Engineering of polymer-surfactant nanoparticles of doxycycline hydrochloride for ocular drug delivery.

http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–14 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.893381

RESEARCH ARTICLE

Engineering of polymer–surfactant nanoparticles of doxycycline hydrochloride for ocular drug delivery Varsha Pokharkar, Vikram Patil, and Leenata Mandpe

Drug Delivery Downloaded from informahealthcare.com by Selcuk Universitesi on 12/24/14 For personal use only.

Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, Maharashtra, India

Abstract

Keywords

Context: Physiologic barriers of the eye, short precorneal drug residence time and poor corneal penetration are the few reasons for reduced ocular bioavailability. Objective: This study was aimed to develop novel polymer–surfactant nanoparticles of hydrophilic drug doxycycline hydrochloride (DXY) to improve precorneal residence time and drug penetration. Materials and methods: Nanoparticles were formulated using emulsion cross-linking method and the formulation was optimized using factorial design. The prepared formulation was characterized for particle size, potential, encapsulation efficiency, in vitro drug release and ex vivo drug diffusion studies. The antibacterial activity studies were also carried out against Escherichia coli and Staphylococcus aureus using the cup-plate method. In vivo eye irritation study was carried out by a modified Draize test in rabbits. Results and discussion: The particle size was found to be in the range of 331–850 nm. About 45–80% of the drug was found to be encapsulated in the nanoparticles. In vitro release demonstrated sustained release profile. Lower flux values in case of nanoparticles as compared to DXY pure drug solution in ex vivo diffusion studies confirmed the sustained release. The nanoparticles were found to be significantly effective (p50.001) than DXY aqueous solution due to sustained release of doxycycline from nanoparticles in both the E. coli and S. aureus strains. The formulation was found to be stable over entire stability period. Conclusion: The developed formulation is safe and suitable for sustained ocular drug delivery.

Emulsion cross-linking method, factorial design, gellan gum, polymer–surfactant nanoparticles, sustained release

Introduction Formulating an ocular drug delivery has remained the most challenging task due to the unique structure of the eye that restricts the entry of the drug molecule at the site of action (Hughes & Mitra, 1993). Various precorneal loss factors that contribute to the reduced ocular bioavailability include rapid tear turnover, nonproductive absorption, transient residence time in the cul-de-sac and the relative impermeability of the drugs to the corneal epithelial membrane (Kaur et al., 2004; Pijls et al., 2005). To achieve the therapeutic effect, frequent administrations are mandatory which in turn lead to poor patient compliance (Gupta et al., 2010). Ocular bioavailability can be enhanced by increasing corneal drug penetration and prolonging precorneal drug residence time (de Campos et al., 2004). Various drug delivery systems that have been reported till date for increasing ocular bioavailability include ocular inserts (Ding, 1998), collagen shields (Hill et al., 1993), colloidal systems such as liposomes (Pleyer et al., 1993;

Address for correspondence: Prof. Varsha B. Pokharkar, Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune 411038, Maharashtra, India. Tel: +91 2025437237. Fax: +91 2025439383. Email: vbpokharkar@ yahoo.co.in

History Received 9 December 2013 Revised 7 February 2014 Accepted 7 February 2014

Bochot et al., 1998), nanoparticles, nanosuspension (Losa et al., 1991; Ding, 1998) and nanocapsules (Losa et al., 1993; de Campos et al., 2003). The literature survey suggests that an appropriate particle size and a narrow size distribution in case of ocular formulation ensure less irritation, adequate bioavailability and compatibility with ocular tissues (Guinedi et al., 2005). Doxycycline hydrochloride (DXY) is a broad-spectrum semisynthetic antibiotic, derived from oxytetracycline. It is bacteriostatic in nature and inhibits the bacterial protein synthesis by blocking the binding of aminoacyl tRNA to the mRNA at the ribosomal sites (Stratton & Lorian, 1996). It is also well recognized for its therapeutic efficacy in treating matrix metalloproteinases (MMPs) mediated ocular surface diseases, such as rosacea, corneal neovascularization, recurrent epithelial erosions and sterile corneal ulcerations (Seedor et al., 1987; Akpek et al., 1997; Dursun et al., 2001; Su et al., 2011). DXY has been found to inhibit MMP-9 activity in vivo in the corneal epithelial cells of experimental dry eye (De Paiva et al., 2006) as well as in vitro corneal epithelial cells in humans (Li et al., 2004; Kim et al., 2005). Treatment with DXY has been shown to be beneficial in attenuating acute and delayed ocular injuries caused by sulfur mustard (SM) exposure (Amir et al., 2004; Kadar et al., 2009). The drug is an inexpensive, FDA approved antibiotic that likely


2

V. Pokharkar et al.

promotes wound healing by reducing inflammation and protease activity. Although DXY is commercially available in a wide variety of dosage formulations such as tablets, capsules and suspensions for oral delivery; topical ocular DXY formulations are not available. Hence there is a critical need for a controlled release DXY delivery system that can be easily applied to the eye to promote wound healing. To the best of our knowledge this is the first report for nanoparticulate ocular sustained drug delivery of DXY. Nanocarriers such as polymer–surfactant nanoparticles are the preferred choice for the ocular drug delivery as they have the capacity to deliver drug at the specific target site at a steady rate for a prolonged period of time. The ability of nanoparticulate system to encapsulate hydrophilic as well as hydrophobic actives makes them an interesting carrier for delivering drugs across the ocular barriers and helps to reduce the toxicity of the encapsulated drug (Wadhwa et al., 2009; Diebold & Calonge, 2010). Further conversion of pure drug into nanoparticulate system provides stability to the system. In this study, we have developed a novel polymer– surfactant nanoparticulate formulation, using the anionic surfactant Aerosol OTÔ (AOT) and a polysaccharide polymer gellan gum for ophthalmic delivery of water-soluble drug DXY. An emulsion cross-linking technique was used to formulate DXY-loaded nanoparticulate system and the formulation was further optimized using 23 factorial design. Three independent variables viz. concentration of gellan gum, AOT and DXY were studied for their influence on dependent variables – particle size, potential and the percentage of encapsulation. The formulation was also evaluated for in vitro drug diffusion studies, antibacterial activity and in vivo eye irritation studies in rabbits. The formulations were also subjected to 3 months stability studies.

Materials and methods Materials DXY was received as a gift sample from Lupin Research Park (Pune, India). Gellan gum (Kelcogel) was obtained as a gift sample from CP Kelco (Mumbai, India). AOT was purchased from Sigma Aldrich (Mumbai, India). Polyvinyl alcohol (PVA) was purchased from Loba Chemie Pvt. Ltd (Mumbai, India). Dichloromethane (DCM), calcium chloride, methanol and ethanol were purchased from E-Merck (Mumbai, India). Nutrient agar and Luria broth were purchased from Himedia Lab. Pvt. Ltd (Mumbai, India). All other chemicals were of analytical grade. Milli Q water (Nanopure Diamond by Barnstead, Dubuque, IA, USA) was used throughout the experiment. Methods Preparation of DXY loaded nanoparticles DXY loaded nanoparticles (DNPs) were prepared by using an emulsion cross-linking technique (Chavanpatil et al., 2007). Briefly, aqueous solution of gellan gum was prepared and weighed quantity of DXY was dissolved in this solution. One milliter of this solution was emulsified into AOT solution prepared in DCM (3 ml) by sonication (U 200S Control, 230v, IKA Labortechnik, Staufen, Germany) over a period of 1 min

Drug Deliv, Early Online: 1–14

on ice bath. The resulting primary emulsion was then emulsified into aqueous solution of PVA (2% w/v, 15 ml) by sonication over a period of 3 min on ice bath, in order to form a secondary water-in-oil-in-water (w/o/w) emulsion. Further 5 ml of aqueous calcium chloride solution (30%, w/v) was added gradually to the above emulsion with stirring. Resulting emulsion was further stirred for 18 h. Formed methylene chloride was removed by rotating the emulsion under vacuum in rotary evaporator (Superfit 01222) at 60 rpm for 1 h. Nanoparticles so formed were recovered by centrifugation (AllegraÔ 64R Centrifuge, Beckman-Coulter India Pvt Ltd, Andheri (East), Mumbai, Maharashtra, India) at 20 000 rpm for 30 min. To remove PVA and unentrapped drug, nanoparticles were washed twice with deionized water and centrifuged. The residue so obtained was resuspended in water and lyophilized (LYOLAB, Lyophilization Systems India Pvt Limited, Hyderabad, India) at 40 C and 0.0010 mbar pressure for 24 h. The lyophilized powder was used for characterization such as differential scanning calorimetry (DSC), X-ray diffractometry (XRD), Fourier transform-infrared spectroscopy (FTIR). All the other studies were performed using liquid sample. Blank batch (void nanoparticles) was prepared without addition of DXY. Aqueous solution of DXY was prepared by dissolving DXY into deionized water. The prepared formulation was analyzed for the presence of residual solvents and found to be within the acceptable limits (data not shown). Effect of variables To study the effect of variables on nanoparticulate performance and characteristics, different batches were prepared using 23 factorial design approach. The concentrations of gellan gum, AOT and DXY were selected as formulation variables. All other formulation and processing variables were kept constant throughout the study. Table 1 summarizes an account of the eight experimental runs studied, values of all variables and the batch codes. A percentage encapsulation efficiency, particle size and potential were selected as response variables. The experimental design and statistical analysis of the data were done using the Design-Expert Software (Version 8.0.5, Stat-Ease, Inc, Minneapolis, USA). Evaluation of DNPs Particle size determination Particle size and particle size distribution of DNPs were determined by laser diffractometer (LD) using Mastersizer 2000 SM version 2.00 (Malvern Instruments, Malvern, UK). Each measurement was performed in triplicate. The Mie theory (dispersant refractive index ¼ 1.33; real particle refractive index ¼ 1.57; imaginary part of the particle refractive index ¼ 0.001) was used for particle size calculation. The samples were diluted with distilled water to obtain an appropriate obscuration. Particle sizes were expressed by the volume-based 90% (d 0.9) diameter percentiles. The width of the size distribution was indicated by polydispersity index (PDI). The optimized batch was also analyzed with photon correlation spectroscopy for particle size (Brookhaven 90 Plus particle sizing software version 3.94, Brookhaven Instruments Corp, Holtsville, NY, USA).

Engineering of polymer–surfactant nanoparticles

DOI: 10.3109/10717544.2014.893381

3

Table 1. 23 Factorial design using formulation variables influencing the percentage of encapsulation (Y1), particle size (Y2) and zeta potential (Y3). Concentration Exp. no. 1 2 3 4 5 6 7 8

Response

Gellan gum (X1, % w/v)

AOT (X2, % w/v)

DXY (X3, mg)

Y1 (%)

Y2 (nm)

Y3 (mV)

(+1) 1.0 (+1) 1.0 (1) 0.5 (1) 0.5 (1) 0.5 (+1) 1.0 (1) 0.5 (+1) 1.0

(+1) 20 (+1) 20 (1) 5.0 (+1) 20 (1) 5.0 (1) 5.0 (+1) 20 (1) 5.0

(1) 5.0 (+1) 10 (1) 5.0 (+1) 10 (+1) 10 (+1) 10 (1) 5.0 (1) 5.0

79.93 ± 2.4 77.86 ± 2.1 56.26 ± 3.7 79.88 ± 3.8 45.73 ± 4.1 53.66 ± 4.2 80.07 ± 4.6 56.7 ± 2.5

483 ± 2.5 595 ± 3.1 488 ± 2.8 383 ± 4.2 590 ± 3.3 850 ± 4.5 331 ± 3.4 630 ± 4.6

17.8 ± 1.5 15.4 ± 2.4 15.1 ± 2.2 22.6 ± 1.5 13.9 ± 3.1 9.88 ± 2.5 25.2 ± 2.9 11.5 ± 3.1


z Potential determination The surface charge of the particles was assessed in triplicates by potential measurements using Zetasizer 300 HSA (Malvern Instruments, Malvern, UK). The potential was measured after dilution of samples (50–100 ml) with 5 ml of distilled water at room temperature. Encapsulation efficiency (%EE) The percentage of encapsulated DXY was determined by centrifugation method. The prepared nanoparticulate formulation was centrifuged at 20 000 rpm for 30 min, supernatant was separated and analyzed spectrophotometrically at 346 nm to get the amount of free drug. The percentage of encapsulation efficiency was determined by following equation. Total amount of DXY Amount of free DXY %EE ¼ 100: ð1Þ Total amount of DXY Determination of pH of the formulation The pH of the prepared formulations was measured by using a pH meter (Toshniwal Instruments Limited, Chennai, Tamilnadu, India).

basis of ANOVA. To study the combined effect of different variables on the response, 3-D response surface plots were generated. The optimum values of dependent variables to achieve the desired response were calculated using the numerical optimization tool along with desirability approach. Powder X-ray diffractometry The powder X-ray diffractometry (PXRD) patterns of pure drug DXY, void nanoparticles and DNPs were recorded using an X-ray diffractometer (PW 1729, Philips, Eindhoven, Netherlands) with Cu as anode material, crystal graphite monochromator operated at a voltage of 30 kV and a current of 30 mA. The samples were exposed to a Cu Ka radiation over a range of 2 angles from 2 to 50 . The range and the chart speed were 5 103 CPS and 10 mm/ 2, respectively. Fourier transform-infrared spectroscopy To investigate the possible chemical interactions between the pure drug DXY and the polymer matrix, FTIR spectra were taken on the JASCO V5300 FTIR (Tokyo, Japan). Samples were crushed with KBr to produce pellets by applying a pressure of 150 kg/cm2. FTIR spectra of pure drug DXY, void nanoparticles and DNPs were scanned in the 4000– 400 cm1 range. Differential scanning calorimetry

Statistical data analysis Polynomial models including interaction and quadratic terms were generated for all the response variables using regression analysis. The general form of the regression analysis model is represented as Equation (2). Y ¼ 0 þ 1 X1 þ 2 X2 þ 3 X3 þ 4 X1 X2 þ 5 X1 X3 þ 6 X2 X3 þ 7 X12 þ 8 X22 þ 9 X32 ,

ð2Þ

where 0 is the intercept representing the arithmetic average of all quantitative outcomes of eight runs; 1–9 are the coefficients estimated from the observed experimental values of response variable Y; X1, X2 and X3 are the coded levels of the independent variables and X1X2, X1X3, X2X3 and X12 , X22 , X32 are the interaction and quadratic terms, respectively. The interaction terms show how the response changes when two factors were simultaneously changed. The second-degree terms (X12 , X22 , and X32 ) help to investigate nonlinearity. Statistical validity of the polynomials was established on the

The DSC measurements were performed using METTLER Toledo DSC 821e module controlled by STARe software (Mettler-Toledo India Private Limited, Powai-Mumbai, Maharashtra, India). Each sample (10–15 mg of pure drug DXY, void nanoparticles and DNPs) was sealed separately in a standard aluminum pan, the samples were purged in DSC with pure dry nitrogen set at a flow rate of 10 ml/min, the temperature speed set at 10 C/min and the heat flow recorded from 0 to 250 C. An empty aluminum pan was used as reference. Transmission Electron Microscopy Morphologic evaluation of the DNPs was performed using transmission electron microscopy (TEM; Philips CM-200). A drop of nanoparticulate suspension was placed on FormvarÕ coated copper grids (Ted Pella, Inc., Redding, CA) and allowed to equilibrate. Excess liquid was removed with a filter paper and dried at room temperature for about half an hour. The dried grid containing the nanoparticles was

4

V. Pokharkar et al.


visualized using TEM. Digital Micrograph and Soft Imaging Viewer software (Olympus Soft Imaging Solutions Pte Ltd, Jurong East St, IMM Building, Singapore) were used to perform the image capture and analysis.


In vitro drug diffusion study In vitro drug diffusion study was performed in triplicate using a Franz-type diffusion cell. The cell consists of two chambers, the donor and the receptor (surface area 7.06 cm2, capacity 22 ml). The receptor chamber was filled with freshly prepared phosphate buffer (pH 7.4) to ensure perfect sink conditions and was provided with a sampling port. The 2 2 cm piece of activated cellophane membrane (cut-off: 12 000 to 14 000 Da) was mounted over diffusion cells and then air bubbles were removed. Recirculating water bath was maintained at 37 ± 0.5 C and the solution in the receptor chambers was stirred at 200 rpm with a small magnetic bar for uniform mixing of the contents. Prior to application of formulations the membrane was allowed to equilibrate for 30 min. Donor compartments were filled with 10 ml of optimized batch of DNPs and aqueous solution of DXY separately and covered with aluminum foil to prevent evaporation of vehicle. At set time interval within 24 h, 2 ml of sample was withdrawn from the receptor chamber and immediately replaced by the equal volume of phosphate buffer solution. The sample was then appropriately diluted and quantitatively analyzed by using U.V. spectrophotometer at 346 nm. By determining the amount of DXY diffused at various time intervals, the percentage of DXY release versus time (h) graphs was plotted. The results of in vitro data were analyzed by Microsoft excel to obtain the best fit kinetic model for in vitro drug release.

Transcorneal permeability of prepared DNPs and DXY pure drug solution was studied with the help of goat cornea. Fresh whole eyeballs of goat were obtained from a local butcher’s shop. Corneas were carefully removed along with the surrounding scleral tissue and stored in freshly prepared artificial tear solution, pH 7.4. Diffusion of DXY across the cornea was measured in triplicate using a Franz-type diffusion cell. The cornea was excised, blotted dry and mounted between donor and receptor chambers. Donor compartments were filled with optimized batch of DNPs (equivalent to 5 mg of DXY) and aqueous solution of DXY (equivalent to 5 mg of DXY), separately. At set time interval within 24 h, 2 ml of sample was withdrawn from the receptor chamber and immediately replaced by the equal volume of phosphate buffer solution. The sample was then appropriately diluted and quantitatively analyzed by using UV spectrophotometer at 346 nm. The apparent corneal permeability coefficients (p) were calculated according to the equation below: dQ=dt , A C0

Animal Male New Zealand albino rabbits weighing 2–3 kg and free of any signs of ocular inflammation or gross abnormality were used in the study. The study was performed in accordance with the Institutional Animal Ethics Committee constituted as per directions of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA/41/ 2010), under the Ministry of Animal Welfare Division, Government of India, New Delhi. The rabbits were divided into three groups (n ¼ 3). The first group was administered with DXY aqueous solution, the second group was administered with void nanoparticles and the third group was administered with DNPs. Ocular tolerability The potential ocular irritancy and/or damaging effects of DXY aqueous solution, void nanoparticles and DNPs test formulations were evaluated according to a modified Draize test (Falahee et al., 1981; McDonald & Shadduck, 1991) using a slit-lamp. The test formulations (50 ml) were topically administered in the left eye every 30 min for 6 h (12 treatments). Right eyes served as controls and were treated with distilled water. Three observations at 10 min, 6 and 24 h after last treatment were carried out to evaluate the ocular tissues. The congestion, swelling, discharge and redness of the conjunctiva were graded on a scale from 0 to 3, 0 to 4, 0 to 3 and 0 to 3, respectively, and irritation and corneal opacity were graded on a scale from 0 to 4 (Pignatello et al., 2002). Evaluation of antibacterial activity on strains of Escherichia coli and Staphylococcus aureus

Ex vivo drug diffusion study

p¼

Eye irritancy evaluation

ð3Þ

where dQ/dt is the steady-state slope of the linear portion of the plot of the percentage drug release (Q) versus time (t); A is the area of exposed corneal surface (3.46 cm2) and C0 is the initial concentration of drug in the donor cell.

A layer of nutrient agar (20 ml) seeded with the test microorganism (0.1 ml) was allowed to solidify in the petri plate. Cups were made on the solidified agar layer with the help of a sterile borer at 10-mm diameter. Then, formulations (DXY aqueous solution and DNPs) containing 1–8 mg/ml of DXY was separately poured into two cups. After keeping petri plates at room temperature for 4 h, the plates were incubated at 37 C for 24 h. The diameter of the zone of inhibition was measured by an antibiotic zone finder. Readings were taken in triplicate. Determination of MIC and minimum bactericidal concentration of DXY The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by microdilution or serial dilution method. Briefly, E. coli and S. aureus bacterial culture containing 0.5 McFarland (1.5 108 CFU/ml) of organisms in Luria broth was added to various concentrations of DXY aqueous solution and DNPs (1–8 mg/ml). The MIC concentration of DXY was defined as the lowest concentration inhibiting visible growth of bacteria after overnight incubation of the cultures at 37 C. The MBC is measured by subculturing the broths used for MIC determination onto fresh agar plates. The MBC is the


DOI: 10.3109/10717544.2014.893381

lowest concentration of a drug that results in killing 99.9% of the bacteria being tested.

and DNPs (Figure 1A(c)) showed complete absence of diffraction peaks.

Statistical data analysis

FTIR

All the data is reported as the mean ± standard deviation. The significance of differences was evaluated using two-way ANOVA followed by Bonferroni post-tests (Graphpad PrismVersion 5.0).

Pure drug DXY showed characteristic bands due to different functional groups and include bands at 3388 cm1 (O–H/N–H stretching vibrations), 2924 and 2855 cm1 (C–H stretching vibrations), 1673 cm1 (primary amide (N–H) bending), 1583 cm1 (aromatic N–H bending vibrations), 1615 cm1 (carbonyl (C¼O) stretching vibrations), 1458 cm1 (CH2 bending), 1328 cm1 (C–H bending vibrations) and 1219 and 1171 cm1 (C–N stretching vibrations) (Figure 1B(a)). DNPs displayed identical bands as that of the void nanoparticles (Figure 1B(b)) in addition to some extra bands due to DXY. Notice that bands appearing at 3412, 2946, 2865, 1725, 1615, 1577, 1461 and 1294 cm1 for DXY are also appearing in DNPs, indicating the chemical stability of DXY on the surface of nanoparticles (Figure 1B(c)).

Stability studies Stability studies were carried out for optimized DNPs suspensions as per ICH guidelines (25 C/60%RH and 40 C/75%RH) for 3 months. DNPs were stored in closed glass vials and each withdrawal of the sample was characterized for particle size, PDI and drug entrapment at 1-, 2- and 3-month time intervals. Drug Delivery Downloaded from informahealthcare.com by Selcuk Universitesi on 12/24/14 For personal use only.

5

Results Particle size determination

DSC

The average particle size of all the batches as measured by laser diffraction was found to be in the range of 331–850 nm (Table 1). The particle size of optimized DNPs was found to be 383 ± 4.2 nm (180 nm by dynamic light scattering) with PDI of 0.128. The difference in the particle size of both the measurements is explained with the help of the principle involved in the particle size analysis. The particle size analysis by lased diffraction is volume based while the mean diameter obtained by dynamic light scattering is light intensity weighted size. Particle size obtained by laser diffraction is usually higher (Jacobs & Mu¨ller, 2002). Similar results were observed by Pokharkar et al. (2013) where the particle size obtained by laser diffraction was 168 nm and by PCS it was 46.3 nm for bicalutamide nanosuspension prepared by antisolvent precipitation method.

DSC studies were performed on DXY, gellan gum, void nanoparticles and DNPs. Gellan gum showed a broad endotherm at 102 C which is characteristic of water loss from gellan gum (Figure 1C(a)). The endothermic peak of DXY was found to be at approximately 198 C (Figure 1C(b)). The DSC curves of void nanoparticles are almost identical to gellan gum polymer curve, indicating no influence of the organic solvents used in the formulations on the thermal properties of gellan gum (Figure 1C(c)). DSC thermogram of DNPs did not show the native DXY peak at 198 C, while the endothermic peak of gellan gum was present distinctly in the formulation (Figure 1C(d)). The absence of detectable crystalline domains of DXY in DNPs clearly indicates that the drug is in the amorphous or solid state solubilized form in the polymer matrix (Misra et al., 2009).

Zeta potential () determination The zeta potential of DNPs was in the range of 10 to 25 mV (Table 1). The zeta potential of optimized DNPs was found to be 22.6 mV. Encapsulation efficiency The encapsulation efficiency (%EE) of prepared DNPs was determined by indirect method. The amount of free DXY present in the supernatant after centrifugation of prepared nanoparticles was determined by UV analysis. About 45.73– 80.07% of drug was found to be encapsulated in to the lipid matrix. pH study pH values for all the formulations were found to be in the range of 5.8–6.5. XRD analysis Pure drug DXY presented several sharp diffraction peaks at 11 , 15.1 , 17.9 , 22.8 and 24.7 2 (Figure 1A(a)). PXRD studies of prepared void nanoparticles (Figure 1A(b))

TEM TEM analysis revealed irregular shape particles with approximate particle size of about 180 nm (Figure 1D). In vitro drug diffusion study To elucidate the release mechanism of DNPs, in vitro drug diffusion studies were carried out using vertical Franz diffusion cells. Phosphate buffer (pH 7.4) was used as diffusion medium. The temperature was maintained at 37 ± 0.5 C. The release was compared with aqueous DXY solution. Developed AOT–gellan gum nanoparticles released 22.12% of DXY in 2 h, 33.38% in 4 h, 44.94% in 6 h and 83.82% in 24 h compared with 68.96%, 86.74% and 96.94% in 2, 4 and 6 h, respectively, of the plain aqueous drug solution (Figure 2). Ex vivo drug diffusion study The ex vivo diffusion studies demonstrated similar release pattern as that obtained using cellophane membrane. The DXY aqueous solution released 98% of the drug at the end of 6 h while DNPs showed 80% release at the end of 24 h. The results depicted good corneal permeation for longer period of


6

V. Pokharkar et al.


Figure 1. (A) X-ray diffractogram: (a) DXY, (b) void nanoparticles and (c) DNPs; (B) FTIR spectra: (a) doxycycline, (b) void nanoparticles and (c) DNPs; (C) DSC thermograms: (a) gellan gum, (b) DXY, (c) void nanoparticles and (d) DNPs; (D) transmission electron microphotographs of DNPs.

(7.85 104 mg/cm2/h) in case of DXY aqueous solution at the end of 6 h corresponds to faster drug release which was achieved within 6 h. Nanoparticles exhibit the property of surface adhesion. This helps to form the depot from which the drug permeates in sustained fashion providing therapeutic effect for longer duration and reducing the frequent administration (Figure 3). Eye irritancy evaluation

Figure 2. In vitro drug release profile of aqueous solution of DXY and optimized DNPs over a period of 24 h by Franz diffusion cells (mean ± SD, n ¼ 3).

There was no difference between control solution, void nanoparticles and DNPs with respect to the degree of irritation on the cornea and conjunctiva irrespective of the time when the degree of irritation was measured (Figure 4 and Table 2). Antibacterial activity of DXY on strains of E. coli and S. aureus

time. The permeability coefficient value of 1.81 104 mg/ cm2/h in case of DNPs at the end of 24 h signifies the slow diffusion of the drug from the lipid matrix thereby maintaining the constant concentration of the drug in the receiver chamber. The higher permeability coefficient

The antibacterial property of pure drug DXY and DNPs was tested on the strains of E. coli (NCIM-2065; Figure 5A) and S. aureus (NCIM-2066; Figure 5B). by cup-plate technique. Both the strains were found to be susceptible to DXY as distinct zone of inhibition was observed around the wells.

DOI: 10.3109/10717544.2014.893381

Determination of MIC and MBC of DXY The MIC of pure drug DXY on strains of E. coli and S. aureus was found to be 4.67 ± 0.58 mg/ml and 3.67 ± 0.58 mg/ml, respectively, whereas MIC of DNPs on strain of E. coli and S. aureus was found to be 2.67 ± 0.58 and 1.67 ± 0.58 mg/ml, respectively (Figure 6A).


7

The MBC is measured by subculturing the broths used for the MIC determinations onto fresh agar plates. MBC was 7.67 ± 0.58 and 6.67 ± 0.58 mg/ml on strain of E. coli and S. aureus, respectively, for native DXY, where as 5.67 ± 0.58 and 4.67 ± 0.58 mg/ml for DNPs on strain of E. coli and S. aureus, respectively (Figure 6B).


Stability studies During 3 months of stability studies no marked differences were observed in the mean particle size of the DNPs. No changes in macroscopic properties were observed. Very small values for PDI ensure stability of the system. Furthermore, drug entrapment remained constant, indicating that no leakage or release occurred in the final concentrated suspension. Thus, from the above results it can be concluded that gellan gum–AOT nanoparticulate suspension showed good stability at 25 C and 60% RH and 40 C and 75% RH (Table 3). Thus, the formulation can be expected to be stable, safe and effective after long-term storage.

Discussion

Figure 3. Ex vivo drug release profile of aqueous solution of DXY and optimized DNPs over a period of 24 h by Franz diffusion cells (mean ± SD, n ¼ 3).

In the present investigation, the DXY loaded polymer– surfactant nanoparticles were prepared by emulsion crosslinking technique. Gellan gum, an anionic polysaccharide, was used as polymer and AOT was used as a surfactant. Gellan gum is water soluble and commercially available as free flowing powder. It is approved by USFDA and European

Figure 4. Results from eye irritation studies of DNPs suspension performed using modified Draize test.

8

V. Pokharkar et al.


Table 2. Results from Draize eye irritation test. Degree of irritation 10 min Parameters Cornea Opaqueness Iris Irritation value Conjunctiva Degree of flare Degree of swelling Degree of redness Congestion Secretion

Control

Blank NPs

6h Test

Control

Blank NPs

24 h Test

Control

Blank NPs

Test

0

0

0

0

0

0

0

0

0

0

0.5

0.5

0

1

1

0

0

0

0 0 0 0 0

0.5 0.5 0 0 0

0.5 0.5 0 0 0

0.5 0 0 0 0

0.5 0.5 1 1 0.5

0.5 0.5 1 1 0.5

0.5 0 0 0 0

0.5 0 0 0 0

0.5 0 0 0 0


Grading scale: irritation and corneal opacity – 0 to 4; congestion – 0 to 3; flare – 0 to 4; swelling – 0 to 4; secretion – 0 to 3; redness of the conjunctiva – 0 to 3.

Figure 5. (A) Zone of inhibition for the aqueous solution of DXY and DNPs on strain of E.coli and (B) zone of inhibition for the aqueous solution of DXY and DNPs on strain of S. aureus.

Figure 6. (A) Comparison of minimum inhibitory concentration of DNPs suspension with DXY aqueous solution on strain of E. coli and S. aureus at 24 h (mean ± SD, n ¼ 3) and (B) comparison of MBC of DNPs suspension with DXY aqueous solution on strain of E. coli and S. aureus at 24 h (mean ± SD, n ¼ 3).

Union for use as food, cosmetic and pharmaceutical additive (Dhar et al., 2008). Gellan gum is capable of gelation in the presence of mono- and divalent ions by forming an ordered gellan chains. Certain unique properties of gellan gum such as

gelling ability with specific ions, thermal stability, need of very low concentrations for preparing the ocular formulations, controlled gel phase transition and effective and efficient delivery make them an ideal polymer for an ophthalmic


DOI: 10.3109/10717544.2014.893381

Table 3. Particle size, PDI and the percentage of encapsulation efficiency of optimized batch subjected to stability studies for 3 months at various stability conditions. Condition Time (months) Particle size (nm) Polydispersity index Encapsulation efficiency (%)


Particle size (nm) Polydispersity index Encapsulation efficiency (%)

0

1

2

3

383 ± 4.2

388 ± 2.3

385 ± 2.1

389 ± 1.5

0.128 ± 0.03

0.134 ± 0.05

0.135 ± 0.03

0.137 ± 0.02

79.88 ± 3.8

77.23 ± 1.7

80.32 ± 2.1

78.26 ± 1.9

40 C/75% RH 3855 ± 2.1 386 ± 1.2

388 ± 4.1

383 ± 4.2

responses Y1, Y2 and Y3 can be represented by Equations (4)–(6), respectively. ðY1 Þ ¼ 66:26 þ 0:78X1 þ 13:17X2 1:98X3 ,

25 C/60% RH

0.128 ± 0.03

0.129 ± 0.01

0.134 ± 0.04

0.126 ± 0.05

79.88 ± 3.8

79.95 ± 2.5

78.09 ± 2.1

77.86 ± 1.6

delivery. The electrolytes of the tear fluid, especially Na+, Ca2+ and Mg2+ cations are particularly suited to initiate gelation of the polymer when instilled as a liquid solution into the cul-de-sac. Increased precorneal residence time and increased ocular bioavailability of timolol has been observed when formulated as an aqueous solution containing gellan gum (Rozier et al., 1989). AOT, also called as di-octyl sodium sulfosuccinate is an anionic surfactant, having a polar sulfosuccinate head group and large branching, di-octyl side chain. AOT forms reverse micelles in non-polar solvents such as methylene chloride, hexane, etc. and being a double chain amphiphilic surfactant, forms bilayer structure in multiple emulsions (Israelachevilli, 1991). Further, anionic AOT also interacts with cationic calcium ions to form insoluble salts. On the addition of calcium ions, gellan gum and AOT get cross-linked forming a core comprising of gellan gum and AOT head groups, surrounded by hydrophobic matrix comprising of AOT side chain with DXY encapsulated in the core. The aqueous core of gellan gum entrapped into the reverse micelles formed by the AOT in methylene chloride was further emulsified in the aqueous phase using PVA as a secondary emulsifier. Preliminary studies demonstrated that the particle size, encapsulation efficiency and zeta potential of nanoparticles were significantly influenced by the concentration of polymer, surfactant and drug. Hence the concentration of gellan gum, concentration of AOT and concentration of DXY were selected as three independent variables and optimization of the formulation was conducted using response surface methodology using 23 factorial design approach. Particle size, zeta potential and encapsulation efficiency were selected as dependent variables (Table 1). The design resulted into total eight batches. The results of response generated using the experimental design were fitted into polynomial models and ANOVA test was applied to estimate their significance. Equations (4)–(6) represent the reduced models having significant responses. The results of this analysis revealed that the response encapsulation efficiency (Y1) fitted best into the quadratic model, while the response for particle size (Y2) and potential (Y3) fitted best into quadratic model with backward elimination. The polynomial models for the

9

ðY2 Þ ¼ 543:75 þ 95:75X1 95:75X2 þ 60:75X3 þ 22:25X1 X3 19:75X2 X3 , ðY3 Þ ¼ 16:42 þ 2:78X1 3:83X2 þ 0:98X3 þ 0:87X1 X2 þ 0:27X2 X3 :

ð4Þ ð5Þ

ð6Þ

Equation (4) quoted above reveals that all the three variables significantly affect encapsulation efficiency (Y1). The concentration of gellan gum (X1) and concentration of AOT (X2) positively affect the response Y1. However, Y1 was antagonistically affected by the linear contributions of concentration of DXY (X3). The response surface plots were also created to investigate the simultaneous effect of two variables on the response variables when one variable was kept at constant level. Encapsulation efficiency is the percentage of the actual mass of drug encapsulated in the polymeric carrier, relative to the initial amount of loaded drugs. Figure 7 shows the combined effect of concentration of AOT and concentration of gellan gum on the percentage of encapsulation. It can be observed that the effect of AOT is more pronounced than the effect of gellan gum. Thus, concentration of AOT is the limiting factor; any subtle variation in AOT concentration will greatly influence the percentage of encapsulation. In the present technique, AOT forms reverse micelles, which along with gellan gum act as nanoreservoir for encapsulation of DXY. Increasing the amount of AOT allows encapsulation of more amount of DXY within the matrix. The maximum encapsulation of drug within the matrix occurs in nanoparticles prepared using lower drug concentration and higher AOT concentration. Thus increased encapsulation of DXY may lead to enhanced therapeutic efficacy for use in the clinical practice. Equation (5) revealed that all the three independent variables significantly affect the particle size either alone or when combined with each other. The particle size analysis demonstrates positive relationship with the concentration of gellan gum (X1) and concentration of DXY (X3) and negative relationship with the concentration of AOT (X2). The interaction effects of X1 and X3 also demonstrate positive relationship while interaction effects of X2 and X3 demonstrate negative relationship. Results from particle size analysis demonstrated that all the particles were in nanometer range and would prove beneficial as the nanosize helps to improve cellular uptake. Figure 8 exhibits the combined effect of concentrations of AOT/gellan gum, DXY/gellan gum and DXY/AOT, respectively, on the particle size. The plot shows an antagonistic relationship between the AOT and particle size (Figure 8a), and a synergistic relationship between the gellan gum and particle size, with the effect of AOT more pronounced than the gellan gum. AOT, an anionic surfactant forms reverse micelles, increasing the concentration of AOT in the system decreases the size of reverse micelles and thus the particle size. A synergistic relationship between the DXY/gellan gum

10

V. Pokharkar et al.



Figure 7. Response surface plot showing combined effect of concentration of (a) AOT and gellan gum; (b) gellan gum and DXY on the percentage of encapsulation of DXY within the nanoparticles.

and particle size can also be observed (Figure 8b). Increasing the concentration of DXY and gellan gum increases the size of the core matrix encapsulating the DXY leading to increase in particle size. Figure 8(c) displays the combined effect of drug concentration and concentration of AOT on particle size, with the effect of AOT more pronounced than the drug concentration. In case of zeta potential (Y3, Equation (6)), the linear effects of X1, X3 and interaction effects of X1 and X2, X2 and X3 contributed to significant increase in zeta potential, while the linear effects of X2 contributed toward significant reduction in zeta potential. Figure 9 shows the combined effect of concentrations of AOT/gellan gum, DXY/AOT and gellan gum/DXY on the zeta potential. It can be observed that AOT, which coats the core comprising DXY matrix and gellan gum, had the most pronounced effect on zeta potential. The AOT and gellan gum being anionic in nature impart negative charge on the nanoparticles, which increases in magnitude with increasing the concentration of AOT. Thus, negative charges on the surface of nanoparticles could contribute to the negative zeta potential of AOT–gellan gum nanoparticles. Table 4 represents the result of ANOVA test on the quadratic regression model and details the model summary statistics for the selected significant models, which indicated that the response surface model developed for three responses, were significant and adequate, without significant ‘‘lack of fit’’. Values of ‘‘Prob4F’’50.05 indicate model terms are significant. It can be observed that, all responses showed R2 value 40.9, which indicates a good correlation between the experimental and predicted responses. In addition, the predicted R2 values are in reasonable good agreement with adjusted R2 values, resulting in reliable models. The relatively lower values of coefficient of variation indicated better precision and reliability of the experiments carried out. The pH of all the formulations was found to be same as that of the pH of the ocular fluid, hence would not cause any irritation upon administration of the formulation to ocular tissues. The prepared nanoparticles were further characterized for XRD, FTIR, DSC and TEM. Complete absence of diffraction peaks in void nanoparticles and DNPs can be attributed to the

dilution factor due to high concentration of polymer without any qualitative fraction. This is in accordance with previous studies (Pignatello et al., 2002; Agnihotri & Vavia, 2009) where author confirmed that the drug is in matrix of poly(DL-lactide-co-glycolide) polymer and hence the diffraction peaks of drug are not observed. FTIR spectroscopy results confirmed the chemical stability and compatibility of DXY with the excipients that are present in the nanoparticles. DSC studies were carried out to determine whether the drug was incorporated in the nanoparticulate system as crystalline, amorphous or bound form. The complete absence of DXY melting peak confirmed that DXY is completely incorporated into the polymer–surfactant matrix. TEM analysis helps to identify the morphological characteristic of the nanoparticles. Figure 1(D) reveals that the DNPs are irregular shape with slightly rough surface. The optimized formulation of DNPs showed initial burst release followed by a second slow-release phase (extended release) (Figure 2). An initial burst release is beneficial in terms of antibacterial activity as it helps to achieve the therapeutic concentration of drug in minimal time followed by sustained release to maintain constant concentration of the drug. The initial burst release or the fast release of the drug may be due to the diffusion of DXY adsorbed at or just beneath the surface of the nanoparticles (Holland & Tighe, 1992). The slow and constant release of DXY after the initial burst release is mainly due to the slow diffusion of drug molecules through the polymeric matrix of the nanoparticles. The sodium–calcium exchange and electrostatic interaction also plays an important role in drug release from nanoparticles (Chavanpatil et al., 2007). When calcium gellan gum is introduced in environment rich in monovalent salts (e.g. sodium), insoluble calcium gellan gum is converted into soluble gellan gum, resulting in solubilization of the delivery system and drug release. If electrostatic interactions between basic drug and anionic matrix contribute to sustained drug release, then the release of an acidic drug from nanoparticles can be expected to be faster than that of a basic drug. Here DXY is a basic drug and gellan gum is an anionic polymer, combination of these two results in the sustained release of drug. Finally, the slower and sustained release of the drug at later stages can be attributed to the

Figure 8. Response surface plots showing combined effect of concentrations of (a) AOT and gellan gum, (b) DXY and gellan gum, (c) DXY and AOT on particle size of nanoparticles.


DOI: 10.3109/10717544.2014.893381

Engineering of polymer–surfactant nanoparticles 11

V. Pokharkar et al.

Figure 9. Response surface plots showing combined effect of concentrations of (a) AOT and gellan gum, (b) DXY and AOT, and (c) gellan gum and DXY on zeta potential of nanoparticles.


12 Drug Deliv, Early Online: 1–14


DOI: 10.3109/10717544.2014.893381

Table 4. Model summary statistics of the quadratic response surface model. Model Response factor F value Prob4F Y1 Y2 Y3

R2

Adj. R2

Pred. Adeq. R2 precision CV %

42.44 0.0017 0.9695 0.9467 0.8781 13.469 121.99 0.0082 0.9967 0.9886 0.9477 33.605 1428.78 0.0007 0.9997 0.9990 0.9955 106.470

5.05 3.19 1.00

Table 5. Kinetic modeling of release data. Release kinetics


Formulation DNPs

Zero order r2

First order r2

Matrix r2

r2

n

Hixson– Crowell r2

0.7754

0.9801

0.9922

0.9960

0.5

0.9386

Peppas

diffusion/erosion of the polymeric matrix and electrostatic interaction between basic drug and anionic matrix. Curve fitting of in vitro release data of the optimized DNPs suspension was compared with a different release model to select the best fitting model. The best fit kinetic model was the Peppas model (R2 ¼ 0.9960) suggesting non-Fickian diffusion process (Table 5). The ex vivo diffusion studies demonstrated the slow diffusion of drug molecules through the polymeric matrix of the nanoparticles. This is suggestive of prolonged drug release, good ocular permeability and increased retention of DNPs that may play a promising role in effective ocular treatment. This will further help in increasing safety and reducing the requisite frequency of administration. The results from the modified Draize test confirmed that all the three samples tested did not produce any discomfort to rabbit eyes. All the irritation study data showed that the values of irritation and opaqueness are almost zero. Thus it can be concluded that void nanoparticles and DNPs produced negligible irritation to rabbit eyes. Hence we can conclude that potency of the polymer to deliver DXY at the ocular surface has not been changed by the processing technique, and the activity of drug was retained also. It is very much suitable for ocular drug delivery. MIC studies revealed that the growth of organisms was reduced or inhibited in presence of both aqueous solution of DXY as well as DNPS which is evident from the graph (Figure 4). Here, the significant difference was observed between the antimicrobial activity of the DXY solution and DNPs in comparison to control (p50.01 and p50.001) after 24 h. The significance of differences was evaluated using twoway ANOVA followed by Bonferroni post-tests at the probability level of 0.05. No increase in the growth was seen during the 24 h incubation with formulation. These results indicate the sustained release characteristics of DNPs suspension that helps to inhibit bacterial growth for a longer period confirming prolonged microbial efficacy on strains of E. coli and S. aureus as compared with aqueous solution of DXY.

13

It is observed that MBC values are higher than the MIC values, suggesting that a higher concentration of drug is required to kill the bacteria completely. However, it is noteworthy that even though a 7.67 ± 0.58 and 6.67 ± 0.58 mg/ml concentration of aqueous solution of DXY killed 99.9% of bacteria E. coli and S. aureus, respectively, it was observed that microbes multiplied when transferred to a fresh medium, thus indicating that even at a higher dose the antibiotics are not able to inhibit growth of bacteria completely. The pure drug gradually loses its effect after 24 h and bacteria that escaped drug action can multiply at a faster rate when given suitable conditions. Therefore, a sustained release formulation is required that can control the growth of bacteria for a longer period of time (Jeong et al., 2008). A significant decrease in the MBC values of DNPs was observed in comparison to control (p50.01) after 24 h. It is worth mentioning that MIC and MBC values in DNPs were less than that of aqueous solution of DXY in our study. It means better penetration of DNPs in bacterial cells and better delivery of DXY to its site of action than DXY aqueous solution. These results are in accordance with the results obtained by Esmaeili et al. where the MIC was found to be 0.008 mg/ml for free rifampicin and 0.002 mg/ml for rifampicin loaded nanoparticles (Esmaeili et al., 2007). The formulation was found to be stable for entire stability period (Table 3).

Conclusion Preparation of gellan gum–AOT nanoparticles using emulsion cross-linking method proved to be a sound approach for efficient encapsulation and sustained release of water soluble drug DXY. Draize test confirmed the non-irritant properties of prepared nanoparticles. The therapeutic efficacy of the developed formulation was examined with the help of ex vivo diffusion and antibacterial studies. The results showed that the nanoparticles effectively released the drug over a sustained period of time and inhibited the bacterial growth at very low concentrations than that of the pure drug. Hence the developed doxycycline-loaded nanoparticles are safe and suitable for sustained ocular drug delivery in the treatment of various ocular bacterial infections.

Acknowledgements The authors thank Lupin Research Park (Pune, India) and CP Kelco (Mumbai, India) for providing gift sample of DXY and gellan gum (Kelcogel), respectively.

Declaration of interest Vikram Patil thanks AICTE (New Delhi, India) for providing financial support in the form of Junior Research Fellowship. Authors report no conflict of interest.

References Agnihotri SM, Vavia PR. (2009). Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application. Nanomedicine 5:90–5. Akpek EK, Merchant A, Pinar V, Foster CS. (1997). Ocular rosacea: patient characteristics and follow-up. Ophthalmology 104:1863–7.


14

V. Pokharkar et al.

Amir A, Turetz J, Brandeis R, et al. (2004). Evaluation of protease inhibitors in sulfur mustard ocular injuries. In: Proceedings of the U.S. Army Medical Defense Bioscience Review, Aberdeen Proving Ground, MD. 197–201. Bochot A, Fattal E, Grossiord JL, et al. (1998). Characterization of a new ocular delivery system based on a dispersion of liposomes in a thermosensitive gel. Int J Pharm 162:119–27. Chavanpatil M, Khdair A, Patil Y, et al. (2007). Polymer–surfactant nanoparticles for sustained release of water-soluble drugs. J Pharm Sci 96:3379–89. de Campos AM, Diebold Y, Carvaiho EL, et al. (2004). Chitosan nanoparticles as new ocular drug delivery system: in vitro stability, in vivo fate, and cellular toxicity. Pharm Res 21:803–10. de Campos AM, Sa´nchez A, Gref R, et al. (2003). The effect of PEG versus a chitosan coating on the interaction of drug colloidal carriers with the ocular mucosa. Eur J Pharm Sci 20:73–81. De Paiva CS, Corrales RM, Villarreal AL, et al. (2006). Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp Eye Res 83:526–35. Dhar S, Maheswara Reddy E, Shiras A, et al. (2008). Natural gum reduced/stabilized gold nanoparticles for drug delivery formulations. Chem Eur J 14:10244–50. Diebold Y, Calonge M. (2010). Applications of nanoparticles in ophthalmology. Prog Retin Eye Res 29:596-609. Ding S. (1998). Recent developments in ophthalmic drug delivery. Pharm Sci Technol Today 1:328–35. Dursun D, Kim MC, Solomon A, Pflugfelder SC. (2001). Treatment of recalcitrant recurrent corneal erosions with inhibitors of matrix metalloproteinase-9, doxycycline and corticosteroids. Am J Ophthalmol 132:8–13. Esmaeili F, Hosseini-Nasr M, Rad-Malekshahi M, et al. (2007). Preparation and antibacterial activity evaluation of rifampicinloaded poly-lactide-co-glycolide nanoparticles. Nanomedicine 3: 161–7. Falahee KJ, Rose CS, Olin SS, Seifried HE. (1981). Eye irritation testing. An assessment of methods and guidelines for testing materials for eye irritancy. Office of Pesticides and toxic Substances, US Environmental Protection Agency, Washington D. C. National Service Center for Environmental Publications (NSCEP). Guinedi AS, Mortada ND, Mansour S, Hathout RM. (2005). Preparation and evaluation of reverse-phase evaporation and multilamellar niosomes as ophthalmic carriers of acetazolamide. Int J Pharm 306: 71–82. Gupta H, Aqil M, Khar RK, et al. (2010). Sparfloxacin loaded PLGA nanoparticles for sustained ocular drug delivery. Nanomedicine 6: 324–33. Hill JM, O’Callaghan RJ, Hobden JA, Kaufman HE. (1993). Corneal collagen shields for ocular drug delivery. In: Mitra AK, ed. Ophthalmic drug delivery systems. New York: Dekker, 261–75. Holland SJ, Tighe BJ. (1992). Biodegradable polymers. In: Garderton D, Jones T, eds. Advances in pharmaceutical science, Vol. 6. London: Academic Press, 101–64. Hughes PM, Mitra AK. (1993). Overview of ocular drug delivery and iatrogenic ocular cytopathologies. In: Ashim K Mitra, ed. Ophthalmic drug delivery systems, 2nd ed. New York: Marcel Dekker Inc, 1–27. Israelachevilli JN. (1991). Intermolecular and surface forces: with applications to colloidal and biological systems (colloid science), 2nd ed. London: Academic Press.


Jacobs C, Mu¨ller RH. (2002). Production and characterization of a budesonide nanosuspension for pulmonary administration. Pharm Res 19:189–94. Jeong YI, Na HS, Seo DH, et al. (2008). Ciprofloxacin-encapsulated poly(DL-lactideco-glycolide) nanoparticles and its antibacterial activity. Int J Pharm 352:317–23. Kadar T, Dachir S, Cohen L, et al. (2009). Ocular injuries following sulfur mustard exposure – pathological mechanism and potential therapy. Toxicology 263:59–69. Kaur IP, Garg A, Singla AK, Aggarwal D. (2004). Vesicular systems in ocular drug delivery: an overview. Int J Pharm 269:1–14. Kim HS, Luo L, Pflugfelder SC, Li DQ. (2005). Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. Invest Ophthalmol Vis Sci 46:840–8. Li DQ, Chen Z, Song XJ, et al. (2004). Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells. Invest Ophthalmol Vis Sci 45:4302–11. Losa C, Calvo P, Castro E, et al. (1991). Improvement of ocular penetration of amikacin sulphate by association to poly (butylcyanoacrylate) nanoparticles. J Pharm Pharmacol 43:548–52. Losa C, Marchal-Heussler L, Orallo F, et al. (1993). Design of new formulations for topical ocular administration: polymeric nanocapsules containing metipranolol. Pharm Res 10:80–7. McDonald T, Shadduck J. (1991). 2. In: Marzulli FM, Maibach HI, eds. Dermatotoxicology and Pharmacology In Advances in modern toxicology, 4th ed. Washington (DC): Hemisphere Publishing Corp., 139–91. Misra R, Acharya S, Dilnawaz F, Sahoo SK. (2009). Sustained antibacterial activity of doxycycline loaded poly(D,L-lactide-coglycolide) and poly(e-caprolactone) nanoparticles. Nanomedicine 4: 519–30. Pignatello R, Bucolo C, Spedalieri G, et al. (2002). Flurbiprofen-loaded acrylate polymer nanosuspensions for ophthalmic application. Biomaterials 23:3247–55. Pijls RT, Sonderkamp T, Daube GW, et al. (2005). Studies on a new device for drug delivery to the eye. Eur J Pharm Biopharm 59:283–8. Pleyer U, Lutz S, Jusko WJ, et al. (1993). Ocular absorption of topically applied FK506 from liposomal and oil formulations in rabbit eye. Invest Ophthalmol Vis Sci 34:2737–42. Pokharkar VB, Malhi T, Mandpe L. (2013). Bicalutamide nanocrystals with improved oral bioavailability: in vitro and in vivo evaluation. Pharm Dev Technol 18:660–6. Rozier A, Manuel C, Groove J, Plazonet B. (1989). Gelrite: a novel ion activated in situ gelling polymer for ophthalmic vehicles, effect on bioavailability of timolol. Int J Pharm 57:163–8. Seedor JA, Perry HD, McNamara TF, et al. (1987). Systemic tetracycline treatment of alkali-induced corneal ulceration in rabbits. Arch Ophthalmol 105:268–71. Stratton CW, Lorian V. (1996). Mechanisms of action for antimicrobial agents: general principles and mechanisms for selected classes of antibiotics. Antibiotics in laboratory medicine, 4th ed. Baltimore (MD): Williams and Wilkins. Su W, Li Z, Lin M, et al. (2011). The effect of doxycycline temperaturesensitive hydrogel on inhibiting the corneal neovascularization induced by BFGF in rats. Graefes Arch Clin Exp Ophthalmol 249: 421–7. Wadhwa S, Paliwal R, Paliwal SR, Vyas SP. (2009). Nanocarriers in ocular drug delivery: an update review. Curr Pharm Des 15:2724–50.

Hydrogels for ocular drug delivery and tissue engineering.

Engineering biodegradable nanoparticles for drug and gene delivery.

Targeted drug delivery to emphysematous lungs: Inhibition of MMPs by doxycycline loaded nanoparticles.

Photoresponsive nanoparticles for drug delivery.

Manufacturing Techniques and Surface Engineering of Polymer Based Nanoparticles for Targeted Drug Delivery to Cancer.

Lipid-based surface engineering of PLGA nanoparticles for drug and gene delivery applications.

Ocular drug delivery for glaucoma management.

Engineering nanolayered particles for modular drug delivery.

Block copolypeptide nanoparticles for the delivery of ocular therapeutics.

Caged protein nanoparticles for drug delivery.

Cell membrane-camouflaged nanoparticles for drug delivery.

Silk fibroin-based nanoparticles for drug delivery.

Ocular drug delivery systems: An overview.

Controlled ocular drug delivery with nanomicelles.

Recent advances in ocular drug delivery system.

Advanced drug delivery and targeting technologies for the ocular diseases.

Development and characterization of nanoparticles for the delivery of gemcitabine hydrochloride.

silica hydrogel for sustained ocular drug delivery.

Drug delivery in aortic valve tissue engineering.

Effect of nanoparticles on transdermal drug delivery.

A review on therapeutic contact lenses for ocular drug delivery.

Colloidal drug delivery system: amplify the ocular delivery.

Core-shell designed scaffolds for drug delivery and tissue engineering.

Gelatin carriers for drug and cell delivery in tissue engineering.