Accepted Manuscript Title: Formulation optimization and in vitro skin penetration of spironolactone loaded solid lipid nanoparticles Author: H.R. Kelidari M. Saeedi J. Akbari K. Morteza-Semnani P. Gill H. Valizadeh A. Nokhodchi PII: DOI: Reference:
S0927-7765(15)00130-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.02.046 COLSUB 6937
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
15-12-2014 3-2-2015 25-2-2015
Please cite this article as: H.R. Kelidari, M. Saeedi, J. Akbari, K. Morteza-Semnani, P. Gill, H. Valizadeh, A. Nokhodchi, Formulation optimization and in vitro skin penetration of spironolactone loaded solid lipid nanoparticles, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.02.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Formulation optimization and in vitro skin penetration of spironolactone loaded solid
ip t
lipid nanoparticles
H.R. Kelidari1, M. Saeedi2, J. Akbari2, K. Morteza-Semnani2, P. Gill3, H. Valizadeh4, A.
Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences,
us
1
cr
Nokhodchi5,*
3
an
Sari, Iran; 2Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran; Department of Pharmacology, Faculty of Medicine, Mazandaran University Of Medical Sciences, Sari, Iran; 4Drug applied research center and Faculty of Pharmacy, Tabriz
M
University of Medical Sciences, Tabriz, Iran; 5School of Life Sciences, University of Sussex,
Ac ce p
te
d
Falmer, Brighton, BN1 9QG, United Kingdom
Corresponding author: (Ali Nokhodchi, [email protected]) Tel: +44 1273872811
Highlights
SLN was prepared by emulsion-solvent evaporation method and ultrasonication DSC study showed that SP encapsulated in SLNs was in the amorphous form Spironolactone-SLNs showed faster release than drug alone Amount of SP penetrated via rat skin from SLNs was 1.6 fold higher than drug alone
1 Page 1 of 26
ip t
Abstract The aim of the current investigation was to prepare and evaluate the potential use of solid
cr
lipid nanoparticles for the dermal delivery of spironolactone (SP). The spironolactone loaded SLN (SP-SLN) was prepared by emulsion-solvent evaporation method followed by
us
ultrasonication. The properties of obtained SLNs were characterized by photon correlation
an
spectroscopy (PCS), scanning tunneling microscopy (STM) and differential scanning calorimetry. FT-IR was also used to investigate any interaction between SP and excipients in
M
the molecular level during the preparation of SLNs. The performance of the formulations was investigated in terms of drug release, skin permeation and also the retention of drug by the
d
skin. The SP-SLNs presented spherical shape with the mean diameter, zeta potential and
te
entrapment efficiency of 88.9 nm, -23.9 mV and 59.86%, respectively. DSC study showed that SP alone encapsulated in SLNs was in the amorphous form. FT-IR analysis revealed that
Ac ce p
there were hydrogen bond interactions between the SP alone and SLN components. The dissolution results revealed that the drug release from SP-SLNs was at least 4.9 times faster than original SP within the first 30 min. The cumulative amount of SP penetrated through rat skin from SP-SLNs was almost 2-fold that of the SP alone in 24 h after the administration. In vitro permeation studies indicated that SP-SLN may be a promising vector for use in the topical treatment. It can be concluded that SLNs provide good skin permeation for SP and may be a promising carrier for topical delivery of spironolactone offering the biphasic release pattern that might be interesting for topical application resulting in an effective treatment for skin disorders such as acne.
2 Page 2 of 26
Keywords: Spironolactone, Solid lipid nanoparticles (SLNs), Acne, Dermal delivery
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Abbreviations: DCM: dichloromethane
cr
DSC: Differential scanning calorimetry EE: entrapment efficiency
us
FT-IR: Fourier transforms infrared
an
PBS: Phosphate buffer saline PI: polydispersity index
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SA: stearic acid SLNs: solid lipid nanoparticles
d
SP: spironolactone
Ac ce p
ZP: zeta potential
te
STM: Scanning Tunneling Microscopy
3 Page 3 of 26
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1. Introduction Spironolactone (SP) has been used for more than 20 years as tablets to treat coetaneous
cr
disorders. However, observed endocrine side effects restricted its clinical application due to its variable oral bioavailability. One promising route of SP administration could be topical
us
delivery which can lessen the systemic side effects and also improve the patient compliance.
an
Topical therapy of skin diseases allow high drug levels at the site of action and reduce
M
systemic side effects [1, 2].
Up until now, various approaches have been performed to increase SP dissolution rate, such
d
as complexation with cyclodextrin, lyophilization, solid dispersion, SP nanoparticles [3, 4],
te
SP loaded nanocapsules [5], liposomes [6] and solid lipid nanoparticles (SLNs) [7]. Solid lipid nanoparticles contain non-irritative and non-toxic lipids and it seems to be well-suited
Ac ce p
formulations for use on inflamed and damaged skin [8-9]. The small particle size of SLN ensures close contact with the stratum corneum and due to the formation of an intact film on the skin surface, increases the amount of encapsulated compounds penetrating into the skin [9]. SLNs have been used to improve the skin/dermal uptake of several drugs such as fluconazole [2], clotrimazole [9], isotretinoin [10] tretinoin [11] and penciclovir [12], which supports that SLNs might be employed as the carrier for the topical delivery of SP. So, in the present research, attempts were made to prepare the SP loaded SLN using solvent emulsification–evaporation techniques followed by ultrasonication. The properties of SLNs were also optimized by the role of surfactants type and surfactant to drug-lipid concentration.
4 Page 4 of 26
The skin permeation and deposition of SP-SLNs through rat skin were examined to explore
ip t
the potential use of SLN of SP in topical formulations.
2. Materials and methods
cr
2.1. Materials
Spironolactone (SP) was purchased from Behdashtkar Co. (Pasdaran, Tehran). Tween 80,
us
Span 80, Span 60 and stearic acid (SA) were purchased from Merck Co. (Germany).
an
Solvents, such as dichloromethane (DCM) and HPLC grade acetonitrile were also obtained from Merck Co (Germany). Distilled water was purified using a Milli-Q system (Millipore,
M
Direct-Q). Highly Ordered Pyrolytic Graphite (HOPG) was purchased from the
te
2.2. Preparation of SP-SLN
d
Nanotechnology Systems Corporation (NATSYCO Co, Tehran, Iran).
The SP-loaded SLNs (SP-SLNs) were prepared according to a modified emulsion/solvent
Ac ce p
evaporation method followed by ultrasonication [4]. In the first step, to evaluate the best formulation based on proportion of lipophilic and hydrophilic surfactants, 0.2g SP, 0.8 g stearic acid (ratio of drug:lipid 1:4), span 60 or span 80 (refer to Table 1 for amounts) were dissolved in 20 mL dichloromethane (the ratio of SA-SP/DCM was 1/20 w/w) (organic phase) at 85◦C. The surfactant solutions containing 0.25 or 0.5 g tween 80 were homogenized
by
high-shear
homogenizer
(D-91126
Schwabach,
Heidolph,
Germany).Then the organic phase was added dropwise to 1/3 of the preheated aqueous surfactant solution (85 ◦C) containing tween 80 for 5 min using sonication (Bandelin sonopuls, Berlin, Germany) to form a coarse pre-emulsion. At the end of sonication, the mixture was dispersed into the remaining 2/3 of the aqueous surfactant solution
5 Page 5 of 26
maintained in an ice bath and homogenized by high-shear homogenizer (D-91126 Schwabach, Heidolph, Germany) at 13000 rpm for 7 min. Thereafter, the organic solvent (DCM) was evaporated by mechanical stirring (600 rpm) for 24 h at room
ip t
temperature (formulations 1, 2, 3, 4) to obtain SLNs. After selection of the best formulation, the amount of surfactant concentration was kept constant while the
cr
concentration of drug and SA was changed (formulations 5 to 14, Table 1). 2.3 Physicochemical characterization
us
SLNs were characterized in terms of mean particle size, polydispersity index (PI) and zeta
an
potential (ZP) using a Zeta Sizer Nano ZS (Malvern Instruments, UK) at 25 ◦C. The results
M
are the means and standard deviations of three determinations which are reported in Table 1.
2.4 Entrapment efficiency
d
To determine the entrapment efficiency (EE%) of SP in the SLNs, the SP-SLNs was
te
subjected to centrifugation for 20 min at 25,000 rpm (HERMLE, Z36HK, Germany), filtered (pore size: 0.22 µm) and the amount of SP in supernatant (free drug) was determined by
Ac ce p
HPLC Agilent 1100 at 238 nm, which was equipped with the Agilent Eclipse XDB-C18 column (5 µm, 4.6×250 mm). The mobile phase, composed of 70% acetonitrile and 30% millipore water (v/v), was delivered at 0.8 ml/min and the retention time of the drug was 7 min. Drug entrapment efficiency (EE%) was calculated by equation 1:
EE%
Winitialdrug Wfreedrug Winitialdrug
100
(Equation 1)
Where Winitial drug is amount of drug added in the formulation and Wfree drug is the amount of drug in supernatant [3].
6 Page 6 of 26
2.5 Scanning Tunneling Microscopy (STM) analysis The specimens were scanned with NAMA-STM SS-1 (Nanotech System Corporation, Natsyco, Iran). Highly Ordered Pyrolytic Graphite (HOPG) was also prepared from the
ip t
Nanotech System Corporation. STM tip was prepared mechanically using platen iridium with 0.35 mm diameter (SPI, USA). All experiments were performed in air at around 25 ◦C with
cr
at a relative humidity of 70%. The constant current mode was used to take images. The images were obtained with a proportional integral differential (PID) frequency of 100 Hz
us
and a current set point of around 0.1 nA. The sample bias voltage for the images was 0.1 V,
an
which during the scans was increased to 1.6 V gradually. During the scan, the current set point was decreased from 0.1 nA to 50 pA. Rough data were processed by using median
M
(middle range) and low pass Gaussian (high range) filters [13].
d
2.6 Fourier transforms infrared (FT-IR) analysis
te
The optimized formulations were freeze-dried and converted into solid form. A Perkin Elmer FT-IR spectrophotometer (Perkin FTIR-One, USA) was used to identify any changes in the
Ac ce p
molecular levels of SP, SA, Physical mixture and SP-SLNs from 400 to 4000 cm−1.The sample was grounded with KBr and compressed into a suitable-size disk (13mm) for measurement [3].
2.7 Differential scanning calorimetry (DSC) Thermal behaviors of samples were studied by differential scanning calorimetry (Pyris 6, PerkinElmer, USA). Prior to heating, approximately 7 mg samples were equilibrated in the DSC pan (hermetic crimped aluminum pans) at 20 ˚C for 30 min and then heated to 250 ˚C at a scanning rate of 10˚C/min under N2 atmosphere.
7 Page 7 of 26
2.8. X-ray powder diffraction (XRD) X-ray diffractometery of the samples (stearic acid, spironolactoon and the optimized SLNs) were performed using a Bruker D8 Advance X-ray diffractometer (Germany)
ip t
(40 kV and 30 mA), a step size of 0.02° and a time per step of 1 s. The cross-section of a sample was exposed to X-ray radiation (Cu Kα) with a wavelength of 1.5406Å. Samples
cr
were placed into a stainless steel holder and the surface of powder was levelled manually for analysis. The sample was scanned between 2 and 40 of 2θ with a step size
an
us
of 0.02° and a step time of 1s.
2.9 Dissolution testing
M
The dissolution behavior of SP from lipid nanoparticles was examined in accordance with the paddle method (type II) mentioned in United States Pharmacopeia (USP XXVIII, 2005).
d
The Erweka dissolution apparatus (DT620, Erweka, Germany) was used to carry out the
te
dissolution tests. The dissolution medium was 900 ml of simulated intestinal fluid pH 6.8 maintained at 37.0 ◦C and paddle rotation speed was 60 rpm. At different time intervals (0.5,
Ac ce p
1, 2, 4, 6, 8 and 24 h) the samples (1.5 ml) were withdrawn, filtered (pore size: 0.22 μm) and concentration of SP was determined using the HPLC method at 238 nm described earlier in the manuscript. The dissolution data were compared to the dissolution of plain (original) SP (pure drug with no additive).
2.10 In vitro skin permeation study The abdominal hair of Wistar male rats, weighing 200-250 g, was shaved using electric and hand razors. After anesthetizing the rats with ether, the abdominal skin was surgically excised. To remove the adhering subcutaneous debris and leachable enzymes, the dermal side of the skin was put in contact with a saline solution for 1 h before starting the diffusion
8 Page 8 of 26
experiment. A system employing Franz diffusion cells was used for permeation studies. The excised rat skin was set in place with the stratum corneum facing the donor compartment and the dermis facing the receptor [14]. The receiver compartment was filled with 5.5 ml of
ip t
phosphate buffer saline (PBS) (pH 7.4). The diffusion cells were maintained at (37±0.5) oC with stirring at 300 rpm throughout the experiment. Lyophilized SP-SLNs equivalent to 10
cr
mg of SP and similar amount of SP alone (plain SP which is pure drug without any additives and was applied in the form of dispersion) as control were dispersed by
an
us
vortexing for 15 min in 5 ml distilled water and applied on the skin surface.
Samples from receiver medium were withdrawn at predetermined time intervals (1, 2, 3, 4, 6,
M
8 and 24 h) and an equivalent volume of fresh PBS maintained at 37 ºC was replaced. All samples were filtered through an aqueous 0.22 µm filter and analyzed by HPLC method as
d
described before. At the end of the permeation study, excess formulation from the skin was
te
removed, washed 3 times with PBS, and then dried with filter paper. To calculate the amount of SP deposited within the skin, the skins were removed and washed three times with
Ac ce p
phosphate buffer solution (PBS, pH 7.4) and residual washing solvent was carefully wiped off from the skin. The skins were minced, transferred to a test tube and digested for 24h in 1 ml of PBS (pH 7.4) and 1 ml of HCL 0.1M. All the samples were then centrifuged at 25000 rpm for 20 min. The supernatant filtered through 0.22 μm membrane and quantified by HPLC at 238 nm for SP content.
2.11 Statistical analysis All the results are expressed as the mean ± standard deviation of at least three determinations (n=3). The treated groups were compared to control by analysis of variance (ANOVA),
9 Page 9 of 26
following Dunnet’s test. The statistical analysis was carried out using software, SPSS. The Pvalue < 0.05 was considered as significant.
ip t
3. Results and discussion 3.1 Characterization of SLNs
cr
The results showed that all formulations were able to form SLN (Table 1). It was found that, generally, with increasing the concentration of surfactant to drug-lipid concentration (Table
us
1: surfactant concentration was constant while drug-lipid concentration decreased), the
an
particle size of SLNs decreased and smaller particle size showed a narrower size distribution compared to larger SLNs (P0.05). Span 60 is solid at room temperature and showed
ip t
the higher phase transition temperature. The Span 60 having the highest transition
cr
temperature provides the highest entrapment [20].
us
3.2 STM analysis
an
Figure 1 shows particles from SP-SLN14 formulation (selected as optimal formulation because of the smallest particle size, 88.9 nm, and relatively high entrapment efficiency of
M
59.86%) before and after graphical process. These micrographs indicated spherical shape. STM micrographs, in the present research, demonstrate the uniform distributions which are in
d
agreement with the micrographs obtained from the scanning electron microscopy [2]), atomic
Ac ce p
[21] of SLN-images.
te
force microscopy [18], fluorescence microscopy [19] and transmission electron microscopy
3.3 FT-IR
Figure 2 illustrates the infrared spectra of SP alone , SA, SP-SLNs and physical mixture (with the same composition). In the case of SP alone, a sharp absorption band appearing around 2950 cm-1 corresponds to C-H stretching, absorption bands around 1768 cm-1 could be attributed to the stretching band of lactone C=O, 1691 cm-1 to C=O of thioacetyl group (stretching), 1673 cm-1 to C=O of C6-ring (stretching), and 1617 cm-1 to C=C stretching. The FT-IR spectrum of solid lipid, revealed the presence of absorption band at 2849 and 2917 cm-1 due to symmetric and asymmetric C-H stretching, wavenumber 1702 cm-1 depicts C=O stretching, and 2400-3400 cm-1 depicts O-H stretching. In the spectra of the physical blend
11 Page 11 of 26
of drug and excipients, the wavenumbers related to carbonyl of lactone, carbonyl of thioacetyl, C=O of ring, and double bond of ring have no changes, but in SP-SLN spectra, the carbonyl groups were shifted from 1673-1768 cm-1 to 1700-1735 cm-1. This confirms
ip t
interaction between SP and nanoparticle vehicle via intermolecular hydrogen bonding
us
3.4 Differential scanning calorimetry (DSC) and XRD
cr
between drug and SLN components [22].
an
The SP alone, SA, the physical mixture and SP-SLNs powder were examined by DSC and their DSC traces are shown in Figure 3. SP alone showed a single sharp endothermic melting
M
peak at about 212 ºC. The thermogram of solid lipid (stearic acid) showed a single sharp endothermic melting peak around 70 ºC. DSC traces of SLN and physical mixture contained
d
the endothermic peak around stearic acid melting point, but the endothermic peak of
te
crystalline form of drug disappeared. The possible explanation is that the lipid (SA) may inhibit the crystallization of SP during the nanoparticle formation [23]. The most probable
Ac ce p
reason for the disappearance of SP peak in the physical mixtures could be due to the dissolution of SP in the molten SA. Similar event has previously been reported for antiviral drug (UC-781) in the presence of PEG 6000 and gelucire [24] where the endothermic peak of the drug disappeared during DSC run. This, in fact, is one of the limitations of DSC analysis as DSC method is an invasive method, and heating the sample during analysis might dissolve the drug crystals in the polymer which has already been into the molten stage before the drug melts. In the case of SP-SLN, it has been reported that when the drug does not show its endothermic peak in the nanoparticulate formulations, it is said to be in the amorphous state [25]. To provide more evidence for the above finding XRD was conducted for spironolactone, SA and SP-SLN 14 and their XRD spectra were shown in Figure 4. The
12 Page 12 of 26
XRD of SP shows diagnostic peaks at 2Ɵ 9.2, 16.6, 17.4 and 20.3 [26] which were shown on the figure. XRD spectrum of SP-SLN 14 shows that the main peaks of spironolactone disappeared or their intensity significantly reduced (highlighted on the Figure) which
ip t
could be an indication of amorphous state or a very fine crystal structure for SP. The relative reduction of diffraction intensity of spironolactone in SLNs at these angles
cr
suggests that spironolactone might present in partially amorphous or microcrystalline
us
form in the SLNs.
an
3.5 Dissolution testing
The dissolution profiles of the selected SP-SLNs (SP-SLN 14) and SP alone are shown in
M
Figure 4. The drug release behavior from the SLN displayed a faster drug release at the initial stage (the first 30 min) followed by sustained release pattern. Faster dissolution of SP from
d
SLNs compared to SP alone could be due to differences in their surface area available for
te
dissolution as the rate of drug dissolution is directly proportional to the surface area of the solute particle. In the SP alone, the drug has crystalline structure (DSC traces and XRD
Ac ce p
showed this) with relatively larger particle size (mean diameter of 131 µm obtained via sieving technique) whereas SLNs have very smaller particles and the drug is partially in amorphous state or very fine crystals which both of these parameters (size and the state of solid particles) may contribute to increase the dissolution rate of SP. Different release profiles from SLNs have been reported [20]. The results obtained in the present study are in close agreement with the data reported by Bourezg et al. [7]. They reported that SP loaded lipid nanoparticles exhibited higher burst release in comparison to the commercially available tablets. They attributed this phenomenon to the surface enrichment of the drug as well as to the large surface area of nanoparticles [7].
13 Page 13 of 26
After the initial fast release within 30-60 min, prolonged-release pattern was observed between 1 to 24 h. The slow release of SP could be due to the slow penetration of the dissolution medium into the core of the nanoparticles (due to hydrophobic lipid nature of the
ip t
core) followed by slow diffusion of SP out of the core. In addition, a further reduction in drug release could occur at later stages of the dissolution due to depletion of SP from the core,
cr
hence a reduced concentration gradient.
The observed biphasic release pattern could be interesting for topical application of
us
spironolactone nanoparticles to treat skin diseases as the initial release improves the
an
penetration of spironolocatone, while the second release phase (steady release) provides the drug for longer time.
M
3.6 In vitro percutaneous absorption study
Skin permeation studies were presented in Figure 5. There was a significant difference
d
between SP-SLN 14 and SP alone in terms of drug penetration (P
S0927-7765(15)00130-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.02.046 COLSUB 6937
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
15-12-2014 3-2-2015 25-2-2015
Please cite this article as: H.R. Kelidari, M. Saeedi, J. Akbari, K. Morteza-Semnani, P. Gill, H. Valizadeh, A. Nokhodchi, Formulation optimization and in vitro skin penetration of spironolactone loaded solid lipid nanoparticles, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.02.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Formulation optimization and in vitro skin penetration of spironolactone loaded solid
ip t
lipid nanoparticles
H.R. Kelidari1, M. Saeedi2, J. Akbari2, K. Morteza-Semnani2, P. Gill3, H. Valizadeh4, A.
Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences,
us
1
cr
Nokhodchi5,*
3
an
Sari, Iran; 2Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran; Department of Pharmacology, Faculty of Medicine, Mazandaran University Of Medical Sciences, Sari, Iran; 4Drug applied research center and Faculty of Pharmacy, Tabriz
M
University of Medical Sciences, Tabriz, Iran; 5School of Life Sciences, University of Sussex,
Ac ce p
te
d
Falmer, Brighton, BN1 9QG, United Kingdom
Corresponding author: (Ali Nokhodchi, [email protected]) Tel: +44 1273872811
Highlights
SLN was prepared by emulsion-solvent evaporation method and ultrasonication DSC study showed that SP encapsulated in SLNs was in the amorphous form Spironolactone-SLNs showed faster release than drug alone Amount of SP penetrated via rat skin from SLNs was 1.6 fold higher than drug alone
1 Page 1 of 26
ip t
Abstract The aim of the current investigation was to prepare and evaluate the potential use of solid
cr
lipid nanoparticles for the dermal delivery of spironolactone (SP). The spironolactone loaded SLN (SP-SLN) was prepared by emulsion-solvent evaporation method followed by
us
ultrasonication. The properties of obtained SLNs were characterized by photon correlation
an
spectroscopy (PCS), scanning tunneling microscopy (STM) and differential scanning calorimetry. FT-IR was also used to investigate any interaction between SP and excipients in
M
the molecular level during the preparation of SLNs. The performance of the formulations was investigated in terms of drug release, skin permeation and also the retention of drug by the
d
skin. The SP-SLNs presented spherical shape with the mean diameter, zeta potential and
te
entrapment efficiency of 88.9 nm, -23.9 mV and 59.86%, respectively. DSC study showed that SP alone encapsulated in SLNs was in the amorphous form. FT-IR analysis revealed that
Ac ce p
there were hydrogen bond interactions between the SP alone and SLN components. The dissolution results revealed that the drug release from SP-SLNs was at least 4.9 times faster than original SP within the first 30 min. The cumulative amount of SP penetrated through rat skin from SP-SLNs was almost 2-fold that of the SP alone in 24 h after the administration. In vitro permeation studies indicated that SP-SLN may be a promising vector for use in the topical treatment. It can be concluded that SLNs provide good skin permeation for SP and may be a promising carrier for topical delivery of spironolactone offering the biphasic release pattern that might be interesting for topical application resulting in an effective treatment for skin disorders such as acne.
2 Page 2 of 26
Keywords: Spironolactone, Solid lipid nanoparticles (SLNs), Acne, Dermal delivery
ip t
Abbreviations: DCM: dichloromethane
cr
DSC: Differential scanning calorimetry EE: entrapment efficiency
us
FT-IR: Fourier transforms infrared
an
PBS: Phosphate buffer saline PI: polydispersity index
M
SA: stearic acid SLNs: solid lipid nanoparticles
d
SP: spironolactone
Ac ce p
ZP: zeta potential
te
STM: Scanning Tunneling Microscopy
3 Page 3 of 26
ip t
1. Introduction Spironolactone (SP) has been used for more than 20 years as tablets to treat coetaneous
cr
disorders. However, observed endocrine side effects restricted its clinical application due to its variable oral bioavailability. One promising route of SP administration could be topical
us
delivery which can lessen the systemic side effects and also improve the patient compliance.
an
Topical therapy of skin diseases allow high drug levels at the site of action and reduce
M
systemic side effects [1, 2].
Up until now, various approaches have been performed to increase SP dissolution rate, such
d
as complexation with cyclodextrin, lyophilization, solid dispersion, SP nanoparticles [3, 4],
te
SP loaded nanocapsules [5], liposomes [6] and solid lipid nanoparticles (SLNs) [7]. Solid lipid nanoparticles contain non-irritative and non-toxic lipids and it seems to be well-suited
Ac ce p
formulations for use on inflamed and damaged skin [8-9]. The small particle size of SLN ensures close contact with the stratum corneum and due to the formation of an intact film on the skin surface, increases the amount of encapsulated compounds penetrating into the skin [9]. SLNs have been used to improve the skin/dermal uptake of several drugs such as fluconazole [2], clotrimazole [9], isotretinoin [10] tretinoin [11] and penciclovir [12], which supports that SLNs might be employed as the carrier for the topical delivery of SP. So, in the present research, attempts were made to prepare the SP loaded SLN using solvent emulsification–evaporation techniques followed by ultrasonication. The properties of SLNs were also optimized by the role of surfactants type and surfactant to drug-lipid concentration.
4 Page 4 of 26
The skin permeation and deposition of SP-SLNs through rat skin were examined to explore
ip t
the potential use of SLN of SP in topical formulations.
2. Materials and methods
cr
2.1. Materials
Spironolactone (SP) was purchased from Behdashtkar Co. (Pasdaran, Tehran). Tween 80,
us
Span 80, Span 60 and stearic acid (SA) were purchased from Merck Co. (Germany).
an
Solvents, such as dichloromethane (DCM) and HPLC grade acetonitrile were also obtained from Merck Co (Germany). Distilled water was purified using a Milli-Q system (Millipore,
M
Direct-Q). Highly Ordered Pyrolytic Graphite (HOPG) was purchased from the
te
2.2. Preparation of SP-SLN
d
Nanotechnology Systems Corporation (NATSYCO Co, Tehran, Iran).
The SP-loaded SLNs (SP-SLNs) were prepared according to a modified emulsion/solvent
Ac ce p
evaporation method followed by ultrasonication [4]. In the first step, to evaluate the best formulation based on proportion of lipophilic and hydrophilic surfactants, 0.2g SP, 0.8 g stearic acid (ratio of drug:lipid 1:4), span 60 or span 80 (refer to Table 1 for amounts) were dissolved in 20 mL dichloromethane (the ratio of SA-SP/DCM was 1/20 w/w) (organic phase) at 85◦C. The surfactant solutions containing 0.25 or 0.5 g tween 80 were homogenized
by
high-shear
homogenizer
(D-91126
Schwabach,
Heidolph,
Germany).Then the organic phase was added dropwise to 1/3 of the preheated aqueous surfactant solution (85 ◦C) containing tween 80 for 5 min using sonication (Bandelin sonopuls, Berlin, Germany) to form a coarse pre-emulsion. At the end of sonication, the mixture was dispersed into the remaining 2/3 of the aqueous surfactant solution
5 Page 5 of 26
maintained in an ice bath and homogenized by high-shear homogenizer (D-91126 Schwabach, Heidolph, Germany) at 13000 rpm for 7 min. Thereafter, the organic solvent (DCM) was evaporated by mechanical stirring (600 rpm) for 24 h at room
ip t
temperature (formulations 1, 2, 3, 4) to obtain SLNs. After selection of the best formulation, the amount of surfactant concentration was kept constant while the
cr
concentration of drug and SA was changed (formulations 5 to 14, Table 1). 2.3 Physicochemical characterization
us
SLNs were characterized in terms of mean particle size, polydispersity index (PI) and zeta
an
potential (ZP) using a Zeta Sizer Nano ZS (Malvern Instruments, UK) at 25 ◦C. The results
M
are the means and standard deviations of three determinations which are reported in Table 1.
2.4 Entrapment efficiency
d
To determine the entrapment efficiency (EE%) of SP in the SLNs, the SP-SLNs was
te
subjected to centrifugation for 20 min at 25,000 rpm (HERMLE, Z36HK, Germany), filtered (pore size: 0.22 µm) and the amount of SP in supernatant (free drug) was determined by
Ac ce p
HPLC Agilent 1100 at 238 nm, which was equipped with the Agilent Eclipse XDB-C18 column (5 µm, 4.6×250 mm). The mobile phase, composed of 70% acetonitrile and 30% millipore water (v/v), was delivered at 0.8 ml/min and the retention time of the drug was 7 min. Drug entrapment efficiency (EE%) was calculated by equation 1:
EE%
Winitialdrug Wfreedrug Winitialdrug
100
(Equation 1)
Where Winitial drug is amount of drug added in the formulation and Wfree drug is the amount of drug in supernatant [3].
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2.5 Scanning Tunneling Microscopy (STM) analysis The specimens were scanned with NAMA-STM SS-1 (Nanotech System Corporation, Natsyco, Iran). Highly Ordered Pyrolytic Graphite (HOPG) was also prepared from the
ip t
Nanotech System Corporation. STM tip was prepared mechanically using platen iridium with 0.35 mm diameter (SPI, USA). All experiments were performed in air at around 25 ◦C with
cr
at a relative humidity of 70%. The constant current mode was used to take images. The images were obtained with a proportional integral differential (PID) frequency of 100 Hz
us
and a current set point of around 0.1 nA. The sample bias voltage for the images was 0.1 V,
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which during the scans was increased to 1.6 V gradually. During the scan, the current set point was decreased from 0.1 nA to 50 pA. Rough data were processed by using median
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(middle range) and low pass Gaussian (high range) filters [13].
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2.6 Fourier transforms infrared (FT-IR) analysis
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The optimized formulations were freeze-dried and converted into solid form. A Perkin Elmer FT-IR spectrophotometer (Perkin FTIR-One, USA) was used to identify any changes in the
Ac ce p
molecular levels of SP, SA, Physical mixture and SP-SLNs from 400 to 4000 cm−1.The sample was grounded with KBr and compressed into a suitable-size disk (13mm) for measurement [3].
2.7 Differential scanning calorimetry (DSC) Thermal behaviors of samples were studied by differential scanning calorimetry (Pyris 6, PerkinElmer, USA). Prior to heating, approximately 7 mg samples were equilibrated in the DSC pan (hermetic crimped aluminum pans) at 20 ˚C for 30 min and then heated to 250 ˚C at a scanning rate of 10˚C/min under N2 atmosphere.
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2.8. X-ray powder diffraction (XRD) X-ray diffractometery of the samples (stearic acid, spironolactoon and the optimized SLNs) were performed using a Bruker D8 Advance X-ray diffractometer (Germany)
ip t
(40 kV and 30 mA), a step size of 0.02° and a time per step of 1 s. The cross-section of a sample was exposed to X-ray radiation (Cu Kα) with a wavelength of 1.5406Å. Samples
cr
were placed into a stainless steel holder and the surface of powder was levelled manually for analysis. The sample was scanned between 2 and 40 of 2θ with a step size
an
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of 0.02° and a step time of 1s.
2.9 Dissolution testing
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The dissolution behavior of SP from lipid nanoparticles was examined in accordance with the paddle method (type II) mentioned in United States Pharmacopeia (USP XXVIII, 2005).
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The Erweka dissolution apparatus (DT620, Erweka, Germany) was used to carry out the
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dissolution tests. The dissolution medium was 900 ml of simulated intestinal fluid pH 6.8 maintained at 37.0 ◦C and paddle rotation speed was 60 rpm. At different time intervals (0.5,
Ac ce p
1, 2, 4, 6, 8 and 24 h) the samples (1.5 ml) were withdrawn, filtered (pore size: 0.22 μm) and concentration of SP was determined using the HPLC method at 238 nm described earlier in the manuscript. The dissolution data were compared to the dissolution of plain (original) SP (pure drug with no additive).
2.10 In vitro skin permeation study The abdominal hair of Wistar male rats, weighing 200-250 g, was shaved using electric and hand razors. After anesthetizing the rats with ether, the abdominal skin was surgically excised. To remove the adhering subcutaneous debris and leachable enzymes, the dermal side of the skin was put in contact with a saline solution for 1 h before starting the diffusion
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experiment. A system employing Franz diffusion cells was used for permeation studies. The excised rat skin was set in place with the stratum corneum facing the donor compartment and the dermis facing the receptor [14]. The receiver compartment was filled with 5.5 ml of
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phosphate buffer saline (PBS) (pH 7.4). The diffusion cells were maintained at (37±0.5) oC with stirring at 300 rpm throughout the experiment. Lyophilized SP-SLNs equivalent to 10
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mg of SP and similar amount of SP alone (plain SP which is pure drug without any additives and was applied in the form of dispersion) as control were dispersed by
an
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vortexing for 15 min in 5 ml distilled water and applied on the skin surface.
Samples from receiver medium were withdrawn at predetermined time intervals (1, 2, 3, 4, 6,
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8 and 24 h) and an equivalent volume of fresh PBS maintained at 37 ºC was replaced. All samples were filtered through an aqueous 0.22 µm filter and analyzed by HPLC method as
d
described before. At the end of the permeation study, excess formulation from the skin was
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removed, washed 3 times with PBS, and then dried with filter paper. To calculate the amount of SP deposited within the skin, the skins were removed and washed three times with
Ac ce p
phosphate buffer solution (PBS, pH 7.4) and residual washing solvent was carefully wiped off from the skin. The skins were minced, transferred to a test tube and digested for 24h in 1 ml of PBS (pH 7.4) and 1 ml of HCL 0.1M. All the samples were then centrifuged at 25000 rpm for 20 min. The supernatant filtered through 0.22 μm membrane and quantified by HPLC at 238 nm for SP content.
2.11 Statistical analysis All the results are expressed as the mean ± standard deviation of at least three determinations (n=3). The treated groups were compared to control by analysis of variance (ANOVA),
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following Dunnet’s test. The statistical analysis was carried out using software, SPSS. The Pvalue < 0.05 was considered as significant.
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3. Results and discussion 3.1 Characterization of SLNs
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The results showed that all formulations were able to form SLN (Table 1). It was found that, generally, with increasing the concentration of surfactant to drug-lipid concentration (Table
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1: surfactant concentration was constant while drug-lipid concentration decreased), the
an
particle size of SLNs decreased and smaller particle size showed a narrower size distribution compared to larger SLNs (P0.05). Span 60 is solid at room temperature and showed
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the higher phase transition temperature. The Span 60 having the highest transition
cr
temperature provides the highest entrapment [20].
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3.2 STM analysis
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Figure 1 shows particles from SP-SLN14 formulation (selected as optimal formulation because of the smallest particle size, 88.9 nm, and relatively high entrapment efficiency of
M
59.86%) before and after graphical process. These micrographs indicated spherical shape. STM micrographs, in the present research, demonstrate the uniform distributions which are in
d
agreement with the micrographs obtained from the scanning electron microscopy [2]), atomic
Ac ce p
[21] of SLN-images.
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force microscopy [18], fluorescence microscopy [19] and transmission electron microscopy
3.3 FT-IR
Figure 2 illustrates the infrared spectra of SP alone , SA, SP-SLNs and physical mixture (with the same composition). In the case of SP alone, a sharp absorption band appearing around 2950 cm-1 corresponds to C-H stretching, absorption bands around 1768 cm-1 could be attributed to the stretching band of lactone C=O, 1691 cm-1 to C=O of thioacetyl group (stretching), 1673 cm-1 to C=O of C6-ring (stretching), and 1617 cm-1 to C=C stretching. The FT-IR spectrum of solid lipid, revealed the presence of absorption band at 2849 and 2917 cm-1 due to symmetric and asymmetric C-H stretching, wavenumber 1702 cm-1 depicts C=O stretching, and 2400-3400 cm-1 depicts O-H stretching. In the spectra of the physical blend
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of drug and excipients, the wavenumbers related to carbonyl of lactone, carbonyl of thioacetyl, C=O of ring, and double bond of ring have no changes, but in SP-SLN spectra, the carbonyl groups were shifted from 1673-1768 cm-1 to 1700-1735 cm-1. This confirms
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interaction between SP and nanoparticle vehicle via intermolecular hydrogen bonding
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3.4 Differential scanning calorimetry (DSC) and XRD
cr
between drug and SLN components [22].
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The SP alone, SA, the physical mixture and SP-SLNs powder were examined by DSC and their DSC traces are shown in Figure 3. SP alone showed a single sharp endothermic melting
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peak at about 212 ºC. The thermogram of solid lipid (stearic acid) showed a single sharp endothermic melting peak around 70 ºC. DSC traces of SLN and physical mixture contained
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the endothermic peak around stearic acid melting point, but the endothermic peak of
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crystalline form of drug disappeared. The possible explanation is that the lipid (SA) may inhibit the crystallization of SP during the nanoparticle formation [23]. The most probable
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reason for the disappearance of SP peak in the physical mixtures could be due to the dissolution of SP in the molten SA. Similar event has previously been reported for antiviral drug (UC-781) in the presence of PEG 6000 and gelucire [24] where the endothermic peak of the drug disappeared during DSC run. This, in fact, is one of the limitations of DSC analysis as DSC method is an invasive method, and heating the sample during analysis might dissolve the drug crystals in the polymer which has already been into the molten stage before the drug melts. In the case of SP-SLN, it has been reported that when the drug does not show its endothermic peak in the nanoparticulate formulations, it is said to be in the amorphous state [25]. To provide more evidence for the above finding XRD was conducted for spironolactone, SA and SP-SLN 14 and their XRD spectra were shown in Figure 4. The
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XRD of SP shows diagnostic peaks at 2Ɵ 9.2, 16.6, 17.4 and 20.3 [26] which were shown on the figure. XRD spectrum of SP-SLN 14 shows that the main peaks of spironolactone disappeared or their intensity significantly reduced (highlighted on the Figure) which
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could be an indication of amorphous state or a very fine crystal structure for SP. The relative reduction of diffraction intensity of spironolactone in SLNs at these angles
cr
suggests that spironolactone might present in partially amorphous or microcrystalline
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form in the SLNs.
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3.5 Dissolution testing
The dissolution profiles of the selected SP-SLNs (SP-SLN 14) and SP alone are shown in
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Figure 4. The drug release behavior from the SLN displayed a faster drug release at the initial stage (the first 30 min) followed by sustained release pattern. Faster dissolution of SP from
d
SLNs compared to SP alone could be due to differences in their surface area available for
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dissolution as the rate of drug dissolution is directly proportional to the surface area of the solute particle. In the SP alone, the drug has crystalline structure (DSC traces and XRD
Ac ce p
showed this) with relatively larger particle size (mean diameter of 131 µm obtained via sieving technique) whereas SLNs have very smaller particles and the drug is partially in amorphous state or very fine crystals which both of these parameters (size and the state of solid particles) may contribute to increase the dissolution rate of SP. Different release profiles from SLNs have been reported [20]. The results obtained in the present study are in close agreement with the data reported by Bourezg et al. [7]. They reported that SP loaded lipid nanoparticles exhibited higher burst release in comparison to the commercially available tablets. They attributed this phenomenon to the surface enrichment of the drug as well as to the large surface area of nanoparticles [7].
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After the initial fast release within 30-60 min, prolonged-release pattern was observed between 1 to 24 h. The slow release of SP could be due to the slow penetration of the dissolution medium into the core of the nanoparticles (due to hydrophobic lipid nature of the
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core) followed by slow diffusion of SP out of the core. In addition, a further reduction in drug release could occur at later stages of the dissolution due to depletion of SP from the core,
cr
hence a reduced concentration gradient.
The observed biphasic release pattern could be interesting for topical application of
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spironolactone nanoparticles to treat skin diseases as the initial release improves the
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penetration of spironolocatone, while the second release phase (steady release) provides the drug for longer time.
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3.6 In vitro percutaneous absorption study
Skin permeation studies were presented in Figure 5. There was a significant difference
d
between SP-SLN 14 and SP alone in terms of drug penetration (P
