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Author(s): Purnima Verma and Munish Ahuja

Article title: Cubic liquid crystalline nanoparticles: optimization and evaluation for ocular delivery of tropicamide

Article no: IDRD_A_1143057

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 http://informahealthcare.com/drd
1 ISSN: 1071-7544 (print), 1521-0464 (electronic) 61
2 62
3 Drug Deliv, Early Online: 1–12 63
! 2016 Taylor & Francis. DOI: 10.3109/10717544.2016.1143057
4 64
5 65
6 66
7
RESEARCH ARTICLE 67
8 68
9 Cubic liquid crystalline nanoparticles: optimization and evaluation for 69
10 70
11
ocular delivery of tropicamide 71
12 72
Q1 Purnima Verma and Munish Ahuja
13 73
14 74
Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar, India
15 75
16 76
17 Abstract Keywords 77
18 The purpose of this study was to investigate the potential of cubic liquid crystalline Central composite design (CCD), cubic 78
19 nanoparticles for ocular delivery of tropicamide. Ultrasound-assisted fragmentation of cubic nanoparticles, ocular delivery, tropicamide, 79
20 liquid crystalline bulk phases resulted in cubic liquid crystalline nanoparticles employing ultrasound 80
Pluronic F127 as dispersant. The effects of process variables such as sonication time, sonication
21 81
amplitude, sonication depth, and pre-mixing time on particle size and polydispersity index History
22 was investigated using central composite design. The morphology of tropicamide-loaded 82
23 nanoparticles was found to be nearly cubical in shape by transmission electron microscopy Received 26 November 2015 83
observation. Further, small angle X-ray scattering experiment confirmed the presence of D and Revised 12 January 2016
24 84
P phase cubic structures in coexistence. The optimized tropicamide-loaded cubic nanoparticles Accepted 13 January 2016
25 85
showed in vitro corneal permeation of tropicamide across isolated porcine cornea comparable
26 86
to its commercial preparation, TropicacylÕ . Ocular tolerance was evaluated by Hen’s egg
27 test–chorioallantoic membrane test and histological studies. The results of in vivo mydriatic 87
28 response study demonstrated a remarkably higher area under mydriatic response curve 88
29 (AUC0!1440 min) values of cubic nanoparticles over TropicacylÕ indicating better therapeutic 89
30 value of cubic nanoparticles. Furthermore, tropicamide-loaded cubic nanoparticles exhibited 90
prolonged mydriatic effect on rabbits as compared to commercial conventional aqueous
31 91
ophthalmic solution.
32 92
33 93
34 94
35 Introduction significant scientific interest recently. These are self- 95
36 assembled bicontinuous liquid crystalline phases comprising 96
Ocular drug delivery is one of the most challenging problem
37 lipid bilayer enclosing water channels providing both 97
facing pharmaceutical researchers due to the unique anat-
38 hydrophilic and hydrophobic region for encapsulation of 98
omy, physiology, and biochemistry of eye. It is reported that
39 drugs with varying solubility (Siekmann et al., 2002). Cubic 99
effectively 5% or even less of the instilled dose reaches
40 liquid crystalline nanoparticles can be easily administered in 100
intraocular tissues (Ahmed 2003; Zhang et al., 2004; Ali
41 a liquid form and seemed to have high diffusivity across the 101
et al., 2014). Most of the topically instilled drugs drain away
42 corneal epithelium (Gan et al., 2010; Li et al., 2013). In 102
quickly from the surfaces of the eyes by various mechan-
43 addition, prolonged drug release, bio-adhesion, biocompati- 103
isms, such as blinking, lacrimation, tear dilution, and tear
44 bility, and biodegradability makes these drug delivery 104
turnover. In addition, different layers of cornea, conjunctiva,
45 systems interesting as new therapeutic tools in ocular 105
and sclera constitute a compact barrier for ocular drug
46 delivery (Engstrom et al., 1995; Spicer et al., 2001; Dian 106
delivery (Gaudana et al., 2010). Several novel formulation-
47 et al., 2013). Despite the astounding properties of cubic 107
based approaches such as emulsions, nanoparticles, and
48 nanoparticles as innovative drug carriers, little research has 108
liposomes are being explored for their potential as ocular
49 so far been conducted on evaluating the potential of cubic 109
drug delivery vehicle to overcome the ocular barriers by
50 nanoparticles for ophthalmic drug delivery (Gan et al., 2010; 110
improving the transcorneal permeation (Law et al., 2000;
51 Li et al., 2013). The aim of this work was to study the 111
Lallemand et al., 2012; Dilbaghi et al., 2013; De Sa et al.,
52 performance of cubic nanoparticles as innovative ocular 112
2015). Cubic liquid crystalline nanoparticles have gained
53 delivery systems for tropicamide, a mydriatic agent, chosen 113
54 as a model drug. Tropicamide is a nonselective antimus- 114
55 carinic agent which is used for eye examinations before and 115
56 after eye surgery, cycloplegic retinoscopy, dilated fundu- 116
57 Address for correspondence: Dr. MunishAhuja, Associate Professor, scopic exam, cycloplegia, etc. Tropicamide blocks the 117
Drug Delivery Research Laboratory, Department of Pharmaceutical
58 receptor in the muscles of the eye that control the size of 118
Sciences, G. J. University of Science and Technology, Hisar 125 001,
59 India. Tel: +91-1662-263515. Fax: +91-1662-276240. Email: pupil and lens shape, thereby, producing dilation of pupil, 119
60 munishahuja17@yahoo.co.in i.e. mydriasis. 120
 2 P. Verma & M. Ahuja Drug Deliv, Early Online: 1–12

121 Several methods have been reported for production of Preparation of cubic liquid crystalline nanoparticles 181
122 cubic nanoparticles (Boyd 2003; Barauskas et al., 2005; 182
The cubic liquid crystalline nanoparticles were prepared by
123 Bei et al., 2009a). However, top down techniques is being 183
top down technique (Guo et al., 2010). Briefly, water was
124 most frequently employed as it can produce nanoparticles by 184
added to molten monoolein (4.5%w/v) in the sample holder
125 consuming less time and energy and more economic (Bei 185
and kept at room temperature until transparent cubic phase gel
126 et al., 2009b). Recently it has been reported that ultrasonic 186
was formed. Thereafter, aqueous solution of Pluronics F127
127 condition such as duration of treatment and amplitude 187
(0.5%w/v) was added and pre-mixing was carried out as given
128 influences the particle size and poly dispersity index of 188
in design protocol using a vortex mixer (Cyclo Mixer VM102,
129 nanoemulsion (Tang et al., 2012). Furthermore, it has also 189
Remi Equipments, India) to form coarse dispersion. The Q2
130 been reported that position of ultrasound source strongly 190
resultant coarse dispersion was subjected to probe sonication
131 affects the particle size of lipid-based vesicles (Silva et al., 191
as per the parameters specified in design protocol using
132 2010). Ultrasound-assisted production of cubic nanoparticles 192
ultrasonic processor to give cubic liquid crystalline
133 has been extensively reported in the literature (Ferreira et al., 193
nanoparticles.
134 2006; Chong et al., 2012; Driever et al., 2013; Verma & 194
135 Ahuja 2015). However, ultrasonic set up is poorly described 195
Experimental design
136 resulting in non-reproducibility of this methodology. 196
137 Therefore, this study was undertaken to optimize operating The central composite design (with a ¼ 1) was employed to 197
138 parameters of probe-type sonicator to engineer the character- find out optimum process parameters. The independent 198
139 istics of cubic liquid crystalline nanoparticles. The influence variables selected were: sonication time (X1), amplitude 199
140 of ultrasonic processing parameters, namely pre-mixing time, (X2), sonication depth (X3), and premixing time (X4). Each 200
141 ultrasonic amplitude, sonication depth, and sonication time on factor was studied at 3 levels (i.e. 1, 0, +1). The dependent 201
142 particle size and polydispersity index (PdI) of cubic liquid variables chosen were: particle size (Y1) and PdI (Y2). The 202
143 crystalline nanoparticles was studied using response surface central point (0, 0, 0) was studied in sextet. The total design 203
144 methodology. The optimized batch of cubic nanoparticles was matrix showed 30 runs (Table 1), to be carried out randomly 204
145 evaluated for ocular drug delivery by loading with tropica- to eliminate the effects of extraneous or nuisance variables. 205
146 mide. The crystalline structure and morphology of tropica- Statistical analysis was performed using Design Expert 206
147 mide-loaded cubic nanoparticles was investigated by small software (Version 7.0.0, Stat-ease Inc., Minneapolis, MN). 207
148 angle X-ray scattering and transmission electron microscopy Analysis of variance (ANOVA) was carried out to predict 208
149 (TEM), respectively. Further, the tropicamide-loaded cubic whether there are significant differences between independent 209
150 nanoparticles were evaluated comparatively with commercial variables or not. 210
151 conventional formulation of tropicamide for in vitro corneal 211
152 permeation characteristics using isolated porcine cornea and Particle size, zeta potential, and polydispersity index 212
153 for in vivo mydriatic response using rabbits. measurement 213
154 214
155
The average particle size (Z-average) and PdI was measured 215
Material and methods using Zetasizer nano ZS (Malvern Instruments, UK). All the 216
156 Q2
157 Chemicals and reagents measurements were performed in triplicate. 217
158 Monoolein (RYLO MG 19, monoglycerides content 495%) 218
Preparation of tropicamide-loaded ophthalmic cubic
159 was a generous gift from Danisco Cultor (Grinsted, 219
nanoformulation
160 Denmark). Tropicamide was obtained as gift sample from 220
161 Optica Pharmaceuticals (Yamunanagar, India). Pluronic F127 The optimized batch of cubic nanoparticles prepared by 221
162 (PEO98-polyPPO67-PEO98) was obtained from Ranbaxy ultrasonic fragmentation was evaluated for ocular drug 222
163 Research Laboratory (Gurgaon, India). SephadexÕ G-50 was delivery using tropicamide as a model drug. Required 223
164 procured from Sigma-Aldrich (USA). Commercial formula- quantity of tropicamide was added to water and pH was 224
Q2
165 tion of eye drop (TropicacylÕ , Sunways Pvt. Ltd. Mumbai, adjusted to 4 for complete dissolution of tropicamide (Kumar 225
166 India) was purchased from local pharmacy (Hisar, India). All & Ahuja 2014). The aqueous solution of tropicamide was then 226
167 other chemicals used were of analytical grade. sterilized by autoclaving at 121  C, for 15 min, at 15 lbs/in2. 227
168 This sterilized solution was added to molten monoolein 228
169 under aseptic conditions to form bulk phase gel, followed by 229
Experimental setup
170 pre-mixing and probesonication to get tropicamide-loaded 230
171 The model of probe sonicator that had been used for the ophthalmic cubic nanoformulation. 231
Q2 172 present study is: Q55 (Qsonica sonicators, USA). It can 232
173 deliver a power output maximum of 55 W. The amplitude can Characterization of tropicamide-loaded ophthalmic 233
174 be varied from 10% to 100%. The probe tip was made up of cubic nanoformulation 234
175 titanium. The sample holder was an open glass test tube 235
Particle size, zeta potential, and polydispersity index
176 having height and inner diameter 120 mm and 14 mm, 236
measurement
177 respectively, which contained 10 ml of the sample. The test 237
178 tube was held straight in a water bath maintained at 25  C The Z-average and PdI was measured using Zetasizer nano ZS 238
179 using water bath. The probe tip was set at the center of test (Malvern Instruments, UK). All the measurements were 239
180 tube. Time of sonication was controlled by stop watch. performed in triplicate. 240
 DOI: 10.3109/10717544.2016.1143057 Potential of cubic liquid crystalline nanoparticles for ocular delivery 3

241 Table 1. Central composite design used to study effect of process variables on particle size and PdI and results of zeta potential. 301
242 302
Sonication Sonication Sonication Pre-mixing Particle size Zeta potential
243 303
Runs time (X1) (min) amplitude (X2) (%) depth (X3) (mm) time (X4) (min) (Y1) (nm) PdI (Y2) (mv)
244 304
245 1 2.5 (0) 30 (0) 38 (+1) 5 (0) 10.73 0.398 10.8 305
2 0.5 (1) 45 (+1) 19 (1) 0 (1) 19.1 0.436 10.1
246 3 2.5 (0) 30 (0) 19 (1) 5 (0) 9.83 0.367 10.5 306
247 4 4.5 (+1) 15 (1) 38 (+1) 10 (+1) 16.97 0.411 8.29 307
248 5 4.5 (+1) 15 (1) 19 (1) 10 (+1) 12 0.372 10.3 308
6 2.5 (0) 45 (+1) 28.5 (0) 5 (0) 10 0.351 5.58
249 309
7 0.5 (1) 15 (1) 38 (+1) 10 (+1) 92.29 0.537 1.69
250 8 2.5 (0) 15 (1) 28.5 (0) 5 (0) 17.16 0.401 3.74 310
251 9 4.5 (+1) 30 (0) 28.5 (0) 5 (0) 7.419 0.269 4.62 311
252 10 0.5 (1) 15 (1) 19 (1) 10 (+1) 105.7 0.633 14.7 312
11 0.5 (1) 15 (1) 38 (+1) 0 (1) 272.5 0.563 13.9
253 313
12 0.5 (1) 45 (+1) 38 (+1) 10 (+1) 62.88 0.56 16.8
254 13 2.5 (0) 30 (0) 28.5 (0) 10 (+1) 12.87 0.399 9.61 314
255 14 4.5 (+1) 45 (+1) 38 (+1) 0 (1) 7.827 0.34 6.53 315
256 15 2.5 (0) 30 (0) 28.5 (0) 5 (0) 12.3 0.382 7.87 316
16 4.5 (+1) 45 (+1) 19 (1) 0 (1) 7.239 0.243 2.64
257 317
17 4.5 (+1) 15 (1) 38 (+1) 0 (1) 18.23 0.432 6.72
258 18 4.5 (+1) 15 (1) 19 (1) 0 (1) 11.29 0.369 7.05 318
259 19 0.5 (1) 45 (+1) 19 (1) 10 (+1) 16.63 0.432 3.51 319
260 20 2.5 (0) 30 (0) 28.5 (0) 5 (0) 13.32 0.397 7.30 320
21 0.5 (1) 45 (+1) 38 (+1) 0 (1) 28.27 0.459 6.36
261 22 0.5 (1) 15 (1) 19 (1) 0 (1) 297.2 0.473 7.46 321
262 23 2.5 (0) 30 (0) 28.5 (0) 5 (0) 10.29 0.369 6.17 322
263 24 2.5 (0) 30 (0) 28.5 (0) 5 (0) 12.36 0.325 7.97 323
264 25 4.5 (+1) 45 (+1) 19 (1) 10 (+1) 8.111 0.311 6.05 324
26 0.5 (1) 30 (0) 28.5 (0) 5 (0) 25.49 0.477 11.8
265 27 2.5 (0) 30 (0) 28.5 (0) 0 (1) 12.09 0.387 10.2 325
266 28 2.5 (0) 30 (0) 28.5 (0) 5 (0) 9.115 0.394 11.6 326
267 29 4.5 (+1) 45 (+1) 38 (+1) 10 (+1) 5.712 0.275 4.4 327
30 2.5 (0) 30 (0) 28.5 (0) 5 (0) 9.637 0.371 11.1
268 328
269 Values in parenthesis indicate the coded values. 329
270 330
271 331
272 332
273 333
274 Encapsulation efficiency Small angle X-ray scattering analysis 334
275 335
Gel filtration chromatographic technique was employed to SAXS measurements were used to identify the inner structure
276 336
separate unentrapped tropicamide from the cubic nanoparti- of tropicamide-loaded cubic nanoformulation. SAXS diffrac-
277 337
cles (Verma & Ahuja 2015). Sephadex G-50 powder was tograms were acquired on a SAXSess mc2 (Anton Paar,
278 338
filled into the column and water was added into the column in Austria) equipped with a line collimation set-up. The
279 339
order to form sephadex gel avoiding bubbles and cracks. An experiments were conducted using Ni-filtered Cu Ka radi-
280 340
aliquot of 1 ml of the tropicamide-loaded cubic nanoformula- ations (1.542 Å) from a copper rotating anode operating at
281 341
tion was passed through a column of sephadex with specific 45 kV and 50 mA. The samples were filled into a 1 mm quartz
282 342
length and inner diameter of 6.5 cm and 2.3 cm, respectively. capillary in a steel sample holder and the sample to detector
283 343
The column contained 27.01 ml of gel. The eluate was distance was 261 mm. Measurements were carried out at
284 344
analyzed for the contents of free tropicamide by UV 25 ± 0.5  C with exposure time of 3 h for nanoparticles. In
285 345
spectrophotometric analysis at max of 257 nm. The percent order to reduce the noise and to cancel out the effect of LC
286 346
encapsulation of tropicamide into nanoparticles was calcu- monodomains contribution in the final SAXS diffractograms,
287 347
lated with the formula: the capillaries were slightly rotated every 10 min. All
288 348
scattering signals were treated with SAXSquant software
289 Trt  Tre 349
EEð%Þ ¼  100 ð1Þ (Anton Paar).
290 Trt 350
291 351
292 where, Trt is the total tropicamide in formulation and Tre is the Evaluation for ocular drug delivery 352
293 amount of tropicamide found in eluant aqueous phase. 353
In vitro corneal permeation study
294 354
295 Corneal permeation characteristics of tropicamide-loaded 355
Transmission electron microscopy
296 cubic ophthalmic nanoformulation were comparatively eval- 356
297 The morphology of tropicamide-loaded cubic nano- uated with the commercially available conventional ophthal- 357
298 formulation was observed by TEM on a Philips CM 10 mic preparation (Tropicacyl 1%, (w/v)) using isolated porcine 358
299 electron microscope operating at an accelerated voltage of eyes cornea as model (Gratieri et al., 2011). Fresh whole 359
300 100 kV. eyeballs were obtained from the local butcher shop 360
 4 P. Verma & M. Ahuja Drug Deliv, Early Online: 1–12

361 immediately after slaughtering and transported to the labora- In vivo mydriatic activity 421
362 tory in cold normal saline within an hour. Cornea was 422
Tropicamide produces a rapid mydriatic response and there-
363 carefully excised along with 2–4 mm of scleral tissue and 423
fore bioavailability was assessed by measuring pupil diameter
364 finely cleaned and washed till free from proteins with cold 424
(Kumar & Ahuja 2014). The protocol for in vivo mydriatic
365 normal saline. Isolated cornea was mounted by clamping 425
study in rabbit was designed and an approval of institutional
366 between the donor and receptor compartments of modified 426
animal ethics committee was obtained. All tests were carried
367 Franz diffusion cell, with endothelial side facing the receptor 427
out on non-anesthetized Albino rabbits procured from disease
368 and epithelial side facing the donor. The receptor compart- 428
free small animal house of Lala Lajpat Rai University of
369 ment contained 11.5 ml of freshly prepared phosphate- 429
Veterinary and Animal Sciences (Hisar, India). Three albino
370 buffered saline (PBS) pH 7.4 maintained at 35 ± 0.5  C 430
rabbits with equivalent pupil light response were used in the
371 under magnetic stirring. Area available for corneal perme- 431
study. Each rabbit was acclimatized to the laboratory testing
372 ation was 0.785 cm2. The test formulation (1 ml) was placed in 432
conditions for 1 h prior to initiating the study. The animals
373 the donor compartment over the cornea. An aliquot of 1 ml of 433
were positioned in restraining boxes in the normal upright
374 the sample was withdrawn from receptor compartment at 434
position in a room with constant light intensity and devoid of
375 fixed time intervals and analyzed for the contents of 435
distractions. Baseline pupil diameter measurements were
376 tropicamide. The study was conducted using paired corneas, 436
taken every minute for 5 min prior to dosing. One drop of the
377 i.e. one cornea of the animal was used for the permeation 437
cubic nanoformulation was carefully instilled into the lower
378 study of cubic nanoparticles and the contra lateral cornea was 438
cul-de-sac region of the right eye whereas one drop of
379 used for conventional commercial formulation of aqueous 439
TropicacylÕ was instilled into the left eye of each rabbit. After
380 drug solution. Corneal hydration level was determined by 440
15 min the second dose was instilled followed by third dose
381 removing the scleral tissue from the cornea at the end of 441
after 30 min. At appropriate time intervals pupil diameter
382 experiment and weighing followed by dehydration by over- 442
measurements. The % increment in pupil diameter was
383 night soaking in methanol and drying in an oven at 90  C and 443
calculated as follows:
384 weighing again. 444
385 It  Io 445
Increment ð%Þ ¼  100 ð4Þ
386 Determination of tropicamide flux and permeability Io 446
387 447
The cumulative amount of tropicamide permeating across the where, Io is average baseline diameter and It the pupil
388 448
porcine cornea was plotted against time and slope of the linear diameter at time t.
389 449
portion of the graph was calculated. The steady state flux (Js,
390 450
mg/h/cm2) and apparent permeability coefficient (Kp, cm/h) Effect on corneal integrity
391 451
were calculated as follows (Peltola et al., 2003):
392 Freshly excised cornea was incubated at 37  C for 1 h with the 452
393 dQ formulation employing PBS pH 7.4 and sodium dodecyl 453
Js ¼ ð2Þ
394 A  dt sulfate (SDS) aqueous solution (0.1% w/v) as negative and 454
395 positive control, respectively. After incubation corneas were 455
396 Js washed with PBS and immediately fixed with a formaline 456
Kp ¼ ð3Þ
397 C0 solution 8% (w/w). The material was dehydrated with an 457
398 alcohol gradient, put in melted paraffin, and solidified in 458
where dQ/dt is the linear portion of the slope (mg/h), A is the
399 block form. Cross sections (51 mm) were cut, stained with 459
corneal surface area (in this study, 0.785 cm2), and C0 is the
400 hematoxylin and eosine (H&E), blinded and microscopically 460
initial drug concentration (mg/ml).
401 observed (Baydoun et al., 2004). Fresh cornea was also 461
402 treated by same staining procedure and observed under light 462
Ocular tolerance microscope for comparison purpose.
403 463
404 Ocular irritancy potential of tropicamide-loaded cubic ocular 464
405 nanoformulation was assessed employing Hen’s Egg Test Results and discussion 465
406 Chorioallantoic Membrane (HET-CAM). HET-CAM study is 466
Preparation and optimization of cubic nanoparticles
407 established alternative technique to the Draize rabbit eye test 467
408 to check potential irritation effects in the eye (Luepke 1985). Monoolein forms stiff and highly viscous bulk cubic phase gel 468
409 Ten-day-old fertilized hen’s eggs were procured from a when added to water. In order to produce nanoparticles, it is 469
410 poultry farm. The eggs were placed in a stand with the desirable to fragment the highly viscous cubic phase gel using 470
411 equatorial side up where a small window was opened to numerous dispersive techniques. It has been reported by Bei 471
412 expose the CAM. Only eggs with an air sac and live embryo and coworkers (Bei et al., 2009a) that characteristics of cubic 472
413 were used for further testing. An aliquot of 0.5 ml of the test nanoparticles prepared employing high speed homogeniza- 473
414 samples were placed directly onto the CAM’s surface and tion, greatly vary with operating parameters such as hom- 474
415 CAM was observed for 5 min for appearance of any of the ogenization speed and time. 475
416 following phenomena: hemorrhage, vasoconstriction, and Ultrasonic sound-assisted production of nanoparticles has 476
417 coagulation for which a score was calculated (Dehelean been widely employed in recent years. Ultrasonic is defined as 477
418 et al., 2011; Kaur et al., 2012). Saline solution and a 0.1 N sound intensity (above 18 Hz) exceeding the threshold of 478
419 sodium hydroxide solution were used as the negative and human hearing. The production of ultrasound entails elastic 479
420 positive controls, respectively. deformation of ferroelectric materials within a high frequency 480
 DOI: 10.3109/10717544.2016.1143057 Potential of cubic liquid crystalline nanoparticles for ocular delivery 5

481 electrical field (Kuttruff 1988; Raichel 2000). It has been Table 2. Summary of each factor effect and p values, for responses Y1 541
and Y2.
482 reported that ultrasound processing parameters strongly affect 542
483 the size of nanoparticles (Floris et al., 2013). The present 1/sqrt Y1 Y2
543
484 work is intended to provide optimum sonication conditions 544
485 in order to formulate optimal monoolein-based cubic liquid Factor Factor effect p Value Factor effect p Value 545
486 crystalline nanoparticles. The effect of process variables such X1 +0.08744 50.0001 0.035 50.0001 546
487 as, sonication time (X1) and sonication amplitude (X2), X2 +0.0136 50.0001 0.002904 50.0001 547
X3 0.001288 0.0752 +0.003548 0.0240
488 sonication depth (X3), and pre-mixing time (X4) on particle X4 – – 0.004264 0.1173 548
489 size (Y1) and PdI (Y2) of cubic liquid crystalline nanoparticles X1X4 – – 0.00154 0.0755 549
490 was studied employing central composite experimental X3X4 – – 0.00031315 0.0848 550
491 design. The preliminary studies were conducted in order to X12 0.00874 0.0337 – – 551
X22 0.000167 0.0195 – –
492 determine the levels of independent variables. X42 – – +0.00195 0.0006 552
493 Response surface methodology is a collection of mathem- 553
494 atical models such as central composite design (CCD), Box– 554
495 Behnken design and three-level factorial design having greater than 0.1000 indicates the insignificant model terms 555
496 different properties and characteristics. Amongst these three (Korbahti & Tanyolac 2008; Bashir et al., 2010). As it can be 556
497 methodologies, CCD is a most popular technique that allow seen from table that response Y1 is affected significantly (p 557
498 optimization, estimation, and evaluation of the main and value50.05) by linear and quadratic contribution of X1 and 558
499 interaction of variables with least number of experiments X2 whereas, response Y2 was influenced by linear contribution 559
500 (Hao et al., 2012). Here, in accordance with design matrix of of X1, X2, and X3 and quadratic contribution of X4. 560
501 CCD, a total of 30 runs were required for predicting the The model F value was found to be 59.97 and 26.44 561
502 variation of the Z-average (Y1) and PdI (Y2) as a function of respectively, for Y1 and Y2, implies that the model is 562
503 the process variables which are: sonication time (X1), significant. There is only a 0.01% chance that a model 563
504 sonication amplitude (X2), sonication depth (X3) and pre- F value could occur due to noise. The magnitude of lack of fit 564
505 mixing time (X4). The combination of process parameters in F value for Y1 and Y2 was 1.54 and 1.76, respectively, 565
506 random order and the experimental obtained values of particle indicating that the ‘‘Lack of Fit’’ is not statistically significant 566
507 size and zeta potential are presented in Table 1. relative to the pure error which further ensures the reliability 567
508 The obtained values of responses was fitted into various of model. As it can be seen that there is a 33.37% and 27.66% 568
509 polynomial models and subjected to multiple regression chance for Y1 and Y2, respectively that this F value could 569
510 analysis. The response Y2 fitted best into response surface occur due to noise. The magnitude of R2, correlation 570
511 reduced quadratic model for (p value is 0.0304) whereas coefficient was 0.9259 (Y1) and 0.8938 (Y2) shows a good 571
512 response Y1 fitted best into reduced quadratic model (p value correlation between experimental and predicted values. 572
513 is 0.0012) with inverse square root transformation. The Further, predicted R2 (0.8795 and 0.7502 for Y1 and Y2, 573
514 regression equations (Equations 5 and 6) in terms of coded respectively) was in reasonable agreement with the adjusted 574
515 values of factors are as follows: R2 (9.105 and 0.8600 for Y1 and Y2, respectively). Adequate 575
516 precision is given by signal to noise ratio. The values of 576
517 1 adequate precision were higher than 4 (25.096 and 18.923 for 577
¼ 0:30 þ 0:090X1 þ 0:054X2
518 SqrtðY1 Þðparticle sizeÞ ð5Þ Y1 and Y2, respectively) indicating an adequate signal. 578
519  0:012X3  0:034X12  0:038X22 Therefore, the model could be used to navigate the 579
520 design space. 580
521 Figures 1 and 2 portray the 3-D response surface plots 581
Y2 ðPdIÞ ¼ 0:38  0:086X1  0:044X2 þ 0:019X3
522 prepared using model generated by response surface meth- 582
523 þ 0:013X4  0:015X1 X4  0:015X3 X4 þ 0:049X42 odology. Figure 1(a) displays the combined effect of sonic- 583
524 ð6Þ ation depth and sonication amplitude on particle size. The 584
525 plot shows the curvilinear relationship between the independ- 585
526 The polynomial equations showed above presents the ent and dependent variables. Notably, when sonication 586
527 quantitative effects of process variables and their interaction amplitude was increased, significant decrease in particle 587
528 effects on the responses Y1 and Y2. The polynomial equations size was observed. Furthermore, it can be inferred from the 588
529 comprise coefficients for intercept, first order main effect plot that at low levels of sonication amplitude, decreasing the 589
530 (i.e. X1), interaction terms (i.e. X1X4) and higher order effects sonication depth led to slight decrease in the particle size. 590
531 (i.e. X12 ). The sign of coefficients indicates positive or However, effect of decrease in size by decreasing depth was 591
532 negative effects on the response. Positive sign shows syner- diminished at higher amplitude levels. Figure 1(b) shows the 592
533 gistic effect indicating the increase in response at higher level combined effect of pre-mixing time and sonication time on 593
534 of independent variable. The negative coefficient presents particle size. It is evident from the plot that the effect of 594
535 antagonistic effect showing increase in response at low levels sonication time is more prominent than the pre-mixing time. 595
536 of independent variable. The magnitude of main effects Furthermore, increasing sonication time resulted in signifi- 596
537 indicates the relative impact of each factor on the response. cant decrease in the particle size of nanoparticles whereas 597
538 The magnitude of actual factor coefficient and p value for pre-mixing time did not result in any change in particle size. 598
539 both responses is shown in Table 2. Generally, model terms is This might be explained by the fact that mechanical energy of 599
540 considered significant when p value50.05 while p value vortex shaking is insufficient to aid dispersion of 600
 6 P. Verma & M. Ahuja Drug Deliv, Early Online: 1–12

601 Figure 1. 3-D response surface plot showing 661

the interaction effect for particle size as a
602 function of (a) sonication amplitude and 662
603 depth (b) sonication time and pre-mixing 663
604 time. 664
605 665
606 666
607 667
608 668
609 669
610 670
611 671
612 672
613 673
614 674
615 675
616 676
617 677
618 678
619 679
620 680
621 681
622 682
623 683
624 684
625 685
626 686
627 687
628 688
629 689
630 690
631 691
632 692
633 693
634 694
635 695
636 696
637 nanoparticles during pre-mixing. It is well known that values. The effect of pre-mixing time and sonication time on 697
638 ultrasonic waves generate gas bubbles in liquids called PdI is presented in Figure 2(b). Pre-mixing time has shown 698
639 cavitation. The formation of the cavitation due to application curvilinear relationship PdI while sonication time has shown a 699
640 of high intensity ultrasound leads to intense mechanical forces linear functionality. As can be seen from plot, in the middle 700
641 in liquids (Karaman et al., 2012). High irradiation power value pre-mixing time, magnitude of PdI is least. However, 701
642 exerts greater shear forces within the dispersion thereby, reason could not be understood. Furthermore, PdI value was 702
643 increasing number of events and the cavity collapse intensity decreased with increase in sonication time. Cubic liquid 703
644 of cavitation. The decrease in particle size could be ascribed crystalline nanoparticles comprising uniform size distribution 704
645 to increase in cavitation effect with increase in sonication could be prepared at higher level of amplitude and time 705
646 amplitude (Silva et al., 2010; Jadhav et al., 2015). Similarly, whereas at low level of sonication depth. 706
647 the decrease in size on increasing sonication time can be To develop liquid crystalline nanoparticles with desired 707
648 attributed to higher shearing with increase in exposure time. parameters, numerical optimization tool along with desirabil- 708
649 These results are contradictory to the previous investigation ity approach was employed. The optimization was performed 709
650 by Tang and coworkers (Bei et al., 2009b) which reported the setting constraints for particle size and PdI to be minimum as 710
651 increase in droplet size and PdI of Cremophore EL-based the goal to locate the optimum values of independent 711
652 nanoemulsion with increase in sonication amplitude and variables. The Design Expert software provided us with 21 712
653 irradiation time. solutions; out of this one with highest desirability was 713
654 Figure 2(a) displays the combined effect of sonication selected for preparing optimized formulation. The optimal 714
655 depth and sonication amplitude on PdI. The plot depicts the calculated parameters obtained were: sonication time 715
656 linear relationship between the independent and dependent (4.5 min), sonication amplitude (40%), sonication depth 716
657 variables. It can be elicited that increases in sonication (19 mm), and pre-mixing time (4.5 min). 717
658 amplitude led to significant decrease in PdI values. To check the reliability of developed mathematical models, 718
659 Decreasing the sonication depth was found to decrease the the response of the optimal sonication conditions and three 719
660 PdI value which was more pronounced at lower amplitude additional checkpoint formulations covering the entire range 720
 DOI: 10.3109/10717544.2016.1143057 Potential of cubic liquid crystalline nanoparticles for ocular delivery 7

721 Figure 2. 3-D response surface plot showing 781

the interaction effect for PdI as a function of
722 (a) sonication amplitude and depth (b) son- 782
723 ication time and pre-mixing time. 783
724 784
725 785
726 786
727 787
728 788
729 789
730 790
731 791
732 792
733 793
734 794
735 795
736 796
737 797
738 798
739 799
740 800
741 801
742 802
743 803
744 804
745 805
746 806
747 807
748 808
749 809
750 810
751 811
752 812
753 813
754 814
755 815
756 816
757 817
758 818
759 Table 3. The observed predicted values and % prediction error at check percentage prediction error (0.415 to 0.040 for Y1, 0.097 819
points for the responses Y1 and Y2. to +0.054 for Y2) is indicative of robustness of applied
760 820
761 mathematical model and high prognostic ability of response 821
Y1 Y2
762 surface methodology. 822
763 Checkpoint % % 823
(X1/X2/X3/X4) Observed Predicted Error Observed Predicted Error
764 824
3/40/19/1 10.94 7.94 0.3778 0.349 0.318 0.097 Characterization of cubic nanoparticles
765 825
4.5/40/10/4.5a 7.06 6.55 0.0787 0.227 0.240 0.054
766
3.5/30/28.5/4 26.45 18.68 0.4159 0.426 0.398 0.070
The zeta potential of the blank liquid crystalline nanoparticles 826
767 4/35/26/6 7.64 7.34 0.0408 0.302 0.294 0.027 was found to be in the range of 16.8 to 2.64 mV as shown 827
768 a
in Table 1. Nonionic monoolein releases subtle amount of 828
Shows optimum composition. oleic acid which is anionic resulting in anionic behavior of
769 829
770 dispersion. Blank liquid crystalline nanoparticles were loaded 830
771 of experimental domain was recorded. For each of the test with tropicamide and studied for ocular drug delivery 831
772 runs, comparative analysis was performed for experimentally applications. The % drug loading was found to be 832
773 observed response and the response predicted by the math- 96.57 ± 0.23 (%) which could be attributed to high internal 833
774 ematical model. Table 3 displays the composition of optimum area of cubic phase nanoparticles. The particle size, PdI, and 834
775 and random check points, their observed and predicted zeta potential of tropicamide-loaded cubic nanoformulation 835
776 response variables and calculated percentage prediction was found to be 54.52 ± 1.12 nm, 0.248 ± 0.05, and 836
777 error. The correlation between observed and predicted  8.76 ± 0.82 mV, respectively. It can be seen from the results 837
778 values was observed to be linear with correlation coefficient, that drug loading led to increase in average size of the 838
779 R2 of 0.988 and 0.983 for particle size and PdI, respectively, nanoparticles while PdI remained unaffected. Although, the 839
780 confirming the model validity. The lower magnitude of absolute value of zeta potential was very low but no 840
 8 P. Verma & M. Ahuja Drug Deliv, Early Online: 1–12

841 the fit of ‘‘s’’ values to the corresponding Miller indices 901
842 (h2 + k2 + l2)1/2 (where h, k, and l are the miller indices) 902
843 identified for both phases in the blank cubic nanoparticles and 903
844 tropicamide-loaded cubic nanoparticles. The linearity of the 904
845 plots and the (0, 0) intercept are the indications of valid space 905
846 group assignment. The value of cubic cell lattice parameter 906
847 (a) was calculated from the slope of the plots (Rodriguez & 907
848 Kunieda 2000). Tropicamide incorporation did not alter the 908
849 unit cell dimension because no difference in lattice parameter 909
850 was observed in the presence and absence of tropicamide in 910
851 cubic phases. Bonnet ratio (Br) was also determined, given by 911
852 the ratio of lattice parameter of two different cubic phases (as 912
853 shown in Equation (7) with periodic minimal surface 913
854 structures. 914
Figure 3. Transmission electron micrograph of tropicamide-loaded
855 915
cubic nanoparticles. aðIm3mÞ
856 Br ¼ ð7Þ 916
857
aðPn3mÞ 917
858 aggregation and visual sign of instability was observed in The Bonnet ratio was also remained unaffected and 918
859 nanoparticles. It is presumed that the hydrophobic polypro- calculated to be 1.16 which is close to the theoretically 919
860 pylene oxide chain adsorbs to the surface of cubic predicted value of 1.279 for both blank cubic nanoparticles 920
861 nanoparticles leading to adhesion of polymer to nanoparticles and tropicamide-loaded cubic nanoparticles (Muller et al., 921
862 while hydrophilic polyethylene oxide chain extend out into 2010). 922
863 aqueous environment in order to provide steric shielding, 923
864 thereby, preventing aggregation (Lancelot et al., 2014). The 924
Evaluation as ocular drug delivery vehicle
865 transmission electron micrograph of tropicamide-loaded 925
866 cubic nanoparticles is shown in Figure 3. It can be observed Figure 5 presents the results of in vitro corneal permeation 926
867 from the figure that the particles are cubical in shape. profile of tropicamide from the cubic nanoparticles and 927
868 SAXS is considered most excellent tool for structural commercial ophthalmic solution (TropicacylÕ ) across the 928
869 determination of cubic liquid crystalline phases. The overlay isolated porcine cornea. It can be seen that permeation 929
870 of SAXS diffraction profile of blank cubic nanoparticles characteristics of tropicamide-loaded cubic nanoparticles are 930
871 and tropicamide-loaded cubic nanoparticles are shown in comparable to that of conventional commercial preparation 931
872 Figure 4(a). The scattering curve of blank cubic nanoparticles (TropicacylÕ ). Furthermore, no remarkable difference in 932
873 prepared at optimal process conditions has shown two set values of flux (Jss) and apparent permeability (Kp) were 933
874 of
pffiffiffi medium
pffiffiffi pffiffiffi intensity
pffiffiffiffiffi ppeaks
ffiffiffi in the characteristics ratio of; seen as listed in Table 5. The commercial formulation, which 934
875 2 , 4 , 6 , 10
pffiffiffi pffiffiffi pffiffiffi pffiffiffi pffiffiffi ( 8 was scarcely observed) and was used for comparative evaluation contained chlorbutol as a 935
876 2, 3, 4, 6, 8, confirming the presence of dispersed preservative. It is reported that chlorbutol enhances the 936
877 cubic phase particles of Im3m (P-type phase) and Pn3m corneal permeability (Dilbaghi et al., 2013). Even though, no 937
878 (D-type phase) symmetry (Table 4). Phase diagram of preservative was added in the cubic nanoparticles, it provided 938
879 monoolein/water system exhibits mainly cubic phase of % permeation of tropicamide comparable to the commercial 939
880 Pn3m symmetry (D-type phase) in excess of water (concen- formulation. This could be attributed to the nanometric size of 940
881 tration above 40%w/w) (Hyde et al., 1984; Qiu & Caffrey the cubic nanoparticles. Earlier it was reported that 941
882 2000). It is reported that Pluronic F127 causes swelling in nanoparticles provide higher corneal permeation due to 942
883 cubic lattice thereby leading to the transition from D phase to endocytic uptake (Calvo et al., 1996; Gupta et al., 2000). In 943
884 P phase (Landh 1994). Possibly, transition in the phases is due addition, lipid monoolein have also been reported to provide 944
885 to the embedment of hydrophobic polypropylene oxide chain corneal penetration-enhancing effect (Li et al., 2013). 945
886 of Pluronic F127 into bilayer of monoolein-based cubic The corneal hydration levels are indicative of corneal 946
887 phases (Dong et al., 2006). The non-uniform distribution of integrity and its normal levels lies in range of 75–80% 947
888 Pluronic F127 into cubic nanoparticles gave rise to formation (Maurice et al., 1970). Since, the corneal hydration levels in 948
889 of two distinct structures: one without Pluronic F127, D phase the present study are within limits (Table 5), the integrity of 949
890 and another with Pluronic F127, P phase. Phase coexistence corneal epithelium and endothelium was not compromised 950
891 in dispersion carrying monoolein and Pluronic F127 in excess by formulation tested. 951
892 water has already been reported (Nakano et al., 2002). The The toxicological and ocular irritation of the cubic 952
893 scattering curve for tropicamide-loaded cubic nanoparticles nanoparticles was evaluated employing the HET-CAM 953
894 was without any remarkable difference when compared to the study. The study depends on individual interpretations of 954
895 SAXS curve of blank nanoparticles. The pffiffiffi ppresence
ffiffiffi pffiffiffi pofffiffiffiffiffiBraggs tissue reactions. Severe coagulation was observed in positive 955
896 peaks
pffiffiffi pffiffiffi atpffiffiffi the
pffiffiffi pspacing
ffiffiffi ratios ( 2 , 4, 6, 10 and control, i.e. 0.1 N NaOH (positive control) and no reaction in 956
897 2, 3, 4, 6, 8) consistent with the formation of P- case of 0.9% NaCl (negative control). The values of potential 957
898 and D-phases in tropicamide-loaded cubic nanoparticles irritation scores were calculated to be 0.85 and 11.12 for 958
899 indicating that tropicamide did not produce any phase tropicamide-loaded cubic nanoparticles and 0.1 N NaOH, 959
900 transition in cubic nanoparticles. Figure 4(b) and (c) illustrate respectively. The tropicamide containing cubic nanoparticles 960
 DOI: 10.3109/10717544.2016.1143057 Potential of cubic liquid crystalline nanoparticles for ocular delivery 9

961 1021
962 1022
963 1023
964 1024
965 1025
966 1026
967 1027
968 1028
969 1029
970 1030
971 1031
972 1032
973 1033
974 1034
975 1035
976 1036
977 1037
978 1038
979 1039
980 1040
981 1041
982 1042
983 1043
984 1044
985 1045
986 1046
987 1047
988 1048
989 1049
990 1050
991 1051
992 1052
993 1053
994 1054
995 1055
996 1056
997 1057
998 1058
999 1059
1000 1060
1001 1061
1002 1062
1003 1063
1004 1064
1005 1065
1006 1066
1007 1067
1008 1068
1009 1069
1010 1070
1011 1071
Figure 4. SAXS results for tropicamide-loaded cubic nanoformulation. (a) Overlay showing scattering curve of cubic nanoparticles and tropicamide-
1012 loaded cubic nanoparticles. (b) Data fitting graphs of blank cubic nanoparticles. (c) Data fitting graphs of tropicamide-loaded cubic nanoparticles. 1072
1013 1073
1014 1074
1015 1075
1016 1076
1017 1077
1018 1078
1019 1079
1020 1080
 10 P. Verma & M. Ahuja Drug Deliv, Early Online: 1–12

1081 were found to be nonirritant as the potential irritation score results of HET-CAM study and therefore, demonstrated a 1141
1082 was less than 1 (Dehelean et al., 2011). preferable ocular tolerance. 1142
1083 Further, the effect of formulation on corneal cell structure Optimized tropicamide-loaded nanoparticles were evalu- 1143
1084 and tissue integrity was determined by histochemical studies. ated for their therapeutic performance by measuring in vivo 1144
1085 The cross section of fresh cornea was compared with cornea mydriatic response in rabbits. The plot between % increments 1145
1086 incubated with cubic nanoparticles, PBS (pH 7.4) and SDS in pupillary diameter of eye as the function of time is 1146
1087 (Figure 6). The corneal cells and tissues appeared to be exemplified in Figure 7. Both formulations showed increase 1147
1088 deformed in cornea exposed to SDS (positive control) when in pupillary response up to a maxima then decrease in 1148
1089 compared to the cross section of fresh cornea. Intercellular response was observed. It can be observed from the results 1149
1090 space widening and separation of superficial epithelium layer that it took 90 min to achieve maximum pupillary response in 1150
1091 was also observed. On the other hand, epithelium and stroma case of tropicamide-loaded cubic nanoparticles while 1151
1092 structure were found to be well maintained in corneal tissues TropicacylÕ elicited maximum response in 330 min. The 1152
1093 exposed to PBS pH 7.4 (negative control) and cubic reason for which could be attributed to rapid absorption 1153
1094 nanoparticles as observed in non-treated eye. The results nanoparticles through ocular tissues. Further, the values of 1154
1095 showed a good corneal biocompatibility corroborating the maximum increment (%) in pupillary diameter were observed 1155
1096 to be 74.52 ± 2.11% and 52.64 ± 1.18% for tropicamide cubic 1156
1097 Table 4. SAXS data for blank cubic nanoparticles and tropicamide- nanoparticles and commercial preparation respectively, 1157
loaded cubic nanoparticles.
1098 revealing higher therapeutic efficiency of nanoparticles 1158
1099
Blank cubic Tropicamide-loaded
in vivo as compared to TropicacylÕ . Area under mydriatic 1159
1100 nanoparticles cubic nanoparticles response curve (AUC0!1440 min) was remarkably higher for 1160
1101 1 1 tropicamide-loaded cubic nanoparticles than TropicacylÕ 1161
Phase q (nm ) d (nm) hkl q (nm ) d (nm) hkl
1102 (45 752.52%.min and 30 514.09%.min for tropicamide- 1162
1103 P type 0.178 35.374 110 0.174 36.092 110 loaded cubic nanoparticles and TropicacylÕ , respectively). 1163
0.254 24.774 200 0.253 24.822 200
1104
0.307 20.437 211 0.307 20.437 211 Further results obtained were suggestive of the prolonged 1164
1105 0.383 16.388 310 0.383 16.388 310 mydriatic response of cubic nanoparticles as compared to 1165
1106 TropicacylÕ . It is presumed that lipid bilayer in cubic 1166
D type 0.199 31.557 110 0.199 31.558 110
1107 0.241 26.077 111 0.241 26.078 111 nanoparticles may serve to prolong contact time with ocular 1167
1108 0.285 22.024 200 0.288 21.783 200 tissues. In addition, nonspecific interactions (hydrophobic and 1168
1109 0.345 18.192 211 0.345 18.192 211 van der Waals) of the cubic nanoparticles with the superficial 1169
0.400 15.700 220 0.400 15.700 220
1110 oily layer of the tear film could be another possible 1170
1111 explanation for this finding (Alany et al., 2006). Cubic 1171
1112 liquid crystalline nanoparticles are therefore a promising 1172
1113 effective system for delivery and prolonged release of 1173
1114 tropicamide with reduced ocular toxicity, to pursue a better 1174
1115 patient compliance. 1175
1116 1176
1117 Conclusions 1177
1118 1178
Ultrasound-assisted preparation of cubic nanoparticles was
1119 1179
optimized employing central composite experimental design.
1120 1180
The optimized parameters were used for preparing tropica-
1121 1181
mide-loaded ophthalmic cubic nanoformulation.
1122 1182
Tropicamide-loaded cubic nanoparticles were observed to
1123 1183
exist in both P and D phase form. The results of comparative
1124 1184
evaluation of tropicamide-loaded cubic nanoparticles with
1125 1185
commercial conventional ophthalmic solution revealed no
1126 1186
significant difference in in vitro corneal permeation charac-
1127 1187
teristics but significantly faster onset and higher intensity of
1128 1188
mydriatic action was observed by in vivo study for the cubic
1129 1189
nanoparticles formulation. According to HET-CAM’s and
1130 Figure 5. In vitro corneal permeation profile tropicamide-loaded cubic 1190
histochemical studies, the developed cubic ophthalmic
1131 nanoformulation and commercially available conventional aqueous 1191
preparation of tropicamide (TropicacylÕ ). nanoformulation should not bring discomfort to the patient.
1132 1192
1133 1193
1134 1194
1135 Table 5. Steady state flux (Jss) and permeability (Kp) of tropicamide through porcine cornea and corneal hydration level. 1195
1136 1196
Apparent permeability
1137 2 1197
Formulations Flux Jss (mg/h/cm ) coefficient Kp (103 cm/h) Corneal hydration (%)
1138 1198
1139 Tropicamide-loaded ophthalmic cubic nanoparticles 68.43 6.84 77.79 ± 1.81 1199
TropicacylÕ 71.23 7.12 77.62 ± 1.56
1140 1200
 DOI: 10.3109/10717544.2016.1143057 Potential of cubic liquid crystalline nanoparticles for ocular delivery 11

1201 1261
1202 1262
1203 1263
1204 1264
1205 1265
1206 1266
1207 1267
1208 1268
1209 1269
1210 1270
1211 1271
1212 1272
1213 1273
1214 1274
1215 1275
1216 1276
1217 1277
1218 1278
1219 1279
1220 1280
1221 1281
1222 1282
1223 1283
1224 1284
1225 1285
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1227 1287
1228 1288
1229 1289
1230 1290
1231 1291
1232 1292
1233 1293
1234 1294
1235 1295
1236 1296
1237 1297
1238 1298
1239 Figure 6. Histological section of (a) non-treated, (b) tropicamide-loaded cubic nanoformulation-treated, (c) phosphate-buffered saline pH 7.4 (negative 1299
1240 control)-treated, and (d) SDS (positive control)-treated corneas. 1300
1241 1301
1242 In conclusion, cubic liquid crystalline nanoparticles can be a 1302
1243 promising vehicle for effective ocular delivery of tropicamide. 1303
1244 1304
1245 Acknowledgements 1305
1246 1306
The authors express gratitude to SAIF, AIIMS, New Delhi
1247 1307
and SMITA Research labs, IIT, Delhi for TEM and SAXS
1248 1308
analysis. The authors are extremely grateful to the
1249 1309
Coordinator, DST FIST, Dept. of Pharmaceutical Sciences
1250 1310
(GJUS&T, Hisar) for particle size analysis.
1251 1311
1252 1312
Declaration of interest
1253 1313
1254 The authors report no conflicts of interest. The authors alone 1314
1255 are responsible for the content and writing of this article. 1315
1256 1316
1257 References 1317
1258 1318
Ahmed I. (2003). The noncorneal route in ocular drug delivery. In: Mitra
1259 AK, ed. Ophthalmic drug delivery systems. New York: Marcel 1319
1260 Figure 7. Pupillary size increment (%) of rabbit eye against the Dekker, 335–63. 1320
commercial preparation (TropicacylÕ ) as a function of time.
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1321 Alany RG, Rades T, Nicoll J, et al. (2006). W/O microemulsions for surface of the gyroid type in the glycerolmonooleat-water system. Z 1381
ocular delivery: evaluation of ocular irritation and precorneal Kristallogr 168:213–19.
1322 1382
retention. J Control Release 111:145–52. Jadhav AJ, Holkar CR, Karekar SE, et al. (2015). Ultrasound assisted
1323 Ali J, Fazil M, Qumbar M, et al. (2014). Colloidal drug delivery system: manufacturing of paraffin wax nanoemulsions: process optimization. 1383
1324 amplify the ocular delivery. Drug Deliv 21:1–17. Ultrason Sonochem 23:201–7. 1384
1325 Barauskas J, Johnsson M, Joabsson F, Tiberg F. (2005). Cubic phase Karaman S, Yilmaz MT, Ertugay MF, et al. (2012). Effect of ultrasound 1385
nanoparticles (cubosome): principles for controlling size, structure, treatment on steady and dynamic shear properties of glucomannan
1326 1386
and stability. Langmuir 21:2569–77. based salep dispersions: optimization of amplitude level, sonication
1327 Bashir MJK, Aziz HA, Yusoff MS, Adlan MN. (2010). Application of time and temperature using response surface methodology. Ultrason 1387
1328 response surface methodology (RSM) for optimization of ammoniacal Sonochem 19:928–38. 1388
1329 nitrogen removal from semi-aerobic landfill leachate using ion Kaur H, Ahuja M, Kumar S, Dilbaghi N. (2012). Carboxymethyl 1389
exchange resin. Desalination 254:154–61. tamarind kernel polysaccharide nanoparticles for ophthalmic drug
1330 Baydoun L, Düvel A, Daniels R, et al. (2004). Comparison of different 1390
delivery. Int J Biol Macromol 50:833–9.
1331 ibuprofen-amino acid compounds with respect to emulsifying and Korbahti BK, Tanyolac A. (2008). Electrochemical treatment of 1391
1332 cytotoxic properties. Int J Pharm 274:157–65. simulated textile wastewater with industrial components and Levafix 1392
1333 Bei D, Marszalek J, Youan BBC. (2009a). Formulation of dacarbazine- Blue CA reactive dye: optimization through response surface meth- 1393
loaded cubosomes – part I: influence of formulation variables. AAPS odology. J Hazard Mater 151:422–31.
1334 PharmSciTech 10:1032–9. 1394
Kumar A, Ahuja M. (2014). Aqueous carboxymethyl gum kondagogu as
1335 Bei D, Marszalek J, Youan BC. (2009b). Formulation of dacarbazine- vehicle for ocular delivery. J Pharm Investig 44:237–42. 1395
1336 loaded cubosomes-part II: influence of process parameters. AAPS Kuttruff H. (1988). Physik und Technik des Ultraschalls. Stuttgart: S. 1396
PharmSciTech 10:1040–7. Hirzel Verlag.
1337 1397
Boyd BJ. (2003). Characterisation of drug release from cubosomes using Lallemand F, Daull P, Benita S, et al. (2012). Successfully improving
1338 the pressure ultrafiltration method. Int J Pharm 260:239–47. 1398
ocular drug delivery using the cationic nanoemulsion, novasorb. J
1339 Calvo P, Vila-Jato JL, Alanson MJ. (1996). Comparative in vitro Drug Deliv 2012:604204. 1399
1340 evaluation of several colloidal system, nanoparticles, nanocapsules Lancelot A, Sierra T, Serrano JL. (2014). Nanostructured liquid 1400
and nanoemulsions as ocular drug carriers. J Pharm Sci 85:30–6. crystalline particles for drug delivery. Expert Opin Drug Deliv 11:
1341 1401
Chong JYT, Mulet X, Waddington LJ, et al. (2012). Highthroughput
1342 547–64. 1402
discovery of novel steric stabilizer for cubic lyotropic liquid drystal-
Landh T. (1994). Phase behavior in the system pine oil monoglycerides-
1343 line nanoparticles dispersions. Langmuir 28:9223–32. 1403
poloxamer 407-water at 20 C. J Phys Chem 98:8453–67.
1344 De Sa FAP, Taveira SF, Gelfuso GM, et al. (2015). Liposomal 1404
Law SL, Huang KJ, Chiang CH. (2000). Acyclovir-containing liposomes
voriconazole (VOR) formulation for improved ocular delivery.
1345 for potential ocular delivery. Corneal penetration and absorption. J 1405
Colloids Surf B 133:331–8.
1346 Dehelean C, Alexa E, Feflea S, et al. (2011). Ochratoxin A: a toxicologic Control Release 63:135–40. 1406
evaluation using in vitro and in vivo bioassays. Ann Oradea Univ Biol Li J, Wu L, Wu W, et al. (2013). A potential carrier based on liquid
1347 1407
Fasc 28:99–103. crystal nanoparticles for ophthalmic delivery of pilocarpine nitrate. Int
1348 J Pharm 455:75–84. 1408
Dian L, Yang Z, Li F, et al. (2013). Cubic phase nanoparticles for
1349 sustained release of ibuprofen: formulation, characterization, and Luepke NP. (1985). Hen’s egg chorioallantoic membrane test for 1409
1350 enhanced bioavailability study. Int J Nanomed 8:845–54. irritation potential. Food Chem Toxicol 23:287–91. 1410
Dilbaghi N, Kaur H, Ahuja M, Kumar S. (2013). Evaluation of Maurice DM, Riley MV, Graymore CN. (1970). Biochemistry of the eye.
1351 1411
tropicamide-loaded tamarind seed xyloglucan nanoaggregates for London. Academic Press, 6–16.
1352 Muller F, Salonen A, Glatter O. (2010). Monoglyceride-based cubo- 1412
ophthalmic delivery. Carbohydr Polym 94:286–91.
1353 Dong YD, Larson I, Hanley T, Boyd BJ. (2006). Bulk and dispersed somes stabilized by laponite: separating the effects of stabilizer, pH 1413
1354 aqueous phase behavior of phytantriol: effect of vitamin E acetate and and temperature. Colloids Surf A Physicochem Eng Asp 358:50–6. 1414
F127 polymer on liquid crystal nanostructure. Langmuir 22:9512–18. Nakano M, Teshigawara T, Sugita A, et al. (2002). Dispersions of liquid
1355 1415
Driever CD, Mulet X, Waddington LJ, et al. (2013). Layer by layer crystalline phases of the monoolein/oleic acid/Pluronic F127 system.
1356 Langmuir 18:9283–8. 1416
polymer coating on discrete particles of cubic liquid crystalline
1357 dispersions. Langmuir 29:12891–900. Peltola S, Saarinen-Savolainen P, Kiesvaara J, et al. (2003). 1417
1358 Engstrom S, Ljusberg-Wahren H, Gustafsson A. (1995). Bioadhesive Microemulsions for topical delivery of estradiol. Int J Pharm 254: 1418
1359 properties of the monoolein-water system. Pharm Tech Eur 7:14–17. 99–107. 1419
Ferreira DA, Bentley M, Karlsson G, Edwards K. (2006). Cryo-TEM Qiu H, Caffrey M. (2000). The phase diagram of the monoolein/water
1360 system: metastability and equilibrium aspects. Biomaterials 21: 1420
investigation of phase behaviour and aggregate structure in dilute
1361 dispersions of monoolein and oleic acid. Int J Pharm 310:203–12. 223–34. 1421
1362 Floris A, Meloni MC, Lai F, et al. (2013). Cavitation effect on chitosan Raichel DR. (2000). The science and applications of acoustics. 1422
1363 nanoparticle size: a possible approach to protect drugs from ultrasonic New York: Springer. 1423
stress. Carbohydr Polym 94:619–25. Rodriguez C, Kunieda H. (2000). Effect of electrolytes on discontinuous
1364 Gan L, Han S, Shen J, et al. (2010). Self-assembled liquid crystalline cubic phases. Langmuir 16:8263–9. 1424
1365 nanoparticles as a novel ophthalmic delivery system for dexametha- Siekmann B, Bunjes H, Koch MH, Westesen K. (2002). Preparation and 1425
1366 sone: improving preocular retention and ocular bioavailability. Int J structural investigations of colloidal dispersions prepared from cubic 1426
1367 Pharm 396:179–87. monoglyceride-water phases. Int J Pharm 244:33–43. 1427
Gaudana R, Ananthula HK, Parenky A, Mitra AK. (2010). Ocular drug Silva R, Ferreira H, Little C, Cavaco-Paulo A. (2010). Effect of
1368 delivery. AAPS J 12:348–60. ultrasound parameters for unilamellar liposome preparation. Ultrason 1428
1369 Gratieri T, Gelfuso GM, de Freitas O, et al. (2011). Enhancing and Sonochem 17:628–32. 1429
1370 sustaining the topical ocular delivery of fluconazole using chitosan Spicer PT, Hayden KL, Lynch ML, et al. (2001). Novel process for 1430
1371
solution and poloxamer/chitosan in situ forming gel. Eur J Pharm producing cubic liquid crystalline nanoparticles (cubosomes). 1431
Biopharm 79:320–7. Langmuir 17:5748–56.
1372 Guo C, Wang J, Cao F, et al. (2010). Lyotropic liquid crystal systems in Tang SY, Manickam S, Wei TK, Nashiru B. (2012). Formulation 1432
1373 drug delivery. Drug Discov Today 15:1032–40. development and optimization of a novel Cremophore EL-based 1433
1374 Gupta AK, Madan S, Majumdar DK, Maitra A. (2000). Ketorolac nanoemulsion using ultrasound cavitation. Ultrason Sonochem 19: 1434
entrapped in polymeric micelles: preparation, characterisation and 330–45.
1375 1435
ocular anti-inflammatory studies. Int J Pharm 209:1–14. Verma P, Ahuja M. (2015). Optimization, characterization and evalu-
1376 Hao J, Wang F, Wang X, et al. (2012). Development and optimization of 1436
ation of chitosan-tailored cubic nanoparticles of clotrimazole. Int J
1377 baicalin-loaded solid lipid nanoparticles prepared by coacervation Biol Macromol 73:138–45. 1437
1378 method using central composite design. Eur J Pharm Sci 47:497–505. Zhang W, Prausnitz MR, Edwards A. (2004). Model of transient drug 1438
Hyde ST, Andersson S, Ericsson B, Larsson K. (1984). A cubic structure diffusion across cornea. J Control Release 99:241–58.
1379 1439
consisting of a lipid bilayer forming an infinite periodic minimum
1380 1440