Preparation and Evaluation of Contact Lenses Embedded with

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Preparation and Evaluation of Contact Lenses Embedded with Polycaprolactone-based Nanoparticles, for Ocular Drug Delivery Farzaneh Hashemi Nasra, Sepideh Khoee, Mohammad Mehdi Dehghan, Sirous Sadeghian Chaleshtori, and Abbas Shafiee Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01387 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 11, 2015

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Preparation and Evaluation of Contact Lenses Embedded with Polycaprolactone-based Nanoparticles, for Ocular Drug Delivery Farzaneh Hashemi Nasr a, Sepideh Khoee*,a, Mohammad Mehdi Dehghanb, Sirous Sadeghian Chaleshtoric, Abbas Shafieed a b

Polymer Laboratory, Chemistry Department, School of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Department of Clinical Science, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

c Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran b Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran Email: [email protected]. Tel.: +98 21 61113301; Fax: +98 21 66495291

ABSTRACT: To improve the efficiency of topical ocular drug administration, we focused on development of a nanoparticles loaded contact lens to deliver the hydrophobic drug over a prolonged period of time. The crosslinked nanoparticles based on PCL (poly ε-caprolactone), 2-hydroxy ethyl methacrylate (HEMA) and poly ethylene glycol diacrylate (PEG-DA) were prepared by surfactant-free miniemulsion polymerization. The lens material was prepared through photopolymerization of HEMA and N-vinylpyrrolidone (NVP) using PEG-DA as the crosslinker. Effects of nanoparticles loading on critical contact lens properties such as transparency, water content, modulus and ion and oxygen permeabilities were studied. Nanoparticles and hydrogel showed high viability indicating the absence of cytotoxicity and stimulatory effect. Drug release studies revealed that the hydrogel embedded with nanoparticles released the drug for a period of 12 days. The results of this study provide evidences that nanoparticles loaded hydrogels could be used for extended delivery of loteprednol etabonate and perhaps other drugs. Keywords: Emulsion polymerization, Nanoparticles, Hydrogel, Ocular drug delivery, Loteprednol etabonate

* Corresponding author

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1. INTRODUCTION Controlled ophthalmic drug delivery is one of the most challenging fields of pharmaceutical research. Low drug-contact time of drugs is mainly attributable to the precorneal loss factors that include rapid tear turnover, nonproductive absorption, transient residence time in the culde-sac, and the relative impermeability of the drugs to the corneal epithelial membrane.1-3 With the development of nanotechnology in drug delivery, drug carriers such as nanoparticles, dendrimers, niosomes, liposomes, insert films and hydrogels have been developed in order to solve mentioned problems.4-7 These novel systems offer manifold advantages over conventional systems as they increase the efficiency of drug delivery by improving the release profile and also reduce drug toxicity.8 Among these carriers, treatment with hydrogels used as contact lenses are particularly attractive because of their good biocompatibility, high water content and soft nature, ability to increase the bioavailability of drugs (up to 35 times compared to eye drops),9-13 ease of control, and convenient use.14 The manipulation of drug release rate could be achieved by variation of hydrogel parameters such as crosslink density and degree of swelling.15 The drugs or macromolecules can be loaded into hydrogels by three approaches : (1) mixing the drug with appropriate monomer(s), crosslinker and initiator and then entrapping it within the formed matrix after polymerization; (2) mixing the drug with a preformed polymer which is then crosslinked;16,17 (3) diffusion of the drug into a pre-formed hydrogels by immersing the hydrogel into a drug’ solution.18 However, these methods have some disadvantages such as having a detrimental effect on the activity of the drug or being time consuming. In addition, the most important drawback of hydrogels relates to the quantity and homogeneity of drug loading, which may be limited, especially in the case of hydrophobic drugs. On the other hand, the high water content and large pores frequently result in relatively rapid drug release. To surmount these limitations, incorporation of drug loaded nanocarriers into


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hydrogels will be an efficient method to overcome mentioned difficulties while providing extended drug delivery. As similar systems of nanoparticles embedded contact lens, Ho and co-workers presented a nanodiamond (ND)-embedded contact lens capable of lysozyme triggered release of timolol maleate (TM) for sustained therapy. The individual NDs were coated with polyethyleneimine and then cross-linked with chitosan, forming a TM loaded ND-nanogel which were then embedded within a poly HEMA matrix and cast into contact lenses.19 Moreover, Chauhan et al. prepared drug loaded particles dispersed in HEMA gels, and studied TM release for 2-4 weeks.20 They also encapsulated the ophthalmic drug formulations in microemulsion drops, and the drug loaded microemulsion drops were dispersed in the poly HEMA hydrogels, which was further inserted into the eyes.21 Also, Kapoor et al. prepared Brij surfactant loaded poly HEMA hydrogels that can release Cyclosporine A at a controlled rate for extended periods of time.22,23 Danion et al. validated the biocompatibility and transmittance properties of liposomes in contact lenses.24 Although interests in the preparation of hydrogels have increased considerably in recent years,25,26 the number of polymers suitable for controlled release is quite limited compared to the total available synthetic polymers due to the lack of certain properties like biodegradability, swellability and none or less toxicity in specific environments. The homo and copolymer hydrogels of HEMA have found extensive applications in the field of biomedicals such as contact lenses because of their well-tolerated safety and biocompatibility, good chemical stability, non-toxicity and non-antigenic properties.27-29 Surfactant-free miniemulsion polymerization is a widely used technique for the synthesis of polymer nanoparticles.30 This method allows the preparation of narrow-sized spherical particles that do not contain any adsorbed surfactant.31 The long-term physical stability of miniemulsions with no apparent flocculation or coalescence makes them unique and they are sometimes referred to as ‘approaching thermodynamic stability’.32


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To achieve molecular level understanding, molecular modeling would be very useful, and there are such molecular modeling approach for poly(HEMA-co-VP) hydrogel system. For instance, Jang and coworkers used molecular modeling for both random and blocky hydrogel networks of poly (VP-co-HEMA) with VP:HEMA=37:13 composition to investigate the effect of the monomeric sequence on the mechanical properties. 33 They observed that in such networks, the water molecules are associated more closely with NVP than with the HEMA moieties, which is consistent with results from quantum mechanical solvation free energy calculations. They also found difference in stress reduction between the random and the blocky sequence networks which was due to the difference in the structural rearrangement of monomers in the presence of water during deformation. They also adopted the mentioned system and used molecular dynamics simulations in order to investigate the effect of the water content on the equilibrium structures and the mechanical properties.34 The results revealed that NVP is more hydrophilic than HEMA and that the random sequence hydrogel is solvated more than the blocky sequence hydrogel at low water content, which disappears with increasing water content. From mechanical deformation simulations, stress–strain analysis showed that the NVP is found to relax more efficiently, especially in the blocky sequence, so that the blocky sequence hydrogel shows less stress levels compared to the random sequence hydrogel. Our study specifically focuses on encapsulation of anti-inflammatory drug into nanoparticles prepared by oil-in-water (O/W) surfactant free miniemulsion, and dispersion of the nanoparticles in poly HEMA based hydrogel. The synthesized nanoparticles were comprised of PEG as hydrophilic outer shell, poly HEMA as hydrophobic inner shell and PCL as hydrophobic core. The nanoparticles properties such as size, morphology and stability were investigated. HEMA/NVP copolymers with varying comonomer compositions were prepared using Irgacure 2959 and PEG-DA as the initiator and crosslinker, respectively. The


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nanoparticles loaded poly (HEMA-co-VP) hydrogels were characterized to explore drug release profiles and all properties relevant to extended wear contact lenses including transparency, modulus, cytotoxicity and ion and oxygen permeabilities.

2. EXPERIMENTAL SECTION 2.1 Materials. ε-caprolactone, Sn(Oct)2, ethylene glycol, acryloyl chloride, diethanolamine, triethylamine, dichloromethane (DCM), toluene, polyethylene glycol with molecular weight of 2000 g/mol (PEG2000), 2-hydroxyethyl methacrylate (HEMA), N-vinylpyrrolidone (NVP) and Irgacure 2959 were purchased from Merck Chemical Co. Loteprednol Etabonate (LPE) eye drop (Bausch and Lomb incorporated) was used as the drug. All the chemicals except ethylene glycol were of analytical grade and used without purification. Ethylene glycol was refluxed for several hours in the presence of sodium hydroxide and toluene. Subsequently, toluene was distilled off to get water free ethylene glycol. It was stored over the molecular sieve 4°A for further use. 2.2. Preparation Methods 2.2.1. Synthesis of branched diacrylated-PCL. Acrylated PCL was synthesized in four steps according to our previous study.35 2.2.2. Synthesis of diacrylated- PEG (PEG-DA). PEG-DA was synthesized according to the recipe given in our previous report.36 2.2.3. Drug free and drug loaded crosslinked nanoparticles preparation. Hybrid nanoparticles of PCL/PHEMA/PEG were prepared by surfactant-free miniemulsion polymerization (SFEP). Three formulations containing different weight percent of hydrophobic acrylated PCL segment with fixed amounts of HEMA to PEG-DA weight ratios using Irgacure 2959 as radical photoinitiator formed the oil phase. Deionized water free of


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any surfactant was used as the aqueous phase. LPE was dissolved in acetone (0.1 ml). Acrylated PCL, HEMA, PEG-DA and Irgacure 2959 with three different compositions were dissolved in DCM (0.4 ml). The LPE in acetone was added to the prepared mixture in DCM to form the final oil phase and then, the reaction mixture was homogenized by a probe sonicator (20 KHz±500 Hz Ultrasonic generator, SONOPULS Ultrasonic homogenizer, Model HF-GM 2200, titanium micro tip Ms-72) employing a pulse mode at 20W for 10 s (2 sec “on” and 3 sec “off”) in an ice bath. Afterward, the miniemulsion was directed into the deionized water (5 ml) at an agitation speed of 500 rpm and purged with nitrogen gas. The final solution was exposed to UV light with stirring at room temperature for 15min. After that, the organic solvent was evaporated at room temperature in order to form nanoparticles consisting of PEG as hydrophilic outer shell, PHEMA as hydrophobic inner shell and PCL as hydrophobic core. Finally, the nanoparticles solution were purified by centrifugal concentrator with hydrosart® membrane (cut off = 10kDa) to remove unreacted materials; nanoparticles were obtained after freeze drying (yield 55%). 2.2.4. Hydrogel preparation. Poly(HEMA-co-VP) based hydrogels were prepared by a free radical photopolymerization at room temperature. Five types of hydrogels were prepared as follows: HEMA, NVP and PEG-DA were mixed in five different weight ratios (10:4:0.3, 10:5:0.3, 10:4:0.8, 10:4:1.5 and 10:6:0.8) followed by adding Irgacure 2959 (0.5 wt%) as a biocompatible UV initiator. These ratios of HEMA/VP/PEG-DA will be designated hereafter as H1, H2, H3, H4 and H5, respectively. Water was used as the solvent (40wt% to the monomers mixture).The reaction mixtures were degassed with nitrogen for 15 min to remove the oxygen which acts as a chain terminating agent. Each mixture was poured in between two glass plates separated by a 200 µm thick plastic spacer. The mold was then subjected to ultraviolet irradiation at 325 nm and irradiated for 30 min. The polymerized hydrogels were then stored overnight in deionized water to remove the residual unreacted monomers and the 6 ACS Paragon Plus Environment

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crosslinker. The molded gels were dried in air overnight before further use. Herein, the hydrogel with proper equilibrium water content, optical clarity and mechanical property was chosen as the matrix for the preparation of nanoparticles loaded hydrogel. 2.2.5. Preparation of nanoparticles loaded hydrogels via adding nanoparticles to the polymerization mixture. Nanoparticles were incorporated in hydrogels by direct entrapment during polymerization process. To load prepared nanoparticles by direct addition to the polymerization mixture, different mass fraction of nanoparticles (3, 5, 7.5, 10 and 15wt %) was added to the monomers mixture before nitrogen bubbling. The procedures described above were then utilized for gel curing. 2.3. Characterization 2.3.1. Polymer, nanoparticles and hydrogel characterization. The synthesized polymers structures were characterized by 1H NMR (Bruker, 500 MHz) using tetramethylsilane as an internal standard and CDCl3 as solvent at 25°C. The nanoparticles and hydrogel structures were also confirmed by FT-IR spectroscopy (Bruker equinoxss). 2.3.2. Particle size measurement. Freshly prepared nanoparticles solution was used for nanoparticles mean diameter and size distribution determination by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instrument, UK) at 25°C. For each sample, the reported diameter ± standard deviation was the mean of five times test.

2.3.3. Stability of synthesized nanoparticles. The prepared PCL/PHEMA/PEG-DA based nanoparticles was placed in deionized water and stored at 4°C for 4 weeks. The size of nanoparticles was measured at the beginning and at each 7-day interval after storage. The ratio of particle size following storage to initial size was calculated. The obtained diameter ± standard deviation was the mean of five times test for each sample.


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2.3.4. Nanoparticles yield, drug loading and encapsulation efficiency. The yield of prepared nanoparticles was calculated gravimetrically after freeze drying. To determine the amount of drug encapsulated in nanoparticles, the nanoparticles solution obtained by SFEP with known amount of drug incorporated (feeding drug: copolymer is 2:10) was centrifuged at 10,000 rpm for 15 minutes. The supernatant solution was separated and the amount of unentrapped drug was evaluated by UV spectrophotometer (Perkin Elmer, lambda 800) at 265 nm using an established calibration curve of LPE in PBS (indirect method). Nanoparticles yield, drug loading content (DL) and encapsulation efficiency (EE) were calculated as follows: Nanoparticles yield% =

Weight of the nanoparticles × 100 (1) Weight of the feeding polymer and drug

DL% =

Weight of feeding drug − Weight of unentrapped drug × 100 (2) Total weight of the loaded nanoparticles

EE% =

Weight of feeding drug − Weight of unentrapped drug × 100 (3) Weight of feeding drug

2.3.5. Surface morphology of nanoparticles, hydrogel and nanoparticles loaded hydrogel. Morphologic evaluation of the nanoparticles was performed using field emissionscanning electron microscopy (FE-SEM) (HITACHI S 4160) and transmission electron microscopy (TEM) technique (LEO 906, Germany). Besides, FE-SEM was used to analyze the microstructure of both free hydrogel and nanoparticles loaded hydrogel. The hydrogel samples were kept overnight in a vacuum oven to remove the volatile components from the gel. The dried samples were cracked in liquid nitrogen and the freshly exposed surfaces were studied by FE-SEM. For FE-SEM analysis, all the samples were sputtered with gold before observation. In TEM analysis, the nanoparticles were diluted with deionized water and placed on a copper grid covered with carbon film. Observation was done at 150 kV.


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2.3.6. Equilibrium water content (EWC) of hydrogels and nanoparticles loaded hydrogels. The swelling kinetics of the hydrogels was measured gravimetrically. The xerogel of weighing (Wdry) was immersed in phosphate buffer solution (PBS, pH=7.4) at 35°C, periodically removed from the water, surface dried lightly and weighed (Wwet). Water uptake capability was calculated using the following equation:

%EWC =

&'() *&+,&'()

× 100 (4)

2.3.7. Transmittance. Optical property of nanoparticles loaded hydrogels with 200 µm thickness was measured using UV–Vis spectrophotometer in the range from 200 to 900 nm. Before the examination, the hydrogels were soaked in PBS overnight for complete hydration. Each sample was mounted in a quartz cuvette. The cuvette was placed in a spectrophotometer and its transmittance was determined. 2.3.8. Mechanical properties. A dynamic mechanical analyzer (DMA Q800, TA instruments) was used to measure the storage and loss modulus of different nanoparticlesloaded systems. Hence, hydrated rectangular gels 400 µm in thickness were mounted in the submersion tension clamp at room temperature. Gel response in the form of storage and loss modulus was evaluated by applying tensile force in the longitudinal direction while keeping the gel tightly screwed between the clamps by applying a preload force of 0.01 N. To determine the linear viscoelastic range, strain tests were first conducted at a frequency of 1 Hz followed by frequency sweep measurements executed for all the samples at 20 µm strain. 2.3.9. Ion permeability. The ionic permeability of prepared nanoparticles loaded hydrogel (H5, 7.5%) in compared to blank hydrogel (H5) was determined by the following experimental procedure. Each gel (1.6 cm diameter, 200 µm thick) was placed in the lensretaining member between the male and female portions. Then, it was positioned between the 9 ACS Paragon Plus Environment

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donor and receiving chambers containing 20 ml of 0.1 molar NaCl solution and 40 ml of deionized water, respectively. A stir bar was added to the receiving chamber and the system was placed in a thermostat to hold the temperature at about 35°C. The conductivity of receptor solution was monitored as a function of time by a conductivity meter (AZ8361 Conductivity Test Pen) and subsequently converted to NaCl concentration using the calibration plot. Measurements of conductivity were taken every 20 minutes for about five hours. The conductivity probe was calibrated before each measurement experiment, using different NaCl solutions of known concentrations. By plotting concentration of Na+ ions in receiving chamber versus time, the apparent Na+ ion permeability was calculated from the slope, using eq. (4):

C. =

/ 0 12,4 56 7

(8 − 89 ) (5)

where CR is the concentration of Na+ ions at time t in the receiving chamber, CL, 0 is the initial concentration of Na+ ions in donor chamber, A is the area of the lens exposed to the salt flux, L represents the average hydrogel thickness in the area exposed, VR is the volume of the receiving chamber compartment and P is the apparent permeability coefficient of Na+ ion.37

2.3.10. Oxygen permeability. Oxygen permeability (OP) is a parameter of a contact lens which expresses the ability of the lens to let oxygen reach the eye by diffusion. Different procedures have been used to determine the oxygen transmissibility and permeability coefficients of contact lenses.38 In this study, we measured the oxygen permeability through the lenses using dual chambers separated by the membrane whose Dk/t can be evaluated. Details of the procedures for measuring oxygen permeability of gels are available elsewhere.39 Briefly, gel (1.6 cm diameter, 200 µm thick) was mounted between donor and receiver chambers. The donor and receiver chambers were filled with deionized water that was equilibrated with air (20 ml) and water which was deoxygenated by bubbling nitrogen 10 ACS Paragon Plus Environment

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for an extended period of time (40 ml), respectively. The oxygen concentration in the receiver was measured with OX-TRAN® Model 2/21 from MOCON and the data was fitted to a diffusion model to determine the oxygen permeability through the gel. 2.3.11. In-vitro drug release of nanoparticles. The in-vitro drug release profile for drug loaded nanoparticles was performed using dialysis bag method (molecular weight cutoff, 12kDa) which was incubated in PBS (pH =7.4) at 35°C under gentle shaking. At regular time intervals, an aliquot amount (1.5 ml) was removed from release media, replaced with fresh PBS and measured at 265 nm according to calibration curve of LPE in PBS using UV spectrophotometry. 2.3.12. In-vitro drug release from the nanoparticle loaded hydrogel. Drug release profile of LPE trapped alone in the hydrogel dispersed in the polymerization feed mixture was compared with optimized LPE containing nanoparticles loaded hydrogel (cut into circular piece of 1.6 cm in diameter) in PBS (pH=7.4) at 35°C. The PBS volume in the release media was 3.5 ml for 200 µm thick gel. The drug concentration was determined periodically by measuring the UV–Vis absorbance at 265 nm and fitting the data to LPE calibration curve. 2.3.13. Cell culture and in vitro cytotoxicity study of nanoparticles and hydrogel. Female New Zealand white rabbit weighing 3 to 4 kg was used for corneal epithelium cell culture. Isolation and cultivation of rabbit corneal were accomplished according to previously published protocol.40 The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco #15140−122) supplemented with 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin). The cells were grown in 5% CO2 at 37 °C. The RCEC were seeded in 96-well plates (5000 cells per well) with culture medium in 5% CO2 at 37 °C overnight to adhere to the plates. Cells were treated with different concentration of nanoparticles (0, 0.1, 0.5, 1 and 2 mg/ml) for 24 h at 37 °C.


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Subsequently, the medium was removed and gently washed with PBS (pH = 7.4). A total of 100 µL of MTT (5 µg/mL in serum free DMEM medium) was supplied to each well followed by incubation at 37 °C for 4 h to allow the soluble yellow MTT to be reduced into dark-blue insoluble formazan crystals by the metabolically active cells. The formazan crystals were subsequently dissolved by the addition of dimethylsulfoxide at room temperature with shaking for 20 min. Finally, the optical density (OD) of individual wells was measured at 570 nm in a microplate reader (Anthos 2020; Anthos Labtec Instruments, Wals, Austria). Nontreated cells assumed to possess 100% viability were used as control. For each concentration, the reported cell viability ± standard deviation was the mean of three times test. The effect of hydrogel on cell viability was also studied as the same procedure described above. The xerogel sample was sterilized by UV radiation. Thereafter, the hydrogel sample was immersed in ultra-purified water for 24 h. During this period, the water was refreshed every several hours. The reported cell viability ± standard deviation was the mean of three times test.

3. RESULTS AND DISCUSSION 3.1. Polymer, nanoparticles and hydrogel characterization. General strategies for PCL/PHEMA/PEG-DA nanoparticles synthesis by SFEP and also preparation of nanoparticles loaded hydrogel are illustrated in Scheme 1. The number average molecular weight of PCL was calculated by 1HNMR with respect to the ratio of peaks integral of “l” (4.2 ppm) to “b” (1.6 ppm) and was found to be 5110 g/mol (Figure S1). Acrylated PCL with dendritic–linear–dendritic copolymer structure was synthesized by acrylation/Michael addition/acrylation of PCL with two hydroxyl end groups using acryloyl chloride and diethanolamine. 1HNMR (Figure S2) and FT-IR spectroscopy were employed to study the


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microstructural changes development of dendrimers, during generations growth based on our previous study35. PEG-DA was prepared through the reaction between acryloyl chloride and PEG 2000 in the presence of trimethylamine in 63% yield. The polymer structure was confirmed by 1HNMR (Figure S3) and FT-IR.

Scheme 1. Schematic illustration of surfactant-free emulsion polymerization for nanoparticles synthesis (a) and nanoparticles loaded hydrogel preparation (b) (The obtained hydrogel is transparent.) Characteristic peaks of PEG-DA appeared at 1101 cm-1 was attributed to C-O etheric bond, 2884.6 cm-1 assigned to C-H stretching bond and the peak related to carbonyl group appeared at 1724 cm-1.The disappearance of O-H stretching bond at 3200-3500 cm-1 on PEG spectrum confirms the acrylation reaction (Figure 1b). Nanoparticles comprised of PCL core, PHEMA inner shell and PEG outer shell were prepared via single step surfactant-free miniemulsion polymerization process using Irgacure 2959 as the photo-initiator (Scheme 1a). All the three different compositions of PCL/PHEMA/PEG-DA nanoparticles were prepared under the same conditions (Table 1). The final structure was confirmed by FT-IR (Figure 1d).


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Figure 1. FT-IR spectra of poly HEMA (a), PEG-DA (b), acrylated PCL (c), PCL/PHEMA/PEG-DA nanoparticles (d) and poly(HEMA-co-VP) hydrogel (e). 14 ACS Paragon Plus Environment

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As presented in Figure 1a, poly HEMA shows typical stretching absorption bands at 1152 cm-1(C-O), 1717 cm-1(C=O), 2949 cm-1 (CH2) and a wide absorption band in 3200-3500 cm-1 region relating to O-H groups. Characteristic peaks of acrylated PCL (Figure 1c) are the absorption bands at 1722, 1175 and 2945 cm-1 which are assigned to the C=O, C-O-C and CH stretching bonds. By considering the FT-IR spectra of pure poly HEMA, PEG-DA and acrylated PCL and compare them with FT-IR spectrum of synthesized nanoparticles (Figure 1d), we found that all the characteristic peaks related to three polymers are present in nanoparticles spectrum. It should be noted that the three polymer components FT-IR absorption peaks overlapped with each other mostly at 1000-1700 cm-1 due to their same functional groups. Overall, we demonstrate the successful preparation of nanoparticles by SFEP. Poly(HEMA-co-VP) hydrogels with different compositions of monomers and crosslinker were prepared by photo-polymerization in the presence of Irgacure 2959 as the initiator (Scheme 1b). The weight ratios of HEMA: NVP: PEG-DA used are as follows: H1 (10:4:0.3), H2 (10:5:0.3), H3 (10:4:0.8), H4 (10:4:1.5) and H5 (10:6:0.8). The FT-IR spectrum of hydrogel is illustrated in Figure 1e. All characteristic bands present in FT-IR spectra of components (HEMA, NVP and PEG-DA) appear in the FT-IR spectrum of the obtained hydrogel. Absorption bands at 1151, 1655, 1720, 2923 and 3200-3400 cm-1 attributed to C-O, HN-C=O, C=O, C-H and O-H groups, respectively.

3.2. Particle size measurement The particle size and polydispersity index (PDI) of all nanoparticles obtained by miniemulsion polymerization were estimated by DLS (Figure S4). In all series, the size distributions were unimodal and the largest particle size was 83.4 nm which belonged to emulsion III. By increasing the weight percent of hydrophobic PCL segment from 8.5-21.5 wt%, the related particle size increases (Table 1). Our purpose of choosing different amounts 15 ACS Paragon Plus Environment

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of PCL in nanoparticles structure was to study its effect on the final particle size. In addition, the increase of PCL content in the feed was continued as far as the particle size did not exceed 100 nm. Hence, emulsion III was chosen for further studies.

Table 1. Experimental design and characterization of synthesized nanoparticles Emulsion type Emulsion I

PCL (wt%) 8.3

HEMA (wt%) 50

PEG-DA (wt%) 41.6

Particle size (nm) a 52.3±2.4

PDI a 0.28

53

17.2

60.3

Emulsion II

15.3

46.1

38.4

67.1±1.7

0.31

52

17.5

63.5

Emulsion III

21.4

42.8

35.7

83.4±3.1

0.35

55

20.4

71.4

a

Yield(%) DL(%) EE%

Data determined using a zetasizer at 25 °C. Data presented as mean ± SD, n = 5.

3.3. Stability of synthesized nanoparticles. Figure 2 displays the change of particle size of emulsion III based nanoparticles following storage in deionized water at 4°C for 4 weeks. If the ratio of particle size of nanoparticles after storage to its initial size were in the range of 1.0 ± 0.3, indicating stable nanoparticles maintained while outside this range, a significant aggregation or dissociation occurred.41 As PEG is a versatile polymer and can be employed as stabilizer,42 or dispersant43 in the field of emulsion polymerization, after 4 weeks of preparation, the particle size measurements by DLS do not show any clear change compared to the result obtained from fresh emulsion. This result indicates that these nanoparticles are very stable because of the PEG chains on their surfaces. Shan and coworkers proved that PEG in the monomer phase has a great effect on breakage of a polymer/monomer mixture when such a mixture is dropped into a stirred aqueous phase. Because PEG in the monomer phase can be adsorbed on the surface of the copolymer in the mixture, it is easier for the copolymer to separate from the viscous mixture matrix through stirring due to the wedge effect of the PEG. In addition, Zhao and coworkers showed that PEG-functionalized samples do not form agglomerates and remain stable for weeks.42 In addition, the nanoparticles stability can also be attributed to their crosslinked structure.44 This actually indicates that 16 ACS Paragon Plus Environment

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PEG-DA which was used as the crosslinker, can effectively act as the emulsifier in order to provide colloidal stability during SFEP and prevent particles from coagulating.45

Figure 2. The change of particle size of emulsion III based nanoparticles over time in deionized water at 4°C

3.4. Nanoparticles yield, drug loading and encapsulation efficiency. Emulsion III based nanoparticles were prepared in 55% yield determined using eq. 1. The drug loading and encapsulation efficiency were calculated by eqs. 2 and 3 described above. Loading and encapsulation efficiency were found to be 20.4% and 71.4%, respectively (Table 1). Drug loading and encapsulation efficiency of LPE as hydrophobic drug depend mainly on the interactions between drug and the hydrophobic parts of nanoparticles.46 Also, the open nature of the dendritic architecture and an abundance of functional groups facilitate drug encapsulation into the branches of dendritic parts.47 In comparison with our previous study,48 it is concluded that increase in the number of interactions between drug and the polyester play an important role to have appropriate drug loading. Moreover, according to another study of our research group,35 the Brunauer–Emmet–Teller (BET) adsorption data revealed that the N2 adsorption/desorption amount of dendritic structure is higher than that of linear structure. As a result, the presence of poly(amino-ester) block conjugated to PCL segment causes more interactions between the hydroxyl and ester groups of LPE and the functional groups of the polyester in the nanoparticles structure. In addition, the cavities inside the 17 ACS Paragon Plus Environment

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dendritic structure can be useful to incorporate the hydrophobic drug. Therefore, the drugpolymer affinity and drug-loading content were improved considerably. Achieving a higher drug loading is beneficial for nanoparticles loaded hydrogel systems for drug delivery as contact lenses. In fact, although nanoparticles incorporation in hydrogels provides extended drug release, but it has undesirable effects on lens properties such as increase in lens modulus and reduction in its ion and oxygen permeabilities. Hence, it is necessary to minimize nanoparticles loading, while maximizing the drug loading in the particles to insure adequate drug loading in the lens and minimize its impact on lens properties. 3.5. Surface morphology of nanoparticles, hydrogel and nanoparticles loaded hydrogel. The size and surface morphology of the formed polymer nanoparticles were investigated using SEM and TEM microscopy in order to further evaluate their properties. Spherical shape and fine round particles were prepared successfully by SFEP (Figure 3a). Difference in particle size obtained by SEM and TEM compared to DLS is due to the nanoparticles hydration during particle size measurement by DLS.


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Figure 3. SEM image of emulsion III nanoparticles (a), hydrogel free (H5) (b), emulsion III nanoparticles loaded hydrogel (c), TEM image of emulsion III nanoparticles (d)

The SEM image of the cross-section of a pure poly (HEMA-co-VP) hydrogel appears smooth and non-porous and does not have any grain boundaries (Figure 3b). The SEM study was also performed to determine the structure of the nanoparticles loaded hydrogel and directly observe the particles entrapped inside the hydrogel matrix. According to Figure 3c, it is concluded that emulsion III based nanoparticles were stable and did not break or aggregate during the polymerization and thus the gels remained transparent. In accordance with observations obtained from SEM studies, TEM micrograph confirmed that polymeric nanoparticles are completely sphere (Figure 3d). The representative TEM image also illustrates that the as-prepared nanoparticles are uniform in shape as well as ordered in size.

3.6. Equilibrium water content (EWC) of hydrogels and nanoparticles loaded hydrogels. It is worth to investigate the swelling behavior of hydrogels which is a very important factor for their application as a contact lens. For conventional poly HEMA based contact lenses, oxygen transport is provided by water contained within the polymer network with an exponential relationship between permeability and EWC.49 In this regard, swelling studies were performed to study the influence of the hydrogel composition on the swelling properties of poly(HEMA-co-VP) hydrogels using PEG-DA as the crosslinker. In addition, the one with higher EWC% was chosen for nanoparticles loading and further studies. Each hydrogel was placed in PBS (pH=7.4) at 35°C in order to induce maximal swelling under physiologic conditions. Table 2 shows the EWC% for hydrogels prepared from different compositions of HEMA, NVP and PEG-DA. The results reveal that the water absorbance characteristics of hydrogels vary depending on two main factors: (1) amount of hydrophilic groups; (2) cross linking density of the polymer system. Herein, the principal backbone material used is based


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on poly HEMA hydrogel which alone has a water content of 38%.50 In the present study, addition of hydrophilic monomer increases water content in all hydrogel types due to stronger binding power of lactam groups such in pyrrolidone than either hydroxyl or ether groups51. H2 in compared to H1 showed more water content owing to the increase of NVP content in the hydrogel. By keeping the NVP amount constant in the hydrogel, increase of PEG-DA as the crosslinker from 0.3 to 1.5 mg, increase final EWC%. Increasing the density of crosslinkages will cause a decrease in water content. At the same time, the absolute amount of bound water increases as the amount of free water decreases, due to the reduced mobility of water molecules in the more ‘rigid structure’.52 At low levels of water content, all water in hydrogels exists in a tightly bound form. This is obvious in view of the available sites for hydrogen bonding and strong ion–dipole interaction. At a medium level of water content, loosely bound water exists in addition to tightly bound water, which already occupies the available sites for strong interaction. Free water begins to appear at higher levels of water content.50 In this study, increase in crosslinking density which cause less water uptake and in contrast increase in PEG content known as ‘‘super absorbent’’ material, are competing with each other. To elaborate on this topic, use of super absorbent polymer as the crosslinker will help the hydrogel to have more water uptake. As a result, the presence of PEG causes a remarkable capability to bind water molecules when immersed directly in water. As shown in Table 2 (fifth entry), H5 showed more water content than H3 through increasing the NVP content while keeping PEG-DA constant.

Table 2. EWC% of poly(HEMA-co-VP) based hydrogels with different compositions Hydrogel type

HEMA(mg)

NVP(mg)

PEG-DA (mg)

EWC%

H1

10

4

0.3

51.4

H2

10

5

0.3

52.8

H3

10

4

0.8

59.68 20

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H4

10

4

1.5

60.79

H5

10

6

0.8

67.11

Figure 4a shows the time-dependent swelling behavior of hydrogels. Almost in all hydrogel types the majority of swelling took place within 30 minutes and equilibrium swelling was achieved within 80 minutes. It means that when the xerogel is immersed in PBS, the water absorption is in such a way that after 30 minutes the amount of water absorbed by the hydrogel is close to its water content in maximum swelling. After 80 minutes, the amount of water absorption is almost equal to the amount of the equilibrium water content of the hydrogel. According to the present study, hydrogels had EWC% between 51.4%-67.11%. The parameters varied to obtain hydrogels with different water content were the weight fraction of NVP as the hydrophilic co-monomer as well as the amount of PEG-DA as the crosslink agent. The results show that H5 formulation consists of HEMA: NVP: PEG-DA with 10:6:0.8 weight ratios exhibits better swelling behavior compared to other formulations.


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Figure 4. Swelling behaviors of poly(HEMA-co-VP) based hydrogels (a), nanoparticles loaded hydrogel (H5) (b) (Data were expressed as mean ±SD, n = 3)

The effect of nanoparticles addition on swelling of H5 hydrogel in PBS is illustrated in Figure 4b and summarized in Table 3. Since the crosslinked nanoparticles as nanogels are expected to absorb water, the degree of swelling in PBS is expected to increase in proportion to the amount of nanoparticles added. While the nanoparticles content increases from 3 wt% up to 7.5 wt%, the amount of water absorption also increases. However, the incorporation of higher nanoparticles amounts in hydrogel matrix (10 wt% and 15 wt%) lead to lower hydrogel swelling. To explain this phenomenon, two reasons are possible: (1) increase in hydrophobic content of nanoparticles; (2) increase in physical crosslinking between nanoparticles and hydrogel which both play a reverse effect on EWC%. The water content for nanoparticles loading of 7.5 wt% was determined to be 74.8% which is within the range for commercial lenses. 22 ACS Paragon Plus Environment

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Table 3. Transmittance and EWC% values of hydrogels loaded with different amounts of nanoparticles Hydrogel type (%wt) EWC% Transmittance (%) H5 (without nanoparticles) 67.11 98.7 H5 (3%) 69.70 98.2 H5 (5%) 73.88 96.5 H5 (7.5%) 74.80 96.1 H5 (10%) 49.90 94.7 H5 (15%) 49.10 94.7

3.7. Transmittance. Transparency of contact lenses is another important characteristic which must be considered. The interaction between nanoparticles and proper dispersion in hydrogel matrix should be tailored to obtain suitable light transmission. The clarity of the nanoparticles loaded hydrogels was characterized by measuring the transmittance spectra of the 200 µm thick gels in the range from 200 to 900 nm. The UV-vis spectrum is divided into four bands: Visible (400-700 nm), UVA (320 to 400 nm), UVB (280 to 320 nm), and UVC (100 to 280 nm). Each UV band has shown to be absorbed differently by ocular tissues.53 For all six lens types at wavelengths longer than 400 nm, there was a high, relatively uniform range of transmittance of approximately 94.7% to 98.2%. For nanoparticle loaded hydrogels, the spectrum showed a sharp drop off beginning at 370 nm with a 94.7% to 98.2% transmittance and ending at 300 nm with a 1 to 1.5% transmittance. The spectrum for these five lens types then showed a sharp spike beginning at 300 nm and ending at 240 nm with a peak of 18 to 25% transmittance (depending on the % wt of nanoparticles) at 270 nm which can be attributed to the presence of nanoparticles which absorbs UV in the 240–300 nm range and is a desirable property for contact lenses. Below 240 nm, there was less than 1% transmittance for these three lens types. For the control gel, the transmittance spectrum did not show a sharp drop until 300 nm. Above 300 nm, there was approximately a 98.7% transmittance for the H5 without nanoparticles. Beginning at 300 nm, the spectrum decreases rapidly until it began to plateau at 260 nm with less than 1% transmittance at 230 nm and below (data not


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shown). To be brief, both control hydrogel (H5) and the nanoparticles loaded gels exhibit high transmittance in the visible range (shown in Figure 5 and summarized in Table 3). This proves that the presence of nanoparticles does not reduce the visual clarity of the gel.

Figure 5. Photographic images of transparent nanoparticles loaded hydrogels with different mass ratios of nanoparticles 3.8. Mechanical properties. In biomedical applications, it is well known that there should be a good balance between swelling and mechanical properties of hydrogels. In other words, although any approach to increase mechanical properties of hydrogel offsets the hydrogel swelling property, the selection of appropriate hydrophilic monomer(s) and crosslinker addition followed by crosslinking density was found to improve both. The mechanical properties of the control and 7.5% nanoparticles loaded hydrogels are shown in Figure 6. The results show that nanoparticles incorporation in hydrogel matrix increases the storage (G´) and loss (G´´) modulus (data not shown) in compared to free hydrogel. The hydrogel with 7.5% nanoparticles loading has a zero frequency modulus of 0.92 MPa, which is more than that of the hydrogel without nanoparticles (0.51 MPa). The increase in modulus could be due to the presence of the nanoparticles and also slightly increase in crosslinking density of the hydrogel matrix owing to the presence of some unreacted double bond due to the incomplete


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reaction of emulsion III type nanoparticles during the particle formation step. The obtained storage modulus for nanoparticles loaded hydrogel is approximately equal to the value for poly HEMA gels and also within the range of typical commercial lenses.

Figure 6. Frequency dependent storage moduli of hydrogels with and without nanoparticles (Data were expressed as mean ±SD, n = 3)

3.9. Ion permeability. High water content hydrogels possess more open and less tortuous pathways, which will have higher transfer rates for both oxygen and ion transport. Ion permeability of contact lenses is a critical parameter for lens movement on the eye required for post-lens tear turnover and metabolic waste removal.37 Ionic diffusion in polymeric membranes presumably involves dissociation of the ions from the salt, subsequent transfer of the anion and cation to the aqueous medium, and finally, diffusion of the ions in the confined water within the polymer matrix. As a result, ionic mobility will depend on water flux which is a critical parameter together with the water uptake by the lens. In this work, the ionic permeability of hydrogel free and containing 7.5% nanoparticles were determined as a function of time using a conductivity meter. In Figure 7a we plot the salt concentration of both hydrogels in the receptor chamber as a function of time. Previously to the characterization of the salt transport across the samples, a calibration curve correlating salt


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concentrations and conductivity has been measured. It can be observed from Figure 7a that the apparent Na+ permeability obtained for nanoparticles loaded hydrogel is higher than free hydrogel. In fact, the salt diffusion coefficient increased significantly with increasing water uptake. The ion permeability (IP), as the product of diffusivity (D) and partition coefficient (K), is 2.83×10−3±0.000373 mm2/min for 7.5% nanoparticles containing hydrogel which is comparable to that for the control gel (1.71× 10-3 ±0.000541 mm2/min), and larger than the critical value of 1.5×10−6 mm2/min required for on-eye lens motion (Figure 7b). Obtaining sufficient ion permeability, a fluid hydrodynamic boundary layer is formed between the lens and the cornea which is vital for reducing direct abrasion to the eye.

Figure 7. Salt concentration in the receiving chamber as a function of time (a) and ion permeability (b) for hydrogel containing 7.5% nanoparticles and control gel (H5) at 35 °C (Data were expressed as mean ±SD, n = 3)

3.10. Oxygen permeability. The cornea is avascular and derives most of its oxygen from the atmosphere. A sufficient oxygen supply to the cornea maintains corneal integrity and provides defense against infection. Any contact lens acts as a potential barrier to oxygen 26 ACS Paragon Plus Environment

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transport to the anterior ocular surface, and the ability of a material to transport oxygen through the lens is a major factor in determining the clinical success of that material. Therefore, in order to determine if contact lenses provide patients with such levels of oxygen, it is necessary to examine how easily oxygen transfers through the lens material. To establish this, lens oxygen transmissibility (Dk/t), which takes into consideration both material oxygen permeability (Dk) and lens thickness (t), must be evaluated. In our study, oxygen introduced into one of the chambers diffuses through the hydrogel from the chamber with the higher partial pressure of oxygen to the second chamber free of oxygen. The close relationship between water content and oxygen permeability for conventional soft lenses is because of the fact that O2 molecules distribute in the hydrophilic pats of the hydrogel materials. The oxygen permeability of the 7.5% nanoparticles loaded hydrogel was 28.3±1.3 [(10−11(cm2/s)·(ml O2/(ml mm Hg)] which is slightly less than that for the control (27.4±2.2). Oxygen transmissivity values of about 20 and 75 [(10−9(cm2/s)·(ml O2/(ml mm Hg)] are recommended to avoid corneal edema for the open and closed eyes, respectively. According to the measured Dk value and a thickness of 80 µm for a contact lens, a Dk/t value of 35.3 will obtain for contact lenses made from the hydrogel loaded with 7.5% nanoparticles. Normally, conventional hydrogels (based on poly HEMA) have higher water contents and lower Dk/t values compared with silicone hydrogels. In comparison with Dk and Dk/t values of common conventional hydrogel contact lens materials,54 the prepared nanoparticles loaded hydrogel in this study showed more water content and oxygen transmissivity and therefore can effectively use as a daily disposable contact lens by meeting the oxygen level that the open eye needs for healthy eyes and also matching the cornea’s water content, at 74.8% water, to provide all-day comfort. 3.11. Estimation of therapeutic dose. LPE ophthalmic suspension is used to treat eye swelling caused by surgery, infection, allergies, and other conditions. The recommended 27 ACS Paragon Plus Environment

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dosage for 0.5% solution of LPE eye drops is 1 drop 4 times daily which amounts to a daily dose of about 1000 µg. Since the bioavailability of LPE delivered through eye drops is only about 1-2%, therefore the therapeutic requirement of LPE from hydrogel contact lens is about 20 µg/day. Supposing 50% bioavailability for contact lenses, the hydrogel lenses require a release of about 40µg/day. However, poly HEMA based contact lenses can only be worn during the day due to the insufficient oxygen transport. Based on 50% bioavailability and 12 h daily wear time, poly HEMA lenses require a release of about 80µg/day from a contact lens. 3.12. In-vitro drug release of nanoparticles and nanoparticle loaded hydrogel. In the present study, the dialysis bag diffusion method was exploited to assess the drug release behavior of drug loaded nanoparticles. The rate of drug release was determined by measuring the concentration of LPE in the surrounding dissolution media as the drug diffused from the nanoparticles. A representative drug release profile is shown in Figure 8, where the percent of drug released from the nanoparticles is plotted against time. As was stated in the introduction, the motivation behind of the present work is to use the hydrogel containing drug loaded nanoparticles with the ability to release a defined amount of encapsulated compound during long period of time. To demonstrate the superiority of this system to incorporation of drug alone into the feed mixture of polymerization, the release behavior of both hydrogels were studied. As illustrated in Figure 8, the release rate of the hydrogel loaded with 7.5% nanoparticles is less than that for the nanoparticles and hydrogel containing free drug suggesting that the presence of nanoparticles in the hydrogel further slows down drug release. The reason is that during the drug release process, the certain amount of hydrophobic drug which is placed inside the nanoparticles has to first diffuse through the nanoparticles into the poly(HEMA-co-VP) hydrogel, and subsequently diffuse through the gel. The results suggest that the LPE release from 7.5% nanoparticles loaded 28 ACS Paragon Plus Environment

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hydrogel is about 80 µg/day for 12 days, which is potentially in the therapeutic range. In comparison with immersion of the xerogel discs in drug solution, hydrogel embedded with drug loaded nanoparticles has several advantages. Firstly, the release time of drug from the hydrogel increases which means an improvement in the control of drug delivery. This fact can be a consequence of a better distribution of the drug in the polymer matrix. Secondly, the amount of drug included in the hydrogel can be known from the beginning of the gel synthesis and the maximum amount of drug included is higher than the one included by immersion.

Figure 8. Cumulative drug release from free drug dispersion in hydrogel (●), drug loaded nanoparticles (▲), and hydrogel embedded with drug loaded nanoparticles (■) (Data were expressed as mean ±SD, n = 3)

3.14. In vitro cytotoxicity study of nanoparticles and hydrogel. Biocompatibility testing of polymeric biomaterials is an important step in the development of systems for drug delivery and other biomedical applications.55 In this study, the cytotoxicity of emulsion III based nanoparticles and poly (HEMA-co-VP) (H5) hydrogel was evaluated using rabbit corneal epithelial cells (RCEC) according to MTT assay. H5 showed cell viability of 96.31% ± 0.02 after 24h incubation. The cytotoxicity studies results for nanoparticles in different concentrations are presented in Figure 9. The results demonstrate that the cell viability of all


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samples was in the range of 95–100%. Hence, the nanoparticles and hydrogel apparently have no toxicity or stimulatory effect on the selected cells and are convenient for the applications as biomaterials.

Figure 9. Variation of cellular viability of the RCEC after 24 h incubation with different concentrations of nanoparticles. The viability of cells incubated in free nanoparticle medium as control was taken as 100% (Data were expressed as mean ±SD, n = 3)

4. CONCLUSIONS The results reported here indicate the possibility of nanoparticles loaded hydrogel for controlling ocular drug delivery. Nanoparticles synthesized by surfactant-free miniemulsion polymerization showed narrow sized distribution and spherical shape determined by DLS and SEM, respectively. Copolymerization of HEMA with hydrophilic monomer (NVP) using PEG-DA as a super absorbent material enhanced the swelling capacity of hydrogel. Dispersion of different weight percent of nanoparticles in hydrogel mixture revealed that nanoparticles

were

successfully

entrapped

in

the

hydrogel

matrices

after

photopolymerization. SEM images and UV results also proved that the emulsion based nanoparticles were stable and did not break or aggregate during the hydrogel preparation and thus all the gels remained transparent. The obtained storage modulus for nanoparticles loaded hydrogel was approximately equal to the value for poly HEMA gels and also within the range 30 ACS Paragon Plus Environment

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of typical commercial lenses. In addition, the hydrogel can use as a daily disposable contact lens by meeting the ion and oxygen level that the open eye needs per day. Moreover, biocompatibility study of both nanoparticles and hydrogel revealed their low cytotoxicity. In vitro release experiments in perfect sink condition demonstrate that the loaded LPE in the lens can be released for about 12 days which was comparable to free nanoparticles. It is noted that in-vivo release studies are needed to fully determine the benefits of LPE release from prepared contact lenses.

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Figure captions: Scheme 1. Schematic illustration of surfactant-free emulsion polymerization for nanoparticles synthesis (a) and nanoparticles loaded hydrogel preparation (b) (The obtained hydrogel is transparent.) Figure 1. FT-IR spectra of poly HEMA(a), PEG-DA (b), acrylated PCL (c), PCL/PHEMA/PEG-DA nanoparticles (d) and poly(HEMA-co-VP) hydrogel (e) Figure 2. The change of particle size of emulsion III based nanoparticles over time in deionized water at 4°C Figure 3. SEM image of emulsion III nanoparticles (a), hydrogel free (H5) (b), emulsion III nanoparticles loaded hydrogel (c), TEM image of emulsion III nanoparticles (d) Figure 4. Swelling behaviors of poly(HEMA-co-VP) based hydrogels (a), nanoparticles loaded hydrogel (H5) (b) (Data were expressed as mean ±SD, n = 3) Figure 5. Photographic images of transparent nanoparticles loaded hydrogels with different mass ratios of nanoparticles Figure 6. Frequency dependent storage moduli of hydrogels with and without nanoparticles (Data were expressed as mean ±SD, n = 3) Figure 7. Salt concentration in the receiving chamber as a function of time (a) and ion permeability (b) for hydrogel containing 7.5% nanoparticles and control gel (H5) at 35 °C (Data were expressed as mean ±SD, n = 3) Figure 8. Cumulative drug release from free drug dispersion in hydrogel (●), drug loaded nanoparticles (▲), and hydrogel embedded with drug loaded nanoparticles (■) (Data were expressed as mean ±SD, n = 3) Figure 9. Variation of cellular viability of the RCEC after 24 h incubation with different concentrations of nanoparticles. The viability of cells incubated in free nanoparticle medium as control was taken as 100% (Data were expressed as mean ±SD, n = 3) 35 ACS Paragon Plus Environment

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Table captions: Table 1. Experimental design and characterization of synthesized nanoparticles Table 2. EWC% of poly(HEMA-co-VP) based hydrogels with different compositions Table 3.Transmittance and EWC% values of hydrogels loaded with different amounts of nanoparticles


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Scheme 1 70x33mm (300 x 300 DPI)


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Figure 1 243x594mm (300 x 300 DPI)


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Figure 2 12x7mm (300 x 300 DPI)


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Biomacromolecules

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Figure 4 116x146mm (300 x 300 DPI)


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Figure 5 113x160mm (300 x 300 DPI)


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Biomacromolecules

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Biomacromolecules

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Graphical abstract 85x47mm (300 x 300 DPI)


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Preparation and Evaluation of Contact Lenses Embedded with

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