about TPGS-based DDS for eye drops (Caruso et al., 2016; Cholkar et al., 2015; Cholkar et al., 2014a;
⁎
Corresponding authors. E-mail addresses: [email protected] (J. Wu), [email protected] (X. Song). 1 These authors contributed equally to this article. https://doi.org/10.1016/j.ijpharm.2018.10.033 Received 8 June 2018; Received in revised form 8 October 2018; Accepted 10 October 2018 Available online 11 October 2018 0378-5173/ © 2018 Published by Elsevier B.V.
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S100. The organic solvents were subsequently removed by a rotary evaporator at 37 °C to form a thin lipid film. Phosphate buffer solution (pH 7.4) was used to hydrate the thin lipid film for 1 h at 60 °C to obtain a colloidal dispersion. T-LPs/Brz were finally obtained after sonicating the colloidal dispersion for 3 min at 100 W in an ice bath and filtering with a 0.22-μm syringe filter for purpose of sterilization. The preparation of the normal liposomes of Brz (LPs/Brz) was performed as described in the previous study (Li et al., 2016a,b). The blank liposomes (LPs and T-LPs) were prepared following the same method without the addition of Brz.
Duan et al., 2015; Fogagnolo et al., 2013; Vadlapudi et al., 2014; Warsi et al., 2014). To our knowledge, it’s still unclear if TPGS can be used to promote the ocular penetration of drug-loaded nanoliposomes. Conventional liposomes, consisting of an outer bilayer of lipids enclosing a central aqueous core, vary in size from 10 nm to 1 mm or greater. They are of sufficient flexibility to be formulated as eye drops (Agarwal et al., 2016). Liposomes can easily solubilize hydrophobic drugs by embedding them in the lipid bilayer (Honda et al., 2013) and enhance the corneal permeability of lipophilic drugs through the close contact with cornea and conjunctiva and prolonged corneal contact time (Agarwal et al., 2016; Rathod and Deshpande, 2010). Our group previously used liposomes to deliver the therapeutic glaucoma drug brinzolamide (Brz) with poor water solubility (Li et al., 2016a; Wang et al., 2018). The topical application presented enhanced intraocular pressure lowering effect mainly because of the increased permeation. However, clinical application using conventional liposomes for topical ophthalmic drug delivery of Brz is hindered by instability and aggregation (Agarwal et al., 2016). Thus, it’s urgent to explore novel liposomes for eye drops. Brz is a highly specific, non-competitive, reversible, and effective inhibitor of carbonic anhydrase II (CA-II), which is able to suppress formation of aqueous humor in the eye and thus decrease IOP. Brz is an effective drug for the treatment of glaucoma (Siesky et al., 2008). Its commercially available formulation AZOPT® (Brz-Sus) is a sterile aqueous suspension due to its high lipophilicity (Siesky et al., 2008). Patients often subsequently feel uncomfortable due to the obvious granular sensation of the suspension, leading to tears and rapid clearance of the administered Brz-Sus. Although we have used an inclusion complex or liposomes to increase patient compliance, further improvement of Brz bioavailability still needs to be explored. In this study, Brz loaded nanoliposomes containing TPGS (T-LPs/Brz) were developed as a novel ocular delivery system for topical administration. We showed, for the first time, that TPGS modified nanoliposomes can improve the cornea penetration and retention time of Brz, thereby enhancing in vivo efficacy for glaucoma treatment.
3. Characterization of T-LPs/Brz 3.1. Particle size and zeta potential The particle size, polydispersity index, and zeta potential of T-LPs/ Brz was determined using a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., UK). The samples were diluted with distilled water 5 times before measurement. All measurements were carried out in triplicate. 3.2. Morphology The morphology of T-LPs/Brz was determined by transmission electron microscopy (TEM, (H-600; Hitachi, Tokyo, Japan). Briefly, a drop of the liposome solution was placed onto on copper grids, and then they were stained with 2% phosphotungstic acid (PTA) for 30 s. Samples were air-dried for 10 min at room temperature and then observed at an acceleration voltage of 100 kV. 3.3. Entrapment efficiency and drug loading efficiency The unencapsulated Brz was removed from the nanoliposomes solution by centrifugation at 50,000 rpm for 1 h at 4 °C, followed by removal of the supernatant. Then, one milliliter of acetonitrile was added into the sediment of the liposomes and sonicated for 20 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was diluted with acetonitrile. The Brz content was quantified by reverse-phase high performance liquid chromatography (HPLC) at 254 nm with a C18 column (4.6 mm × 150 mm–5 μm, Atlantis column) using a combination of two solvent systems of acetonitrile/water (40/60, v/v) as a mobile phase. Entrapment efficiency (EE%) and drug loading (DL%) was calculated by the following calculation Eq. (1) and Eq. (2):
2. Materials and methods 2.1. Materials Brz (purity > 99%) was purchased from Dalian Meilun Biology Technology Company (Dalian, China). TPGS was bought from Sigma Co. (St Louis, MO, USA). Soybean phosphatidylcholine (S100) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol was obtained from Shanghai Yuanju Biology Technology Company (Shanghai, China). AZOPT® was received from Alcon Laboratories (Puurs, Belgium). All other reagents were of analytical grade.
EE% = amount of the Brz in nanoliposomes/initial Brz amount ∗ 100% (1)
DL% = amount of the Brz in nanoliposomes/total amount of the liposome∗ 100%
(2)
2.2. Cell culture and animals 3.4. In vitro Brz release Murine fibroblasts L929 cells were obtained from the American Type Culture Collection (ATCC) and were cultured at 37 °C, 5% CO2 incubator in a phenol-red free RPMI 1640 with L-glutamine, 10% fetal bovine serum and 1% penicillin–streptomycin (Lonza, Germany). New Zealand rabbits (2.0–2.5 kg) were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, Sichuan, China). All animal procedures were approved and supervised by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, Sichuan, China).
The release rate of Brz from T-LPs/Brz was investigated by dynamic dialysis techniques. In short, 2 mL of T-LPs/Brz, LPs/Brz and Brz solutions were placed in different dialysis bags (MWCO = 144,000 Da). Each dialysis bag was completely immersed in 40 mL of simulated tear fluid (STF) at 37 °C under mild stirring. At pre-determined time intervals, 0.5 mL of the samples were taken from the solution for drug content analysis and replaced with 0.5 mL of the fresh release STF. The amount of Brz released into the STF was quantified by the HPLC method described above. All the data was presented as the mean of the results of experiments run in triplicate.
2.3. Preparation of T-LPs/Brz
3.5. Corneal permeability study and corneal hydration level
T-LPs/Brz were prepared using a modified thin-film hydration method (Li et al., 2016a,b). Briefly, S100 and cholesterol were dissolved in chloroform/methanol (4:1, v/v) solution. Then, Brz and TPGS, dissolved in acetone, were added to the mixture solution of cholesterol and
The transcorneal transport of Brz was studied using a Franz diffusion chamber (Gupta et al., 2013) which consisted of a donor and a 22
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were done thrice, and the mean values were taken. The mean percentage decrease in IOP was calculated by the following Eq. (6)
receiver compartment. Glutathione Bicarbonate Ringer (GBR) buffer was used as the medium and the temperature was maintained at 37 ± 1 °C under mixing conditions by using a magnetic stirrer. The cornea and 2 mm ring sclera were cut and stored in GBR buffer within 20 min after the rabbits were sacrificed. The obtained cornea was first mounted between the donor and receptor compartments through the sclera while the epithelium of the cornea was kept facing the donor compartment. After that, 1 mL of the GBR solution was added to the receiver compartment, while 0.5 mL of T-LPs/Brz or LPs/Brz was added to the donor compartment. Next, 0.3 mL of the samples were taken out at 15, 30, 60, 90, 120, 180, 240, 300, and 360 min from the receiver compartment and replaced with fresh GBR buffer. The total content of Brz permeating across the cornea was quantified by the HPLC method as described above. The experiment was done in triplicate. The apparent permeation coefficient (Papp, cm/s) of each formulation was calculated by Eq. (3):
Papp = (ΔQ/Δt )/(C0 ∗A∗60)
ΔIOP% = (IOPcontroleye−IOPtreatmenteye)/IOPcontroleye × 100% 4. Preliminary safety evaluation 4.1. In vitro cytotoxicity
L929 cells were seeded in a 96-well plate (2 × 104 cells per well). After 24 h of culture, the cells were washed with PBS and incubated with increasing concentrations of Brz, LPs/Brz, T-LPs and T-LPs/Brz (0.1–1000 μg/mL) for 24 h. The medium was then removed, and cell viability was determined by MTT assay (Oka et al., 1992; Vijayakumar and Ganesan, 2012). Untreated cells were used as reference for cell viability determination. The experiment was conducted in triplicate and results were expressed as mean ± standard deviation.
(3)
4.2. Eye irritation study of T-LPs/Brz
where C0 is the initial concentration of Brz in the donor chamber (μg/ mL) and A is the corneal surface area for diffusion (cm2), ΔQ/Δt is the permeation rate (μg/min) of Brz across the cornea as calculated from the slope of the straight line relating the cumulative infiltration of Brz to time. Corneal hydration levels (HL, %) were investigated by measuring total water content. Each corneal sample was carefully removed from the scleral ring and weighed (WW). The dry corneal weight (Wd) was then obtained after the cornea was dessicated at 100 °C for 6 h and weighed to determine HL% was determined by Eq. (4):
HL% = (Ww−Wd)/Ww ∗100
18 male healthy New Zealand rabbits were used as testing animals and randomly divided into three groups. The self-contrast method was used. The right eye was directly given 50 μL of the drug solution (1 mg/ mL Brz in LPs/Brz and T-LPs/Brz, 10 mg/mL Brz in AZOPT®) and the left eye was given the same amount of isotonic sodium chloride solution as a control. The rabbit eyes were passively closed for 30 s after administration. Once a day for 2 weeks, the topical state of the eyes was observed before each administration and 1, 2, 24, 48 and 72 h after the last administration. According to scoring criterion (Alany et al., 2006), the cornea, iris, edema, secretions and conjunctival scores were assessed respectively and the irritation score of each eye was calculated as the sum of the five parts. The final irritation score was calculated as the average of the sum of the scores between the 6 rabbits in each respective group. The relationship between response scores and irritation potential was shown in Table 1.
(4)
3.6. Study on the precorneal residence time According to the methods previously described (Ding et al., 1992), the study was performed by using Schimer Test with some modifications. Briefly, 50 μL of each prepared formulation or a control commercial formulation were added into the conjunctiva of the eye of New Zealand rabbits using a micro sampler. Pre-weighed filter paper with a diameter of 8 mm was then inserted into the lower eyelid position at 0.5, 1, 2 and 3 h after the eye drop administration. The filter paper was removed immediately after tear absorption and weighed again. The weight difference of filter paper was recorded and the density of tear fluid was regarded as 1.005 g/mL. The filter paper containing tear fluid was placed into a 1.5 mL eppendorf tube with 200 μL methanol, and Brz was extracted by vortexing for 30 min at room temperature. After centrifugation at 13000 rpm for 10 min, the supernatant containing Brz was quantified by HPLC. The concentration of Brz was calculated by the following equation:
C0 =
C×V×ρ Δm
(6)
4.3. H&E staining For the histopathological analysis, the excised cornea were immersed in a 4% paraformaldehyde solution, embedded in paraffin, sectioned, and processed for hematoxylin and eosin (H&E) staining. Images were acquired on a light microscope (Olympus, Tokyo, Japan). 4.4. Statistical analysis Statistical analysis was performed by one-way ANOVA. p values < 0.05 were considered statistically significant, and extreme significance levels were set at p < 0.01 and p < 0.001. 5. Results
(5)
where C0 is the initial concentration of Brz, C is the concentration of Brz diluted by methanol, V is the volume of methanol, ρ is the density of tear fluid, nearly 1.005 g/mL, Δm is the weight difference of filter paper between the twice weighing.
5.1. Preparation of T-LPs/Brz The processing parameters of T-LPs/Brz were optimized according to our preliminary screening studies. This included the lipid-to-cholesterol (L/C) molar ratio, lipid-to-drug (L/D) molar ratio, and the
3.7. In vivo efficacy investigation Table 1 The relationship between response scores and irritation potential.
Three rabbits were used for each formulation. Each formulation (50 µL, 1 mg/mL Brz in LPs/Brz and T-LPs/Brz, 10 mg/mL Brz in AZOPT®) was administered topically into the upper quadrant of the right eye, which was then immediately followed by manual blinking for three times. The left eye was left to serve as a control. The IOP was measured at different time intervals after dosing (viz. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h post-dosing) by using a Schiotz Tonometer (Rudolf Riester GmbH and Co., KG, Germany). All the measurements 23
Cumulative score
Assessment
0–3 4–8 9–12 13–16
Nonirritant Slightly irritant Moderately irritant Seriously irritant
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Fig. 1. Effects of various processing parameters on the mean diameter and drug entrapment efficiency of T-LPs/Brz. A, the lipid-to-cholesterol (L/C) molar ratio; B, lipid-to-drug (L/D) molar ratio; C, concentrations of TPGS. All data is expressed as mean ± SD for n = 3. *P < 0.05, **P < 0.01.
but a dramatic distinction appeared at 120–360 min. As presented in Table 2, the apparent permeation coefficients (Papp) of the T-LPs/Brz was 1.38 folds that of the LPs /Brz (p < 0.05) and 10.2 folds that of AZOPT® (p < 0.001). Precorneal residence time was also studied and displayed in Fig. 3B. At 60 min, there was no significant difference in Brz concentration in tear fluid among the three groups. However, TLPs/Brz achieved the highest drug concentration at 120 min. Compared to AZOPT®, the drug concentration of T-LPs/Brz was 3.18 folds higher (p < 0.05) at 180 min, which indicated that the novel nanoliposomes dramatically prolonged the corneal residence time of Brz and thus improved the drug absorption.
percentage of TPGS. As the molar percent of cholesterol increased, EE% increased at first and then reached a peak (at about 100%) when the L/ D was held constant at a molar ratio of 7:7 (Fig. 1A). The L/D molar ratio was closely related to the encapsulation rate of Brz. As shown in Fig. 1B, 95% EE was achieved when the L/D molar ratio was 10:1. However, EE% did not change significantly with a further increase of the L/D molar ratio to 20:1. In order to get optimal T-LPs/Brz with high EE% and small size, the L/C and L/D molar ratios were set at 7:7 and 10:1, respectively. Furthermore, various amounts of TPGS were introduced to assess the penetration profiles of TPGS modified liposomes. Fig. 1C displayed that 1% TPGS (w/w) in T-LPs/Brz was enough to achieve the strongest permeation of Brz. As such, the optimal process for preparing T-LPs/Brz was determined to be a L/C molar ratio of 7:7 and a L/D molar ratio of 10:1 with a concentration of 1% TPGS (w/w).
5.4. Intraocular pressure lowering effect It was reported that 15% or higher average reduction in IOP was considered as effective in IOP control (Akman et al., 2005). IOP in both eyes of rabbits was monitored for 12 h. The maximal IOP reduction (up to 35.17%) was achieved 4 h after instillation of T-LPs/Brz, while LPs/ Brz showed the maximal IOP reduction of 28.97% 5 h after administration. As seem in Fig. 4, T-LPs/Brz caused 26.38% reduction of IOP 2 h after administration. Whereas, only a 21.19% and 24.87% IOP reduction was in AZOPT® and LPs/Brz groups, respectively. T-LPs/Brz, similar to LPs/Brz, displayed an effective IOP reduction for about 11 h. In contrast, AZOPT® maintained an effective IOP reduction only over a period of 6 h. In summary, T-LPs/Brz showed significantly lower IOP than the control groups after treatment for 4–10 h. These results clearly demonstrated that T-LPs/Brz were the most effective prepared treatment for IOP reduction.
5.2. Pharmaceutical properties The optimum T-LPs/Brz presented an EE% of 95.41 ± 3.03% (n = 3), DL% of 4.00 ± 0.13% (n = 3) and a mean particle size of 96.87 ± 4.43 nm (n = 3) (Fig. 2A) with a narrow size distribution (the polydispersity index was 0.188 ± 0.011, n = 3). The zeta potential value was about neutral, with the value of -1.17 ± 1.91 mV (n = 3). No significant difference in EE, size, and zeta potential among the three batches of T-LPs/Brz was observed, indicating a reproducible preparation process. As shown in Fig. 2B, T-LPs/Brz had slightly blue opalescence and evident Tyndall effect. The morphology of the T-LPs/Brz was observed by TEM. Most of particles were nearly spherical in shape with an average diameter of approximate 90 nm (Fig. 2C). This was in accordance with the results obtained by photon correlation spectroscopy using a Zetasizer Nano ZS90. Moreover, despite the small absolute value of the zeta potential, our preliminary stability study proved that the T-LPs/Brz were stable for at least 10 days at 4 °C and 25 °C (Fig. 2D). A significant improvement over LPs/Brz which are only physically stable for 3 days at 4 °C (Li et al., 2016a). The in vitro release profile of T-LPs/Brz was illustrated in Fig. 2E, which differed from Brz or LPs/Brz. T-LPs/Brz displayed no burst release in the first 1 h. Furthermore, compared to Brz and the LPs/Brz, TLPs/Brz delayed the drug release to a large extent. Interestingly, AZOPT® also exhibited the same slow release properties as T-LPs/Brz for the first 4 h. The release rate of T-LPs/Brz was significantly lower than that of AZOPT®, Brz and LPs/Brz after 4 h, which demonstrated the better sustained-release properties of T-LPs/Brz.
5.5. Preliminary safety evaluation The development of an effective and non-toxic treatment strategy is still a challenging task for ocular drug delivery. In this study, an MTT assay was employed to test the influence of different formulations on the metabolic activity of L929 cells (Bauer et al., 2012; Luo et al., 2016). As a standard assay of cell viability, this method was based on the colorimetric analyses of living cells. As shown in Fig. 5A, T-LPs/Brz, LPs/Brz, Brz, T-LPs and LPs, had no apparent influence on cell viability (near 100%) of L929 cells even with extremely high concentrations of Brz of up to 1000 μg/mL. This study leads to the conclusion that the nanoliposomes could be non-toxic as eye drops. HL (Corneal hydration levels) is a parameter frequently used to evaluate cornea damage. Normal HL of the cornea is between 75% and 80%. Change in this level demonstrates damage to the epithelium and/ or endothelium (Vega et al., 2008). As seen in Table 3, the HL of the TLPs/Brz was 78.56%, indicating that the T-LPs/Brz are safe for corneal tissue. To examine the ocular tolerability of T-LPs/Brz, an eye irritation experiment was performed by using the Draize test in rabbit eyes (Li et al., 2013). The in vivo results were summarized in Table 4. AZOPT®
5.3. Ex vivo corneal permeation A corneal penetration study was carried out to evaluate the effect of T-LPs/Brz on the drug transcorneal transportation. As shown in Fig. 3A, the transcorneal drug permeation of the T-LPs/Brz was 2 times and 5 times higher than LPs/Brz and AZOPT®, respectively. Initially, there was no significant difference among AZOPT®, LPs/Brz and T-LPs/Brz, 24
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Fig. 2. Characterization of T-LPs/Brz. A, the size and Zeta Potential of T-LPs/Brz; B, the appearance of T-LPs/Brz demonstrating obvious Tyndall effect; C, Transmission electron microscopy (TEM) image of T-LPs/Brz. Scale bar, 100 nm; D, the stability of T-LPs/Brz in 4 °C (left) and 25 °C (right); E, Release profile of TLPs/Brz at 37 °C in STF. All data is expressed as mean ± SD for n = 3.
nanoliposomes encapsulating Brz for glaucoma therapy. The therapeutic effects and preliminary safety profile of the T-LPs/Brz was investigated to assess the potential application of this new preparation of Brz as eye drops. T-LPs/Brz with good EE and particle size were successfully prepared after optimization of processing factors including L/C molar ratio, L/D molar ratio and TPGS content. As presented in Fig. 1, the mean size and EE were influenced significantly by the content of lipid and cholesterol. It is well known that cholesterol reduces the flexibility of membranes in the preparation process (Hathout et al., 2007; Kirby et al., 1980; Senior and Gregoriadis, 1982). Excessive cholesterol content hinders Brz uptake into the phospholipid bilayer space, resulting in reduced encapsulation efficiency. Moreover, a large amount of cholesterol might also compete with Brz for the hydrophobic space inside the
caused relatively severe eye irritation including conjunctival congestion, corneal opacification and secretions. While T-LPs/Brz, similar to LPs/Brz, only resulted in small amounts of conjunctival inflammation and no negative clinical signs such as corneal opacity and conjunctival redness or abnormality. This demonstrates that the T-LPs/Brz have little short or long term irritant effects in rabbits. The results of a histological analysis using a light microscope were shown in Fig. 5B. The corneal epithelium of the rabbits treated by TLPs/Brz retained an intact structure, providing further proof that T-LPs/ Brz caused no eye irritation.
6. Discussion The goal of this study was to construct TPGS modified 25
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Fig. 3. Corneal permeation of T-LPs/Brz in vitro. A, drug permeation of T-LPs/Brz at different times, B. Brz concentration in tear fluid at different times. All data is expressed as mean ± SD for n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. Table 2 The apparent permeation coefficients (Papp). Papp(*10−6cm/s) T-LPs/Brz LPs/Brz AZOPT®
3.55 ± 0.12*** 2.58 ± 0.04*** 0.35 ± 0.01
&
Date are mean ± SD. *** P < 0.001 versus AZOPT® group. & P < 0.05 versus LPs/Brz group.
nanoliposomes. Therefore, further increasing the cholesterol content (when the L/C molar ratio was set at 7:14) did not enhance Brz entrapment. Fig. 1A showed that more lipids resulted in a bigger size. This is probably due to incomplete hydration of the lipid film caused by the high viscosity of lipids (Mourtas et al., 2009). The amount of TPGS was also optimized to enhance the permeation of Brz. T-LPs/Brz exhibited the strongest corneal penetration when the concentration of TPGS was 1% (w/w) and it was observed that, more TPGS cannot further improve drug permeation. Furthermore, it has been reported that P-gp expression in rabbit corneas inhibits drug penetration (Dey et al., 2003). A 1% (w/w) concentration of TPGS is enough to inhibit P-gp expression, and as a result, leading to improved permeation of Brz. It is known that high concentrations of nonionic surfactants are not advised for ophthalmic formulations (Mu et al., 2005). Therefore, the optimal amount of TPGS in T-LPs/Brz was set at 1% (w/w) in this study. The size of the optimized T-LPs/Brz was < 100 nm with a slightly negative surface charge, which probably resulted from the phosphatidylcholine head group on the outer nanoliposome surface (Ascenso et al., 2013). The introduction of TPGS might contribute to improved stability of the T-LPs/Brz when compared with LPs/Brz at 4 °C and 25 °C. Vitamin E succinate and PEG, two parts of TPGS, each provide large steric hindrance and a hydrophilic chain on the surface of the TLPs/Brz, respectively. This can prevent aggregation of the nanoliposomes (Guo et al., 2013). Despite the relative improvement in stability, the T-LPs/Brz are still only stable for < 20 days. Thus, freezing the preparation should be further investigated for longer-term storage of TLPs/Brz. T-LPs/Brz displayed a better sustained release profile when compared with LPs/Brz. There was no burst release of Brz in T-LPs/Brz in the first 1 h. Which is indicative of complete entrapment of Brz in TLPs/Brz without any drug free in solution or adherent on the liposome surface. It has been reported that TPGS affects the release behavior of nanoparticle drug delivery systems (Li et al., 2016b). This makes the Brz slowly diffuse from the liposome vehicle which contains TPGS and phospholipids. Further slowing the release rate of Brz is the hydrophilic PEG chains in the TPGS on the surface of the T-LPs/Brz. These chains retard the release of the hydrophobic Brz into the diffusion medium (Raju et al., 2013). As such, T-LPs/Brz released < 30% of their Brz after
Fig. 4. The IOP reduction of white New Zealand rabbits treated by T-LPs/Brz, LPs/Brz and AZOPT®. All data is expressed as mean ± SD for n = 6. * P < 0.05, **P < 0.01, ***P < 0.001.
26
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Fig. 5. Preliminary Safety Evaluation of the T-LPs/Brz. A, cell viability at different concentrations treated with different formulations, namely LPs, TPGS-LPs, Brz, LPs/Brz and T-LPs/Brz; B, representative H&E images (200X) of the cornea of New Zealand rabbits treated with PBS(Control), AZOPT®, LPs/Brz and Brz-TPGS-LPs.
biomechanical and biochemical features, and conjunctiva cavity volume (York and Steiling, 1998). As expected, T-LPs/Brz exhibited enhanced and sustained IOP reduction effect when compared with LPs/ Brz and AZOPT® as shown in Fig. 4. The promising efficacy of T-LPs/Brz on glaucoma treatment may be mainly attributed to the improved precorneal retention and transcorneal transportation. The effective inhibition of P-gp by TPGS can avoid clearance in the cornea and promote quick drug penetration and cornea absorption (Ostacolo et al., 2013; Wempe et al., 2009). Moreover, the sustained-release profile of the TLPs/Brz is important for glaucoma treatment due to the maintenance of a constant Brz concentration in the cornea (Feng et al., 2007). Ocular tolerability is vital to the potential application for novel eye preparations. In this study, several experiments were employed to investigate the preliminary safety of T-LPs/Brz. The in vitro cytotoxicity test showed that T-LPs/Brz were non-toxic on L929 cells, because both the vector T-LPs and drug Brz were safe. Brz can be absorbed systemically after topical administration. Excessive concentration of Brz in the circulatory system may cause side effects of sulfonamides. But due to the exist of the blood-ocular barrier (Maurice, 1984), the amount of the Brz into the circulatory system through local administration is even less. In view of this, we speculate that its biodistribution may not affect the safety of the formulation. Eye irritation experiments and histological analysis demonstrated that T-LPs/Brz had no long or short-term irritant effects. Consistent with literature (Khalil et al., 2017; Lajunen et al., 2016), T-LPs/Brz with nearly neutral surface charge and small size do not induce eye inflammation. Generally, rabbit eyes are more susceptible to extraneous substances than human’s, it is therefore reasonable to believe that T-LPs/Brz possess excellent ocular tolerability.
Table 3 The HLs of AZOPT®, LPs/Brz and T-LPs/Brz. HL (%) AZOPT® LPs/Brz T-LPs/Brz
80.23 ± 2.20 78.35 ± 2.35 78.56 ± 1.10
Table 4 The results of the eye irritation score. The degree of stimulation
Cornea Iris Conjunctival Edema Secretions Sum score
Physiological saline
AZOPT®
LPs/Brz
T-LPs/Brz
0 0 0 0 0 0
2 0 1 0 2 5
0 0 1 1 0 2
0 0 1 1 0 2
1 h. This sustained release performance might be conducive to the retention in the cornea and thus provide potent and prolonged therapeutic efficacy. Although it looks like the plateau phase appeared when Brz was released around 80% out of TLPs/Brz, a slight increase of the percentage of Brz release can be observed. So it can be inferred that 100% release of Brz out of TLPs/Brz might be achieved if the time was prolonged. Drug trans-corneal transportation and precorneal retention are widely used to evaluate ophthalmic drug delivery systems, providing vital information which help to predict the bioactivity of eye drops. Both human and rabbit corneas express P-gp (Katragadda et al., 2006), which is an ATP-dependent efflux pump and can actively remove previously absorbed drugs (Warsi et al., 2014). TPGS was reported to interact with P-gp and inhibit its efflux function of exogenous drugs (Ostacolo et al., 2013; Wempe et al., 2009). In this study, T-LPs/ Brz enhanced Brz permeation and improved Brz concentration in tear fluid (as shown in Fig. 3 and Table 2) in contrast to LPs/Brz. This effect is probably due to the TPGS in T-LPs/Brz efficiently inhibiting the efflux of Brz mediated by P-gp. Moreover, T-LPs/Brz can release Brz slowly and protect Brz against drainage through tears, hence enhancing the precorneal retention and trans-corneal transportation. New Zealand rabbits were selected as model animals to evaluate the efficacy of IOP reduction and safety of T-LPs/Brz in this study, because they have relatively large eyes, sharing many anatomical features with humans such as eyeball size, internal structure, optical system,
7. Conclusions We successfully developed an innovative ocular delivery system (TLPs) which can efficiently incorporate hydrophobic molecules (Brz) and improve their therapeutic efficacy. Brz is entrapped by T-LPs to form novel nanoliposomes (T-LPs/Brz) with high efficiency by a simple preparation method. T-LPs/Brz with a nearly-neutral surface charge displayed a better sustained release performance and storage stability when compared with LPs/Brz. Ex vivo corneal transport experiments demonstrated that T-LPs/Brz improve the penetration of Brz across the cornea to a large extent. In vivo experiments found that T-LPs/Brz achieved extended pre-corneal retention of Brz and prolonged reduction of IOP with high efficacy. Moreover, no eye irritation or lesions were observed in healthy white rabbits. Cumulatively, these results support the conclusion that TPGS modified nanoliposomes are an 27
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effective delivery system for Brz to treat glaucoma.
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