Ex vivo localization and permeation of cisplatin from novel topical formulations through excised pig, goat, and mice skin and in vitro characterization for effective management of skin-cited malignancies

Abstract

Context: It would be advantageous to administer cisplatin topically for treatment of cutaneous malignancies. Objectives: Present work focuses on ex vivo and in vitro characterization of proultraflexible topical formulations. Materials and methods: Permeation of cisplatin through the excised pig, goat, and mice skin was quantitatively determined. Results: Data indicate that protransfersome carbopol gel (pcg) formulation clearly delayed drug permeation through skin. Permeation of cisplatin from protransfersome system (ps) formulation was enhanced by approximately 1.5 fold compared with pcg for pig and goat skin. Discussion: Localization of drug from pcg was higher and showed less permeation. Conclusion: Cisplatin-loaded pcg formulation is better to treat cutaneous malignancies.

Keywords::

• A-431 cell line
• cutaneous squamous cell carcinoma
• cytotoxic activity
• fluorescence microscopy
• local accumulation efficiency
• proultraflexible topical formulations

Introduction

Cisplatin, classified as a potent antineoplastic drug, is approved by the FDA to treat various types of solid tumors or carcinoma, including locally advanced squamous cell carcinoma of epithelial origin (Martindale 2002). Although its beneficial effects are counteracted by significant systemic toxicities. Topical chemotherapy for the treatment of cutaneous squamous cell carcinoma (cSCC) could be an alternative to reduce cisplatin systemic toxicity (Gupta et al. 2012).

cSCC is a common non-melanoma cancer arising from the malignant proliferation of the keratinocytes of the epidermis. It may arise on any part of the skin and mucous membranes lined by the squamous epithelium, but this is more likely to occur on parts exposed to the sun (Gupta et al. 2011, Johnson et al. 1992).

Topical therapy is a preferable route for direct, site- specific, local treatment of skin-related malignancies, and it is a noninvasive method of treatment as compared to parenteral route. Further, it can eliminate the systemic toxicities associated with the antineoplastic agents. Various topical chemotherapeutic approaches were reported in the literature such as topical medication of actinic keratosis, actinic cheilitis, Bowen's disease by 5-fluorouracil and tretinoin, imiquimod topical delivery for the management of nonfacial, nodular and superfacial basal cell carcinoma (Alfred 2001, Newman and Weinberg 2007, Price 2007, Torres et al. 2007, Leiter and Garbe 2008).

Protransfersome are liquid crystalline proultraflexible lipid vesicles, which will be converted to ultraflexible vesicles transfersome in situ by absorbing water from the skin (Jain et al. 2003). Further, to stabilize the protransfersome system (ps) for their use at clinical and industrial levels, we can modify the system with block polymers and carbopol gel base to get colloidal semisolid formulations. In addition, colloidal gels were found to enhance the skin retention of drugs, that is, they provide higher and sustain skin concentration of drugs, without enhancing the systemic absorption and thus reducing drugs concentration (Kim et al. 1998, Seth et al. 2004, Padamwar and Pokharkar 2006). Further, in the case of drugs that should act topically, colloidal semisolid formulations are known to provide a localized and controlled drug delivery, acting as a drug reservoir for continuous delivery of drug (Singh and Vyas 1996, Glavas-Dodov et al. 2002, Puglia et al. 2004). Formulation development part of the research work was already done and published (Gupta and Trivedi 2012).

One of our driving goals, hereby, has been to compare the percutaneous penetration and localization of cisplatin using pig-ear, goat-ear, and mice-skin models. On account of inconsistent human skin availability, however, pig skin (Dalton et al. 2006, Dorandeu et al. 2007) and preferentially pig-ear skin (Chilcott et al. 2005) is used as a relevant anatomical site. Excised human skin is properly regarded as the “gold standard” for in vitro penetration experiments related to human dermal risk assessment. However, it is often neither timely nor sufficiently available and there is variability among samples due to differences in gender, race, age, and anatomical site of the donor (Bronaugh et al. 1982, Bronaugh and Stewart 1985, Sato and Sugibayashi 1991, Panchagnula et al. 1997, Schmook et al. 2001, Vallet et al. 2008). Numerous animal skin models from various mammals, rodents, and reptiles have been developed as surrogates for human skin (Walker et al. 1983, Vecchia and Bunge 2006). For ethical reasons, primate research is restricted, thus pig and goat skin is preferred as it can be readily obtained as waste from animals slaughtered for food.

Methods

Materials

Cisplatin was procured as a gift sample from Cipla limited (Mumbai, India). Soya lecithin (Phosphatidylcholine) was supplied by ACROS ORGANICS (New Jersey, USA). Sodium cholate, Pluronic F-68 (PF-68), Sodium diethyldithiocarbamate (DDTC) were procured from Sigma Chemical Co (St. Louis, MO, USA). Carbopol 940 (CP-940) was purchased from Himedia (Mumbai, India). Reagents and organic solvents such as disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sodium hydroxide, and isopropanol were purchased from CDH Pvt. Ltd. (New Delhi, India). Acetonitrile, methanol, and Chloroform (HPLC grade) were purchased from Merck Ltd. (Delhi, India). All other reagents used were of analytical or HPLC grade.

Animal skin

Pig and goat skin were obtained from the local slaughterhouse. Full thickness swiss albino mice skin was obtained from a native pharmacological laboratory, which were already killed for the routine pharmacological experiment. All protocols using animals were reviewed and approved by institutional ethical committee.

Preparation of cisplatin-loaded formulations

Cisplatin-loaded ps formulation was prepared by the reported method (Jain et al. 2005) with minor modifications. Soya lecithin (1.7 g), surfactant (sodium cholate, 0.3 g), and isopropanol (2 ml) were taken in a round-bottom flask and warmed at 60–70°C in a water bath until the ingredients were dissolved. The cisplatin (0.01 g) in 2 ml of phosphate buffer saline (pH 7.4) was added dropwise to it. Cisplatin-loaded pluronic protransfersome system (pps) formulation was prepared exactly as above. The method of preparation was designed as such that PF-68 is able to incorporate inside the lipid bilayer, by initially mixing the PF-68 (0.2 g) with the lecithin. For the preparation of cisplatin-loaded protransfersome carbopol gel (pcg) formulation, the best-achieved pps formulation was incorporated into 2% (w/v) carbopol gel.

Skin permeation studies

Pig and goat skin from ear pinna and swiss albino mice skin of full thickness were taken for studies. After cleaning with cold tap water, full thickness, non-dermatome skin was removed with the help of a scalpel. Locally fabricated Franz diffusion cell was used for the skin permeation studies. Cells had an effective diffusion area of 0.785 cm². The receptor compartment was filled with 10 ml of phosphate buffer saline (PBS), pH 7.4, and stirred with a magnetic bar. The temperature of the receptor compartment was maintained at 37 ± 1°C with an external, constant temperature circulator water bath.

The skins were sandwiched between the donor and receptor compartments of the Franz diffusion cell with the stratum corneum (SC) facing the donor compartment. Formulation equivalent to 200 μg of cisplatin were placed in the donor compartment. At predetermined time intervals, samples (1.0 ml) were taken from the receptor compartment and replaced with an equivalent amount of fresh buffer solution to maintain sink condition.

Skin retention/deposition studies

At the end of the permeation study, excess formulation was washed and the SC was removed by striping with adhesive tape. The remaining skin was cut into small pieces to determine the amount of drug in the viable skin (epidermis and dermis). Tape strips and viable part of the skin were pooled in a tube containing PBS (pH 7.4) and further processed (vortexes, sonicated, and centrifuged) to extract the drug for estimation.

To quantify the cisplatin, a HPLC method was used. This method was based on other previously reported literature (Augey et al. 1995, Lopez-Flores et al. 2005) with slight modifications. In brief, an aliquot of 90 μl of the sample was placed in a 1.5-mL eppendorf tube and mixed with 10 μl of a solution of sodium DDTC (10% [wt/vol] prepared with 0.1 N NaOH). Samples were incubated at 37°C in a water bath for 1 h and cooled on ice for 10 min to stop the reaction (formation of chelates, i.e., cisplatin–DDTC). These chelates were extracted with 100 μl of chloroform by vortexing at maximal speed for 1 min and centrifuged at 5000 rpm for 5 min at 5°C. Then, 10 μl of the chloroform layer was injected into the chromatographic system which consisted of a Shimadzu-LC-10ATVP equipped with a double reciprocating plunger pump with PDA detector (SPD-M10 AVP).

HPLC analysis of cisplatin

The analytical separation was performed at 30°C by a Phenomenex C-18 (25 × 0.46 cm i.d. of 5 μm particle size). The mobile phase fixed at 1ml/min flow rate at a concentration of 80:20 (methanol/water). Detection was performed at 340 nm. Retention times of 5 and 7 min were found for DDTC and DDTC–Platinum complex, respectively (Figure 1). The assay was calibrated with standards prepared previously, following the procedure described above.

Figure 1. Cisplatin determination by HPLC method. Chromatogram shows the peak of chelating agent, diethyldithiocarbamate (DDTC), and cisplatin in terms of DDTC–platinum complex.

Data plot and parameter calculations

The cumulative amount of cisplatin permeated per centimeter square of skin was calculated and plotted as a function of time. The permeation parameters were calculated from the linear part of the plot, which corresponds to the steady state, being the flux (J), the slope, and the lag time (T_lag). The permeability coefficient (K_p) was obtained dividing the flux by the drug concentration in the donor compartment. The correlation between the steady state flux data was determined. The local accumulation efficiency (LAC) was calculated by the following formula:

Ex vivo vesicle–skin interaction studies

Sections of pig, goat, and mice skin used for the permeation studies were examined under the fluorescence microscope. For the purpose, a hydrophilic fluorescence probe (6-carboxyfluorescein) was added during preparation of the various formulations. After a 10-h incubation period at 37 ± 1°C with each formulation (placed on the skin surface) in the Franz diffusion cell, skins were washed with PBS (pH 7.4). Sections of the skin were cut (25 μm thickness) with a cryostat microtome and mounted on a glass microscope slide with a permanent aqueous mounting medium and examined to investigate the fluorescent probe (6-carboxyfluorescein) distribution in the different skin strata under the Fluorescence microscope (ProgRes CP-SCAN, LASER OPTIC SYSTEM, USA).

Drug–excipients interaction (FT-IR studies)

The drug–excipients interaction was observed using FT-IR. The IR spectra of cisplatin, blank unloaded ps formulation, cisplatin-loaded ps, pps, and pcg formulations were recorded by using Fourier transform infrared spectrophotometer (JASCO FT/IR-470 plus, Japan) and analyzed for characteristic absorption of various functional groups.

Cell lines, growth conditions, and treatment

The cell line A-431 (human cutaneous squamous cell carcinoma) was obtained from the National Center for Cell Sciences, Pune, India. Cells were maintained in a 96-well microtiter plate containing RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FCS), containing 5% of the mixture of Gentamycin, Penicillin (100 Units/ml) and Streptomycin (100 μg/ml) in the presence of 5% CO₂ at 37°C for 3–4 days. After 3–4 days supernatant was removed and RPMI-1640 medium was replaced with Hank's balanced solution supplemented with Gentamycin, Penicillin, and Streptomycin and incubated overnight.

In vitro anticancer activity

In vitro anticancer activity of the tested compound was assessed by colorimetric determination of reduction of a yellow tetrazolium salt MTT to dark purple formazan by an enzyme succinate dehydrogenase, mainly in mitochondria. Reduction of MTT to formazan happens only in metabolically active cells and thus can be used to measure the viability of the cells.

The cytotoxicity of the ps, pps, and pcg was studied in the human A-431 cell line using MTT assay. Cells at 1 × 10⁵/mL were seeded in 96-well plates and incubated for 24 h. The growth medium was then replaced with different concentration of the ps, pps, and pcg formulations. Untreated cells were used as the control group. The cells were further incubated for 48 h, and the relative anticancer activity was assessed using MTT assays. In brief, MTT solutions were added after the treatments and incubated for an additional 4 h. Dimethyl sulfoxide was added to solubilize the formazan crystal, and optical density (OD) at 492 nm was recorded. The OD was determined at 492 nm using an ELISA plate reader (BioTek, USA). The cell viability fraction (%) was calculated as follows:

Statistical analysis

All formulations were prepared and analyzed in triplicate (n = 3). Results are expressed as mean ± SD (standard deviation). The correlation between the steady-state flux data of pig, goat, and mice skin were determined. Student's t-test and ANOVA were performed to determine the level of significance between the groups.

Results

Preparation of cisplatin-loaded formulation

The ps formulation is a proultraflexible lipid vesicular drug delivery system which contains edge activator, that is, sodium cholate. The pps formulation contains PF-68 which provide steric stabilization to the system by adsorbing on the surface of the vesicles. The pps formulation was converted into a high viscosity formulation when incorporated into 2% carbopol-940 gel base, that is, pcg formulation, which forms a gel matrix dosage form (Figure 2).

Figure 2. Illustrative representation of the strategy of developing cisplatin (CDDP)-loaded pluronic (PF-68)-modified carbopol (CP-940) protransfersome gel.

Ex vivo drug permeation and retention studies

Figure 3 shows the permeation profile of cisplatin from ps, pps, and pcg formulations through pig, goat, and mice skin model ex vivo. The data indicate that pcg formulation clearly delayed the drug permeation through the skin. The permeation of cisplatin from ps formulation for pig and goat skin model was reduced by approximately 2.5 times compared with that for the mice skin model. Further, the permeation of cisplatin from ps formulation was enhanced by approximately 1.5 fold compared with that from pcg formulation for pig and goat skin model. However, mice skin was 3.5 times more permeable for cisplatin from ps formulation as compared to pcg formulation. There was no significant difference (p > 0.5) among pig and goat skin model for the permeation profile of cisplatin, but there was statistically significant difference between mice versus pig (p < 0.001) and mice versus goat (p < 0.001) skin model for the permeation profile of cisplatin from ps formulation. Further, there was no significant difference between pig versus goat (p > 0.5), goat versus mice (p > 0.5) and pig versus mice (p > 0.5) skin model for the permeation of cisplatin from pcg formulation. Table I summarizes the data for steady-state flux (J), Lag Time (T_lag), permeability coefficient (K_P). LAC values were found to be higher for pcg formulation as compared to other two formulations (Table II) which reveals that cisplatin accumulation is high in the skin from pcg formulation. A positive correlation between the steady-state flux values of pig and goat skins were observed, however, mice skin showed significantly different permeation and accumulation profile (Figure 4). The percentage of cisplatin permeated the skin and accumulated into the SC as well as viable skin has been compiled in Figure 5. It shows the permeation and partitioning of the cisplatin into different compartment of the skin (i.e., SC and viable skin). Results revealed that the drug is more permeable for ps formulation in case of mice skin model in spite of retention in the skin. The retention profiles and LAC values (Table II) were observed after 24 h of permeation studies ex vivo in SC and viable part of the skin. The data of LAC values show that the maximum drug was accumulated in the viable part of the pig and goat skin model from pcg formulation than other two formulations (i.e., ps and pps) as compared to mice skin model. Figure 6 shows the morphology of the skin before (Figure 6a) and after (Figure 6b–d) permeation studies. A change in skin integrity was observed after permeation, that is, skin became microporous after studies.

Figure 3. Permeation profile of cisplatin. Ex-vivo permeation of drug for ps, pps, and pcg formulations using pig, goat, and mice skin. Values are expressed as mean ± standard deviation (n = 3).

Figure 4. Correlation between various skin models in terms of cisplatin permeation parameter. Correlation between steady-state flux values of cisplatin across pig-ear skin versus goat-ear skin, mice skin versus goat-ear skin and mice skin versus pig-ear skin.

Figure 5. Percent cisplatin permeation and skin-retention in stratum corneum (SC) and viable part of the skin ex-vivo from ps, pps and pcg formulation. Values are expressed as mean ± standard deviation (n = 3).

Figure 6. Morphology of pig, goat, and mice skin before (a) and after permeation study with the formulation ps (b), pps (c), and pcg (d). A longitudinal section of the skin showing epidermal part before permeation study (saturated with buffer media) and 24 h after permeation study.

Table I. Results of ex vivo drug permeation parameters for pig- and goat-ear pinna skin and mice skin.

	Steady-state flux (J) (μg/cm^2/h)			Permeability coefficient (KP) (μg × 10^− 3)			Lag time (Tlag) (h)
Skin model	ps	pps	Pcg	ps	pps	pcg	ps	pps	pcg
Pig skin	13.07 ± 0.6	01.97 ± 0.1	02.68 ± 0.2	65.35 ± 1.8	09.71 ± 0.6	13.40 ± 1.2	0.8 ± 0.06	0.8 ± 0.04	0.2 ± 0.02
Goat skin	17.29 ± 0.5	06.08 ± 0.3	03.65 ± 0.2	86.45 ± 2.2	30.40 ± 1.6	18.25 ± 1.4	1.0 ± 0.04	0.5 ± 0.03	1.5 ± 0.06
Mice skin	16.86 ± 1.1	12.71 ± 1.4	03.96 ± 0.4	84.30 ± 2.6	63.55 ± 2.4	19.80 ± 2.2	0.2 ± 0.06	0.1 ± 0.02	0.2 ± 0.04

Results are represented as mean ± SD (n = 3).

Table II. Results of ex vivo drug accumulation parameter for pig- and goat-ear pinna skin and mice skin.

	LAC (after 24 h)
	SC			Viable
Skin model	ps	Pps	pcg	ps	pps	pcg
Pig-ear skin	0.90 ± 0.08	1.54 ± 0.11	1.05 ± 0.06	0.88 ± 0.06	1.52 ± 0.10	1.84 ± 0.12
Goat-ear skin	0.97 ± 0.07	1.50 ± 0.10	1.70 ± 0.12	1.18 ± 0.11	1.41 ± 0.18	1.90 ± 0.12
Mice skin	0.10 ± 0.01	0.47 ± 0.02	1.75 ± 0.10	0.11 ± 0.03	0.30 ± 0.03	1.24 ± 0.06

Results are represented as mean ± SD (n = 3).

Ex vivo vesicle–skin interaction studies by fluorescence microscopy

In order to visualize the skin delivery of the different formulations tested (ps, pps, and pcg formulations), they were made fluorescent by the hydrophilic dye (6-carboxyfluorescein). Fluorescence was more intense in the inter-corneocytes spaces in the case of pcg formulation (Figure 7), may be because of colloidal semisolid formulations which are known to provide a localized and controlled drug delivery and acting as a drug reservoir.

Figure 7. Fluorescence micrographs of pig- and goat-ear pinna skin and mice skin treated with the formulation ps (a) pps (b) pcg (c). Various formulations labeled with hydrophilic fluorescence probe (6-carboxyfluorescein) to mark the penetration of the drug to deep skin strata.

Drug–excipients interactions studies using FT-IR

Drug–excipients interactions during the formulation development can be revealed using FT-IR by examination of wavelength shifts in the characteristic peak positions of either the drug or the excipient. The IR spectrum of cisplatin, blank formulation, and drug-loaded formulations shows the positions of characteristic absorption of functional groups, that is, absorption spectrum of NH stretch peak of aliphatic amine in cisplatin at 3298.28 cm^{− 1}, which was also observed in ps, pps, and pcg formulations between 320 and 3300 cm^{− 1}. The absorption spectrum of tertiary amines of lecithin was also present in blank, ps, pps, and pcg formulations between 2300 and 2400 cm^{− 1}. A unique peak of a CO ester of sodium cholate was present in blank, ps, pps, and pcg formulations in the range of 1700–1750 cm^{− 1}. Spectrum of primary hydroxyl group of PF-68 was present in pps and pcg formulation between 3200 and 3450 cm^{− 1}. Finally the spectrum of a COO of carboxylic acid in acrylate for carbopol 940 was present at 1645.28 cm^{− 1} in pcg formulation (Figure 8).

Figure 8. FT-IR study of cisplatin, blank, ps, pps, and pcg formulations and their characteristics absorption spectrum. Spectra show the characteristics absorption peaks of various functional group present in drug and excipients in terms of wave number.

In vitro anticancer studies

The cytotoxic activity of various formulations was tested in vitro against A-431 skin carcinoma cell line using MTT assay. The concentration of cisplatin in the ps, pcg formulation ranged from 40 to 400 μg caused a strong cytotoxicity, that is, the viability of A-431 cells treated by cisplatin-loaded ps, pcg markedly decreased. However, pps formulation showed anticancer activity at the concentration range 120–400 μg (Figure 9). The percentage of cell lysis and IC50 values of the various cisplatin-loaded formulations are given in Table III.

Figure 9. In vitro antiproliferative/cytotoxicity activity of ps, pps, and pcg formulations against cutaneous squamous cell carcinoma cell line (A-431). The antiproliferative/cytotoxic effect is expressed in terms of the percentage of cell viability fraction as a function of amount of drug (μg).

Table III. Results of in vitro anticancer activity of drug-loaded ps, pps, and pcg formulations with respect to control against A-431 skin squamous cell carcinoma cell line.

Formulation	Concentration of drug (μg)	Optical density* (at 492 nm)	cell viability fraction (%)	IC50 (μg)
Control	−	2.211 ± 0.121	100.00	–
ps	40	0.548 ± 0.113	24.78
	80	0.501 ± 0.102	22.65
	120	0.052 ± 0.010	02.35	< 40
	200	0.012 ± 0.001	0.54
	400	0.010 ± 0.001	0.45
pps	40	2.011 ± 0.130	90.95
	80	1.980 ± 0.123	89.55
	120	1.020 ± 0.131	46.13	120
	200	0.602 ± 0.111	27.22
	400	0.012 ± 0.002	0.54
pcg	40	1.023 ± 0.121	46.26
	80	0.511 ± 0.101	23.11
	120	0.011 ± 0.010	0.49	40
	200	0.010 ± 0.002	0.45
	400	0.009 ± 0.001	0.40

Compared with vehicle control, ps, pps, and pcg formulations treatment showed statistically significant results (P < 0.01).

*Results are represented as mean ± SD (n = 3).

Discussion

Edge activator containing vesicles is flexible in nature. These vesicles are able to penetrate the skin in a spontaneous manner throughout the intercellular domain. At optimum concentration, PF-68 acts as a stabilizer to the vesicular system. Pluronic is an A-B-A block copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO) in which one type of repeated unit (PPO) can act as an anchor to lipid vesicles and the other type (PEO) can act in the moieties extending into an aqueous environment. Further, cisplatin is unstable in aqueous environment and carbopol gel base provide a matrix like structure to the system by absorbing water from the system.

Carbopol gel network system may be responsible for the rate-limiting effect in drug permeation from the pcg formulation. Carbomer gel absorbs water, which is accompanied by a polymer chain relaxation in which the vesicle is entrapped in the gel intramatrix space and releases the drug over a long period of time. A positive correlation was obtained for permeation parameters between pig and goat skin model as compared to mice skin model which may be due to structural and anatomical similarities in skin of pig and goat. Further, human skin for research purposes is not readily available. As per various researchers (Dick and Scott 1992, Lin et al. 1992, Jacobi et al. 2007) the skin of pigs and goats is composed of an epidermis and dermis with characteristics like those of human skin and there is no need of ethical permission because it can be easily obtained from the slaughter house. However, the skin of laboratory animals, for example, mice, rat, guinea pig, rabbits, etc., shows marked anatomic differences from human skin. In particular, the epidermis of these animals is too thin and the flat epidermal–dermal interface does not have rete ridges and papillary projections. For ethical reasons, primate research is restricted and pig skin is preferred and based on morphological and functional data, domestic pig skin seems to be the closest to human skin (Meyer et al. 1978, Calabrese 1984, Monteiro-Riviere and Riviere 1996).

Furthermore, in these animals, dermal structures are relatively loose, and the vascular system is underdeveloped, therefore, the skin of most animals presents a much weaker barrier than human skin. The increase in cisplatin retention from the pcg formulation in SC and viable part of the skin demonstrates the efficiency of the pcg formulation developed with carbopol-940 gel base in association with PF-68 block copolymer.

A high accumulation of fluorescence probe in deep skin strata from pcg formulation may be considered due to carbopol. Carbopol is a cross-linked polymer which forms a matrix-type reservoir during gel preparation and release drug slowly for a prolonged period of time (Han et al. 2012). Lipid vesicles may entrap into the intra-matrix system of the carbopol gel and responsible for the drug accumulation in deep skin strata. It means carbopol gel (pcg formulation) was found to enhance the skin retention of drugs, that is, they provide higher and sustained skin concentrations of drugs, without enhancing the systemic absorption of drugs.

On comparison, of the IR spectra, it is clear that there is no significant interaction between the incorporated drug and the other components of the formulation. All the absorption spectra of characteristic functional groups are present within the range. Thus, drug and excipients are compatible with each other.

Generally, the cytotoxicity is the first test used to investigate biocompatibility. The evaluation of new formulations using in vitro methods should be done initially to avoid unnecessary animal sacrifice. Cytotoxicity testing of a novel biomaterial is the first-level evaluation before its biomedical application. Results indicate that lethality increases with increasing concentrations of cisplatin, suggesting a dose-dependent effect in vitro. This increased cytotoxicity may be due to improved cisplatin cellular uptake by novel biocomposites through the endocytosis pathway, which is a common characteristic of vesicular drug delivery systems. Therefore, a sufficiently high concentration of cisplatin can be generated within tumor cells by proultraflexible vesicular drug delivery system, thereby greatly promoting the cytotoxic effects (Yoo et al. 2000, Sabitha et al. 2013).

Conclusion

The permeability and localization of pig skin for cisplatin was quantitatively similar to goat skin but different with mice skin. The results obtained indicate that the localization of the drug from the pcg formulation in the viable skin was higher than other two formulations and also showed the less permeation which mimics the reduced systemic toxicity of cisplatin. Further, on the basis of in vitro cell lysis assay it was clear that cisplatin-loaded pcg formulation is better drug delivery system to treat skin-cited nonmelanoma cancer-like cSCC. However, human skin permeation data and study with abnormal skin are needed to evaluate the risk versus benefit ratio.

Acknowledgment

The authors would like to express their gratitude to the National Institute of Cell Sciences for providing cell line and, Prof (Dr) Kishore G Bhat, Dept of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences & Research Centre, Belgaum, Karnataka, for providing in vitro anticancer activity. The authors are grateful to Department of Science and Technology (DST), Govt. of India, New Delhi for financial support under Women Scientist Scheme (WOS-A) grant for research work.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.