Bromelain nanoparticles protect against 7,12-dimethylbenz[a]anthracene induced skin carcinogenesis in mouse model

Bromelain nanoparticles protect against 7,12-dimethylbenz[a]anthracene induced skin carcinogenesis in mouse model

EJPB 11809 No. of Pages 12, Model 5G 28 January 2015 European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx 1 Contents lists ava...

2MB Sizes 0 Downloads 0 Views

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx 1

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

2

Research paper

6 4 7

Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogenesis in mouse model

5

Q1

8

Q2

9 10 11

Q3

a b

13 12 14

CSIR-Institute of Genomics and Integrative Biology, Delhi, India CSIR-Indian Institute of Toxicology Research, Lucknow, India

a r t i c l e

1 2 6 8 17 18 19 20 21 22 23 24 25 26 27

Priyanka Bhatnagar a, Aditya B. Pant b, Yogeshwer Shukla b, Bhushan Chaudhari b, Pradeep Kumar a, Kailash C. Gupta a,b,⇑

i n f o

Article history: Received 4 August 2014 Accepted in revised form 14 January 2015 Available online xxxx

Q4

Keywords: Bromelain Nanoparticles DMBA Skin tumorigenesis Poly (lactic-co-glycolic acid)

a b s t r a c t Conventional cancer chemotherapy leads to severe side effects, which limits its use. Nanoparticles (NPs) based delivery systems offer an effective alternative. Several evidences highlight the importance of Bromelain (BL), a proteolytic enzyme, as an anti-tumor agent which however has been limited due to the requirement of high doses at the tumor site. Therefore, we illustrate the development of BL loaded poly (lactic-co-glycolic acid) NPs that show enhanced anti-tumor effects compared to free BL. The formulated NPs with a mean particle size of 130.4 ± 8.81 nm exhibited sustained release of BL. Subsequent investigation revealed enhanced anti-tumor ability of NPs in 2-stage skin tumorigenesis mice model. Reduction in average number of tumors (2.3 folds), delay in tumorigenesis (2 weeks), percent tumorigenesis (4 folds), and percent mortality rate as well as a reduction in the average tumor volume (2.5 folds) in mice as compared to free BL were observed. The NPs were found to be superior in exerting chemopreventive effects over chemotherapeutic effects at 10 fold reduced dose than free BL, validated by the enhanced ability of NPs (1.8 folds) to protect the DNA from induced damage. The effects were also supported by histopathological evaluations. NPs were also capable of modulating the expression of pro-apoptotic (P53, Bax) and anti-apoptotic (Bcl2) proteins. Therefore, our findings demonstrate that developed NPs formulation could be used to improve the efficacy of chemotherapy by exerting chemo-preventive effects against induced carcinogenesis at lower dosages. Ó 2015 Published by Elsevier B.V.

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

48 49

1. Introduction

50

Cancer is one of the leading causes of morbidity and accounts for almost 7.6 million deaths annually worldwide [1]. Amongst cancers of all types, instances of skin cancer are reportedly increasing as their early detection is comparatively easier [2]. Surgery is often the most frequent approach to remove a localized tumor, but in the later stage of a metastasized cancer, use of chemotherapeutic agents, radiotherapy, or a combination of both is required to completely eliminate the tumor but does not work in most of the cases [3]. Chemotherapy usually involves long term chronic exposure to chemotherapeutic drugs, which leads to various physiological complications, severe/moderate distress and cytotoxicity [4,5]. Recently, topical application of anti-cancer drugs has gained tremendous importance as they offer several benefits such as ease

51 52 53 54 55 56 57 58 59 60 61 62

⇑ Corresponding author. CSIR-Indian Institute of Toxicology Research, M.G. Marg, Lucknow 226001, Uttar Pradesh, India. Tel.: +91 522 2621856; fax: +91 522 2628227. E-mail addresses: [email protected], [email protected] (K.C. Gupta).

of availability, application and reduced systemic side effects with no drug degradation in the gastrointestinal tract [6,7]. Thus, topical administration of anti-cancer drugs provides an attractive alternative to increase drug targeting and penetration of drugs in sufficient levels in the tumor tissues [8]. Contrary to this, reports suggest that the stratum corneum, the outermost layer of the epidermis, acts as a skin barrier, preventing entry of the majority of drugs into the viable skin [9]. In addition, the ability of tumor cells to develop multidrug resistance due to P-glycoprotein on their surface results in exudation of drug from tumor cells thus increasing the difficulties associated with conventional chemotherapy [10,11]. Several formulations have, therefore, been developed to overcome these barriers and to reach skin malignancies by favoring drug penetration into the deep layers of the epidermis [12,13]. In this regard, scientists have generated great interest in the nanoparticles (NPs) based delivery systems owing to their ability to have enhanced penetration and retention (EPR) effect at the tumor site [14,15]. Several reports have demonstrated the ability of NPs to release anti-cancer drugs into cells without triggering the P-glycoprotein pump as they internalize into the target cells by

http://dx.doi.org/10.1016/j.ejpb.2015.01.015 0939-6411/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 2 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

endocytosis [16,17]. In addition, NPs also provide protection to the drug against degradation and therefore, sustained release of drug molecules from the NPs surface could be achieved at the tumor site with high therapeutic efficiency and low cytotoxicity. Moreover, nano-carriers can reduce skin irritation by avoiding direct contact of the drug with the skin surface [18]. Various nano-carriers based on liposomes, biodegradable polymers, lipids, chitosan, dextrans, etc. have been studied extensively for the topical application of anti-cancer drugs such as 5-fluorouracil [19–21]. Recent studies strongly indicate that certain commonly consumed dietary photochemicals have potent cancer protective effects against a variety of human cancers [22]. More than 25% of drugs used during the last 20 years are directly derived from plants, while the other 25% are the chemically altered natural products [23]. The advantage of using such compounds for cancer treatment is that they are relatively non-toxic/less toxic at therapeutic doses. Several molecules, including paclitaxel (obtained from the bark of the Pacific yew tree), vincristine (from Catharanthus roseus), topotecan, curcumin (from Indian spice turmeric), epigallocatechin gallate (from green tea), etc. have been shown to possess excellent anti-cancer properties against a variety of human cancers and some of them are already in clinical use [24]. Among the various natural phytochemicals, Bromelain (BL), which belongs to a family of sulfhydryl proteolytic enzymes obtained from the pineapple plant (Ananas comosus), has been shown to possess anti-proliferative and anti-metastatic activities in tumor models in vitro and in vivo [25,26]. Presumably, BL is known to impart its anti-cancerous activity via immune, inflammatory, and homeostatic pathways, as well as it exerts its influence on a variety of molecules involved in cell signaling [26]. Although the mechanism of anti-cancer activity of BL is known, its therapeutic efficacy is low due to necessity of high doses of the drug at the tumor site. In order to further increase the potency of BL against solid tumors, a concept of nano-chemoprevention has been made. We hypothesized that formation of safe and biocompatible BL encapsulated stable NPs would provide a prolonged release of BL at the tumor site, hereby coupled with the EPR effect exerted by NPs could offer more consistent biological results. Thus, in the present investigation, BL was encapsulated in an FDA approved biodegradable and biocompatible, poly (lactic-co-glycolic acid) (PLGA) polymer, formulating NPs named as, BL-PLGA NPs. Formulated BL-PLGA NPs were characterized and then evaluated for their anti-cancer efficacy in 7,12-dimethylbenz[a]anthracene (DMBA) induced and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted 2-stage skin tumorigenesis model in Swiss albino mice. The experiments were carried out to investigate the chemo-preventive and chemotherapeutic effects of BL-PLGA NPs and compared with that of free BL.

130

2. Materials and methods

131

2.1. Materials

132

139

Poly (lactic-co-glycolic acid) (PLGA) 50:50 (m.wt. 30–60 kDa), Bromelain from pineapple stem, poly vinyl alcohol (PVA) (m.wt. 30 kDa), sodium bicarbonate, sucrose, casein, 7,12-dimethylbenz [a]anthracene (DMBA), 12-O-tetradecanoylphorbol-13-acetate (TPA), sodium dodecylsulphate (SDS) and bicinchoninic acid (BCA) kit for protein estimation were purchased from Sigma– Aldrich (St. Louis, USA). Other chemicals and reagents were purchased locally and were of highest purity grade.

140

2.2. Preparation of BL-PLGA NPs

141

BL-PLGA NPs were prepared using double emulsion solvent evaporation technique, as described previously in the literature

133 134 135 136 137 138

142

by our group [27]. The formulated NPs with 84 ± 3.4% yield were obtained and stored at 4 °C until further use.

143

2.3. Characterization of BL-PLGA NPs

145

2.3.1. Particle size analysis by Dynamic Light Scattering The formulated NPs were evaluated for their mean particle size and distribution by Dynamic Light Scattering (DLS) technique (Zetasizer Nano-ZS, Malvern Instruments, UK), employing a nominal 5 mW He–Ne laser operating at 633 nm as described here. Briefly, BL-PLGA NPs were suspended in double distilled water at a concentration of 0.5 mg/mL and the particle size was measured. The measurements were carried out at 25 ± 2 °C with the following settings: 14 measurements per sample, refractive index of water: 1.33, viscosity for water: 0.89 cP. The particle size reported was the average of three samples. The particle size of the NPs was computed from the intensity–intensity correlation function using the Malvern software package based on the theory of Brownian motion and Stroke’s equation.

146

2.3.2. Transmission Electron Microscopy The morphology and size of the BL-PLGA NPs were evaluated using Transmission Electron Microscopy (TEM). Briefly, BL-PLGA NPs were suspended in double distilled water at a concentration of 0.5 mg/mL, and a drop of the suspension and a drop of 1% uranyl acetate were placed gently on a formvar-coated TEM grid surface. Q5 After 30 min of incubation, excess fluid was removed and the grid surface was allowed to air dry at 25 ± 2 °C. The particles were visualized under the microscope (FEI Company, Hillsboro, OR) operated at 80 kV and attached to a Gatan Digital Micrograph™ (Gatan Inc, Pleasanton, CA).

160

2.3.3. Encapsulation efficiency and drug loading The encapsulation efficiency (% EE) and drug loading (% DL) of BL in the BL-PLGA NPs were evaluated as done previously [27].

171

2.3.4. In vitro release study In vitro release profile of BL from BL-PLGA NPs was evaluated in phosphate buffer saline (PBS) at the physiological pH (7.2–7.4) and acidic pH (6.2–6.5). In brief, 10 mg BL-PLGA NPs were suspended in 1 mL of respective buffers and kept in an incubator shaker pre-maintained at 37 ± 0.5 °C with constant stirring. At predetermined time intervals, the suspension was centrifuged at 100g for 10 min at 4 °C; the supernatant was analyzed for total BL content by BCA assay. Equal amount of fresh buffer was added to the NPs pellet and the release study was continued. The amount of the BL release was then calculated using a previously drawn standard curve of BL in the same buffer.

174

2.3.5. Proteolytic activity of BL in BL-PLGA NPs: Casein Digesting Unit The enzymatic activity of BL encapsulated in BL-PLGA NPs was measured by estimating its ability to act on its substrate, casein and measured Casein Digestion Units (CDU), as described earlier [27]. The experiment was carried out in triplicate.

186

2.3.6. Colloidal stability of BL-PLGA NPs The colloidal stability of BL-PLGA NPs was examined by measuring change in their hydrodynamic diameter during storage using DLS technique. Briefly, 0.5 mg BL-PLGA NPs were dispersed in 1 mL of PBS pH 7.2–7.4 and 6.2–6.5, respectively. The NPs suspension was stored at 4 °C. At regular intervals of time, the particle size was measured by DLS using a Zetasizer Nano-ZS instrument. The experiment was carried out in triplicate.

191

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

144

147 148 149 150 151 152 153 154 155 156 157 158 159

161 162 163 164 165 166 167 168 169 170

172 173

175 176 177 178 179 180 181 182 183 184 185

187 188 189 190

192 193 194 195 196 197 198

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 3

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx 199

2.4. In vivo anti-tumor efficacy of BL-PLGA NPs

200

2.4.1. Experimental mice and development of 2-stage skin tumorigenesis model Male Swiss albino mice (20–22 g body weight), obtained from animal breeding colony of CSIR-Indian Institute of Toxicology Research (Lucknow, India), were acclimatized for one week and housed in plastic cages with rice husk bedding under standard laboratory conditions of 12 h light/dark cycle, temperature 22 ± 2 °C, relative humidity 55 ± 5%. These were fed with the commercial solid pellet diet (Nutrilab Rodent, Tetragon Chinese, Bangalore, India) and water ad libitum. The ethical approval for the experiment was obtained from the Institutional Ethical Committee. These mice were used to develop 2-stage skin tumorigenesis model in which DMBA was used as a tumor initiator and was applied topically at a single dose; TPA was used as a tumor promoter and was applied topically thrice a week for 22 weeks. The experiment was carried out for 22 weeks to evaluate the entire period of skin tumorigenicity.

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229

2.4.2. Experimental design and doses The mice were randomly divided into 8 groups, each consisting of 10 mice. The dorsal skin of mice (2  2 cm) was shaved off with proper care using an electrical clipper 48 h prior to the start of the experiment. DMBA was applied at the dose: 100 lg in 200 lL acetone, TPA: 25 lg in 200 lL acetone, free BL suspension: 1.5 mg in 200 lL normal saline and BL-PLGA NPs: 0.15 mg in 200 lL normal Q6 saline were applied topically to the shaved dorsal skin of mouse. This study was divided into 2 set of experiments where the mice received the treatment at different stages of tumorigenesis: i.e. (1) pre-TPA: served as an experimental set to evaluate chemopreventive effects, (2) post-TPA: served as an experimental set to evaluate chemo-therapeutic effects. The experimental design, treatment groups and treatment schedule are described as below: Group

Treatment

I: Vehicle control

II: Positive control

Free BL groups III:

IV:

V:

DMBA + TPA

DMBA + {BL + TPA}

DMBA + {TPA + BL}

DMBA + BL

Schedule Topical application of 200 lL acetone. Single application of DMBA, 1 week followed by topical application of TPA thrice a week Single application of DMBA, 1 week followed by thrice a week application of BL 1 h prior to TPA application Single application of DMBA, 1 week followed by thrice a week application of TPA 1 h prior to BL application Single application of DMBA, 1 week followed by thrice a week BL application

Group

Treatment

BL-PLGA-NPs groups DMBA + {NPs + TPA} VI:

VII:

DMBA + {TPA + NPs}

VIII:

DMBA + NPs

Schedule

Single application of DMBA, 1 week followed by thrice a week application of NPs 1 h prior to TPA application Single application of DMBA, 1 week followed by thrice a week application of TPA 1 h prior to NPs application Single application of DMBA, 1 week followed by thrice a week NPs application 311

DMBA + TPA group II served as carcinogen treated positive control group. Groups III, VI served as the groups for chemo-preventive studies. Groups IV, VII served as groups for chemo-therapeutic studies. Group V and group VIII served as control groups to evaluate the toxicity of free BL and BL-PLGA NPs alone.

312

2.4.3. Morphological observations of developed papillomas Throughout the experiment, the mice were kept under observation for any change in body weight, development of skin tumors, latency in tumor formation, percent tumor incidence and cumulative number of tumors. Tumor volume was also calculated for every mouse of each group using the formula V = D  d2  p/6, where V = average tumor volume, D = biggest dimension and d = smallest dimension. Tumors of at least 1.0 mm diameter were counted every week. After completion of 22 weeks of treatment, the regression pattern in tumors in terms of both tumor number and tumor volume was recorded. The survival rate of mice of every group was also noted.

317

2.4.4. Histopathological analysis At the end of 20 weeks, the mice of each group were sacrificed and the skin from the treated area was excised, cleaned, stored in formalin solution before processing for histopathological examinations. The tissue sections were fixed in 4% formaldehyde and then embedded in paraffin. Sections of 2–4 lm thick were cut using automatic Rotary Microtome 125 (Leica Microsystems, Germany) and processed as per the standard procedure and were subjected to hematoxylin and eosin staining. The slides were examined Q7 under the light microscope (Leica, Wetzlar, Germany).

329

2.5. Chemo-preventive potential of BL-PLGA NPs

339

2.5.1. Animals and treatments To evaluate the mechanism of tumor cell killing and anti-mutagenic potential of BL-PLGA NPs, another set of experiment was performed. The Swiss albino mice were divided into 7 groups consisting of 6 mice each. The dorsal skin of mice (2  2 cm) was shaved with the help of an electrical clipper 48 h prior to the treatment. The experimental design and the treatment groups are as described below.

340

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

313 314 315 316

318 319 320 321 322 323 324 325 326 327 328

330 331 332 333 334 335 336 337 338

341 342 343 344 345 346 347

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 4 348 349 350 351 352 353 354

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

Mice were administered with free BL and BL-PLGA NPs followed by treatment with DMBA, which is a known mutagen. The ability of NPs to protect the DNA from DMBA induced damage was evaluated and compared with that of free BL. Post 24 h of administration, the mice from all the groups were sacrificed by cervical dislocation and the skin from the treated area was excised, cleaned, frozen in liquid nitrogen and stored at 80 °C until further use.

Group

Treatment

Vehicle control:

Single topical application of 200 lL of acetone Single topical application of DMBA at 100 lg in 200 lL acetone Single topical application of BL solution at 1.5 mg in 200 lL normal saline Single topical application of NPs at 1.5 mg in 200 lL normal saline. Single topical application of BL at 1.5 mg in 200 lL normal saline 1 h prior to DMBA application at 100 lg in 200 lL acetone Single topical application of NPs at 75 lg in 200 lL normal saline 1 h prior to DMBA application at 100 lg in 200 lL acetone Single topical application of BL-PLGA NPs at 150 lg in 200 lL normal saline 1 h prior to DMBA application at 100 lg in 200 lL acetone

DMBA: Free BL only:

NPs only: Free BL + DMBA:

NPs (1/20) + DMBA:

NPs (1/10) + DMBA:

388

389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410

411 413 414 415 416 417 418

419 421

2.5.2. DNA alkaline unwinding assay The DNA was isolated from the skin samples of mice of each group using the procedure, as described earlier [28]. The concentration and integrity of DNA isolated from skin samples of each group were measured by taking absorbance at 260 nm. The alkali unwinding assay was performed as described previously in the literature [29]. Briefly, 100 lg DNA from each group was loaded onto hydroxyapatite columns and was subjected to alkaline unwinding for 1 h in a dark chamber in presence of 0.06 N NaOH in 0.1 M phosphate buffer, pH 12. The pH of the reaction mixture was neutralized to pH 7.0 with 0.07 N HCl. To this solution was added 150 lL of 10 mM phosphate buffer with 10% DMF, followed by addition of 150 lL of 0.02 M EDTA containing 2% SDS. The columns were kept at 4 °C overnight and then incubated at 60 ± 2 °C for 2 h followed by centrifugation at 100g for 20 min at 22 ± 0.5 °C. The supernatant was discarded and the DNA was selectively eluted from the hydroxyapatite column using 0.125 M potassium phosphate buffer, pH 7.0, containing 20% DMF. The fraction was incubated in a water bath maintained at 60 °C for 30 min and centrifuged at 100g for 20 min. The DNA was quantified by measuring absorbance at 260 nm. The strand breaks in DNA were calculated by the formula given below:

ln F ¼ ðk=Mn Þt

b

where F is the fraction of remaining ds-DNA after alkali treatment for the time t, Mn is the number-average molecular weight between two breaks, and b is a constant that is less than 1. The number of unwinding points (P) per alkaline unwinding unit of DNA was measured by using the following equation:

P ¼ ln F x = ln F 0

where Fx and F0 are the fractions of dsDNA remaining after alkaline denaturation of treated and control samples, respectively. The number of breaks (n) per unit DNA was then determined using the equation:

n¼P1

422 423 424 425

426 428

2.5.3. Western blot analysis The Swiss albino mice treated with DMBA, free BL and BL-PLGA NPs at indicated doses were sacrificed after 24 h of treatment and the skin from the treated area was excised, cleaned and snap frozen. The total protein content was harvested by the conventional method as described here. Briefly, 100 mg of the tissue samples were homogenized with 1 mL of RIPA buffer (pH 7.6) containing 1 M sodium dihydrogen phosphate, 10 mM disodium hydrogen phosphate, 154 mM sodium chloride, 1% Triton X-100, 12 mM sodium deoxycholate, 0.2% sodium azide, 2 mM phenylmethylsulfonyl fluoride, 50 mg/mL aprotinin and 0.1% SDS at 0 °C. The tissue lysates were centrifuged at 15,000g for 20 min at 4 °C and the supernatants were collected. Amount of protein was quantified by BCA assay and stored at 80 °C for Western blot analysis. The altered expression of marker proteins of apoptosis and cell death (P53, P21, Bax and Bcl2) were studied in lysates as described earlier [30]. Briefly, an equal amounts (40 lg/well) of denatured proteins were loaded onto 10% SDS gel and blotted to polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by wet transfer method using transfer buffer [25 mM Tris (pH 8.3), 190 mM Glycine, 20% methanol] at 250 mA current for 2 h. Non-specific binding was blocked with 2% BSA and 3% non-fat dry milk powder in TBST [20 mM Tris–HCl (pH 7.4), 137 mM NaCl and 0.1% Tween 20] for 2 h at 37 °C. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies specific for P53, Bax, Bcl2, P21 (1:1000, Chemicon, USA) in blocking buffer (pH 7.5). The membrane was then incubated for 2 h at 22 ± 2 °C with secondary anti-primary immunoglobulin G (IgG)-conjugated with horseradish peroxidase (Chemicon, USA). The blots were developed using chemiluminescence kit (Millipore, Bedford, USA) and visualized by Versa Doc Imaging System (Bio-Rad, CA, USA). Densitometry measurements of the bands were done with digitized scientific software program UN-SCAN-IT (Silk Scientific Corporation, Orem, USA).

429

2.6. Statistical analysis

463

All the results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by one-way ANOVA using IBM SPSS Statistical software version 20. To find out whether the two values of interest were statistically significant, a Post hoc Tukey’s test was performed after ascertaining homogeneity of variance and normalization of data. All the comparisons with p value less than 0.05 were considered to be statistically significant.

464

3. Results

471

In the present work, BL loaded PLGA NPs were synthesized, characterized and used as an anti-carcinogenic agent against DMBA induced skin cancer in mouse model. The mechanism and efficacy analysis was done by comparing BL-PLGA NPs formulation with that of free BL. The present laboratory scale investigations are considered as a proof of concept for the potential applicability of BL loaded PLGA NPs to treat skin cancers.

472

3.1. Preparation and characterization of BL-PLGA NPs

479

BL-PLGA NPs were prepared using double emulsion solvent evaporation method, where PVA was used as a stabilizer. The

480

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462

465 466 467 468 469 470

473 474 475 476 477 478

481

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 5

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

491

3.2. In vitro release profile

492

501

The in vitro release of the BL from the BL-PLGA NPs was investigated in PBS for 8 days at physiological pH (7.2–7.4) and acidic pH (6.2–6.5) Fig. 2A. The formulation exhibited the burst release of BL releasing 17 ± 2.34 and 21 ± 3.17% of the encapsulated BL at pH 7.4 and 6.5, respectively, in the initial 12 h. The release profile was followed by sustained release of BL providing 87 ± 3.27% of the encapsulated BL in 192 h at pH 7.2. On the other hand, in acidic conditions, it took only 120 h to completely (90 ± 4.36%) release the BL from the polymer matrix. The results demonstrated the faster release of BL at acidic pH.

502

3.3. Colloidal stability of BL-PLGA NPs

503

In order to determine the time dependent colloidal stability of the developed BL-PLGA NPs, the hydrodynamic diameter of the NPs was measured in PBS as a function of time for a period of 8 days. Fig. 2B represents the hydrodynamic diameter of BL-PLGA NPs at pre-determined intervals of time measured by DLS technique at physiological pH 7.2–7.4 and acidic pH 6.2–6.5. In the beginning of the experiment, the mean particle size of the NPs was 192.2 ± 10.5 and 187.19 ± 11.81 nm at physiological pH and acidic pH, respectively. At the end of 8 days, the particle size was

484 485 486 487 488 489

493 494 495 496 497 498 499 500

504 505 506 507 508 509 510 511

found to be 201.9 ± 12.18 and 218.81 ± 13.8 nm, at pH 7.2 and 6.5, respectively. The results showed non-significant (p > 0.05) changes in hydrodynamic diameter of NPs throughout the period i.e., 8 days at both pH values rendering the NPs stable.

512

3.4. Proteolytic activity: Casein Digesting Units

516

The loss, if any, in the proteolytic activity of BL in the nanotized formulation of BL was evaluated by calculating the Casein Digesting Units (CDU). CDU values were calculated for both free BL and BL extracted from BL-PLGA NPs. The CDU value for free BL was 564 ± 7.43 units/mg, while CDU for BL extracted from BL-PLGA NPs was 525 ± 13.01 units/mg. The results revealed an insignificant loss in proteolytic activity of BL encapsulated in the PLGA NPs. Hence, the NPs could effectively protect the BL from degradation or distortion.

517

3.5. In vivo anti-tumor efficacy

526

3.5.1. Onset of tumorigenesis and percent tumorigenicity As an initial approach, the anti-tumor efficacy of free BL and formulated BL-PLGA NPs was evaluated by monitoring the onset of tumorigenesis and percent tumorigenicity of the treated groups. The incidence of tumors in the carcinogen treated positive control DMBA + TPA group II was observed on 56 ± 4.01 day of experiment (Fig. 3A), whereas BL was found to be significantly effective in delaying the onset of tumorigenesis when compared to group II.

527

(A)

120 100 80 60 40

at pH 7.2-7.4 20

at pH 6.2-6.5 0 0

24

48

72

96

120

144

168

192

Time in hr

(B) 300 Hydrodynamic Diameter (nm)

483

%Drug Release

490

%EE and %DL of BL as evaluated by BCA assay, were found to be 52 ± 3.34 and 4.7 ± 0.42%, respectively. The mean particle size of BL-PLGA NPs measured by DLS was 130.4 ± 10.5 nm (Fig. 1A). The low poly-dispersity (PDI) index (0.095) of NPs confirmed the non-existence of aggregated particles. The TEM analysis showed smooth and spherical morphology of NPs with a particle size in the range of 20–35 nm (Fig. 1B). The images also revealed that the particles have a unimodal size distribution with low polydispersity index.

482

250 200 150 100

at pH 7.2-7.4

50

at pH 6.2-6.5 0 0

1

2

3

4

5

6

7

8

No. of Days

Fig. 1. (A) Size distribution of BL-PLGA NPs by Dynamic Light Scattering. (B) TEM image for BL-PLGA NPs. Image was taken at magnification of 21,000 and is represented at the bar scale of 100 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. (A) In vitro release profile of BL-PLGA NPs in PBS at physiological pH 7.2–7.4 and acidic pH 6.2–6.5. (B) The stability of BL-PLGA NPs in PBS at physiological pH 7.2–7.4 and acidic pH 6.2–6.5. The average hydrodynamic diameter (nm) of BLPLGA NPs was measured as functions of time. The experiment was carried out three times and data represented as mean ± standard deviation (SD) from the obtained values.

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

513 514 515

518 519 520 521 522 523 524 525

528 529 530 531 532 533 534

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015

538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558

(A)

NPs (groupVII)

**$

BL (groupIV)

**

NPs (groupVI)

570

(A) 100

30

60

90

120

No. of Days 30

*

*

DMBA+TPA (Group II) BL (Group III)

80

NPs (Group VI)

60

BL (Group IV) NPs (Group VII)

40 20 0

(B)

*

0

120 100

* **

80

** ## **$

60 40 20 0 DMBA+TPA Group (II)

BL NPs Group (III) Group (VI)

BL NPs Group (IV) Group (VII)

**$

20 15 10

3.5.2. Tumor free survival The enhanced anti-tumor efficacy of BL-PLGA NPs as compared to free BL was also evident by the significant (p < 0.05) increase in the percentage of mice that remained tumor free. The outcome of the experiment is shown in Fig. 4A, which revealed the percent tumor free survival of mice of each treated group. For DMBA + TPA treated group II, at the onset of tumorigenesis, 75 ± 3.60% of the mice remained tumor free. The percent tumor free survival was

4 week 6 week 8 week 10 week 12 week 14week 16week 18week 20week 22week

DMBA+TPA (groupII)

25

559

**#

BL (groupIII)

(B)

decreased by 5% in case of BL-PLGA NPs applied after TPA i.e. group VII. Therefore, on comparing the chemopreventive and therapeutic potentials of formulated NPs, we found that the NPs were found to be more efficient in exerting their chemo-preventive effects rather than chemo-therapeutic effects. The results clearly showed that both free BL and BL-PLGA NPs were able to produce significant (p < 0.05) delay in onset of tumorigenesis and decrease in %tumorigenicity, but the results were more significant (p < 0.01) in case of BL-PLGA NPs. It is also evident from the results that the BL-PLGA NPs were more efficient in rendering their chemo-preventive effects.

Average Tumor Volume (mm3)

537

The onset of tumorigenesis was observed on 70 ± 4.12 and 84 ± 5.23 days for free BL groups, i.e. groups III and IV, respectively. The delay in onset of tumorigenesis was further enhanced significantly (p < 0.01) in animals treated with BL-PLGA NPs i.e., 84 ± 4.90 and 98 ± 6.61 days for groups VI and VII, respectively. Thus, the results showed a significant delay in tumorigenesis due to free BL treatment when compared with DMBA + TPA groups and this delay was further increased significantly in the animals treated with BL-PLGA NPs. Moreover, the enhanced anti-tumor efficacy of BL-PLGA NPs as compared to free BL was also evident from the significant (p < 0.01) decrease in the percentage of mice with tumors at the onset of tumorigenesis, referred as percent tumorigenicity. DMBA + TPA control group II showed the high percent tumorigenicity of 25 ± 2.13% (Fig. 3B). However, the percent tumorigenicity significantly (p < 0.01) decreased to 5 ± 1.60% and 15 ± 2.18% for NPs treated groups i.e., groups VI and VII, respectively, as compared to that of 20% for the corresponding free BL treated groups III and IV. Also, BL-PLGA NPs group VI was found to be more efficient than BL-PLGA NPs group VII in producing more pronounced decrease in percent tumorigenicity. Clearly, the percent tumorigenicity in the group where NPs were applied prior to tumor promoter TPA i.e. BL-PLGA NPs group VI, was reduced by 15% as compared to corresponding free BL treated group III. It was

Group (III) Group (VI)

(C) **##

5 0

Fig. 3. Inhibitory effects of BL-PLGA NPs on induced mouse skin tumorigenesis. Representative bar diagram for (A) Average time for onset of tumorigenesis in every group. (B) Percent tumorigenicity at the time of tumor incidence. Each data point is represented as mean ± SD of 10 independent mice in every experimental group (⁄p < 0.05, ⁄⁄p < 0.01 vs. DMBA + TPA group II, #p < 0.05, ##p < 0.01 vs. free BL group III, $p < 0.05, $$p < 0.01 vs. free BL group IV). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

BL

%Reduction in Tumor Volume

536

%Tumorigenicity

535

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

% Tumor free Survival

6

NPs

Group (IV) Group (VII) BL

NPs

0 -10 -20 -30 -40 -50

## $$

-60

Fig. 4. Representative bar diagram showing the effect of BL-PLGA NPs on mouse skin tumorigenesis in terms of (A) Percent tumor free survival of mice throughout the duration of experiment i.e. 22 weeks. (B) Average tumor volume of all experimental groups. (C) Percent reduction in average tumor volume with respect to DMBA + TPA group II. Each data point is represented as mean ± SD of 10 independent mice in every experimental group (⁄p < 0.05, ⁄⁄p < 0.01 vs. DMBA + TPA group II, #p < 0.05, ##p < 0.01 vs. free BL group III, $p < 0.05, $$p < 0.01 vs. free BL group IV). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

560 561 562 563 564 565 566 567 568 569

571 572 573 574 575 576 577

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

580 581 582 583 584 585 586 587 588 589 590 591 592 593

594 595 596 597

significantly (p < 0.01) increased to 95 ± 3.50 and 85 ± 6.34% for BLPLGA NPs groups VI and VII, respectively, as compared to the corresponding free BL treated groups (80% for both groups III and IV). However, by the end of the 16th week, all the mice of DMBA + TPA treated group II developed tumors i.e. 0% tumor free. Contrary to this, for the groups that were treated with BL-PLGA NPs, a significant (p < 0.01) increase in time where all the mice of groups developed tumors was observed. 0% tumor free survival condition was delayed by 6 and 4 weeks for NPs treated groups i.e., groups VI and VII, respectively, keeping DMBA + TPA group as standard. The results indicated the decreased tendency of mice to develop tumor in case they are treated with NPs. Additionally, the results further confirmed the enhanced chemo-preventive efficacy of NPs over their chemo-therapeutic effects as NPs were more efficient in preventing the development of tumors (group VI) rather alleviate/aggravate them any further (group VII). 3.5.3. Average tumor volume and cumulative number of tumors We also observed the regression in tumor volume and cumulative number of tumors (CNT) throughout the course of experiment. At the end of 22 weeks, the tumor volume of the DMBA + TPA trea-

(A)

Cummulative no. of Tumors

579

ted group reached up to 106 ± 7.64 mm3 (Fig. 4B). Interestingly, on treatment with both, free BL and BL-PLGA NPs, a significant decrease in average tumor volume was observed as compared to DMBA + TPA group but the results were more significant in case of NPs. The average tumor volume in free BL treated group was 87.16 ± 6.45 and 74.31 ± 8.24 mm3 for groups III and IV, respectively, which was further reduced to 62.18 ± 9.29 and 56.38 ± 12.18 mm3 for NPs treated groups i.e. groups VI and VII, respectively. These observations corroborate our hypothesis that NPs were more efficient in imparting their anti-tumor effects. The results can also be seen as the percentage reduction in tumor volume (Fig. 4C). It is evident from the bar diagram that NPs showed excellent anti-tumor property as compared to free BL as more pronounced reduction in tumor volumes was observed. The percent reduction in average tumor volume was as high as 41 ± 4.16% and 46 ± 2.01% for NPs treated groups, i.e., groups VI and VII, respectively, whereas it was 17.78 ± 3.17 and 29.84 ± 4.21% in case of free BL treated groups III and IV, respectively, keeping DMBA + TPA control group II as a standard. BL in the form of NPs was found to be more efficient in inducing anti-tumor effects and also producing regression in average tumor volume.

100 80 **$

**#

60 40 20

0 DMBA+TPA

BL

Group(II)

(B)

NPs

Group(III) Group(VI)

Group(III) Group(VI) BL

NPs

BL

NPs

Group(IV) Group(VII)

Group(IV)

Group(VII)

BL

NPs

0

%Reduction in CNT

578

7

-10 -20 -30 -40 -50

$$ ##

(C)

Fig. 5. Representative bar diagram showing the effect of BL-PLGA NPs on mouse skin tumorigenesis in terms of (A) Cumulative number of tumors (CNT) on mice of every experimental group. (B) Percent reduction in cumulative number of tumors with respect to DMBA + TPA group II. Each data point is represented as mean ± SD of 10 independent mice in every experimental group (⁄p < 0.05, ⁄⁄p < 0.01 vs. DMBA + TPA group II, #p < 0.05, ##p < 0.01 vs. free BL group III, $p < 0.05, $$p < 0.01 vs. free BL group IV). (C) Pictures of mouse of every experimental group after 20 week of experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 8

621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646

647 648 649

The enhanced chemo-preventive potential of BL-PLGA NPs was also evident by measuring cumulative number of tumors (CNT) on mice (Fig. 5A). The mice of the DMBA + TPA group developed an average of 86 ± 4.24 papillomas by the end of 22 weeks. Alternatively, the CNT on mice was reduced significantly (p < 0.05) to 68 ± 6.35 and 72 ± 5.26 in case of free BL i.e. groups III and IV, respectively, due to anti-tumor effects of BL, whereas the CNT were significantly (p < 0.01) reduced to 51 ± 7.23 and 54 ± 3.15 for BL-PLGA NPs groups VI and VII, respectively, due to the enhanced anti-tumor efficacy of BL-PLGA NPs compared to free BL. The anti-tumor efficacy of NPs was superior as also evident from the percentage reduction in CNT (Fig. 5B). The CNT was reduced by 40 ± 8.18 and 37 ± 4.15% in case of BL-PLGA NPs groups VI and VII, respectively, and keeping DMBA + TPA group II as standard. Although, the percent reduction in CNT was less significant in case of mice treated with free BL, it was found out to be 20.9 ± 3.18 and 16.7 ± 2.74% for groups III and IV, respectively. These results clearly indicate the superiority of our formulated BL-PLGA NPs compared to free BL in suppressing tumorigenesis. CNT data also corroborate the improved chemo-preventive nature of BL in nano-particulate form than chemo-therapeutic effects. Moreover, the mice of the vehicle control group I and those that were treated with only free BL and BL-PLGA NPs after DMBA i.e. groups V and VIII, respectively, had no appearance of papillomas, scars and fur loss, suggesting the safety of these particles. Fig. 5C shows the images of the mice of all the experimental groups which also suggest that the anti-tumor effects of the BL were much more prominent if BL is administered in the form of NPs.

The results are shown in Fig. 6, which revealed that the mortality among the mice of the DMBA + TPA group II started after 10 weeks of the commencement of the experiment and continued till the end of 22 weeks, only 10% mice were left alive. On the other hand, when the mice were treated with free BL and BL-PLGA NPs, a significant delay in deaths was observed. The mortality started at the 12th, 16th, 16th and 14th week after the commencement of the experiment for groups III, VI, IV and VII, respectively. It was delayed by 4 weeks in case of BL-PLGA NPs group VI and by 2 weeks in case of BL-PLGA NPs group VII as compared to their corresponding free BL groups i.e. groups III and IV, respectively, hereby confirming the enhanced ability of BL-PLGA NPs to act as a chemo-preventive agent. Also, the overall mortality rate throughout the experiment was significantly reduced by treatment with NPs as compared to their corresponding free BL groups and DMBA + TPA group. NPs gave only 60% and 70% mortality for groups VI and VII at the end of 22 weeks, respectively, as compared to that of 80% for both free BL groups III and IV. The outcomes were also evaluated using the Kaplan–Meier survival curve. Survival curves for the DMBA + TPA and other experimental groups were generated based on death from tumorigenesis for each group (Fig. 7). The overall survival rates among the DMBA + TPA treated group and other groups differed statistically, as there was a tendency of earlier deaths in the DMBA + TPA group compared to the other groups. From the plot, it was revealed that both, free BL and BL-PLGA NPs, were efficient in increasing the survival time of the mice, NPs being more efficient in the same. It can be clearly noticed from the plots that the BL-PLGA NPs were able to produce significant decrease in number of deaths with subsequently delay in deaths from tumorigenesis.

3.5.4. Mortality rate The mortality rate of the animals in all the treated groups was also noted throughout the development of tumors, 0–22 weeks.

(A )

DMBA+TPA (Group II)

100

BL (Group III) 80

%Mortality

620

NPs (Group VI)

60 40 20 0 4

6

8

10

12

14

16

18

20

22

No. of Weeks

(B )

100

DMBA+TPA (Group II) BL(Group IV)

80

%Mortality

619

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

NPs (Group VII)

DMBA+TPA (Group II) 60

BL (Group III) 40

NPs (Group VI)

20

BL (Group IV) 0 4

6

8

10

12

14

16

18

20

22

NPs (Group VII)

No. of Weeks Fig. 6. Line graphs showing percent mortality of mice of (A) chemo-preventive (B) chemo-therapeutic experimental groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Kaplan–Meier survival curve analysis of the experimental groups. Plot shows the data for each of the 10 mouse of every experimental group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx 680 681 682 683 684 685 686

3.5.5. Histopathological analysis On application of formulated BL-PLGA NPs and free BL, a significant restoration in the TPA-induced epidermal hyperplasia and infiltration of leukocytes in dermis was observed (Fig. 8). Histological examinations revealed a normal architecture of the skin consisting of the dermis and epidermis in the acetone-treated control group (Fig. 8A). Application of TPA resulted in an induction

9

of hyperplasia and severe infiltration of leukocytes in the dermis of treated mouse skin (Fig. 8B). The groups pre and post TPA treated with free BL showed only mild epidermal hyperplasia, smaller squamous cell papillomas, moderate cystic dilatation of hair follicle and mild hyperkeratosis (Fig. 8C and D). Interestingly, an enhanced inhibitory effect was observed in the groups that were treated with NPs, where squamous cell papillomas, keratin pearl,

(A) Group I

(B) Group II

(C) Group III

(D) Group IV

(E) Group VI

(F) Group VII

Fig. 8. Histology images of tissues sections from all the experimental groups (A) vehicle control, group I. (B) Positive control: DMBA + TPA treated, group II. (C) Free BL, group III. (D) Free BL, group IV. (E) BL-PLGA NPs, group VI. (F) BL-PLGA NPs, group VII. Representative sections stained with hematoxylin and eosin. All magnifications were at 50 with the inset showing at 125. Here white arrow shows epidermal hyperplasia in the form of figure like projection, blue arrow shows hyperkeratosis, black arrow shows infiltration of inflammatory cells whereas red arrow shows keratin pearl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

687 688 689 690 691 692 693

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 10

696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726

infiltration of inflammatory cells, epidermal hyperplasia and hyperkeratosis were further reduced as illustrated in Fig. 8E and F, indicating the enhanced anti-tumor potential of BL-PLGA NPs in skin carcinogenesis. 3.5.6. DNA alkaline unwinding assay So far, we have observed the enhanced chemo-preventive effects exerted by BL-PLGA NPs in induced 2-stage skin tumorigenesis model. The method has been used extensively to investigate the epithelial carcinogenesis, which consists of initiation phase and promotion phase. Initiation stage is caused by a single topical treatment with DMBA, which produces irreversible damage and/or mutations in the DNA and leads to malignancy. In order to validate the observations showing chemo-preventive efficacy of BL-PLGA NPs, we evaluated the potency of NPs in shielding the DNA from DMBA induced damage by DNA alkaline unwinding assay. In this assay, the strand breaks in DNA were detected when DNA denatures and unwinds slowly in presence of an alkali, which was pre-treated with our drug molecule. More disruption in DNA causes a change in the configuration of the sugar-phosphate backbone of DNA, which reduces the ability of DNA to bind to hydroxyapatite column; hence the number of DNAs which appears to be single stranded is less. Both free BL and BL-PLGA NPs were found to suppress the DMBA-induced DNA damage (Fig. 9A). The results revealed that topically applied DMBA on mouse skin produced significant (p < 0.05) DNA damage, as it shows high number of strand breaks in DNA i.e. 20.28 ± 3.29. However, the number of strand breaks was reduced to 13.55 ± 0.35 in the group where free BL was applied prior to DMBA application i.e. free BL + DMBA group. Interestingly, the results were more significant (p < 0.01) in case of NPs, wherein the number of strand breaks was reduced to 9.63 ± 0.35 and 8.70 ± 0.73, for NPs at 1/20th and 1/10th dose, respectively, hereby showing the enhanced chemo-protective properties of BL-PLGA NPs.

(A)

No. of Strand Breaks

695

727

3.5.7. Effect of BL-PLGA NPs on the expression of pro- and antiapoptotic proteins Once it was confirmed that BL-PLGA NPs produced a significant reduction in induced tumorigenesis, we next attempted to unveil the underlying mechanism. For the set purpose, we further studied the modulation of marker proteins involved in the induction of apoptosis as it is well recognized that various pro- and antiapoptotic proteins play a crucial role in programmed cell death. BL is reported to induce tumor cell apoptosis by increasing the expression of pro-apoptotic protein P53 and Bax with subsequent decrease in the expression of anti-apoptotic protein Bcl-2 in mouse skin tumorigenesis model [25]. We wanted to observe in this experiment whether BL after encapsulation in PLGA NPs was able to induce tumor cell apoptosis in the same manner. We, therefore, administered free BL and BL-PLGA NPs in mice prior to application of DMBA and evaluated the expression of marker proteins related to cell death. DMBA, a DNA damaging agent is known to hinder action of tumor suppressor P53 protein [31]. Our results of western blot supported the earlier evidences, and also demonstrated that

741

20 *

15

**

**

10 5 0 Only

(B)

Also, the data showing the percent prevention from DNA damage are presented in Fig. 9B. The results indicate that pre-treatment with NPs at much lower doses of 1/20th and 1/10th of the dose of free BL significantly (p < 0.01) prevented the DMBA induced DNA damage by 52.58 ± 5.97 and 56.99 ± 3.04%, respectively, keeping the DMBA treated group as standard, whereas the percentage prevention was only 33.00 ± 4.72% for free BL treated group. On the other hand, control untreated group, only free BL and only NPs treated groups did not show any significant (p > 0.05) decrease in the amount of duplex DNA indicating negligible toxicity. The results clearly demonstrate the enhanced ability of BL-PLGA NPs in protecting the DNA from chemically induced damage and show anti-mutagenic properties and hence, chemo-protective effects in a more efficient manner than free BL at much reduced doses.

25

DMBA

%Prevention

694

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

Free BL

NPs Only +DMBA

Free BL

NPs (1/20)

+DMBA

+DMBA

(C)

70 60 50 40 30 20 10 0

**

1

1.06

NPs (1/10) +DMBA 1.22

1.59

**

p53 1

1.15

1.41

1.78

Bax 1

0.95

0.81

0.69

Bcl-2

Free BL +DMBA

NPs (1/20) +DMBA

NPs (1/10) +DMBA

β-Actin

Lane:

A

B

C

D

Fig. 9. (A) Representative bar diagram of reduction in DMBA-induced strand breaks by free BL and BL-PLGA NPs treated groups (⁄p < 0.05, ⁄⁄p < 0.01 vs. DMBA group). (B) Representative bar diagram of percentage prevention in DMBA-induced strand break (⁄p < 0.05, ⁄⁄p < 0.01 vs. Free BL + DMBA group). Each data point is represented as mean ± SD of all the 6 mice of the group. (C) Western blot analysis to study the altered expression of marker proteins associated with apoptosis and cell death in skin tissue samples exposed to free BL and BL-PLGA NPs. (Lane A: DMBA group, B: Free BL + DMBA, C: NPs(1/20) + DMBA, D: NPs(1/10) + DMBA). b-actin was used as internal control to normalize the data. Each experiment was done in triplicate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

728 729 730 731 732 733 734 735 736 737 738 739 740

742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776

P53 expression was down-regulated by DMBA treatment (Fig. 9C). An increase in P53 response was noticed in the group where free BL and BL-PLGA NPs were applied prior to DMBA. DMBA treatment also resulted in the down-regulation of pro-apoptotic protein Bax which had been up-regulated in the case of free BL and NPs treated mice. Both free BL and NPs at both the doses were found to increase the levels of Bax but the up-regulation seemed more significant in the case of NPs treated groups. Besides, a significant downregulation of anti-apoptotic protein (Bcl2) by NPs was observed at much reduced doses i.e. 1/20th and 1/10th than that of free BL. Western blot analysis herein confirmed that NPs treatment was able to maintain a balance between positive and negative regulators of apoptosis and trigger more cells to apoptosis. Thus, our data confirm the induction of apoptosis and subsequent increase in the anti-cancerous activity of BL-PLGA NPs in the same way as that of free BL, however, in a more efficient/effective manner than free BL and that too at much reduced doses.

777

4. Discussion

778

The medicinal properties of the pineapple plant (Ananas comosus) were recognized traditionally in South America, China and Southeast Asia. These properties were attributed to BL, a cysteine proteinase found in the pineapple plant. Among various medicinal properties of BL, its anti-cancer activity has gained tremendous attention among researchers. It has been shown to induce tumor cell apoptosis by increasing the expression of activators of apoptosis, P53 and Bax in mouse skin papillomas. In order to further increase the efficacy of BL, BL loaded PLGA NPs were synthesized and their anti-cancer activity was shown by topical application on mice. Topical application was investigated that has been considered as an effective alternative for drug administration, which reduces systemic side effects. NPs possess a wide range of benefits, which make them particularly useful for topical applications in anti-cancer therapy along with other drug. Being smaller in size, we can make use of the EPR effect and can preferentially penetrate and accumulate more NPs at the porous tumor site [14,32]. The sustained release of proteins in their native form is often a challenging task. Since, proteins are very susceptible to denaturation and chemical degradation during particle preparation, it is difficult to encapsulate and deliver proteins without loss of their biological activity [33]. Protein formulations are well explored with the use of water/oil/water double-emulsion–solvent evaporation method which provides stable NPs [34]. The process involves the generation of hydrophilic/hydrophobic interface that may result in the unfolding or denaturing of protein molecules, an internal stabilizer sucrose was used along with BL, which has been known to form a hydration layer around the protein and reduces its interaction with the organic solvent, thus protecting the BL from the organic solvent induced denaturation resulting in stabilization of the compact native state [35]. Hence, the process leads to the development of stable NPs with optimum BL loading. Particle size and surface morphology of the NPs play a crucial role with respect to the physical stability, bio-distribution, drug release and uptake of NPs by the tumor cells. It is worth mentioning that NPs within the size range of 60–400 nm can exert their effect by passive targeting, which takes the advantage of the inherent size of NPs and exploits the unique anatomical and patho-physiological abnormalities of tumor vasculature to accumulate NPs more at the tumor site by EPR effect. Therefore, we have deliberately kept the size of NPs below 200 nm. TEM analysis revealed the formation of smooth and spherical NPs with unimodal distribution. Any alterations in the proteolytic activity of BL evaluated by measuring CDU, showed the non-significant change in CDU values of BL encapsulated in NPs. It is indispensible for designing efficient NPs

779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822

11

based delivery system to establish a sustained release of encapsulated molecule on reaching the site. The requirement was accomplished by BL-PLGA NPs which showed a biphasic release pattern explained by diffusion-cum-degradation dependent release of an encapsulated drug from PLGA matrix [36]. In vitro release profile also demonstrated rapid release of BL at acidic pH. The observation could be attributed to the accelerated rupturing of ester bonds in acidic pH, degrading PLGA and hence faster release of encapsulated molecule. This property was advantageous in achieving high drug concentrations at the tumor site since solid tumors possess an acidic extracellular environment across their cellular compartments [37]. Chemically induced multistage mouse skin tumorigenesis represents an excellent in vivo model to study the development and chemoprevention of cancer. Hence, we evaluated the antitumor efficacy of developed BL-PLGA NPs and the results were compared with free BL in induced 2-stage skin tumorigenesis model. Our results revealed that the NPs treated group produced a significant (p < 0.01) delay in the onset of tumorigenesis in comparison with both, free BL and DMBA + TPA group. In addition the percent tumorigenicity at the time of tumor incidence was significantly (p < 0.01) reduced in BL-PLGA NPs treated groups VI and VII. On the other hand, if we compare the ability of BL to act as a chemo-preventive or chemo-therapeutic molecule, the results were found to be more skewed toward chemo-preventive effects. BL-PLGA NPs group VI, where NPs were applied before tumor initiator TPA, was found to be more effective in delaying tumor incidence and percent tumorigenicity than BL-PLGA NPs group VII, where NPs were applied after TPA. Improved anti-tumor efficacy of NPs compared to free BL with subsequent chemo-preventive potency was also corroborated by a significant increase in percent tumor free survival of mice, significant reduction in the average tumor number and tumor volumes. The NPs were 2.5 and 1.5 folds efficient in reducing average number of tumors than free BL in inducing its chemo-preventive [group III vs. group VI] and chemo-therapeutic effects [group IV vs. group VII], respectively. The observed improved anti-tumor efficacy of NPs was attributed to the increased bioavailability of BL by BL-PLGA NPs since there was sustained and continuous release of BL from NPs. Additionally, increased preferential localization of NPs at the tumor site through EPR effect resulted in maintaining the therapeutic concentration of BL at the site. The results were also supported by histopathological analysis done by Hematoxylin and Eosin staining. As well, the enhanced tendency of NPs to reduce the mortality rate among mice was observed. It was delayed by 4 and 2 weeks by BL-PLGA NPs groups VI and VII, respectively, as compared to their corresponding free BL groups III and IV, respectively. It can be clearly observed from the Kaplan–Meier survival plot, that the BL-PLGA NPs were able to produce a significant decrease in the number of deaths with subsequent delay in deaths from tumorigenesis. Further, we revealed the mechanism of tumor cell killing by studying the expression of pro-apoptotic protein P53, Bax and anti-apoptotic protein Bcl-2. Both free BL and BL-PLGA NPs were found to induce tumor cell killing by enhancing the expression of P53 and Bax proteins with subsequent decrease in Bcl-2 expression, but the magnitude of alterations was more significant in case of NPs. Conclusively, NPs were able to maintain a balance between the positive and negative regulators of apoptosis and triggered more cells to apoptosis than free BL. Moreover, to further confirm the chemo-preventive efficacy of BL, the potency of BL to protect the DNA from DMBA induced damage was evaluated by DNA alkaline unwinding assay. This method provides a sensitive, robust and convenient way for the detection and quantification of DNA damage and repair. A single topical application of DMBA on the mouse skin pre-treated with free BL and BL-PLGA NPs showed a significant (p < 0.05) protection from

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015

823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888

EJPB 11809

No. of Pages 12, Model 5G

28 January 2015 12

P. Bhatnagar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx

902

DNA damage, the protection being more significant (p < 0.01) in case of NPs given at much reduced doses. A single topical application of BL-PLGA NPs alone, did not induce any significant DNA damage, conferring the safety and biocompatibility of these NPs. Increased anti-tumor activity of our formulated BL-PLGA NPs against free BL is attributed to the ability of NPs to accumulate more at the tumor site through EPR effect. Besides, the sustained and continuous release of the encapsulated BL from the polymer matrix is the reason for greater accumulation at the tumor site, thus improving anti-tumor efficacy of formulated BL-PLGA NPs. Our findings are consistent with our hypothesis as enhanced ability of BL released from formulated NPs suggested improved chemotherapy by exerting chemo-preventive effects against experimentally induced carcinogenesis at lower dosages.

903

5. Conclusion

889 890 891 892 893 894 895 896 897 898 899 900 901

904 905 906 907 908 909 910 911 912 913 914 915

The results of the present study conclude the enhanced antitumor property of the formulated BL loaded PLGA NPs than free BL at lower doses. BL encapsulated PLGA NPs were developed which showed controlled BL release pattern, beneficial for effective drug delivery. Further, the NPs were able to inhibit initiation of tumor formation in skin tumor mice model. Delay in onset of tumorigenesis, reduction in percent tumorigenicity with marked reduction in tumor volume and tumor numbers projected the superiority of formulated NPs over free BL. The positive results of the study promoted that the developed formulation can be fruitfully exploited to improve delivery of anti-cancer drugs across Q8 the skin and can open new vistas for skin cancer therapy.

916

Acknowledgments

917

923

Financial support from CSIR, Delhi, India [Grant No. 0112NanoSHE] is acknowledged. PB gratefully acknowledges the Council of Scientific & Industrial Research (CSIR), New Delhi, India, for award of Senior Research Fellowship to carry out this work. The authors would like to thank Dr. L.K.S. Chauhan, CSIR-IITR, Lucknow, for TEM analysis and Mr. Syed H.N. Naqvi, Technical Staff, CSIR-IITR Lucknow for providing assistance in animal bioassay.

924

References

925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948

[1] GLOBOCAN 2008: Cancer Incidence and Mortality Worldwide, IARC, WHO, 2010. [2] R.J. Friedman, D.S. Rigel, A.W. Kopf, Early detection of malignant melanoma: the role of physician examination and self-examination of the skin, CA Cancer J. Clin. 35 (2008) 130–151. [3] J.A. Neville, E. Welch, D. Leffell, Management of non-melanoma skin cancer in 2007, Nat. Clin. Pract. Oncol. 4 (2007) 462–469. [4] M. Das, C. Mohanty, S.K. Sahoo, Ligand-based targeted therapy for cancer tissue, Exp. Opin. Drug Deliv. 6 (2009) 285–304. [5] S.K. Sahoo, V. Labhasetwar, Nanotech approaches to drug delivery and imaging, Drug Discov. Today 8 (2003) 1112–1120. [6] S.F. Taveira, R.F.V. Lopez, in: Caterina LaPorta (Ed.), Topical Administration of Anti-cancer Drugs for Skin Cancer Treatment, Skin Cancers – Risk Factors, Prevention and Therapy, 2011. ISBN 978-953-307-722-2. [7] C. Unger, M. Peukert, H. Sindermann, P. Hilgard, G. Nagel, H. Eibl, Hexadecylphosphocholine in the topical treatment of skin metastases in breast cancer patients, Cancer Treat. Rev. 17 (1990) 243–246. [8] C.H. Smorenburg, C. Seynaeve, M. Bontenbal, H. Sindermann, J. Verweij, Phase II study of miltefosine 6% solution as topical treatment of skin metastases in breast cancer patients, Anticancer Drugs 11 (2000) 825–828. [9] M.R. Prausnitz, P.M. Elias, T.J. Franz, M. Schmuth, J.C. Tsai, Skin barrier and transdermal drug delivery, Med. Ther. 19 (2008) 2065–2073. [10] M.V. Blagosklonny, Targeting cancer cells by exploiting their resistance, Trends Mol. Med. 9 (2003) 307–312.

918 919 920 921 922

[11] R. Krishna, L.D. Mayer, Multidrug resistance (MDR) in cancer: mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs, Eur. J. Pharm. Sci. 11 (2000) 265–283. [12] L.D.D. Simonetti, G.M. Gelfuso, J.C.R. Barbosa, R.F.V. Lopez, Assessment of the percutaneous penetration of cisplatin: the effect of monoolein and the drug skin penetration pathway, Eur. J. Pharm. Biopharm. 73 (2009) 90–94. [13] N.A. Charoo, Z. Rahman, M.A. Repka, S.N. Murthy, Electroporation: an avenue for transdermal drug delivery, Curr. Drug Deliv. 7 (2010) 125–136. [14] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting, Adv. Enzyme Regul. 41 (2001) 189–207. [15] R. Haag, Supramolecular drug-delivery systems based on polymeric core–shell architectures, Angew. Chem. Int. Ed. Engl. 43 (2004) 278–282. [16] J.M. Koziara, T.R. Whisman, M.T. Tseng, R.J. Mumper, In-vivo efficacy of novel paclitaxel nanoparticles in paclitaxel-resistant human colorectal tumors, J. Control. Release 112 (2006) 312–319. [17] Y. Miyamoto, T. Oda, H. Maeda, Comparison of the cytotoxic effects of the high-and low-molecular-weight anticancer agents on multidrug-resistant Chinese hamster ovary cells in vitro, Cancer Res. 50 (1990) 1571–1575. [18] M.H. Schmid, K.C. Korting, Therapeutic progress with topical liposome drugs for skin disease, Adv. Drug Deliv. Rev. 18 (1996) 335–342. [19] M.V. Barrera, E. Herrera, Topical chemotherapy of actinic keratosis and nonmelanoma skin cancer: current options and future perspectives, Actas Dermo-Sifiliográfica 98 (2007) 556–562. [20] Y. Fang, P. Tsai, H. Wu, Y.B. Huang, Comparison of 5-aminolevulinic acidencapsulated liposome versus ethosome for skin delivery for photodynamic therapy, Int. J. Pharm. 356 (2008) 144–152. [21] S.D. Mandawgade, V.B. Patravale, Development of SLNs from natural lipids: application to topical delivery of tretinoin, Int. J. Pharm. 363 (2008) 132–138. [22] Y.J. Surh, Cancer chemoprevention with dietary photochemical, Nat. Rev. Cancer 3 (2003) 768–780. [23] A. Amin, H.G. Muhtasib, M. Ocker, R.S. Schneider, Overview of major classes of plant-derived anticancer drugs, Int. J. Biol. Sci. 5 (2009) 1–11. [24] .M. Russo, I. Tedesco, G. Iacomino, R. Palumbo, G. Galano, G.L. Russo, Dietary phytochemicals in chemoprevention of cancer, Curr. Med. Chem. – Immun. Endoc. Metab. Agents 5 (2005) 61–72. [25] K. Bhui, S. Prasad, J. George, Y. Shukla, Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumorinitiation triggering mitochondrial death pathway, Cancer Lett. 282 (2009) 167–176. [26] K. Chobotova, A.B. Vernallis, F.A. Majid, Bromelain’s activity and potential as an anti-cancer agent: current evidence and perspectives, Cancer Lett. 290 (2010) 148–156. [27] P. Bhatnagar, S. Patnaik, A.K. Srivastava, M.K.R. Mudiam, Y. Shukla, A.K. Panda, A.B. Pant, P. Kumar, K.C. Gupta, Anti-cancer activity of bromelain nanoparticles by oral administration, J. Biomed. Nanotechnol. 10 (2014) 1–17. [28] P. Chomczynski, N. Sacchi, The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on, Nat. Protoc. 1 (2006) 58–85. [29] A.K. Srivastava, P. Bhatnagar, M. Singh, S. Mishra, P. Kumar, Y. Shukla, K.C. Gupta, Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage, Int. J. Nanomed. 8 (2013) 1451–1462. [30] H. Towbin, T. Staehelint, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 4350–4354. [31] S. Wei, K. Kito, A. Miyoshi, S. Matsumoto, A. Kauzi, T. Aramoto, Y. Abe, N. Ueda, Incidence of p53 and ras gene mutations in DMBA-induced rat leukemias, J. Exp. Clin. Cancer Res. 21 (2002) 389–396. [32] J. Fang, H. Nakamura, H. Maeda, The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect, Adv. Drug Deliv. Rev. 63 (2011) 136–151. [33] M.L. Houchin, E.M. Topp, Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms, J. Pharm. Sci. 97 (2008) 2395–2404. [34] U. Bilati, E. Allemann, E. Doelker, Protein-loaded nanoparticles prepared by the double emulsion method – processing and formulation issues for enhanced entrapment efficiency, J. Microencapsul. 22 (2005) 205–214. [35] J.L. Cleland, M.F. Powell, S.J. Shire, The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation, Crit. Rev. Ther. Drug Carrier Syst. 10 (1993) 307–377. [36] J. Panyam, M.M. Dali, S.K. Sahoo, W. Ma, S.S. Chakravarthi, G.L. Amidon, R.J. Levy, V. Labhasetwar, Polymer degradation and in vitro release of a model protein from poly (D,L-lactide-co-glycolide) nano-and micro particles, J. Control. Release 92 (1979) 173–187. [37] A. Riemann, B. Schneider, A. Ihling, M. Nowak, C. Sauvant, O. Thews, M. Gekle, Acidic environment leads to ROS-induced MAPK signaling in cancer cells, PLoS One 6 (2011) e22445.

949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028

Please cite this article in press as: P. Bhatnagar et al., Bromelain nanoparticles protect against 7,12-dimethylbenz[a] anthracene induced skin carcinogen-

Q1 esis in mouse model, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.01.015