Article pubs.acs.org/molecularpharmaceutics
Colloidal Vesicular System of Inositol Hexaphosphate to Counteract DMBA Induced Dysregulation of Markers Pertaining to Cellular Proliferation/Differentiation and Inflammation of Epidermal Layer in Mouse Model Malti Arya,# Prakash Tiwari,† Chandra Bhushan Tripathi,# Poonam Parashar,# Mahendra Singh,# Priyam Sinha,§ Narayan P. Yadav,§ Gaurav Kaithwas,# Krishna P. Gupta,† and Shubhini A. Saraf*,# #
Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow-226025, U.P., India † Environmental Carcinogenesis Division, CSIR-Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, U.P., India § CSIR-Central Institute of Medicinal and Aromatic Plants, PO CIMAP, Lucknow-226015, U. P., India S Supporting Information *
ABSTRACT: Cancer is a global health problem and chemoprevention is a promising approach for reducing cancer burden. Inositol hexaphosphate (IP6), a natural bioactive constituent of cereals, legumes, etc., has momentous potential as an antiangiogenic agent, that specifically affects malignant cells. The shortcoming is its quick absorption on oral/topical administration. Niosomes are flexible carriers for topical drug delivery. The central venture of current research was to optimize and characterize niosomal delivery system of IP6 for treatment of skin cancer. Thin film hydration method was utilized to prepare IP6 niosomes, and these were dispersed as a suspension in a suitable base. Developed formulations were analyzed for various physicochemical and pharmacological parameters such as particle size, encapsulation efficiency, morphology, drug release, texture analysis, irritability, cell line studies, Western blotting, RT-PCR, and histopathology. IP6 niosomal suspension and IP6 in acetone displayed IC50 value at the concentration of 0.96 mM (0.63 mg/mL) and 1.39 mM (0.92 mg/mL), respectively. IP6 niosomal suspension showed significantly higher (p < 0.05) activity and showed cytotoxic effect in SK-MEL-2 cancer cell line. Crucial events of cellular proliferation and differentiation, like expression of ornithine decarboxylase (ODC), proliferating cell nuclear antigen (PCNA), cycloxygenase-2 (COX-2) and Cyclin D1 were initiated from the fourth hour through application of 7,12dimethylbenzanthracene (DMBA) on albino mice. The DMBA altered expression of aforesaid enzymes was significantly (P < 0.001) prevented by concomitant application of niosomal formulations. Results of cell line study, Western blotting, RT-PCR, and histopathology suggested that IP6 niosomal suspension could constitute a promising approach for prevention of cellular proliferation as well as DMBA induced dysregulation of cellular proliferation/differentiation and inflammation. KEYWORDS: skin cancer, phytic acid, niosomes, suspension, SK-MEL-2 cell line, histopathology, Western blotting, RT-PCR
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cereal products.5,6 Chemically, IP6 is a simple carbohydrate with six phosphates attached to each carbon (inositol1,2,3,4,5,6-hexaphosphate) and a principal storage of phosphorus in several plant tissues.7,8 It possesses various health benefits such as lowering of serum cholesterol, strong antioxidant properties, etc. It has also been revealed to have significant potential as an antiangiogenic agent that only affects malignant
INTRODUCTION Cancer remains a life threatening disease, and various efforts to contain the same have hitherto proved almost futile.1 On an average, 3−8% increase in skin cancer per year has been recorded, with >1 million new cases every year.2 Globally, people still rely on herbal medicines as first line treatment, owing to their perception of safety and general acceptance for cancer prevention as well as treatment. The wide investigation has identified numerous dietary, botanical, and natural compounds that have chemopreventive properties.3,4 Inositol hexaphosphate (IP6), commonly known as phytic acid, is a natural bioactive constituent of grains, legumes, and © 2017 American Chemical Society
Received: Revised: Accepted: Published: 928
December 21, 2016 February 2, 2017 February 7, 2017 February 7, 2017 DOI: 10.1021/acs.molpharmaceut.6b01147 Mol. Pharmaceutics 2017, 14, 928−939
Molecular Pharmaceutics
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cells and does not affect normal cells and tissues of the body. A number of in vitro and in vivo studies have proved chemopreventive and anticancer properties of IP6 in the prostate, colon, lung, skin tumor, metastatic, and mammary cancers.7−9 IP6 is available in the form of dietary powder and capsules. Metallic nanoparticle based IP6 micelles have been prepared to improve its stability.10 IP6 forms tight chelates with many polyvalent and nutritionally important minerals (calcium, copper, magnesium, iron, etc.). It is therefore considered that IP6 diminishes the bioavailability of many dietary minerals and is as an antinutrient.11 IP6 gets quickly absorbed when administered orally and is distributed to various organs, within an hour following its administration.12 Administration via topical route would also lead to its systemic circulation following chelation and fast elimination. Niosomes can embed both lipophilic and hydrophilic substances in their structure. Recently, it received much consideration as a prospective drug delivery system over conventional vesicular systems. In comparison to liposomes, niosomes render high chemical as well as physical stability with lesser cost and larger availability of surfactants. Niosomes further show enhanced residence time of drugs in the epidermis and diminish the systemic absorption. These are flexible, colloidal carriers that are pertinent for systemic as well as topical applications.13,14 7,12-Dimethylbenzanthracene (DMBA) is frequently employed to induce skin carcinogenesis in mice and consequently corroborates the chemopreventive nature of many natural as well as synthetic materials. It is a procarcinogen and therefore entails a metabolic activation to be converted into a vital carcinogen metabolite (dihydrodiol epoxide). This active metabolite fastens to DNA resulting in its damage. The metabolic activation of DMBA generates numerous reactive oxygen species (ROS). It is therefore extensively exploited as an initiator as well as promoter to instigate skin carcinogenesis in mice.15−17 Ornithine decarboxylase (ODC), proliferating cell nuclear antigen (PCNA), and cyclins are implicated in cell proliferation. ODC plays an imperative role in various biological processes of cell growth, differentiation, apoptosis, cell proliferation, and cancer development.18,19 PCNA, which is synthesized in early G1 and S phases of cell cycle, functions in cell cycle progression, replication, and repair of DNA.19−22 Cyclin D1 is concerned with G1/S checkpoint of the cell cycle and forms complexes with CDK4 or CDK6, which phosphorylates RB tumor suppressor protein and results in the release of E2F transcription factors that allow the cell to enter into S phase. The G1/S checkpoint recurrently distorts in several epithelial tumors and may result in enhanced tumorigenesis.23,24 Cycloxygenase-2 (COX-2) is concerned with prostaglandin biosynthesis and, as a result, plays a key role in inflammatory events and also prognoses in epithelial malignancies.25,26 Augmented expressions of PCNA, ODC, COX, and cyclins, respectively, are always observed during tumor progression. IP6 has significant potential as an antiangiogenic agent. Besides having great potential, it is underutilized because of its flaw of being rapidly absorbed and excreted due to biotransformation into inactive chelates. This necessitates developing a delivery system that can control biotransformation of IP6. The focal endeavor of present work was to develop a niosomal delivery system of IP6 for deterrence of preneoplastic skin damage.
Article
EXPERIMENTAL SECTION
Materials. Surfactant and cholesterol were procured from Himedia Laboratories, Mumbai, India. DMBA, IP6, sodium metabisulfite, and hydroxy quinone were purchased from Sigma Co. (St. Louis, MO, USA). Taq DNA polymerase, RT-PCR kit, dNTPs, and PCR primers were purchased from Banglore Genei (India), [methyl-3H] S-adenosyl methionine from AmershamBiosciences-GE Healthcare (USA). All other chemicals were procured from local commercial sources and were of analytical grade, else otherwise stated in the text. Animals. Female Swiss albino mice procured from the inhouse animal breeding colony at CSIR-IITR, Lucknow (IAEC approval IITR/IAEC/04/2015) were utilized for the experiment and fed with synthetic pellet diet (M/S Provimi Animal Nutrition India Pvt. Ltd. Bangalore) and water ad libitum. Animals used in present study were handled as per norms of institutional animal ethics committee. Experimental Design. A two-level three-factor (23) full factorial design of experiment was used to statistically optimize the formulation variables involved in the preparation of IP6 niosomes, to facilitate higher encapsulation efficiency and optimize vesicle size through Design Expert software. A total of nine experiments were run. Three independent variables, namely, cholesterol/surfactant ratio (X1), sonication time (X2), and DCP concentration (X3), were assessed. The particle size (Y1) and encapsulation efficiency (Y2) were the dependent variables. The compositions of the IP6 niosomes prepared according to factorial design are shown in Table 1. Table 1. Factorial Design for Optimization of IP6 Niosomes levels factors (independent variables)
low
X1, cholesterol/surfactant ratio (molar ratio) X2, sonication time (minute) X3, DCP (μM) responses (dependent variables) Y1, particle size (nm) Y2, encapsulation efficiency (%)
high
1:1 1:2.5 1 2 0 5 constraints minimize maximize
Preparation and Characterizations of IP6 Loaded Niosomes. Unilamellar niosomal vesicles were prepared from Span 80-Cholesterol-Dicetyl phosphate (DCP) dispersion according to the subsequent modus operandi. Precalculated quantities of cholesterol and surfactant (Span 80) in molar ratios (Table 1) were dissolved in 10 mL of chloroform in a round-bottomed flask. DCP was added to the solution as a negative charge inducing means (in requisite batches). Chloroform was completely evaporated at 45 °C and 120 rpm under reduced pressure using rotary evaporator (IKA Rotavapor, Bangalore, India) to form a thin film. Thus, formed thin film was hydrated with 5 mL of distilled water containing drug (3/5 mg) at 45 °C and 120 rpm. Subsequently, the dispersion was sonicated for 1 min at 80% energy by using probe-type sonicator (Labsonic-M, Sartorius Stedium) and kept at 4 °C for 24 h for maturation. Prepared formulation was stored at 4 °C in the dark until use for further assessments. Particle Size and Size Distribution. Particle size and width of the size distribution of IP6 loaded niosomes was determined through dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS (Malvern Instruments, UK) at 25 929
DOI: 10.1021/acs.molpharmaceut.6b01147 Mol. Pharmaceutics 2017, 14, 928−939
Article
Molecular Pharmaceutics °C by appraising the autocorrelation function at 90°, in triplicate. Encapsulation Efficiency. The percentage of encapsulated IP6 was determined indirectly, by ultracentrifugation of niosomal suspension, using cooling centrifuge, as described in the Supporting Information. In Vitro Drug Release Study. In vitro drug release of IP6 from niosomes was determined via pretreated dialysis bag, detailed in the Supporting Information. Morphological Evaluation of Niosomes. For shape and morphology, niosomes suspension was evaluated by optical microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), detailed in the Supporting Information. Preparation and Characterizations of IP6 Loaded Niosomal Suspension. Preparation of Different Phases. Phase A (oil phase) was prepared by taking light liquid paraffin (5% w/w) (blending base) and isopropyl myristate (2.5% w/w) (emollient, lubricant) in a beaker and heated up to 80 °C on a hot plate, mixed gently until a clear solution was obtained. To this solution ethylene glycol monostearate (1% w/w) (pearlising agent) and myristyl myristate (1% w/w) (emollient, skin conditioner) was added one by one with gentle stirring and mixed to obtain a clear solution. Phase B (aqueous phase) was prepared by heating water (q.s.) to 80 °C, and disodium EDTA (0.05% w/w) (copreservative) was added with continuous stirring. Carbopol 934 (1% w/w) was dispersed with high speed stirring (1000−1500 rpm) until a uniform dispersion was obtained. Phase C was prepared in a separate beaker by taking propylene glycol (2% w/w) (humectant, plasticizer), methylparaben (0.2% w/w) (water-soluble preservative), and propylparaben (0.3% w/w) (oil soluble preservative) mixed with gentle stirring and gentle heating. Glycerin (3% w/w) (humectant, plasticizer) was added and mixed well. Emulsification. When both phases (phase A and B) were at 80 °C, the oil phase was added slowly to the aqueous phase with high stirring (approx 1500 to 2000 rpm) for 5 min and then stirring speed was stepped down (500 to 1000 rpm) for 10 min. The formed emulsion was allowed to cool to room temperature and phase C added with gentle stirring. The formed emulsion was neutralized with triethanolamine to adjust the pH to the range of 6.5 to 7. Then, the niosomal suspension of IP6 was dispersed and mixed gently. For plain IP6 suspension, weighed quantity of IP6 was dispersed homogeneously in Carbopol suspension base.27 Determination of pH. The pH of niosomal suspension was determined via digital pH meter, NIG-333 (Naina solar limited, New Delhi, India). Measurements were carried out in triplicate.28 Determination of Viscosity. A Brookfield digital viscometer (Labtronics, model LT-730, India) with spindle no. 3 at 60 rpm was used to determine the viscosity in cps of the suspension formulation, in triplicate. Experiment was performed at room temperature. Texture Analysis. Texture analysis of suspension was performed via CT3 Texture Analyzer (Brookfield Engineering Laboratories, USA). Assessment of firmness, spreadability, and extrudability collectively accounts for texture profile of formulations. For estimation of firmness, TA 10 probes, fixture TA-BT-KI, withhold time 2 s, and trigger load 5 g were used. Male and female cone probe with trigger load 2 g and TA DEC (dual extrusion cell) were utilized for assessment of spread-
ability and extrudability. Settings of equipment were made according to recommended stipulations.28,29 In Vitro Skin Permeation Study. In vitro permeation of IP6 from niosomal suspension formulations were estimated by utilizing full thickness abdominal skin, obliterated from Swiss albino mice, explained in the Supporting Information. Irritation Test: Hen’s Egg Test on the Chorioallantoic Membrane (HET-CAM). For estimating irritation tolerability of developed niosomal formulation of IP6, a modified HET-CAM test was performed as reported by Gupta et al.30 Detailed in the Supporting Information. In Vitro Cell Proliferation/Cytotoxicity against Human Cancer Cell Lines. Anticytotoxicity and anti-cell proliferation activity of niosomal suspension of IP6 and IP6 in acetone was examined against control (normal saline) by sulforhodamine-B (SRB) assay on SK-MEL-2 (human melanoma cell line). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 2% inactivated fetal bovine serum to get required concentration of the stock solution, which was filtered and centrifuged. Serial dilutions of 0.5, 1, 1.5, and 2 mM (equivalent to 0.33, 0.66, 0.99, and 1.32 mg/mL, respectively) were prepared from the stock solution. Cell suspension used had a density of about 10,000 cells/0.1 mL, which was instilled in a 96-well plate. After incubation for 24 h, 100 μL of various concentrations of test samples were added to the wells and were incubated at 37 °C for 72 h. Cells were fixed to the bottom of the wells by using cold trichloroacetic acid (TCA) for 1 h at 4 °C and then rinsed with distilled water and kept for air drying. Thereafter, 50 μL of SRB solution was added to wells and allowed to stain for 30 min. Then the plate was washed with 1% v/v acetic acid to remove unbound dye and air-dried. The plate was gently shaken for 5 min after addition of 100 μL of Tris buffer base (pH 10.5;10 mM). The optical density (OD) of the plate wells was measured with a microplate reader (ELx800, BioTek, U.S.A.). Percent growth inhibition of IP6 in acetone and formulation were calculated by using subsequent equation. Inhibitory concentration-50 (IC50) values were calculated by linear regression.17 %growth inhibition ⎡ OD(test) − OD(blank) ⎤ = 100 − ⎢ ⎥ × 100 ⎣ OD(control) − OD(blank) ⎦
(1)
In Vivo Studies. Female Swiss albino mice procured from the in-house animal breeding colony at CSIR-IITR, Lucknow, were used for the experiment. They were fed with synthetic pellet diet (M/S Provimi Animal Nutrition India Pvt. Ltd. Bangalore) and water ad libitum. The study was approved by the Institutional Animal Ethics committee of Indian Institute of Toxicology Research, Lucknow (IAEC approval IITR/IAEC/ 04/2015) as per Government of India norms. Animals were kept in polypropylene cages in a well-ventilated animal house at temperature 22 °C ± 2 °C, maintained under photoperiodic condition. All animals were handled as per norms of institutional animal ethics, and care was taken that all guidelines were followed with a humane approach. Treatment Schedule for Animals. Animals were divided into 6 groups, of 12 animals each. The skin (dorsal side) was shaved by using an electric clipper, in interscapular region over an area of 2 cm2, 2 days prior to the experiment. DMBA (dose = 0.005 μg/kg of mice weight) dissolved in 100 μL of acetone was applied topically on shaved back of animals. All other treatments (Table 2) were given similarly, subsequent to 930
DOI: 10.1021/acs.molpharmaceut.6b01147 Mol. Pharmaceutics 2017, 14, 928−939
Article
Molecular Pharmaceutics
quantification of particular mRNA through mouse specific primers (MWG Bio Tech, Germany) (Table 3). The reaction mixture (20 μL) restrained 100 ng of c-DNA, 1.5 mM of MgCl2, 10 pM each primer, 1.5 mM of dNTPs, and 1 unit Ampli Taq DNA polymerase enzyme (Bangalore Genie). The product was then augmented through thermal cycles (denaturation at 95 °C for 5 min, 95 °C for 60 s, annealing temperature for 60 s, and at 72 °C for 60 s) ×35 subsequently final extensions of 72 °C for 4 min. Thus, obtained PCR products were resolved and visualized over 1.5% agarose gel containing ethidium bromide. Quantification was done via gene tool Syngene software.15 Histopathology. Skin tissues were stored in 10% buffered formalin for histopathology. The tissues were usually processed, entrenched with paraffin wax, and sectioned (3−5 μm) via rotary Microtome (YSI-060 Yorco, Ghaziabad, India). Sections thus obtained were unflustered over the glass slide, deparaffinized, and stained with hematoxylin and eosin. Solitary sections of the specimen were observed (Leica DFC 295 camera under Leica DM 1000 microscope). Statistical Analysis. All the data was subjected to statistical analysis using one-way ANOVA following Student−Newman− Keuls tests for post hoc analysis. Values are expressed as mean ± SE. Significance was determined in terms of p values. A p value of
