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In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis
Journal Pre-proof In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis Wenying Ran, Haidong Ma, Miao Li PII:
S0022-3549(20)30081-2
DOI:
https://doi.org/10.1016/j.xphs.2020.02.008
Reference:
XPHS 1877
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 7 January 2020 Revised Date:
3 February 2020
Accepted Date: 11 February 2020
Please cite this article as: Ran W, Ma H, Li M, In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2020.02.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis Wenying Ran a, Haidong Ma b, Miao Li * c a
Department of Ophthalmology, Zhengzhou Central Hospital Affiliated to Zhengzhou
University, Zhengzhou City, Henan Province, 450007, China b
Department of Ophthalmology, Jiaoyu Ophthalmology Clinic, Xingqing South Road,
Southeast Gate of Xi'an Jiaotong University, Beilin District, Xi'an, Shaanxi, 710049, China c
Department of Ophthalmology, Xi'an International Medical Center, 50 meters east of
Xitai First-class Highway, Chang'an District, Xi'an, Shaanxi, 710100, China
* Corresponding author at: Department of Ophthalmology, Xi'an International Medical Center, 50 meters east of Xitai First-class Highway, Chang'an District, Xi'an, Shaanxi, 710100, China
Tel.: +86-029-68301138 E-mail address: [email protected] (M. Li)
Abstract Currently, conjunctivitis is treated by frequent high dose administration of sparfloxacin eye drop solution. However, the eye drops are inconvenient due to low bioavailability, short ocular drug residence time and need frequent instillation, which lead to patient non-compliance affecting the routine life style of patients. Silicone contact lenses can be used to sustain the release of sparfloxacin. However, the presence of sparfloxacin alters the optical and physical properties of the contact lens. To overcome the issues, a novel PVP (polyvinyl pyrrolidone) coated sparfloxacin laden ring contact lens was designed to provide sustained ocular drug delivery without altering the optical and swelling properties of contact lens. The ring was implanted within the periphery of the lens. Sparfloxacin was loaded by soaking (Sp-S), direct loading (Sp-L) and ring casting method (Sp-R). PVP (comfort agent) was coated on the surface of contact lens by novel short surface curing technique. The in vitro sparfloxacin release data of Sp-S (up to 12-36 h) and Sp-L batches (up to 12-24 h) showed high burst release, while Sp-R batch showed sustained release up to 36-48 h without significant (p> 0.05) alteration of the optical and swelling properties. All the batches showed sustained release of PVP up to 48 h. The in vivo release studies in the rabbit tear fluid showed improvement in the sparfloxacin [>MIC for Staphylococcus aureus] and PVP retention time in comparison to eye drop solution. The in vivo efficacy study in the staphylococcus aureus induced conjunctivitis showed improved healing effect with the single PVP coated Sp-R-300 contact lens in comparison to the frequent high dose sparfloxacin eye drop therapy. The study demonstrated the successful application to codeliver sparfloxacin and PVP from the contact lens for the extended period of time to treat conjunctivitis.
Key words: Sparfloxacin, polyvinyl pyrrolidone, ring contact lens, conjunctivitis, animal studies.
1. Introduction Currently, allergic conjunctivitis is managed by multiple administration of eye drop solution, which shows poor ocular bioavailability (< 5%) due to short drug residence time [1-5]. The frequent dosing (4 times/day) affect the routine life style of patients [6-8]. Therefore, a novel approach/medical device is needed to increase the ocular-drug residence time via sustained drug delivery system. The use of contact lens to sustain/control the release of ocular drugs appear to be natural choice because they are versatile, biocompatible, and economic [9-11]. The therapeutic contact lens showed many fold higher drug residence time in the post lens tear film in comparison to eye drop solution [12, 13]. Scientist proposed the application of soaking method, drug-laden nanoparticles, imprinting, drug-polymeric film, and supercritical fluid to load ophthalmic drugs in the matrix of contact lens to achieve sustained drug delivery [19-26]. However, many issues like high burst release, changes in the optical and swelling properties due to the presence of drug in the matrix of contact lens are yet to address [14, 15]. M. Patel et al., 2017 fabricated timolol loaded nanofibers by electrospinning method, which was coated on the external surface of contact lens with permeation enhancers. The system showed burst release (> 85%) within 24 h [16]. Viviana P. Costa et al., 2010 loaded acetazolamide and timolol in the silicon contact lenses by discontinuous supercritical impregnation technology. The system showed low drug loading in the core of the contact lens and failed to sustain the release of drug [17]. C-Chung et al., 2005 soaked contact lenses in the timolol soaking solution to increase the drug retention time, however, the flux data showed burst release of timolol in the initial hours [18]. Jinku X et al., 2010 developed puerarin loaded β-cyclodextrin contact lens (p-HEMA based), which showed increase in swelling index and tensile strength with high burst release [19]. Maryam Shayani et al., casted ciprofloxacin and betamethasone loaded extended wear contact lens, which result in significant reduction in the water content due to the inclusion of vitamin E [20]. Hyun J. et al., 2013 developed timolol-propoxylated glyceryl triacylate nanoparticles laden silicon contact lenses to deliver timolol for one month. However, the critical lens properties were altered due to the presence of nanoparticles [21]. Sivanaga S. et al., 2009 casted pilocarpine-contact lenses using poly(ethylene glycol) copolymer with multiple thiol groups. The data indicate that, increase in the
copolymer concentration decreases the optical and swelling properties of contact lenses [22]. Derya G et al., 2005 observed lost in the optical transmittance due to aggregation of drug-microemulsion in the contact lens [23]. Similar observation was noted in lidocaine contact lens [24], timolol contact lens [25], polymyxin contact lens [26] and levofloxacin contact lens [27]. Ciolino et al., 2014 casted latanoprost loaded film in the contact lens by spin casting method, the system showed sustained release for one month. However, before the clinical studies the critical lens properties of the contact lens need to be addressed [28, 29]. The literature showed that the therapeutic contact lenses can be used to sustain the release of ophthalmic drugs, however, the issues like high burst release, changes in the optical and swelling properties of the contact lens, and pink eye syndrome need to be addressed [30-32]. In this paper, we have designed a PVP coated drug-ring contact lens to sustain the release of sparfloxacin and PVP without altering the optical and swelling properties of contact lenses. The shape of sparfloxacin loaded ring will bypass the changes in the optical property for clear vision, while the swelling property will not be affected as the ring occupies the limited area of the contact lens. The PVP coating over the surface of contact lens will provide comfort to the patient eye, avoiding pink eye syndrome. Thus, the novel PVP coated sparfloxacin laden ring contact lens will provide a better option to treat conjunctivitis without affecting the critical lens properties.
2. Materials and Methods 2.1. Materials Sparfloxacin (Sp) was procured from Chengdu Brilliant Pharmaceutical Co., Ltd. (Sichuan Sheng, China). Polyvinyl pyrrolidone (PVP-K30), hydroxyl ethylmethacrylate (HEMA), ethyleneglycol dimethacrylate (EGDMA), Darocur®, and 3-[Tris(trimethylsiloxy) silyl] propyl methacrylate (siloxane) were bought from Sigma-Aldrich Chemicals (MO, USA). All other reagents were bought from Sigma-Aldrich Chemical (MO, USA).
2.2. Fabrication of the contact lenses 2.2.1. Fabrication of the contact lenses for soaking method The conventional soaking method (Sp-S) was performed and compared with the direct sparfloxacin loading method (Sp-L) and sparfloxacin-ring casting method (Sp-R). To fabricate contact lenses for soaking method, the excess monomer mixture composed of EGDMA (10 µL), Darocur® (20 mg), siloxane (100 µL), and HEMA (up to 1 ml) was pipetted in the female mold and male mold was joined [33, 34]. The mold was transferred to Ultraviolet transilluminator and polymerized (350-380 nm) for 15 minutes. The fabricated contact lens was removed from the mold and extracted using 50 ml of boiling water for 30 minutes to remove the unreacted monomers. The soaking method was performed by soaking the extracted contact lens in the simulated tear fluid (STF, 2 ml, pH 7.4) with 0.5 % w/v PVP concentration (fixed based on the preliminary data for 2 day release) and varying concentration of sparfloxacin [2 mg/ml, 4 mg/ml and 6 mg/ml coded as Sp-S-2, Sp-S-4 and Sp-S-6 respectively]. The contact lenses were sterilized using autoclaved (121oC, 15 psi for 30 minutes) in their respective soaking solution (PVP and sparfloxacin) and thereafter soaked for a period of 7 days [35], thus the drug is present in the solubilized state in the contact lens matrix. After 7 days of soaking, the PVP was coated on the surface by short curing process discussed in section 2.2.4. The contact lenses (supplementary material part I, Fig. S-1, A) were blotted to remove the excess of soaking solution and were subjected for evaluation studies. The concentration of sparfloxacin soaking solution was selected based on the preliminary data to load > 150 µg of sparfloxacin in the contact lens equivalent to one drop of 0.3 % w/v sparfloxacin eyed drop (Zospar, FDC Ltd.) solution (1 drop ≈ 50 µL ≈ 150 µg sparfloxacin).
2.2.2. Fabrication of the direct sparfloxacin loaded contact lenses The PVP coated direct sparfloxacin-loaded contact lenses were fabricated by adding the required quantity of sparfloxacin (6.66 mg/ml, 10 mg/ml and 13.33 mg/ml coded as Sp-L-100, Sp-L-150 and Sp-L-200 respectively) directly in the monomer mixtures (up to 1 ml monomer mixture) to achieve 100 µg, 150 µg and 200 µg sparfloxacin/15 mg contact lens respectively [34]. The direct sparfloxacin-laden contact lenses were
fabricated by using above sparfloxacin-monomer mixture and plastic lens molds. The fabrication process is same as discussed in section 2.2.1. The direct sparfloxacinloaded contact lenses (supplementary material Part I, Fig. S-1, B) were removed and extracted using 50 ml of boiling water for 30 minutes to remove the unreacted monomers, followed by sterilization in 2 ml STF containing 0.5 % w/v PVP. The drug is present in their microparticles forms in the matrix of the contact lens. The PVP was coated by soaking method and surface curing as discussed in section 2.2.4. The amount of sparfloxacin leached during the extraction and sterilization steps were quantified by HPLC method at 292 nm after suitable dilution [36, 37].
2.2.3. Fabrication of sparfloxacin laden ring contact lenses The sparfloxacin laden ring was fabricated at two different doses of 150 µg (coded as Sp-R-150) and 300 µg (coded as Sp-R-300) sparfloxacin. Briefly, the ring (5 mg weight) of 50 µm thickness was casted using sparfloxacin-monomer mixture solution. The sparfloxacin-monomer mixture was pipetted between the glass slides separated by Teflon spacer (50 µm), followed by the exposure to Ultraviolet transilluminiator (360-370 nm) for 15 minutes [38]. The sparfloxacin laden sheet was cut into a ring shaped ring (5 mg weight, 9 mm outer diameter and 6 mm inner diameter) using a borer. The sparfloxacin ring contact lenses were casted using plastic mould (14.2 mm outer diameter and 6.5 mm base curve). The ring was placed in the female mold cavity, followed by addition of excess monomer mixture (blank contact lens). The male mold was joined and transferred to the Ultraviolet transilluminator for 30 minutes curing. The ring implanted contact lens was removed and processed for extraction and sterilization. The drug is present in their microparticles forms in the matrix of contact lens. The PVP was loaded by soaking method and surface curing (discussed in section 2.2.4). The amount of sparfloxacin leached during the extraction and sterilization steps was quantified by HPLC method. The optical transmittance of the ring laden contact lens was not altered as the ring was placed in the periphery of the lens away from the center for clear vision (supplementary material Part I, Fig. S-1, C).
2.2.4. PVP loading on the sparfloxacin loaded contact lenses The PVP was loaded by soaking sparfloxacin loaded contact lenses (all the three batches) in 0.5 % w/v PVP concentration for 7 days followed by coating via Ultraviolet transilluminator for 30 seconds. The short exposure of UV light cause weak polymerization of PVP on the surface of the contact lens, to form a layer/coating of PVP. The process will retard the release of PVP from the contact lens, to provide comfort for prolong period of time. The above method showed high PVP release for prolong period of time, in comparison to the conventional PVP soaking method (see supplementary material Part I, Fig S-2).
2.3. Characterization of the sparfloxacin-PVP coated contact lenses 2.3.1. Swelling study The dry contact lenses after curing were removed from the molds and accurately weighed (dry weight = W D). The dry contact lenses were hydrated (extraction and sterilization) using their respective packaging/soaking solutions, thereafter blotted using filter paper and weighed (W S) again. The percentage swelling of the contact lens was determined by the formula [37]: % Swelling =
−
× 100
2.3.2. Optical transparency The sparfloxacin-PVP loading in the contact lens should not alter the optical transmittance of the contact lens. A UV-vis spectrophotometer was used to study the optical transmittance of the control contact lens and sparfloxacin-PVP loaded contact lenses. The hydrated contact lens (center portion) were fixed on the inner surface of the quartz cuvette containing 2 ml of distilled water and the optical transmittance of the centre portion of the contact lens was measured at 610 nm. The experiment was repeated three times.
2.3.3. Quantification of the sparfloxacin and PVP loaded in the contact lenses The amount of sparfloxacin and PVP present in the contact lens was quantified by HPLC and colorimetric method [39] respectively. The contact lenses were shifted to an
individual glass vial (screw capped) containing 25 ml of water to extract sparfloxacin and PVP. The glass vials were agitated at 100 RPM for 10 days at room temperature, followed by analysis of drugs in water by developed and validated analysis method. All readings were recorded in triplicate.
2.4. In vitro release study The in vitro flux studies of the PVP coated Sp-S, Sp-L and Sp-R contact lenses were carried out by immersing the lens in the glass vial containing 2 ml of STF at 34oC with constant stirring (100 RPM) [40]. At the pre-determined time intervals, 2 ml of STF was withdrawn from the vial and replaced with the same volume of fresh STF to maintain the prefect sink condition. The amount of sparfloxacin and PVP released in the STF was quantified by HPLC and colorimetric method [41] respectively. The procedure was continued till no release of the drug concentration was observed in the two successive measurements. All the readings were recorded in triplicate.
2.5. Effect of packaging solution (Shelf life study) The shelf life study of selected Sp-S-4, Sp-L-200, and Sp-R-300 batches were performed to estimate the effect of time on in vitro sparfloxacin release profile and leaching of sparfloxacin in the packaging solution. The contact lenses after 3 months (stored at room temperature) were analyzed for in vitro sparfloxacin release kinetics and the sparfloxacin leaching in the STF was quantified by HPLC. The preliminary shelf life data of in vitro PVP release, suggest no significant change in the PVP release kinetic with time, which was expected (data not shown here).
2.6. Animal studies The study protocol for white New Zealand rabbits were approved by Xi'an International Medical Center (2019-1118).
2.6.1. In-vivo drug release study The New Zealand rabbits (male and female) were used to investigate the release profiles of sparfloxacin and PVP in the tear fluid from the selected Sp-R-300 contact
lens [128.9 ± 3.42 µg sparfloxacin (cumulative release) and 35.31 ± 1.47 µg PVP] contrasting with the eye drop solution [0.3 % w/v sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)] [42]. The sterile contact lens was placed on the right eye (n=6) of rabbits (left eye was kept control) without local or general anaesthesia. In case of the eye drop group, the rabbit’s right eye received single drop of sparfloxacin eye drop, followed by Protear eye drop (same time). The left eye was kept control. The rabbit tear fluid was collected using disposable glass capillary from the cul de sac and preserved at -20◦C until analysis. The drug-tear fluid samples were treated with 1 ml of methanol to precipitate proteins, followed by freeze (5oC) centrifugation (Remi freeze-centrifuge) for 1 h at 8000 RPM. The collected supernatant was analyzed for sparfloxacin and PVP by HPLC and colorimetric method respectively. Rabbits were euthanized at the end of the study for histopathological analysis. The eyes were enucleated and fixed in 10% formalin buffered solution. The paraffin embedded corneas were cut into sections using microtome and stained using hematoxylin. The structure of cornea including basement membrane, epithelium and stroma were investigated using light microscopy at ‘×400 magnification’ [43].
2.6.2. Efficacy study The efficacy of the PVP-Sp-R-300 contact lens was evaluated using the New Zealand white rabbits, and was compared with the conventional eye drop therapy. The conjunctivitis was induced using young culture of the Staphylococcus aureus (ATCC 25923, 100 CFU/ml) [44]. Rabbits’ eyes were examined using portable slit lamp prior to the induction of the conjunctivitis to exclude any abnormalities. The study includes 4 groups (n=6 rabbits in each group) and both the eyes of rabbits were utilized for the study. Group 1: Uninfected and untreated (i.e. conjunctivitis was not induced) Group 2: Conjunctivitis was induced and treated with sterile saline (0.9 %w/v NaCl) solution (Negative control). Group 3: Conjunctivitis was induced and treated with commercial eye drop solution containing (0.3 % w/v, Spar eye drop, Cipla) sparfloxacin (Positive control).
Group 4: Conjunctivitis was induced and treated with contact lenses (PVP-Sp-R-300, 129 µg loading of sparfloxacin, Test group).
In this study, the group 1 rabbits were caged and neither the conjunctivitis was induced nor they were treated with any of the formulation; while the conjunctivitis was induced in both the eyes of group 2 to 4 using the young culture of the staphylococcus aureus. The 50 µl of culture solution (102 CFU in 10 µL of tryptic soy broth [45]) was gently instilled in the lower conjunctival sac of the rabbit eye and holded for few seconds to ensure the uniform spearing of the bacteria. After 48 h, the rabbits’ eyes showed the symptoms of discharge, chemosis, conjunctival congestion and palpebral fissure, indicating the successful induction of the conjunctivitis. The treatment was started from the day 3, i.e. after successful induction of conjunctivitis. In group 2, the rabbit’s eyes were treated with one drop of the sterile saline solution every 4 h, which served as the negative control group. In the group 3 (positive control group), rabbit’s eyes were treated with one drop of the sparfloxacin eye drop solution (0.3 % w/v) in 4 h interval. The PVP-Sp-R-300 contact lenses (129 µg loading of sparfloxacin) were carefully placed on the cornea of the group 4 (test group) without anaesthesia. The rabbits were kept under observation and symptoms like the discharge, chemosis, conjunctival congestion and palpebral fissure were observed at every 12 h [46]. The treatment was conducted for 4 days after the induction of the conjunctivitis.
2.7. Statistical analysis Statistical analysis was carried out using SPSS 21.0 for Windows. T-test (2 tailed) and One-way analysis of variance (ANOVA) was used to compare the different groups, after confirming the normality and the homogeneity of variance.
3. Results and discussion 3.1. Characterization of the PVP coated sparfloxacin-contact lenses 3.1.1. Swelling study The percentage swelling data of the contact lenses are shown in Table 1. The swelling property of the contact lens has direct relationship with the oxygen and ion
permeability and final dimensions of the contact lens [47-49]. Thus, the presence of drug should not alter the swelling property of the contact lens. The soaked contact lenses [Sp-S] did not showed significant reduction (p>0.05) in the % swelling (88.2 to 68.4 %) in comparison to the blank contact lenses (90.1 %). However, the direct sparfloxacin loaded contact lens [Sp-L] showed proportional reduction (77.9 to 68.5 %) in the swelling property with increased in the level of sparfloxacin inside the matrix structure of the contact lens, which could be due to drug-polymer interaction during curing process. The sparfloxacin laden ring contact lens (Sp-R) did not showed significant (p>0.05) alteration in the % swelling in comparison to the blank contact lens, as the ring occupied the limited area of the contact lens.
3.1.2. Optical transparency The optical transparency of the contact lenses are shown in table 1. The soaked contact lenses showed > 95 % optical transparency (expect Sp-S-6), while the direct sparfloxacin loaded contact lens [Sp-L] showed proportional reduction (70.4 to 54.6 %) in the optical transparency values with increased in the level of sparfloxacin. Thus, SpS-6 and Sp-L batches cannot be used for therapeutic purpose, as the % optical transparency was < 95%. The optical transparency of the ring-contact lenses (Sp-R) were > 99 %, which was expected as the ring was placed at the periphery of the contact lens for clear vision (supplementary material Part I, Fig S-1, B).
3.1.3. Quantification of the sparfloxacin and PVP loaded in the contact lenses The loading of sparfloxacin and PVP in the contact lenses (supplementary material, Part I, Table S-1) were quantified to investigate the uniform dispersion of drugs in the contact lens and reproducibility of the methodology. The PVP loading was common for all the methodologies/batches. The uptake of PVP from the packaging solution (0.5 % PVP in STF) followed by short curing process showed 32.2 to 35.4 µg of PVP loading, which should be enough to show prolong comfort to the patient eyes during contact lens wear (further discussed in section 3.5). The uptake of sparfloxacin from the drug-soaking solution was found to be 91.7 µg, 106.1 µg and 210.2 µg for Sp-S-2, Sp-S-4 and Sp-S-6 respectively, which was sufficient
to treat conjunctivitis. However, the drug release rate was very high from the contact lens and failed to show sustain release (discussed in section 3.3.1). The loading of sparfloxacin in Sp-L-100, Sp-L-150 and Sp-L-200 batches was found to be 103.7 µg, 151.7 µg and 201.8 µg respectively. The ring contact lens Sp-R-150 and Sp-R-300 shows 151.9 µg and 302.1 µg sparfloxacin loading respectively. The standard deviation values were low enough to confirm the reproducibility of sparfloxacin loading in the contact lenses. Thus, the target dose of single eye drop (0.3 % w/v, 50 µL = 150 µg) was loaded in all the batches.
3.2. Sparfloxacin leached during monomer extraction and sterilization The Sp-L and Sp-R batches were extracted to remove the unreacted monomers from the contact lenses. The data (Table 2) of Sp-L-100, Sp-L-150, and Sp-L-200 batches showed 51.2 µg (49.3 %), 75.3 µg (49.6 %), and 102.1 µg (50.5 %) sparfloxacin leaching during monomer extraction step in boiling water. While, Sp-R-150 and Sp-R-300 batches showed low drug leaching (1.3 fold reduction) of 55.9 µg (36.8 %), and 105.0 µg (34.7 %) respectively. Following monomer extraction step, the contact lenses (Sp-R-150 and Sp-R-300 batches) were sterilized in 2 ml STF containing 0.5 %w/v PVP. The Sp-L-100, Sp-L-150, and Sp-L-200 batches showed 17.6 µg (17.0 %), 28.0 µg (18.5 %), and 36.1 µg (17.9 %) of sparfloxacin lost during sterilization. While, Sp-R-150 and Sp-R-300 batches showed low drug leaching of 22.4 µg (14.7 %), and 39.1 µg (12.9 %) respectively, due to the tight packing of drug in the matrix of ring. Thus, sparfloxacin laden ring contact lens showed better performance in comparison to direct sparfloxacin-laden contact lenses.
3.3. In vitro release studies 3.3.1. In vitro release of sparfloxacin The cumulative release of sparfloxacin from the contact lenses are shown in Fig. 1. The release rate profiles are shown in supplementary material Part I, Fig S-3. The data showed high cumulative release of 90.9 µg, 156.5 µg, and 208.9 µg from Sp-S-2, Sp-S4 and Sp-S-6 batches respectively. The soaked contact lenses do showed sufficient drug uptake due to high solubility of drug in STF. However, the release rate was high
(no drug detected after 24, expect Sp-S-6 batch), making it unsuitable technique to load water soluble drug to produce extended release therapeutic contact lens. In Sp-L batches, the sparfloxacin was directly incorporated in the matrix of the contact lenses during fabrication, which on extraction and sterilization steps showed major loss of drug. The Sp-L-100, Sp-L-150 and Sp-L-200 batches showed 68.9 µg (66.4 %), 103.4 µg (68.1 %), and 138.2 µg (68.5 %) sparfloxacin loss respectively. The cumulative release from direct sparfloxacin-laden contact lenses [Sp-L-100, Sp-L-150 and Sp-L-200 lenses showed 32.29 µg, 40.27 µg, and 52.8 µg respectively] was low, as the major drug was lost during extraction and sterilization. The method failed to improve the release rate kinetics, even with increase in the drug loading amount. This was due to high water solubility of drug in STF, causing high release rate from the aqueous channel of the contact lens matrix. In Sp-R batches, the sparfloxacin was entrapped in the matrix of ring, followed by implantation in the contact lenses. In comparison to Sp-L batches, the Sp-R-150 and Sp-R-300 batches showed relatively low drug leaching of 78.3 µg (51.6 %) and 144.1 µg (47.7 %) respectively, during extraction and sterilization. The Sp-R contact lenses showed sustained release of sparfloxacin up to 36-48 h, in comparison to Sp-S and SpL lenses which showed drug release up to 12-36 h and 24-48 h respectively. The sustained release was due to slow diffusion of drug from the matrix structure of ring, followed by diffusion through the contact lens matrix in the media. The release rate at 24 h from Sp-L contact lenses was in the range of 104 to 171 ng/h, while Sp-R contact lens shows high release rate of 751 to 1235 ng/h. The data clearly indicate the advantage of ring implanted contact lenses in comparison to direct and soaked contact lenses.
3.3.2. In vitro release of PVP The PVP coating on the surface of contact lenses was common for all the batches. The cumulative release of PVP from the selected batches (Sp-S-6, Sp-L-200 and Sp-R300) are shown in Fig. 2. The release rate profiles are shown in supplementary material Part I, Fig S-4. The data indicate initial burst release of 7.7 to 9.3 µg at 1 h, followed by sustained release up to 48 h. The release rate of PVP between 2 to 48 h was 2000 to
100 ng/h. The coated contact lenses showed improved release rate profile in comparison to conventional soaking method (see Supplementary material Part I, Fig S2). The data suggest bonding (due to weak polymerization) of PVP over the surface of the contact lens, resulting in prolong (slow) release. The PVP coat is mechanical less stability, as noted in the in vitro release study. In future studies, we are working to understand the nature of interaction between the coating and the contact lens. The conventional soaking method shows only surface adsorption of PVP which lead to rapid fall in release rate profiles.
3.4. Mathematical models Different mathematical models, i.e., zero order, first order, Higuchi, Korsmeyer– Peppas equation were applied for describing the kinetics of the drug release process from the contact lens, and the most suited being the one which fitted best the experimental results. The drug release patterns were examined according to different mathematical equations from release kinetics theories are shown in supplementary material part-II. The Korsmeyer-Peppas model was found to be best fit, suggesting anomalous transport of drug release mechanism i.e., drug release was controlled by more than one process.
3.4. Effect of packaging solution (Shelf life study) On the first day, the sparfloxacin lost during extraction and sterilization was 138.2 µg (68.5 %) and 144.1 µg (47.7 %) for Sp-L-200 and Sp-R-300 lens respectively. After three months of storage, the contact lenses (Sp-S-6, Sp-L-200 and Sp-R-300) were removed from the packaging solution and analyzed for sparfloxacin (leached) content by HPLC. The data showed further leaching of drug [9.5 µg (9.15 %) and 11.1 µg (7.3 %) from Sp-L-200 and Sp-R-300 lens respectively] in the packaging solution (during three months) suggesting the limitation of therapeutic contact lenses. The in vitro release of sparfloxacin after sterilization (day 1) and after three months of storage are shown in supplementary material Part I, Fig S-5. The soaked contact lenses (Sp-S-6) after three months of storage did not showed significant difference in the release rate profiles (f2 value = 83.43) which was expected, as the lenses were
soaked in the fixed concentration of sparfloxacin. While, a significant drop in the cumulative and release rate profiles were observed with Sp-L-200 lens and Sp-R-300 lenses (f2 value = 49.01) after 3 months. However, statistically the Sp-L-200 lens showed similarity in release rate profile after 3 months (f2 value = 69.0). Thus, it is not advisable to store the lens in wet condition, due to leaching of drug with time in the packaging solution. In future studies, the lenses will be sterilized and investigated by radiation technique (dry method) to overcome the issue of leaching with time.
3.5. In vivo drug release study The in vivo tear fluid analysis was performed using Sp-R-300 contact lens [128.9 ± 3.42 µg sparfloxacin (cumulative release) and 35.31 ± 1.47 µg PVP] and eye drop solution [0.3 %w/v Sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)] to investigate the drug retention pattern. The Cmax (5 minutes) of sparfloxacin was found to be 1210.6 µg/ml and 842.3 µg/ml for eye drop solution and Sp-R-300 contact lens respectively (Fig. 3). The eye drop solution showed rapid fall in drug concentration (up to 1 h), while Sp-R-300 contact lens showed improvement in drug retention up to 24 h. The drug was not detected later due to limited LOQ of HPLC. The Cmax (5 minutes) of PVP was found to be 244.9 µg/ml and 133.3 µg/ml for eye drop solution and Sp-R-300 contact lens respectively (Fig. 4). The eye drop solution showed rapid fall in PVP concentration (up to 1 h), while Sp-R300 contact lens showed major improvement in drug retention up to 24 h with even 8.5 fold lower dose. The contact lens showed sustained drug release and high drug-tear fluid concentration in comparison to eye drop solution, thus the single contact lens will be enough to treat allergic conjunctivitis. The data of in vivo % cumulative drug retained in the tear fluid and in vitro % cumulative drug released was plotted to investigate in vitro-in vivo correlation (IVIVC, Levy plot, level A, supplementary material Part I, Fig S-6). The R2 (correlation coefficient) value for sparfloxacin and PVP was found to be 0.696 and 0.764 respectively. The poor IVIVC was due to binding of drug to lipid (in tear), which could enhance the solubility of drug in tear causing high in vivo initial release. The corneal histopathological images of control and Sp-R-300 contact lens (supplementary material,
Part I, Fig S-7) showed normal squamous epithelium cells with normal pattern of collagen fibers in the stroma with no obvious changes. However, a long term three weeks data is needed to assure the safety of contact lens for clinical use.
3.6. Efficacy study The conjunctivitis was successfully induced in the rabbit eyes after 48 h using the young culture of the Staphylococcus aureus, as confirmed by the symptoms of conjunctival edema of the eyelids, beefy red conjunctiva and the swollen eye lids. No significant difference was observed in the symptoms among the group 2, 3 and 4. On day 3, the treatment with eye drop and the contact lens was started in the group 3 and 4 respectively. After 24 h of treatment, i.e. on the 4th day, the discharge was reduced in the group 4, while no improvement was observed in the group 2 (saline treated) and the group 3 (eye drop treatment). On the 5th day, the inflammatory symptoms in the group 3 and 4 was significantly reduced in comparison to the group 2 (Negative control). On the third day of the treatment, i.e. 6th day, the rabbits’ eye in the group 3 and 4 showed no discharge along with the minimal congestion and edema. The eyes were completely cured on the 4th day of the treatment in the group 3 and 4 (supplementary material Part I, Fig. S-8). While the inflammatory symptom still persisted in the group 2. The outcome of the study suggests that, a single low dose (128 µg of sparfloxacin) implant contact lens was as effective as the frequent high dose eye drop therapy.
4. Conclusion The study demonstrate the successful simultaneous delivery of sparfloxacin and PVP form the novel PVP coated sparfloxacin laden ring contact lens (Sp-R) without affecting the critical lens properties. The soaked contact lenses (Sp-S) did not alter the optical and swelling properties, however, the release rate was very high. The direct sparfloxacin laden contact lens (Sp-L) showed reduction in the optical and swelling properties. The ring contact lens did not altered the optical and swelling properties, as ring covered the limited area of lens. The sparfloxacin lost during the extraction and sterilization from Sp-L lens was 66-68 %, while a significant reduction was observed with Sp-R lens (47-51 %). The flux data showed sustained release of sparfloxacin in
following order: Sp-L (12-24 h) > Sp-S (12-36 h) > Sp-R (36-48 h). The sustained release of sparfloxacin from the Sp-R lens was achieved due to tight packing of drug in the ring implant. The PVP flux data showed sustained release of PVP up to 48 h. The shelf life study in the packaging solution showed leaching of sparfloxacin from the contact lens. Thus, it is not advisable to store the lens in wet condition. In future studies, the lenses will be sterilized and investigated by radiation technique (dry method) to overcome the issue of leaching with time. Furthermore, intense and in-depth research is needed to further reduce the possible burst release of drug from the dry ring implant contact lens sterilized by radiation technique. The in vivo drug release data in the rabbit tear fluid showed sustained drug release and high drug-tear fluid concentration in comparison to eye drop solution, thus the single contact lens will be enough to treat conjunctivitis. The in vivo efficacy study in the conjunctivitis induced rabbits’ eye showed equivalent healing effect with the single implant contact lens in comparison to the frequent high dose eye drop therapy. The study demonstrated the successful delivery of sparfloxacin and PVP from the ring contact lens for the extended period of time without altering the optical and physical properties of contact lens. The platform technology can be applied for similar other potent antimicrobial agents (BCS class-II) for ocular delivery.
Disclosures There are no potential conflicts of interest to disclose for this work.
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FIGURE CAPTION
Fig. 1. Cumulative release of sparfloxacin from contact lenses. Values are shown as mean ± standard deviations (n=3).
Fig. 2. Cumulative release of PVP from the selected contact lenses (Sp-S-6, Sp-L-200 and Sp-R-300). Values are shown as mean ± standard deviations (n=3).
Fig. 3. Sparfloxacin tear fluid concentration from Sp-R-300 contact lens [128.9 µg sparfloxacin (cumulative release) and 32.2 µg PVP] and eye drop solution [0.3 %w/v sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)].
Fig. 4. PVP tear fluid concentration from Sp-R-300 contact lens and eye drop solution. Each time point represents the mean ± standard deviation (n = 6).
LIST OF TABLES
Table 1 Percentage swelling and transmittance data. Values are shown as mean ± standard deviations (n = 3). Transmittance (%)
Swelling (%)
99.2 ± 0.2
90.1 ± 1.5
Sp-S-2
97.9 ± 0.4
88.2 ± 1.9
Sp-S-4
96.1 ± 0.7
87.1 ± 1.9
Sp-S-6
86.4 ± 1.8*
86.4 ± 1.8*
Sp-L-100
70.4 ± 2.0**
77.9 ± 3.5**
Sp-L-150
61.6 ± 1.2**
72.1 ± 3.3**
Sp-L-200
54.6 ± 4.1**
68.5 ± 3.5**
Sp-R-150
99.1 ± 0.5
87.7 ± 2.2
Sp-R-300
99.3 ± 0.4
86.2 ± 2.6*
Codes Control contact lens
* indicate significant difference (p<0.05) with control contact lens. ** indicate very significant difference (p<0.01) with control contact lens.
Table 2 Data of sparfloxacin leached during the monomer extraction and sterilization step (Mean ± SD, n=3). Code
Drug Leaching during
Drug Leaching
Total Sparfloxacin
monomer extraction
during sterilization
leached
µg
%
µg
%
µg
%
Sp-L-100
51.2
49.3
17.6
17.0
68.9
66.4
Sp-L-150
75.3
49.6
28.0
18.5
103.4
68.1
Sp-L-200
102.1
50.5
36.1
17.9
138.2
68.5
Sp-R-150
55.9
36.8
22.4
14.7
78.3
51.6
Sp-R-300
105.0
34.7
39.1
12.9
144.1
47.7
S0022-3549(20)30081-2
DOI:
https://doi.org/10.1016/j.xphs.2020.02.008
Reference:
XPHS 1877
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 7 January 2020 Revised Date:
3 February 2020
Accepted Date: 11 February 2020
Please cite this article as: Ran W, Ma H, Li M, In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2020.02.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
In vitro and in vivo studies of polyvinyl pyrrolidone coated sparfloxacin loaded ring contact lens to treat conjunctivitis Wenying Ran a, Haidong Ma b, Miao Li * c a
Department of Ophthalmology, Zhengzhou Central Hospital Affiliated to Zhengzhou
University, Zhengzhou City, Henan Province, 450007, China b
Department of Ophthalmology, Jiaoyu Ophthalmology Clinic, Xingqing South Road,
Southeast Gate of Xi'an Jiaotong University, Beilin District, Xi'an, Shaanxi, 710049, China c
Department of Ophthalmology, Xi'an International Medical Center, 50 meters east of
Xitai First-class Highway, Chang'an District, Xi'an, Shaanxi, 710100, China
* Corresponding author at: Department of Ophthalmology, Xi'an International Medical Center, 50 meters east of Xitai First-class Highway, Chang'an District, Xi'an, Shaanxi, 710100, China
Tel.: +86-029-68301138 E-mail address: [email protected] (M. Li)
Abstract Currently, conjunctivitis is treated by frequent high dose administration of sparfloxacin eye drop solution. However, the eye drops are inconvenient due to low bioavailability, short ocular drug residence time and need frequent instillation, which lead to patient non-compliance affecting the routine life style of patients. Silicone contact lenses can be used to sustain the release of sparfloxacin. However, the presence of sparfloxacin alters the optical and physical properties of the contact lens. To overcome the issues, a novel PVP (polyvinyl pyrrolidone) coated sparfloxacin laden ring contact lens was designed to provide sustained ocular drug delivery without altering the optical and swelling properties of contact lens. The ring was implanted within the periphery of the lens. Sparfloxacin was loaded by soaking (Sp-S), direct loading (Sp-L) and ring casting method (Sp-R). PVP (comfort agent) was coated on the surface of contact lens by novel short surface curing technique. The in vitro sparfloxacin release data of Sp-S (up to 12-36 h) and Sp-L batches (up to 12-24 h) showed high burst release, while Sp-R batch showed sustained release up to 36-48 h without significant (p> 0.05) alteration of the optical and swelling properties. All the batches showed sustained release of PVP up to 48 h. The in vivo release studies in the rabbit tear fluid showed improvement in the sparfloxacin [>MIC for Staphylococcus aureus] and PVP retention time in comparison to eye drop solution. The in vivo efficacy study in the staphylococcus aureus induced conjunctivitis showed improved healing effect with the single PVP coated Sp-R-300 contact lens in comparison to the frequent high dose sparfloxacin eye drop therapy. The study demonstrated the successful application to codeliver sparfloxacin and PVP from the contact lens for the extended period of time to treat conjunctivitis.
Key words: Sparfloxacin, polyvinyl pyrrolidone, ring contact lens, conjunctivitis, animal studies.
1. Introduction Currently, allergic conjunctivitis is managed by multiple administration of eye drop solution, which shows poor ocular bioavailability (< 5%) due to short drug residence time [1-5]. The frequent dosing (4 times/day) affect the routine life style of patients [6-8]. Therefore, a novel approach/medical device is needed to increase the ocular-drug residence time via sustained drug delivery system. The use of contact lens to sustain/control the release of ocular drugs appear to be natural choice because they are versatile, biocompatible, and economic [9-11]. The therapeutic contact lens showed many fold higher drug residence time in the post lens tear film in comparison to eye drop solution [12, 13]. Scientist proposed the application of soaking method, drug-laden nanoparticles, imprinting, drug-polymeric film, and supercritical fluid to load ophthalmic drugs in the matrix of contact lens to achieve sustained drug delivery [19-26]. However, many issues like high burst release, changes in the optical and swelling properties due to the presence of drug in the matrix of contact lens are yet to address [14, 15]. M. Patel et al., 2017 fabricated timolol loaded nanofibers by electrospinning method, which was coated on the external surface of contact lens with permeation enhancers. The system showed burst release (> 85%) within 24 h [16]. Viviana P. Costa et al., 2010 loaded acetazolamide and timolol in the silicon contact lenses by discontinuous supercritical impregnation technology. The system showed low drug loading in the core of the contact lens and failed to sustain the release of drug [17]. C-Chung et al., 2005 soaked contact lenses in the timolol soaking solution to increase the drug retention time, however, the flux data showed burst release of timolol in the initial hours [18]. Jinku X et al., 2010 developed puerarin loaded β-cyclodextrin contact lens (p-HEMA based), which showed increase in swelling index and tensile strength with high burst release [19]. Maryam Shayani et al., casted ciprofloxacin and betamethasone loaded extended wear contact lens, which result in significant reduction in the water content due to the inclusion of vitamin E [20]. Hyun J. et al., 2013 developed timolol-propoxylated glyceryl triacylate nanoparticles laden silicon contact lenses to deliver timolol for one month. However, the critical lens properties were altered due to the presence of nanoparticles [21]. Sivanaga S. et al., 2009 casted pilocarpine-contact lenses using poly(ethylene glycol) copolymer with multiple thiol groups. The data indicate that, increase in the
copolymer concentration decreases the optical and swelling properties of contact lenses [22]. Derya G et al., 2005 observed lost in the optical transmittance due to aggregation of drug-microemulsion in the contact lens [23]. Similar observation was noted in lidocaine contact lens [24], timolol contact lens [25], polymyxin contact lens [26] and levofloxacin contact lens [27]. Ciolino et al., 2014 casted latanoprost loaded film in the contact lens by spin casting method, the system showed sustained release for one month. However, before the clinical studies the critical lens properties of the contact lens need to be addressed [28, 29]. The literature showed that the therapeutic contact lenses can be used to sustain the release of ophthalmic drugs, however, the issues like high burst release, changes in the optical and swelling properties of the contact lens, and pink eye syndrome need to be addressed [30-32]. In this paper, we have designed a PVP coated drug-ring contact lens to sustain the release of sparfloxacin and PVP without altering the optical and swelling properties of contact lenses. The shape of sparfloxacin loaded ring will bypass the changes in the optical property for clear vision, while the swelling property will not be affected as the ring occupies the limited area of the contact lens. The PVP coating over the surface of contact lens will provide comfort to the patient eye, avoiding pink eye syndrome. Thus, the novel PVP coated sparfloxacin laden ring contact lens will provide a better option to treat conjunctivitis without affecting the critical lens properties.
2. Materials and Methods 2.1. Materials Sparfloxacin (Sp) was procured from Chengdu Brilliant Pharmaceutical Co., Ltd. (Sichuan Sheng, China). Polyvinyl pyrrolidone (PVP-K30), hydroxyl ethylmethacrylate (HEMA), ethyleneglycol dimethacrylate (EGDMA), Darocur®, and 3-[Tris(trimethylsiloxy) silyl] propyl methacrylate (siloxane) were bought from Sigma-Aldrich Chemicals (MO, USA). All other reagents were bought from Sigma-Aldrich Chemical (MO, USA).
2.2. Fabrication of the contact lenses 2.2.1. Fabrication of the contact lenses for soaking method The conventional soaking method (Sp-S) was performed and compared with the direct sparfloxacin loading method (Sp-L) and sparfloxacin-ring casting method (Sp-R). To fabricate contact lenses for soaking method, the excess monomer mixture composed of EGDMA (10 µL), Darocur® (20 mg), siloxane (100 µL), and HEMA (up to 1 ml) was pipetted in the female mold and male mold was joined [33, 34]. The mold was transferred to Ultraviolet transilluminator and polymerized (350-380 nm) for 15 minutes. The fabricated contact lens was removed from the mold and extracted using 50 ml of boiling water for 30 minutes to remove the unreacted monomers. The soaking method was performed by soaking the extracted contact lens in the simulated tear fluid (STF, 2 ml, pH 7.4) with 0.5 % w/v PVP concentration (fixed based on the preliminary data for 2 day release) and varying concentration of sparfloxacin [2 mg/ml, 4 mg/ml and 6 mg/ml coded as Sp-S-2, Sp-S-4 and Sp-S-6 respectively]. The contact lenses were sterilized using autoclaved (121oC, 15 psi for 30 minutes) in their respective soaking solution (PVP and sparfloxacin) and thereafter soaked for a period of 7 days [35], thus the drug is present in the solubilized state in the contact lens matrix. After 7 days of soaking, the PVP was coated on the surface by short curing process discussed in section 2.2.4. The contact lenses (supplementary material part I, Fig. S-1, A) were blotted to remove the excess of soaking solution and were subjected for evaluation studies. The concentration of sparfloxacin soaking solution was selected based on the preliminary data to load > 150 µg of sparfloxacin in the contact lens equivalent to one drop of 0.3 % w/v sparfloxacin eyed drop (Zospar, FDC Ltd.) solution (1 drop ≈ 50 µL ≈ 150 µg sparfloxacin).
2.2.2. Fabrication of the direct sparfloxacin loaded contact lenses The PVP coated direct sparfloxacin-loaded contact lenses were fabricated by adding the required quantity of sparfloxacin (6.66 mg/ml, 10 mg/ml and 13.33 mg/ml coded as Sp-L-100, Sp-L-150 and Sp-L-200 respectively) directly in the monomer mixtures (up to 1 ml monomer mixture) to achieve 100 µg, 150 µg and 200 µg sparfloxacin/15 mg contact lens respectively [34]. The direct sparfloxacin-laden contact lenses were
fabricated by using above sparfloxacin-monomer mixture and plastic lens molds. The fabrication process is same as discussed in section 2.2.1. The direct sparfloxacinloaded contact lenses (supplementary material Part I, Fig. S-1, B) were removed and extracted using 50 ml of boiling water for 30 minutes to remove the unreacted monomers, followed by sterilization in 2 ml STF containing 0.5 % w/v PVP. The drug is present in their microparticles forms in the matrix of the contact lens. The PVP was coated by soaking method and surface curing as discussed in section 2.2.4. The amount of sparfloxacin leached during the extraction and sterilization steps were quantified by HPLC method at 292 nm after suitable dilution [36, 37].
2.2.3. Fabrication of sparfloxacin laden ring contact lenses The sparfloxacin laden ring was fabricated at two different doses of 150 µg (coded as Sp-R-150) and 300 µg (coded as Sp-R-300) sparfloxacin. Briefly, the ring (5 mg weight) of 50 µm thickness was casted using sparfloxacin-monomer mixture solution. The sparfloxacin-monomer mixture was pipetted between the glass slides separated by Teflon spacer (50 µm), followed by the exposure to Ultraviolet transilluminiator (360-370 nm) for 15 minutes [38]. The sparfloxacin laden sheet was cut into a ring shaped ring (5 mg weight, 9 mm outer diameter and 6 mm inner diameter) using a borer. The sparfloxacin ring contact lenses were casted using plastic mould (14.2 mm outer diameter and 6.5 mm base curve). The ring was placed in the female mold cavity, followed by addition of excess monomer mixture (blank contact lens). The male mold was joined and transferred to the Ultraviolet transilluminator for 30 minutes curing. The ring implanted contact lens was removed and processed for extraction and sterilization. The drug is present in their microparticles forms in the matrix of contact lens. The PVP was loaded by soaking method and surface curing (discussed in section 2.2.4). The amount of sparfloxacin leached during the extraction and sterilization steps was quantified by HPLC method. The optical transmittance of the ring laden contact lens was not altered as the ring was placed in the periphery of the lens away from the center for clear vision (supplementary material Part I, Fig. S-1, C).
2.2.4. PVP loading on the sparfloxacin loaded contact lenses The PVP was loaded by soaking sparfloxacin loaded contact lenses (all the three batches) in 0.5 % w/v PVP concentration for 7 days followed by coating via Ultraviolet transilluminator for 30 seconds. The short exposure of UV light cause weak polymerization of PVP on the surface of the contact lens, to form a layer/coating of PVP. The process will retard the release of PVP from the contact lens, to provide comfort for prolong period of time. The above method showed high PVP release for prolong period of time, in comparison to the conventional PVP soaking method (see supplementary material Part I, Fig S-2).
2.3. Characterization of the sparfloxacin-PVP coated contact lenses 2.3.1. Swelling study The dry contact lenses after curing were removed from the molds and accurately weighed (dry weight = W D). The dry contact lenses were hydrated (extraction and sterilization) using their respective packaging/soaking solutions, thereafter blotted using filter paper and weighed (W S) again. The percentage swelling of the contact lens was determined by the formula [37]: % Swelling =
−
× 100
2.3.2. Optical transparency The sparfloxacin-PVP loading in the contact lens should not alter the optical transmittance of the contact lens. A UV-vis spectrophotometer was used to study the optical transmittance of the control contact lens and sparfloxacin-PVP loaded contact lenses. The hydrated contact lens (center portion) were fixed on the inner surface of the quartz cuvette containing 2 ml of distilled water and the optical transmittance of the centre portion of the contact lens was measured at 610 nm. The experiment was repeated three times.
2.3.3. Quantification of the sparfloxacin and PVP loaded in the contact lenses The amount of sparfloxacin and PVP present in the contact lens was quantified by HPLC and colorimetric method [39] respectively. The contact lenses were shifted to an
individual glass vial (screw capped) containing 25 ml of water to extract sparfloxacin and PVP. The glass vials were agitated at 100 RPM for 10 days at room temperature, followed by analysis of drugs in water by developed and validated analysis method. All readings were recorded in triplicate.
2.4. In vitro release study The in vitro flux studies of the PVP coated Sp-S, Sp-L and Sp-R contact lenses were carried out by immersing the lens in the glass vial containing 2 ml of STF at 34oC with constant stirring (100 RPM) [40]. At the pre-determined time intervals, 2 ml of STF was withdrawn from the vial and replaced with the same volume of fresh STF to maintain the prefect sink condition. The amount of sparfloxacin and PVP released in the STF was quantified by HPLC and colorimetric method [41] respectively. The procedure was continued till no release of the drug concentration was observed in the two successive measurements. All the readings were recorded in triplicate.
2.5. Effect of packaging solution (Shelf life study) The shelf life study of selected Sp-S-4, Sp-L-200, and Sp-R-300 batches were performed to estimate the effect of time on in vitro sparfloxacin release profile and leaching of sparfloxacin in the packaging solution. The contact lenses after 3 months (stored at room temperature) were analyzed for in vitro sparfloxacin release kinetics and the sparfloxacin leaching in the STF was quantified by HPLC. The preliminary shelf life data of in vitro PVP release, suggest no significant change in the PVP release kinetic with time, which was expected (data not shown here).
2.6. Animal studies The study protocol for white New Zealand rabbits were approved by Xi'an International Medical Center (2019-1118).
2.6.1. In-vivo drug release study The New Zealand rabbits (male and female) were used to investigate the release profiles of sparfloxacin and PVP in the tear fluid from the selected Sp-R-300 contact
lens [128.9 ± 3.42 µg sparfloxacin (cumulative release) and 35.31 ± 1.47 µg PVP] contrasting with the eye drop solution [0.3 % w/v sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)] [42]. The sterile contact lens was placed on the right eye (n=6) of rabbits (left eye was kept control) without local or general anaesthesia. In case of the eye drop group, the rabbit’s right eye received single drop of sparfloxacin eye drop, followed by Protear eye drop (same time). The left eye was kept control. The rabbit tear fluid was collected using disposable glass capillary from the cul de sac and preserved at -20◦C until analysis. The drug-tear fluid samples were treated with 1 ml of methanol to precipitate proteins, followed by freeze (5oC) centrifugation (Remi freeze-centrifuge) for 1 h at 8000 RPM. The collected supernatant was analyzed for sparfloxacin and PVP by HPLC and colorimetric method respectively. Rabbits were euthanized at the end of the study for histopathological analysis. The eyes were enucleated and fixed in 10% formalin buffered solution. The paraffin embedded corneas were cut into sections using microtome and stained using hematoxylin. The structure of cornea including basement membrane, epithelium and stroma were investigated using light microscopy at ‘×400 magnification’ [43].
2.6.2. Efficacy study The efficacy of the PVP-Sp-R-300 contact lens was evaluated using the New Zealand white rabbits, and was compared with the conventional eye drop therapy. The conjunctivitis was induced using young culture of the Staphylococcus aureus (ATCC 25923, 100 CFU/ml) [44]. Rabbits’ eyes were examined using portable slit lamp prior to the induction of the conjunctivitis to exclude any abnormalities. The study includes 4 groups (n=6 rabbits in each group) and both the eyes of rabbits were utilized for the study. Group 1: Uninfected and untreated (i.e. conjunctivitis was not induced) Group 2: Conjunctivitis was induced and treated with sterile saline (0.9 %w/v NaCl) solution (Negative control). Group 3: Conjunctivitis was induced and treated with commercial eye drop solution containing (0.3 % w/v, Spar eye drop, Cipla) sparfloxacin (Positive control).
Group 4: Conjunctivitis was induced and treated with contact lenses (PVP-Sp-R-300, 129 µg loading of sparfloxacin, Test group).
In this study, the group 1 rabbits were caged and neither the conjunctivitis was induced nor they were treated with any of the formulation; while the conjunctivitis was induced in both the eyes of group 2 to 4 using the young culture of the staphylococcus aureus. The 50 µl of culture solution (102 CFU in 10 µL of tryptic soy broth [45]) was gently instilled in the lower conjunctival sac of the rabbit eye and holded for few seconds to ensure the uniform spearing of the bacteria. After 48 h, the rabbits’ eyes showed the symptoms of discharge, chemosis, conjunctival congestion and palpebral fissure, indicating the successful induction of the conjunctivitis. The treatment was started from the day 3, i.e. after successful induction of conjunctivitis. In group 2, the rabbit’s eyes were treated with one drop of the sterile saline solution every 4 h, which served as the negative control group. In the group 3 (positive control group), rabbit’s eyes were treated with one drop of the sparfloxacin eye drop solution (0.3 % w/v) in 4 h interval. The PVP-Sp-R-300 contact lenses (129 µg loading of sparfloxacin) were carefully placed on the cornea of the group 4 (test group) without anaesthesia. The rabbits were kept under observation and symptoms like the discharge, chemosis, conjunctival congestion and palpebral fissure were observed at every 12 h [46]. The treatment was conducted for 4 days after the induction of the conjunctivitis.
2.7. Statistical analysis Statistical analysis was carried out using SPSS 21.0 for Windows. T-test (2 tailed) and One-way analysis of variance (ANOVA) was used to compare the different groups, after confirming the normality and the homogeneity of variance.
3. Results and discussion 3.1. Characterization of the PVP coated sparfloxacin-contact lenses 3.1.1. Swelling study The percentage swelling data of the contact lenses are shown in Table 1. The swelling property of the contact lens has direct relationship with the oxygen and ion
permeability and final dimensions of the contact lens [47-49]. Thus, the presence of drug should not alter the swelling property of the contact lens. The soaked contact lenses [Sp-S] did not showed significant reduction (p>0.05) in the % swelling (88.2 to 68.4 %) in comparison to the blank contact lenses (90.1 %). However, the direct sparfloxacin loaded contact lens [Sp-L] showed proportional reduction (77.9 to 68.5 %) in the swelling property with increased in the level of sparfloxacin inside the matrix structure of the contact lens, which could be due to drug-polymer interaction during curing process. The sparfloxacin laden ring contact lens (Sp-R) did not showed significant (p>0.05) alteration in the % swelling in comparison to the blank contact lens, as the ring occupied the limited area of the contact lens.
3.1.2. Optical transparency The optical transparency of the contact lenses are shown in table 1. The soaked contact lenses showed > 95 % optical transparency (expect Sp-S-6), while the direct sparfloxacin loaded contact lens [Sp-L] showed proportional reduction (70.4 to 54.6 %) in the optical transparency values with increased in the level of sparfloxacin. Thus, SpS-6 and Sp-L batches cannot be used for therapeutic purpose, as the % optical transparency was < 95%. The optical transparency of the ring-contact lenses (Sp-R) were > 99 %, which was expected as the ring was placed at the periphery of the contact lens for clear vision (supplementary material Part I, Fig S-1, B).
3.1.3. Quantification of the sparfloxacin and PVP loaded in the contact lenses The loading of sparfloxacin and PVP in the contact lenses (supplementary material, Part I, Table S-1) were quantified to investigate the uniform dispersion of drugs in the contact lens and reproducibility of the methodology. The PVP loading was common for all the methodologies/batches. The uptake of PVP from the packaging solution (0.5 % PVP in STF) followed by short curing process showed 32.2 to 35.4 µg of PVP loading, which should be enough to show prolong comfort to the patient eyes during contact lens wear (further discussed in section 3.5). The uptake of sparfloxacin from the drug-soaking solution was found to be 91.7 µg, 106.1 µg and 210.2 µg for Sp-S-2, Sp-S-4 and Sp-S-6 respectively, which was sufficient
to treat conjunctivitis. However, the drug release rate was very high from the contact lens and failed to show sustain release (discussed in section 3.3.1). The loading of sparfloxacin in Sp-L-100, Sp-L-150 and Sp-L-200 batches was found to be 103.7 µg, 151.7 µg and 201.8 µg respectively. The ring contact lens Sp-R-150 and Sp-R-300 shows 151.9 µg and 302.1 µg sparfloxacin loading respectively. The standard deviation values were low enough to confirm the reproducibility of sparfloxacin loading in the contact lenses. Thus, the target dose of single eye drop (0.3 % w/v, 50 µL = 150 µg) was loaded in all the batches.
3.2. Sparfloxacin leached during monomer extraction and sterilization The Sp-L and Sp-R batches were extracted to remove the unreacted monomers from the contact lenses. The data (Table 2) of Sp-L-100, Sp-L-150, and Sp-L-200 batches showed 51.2 µg (49.3 %), 75.3 µg (49.6 %), and 102.1 µg (50.5 %) sparfloxacin leaching during monomer extraction step in boiling water. While, Sp-R-150 and Sp-R-300 batches showed low drug leaching (1.3 fold reduction) of 55.9 µg (36.8 %), and 105.0 µg (34.7 %) respectively. Following monomer extraction step, the contact lenses (Sp-R-150 and Sp-R-300 batches) were sterilized in 2 ml STF containing 0.5 %w/v PVP. The Sp-L-100, Sp-L-150, and Sp-L-200 batches showed 17.6 µg (17.0 %), 28.0 µg (18.5 %), and 36.1 µg (17.9 %) of sparfloxacin lost during sterilization. While, Sp-R-150 and Sp-R-300 batches showed low drug leaching of 22.4 µg (14.7 %), and 39.1 µg (12.9 %) respectively, due to the tight packing of drug in the matrix of ring. Thus, sparfloxacin laden ring contact lens showed better performance in comparison to direct sparfloxacin-laden contact lenses.
3.3. In vitro release studies 3.3.1. In vitro release of sparfloxacin The cumulative release of sparfloxacin from the contact lenses are shown in Fig. 1. The release rate profiles are shown in supplementary material Part I, Fig S-3. The data showed high cumulative release of 90.9 µg, 156.5 µg, and 208.9 µg from Sp-S-2, Sp-S4 and Sp-S-6 batches respectively. The soaked contact lenses do showed sufficient drug uptake due to high solubility of drug in STF. However, the release rate was high
(no drug detected after 24, expect Sp-S-6 batch), making it unsuitable technique to load water soluble drug to produce extended release therapeutic contact lens. In Sp-L batches, the sparfloxacin was directly incorporated in the matrix of the contact lenses during fabrication, which on extraction and sterilization steps showed major loss of drug. The Sp-L-100, Sp-L-150 and Sp-L-200 batches showed 68.9 µg (66.4 %), 103.4 µg (68.1 %), and 138.2 µg (68.5 %) sparfloxacin loss respectively. The cumulative release from direct sparfloxacin-laden contact lenses [Sp-L-100, Sp-L-150 and Sp-L-200 lenses showed 32.29 µg, 40.27 µg, and 52.8 µg respectively] was low, as the major drug was lost during extraction and sterilization. The method failed to improve the release rate kinetics, even with increase in the drug loading amount. This was due to high water solubility of drug in STF, causing high release rate from the aqueous channel of the contact lens matrix. In Sp-R batches, the sparfloxacin was entrapped in the matrix of ring, followed by implantation in the contact lenses. In comparison to Sp-L batches, the Sp-R-150 and Sp-R-300 batches showed relatively low drug leaching of 78.3 µg (51.6 %) and 144.1 µg (47.7 %) respectively, during extraction and sterilization. The Sp-R contact lenses showed sustained release of sparfloxacin up to 36-48 h, in comparison to Sp-S and SpL lenses which showed drug release up to 12-36 h and 24-48 h respectively. The sustained release was due to slow diffusion of drug from the matrix structure of ring, followed by diffusion through the contact lens matrix in the media. The release rate at 24 h from Sp-L contact lenses was in the range of 104 to 171 ng/h, while Sp-R contact lens shows high release rate of 751 to 1235 ng/h. The data clearly indicate the advantage of ring implanted contact lenses in comparison to direct and soaked contact lenses.
3.3.2. In vitro release of PVP The PVP coating on the surface of contact lenses was common for all the batches. The cumulative release of PVP from the selected batches (Sp-S-6, Sp-L-200 and Sp-R300) are shown in Fig. 2. The release rate profiles are shown in supplementary material Part I, Fig S-4. The data indicate initial burst release of 7.7 to 9.3 µg at 1 h, followed by sustained release up to 48 h. The release rate of PVP between 2 to 48 h was 2000 to
100 ng/h. The coated contact lenses showed improved release rate profile in comparison to conventional soaking method (see Supplementary material Part I, Fig S2). The data suggest bonding (due to weak polymerization) of PVP over the surface of the contact lens, resulting in prolong (slow) release. The PVP coat is mechanical less stability, as noted in the in vitro release study. In future studies, we are working to understand the nature of interaction between the coating and the contact lens. The conventional soaking method shows only surface adsorption of PVP which lead to rapid fall in release rate profiles.
3.4. Mathematical models Different mathematical models, i.e., zero order, first order, Higuchi, Korsmeyer– Peppas equation were applied for describing the kinetics of the drug release process from the contact lens, and the most suited being the one which fitted best the experimental results. The drug release patterns were examined according to different mathematical equations from release kinetics theories are shown in supplementary material part-II. The Korsmeyer-Peppas model was found to be best fit, suggesting anomalous transport of drug release mechanism i.e., drug release was controlled by more than one process.
3.4. Effect of packaging solution (Shelf life study) On the first day, the sparfloxacin lost during extraction and sterilization was 138.2 µg (68.5 %) and 144.1 µg (47.7 %) for Sp-L-200 and Sp-R-300 lens respectively. After three months of storage, the contact lenses (Sp-S-6, Sp-L-200 and Sp-R-300) were removed from the packaging solution and analyzed for sparfloxacin (leached) content by HPLC. The data showed further leaching of drug [9.5 µg (9.15 %) and 11.1 µg (7.3 %) from Sp-L-200 and Sp-R-300 lens respectively] in the packaging solution (during three months) suggesting the limitation of therapeutic contact lenses. The in vitro release of sparfloxacin after sterilization (day 1) and after three months of storage are shown in supplementary material Part I, Fig S-5. The soaked contact lenses (Sp-S-6) after three months of storage did not showed significant difference in the release rate profiles (f2 value = 83.43) which was expected, as the lenses were
soaked in the fixed concentration of sparfloxacin. While, a significant drop in the cumulative and release rate profiles were observed with Sp-L-200 lens and Sp-R-300 lenses (f2 value = 49.01) after 3 months. However, statistically the Sp-L-200 lens showed similarity in release rate profile after 3 months (f2 value = 69.0). Thus, it is not advisable to store the lens in wet condition, due to leaching of drug with time in the packaging solution. In future studies, the lenses will be sterilized and investigated by radiation technique (dry method) to overcome the issue of leaching with time.
3.5. In vivo drug release study The in vivo tear fluid analysis was performed using Sp-R-300 contact lens [128.9 ± 3.42 µg sparfloxacin (cumulative release) and 35.31 ± 1.47 µg PVP] and eye drop solution [0.3 %w/v Sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)] to investigate the drug retention pattern. The Cmax (5 minutes) of sparfloxacin was found to be 1210.6 µg/ml and 842.3 µg/ml for eye drop solution and Sp-R-300 contact lens respectively (Fig. 3). The eye drop solution showed rapid fall in drug concentration (up to 1 h), while Sp-R-300 contact lens showed improvement in drug retention up to 24 h. The drug was not detected later due to limited LOQ of HPLC. The Cmax (5 minutes) of PVP was found to be 244.9 µg/ml and 133.3 µg/ml for eye drop solution and Sp-R-300 contact lens respectively (Fig. 4). The eye drop solution showed rapid fall in PVP concentration (up to 1 h), while Sp-R300 contact lens showed major improvement in drug retention up to 24 h with even 8.5 fold lower dose. The contact lens showed sustained drug release and high drug-tear fluid concentration in comparison to eye drop solution, thus the single contact lens will be enough to treat allergic conjunctivitis. The data of in vivo % cumulative drug retained in the tear fluid and in vitro % cumulative drug released was plotted to investigate in vitro-in vivo correlation (IVIVC, Levy plot, level A, supplementary material Part I, Fig S-6). The R2 (correlation coefficient) value for sparfloxacin and PVP was found to be 0.696 and 0.764 respectively. The poor IVIVC was due to binding of drug to lipid (in tear), which could enhance the solubility of drug in tear causing high in vivo initial release. The corneal histopathological images of control and Sp-R-300 contact lens (supplementary material,
Part I, Fig S-7) showed normal squamous epithelium cells with normal pattern of collagen fibers in the stroma with no obvious changes. However, a long term three weeks data is needed to assure the safety of contact lens for clinical use.
3.6. Efficacy study The conjunctivitis was successfully induced in the rabbit eyes after 48 h using the young culture of the Staphylococcus aureus, as confirmed by the symptoms of conjunctival edema of the eyelids, beefy red conjunctiva and the swollen eye lids. No significant difference was observed in the symptoms among the group 2, 3 and 4. On day 3, the treatment with eye drop and the contact lens was started in the group 3 and 4 respectively. After 24 h of treatment, i.e. on the 4th day, the discharge was reduced in the group 4, while no improvement was observed in the group 2 (saline treated) and the group 3 (eye drop treatment). On the 5th day, the inflammatory symptoms in the group 3 and 4 was significantly reduced in comparison to the group 2 (Negative control). On the third day of the treatment, i.e. 6th day, the rabbits’ eye in the group 3 and 4 showed no discharge along with the minimal congestion and edema. The eyes were completely cured on the 4th day of the treatment in the group 3 and 4 (supplementary material Part I, Fig. S-8). While the inflammatory symptom still persisted in the group 2. The outcome of the study suggests that, a single low dose (128 µg of sparfloxacin) implant contact lens was as effective as the frequent high dose eye drop therapy.
4. Conclusion The study demonstrate the successful simultaneous delivery of sparfloxacin and PVP form the novel PVP coated sparfloxacin laden ring contact lens (Sp-R) without affecting the critical lens properties. The soaked contact lenses (Sp-S) did not alter the optical and swelling properties, however, the release rate was very high. The direct sparfloxacin laden contact lens (Sp-L) showed reduction in the optical and swelling properties. The ring contact lens did not altered the optical and swelling properties, as ring covered the limited area of lens. The sparfloxacin lost during the extraction and sterilization from Sp-L lens was 66-68 %, while a significant reduction was observed with Sp-R lens (47-51 %). The flux data showed sustained release of sparfloxacin in
following order: Sp-L (12-24 h) > Sp-S (12-36 h) > Sp-R (36-48 h). The sustained release of sparfloxacin from the Sp-R lens was achieved due to tight packing of drug in the ring implant. The PVP flux data showed sustained release of PVP up to 48 h. The shelf life study in the packaging solution showed leaching of sparfloxacin from the contact lens. Thus, it is not advisable to store the lens in wet condition. In future studies, the lenses will be sterilized and investigated by radiation technique (dry method) to overcome the issue of leaching with time. Furthermore, intense and in-depth research is needed to further reduce the possible burst release of drug from the dry ring implant contact lens sterilized by radiation technique. The in vivo drug release data in the rabbit tear fluid showed sustained drug release and high drug-tear fluid concentration in comparison to eye drop solution, thus the single contact lens will be enough to treat conjunctivitis. The in vivo efficacy study in the conjunctivitis induced rabbits’ eye showed equivalent healing effect with the single implant contact lens in comparison to the frequent high dose eye drop therapy. The study demonstrated the successful delivery of sparfloxacin and PVP from the ring contact lens for the extended period of time without altering the optical and physical properties of contact lens. The platform technology can be applied for similar other potent antimicrobial agents (BCS class-II) for ocular delivery.
Disclosures There are no potential conflicts of interest to disclose for this work.
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FIGURE CAPTION
Fig. 1. Cumulative release of sparfloxacin from contact lenses. Values are shown as mean ± standard deviations (n=3).
Fig. 2. Cumulative release of PVP from the selected contact lenses (Sp-S-6, Sp-L-200 and Sp-R-300). Values are shown as mean ± standard deviations (n=3).
Fig. 3. Sparfloxacin tear fluid concentration from Sp-R-300 contact lens [128.9 µg sparfloxacin (cumulative release) and 32.2 µg PVP] and eye drop solution [0.3 %w/v sparfloxacin eye drop (1 drop = 50 µl = 150 µg sparfloxacin) and 0.6 % w/v Protear eye drop (1 drop = 50 µl = 300 µg PVP)].
Fig. 4. PVP tear fluid concentration from Sp-R-300 contact lens and eye drop solution. Each time point represents the mean ± standard deviation (n = 6).
LIST OF TABLES
Table 1 Percentage swelling and transmittance data. Values are shown as mean ± standard deviations (n = 3). Transmittance (%)
Swelling (%)
99.2 ± 0.2
90.1 ± 1.5
Sp-S-2
97.9 ± 0.4
88.2 ± 1.9
Sp-S-4
96.1 ± 0.7
87.1 ± 1.9
Sp-S-6
86.4 ± 1.8*
86.4 ± 1.8*
Sp-L-100
70.4 ± 2.0**
77.9 ± 3.5**
Sp-L-150
61.6 ± 1.2**
72.1 ± 3.3**
Sp-L-200
54.6 ± 4.1**
68.5 ± 3.5**
Sp-R-150
99.1 ± 0.5
87.7 ± 2.2
Sp-R-300
99.3 ± 0.4
86.2 ± 2.6*
Codes Control contact lens
* indicate significant difference (p<0.05) with control contact lens. ** indicate very significant difference (p<0.01) with control contact lens.
Table 2 Data of sparfloxacin leached during the monomer extraction and sterilization step (Mean ± SD, n=3). Code
Drug Leaching during
Drug Leaching
Total Sparfloxacin
monomer extraction
during sterilization
leached
µg
%
µg
%
µg
%
Sp-L-100
51.2
49.3
17.6
17.0
68.9
66.4
Sp-L-150
75.3
49.6
28.0
18.5
103.4
68.1
Sp-L-200
102.1
50.5
36.1
17.9
138.2
68.5
Sp-R-150
55.9
36.8
22.4
14.7
78.3
51.6
Sp-R-300
105.0
34.7
39.1
12.9
144.1
47.7