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IJP 14846 1–10 International Journal of Pharmaceutics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
1
Pharmaceutical nanotechnology
2
Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery
3
4 Q1 5 6 7 8 9 10 11 12
Donato Cosco a,e,1, Donatella Paolino a,e,1, Jessica Maiuolo a , Luisa Di Marzio b , Maria Carafa c, Cinzia A. Ventura d , Massimo Fresta a,e, * a Department of Health Sciences, University “Magna Græcia” of Catanzaro, Campus Universitario “S. Venuta”, Viale S. Venuta, Germaneto, I-88100 Catanzaro, Italy b Department of Pharmacy, University “G. d'Annunzio” of Chieti-Pescara, Via dei Vestini 31, I-66100 Chieti, Italy c Department of Drug Chemistry and Technologies, University “Sapienza” of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy d Department of Drug Science and Health Products, University of Messina, Viale Annunziata, 98168 Messina, Italy e IRC FSH – Interregional Research Center for Food Safety & Health, University of Catanzaro “Magna Græcia”, Campus Universitario “S. Venuta” – Building of BioSciences, Viale S. Venuta, I-88100 Germaneto, Catanzaro, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 March 2015 Received in revised form 15 April 2015 Accepted 16 April 2015 Available online xxx
Ultradeformable liposomes represent useful formulations able to increase the skin permeation of drug compounds. In this study, resveratrol- and 5-fluorouracil-loaded ultradeformable liposomes were investigated for the potential treatment of non-melanoma skin cancer. The in vitro anticancer activity of ultradeformable liposomes was tested on human skin cancer cells through viability-, cell cycle- and apoptosis-analysis. Furthermore, we tested the percutaneous permeation of ultradeformable liposomes using human stratum corneum and viable epidermis. The co-encapsulation of resveratrol and 5fluorouracil (multi-drug carrier) in ultradeformable liposomes improved their anticancer activity on skin cancer cells as compared to both the free drug form and the single entrapped agents. These multi-drug ultradeformable liposomes arrest cell proliferation in G1/S, thus modifying the action of 5-fluorouracil and increasing the activity of resveratrol. This effect might depend on the ultradeformable liposomes, which may accumulate in deeper skin layers, thus generating a cutaneous depot from which resveratrol and 5-fluorouracil are gradually released. Resveratrol and 5-fluorouracil co-loaded ultradeformable liposomes could be a new nanomedicine for the treatment of squamous cell carcinoma, i.e., actinic keratosis, Bowen’s disease, and keratoacanthoma. ã 2015 Published by Elsevier B.V.
Keywords: Multi-drug colloidal carriers Skin cancer Skin delivery Supramolecular therapeutics Ultradeformable liposomes
13
1. Introduction
14
Lipid-based nanocarriers possess ideal properties which permit them to modulate and improve the biopharmaceutical characteristics of encapsulated drugs. During the last decade, in particular, there has been an increase in investigations concerning the topical delivery of active compounds by innovative vesicular systems (Paolino et al., 2008; González-Paredes et al., 2010; Marianecci et al., 2012). Liposomes were the first nanocarriers used to increase the permeation rate of the encapsulated compounds through the skin, but their use was limited by their localization in the skin stratum corneum (Sinico and Fadda, 2009). For this reason novel
15 16 17 18 19 20 21 22 23
Q2
* Corresponding author at: Department of Health Sciences, University “Magna Græcia” of Catanzaro, Campus Universitario “S. Venuta”, Viale S. Venuta, Germaneto, I-88100 Catanzaro, Italy. Tel.: +39 961 369 4118; fax: +39 961 369 4237. E-mail address: [email protected] (M. Fresta). 1 These two authors equally contributed.
vesicular systems have been investigated, such as ultradeformable liposomes and ethosomes1 which have furnished the best results in terms of increased permeation of drug following their topical administration (Elsayed et al., 2007a; Ainbinder et al., 2010; Celia Q3 et al., 2012). Ultradeformable liposomes, topically administered under nonocclusive conditions, are made up of phospholipids and edge activators, capable of destabilizing the lipid bilayer of the vesicles and increasing the deformability of the bilayer and affecting its interfacial tension (Cevc, 2004; Cevc and Vierl, 2010; El Zaafarany et al., 2010). In fact, their transport across the skin is driven by the osmotic gradient and occlusion could eliminate it, thus compromising the action of the deformable vesicles (Elsayed et al., 2007b). Ultradeformable liposomes were used to increase the skin permeation of different bioactives (i.e., corticosteroids, diclofenac, insulin, etc.) allowing significant drug localization even in the blood stream (Cevc and Blume, 2001; Cevc, 2003; Jain et al., 2005; Benson, 2006).
http://dx.doi.org/10.1016/j.ijpharm.2015.04.056 0378-5173/ ã 2015 Published by Elsevier B.V.
Please cite this article in press as: Cosco, D., et al., Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.04.056
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IJP 14846 1–10 2 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
D. Cosco et al. / International Journal of Pharmaceutics xxx (2015) xxx–xxx
Oxidative stress is a key component in the multistage carcinogenic process. Reactive oxygen species (ROS) are generated in response to both endogenous and exogenous stimuli. However, endogenous antioxidants prevent ROS-mediated injury. In addition, the reduction of oxidative stress coupled with the use of chemotherapeutic drugs produces a large amount of free radicals (Hwang et al., 2001). Many experiments showed that a supplement of antioxidant drug during anticancer therapy may reduce adverse reactions and improve the potential for success in terms of tumor response. It has recently been demonstrated that resveratrol (RSV), a polyphenol largely used as antioxidant, synergistically promotes 5-fluorouracil (5-FU)-mediated apoptosis of cancer cells irrespective of p53 (Gross et al., 2007; Chan et al., 2008; Gatouillat et al., 2010). The aim of this investigation was to co-encapsulate two active compounds having different physicochemical properties within ultradeformable liposomes, in order to obtain a synergistic action against lesions related to squamous cell carcinoma such as actinic keratosis, Bowen’s disease, and keratoacanthoma. This strategy has already been used by many research teams with the aim of increasing the anticancer efficacy of the obtained formulation with respect to the single agents or a physical mixture of them in in vitro and in vivo tumor models (Dicko et al., 2010; Cosco et al., 2011, 2012). 5-FU and RSV were chosen as model drugs because the first is a hydrophilic compound which has a significant antitumor effect on skin cancers, while the second is an antioxidant able to synergistically induce apoptosis in cancer cells. Ultradeformable liposomes, containing sodium cholate as edge activator and different amounts of the aforesaid compounds, were prepared in order to investigate their physicochemical and technological characteristics. The anticancer action of the most suitable ultradeformable liposome formulation was then evaluated in vitro on human skin cancer cells through viability testing, cell cycle- and apoptosis analysis. The interaction rate between the vesicles and the cells was also evaluated as well as the percutaneous permeation of the multi-drug ultradeformable liposomes.
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2. Materials and methods
81
2.1. Chemicals
82
Phospholipon 90G1 (PL 90G1), was a kind gift by Nattermann Phospholipid Gmbh (Cologne, Germany). Cholesterol (Chol), 5-fluorouracil (5-FU), resveratrol (RSV), sodium cholate (SC), phosphate buffered saline (PBS) tablets, 3-[4,5-dimethylthiazol2-yl]-3,5-diphenyltetrazolium bromide salt (MTT), propidium iodide, RNase A, dimethyl sulfoxide, amphotericin B solution (250 mg/ml) were purchased from Sigma–Aldrich (Milan, Italy). Cellulose membrane Spectra/Por MWCO 12,000–14,000 Da were obtained from Spectrum Laboratories Inc. (Eindhoven, The Netherlands). Lissamine rhodamine B 1,2 dihexadecanoyl-snglycero-3-phosphoethanolamine triethylammonium salt (rhodamine DHPE) and Hoechst 33344 were InvitrogenTM products (Life Technologies, Monza, Italy). Human skin cancer cells (SK-MEL28 and Colo-38) were provided by the Instituto Zooprofilattico of Modena and Reggio Emilia. RPMI 1640 Medium with glutamax, trypsin/EDTA (1) solution, foetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco (Invitrogen Corporation, Paisley, UK). Ethanol was obtained from Carlo Erba SpA (Rodano (MI), Italy). The TdT-mediated dUTP nick-end labeling assay (Tunel assay) was purchased as a kit from Promega (Madison, WI, USA). Double-distilled pyrogen-free water was obtained from Sifra SpA (Verona, Italy).
83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
Table 1 Physicochemical parameters of ultradeformable liposomes prepared both in presence and absence of 5-FU and RSV.a Sample
Drug (% w/w)
Mean sizes (nm)
Polydispersity Index
Zeta potential (mV)
Empty ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs MD-ULs MD-ULs MD-ULs MD-ULs MD-ULs MD-ULs
– 1 2 3 4 5 10 1 2 3 4 5 10 1 2 3 4 5 10
307.4 8.6 618.4 26.4 530.8 6.3 478.3 8.0 383.8 9.4 422.2 13.2 595.8 25.8 345.0 12.5 359.7 15.7 432.1 20.4 449.5 12.7 538.6 25.9 573.6 11.8 316.6 4.8 356.5 13.3 370.5 12.2 458.7 23.1 445.6 19.5 663.8 184.8
0.38 0.02 0.28 0.04 0.45 0.03 0.44 0.03 0.31 0.05 0.17 0.07 0.48 0.06 0.30 0.04 0.32 0.04 0.39 0.08 0.42 0.07 0.45 0.04 0.52 0.03 0.36 0.03 0.39 0.04 0.34 0.07 0.31 0.06 0.32 0.09 0.60 0.21
27.4 0.8 24.2 0.4 23.4 0.7 21.3 0.3 25.3 0.3 18.9 0.7 24.1 0.5 24.7 0.4 27.1 1.0 23.7 0.4 26.4 0.7 29.2 1.3 28.7 1.0 28.2 0.7 32.8 0.7 29.8 1.0 27.3 0.4 25.5 0.5 28.5 0.9
a Particle suspensions were suitably diluted and analysis was carried out at 20 C. Each value was the mean of six different experiments SD.
2.2. Vesicle preparation and purification
104
The compositions of the investigated ultradeformable liposomal formulations (ULs) are reported in Table 1. The maximum amount of the active compounds was of 10% (w/w) with respect to the total amount of lipids. The ULs were prepared as previously described (Celia et al., 2009), by dissolving PL 90G1 (88 mg) and SC (12 mg) in 3 ml of ethanol in a Pyrex1 glass vial. The organic solvent was firstly removed by a Rotavapor1 210 (Büchi Italia, Milan, Italy) under a nitrogen flux and then by overnight storage at 30 C in a Büchi T51 glass drying-oven under vacuum. The lipid film was hydrated with 6 ml of a water/ethanol (93:7%, v/v) solution and vortex-mixed at 700 rpm for 15 min at room temperature. The ULs were left at 40 C for 1 h and sonicated with a SONOPULS GM 70 probe sonicator (Bandelin Electronic, Berlin, Germany) at 50 cycle/s for 3 min at 60% of its maximum power. RSV-loaded ULs (RSV-ULs) and rhodamine-labeled ULs were prepared by solubilizing the drug or fluorescent probe (rhodamine-DHPE, 0.1% molar) in the organic phase, respectively, while 5-FU-loaded ULs (5-FUULs) were obtained by adding the hydrophilic drug to the aqueous phase. The multi-drug ultradeformable liposomal formulations (MD-ULs) were obtained following the co-encapsulation of the two drugs, which were respectively dissolved in their suitable environments. In order to remove the free drug from the entrapped one, the drug loaded-ULs were centrifuged at 80,000 g at 4 C for 1 h using a Beckman OptimaTM ultracentrifuge equipped with a TL S55 fixedangle rotor (Bechman Coulter Inc., Fullerton, CA, USA). The drugloaded UL pellets were washed twice with PBS and centrifuged (80,000 g at 4 C for 1 h), while the supernatants were collected and stored at 20 C until analysis. The obtained pellets were then dissolved in PBS buffer (6 ml) and used for in vitro and in vivo experiments.
105 106 107 108 109 110
Q4 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
2.3. Physicochemical characterization
136
Mean size and size distribution were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, United Kingdom). The third-order cumulant fitting correlation function was applied. A 4.5 mW laser diode operating at 670 nm was used as a light source for size analysis and the back-scattered photons were detected at 173 . The
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Please cite this article in press as: Cosco, D., et al., Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.04.056
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real and imaginary refractive indexes were set at 1.59 and 0.0, respectively. The medium refractive index (1.330), medium viscosity (1.0 mPa s), and dielectric constant (80.4) were set before the experiments. The Z-potential of the colloidal suspensions was also measured by a Zetasizer Nano ZS. A Smoluchowsky constant F (Ka) of 1.5 was used to calculate this parameter as a function of the electrophoretic mobility of ultradeformable lipid vesicles. The various ULs were placed into cylindrical glass tubes and submitted to a Turbiscan Lab1 Expert apparatus to evaluate their stability by analyzing the variation of their backscattering (DBS) profiles as a function of time (Celia et al., 2009) (see Supplementary materials for further details).
156
2.4. Evaluation of drug entrapment efficiency
157
The amount of 5-FU and/or RSV entrapped in the ULs was evaluated by HPLC after vesicle disruption by methanol (Cosco et al., 2012). HPLC analysis showed that no interference was determined by the various components of the vesicles (see Supplementary materials for further details). The entrapment efficiency was quantified as the percentage of the added drug that became associated within the ULs, according to the following equation:
144 145 146 147 148 Q5 149 150 151 152 153 154
158 159 160 161 162 Q6 163 164
EE ¼ 166 165 167
DE 100 DT
(1)
169
where DE is the amount of drug associated within the ULs and DT is the total amount of drug added during the preparation procedure. The recovery of 5-FU and RSV both as entrapped ULs and unentrapped drug was always greater than 96%.
170
2.5. Drug release from ULs
171
Drug release was evaluated according to the dialysis method (Cosco et al., 2012) by using cellulose acetate dialysis tubing with molecular cut-off 12,000–14,000 Da sealed at both ends with clips. A water/ethanol mixture (70:30 v/v) was used as the release medium, which was constantly stirred and warmed (GR 150 thermostat, Grant Instruments Ltd., Cambridge, UK) to 37 0.1 C throughout the release experiments. Before dialysis, the tubing was kept overnight in the release medium to allow thorough soaking. The drug-loaded ULs (1 ml) were placed into dialysis bags, which were then transferred into beakers containing 200 ml of the release medium, thus observing sink conditions for 48 h experiments. At predetermined time intervals, 1 ml of the release medium was withdrawn and replaced with fresh. Samples were analyzed by HPLC. No interference peak was observed. The percentage of released drug was calculated according to the following equation:
168
172 173 174 175 176 177 178 179 180 181 182 183 184 185 Q7 186
drugrel Drugrelease ð%Þ ¼ 100 drugload
(2)
187
where drugrel is the amount of drug released at the time t and drugload is the amount of drug entrapped within the ULs.
188
2.6. Percutaneous permeation experiments and dermal delivery of ULs
189
The percutaneous permeation profiles of the various ULs were evaluated using human stratum corneum and viable epidermis (SCE) membranes (Kligman and Christophers, 1963) (see Supplementary materials for further details). Dynamic Franz diffusion cells were used for percutaneous permeation experiments. A mixture of water/ethanol (70:30 v/v) was used as receptor. Experiments were carried out under non-occlusive conditions, and the SCE membranes were mounted between the donor and the
190 191 192 193 194 195 196
3
Fig. 1. Kinetic stability profiles of empty and drug-loaded ULs using Turbiscan Lab1: panel a, 5-FU; panel b, RSV; panel c, MD. The result was a representative experiment of three independent experiments.
Please cite this article in press as: Cosco, D., et al., Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.04.056
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221
receptor compartments. Sink conditions were maintained throughout the percutaneous permeation experiments. The amount of 5-FU and RSV that permeated through the SCE membranes was determined by HPLC. In order to evaluate the ability of the ULs to improve the delivery of the drugs to the dermis, human full-thickness skin (FTS) was also used for the experiments. FTS membranes were used after explantation and pre-equilibrated in physiological solution at 25 C for 2 h before the experiments and mounted between the donor and the receptor compartments with the SC side up. The amounts of 5-FU and RSV retained in the dermis were determined after 24 h at the end of the permeation experiment. In this case, the SC side of the FTS membranes was washed ten times using a cotton cloth immersed in methanol to remove any trace of the formulations. The SC was removed by stripping ten times with Tesa1 AG adhesive tape (Hamburg, Germany) (Saija et al., 2000). The epidermis was separated from the dermis using a sterile surgical scalpel, and the dermis was then cut with scissors, placed in a glass homogenizer containing 1 ml of methanol, homogenized for 5 min, and sonicated using a probe type sonicator (Sonopolus GH70, Bandelin-Electonic, Berlin, Germany) at 50 cycle/s. The resulting tissue suspension was centrifuged for 10 min at 10,000 g, and the supernatant was analyzed by HPLC to determine the amounts of 5FU and RSV present. The recovery of the 5-FU and RSV was, respectively, 97% and 93% of the amount applied to the skin.
222
2.7. Cell cultures and cytotoxic activity
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234
SK-MEL-28 and Colo-38 cells were grown in RPMI1640 medium with glutamate, penicillin (100 UI/ml), streptomycin (100 mg/ml) and FBS (10% v/v) at 37 C (5% CO2). The medium was replaced with fresh every 48 h. When 80% confluence was reached, the cells were seeded in culture dishes before in vitro investigations. The cytotoxic effects of empty vesicles, 5-FU and RSV (as free or encapsulated form and as single agent or in association) were evaluated by MTT-test (cell viability) as previously described (Paolino et al., 2008). The interaction between melanoma cells and fluorescent ULs was evaluated through confocal laser scanning microscopy (CLSM) (see Supplementary materials for further details).
235
2.8. Cell cycle analysis and in vitro evaluation of cell apoptosis
236
252
Melanoma cells at 80% confluence were treated with the empty and drug-loaded ULs at two different drug concentrations (5 and 10 mM) for 24 h (see Supplementary materials for further details). To ensure adequate DNA measurements, assessed by the coefficient of variation of the peaks (CVs), the interval of CVs was maintained at a value of less than 10% in accordance with the DNA Cytometry Consensus Conference (Haroske et al., 2001). The evaluation of the apoptotic induction was carried out following the treatment of melanoma cells for 24 h with different ultradeformable liposomal formulations at a final drug concentration of 5 mM. The apoptotic activity of the various formulations was evaluated using three different and independent methods: the COMET assay, the spectrofluorometric Tunel assay and the Caspase-3 assay. The COMET and caspase-3 assays were evaluated as previously reported (Palumbo et al., 2002), while the Tunel assay was performed according to manufacture instruction (see Supplementary materials for further details).
253
2.9. Statistical analysis
254
One-way ANOVA was used for statistical analysis of the various experiments. A posteriori Bonferroni t-test was carried out to check the ANOVA test. A p value
IJP 14846 1–10 International Journal of Pharmaceutics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
1
Pharmaceutical nanotechnology
2
Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery
3
4 Q1 5 6 7 8 9 10 11 12
Donato Cosco a,e,1, Donatella Paolino a,e,1, Jessica Maiuolo a , Luisa Di Marzio b , Maria Carafa c, Cinzia A. Ventura d , Massimo Fresta a,e, * a Department of Health Sciences, University “Magna Græcia” of Catanzaro, Campus Universitario “S. Venuta”, Viale S. Venuta, Germaneto, I-88100 Catanzaro, Italy b Department of Pharmacy, University “G. d'Annunzio” of Chieti-Pescara, Via dei Vestini 31, I-66100 Chieti, Italy c Department of Drug Chemistry and Technologies, University “Sapienza” of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy d Department of Drug Science and Health Products, University of Messina, Viale Annunziata, 98168 Messina, Italy e IRC FSH – Interregional Research Center for Food Safety & Health, University of Catanzaro “Magna Græcia”, Campus Universitario “S. Venuta” – Building of BioSciences, Viale S. Venuta, I-88100 Germaneto, Catanzaro, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 March 2015 Received in revised form 15 April 2015 Accepted 16 April 2015 Available online xxx
Ultradeformable liposomes represent useful formulations able to increase the skin permeation of drug compounds. In this study, resveratrol- and 5-fluorouracil-loaded ultradeformable liposomes were investigated for the potential treatment of non-melanoma skin cancer. The in vitro anticancer activity of ultradeformable liposomes was tested on human skin cancer cells through viability-, cell cycle- and apoptosis-analysis. Furthermore, we tested the percutaneous permeation of ultradeformable liposomes using human stratum corneum and viable epidermis. The co-encapsulation of resveratrol and 5fluorouracil (multi-drug carrier) in ultradeformable liposomes improved their anticancer activity on skin cancer cells as compared to both the free drug form and the single entrapped agents. These multi-drug ultradeformable liposomes arrest cell proliferation in G1/S, thus modifying the action of 5-fluorouracil and increasing the activity of resveratrol. This effect might depend on the ultradeformable liposomes, which may accumulate in deeper skin layers, thus generating a cutaneous depot from which resveratrol and 5-fluorouracil are gradually released. Resveratrol and 5-fluorouracil co-loaded ultradeformable liposomes could be a new nanomedicine for the treatment of squamous cell carcinoma, i.e., actinic keratosis, Bowen’s disease, and keratoacanthoma. ã 2015 Published by Elsevier B.V.
Keywords: Multi-drug colloidal carriers Skin cancer Skin delivery Supramolecular therapeutics Ultradeformable liposomes
13
1. Introduction
14
Lipid-based nanocarriers possess ideal properties which permit them to modulate and improve the biopharmaceutical characteristics of encapsulated drugs. During the last decade, in particular, there has been an increase in investigations concerning the topical delivery of active compounds by innovative vesicular systems (Paolino et al., 2008; González-Paredes et al., 2010; Marianecci et al., 2012). Liposomes were the first nanocarriers used to increase the permeation rate of the encapsulated compounds through the skin, but their use was limited by their localization in the skin stratum corneum (Sinico and Fadda, 2009). For this reason novel
15 16 17 18 19 20 21 22 23
Q2
* Corresponding author at: Department of Health Sciences, University “Magna Græcia” of Catanzaro, Campus Universitario “S. Venuta”, Viale S. Venuta, Germaneto, I-88100 Catanzaro, Italy. Tel.: +39 961 369 4118; fax: +39 961 369 4237. E-mail address: [email protected] (M. Fresta). 1 These two authors equally contributed.
vesicular systems have been investigated, such as ultradeformable liposomes and ethosomes1 which have furnished the best results in terms of increased permeation of drug following their topical administration (Elsayed et al., 2007a; Ainbinder et al., 2010; Celia Q3 et al., 2012). Ultradeformable liposomes, topically administered under nonocclusive conditions, are made up of phospholipids and edge activators, capable of destabilizing the lipid bilayer of the vesicles and increasing the deformability of the bilayer and affecting its interfacial tension (Cevc, 2004; Cevc and Vierl, 2010; El Zaafarany et al., 2010). In fact, their transport across the skin is driven by the osmotic gradient and occlusion could eliminate it, thus compromising the action of the deformable vesicles (Elsayed et al., 2007b). Ultradeformable liposomes were used to increase the skin permeation of different bioactives (i.e., corticosteroids, diclofenac, insulin, etc.) allowing significant drug localization even in the blood stream (Cevc and Blume, 2001; Cevc, 2003; Jain et al., 2005; Benson, 2006).
http://dx.doi.org/10.1016/j.ijpharm.2015.04.056 0378-5173/ ã 2015 Published by Elsevier B.V.
Please cite this article in press as: Cosco, D., et al., Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.04.056
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IJP 14846 1–10 2 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
D. Cosco et al. / International Journal of Pharmaceutics xxx (2015) xxx–xxx
Oxidative stress is a key component in the multistage carcinogenic process. Reactive oxygen species (ROS) are generated in response to both endogenous and exogenous stimuli. However, endogenous antioxidants prevent ROS-mediated injury. In addition, the reduction of oxidative stress coupled with the use of chemotherapeutic drugs produces a large amount of free radicals (Hwang et al., 2001). Many experiments showed that a supplement of antioxidant drug during anticancer therapy may reduce adverse reactions and improve the potential for success in terms of tumor response. It has recently been demonstrated that resveratrol (RSV), a polyphenol largely used as antioxidant, synergistically promotes 5-fluorouracil (5-FU)-mediated apoptosis of cancer cells irrespective of p53 (Gross et al., 2007; Chan et al., 2008; Gatouillat et al., 2010). The aim of this investigation was to co-encapsulate two active compounds having different physicochemical properties within ultradeformable liposomes, in order to obtain a synergistic action against lesions related to squamous cell carcinoma such as actinic keratosis, Bowen’s disease, and keratoacanthoma. This strategy has already been used by many research teams with the aim of increasing the anticancer efficacy of the obtained formulation with respect to the single agents or a physical mixture of them in in vitro and in vivo tumor models (Dicko et al., 2010; Cosco et al., 2011, 2012). 5-FU and RSV were chosen as model drugs because the first is a hydrophilic compound which has a significant antitumor effect on skin cancers, while the second is an antioxidant able to synergistically induce apoptosis in cancer cells. Ultradeformable liposomes, containing sodium cholate as edge activator and different amounts of the aforesaid compounds, were prepared in order to investigate their physicochemical and technological characteristics. The anticancer action of the most suitable ultradeformable liposome formulation was then evaluated in vitro on human skin cancer cells through viability testing, cell cycle- and apoptosis analysis. The interaction rate between the vesicles and the cells was also evaluated as well as the percutaneous permeation of the multi-drug ultradeformable liposomes.
80
2. Materials and methods
81
2.1. Chemicals
82
Phospholipon 90G1 (PL 90G1), was a kind gift by Nattermann Phospholipid Gmbh (Cologne, Germany). Cholesterol (Chol), 5-fluorouracil (5-FU), resveratrol (RSV), sodium cholate (SC), phosphate buffered saline (PBS) tablets, 3-[4,5-dimethylthiazol2-yl]-3,5-diphenyltetrazolium bromide salt (MTT), propidium iodide, RNase A, dimethyl sulfoxide, amphotericin B solution (250 mg/ml) were purchased from Sigma–Aldrich (Milan, Italy). Cellulose membrane Spectra/Por MWCO 12,000–14,000 Da were obtained from Spectrum Laboratories Inc. (Eindhoven, The Netherlands). Lissamine rhodamine B 1,2 dihexadecanoyl-snglycero-3-phosphoethanolamine triethylammonium salt (rhodamine DHPE) and Hoechst 33344 were InvitrogenTM products (Life Technologies, Monza, Italy). Human skin cancer cells (SK-MEL28 and Colo-38) were provided by the Instituto Zooprofilattico of Modena and Reggio Emilia. RPMI 1640 Medium with glutamax, trypsin/EDTA (1) solution, foetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco (Invitrogen Corporation, Paisley, UK). Ethanol was obtained from Carlo Erba SpA (Rodano (MI), Italy). The TdT-mediated dUTP nick-end labeling assay (Tunel assay) was purchased as a kit from Promega (Madison, WI, USA). Double-distilled pyrogen-free water was obtained from Sifra SpA (Verona, Italy).
83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
Table 1 Physicochemical parameters of ultradeformable liposomes prepared both in presence and absence of 5-FU and RSV.a Sample
Drug (% w/w)
Mean sizes (nm)
Polydispersity Index
Zeta potential (mV)
Empty ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs RSV-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs 5-FU-ULs MD-ULs MD-ULs MD-ULs MD-ULs MD-ULs MD-ULs
– 1 2 3 4 5 10 1 2 3 4 5 10 1 2 3 4 5 10
307.4 8.6 618.4 26.4 530.8 6.3 478.3 8.0 383.8 9.4 422.2 13.2 595.8 25.8 345.0 12.5 359.7 15.7 432.1 20.4 449.5 12.7 538.6 25.9 573.6 11.8 316.6 4.8 356.5 13.3 370.5 12.2 458.7 23.1 445.6 19.5 663.8 184.8
0.38 0.02 0.28 0.04 0.45 0.03 0.44 0.03 0.31 0.05 0.17 0.07 0.48 0.06 0.30 0.04 0.32 0.04 0.39 0.08 0.42 0.07 0.45 0.04 0.52 0.03 0.36 0.03 0.39 0.04 0.34 0.07 0.31 0.06 0.32 0.09 0.60 0.21
27.4 0.8 24.2 0.4 23.4 0.7 21.3 0.3 25.3 0.3 18.9 0.7 24.1 0.5 24.7 0.4 27.1 1.0 23.7 0.4 26.4 0.7 29.2 1.3 28.7 1.0 28.2 0.7 32.8 0.7 29.8 1.0 27.3 0.4 25.5 0.5 28.5 0.9
a Particle suspensions were suitably diluted and analysis was carried out at 20 C. Each value was the mean of six different experiments SD.
2.2. Vesicle preparation and purification
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The compositions of the investigated ultradeformable liposomal formulations (ULs) are reported in Table 1. The maximum amount of the active compounds was of 10% (w/w) with respect to the total amount of lipids. The ULs were prepared as previously described (Celia et al., 2009), by dissolving PL 90G1 (88 mg) and SC (12 mg) in 3 ml of ethanol in a Pyrex1 glass vial. The organic solvent was firstly removed by a Rotavapor1 210 (Büchi Italia, Milan, Italy) under a nitrogen flux and then by overnight storage at 30 C in a Büchi T51 glass drying-oven under vacuum. The lipid film was hydrated with 6 ml of a water/ethanol (93:7%, v/v) solution and vortex-mixed at 700 rpm for 15 min at room temperature. The ULs were left at 40 C for 1 h and sonicated with a SONOPULS GM 70 probe sonicator (Bandelin Electronic, Berlin, Germany) at 50 cycle/s for 3 min at 60% of its maximum power. RSV-loaded ULs (RSV-ULs) and rhodamine-labeled ULs were prepared by solubilizing the drug or fluorescent probe (rhodamine-DHPE, 0.1% molar) in the organic phase, respectively, while 5-FU-loaded ULs (5-FUULs) were obtained by adding the hydrophilic drug to the aqueous phase. The multi-drug ultradeformable liposomal formulations (MD-ULs) were obtained following the co-encapsulation of the two drugs, which were respectively dissolved in their suitable environments. In order to remove the free drug from the entrapped one, the drug loaded-ULs were centrifuged at 80,000 g at 4 C for 1 h using a Beckman OptimaTM ultracentrifuge equipped with a TL S55 fixedangle rotor (Bechman Coulter Inc., Fullerton, CA, USA). The drugloaded UL pellets were washed twice with PBS and centrifuged (80,000 g at 4 C for 1 h), while the supernatants were collected and stored at 20 C until analysis. The obtained pellets were then dissolved in PBS buffer (6 ml) and used for in vitro and in vivo experiments.
105 106 107 108 109 110
Q4 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
2.3. Physicochemical characterization
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Mean size and size distribution were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, United Kingdom). The third-order cumulant fitting correlation function was applied. A 4.5 mW laser diode operating at 670 nm was used as a light source for size analysis and the back-scattered photons were detected at 173 . The
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real and imaginary refractive indexes were set at 1.59 and 0.0, respectively. The medium refractive index (1.330), medium viscosity (1.0 mPa s), and dielectric constant (80.4) were set before the experiments. The Z-potential of the colloidal suspensions was also measured by a Zetasizer Nano ZS. A Smoluchowsky constant F (Ka) of 1.5 was used to calculate this parameter as a function of the electrophoretic mobility of ultradeformable lipid vesicles. The various ULs were placed into cylindrical glass tubes and submitted to a Turbiscan Lab1 Expert apparatus to evaluate their stability by analyzing the variation of their backscattering (DBS) profiles as a function of time (Celia et al., 2009) (see Supplementary materials for further details).
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2.4. Evaluation of drug entrapment efficiency
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The amount of 5-FU and/or RSV entrapped in the ULs was evaluated by HPLC after vesicle disruption by methanol (Cosco et al., 2012). HPLC analysis showed that no interference was determined by the various components of the vesicles (see Supplementary materials for further details). The entrapment efficiency was quantified as the percentage of the added drug that became associated within the ULs, according to the following equation:
144 145 146 147 148 Q5 149 150 151 152 153 154
158 159 160 161 162 Q6 163 164
EE ¼ 166 165 167
DE 100 DT
(1)
169
where DE is the amount of drug associated within the ULs and DT is the total amount of drug added during the preparation procedure. The recovery of 5-FU and RSV both as entrapped ULs and unentrapped drug was always greater than 96%.
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2.5. Drug release from ULs
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Drug release was evaluated according to the dialysis method (Cosco et al., 2012) by using cellulose acetate dialysis tubing with molecular cut-off 12,000–14,000 Da sealed at both ends with clips. A water/ethanol mixture (70:30 v/v) was used as the release medium, which was constantly stirred and warmed (GR 150 thermostat, Grant Instruments Ltd., Cambridge, UK) to 37 0.1 C throughout the release experiments. Before dialysis, the tubing was kept overnight in the release medium to allow thorough soaking. The drug-loaded ULs (1 ml) were placed into dialysis bags, which were then transferred into beakers containing 200 ml of the release medium, thus observing sink conditions for 48 h experiments. At predetermined time intervals, 1 ml of the release medium was withdrawn and replaced with fresh. Samples were analyzed by HPLC. No interference peak was observed. The percentage of released drug was calculated according to the following equation:
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172 173 174 175 176 177 178 179 180 181 182 183 184 185 Q7 186
drugrel Drugrelease ð%Þ ¼ 100 drugload
(2)
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where drugrel is the amount of drug released at the time t and drugload is the amount of drug entrapped within the ULs.
188
2.6. Percutaneous permeation experiments and dermal delivery of ULs
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The percutaneous permeation profiles of the various ULs were evaluated using human stratum corneum and viable epidermis (SCE) membranes (Kligman and Christophers, 1963) (see Supplementary materials for further details). Dynamic Franz diffusion cells were used for percutaneous permeation experiments. A mixture of water/ethanol (70:30 v/v) was used as receptor. Experiments were carried out under non-occlusive conditions, and the SCE membranes were mounted between the donor and the
190 191 192 193 194 195 196
3
Fig. 1. Kinetic stability profiles of empty and drug-loaded ULs using Turbiscan Lab1: panel a, 5-FU; panel b, RSV; panel c, MD. The result was a representative experiment of three independent experiments.
Please cite this article in press as: Cosco, D., et al., Ultradeformable liposomes as multidrug carrier of resveratrol and 5-fluorouracil for their topical delivery. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.04.056
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receptor compartments. Sink conditions were maintained throughout the percutaneous permeation experiments. The amount of 5-FU and RSV that permeated through the SCE membranes was determined by HPLC. In order to evaluate the ability of the ULs to improve the delivery of the drugs to the dermis, human full-thickness skin (FTS) was also used for the experiments. FTS membranes were used after explantation and pre-equilibrated in physiological solution at 25 C for 2 h before the experiments and mounted between the donor and the receptor compartments with the SC side up. The amounts of 5-FU and RSV retained in the dermis were determined after 24 h at the end of the permeation experiment. In this case, the SC side of the FTS membranes was washed ten times using a cotton cloth immersed in methanol to remove any trace of the formulations. The SC was removed by stripping ten times with Tesa1 AG adhesive tape (Hamburg, Germany) (Saija et al., 2000). The epidermis was separated from the dermis using a sterile surgical scalpel, and the dermis was then cut with scissors, placed in a glass homogenizer containing 1 ml of methanol, homogenized for 5 min, and sonicated using a probe type sonicator (Sonopolus GH70, Bandelin-Electonic, Berlin, Germany) at 50 cycle/s. The resulting tissue suspension was centrifuged for 10 min at 10,000 g, and the supernatant was analyzed by HPLC to determine the amounts of 5FU and RSV present. The recovery of the 5-FU and RSV was, respectively, 97% and 93% of the amount applied to the skin.
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2.7. Cell cultures and cytotoxic activity
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SK-MEL-28 and Colo-38 cells were grown in RPMI1640 medium with glutamate, penicillin (100 UI/ml), streptomycin (100 mg/ml) and FBS (10% v/v) at 37 C (5% CO2). The medium was replaced with fresh every 48 h. When 80% confluence was reached, the cells were seeded in culture dishes before in vitro investigations. The cytotoxic effects of empty vesicles, 5-FU and RSV (as free or encapsulated form and as single agent or in association) were evaluated by MTT-test (cell viability) as previously described (Paolino et al., 2008). The interaction between melanoma cells and fluorescent ULs was evaluated through confocal laser scanning microscopy (CLSM) (see Supplementary materials for further details).
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2.8. Cell cycle analysis and in vitro evaluation of cell apoptosis
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Melanoma cells at 80% confluence were treated with the empty and drug-loaded ULs at two different drug concentrations (5 and 10 mM) for 24 h (see Supplementary materials for further details). To ensure adequate DNA measurements, assessed by the coefficient of variation of the peaks (CVs), the interval of CVs was maintained at a value of less than 10% in accordance with the DNA Cytometry Consensus Conference (Haroske et al., 2001). The evaluation of the apoptotic induction was carried out following the treatment of melanoma cells for 24 h with different ultradeformable liposomal formulations at a final drug concentration of 5 mM. The apoptotic activity of the various formulations was evaluated using three different and independent methods: the COMET assay, the spectrofluorometric Tunel assay and the Caspase-3 assay. The COMET and caspase-3 assays were evaluated as previously reported (Palumbo et al., 2002), while the Tunel assay was performed according to manufacture instruction (see Supplementary materials for further details).
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2.9. Statistical analysis
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One-way ANOVA was used for statistical analysis of the various experiments. A posteriori Bonferroni t-test was carried out to check the ANOVA test. A p value
