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Topical Amphotericin B solid lipid nanoparticles: Design and development
Accepted Manuscript Title: Topical Amphotericin B solid lipid nanoparticles: Design and development Author: Dhruv Butani Chetan Yewale Ambikanandan Misra PII: DOI: Reference:
S0927-7765(15)30066-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.07.032 COLSUB 7236
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
4-7-2014 10-7-2015 13-7-2015
Please cite this article as: Dhruv Butani, Chetan Yewale, Ambikanandan Misra, Topical Amphotericin B solid lipid nanoparticles: Design and development, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.07.032 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.
Topical Amphotericin B solid lipid nanoparticles: Design and development Dhruv Butani, Chetan Yewale, Ambikanandan Misra*
Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara - 390 001, Gujarat State, INDIA
*Address for correspondence Ambikanandan Misra, Professor in Pharmacy and Dean, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Post Box No.: 51, Kalabhavan, Vadodara - 390 001, Gujarat state, INDIA Telephone no.: +91-265-2419231 (Direct -O) +91-9426074870 (M) Fax no.: +91-265-2418927 Email: [email protected], [email protected]
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Graphical abstract
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Article Highlight Points
Topical amphotericin B solid lipid nanoparticles were developed to improve the therapeutic antifungal activity.
SLNs were evaluated for ex vivo permeation, retention and skin irritation.
Antifungal activity of SLNs was evaluated in Trichophyton rubrum fungal species.
Relatively better antifungal activity, high skin deposition, and distribution of the Amphotericin B with low skin irritation was observed in optimized topical SLNs.
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Abstract The present work is focused on design and development of topical Amphotericin B solid lipid nanoparticles (SLNs) to improve the therapeutic antifungal activity. Amphotericin B loaded SLNs were prepared by novel solvent diffusion method and were characterized for particle size, zeta potential, drug entrapment, surface morphology, in vitro antifungal activity, ex vivo permeation, retention and skin-irritation. Optimized SLNs were spherical with average size of 111.1 ± 2.2 nm, zeta potential of -23.98 ± 1.36 mV and 93.8 ± 1.8 % of drug entrapment. Characterization of Amphotericin B SLNs by Differential scanning calorimetry, Fourier transform infrared spectroscopy and Powder X-ray diffraction studies revealed absence of interaction between Amphotericin B and lipid. Amphotericin B is well dispersed in the lipid matrix without any crystallization. The SLNs were lyophilized with and without cryoprotectants to evaluate the stability and it was observed that the particle size of the SLNs significantly increased in SLN formulations lyophilized without cryoprotectant.
The optimized SLN 5
formulation exhibited 2 fold higher drug permeation as compared to plain drug dispersion and higher zone of inhibition in Trichophyton rubrum fungal species. Formulation was found to be stable at 2–8°C and 25 ± 2°C for the period of three months. Results of present study indicate that SLNs are suitable carriers for entrapment of poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drug.
Keywords: SLN, Amphotericin B, Antifungal, Solvent Diffusion, Drug Retention, Drug Permeation
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Introduction Lipid nanoparticles are colloidal drug carriers composed of a solid lipophilic matrix in which active molecules can be incorporated. Owing to the biocompatibility of lipids, the lipid nanoparticles are attracting attention of formulation scientists, as carriers for poorly soluble drugs [1]. Lipid nanoparticles have gained huge popularity and it includes the solid lipid nanoparticles (SLNs) as well as nanostructured lipid carriers (NLCs) [2]. Recent attention of the SLNs and NLCs research is more inclined towards the topical (specifically dermal) application both for pharmaceutical and cosmetic purposes [3]. These lipid nanoparticulate systems consist of physiological and biodegradable lipids suitable for incorporation of lipophilic and hydrophilic molecules within the lipid matrix [4]. SLNs are beneficial in many aspects [2, 5] such as negligible toxicity, easy to incorporate and useful for improving bioavailability of lipophilic compounds, prevent degradation of molecules sensitive to chemical, light, moisture and oxidation, sustained release of drug and minimum adverse effects of encapsulated drug molecule. The standard production method used for the preparation of lipid nano dispersions, based on solid lipids is high pressure homogenization, precipitation both from microemulsions and emulsions containing organic solvents [6]. The preparation of SLNs with these methods involves several critical process parameters like high temperatures, high pressures, toxic solvents, high emulsifier concentrations etc. Thus, an alternative method, offering clear advantages over the existing methods was selected which involves the use of pharmaceutically acceptable organic solvents, eliminating high pressure homogenization, easy handling and a fast production process without the use sophisticated equipment. It is based on principle of lipid precipitation briefly; a solution of lipid in a water-miscible solvent is rapidly injected into an aqueous phase containing low concentration of surfactant [7]. Amphotericin B is a broad-spectrum antifungal and antiprotozoal macrolide polyene antibiotic. Purpose of topical and dermatological dosage forms is to conveniently deliver drug molecules across localized area of skin and sustained release of drug [8]. Amphotericin B exerts its toxic effect on fungal cells by forming complexes with membrane sterols (ergosterol and episterol, which are found in fungal and leishmania cells, respectively) that act as transmembrane channels, allowing the increase in permeability of the fungal membrane to small cations promoting the rapid depletion of intracellular potassium and fungal cell death [9]. Amphotericin B also possesses activity against experimental cutaneous leishmaniasis and used in clinical 5
mucocutaneous leishmaniasis and a cutaneous leishmeniasis infection resistant to antimony treatment [10]. To overcome the problem of dermatopharmacotherapy such as limited local activity, there is a need to develop a selective delivery system that enhances penetration of bioactive moiety into the skin. This may help in localizing the drug at application site by serving as regional depot or reservoir and reducing the effective dose, dosing frequency, as well as systemic side effects associated with conventional topical therapy. Stratum corneum is the main barrier in percutaneous absorption of topically applied drugs. Small size and relatively narrow size distribution of SLN permits site specific delivery to the skin and thus intensify the concentration of these agents in the skin. [11]. 2. Materials and Methods 2.1. Materials Amphotericin B was obtained as a gift sample from Lyka Labs Pvt. Ltd., Ankleshwar, India. Compritol 888 ATO (Glyceryl Behenate) and Precirol ATO 5 (Glycerol Palmitostearate) were obtained as gift sample from Gattefosse, Mumbai, India. HPMC K100M was provided by Dow Chemical Pvt. Ltd., Mumbai, India. Surfactant Poloxamer F-127 and Poloxamer F-68 were obtained as a gift sample from BASF, Germany. Stearic Acid and Glycerol were purchased from S. D. Fine Chem. Pvt. Ltd., Mumbai, India. Sodium Carboxymethyl Cellulose and Tween 80 were purchased from Loba Chemie. Pvt. Ltd. Mumbai, India. All other chemicals and reagents used in this study were of analytical grade. 2.2. Methods 2.2.1. Partitioning behaviour of Amphotericin B in various lipids [12] 10 mg of Amphotericin B was dispersed in 1 g melted lipid (at 60°C) and 5 ml double distilled water heated to 60°C and added in melted lipid drug mixture. The above mixture was stirred for 30 minutes in a hot water bath and cooled. After cooling, the aqueous phase was separated and centrifuged. Supernatant was withdrawn after centrifugation and analysed for drug content. 2.2.2. Preparation of drug loaded SLNs Amphotericin B loaded SLNs were prepared by solvent diffusion method in an aqueous system as per reported methods with slight modification [13]. Briefly, Amphotericin B and lipid were dissolved in methanol at 60 ºC in water bath. This solution was emulsified with aqueous solution containing different surfactants at 60 ºC given in Table 1 by high-speed homogenization 6
using Ultra-Turrax T 25 (IKA-Werke, Staufen, Germany) at 10,000 rpm for 10 min. The obtained dispersion was allowed to cool to room temperature leading to formation of solid lipid nanoparticles by recrystallization of the dispersed lipid. Dispersion was probe sonicated to reduce the size of SLNs and was centrifuged at 9000 rpm for 30 min at 4 °C. Then, the supernatant was separated as SLN dispersion. 2.2.3. Incorporation of SLN in gel base [14] For the preparation of hydrogels, glycerol (7% w/w) was added to SLNs dispersion and mixed. Then a gelling agent (Na CMC high viscosity, HPMC K100M, 2% w/w) was added to the SLNs dispersion and the resulting mixture was stirred to yield gel containing 0.1% (w/w) of Amphotericin B. Methylparaben and propylparaben were used as preservatives. The drug loaded SLNs hydrogel formulation was stored at 2-8 °C until use. 3. Characterizations of SLNs 3.1. Evaluation of physical properties The SLNs were characterized for physical properties such as color, odor and stability and the gel formulations were evaluated for color, odor, and pH. 3.2. Particle size, polydispersity index and zeta potential measurement Particle size and polydispersity index (PDI) of the SLNs were measured by dynamic light scattering technique using Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Dispersions were diluted with double distilled water to ensure that the light scattering intensity was within the instruments sensitivity range. Each sample was analyzed in triplicate and the results were shown as mean ± standard deviation. 3.3. % Entrapment Efficiency [25] The entrapment efficiency (% EE) was determined by measuring the concentration of unentrapped drug in the lipidic dispersion [21]. Briefly, the SLNs dispersion was subjected to centrifugation for 30 min, 4°C at 9,000 rpm (Remi Centrifuge Pvt. Ltd., India) and the amount of Amphotericin B in supernatant was determined by dissolving supernatant in methanol: chloroform (6:4) mixture at 406.5 nm by UV Spectrophotometry. The entrapment efficiency was calculated by the equation (1). % EE
Mi Mf
/ M i *100
……………….Equation (1)
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Where “Mi” is the mass of initial drug used and “Mf” is the mass of free drug detected in the sediment after centrifugation of the aqueous dispersion. Each value was measured in triplicate. The results are shown as mean ± standard deviation. 3.4. Differential Scanning Calorimetry (DSC) analysis Different components of the formulation, placebo SLNs, drug loaded SLNs and physical mixtures were subjected to DSC (DSC-60, Shimadzu, Japan) analysis. Briefly, samples (4–5 mg) were kept in the standard aluminum pans and sealed. Then the pans were placed under isothermal condition at 25 ± 1 °C for 10 min. DSC analysis was performed at 10 °C/min from 25 to 300 °C under an inert environment. An empty sealed pan was used as a reference. 3.5. FTIR Study A Fourier transform infrared (FTIR) spectrophotometer (Bruker, Germany) was used for infrared analysis of samples. Drug, drug loaded-SLNs, blank SLNs and physical mixtures were analyzed by FTIR. About 1–2 mg of sample was mixed with dry potassium bromide and the samples were examined at transmission mode over wavenumber range of 4,000 to 400 cm−1. 3.6. Powder X-ray diffraction (PXRD) PXRD analysis of Amphotericin B and Amphotericin B loaded SLNs was carried out by a powder X-ray diffractometer (Philips PW 1710, Tokyo, Japan), where CuKα radiation was used as X-ray source. For the analysis, samples were placed in the glass sample holders and scanned from 10º to 40º with a scan angular speed (2θ/min) of 2º/min at 40 kV operating voltage and 30 mA current. The diffraction spectra were recorded. 3.7. Scanning Electrons Microscopy (SEM) SEM was used to characterize the microstructure of Amphotericin B lyophilized SLNs. SEM analysis was performed using JSM-5610LV (JEOL Ltd, Tokyo, Japan). Samples were attached to sample stubs and then viewed using an accelerating voltage at the magnification. In SEM study, electrons are transmitted from specimen surfaces. The picture was taken under inert condition in the electron microscope. 3.8. In vitro antifungal activity test [16] The various formulations SLN4 gel, SLN5 gel, SLN6 gel and plain drug gel were assayed for antifungal activity against the fungal strain Trichophyton rubrum, ATCC Code 28188TM (KWIK-STIK 0444 P), obtained from Microbiologics, Minnesota, USA. This fungus was grown on Sabouraud’s agar plate at 25 ± 2 °C. The plates were first sterilized in hot air oven at 160 °C 8
for 60 min. The fungal culture suspension was made according to the protocol of Microbiologics [17] and was allowed to stand for 20 min before transferring onto the solid agar medium, as the strain was available in lyophilized form. To the sterile petri-dishes in which solidified agar growth medium were taken, the fungal culture suspension was spread with the help of a spreader. The inoculums were spread uniformly over the solid agar surface by spreader glass rod and incubated at 25± 2°C for 7 days to allow fungal growth. Then wells were made in the middle of the plates with the help of a sterile cork borer and were filled with the formulations and then the plates were incubated at 25 ± 2°C in the incubator. The clear rings appeared around the dishes in 48 hrs. The rings are known as the zone of inhibition. The larger the zone of inhibition, the more effectively the formulation works. The antifungal activity was evaluated by measuring zones of inhibition (measured in millimetre) of fungal growth surrounding the formulations. The complete antifungal analysis was carried out under strict aseptic conditions and the mean inhibition zone from three plates was calculated. The results were shown as mean ± standard deviation. 3.9. Ex vivo permeation studies and retention study [18] The full-thickness of abdomen skin of female Albino Wistar rats weighing 200 ± 30 g was used for all the permeation experiments. After removing hair, the skin was excised and examined for integrity using a lamp-inspecting method. The skin was rinsed with physiological saline. The fat tissues below skin were carefully chopped. The thickness of each skin was similar. The skins were clamped between the donor and the receptor chamber of Keshary Chien diffusion cells with an effective diffusion area of 1.54 cm2 and a cell volume of 20 ml. The receptor chambers were filled with freshly prepared mixtures of physiological buffer solution pH 6.8 and methanol (7:3 v/v). Methanol was used to solubilise Amphotericin B and sink condition was maintained. The diffusion cells were maintained at 32 ± 0.5°C using a recirculating water bath and the fluid in the receptor chambers was stirred continuously at 300 rpm. The formulations (0.5 g) were placed in the donor chambers. At 1, 2, 3, 4, 5, 6 and 24 h, 0.5 mL of the fluid in the receptor chambers was withdrawn for UV spectrophotometry analysis and replaced immediately with an equal volume of fresh mixtures of physiological buffer solution pH 6.8 and methanol (7:3 v/v). The cumulative amounts of Amphotericin B permeated through rat skins were plotted as a function of time. The permeation rate (Jss, μg·cm-2˙h-1) of Amphotericin B at a steady-state through rat skin was calculated from the slope of linear portion of the plots of Qt versus time. The experiments were
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carried out in triplicate for each sample, and the results are presented as mean ± standard deviation. The permeability coefficient (Kp, cm/h) was calculated according to Equation (2). K p Jss / C o
……………….Equation (2)
Where, Kp is the permeability coefficient, Jss is the flux calculated at steady-state and C0 represents the drug concentration which remains constant in the vehicle. At the end of the experiments, the skins were removed and rinsed with distilled water. Then, the skins were soaked in 5 ml of methanol for 24 hrs and were subjected to five sonication cycles for 15 min each in ultrasound bath, followed by centrifugal separation. The skin accumulative amount, namely the total amount of Amphotericin B extracted from skin at the end of the permeation study (at 24 hrs) could be obtained from the concentration of Amphotericin B in supernatant methanol. The extracted Amphotericin B from skins was analyzed by UV spectrophotometry. 3.10. Skin-irritation test [19] The irritation potential of the Amphotericin B SLNs gel formulations were evaluated by carrying out the Draize patch test on the rabbits. Animal care and handling was performed in accordance to the CPCSEA guidelines. White New Zealand rabbits weighing 2.5–3 kg were obtained from Biochemistry Department, The Maharaja Sayajirao University of Baroda, Vadodara, India. Animals were divided into Six groups (n = 3) as follows: Group 1: Conventional plain drug gel. Group 2: Negative control: SLN based gel without Amphotericin B (Placebo gel). Group 3: SLN4 gel formulation containing Amphotericin B (0.1%, w/w). Group 4: SLN5 gel formulation containing Amphotericin B (0.1%, w/w). Group 5: SLN6 gel formulation containing Amphotericin B (0.1%, w/w). Group 6: Positive control: Formalin. The back of the animals was clipped free of hairs 24 hrs prior to the application of the formulations and 0.5 g of formulation was applied on the hair-free skin with uniform spreading within the area of 4 cm2. The skin was observed for any visible change, such as erythema at 24 hrs, 48 hrs and 72 hrs after the application of formulations. Formalin was used as positive control and SLNs based gel without Amphotericin B was used as negative control in the study. The mean erythema scores were recorded (ranging from 0 to 4), depending on the degree of erythema, as follows: no erythema = 0; slight erythema (barely perceptible-light pink) = 1; 10
moderate erythema (dark pink) = 2; moderate to severe erythema (light red) = 3 and severe erythema (extreme redness) = 4 grade. The experimental study was approved by the Institutional Ethics Committee (IAEC, The Maharaja Sayajirao University of Baroda, Vadodara, India). 3.11. Stability study of SLNs [20] The chemical and physical stability of Amphotericin B SLNs were evaluated at 2-8°C and at 25 °C for 3 months for clarity, particle size, zeta potential and drug content. The centrifuge tests were also carried out to assess the physical stability of the Amphotericin B loaded SLN. The Amphotericin B loaded SLNs were centrifuged for 30 min at 2000 rpm in the centrifuge tests. 3.12. Statistical analysis Analysis of variance test (ANOVA) was applied to determine noteworthy results. Data obtained from skin permeation experiments were expressed as mean ± standard error (3 independent samples). The statistical analysis was carried out using Instat 2.1 software (Graph Pad Software Corp., San Diego, CA, USA). The P-value < 0.05 was considered as significant. 4. Results and discussion 4.1. Partitioning behaviour of Amphotericin B Partition coefficients of Amphotericin B were obtained by analysing drug content in aqueous phase. Drug content was found to be 90.2 ± 1.1 %, 96.5 ± 1.4 % and 72.1 ± 2.7 % in compritol ATO 888, Precirol ATO 5 and stearic acid, respectively. Precirol ATO5 in which Amphotericin B exhibited higher partition coefficient was selected for preparation of SLNs gel. 4.2. Evaluation of SLNs gel physical properties HPMC K100M yielded gel with very high viscosity and exhibited very high thickness with tackiness. Na CMC gels loaded with nanoparticulate dispersion were of medium viscosity, yellowish in color, odourless with smooth appearance. The pH of the gels was found in the range of 7.32 to 7.45. On the basis of above observations the Amphotericin B SLN gel (gelled with Na CMC) was used for further evaluations. 4.3. Particle size, Zeta potential and Polydispersity Index (PDI) Particle size, zeta potential and PDI of different Amphotericin B SLN formulations are given in Table 1. The effect of poloxamer F 68, poloxamer F 127 and tween 80 on the particle size is evident from the particle size analysis of samples, in which Poloxamer F127 showed lower particle size whereas Tween 80 showed higher particle size. The average size of all the 11
formulations was found between 111.1±2.2 to 415.8 ± 4.4 nm. Amphotericin B SLN5 has lowest particle size of 111.1±2.2 nm and the narrowest size distribution of 0.1±0.05 compared with the other formulations. In addition, no significant difference of the distribution between Amphotericin B SLNs and drug free SLNs were observed. The incorporation of Amphotericin B into SLNs only resulted in a slight change of average size this may be due to the extremely low amount of drug. The zeta potential of all the formulations was found in the range of –5 to – 23 mV. The incorporation of Amphotericin B into SLNs showed no influence on the zeta potentials of nanoparticles. SLNs prepared by tween 80 showed lower zeta potentials which denote that the SLNs dispersion system was less sterically stabilized in tween 80 surfactant systems. Even though a high zeta potential can provide an electric repulsion, poloxamer F127 provided steric stability for maintaining the stability of SLNs dispersions and produced highly stable dispersion system. 4.4. % Entrapment Efficiency Drug encapsulation efficiency (% EE) was highest and lowest when SLNs were prepared with poloxamer F 127 (>85 %) and tween 80 (<82 %) respectively as given in Table 1. SLNs prepared with poloxamer F 68 also exhibited EE > 80%. % EE decreased as follows: poloxamer F 127 > poloxamer F 68 > tween 80. However, % EE of SLNs significantly increased with increasing surfactant concentration till 0.25%. This might be due to the efficient loading and retention of drug molecules within the nanoparticle matrix or nanoparticle surface at higher surfactant concentration. % EE of SLNs prepared at drug: lipid ratio 1: 10 were not significantly different at 0.25% and 0.50% surfactant concentration. This might be due to the presence of enough lipids to encapsulate the drug molecules. % EE increased with increasing lipid concentration. As the drug: lipid ratio decreases, the lipid concentration increases, so there was an increase in % EE till the ratio of drug: lipid was 1:10 after which there was no significant change. This is reasonable, as higher amount of lipid was available to encapsulate drug molecules at higher lipid concentration. % EE of SLN5 was very high (93.8 ± 1.8%). The high % EE might be beneficial to reduce the skin irritation of drug by avoiding the direct contact between drug and skin surface. 4.5. Differential scanning calorimetry (DSC) analysis
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DSC analysis was used to characterize the state and degree of crystallinity of lipid dispersions, semi-solid systems, polymers and liposomes. It allows the study of the melting and crystallization behaviour of crystalline materials like solid lipid nanoparticles. As illustrated in Fig.1. The thermogram of Amphotericin B (Fig.1 A, endothermic peak at 211.69°C) showed Thermogram of Precirol ATO 5 (Fig.1 B, endothermic peak of melting at 56.98°C and 61.81°C), Poloxamer F 127 (Fig.1 C, endothermic peak of melting at 54.79°C ), sucrose (Fig.1 D, endothermic peak of melting at 190.31°C and 230.94°C), blank SLNs lyophilized with sucrose (Fig.1 E, endothermic peak of melting at 52.52°C, 187.96°C and 227.36°C), Amphotericin B loaded SLNs lyophilized with sucrose (Fig.1 F, endothermic peak of melting at 56.59°C, 187.13°C and 224.25°C), Amphotericin B loaded SLNs lyophilized without sucrose (Fig.1 G, endothermic peak of melting at 53.33°C and 57.14°C) and physical mixture of drug, Precirol ATO 5 and poloxamer F127 (Fig.1 H, endothermic peak of melting at 55.88°C). Endothermic peak of melting of drug was not seen in Amphotericin B loaded SLNs lyophilized without sucrose and physical mixture, indicating that the physical state of Amphotericin B changed from crystalline to amorphous. Hence it could be concluded that in Amphotericin B SLNs, drug was present in amorphous state and may have been homogeneously dispersed in the lipid matrix [21]. However, when SLNs were lyophilized with sucrose, it showed interference of sucrose in SLNs formulation. So, Fig. 3 F showed the endothermic peak of sucrose which indicates the crystalline state of the formulation. From the Fig. 3 G it can be seen that during the formulation of Amphotericin B SLNs, reduction in the endothermic peak of Precirol ATO 5 was observed. This can be attributed to the change in crystal lattice and increased number of lattice defects in the lipid crystal [22]. 4.6. FTIR study The FTIR spectrum showed characteristic peaks of Amphotericin B such as -OH stretch (strongly H- bond) (3368 cm-1), CH stretch (polyene) (3009 cm-1), N-H2 in plane band (1627 cm1
) and Polyene C=C bond (1554 cm-1) shown in Fig. 2A. In the IR spectrum of lyophilized
SLNs, peaks corresponding to Amphotericin B disappeared or broad peaks of Amphotericin B – OH groups shortened. This indicated drug entrapment in lipid matrix as shown in 2B. 4.7. PXRD study The powder X-ray diffractogram of Amphotericin B exhibits sharp peak at 2θ scattered angle of 13.88º, 21.29º and 21.58º, which indicates crystalline nature of Amphotericin B shown 13
in Fig. 3A [23]. The characteristic peaks for Amphotericin B were absent in the SLNs PXRD pattern suggesting that Amphotericin B was not in crystalline form in SLNs shown in Fig. 3B. Therefore, the study demonstrates that the crystalline structure of the Amphotericin B was changed after formation of SLNs with lipid and surfactant. 4.8. Scanning Electrons Microscope (SEM) SEM image of Amphotericin B lyophilized SLNs shown in Fig. 4. SEM image shows that the Amphotericin B SLNs are discrete, spherical, and regular in shape. There was a large particle due to aggregation of lyophilized SLNs formulation. 4.9. In vitro antifungal test The antifungal activity was measured by Trichophyton rubrum fungal species by growing it on Sabouraud’s agar. The antifungal activity results are given in Table 2. The zone of inhibition after 72 hrs was 1.28 ± 0.07, 1.76 ± 0.09, 2.81 ± 0.13 and 2.0 ± 0.10 mm for plain drug gel, SLN4 gel, SLN5 gel and SLN6 gel, respectively. The zone of inhibition was greater for SLN5 due to the higher concentration of drug as well as higher penetration power of SLNs. The zone of inhibition was according to SLN5 > SLN6 > SLN4 > plain drug gel. This indicated the SLN5 gel was more effective as compared to other formulations. 4.10.Ex vivo drug release and skin retention study To assess the influence of the lipidic nanoparticles on the permeation and accumulation of drug into the skin, ex vivo skin permeation and retention studies were performed using hairless rat skin by Keshary chein diffusion cells. All SLN gel formulations followed Higuchi model indicating that the drug released from SLN gel was by diffusion in sustained manner. In dermatological treatment, improving the efficacy demands high drug levels in the skin. With solid nanoparticle dispersion gels, a greater quantity of drug remained localized in the skin, with more amounts penetrating into the receptor compartment as compared with conventional gel. SLN5 gel had the highest uptake of Amphotericin B in skins when compared with conventional gel, SLN4 gel and SLN6 gel. Thus, drug localizing effect in the skin seems possible with SLNs. The amount of permeated drug was lower in case of conventional gel, while SLNs based formulation represented the more amount of permeated drug (Fig. 5 A). The data indicates that lipidic nanoparticles clearly increased the drug permeation through the skin. It may appear that the structural composition of solid lipid nanoparticles was responsible for rate limiting effect in drug permeation. The cumulative amounts of Amphotericin B from conventional gel, SLN4 gel, 14
SLN5 gel, and SLN6 gel at 24 hrs were 9.24 µg/cm2, 19.84 µg/cm2, 22.34 µg/cm2 and 20.72 µg/cm2 respectively. In other words, conventional gel showed lower drug permeation, in comparison to lipidic nanoparticular systems (Fig. 5A). Permeation parameters are given in Table 2. Data indicates that drug flux were 0.39± 0.01 μg/cm2/hr, 0.83± 0.01 μg/cm2/hr, 0.93± 0.01μg/cm2/hr and 0.86± 0.02 μg/cm2/hr for conventional gel, SLN4 gel, SLN5 gel, and SLN6 gel respectively. Amp B SLN5 gel shown much better drug retention in skin compared Amp B microemulsion [16]. Drug retained in skin was approximately 23 % after 24 hrs in microemulsion whereas in Amp SLN5 gel it was 36.48 after 24 hrs. In fact, lipidic nanoparticles have shown to enhance the penetration (dermal delivery) especially into the upper skin layers [24], hence creating a reservoir which is able to prolong the skin residence time. Amp B SLN5 gel showed more drug deposition as compared Amp B microemulsion. This indicate that the Amp B SLN5 gel was more beneficial for topical fungal diseases because of more amount of drug reserved on skin, so more contact with the fungal species and therefore more effectively treat the fungal species on the skin layer. The increased skin delivery by SLNs include the large surface area due to small particle sizes, an occlusive effect and a penetration enhancer effect due to presence of surfactants. This property of the nanoparticles providing a better permeation condition by maintaining the skin hydration level at higher point. Bouwstra [25] reported that this behavior might be expected, as SLNs system based on lipid composition are more similar to subcutaneous lipids, in contrast to conventional gel. Thus, localized delivery of Amphotericin B from SLNs into skin tissue is affected, leading to an effective treatment of cutaneous fungal infection. The mechanism by which lipids increase skin permeability appears to involve disruption of the densely packed lipids which fill the extracellular spaces of the subcutaneous layer [26]. Their effect as penetration enhancers, after addition to semisolid vehicles, have been studied on a variety of drugs [27], In order to assess the skin uptake and penetration of Amphotericin B from SLNs, the in vitro permeation ability through skins and into skins were performed using diffusion cells [28]. SLNs can increase the uptake of drug in skin [29] and the skin targeting effect was disclosed by fluorescence microscopy [30]. SLNs with small diameters are advantageous to improve the penetration of nanoparticles into skins and the controlled release of SLNs may induce the increase of drug accumulation [31]. 15
4.11. Skin irritation study Conventional gel therapy is associated with noticeable skin irritation, which strongly restricts its applicability and acceptability by the patients. Basically, the Amphotericin B delivery system should be able to reduce irritation. The skin-irritation studies indicated that SLN4 gel, SLN5 gel and SLN6 gel exhibited considerably no irritation as compared to conventional gel, even after 48 hrs of application. Results of skin irritation study are given in Table 3. The primary irritation index (PII) was found to be 0.33 ± 0.33, 0.11 ± 0.19, 0.22 ± 0.38 for SLN4 gel, SLN5 gel and SLN6 gel, respectively showing no irritation. The primary irritation index (PII) was found to be 0.00 ± 0.00, 2.11 ± 1.01 and 2.89 ± 1.71 for control gel (placebo), formalin and conventional gel, respectively. Therefore, the developed SLNs formulation resulted in no erythema or edema on the intact and abraded rabbit skin, as compared to the conventional gel. Thus the formulation can be classified as a non-irritant to the rabbit skin. 4.12. Stability study Amphotericin B SLN exhibited a good stability during the period of 3 months. No significant change of clarity and phase separation was observed. Amphotericin B SLN had a good physical stability. This may imply that the transition of dispersed lipid in SLNs dispersion from β’ form to stable β form might occur extremely slowly. Generally, the SLNs are prepared from solid lipids or blends of solid lipids and, after the preparation by solvent diffusion technique; the particle crystallizes, at least partially, in higher energy modifications α and β’. During storage, these modifications can transform to the low-energy, more-ordered β modification. Due to its high degree of order, the number of imperfections in the crystal lattice is reduced thus leading to drug expulsion. No degradation of Amphotericin B in the formulations was observed. 5. Conclusion Amphotericin B SLNs were formulated for topical application and it was clearly demonstrated that SLNs were effective for topical delivery of Amphotericin B. SLN5 showed higher skin deposition, lower skin irritation and better anti-fungal activity. The developed system may provide better remission from the disease due to localized delivery with minimal side effects and the data from in vitro study were promising but further evaluation is needed to elucidate the clinical efficacy of this topical dosage form.
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Figure captions
Fig. 1. DSC analysis of (A) Amphotericin B, (B) Precirol ATO 5, (C) Poloxamer F 127, (D) Sucrose, (E) Drug free SLNs lyophilized with sucrose, (F) Drug loaded SLNs lyophilized with sucrose, (G) Drug loaded SLNs lyophilized without sucrose and (H) Physical mixture
20
Fig. 2. FTIR study of (A) Amphotericin B, (B) Lyophilized SLNs
21
Fig. 3. PXRD study of (A) Amphotericin B and (B) Amphotericin B lyophilized SLNs
22
Fig. 4. SEM images of Amphotericin B SLNs
23
Fig. 5. (A) Drug permeation study (B) Skin retention study of SLN4 gel, SLN5 gel, SLN6 gel and conventional gel (Mean ± SD (Standard Deviation), n=3)
24
Table 1 Different SLN formulations of Amphotericin B with different evaluation parameters. Formulation Drug : Surfactant
Surfactant
Solvent
Purified Particle Size* Zeta
PDI*
%
Code
Lipid
Conc.
Volume
Water
(nm)
Entrapment
Ratio
(% w/v)
(ml)
(nm)
Potential* (mV)
Efficiency*
SLN1
1 : 7.5
Pluronic F 68
0.25 %
10
40
268.3 ± 3.0
-16.02 ± 1.13 0.25 ± 0.05
80.1 ± 1.3
SLN2
1 : 10
Pluronic F 68
0.25 %
10
40
242.0 ± 3.3
-12.64 ± 1.93 0.23 ±0.03
86.4 ± 0.5
SLN3
1 : 12.5
Pluronic F 68
0.25 %
10
40
224.6 ± 2.3
-09.17 ± 1.26 0.27 ± 0.10
83.2 ± 1.0
SLN4
1 : 7.5
Pluronic F 127
0.25 %
10
40
163.1 ± 2.0
-20.95 ± 1.58 0.29 ± 0.05
85.6 ± 2.1
SLN5
1 : 10
Pluronic F 127
0.25 %
10
40
111.1 ± 2.2
-23.98 ± 1.36 0.13 ± 0.04
93.8 ± 1.8
SLN6
1 : 12.5
Pluronic F 127
0.25 %
10
40
133.9 ± 3.0
-15.73 ± 2.72 0.19 ± 0.09
90.0 ± 1.7
SLN7
1 : 7.5
Tween 80
0.25 %
10
40
415.8 ± 4.4
-09.55 ± 2.37 0.31 ± 0.06
72.7 ± 3.8
SLN8
1 : 10
Tween 80
0.25 %
10
40
373.2 ± 4.9
-06.12 ± 1.75 0.34 ± 0.08
81.9 ± 2.4
SLN9
1 : 12.5
Tween 80
0.25 %
10
40
399.2 ± 3.6
-05.69 ± 1.09 0.28 ± 0.04
78.6 ± 3.0
*(Mean ± Standard Deviation, n=3)
18
Table 2 In vitro antifungal activity and percutaneous permeation parameters of various Amphotericin B SLN gel formulations Formulation Code
Zone of Inhibition (diameter in mm)*
Percutaneous permeation parameters*
24 hrs
Jss flux (μg/cm2/hr)
48 hrs
72 hrs
Permeability Co-efficient Kp (cm/hr) × 10-3
Plain Drug Gel
0.53 ± 0.04
0.86 ± 0.06
1.20 ± 0.07
0.39± 0.01
0.78± 0.02
SLN4 Gel
0.61 ± 0.06
1.19 ± 0.05
1.76 ± 0.09
0.83± 0.01
1.65± 0.01
SLN5 Gel
1.22 ± 0.11
1.88 ± 0.09
2.81 ± 0.13
0.93± 0.01
1.86± 0.02
SLN6 Gel
0.94 ± 0.08
1.70 ± 0.06
2.05 ± 0.10
0.86± 0.02
1.73± 0.02
*(Mean ± Standard Deviation, n=3)
19
Table 3 Mean erythemal scores and PII observed for various Amphotericin B formulations obtained at the end of 24, 48 and 72 h. Formulation Code
Erythemal Score* 24 hrs
48 hrs
72 hrs
PII
Conventional Gel
1.00 ± 0.00
2.33 ± 0.58
3.00 ± 1.00
2.11 ± 1.01
SLN 4 Gel
0.00 ± 0.00
0.33 ± 0.58
0.66 ± 0.58
0.33 ± 0.33
SLN 5 Gel
0.00 ± 0.00
0.00 ± 0.00
0.33 ± 0.58
0.11 ± 0.19
SLN 6 Gel
0.00 ± 0.00
0.00 ± 0.00
0.66 ± 0.58
0.22 ± 0.38
Control Gel (Placebo)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
Formalin
1.00 ± 0.00
3.33 ± 0.58
4.33 ± 0.58
2.89 ± 1.71
*(Mean ± Standard Deviation, n=3)
20
S0927-7765(15)30066-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.07.032 COLSUB 7236
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
4-7-2014 10-7-2015 13-7-2015
Please cite this article as: Dhruv Butani, Chetan Yewale, Ambikanandan Misra, Topical Amphotericin B solid lipid nanoparticles: Design and development, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.07.032 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.
Topical Amphotericin B solid lipid nanoparticles: Design and development Dhruv Butani, Chetan Yewale, Ambikanandan Misra*
Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara - 390 001, Gujarat State, INDIA
*Address for correspondence Ambikanandan Misra, Professor in Pharmacy and Dean, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Post Box No.: 51, Kalabhavan, Vadodara - 390 001, Gujarat state, INDIA Telephone no.: +91-265-2419231 (Direct -O) +91-9426074870 (M) Fax no.: +91-265-2418927 Email: [email protected], [email protected]
1
Graphical abstract
2
Article Highlight Points
Topical amphotericin B solid lipid nanoparticles were developed to improve the therapeutic antifungal activity.
SLNs were evaluated for ex vivo permeation, retention and skin irritation.
Antifungal activity of SLNs was evaluated in Trichophyton rubrum fungal species.
Relatively better antifungal activity, high skin deposition, and distribution of the Amphotericin B with low skin irritation was observed in optimized topical SLNs.
3
Abstract The present work is focused on design and development of topical Amphotericin B solid lipid nanoparticles (SLNs) to improve the therapeutic antifungal activity. Amphotericin B loaded SLNs were prepared by novel solvent diffusion method and were characterized for particle size, zeta potential, drug entrapment, surface morphology, in vitro antifungal activity, ex vivo permeation, retention and skin-irritation. Optimized SLNs were spherical with average size of 111.1 ± 2.2 nm, zeta potential of -23.98 ± 1.36 mV and 93.8 ± 1.8 % of drug entrapment. Characterization of Amphotericin B SLNs by Differential scanning calorimetry, Fourier transform infrared spectroscopy and Powder X-ray diffraction studies revealed absence of interaction between Amphotericin B and lipid. Amphotericin B is well dispersed in the lipid matrix without any crystallization. The SLNs were lyophilized with and without cryoprotectants to evaluate the stability and it was observed that the particle size of the SLNs significantly increased in SLN formulations lyophilized without cryoprotectant.
The optimized SLN 5
formulation exhibited 2 fold higher drug permeation as compared to plain drug dispersion and higher zone of inhibition in Trichophyton rubrum fungal species. Formulation was found to be stable at 2–8°C and 25 ± 2°C for the period of three months. Results of present study indicate that SLNs are suitable carriers for entrapment of poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drug.
Keywords: SLN, Amphotericin B, Antifungal, Solvent Diffusion, Drug Retention, Drug Permeation
4
Introduction Lipid nanoparticles are colloidal drug carriers composed of a solid lipophilic matrix in which active molecules can be incorporated. Owing to the biocompatibility of lipids, the lipid nanoparticles are attracting attention of formulation scientists, as carriers for poorly soluble drugs [1]. Lipid nanoparticles have gained huge popularity and it includes the solid lipid nanoparticles (SLNs) as well as nanostructured lipid carriers (NLCs) [2]. Recent attention of the SLNs and NLCs research is more inclined towards the topical (specifically dermal) application both for pharmaceutical and cosmetic purposes [3]. These lipid nanoparticulate systems consist of physiological and biodegradable lipids suitable for incorporation of lipophilic and hydrophilic molecules within the lipid matrix [4]. SLNs are beneficial in many aspects [2, 5] such as negligible toxicity, easy to incorporate and useful for improving bioavailability of lipophilic compounds, prevent degradation of molecules sensitive to chemical, light, moisture and oxidation, sustained release of drug and minimum adverse effects of encapsulated drug molecule. The standard production method used for the preparation of lipid nano dispersions, based on solid lipids is high pressure homogenization, precipitation both from microemulsions and emulsions containing organic solvents [6]. The preparation of SLNs with these methods involves several critical process parameters like high temperatures, high pressures, toxic solvents, high emulsifier concentrations etc. Thus, an alternative method, offering clear advantages over the existing methods was selected which involves the use of pharmaceutically acceptable organic solvents, eliminating high pressure homogenization, easy handling and a fast production process without the use sophisticated equipment. It is based on principle of lipid precipitation briefly; a solution of lipid in a water-miscible solvent is rapidly injected into an aqueous phase containing low concentration of surfactant [7]. Amphotericin B is a broad-spectrum antifungal and antiprotozoal macrolide polyene antibiotic. Purpose of topical and dermatological dosage forms is to conveniently deliver drug molecules across localized area of skin and sustained release of drug [8]. Amphotericin B exerts its toxic effect on fungal cells by forming complexes with membrane sterols (ergosterol and episterol, which are found in fungal and leishmania cells, respectively) that act as transmembrane channels, allowing the increase in permeability of the fungal membrane to small cations promoting the rapid depletion of intracellular potassium and fungal cell death [9]. Amphotericin B also possesses activity against experimental cutaneous leishmaniasis and used in clinical 5
mucocutaneous leishmaniasis and a cutaneous leishmeniasis infection resistant to antimony treatment [10]. To overcome the problem of dermatopharmacotherapy such as limited local activity, there is a need to develop a selective delivery system that enhances penetration of bioactive moiety into the skin. This may help in localizing the drug at application site by serving as regional depot or reservoir and reducing the effective dose, dosing frequency, as well as systemic side effects associated with conventional topical therapy. Stratum corneum is the main barrier in percutaneous absorption of topically applied drugs. Small size and relatively narrow size distribution of SLN permits site specific delivery to the skin and thus intensify the concentration of these agents in the skin. [11]. 2. Materials and Methods 2.1. Materials Amphotericin B was obtained as a gift sample from Lyka Labs Pvt. Ltd., Ankleshwar, India. Compritol 888 ATO (Glyceryl Behenate) and Precirol ATO 5 (Glycerol Palmitostearate) were obtained as gift sample from Gattefosse, Mumbai, India. HPMC K100M was provided by Dow Chemical Pvt. Ltd., Mumbai, India. Surfactant Poloxamer F-127 and Poloxamer F-68 were obtained as a gift sample from BASF, Germany. Stearic Acid and Glycerol were purchased from S. D. Fine Chem. Pvt. Ltd., Mumbai, India. Sodium Carboxymethyl Cellulose and Tween 80 were purchased from Loba Chemie. Pvt. Ltd. Mumbai, India. All other chemicals and reagents used in this study were of analytical grade. 2.2. Methods 2.2.1. Partitioning behaviour of Amphotericin B in various lipids [12] 10 mg of Amphotericin B was dispersed in 1 g melted lipid (at 60°C) and 5 ml double distilled water heated to 60°C and added in melted lipid drug mixture. The above mixture was stirred for 30 minutes in a hot water bath and cooled. After cooling, the aqueous phase was separated and centrifuged. Supernatant was withdrawn after centrifugation and analysed for drug content. 2.2.2. Preparation of drug loaded SLNs Amphotericin B loaded SLNs were prepared by solvent diffusion method in an aqueous system as per reported methods with slight modification [13]. Briefly, Amphotericin B and lipid were dissolved in methanol at 60 ºC in water bath. This solution was emulsified with aqueous solution containing different surfactants at 60 ºC given in Table 1 by high-speed homogenization 6
using Ultra-Turrax T 25 (IKA-Werke, Staufen, Germany) at 10,000 rpm for 10 min. The obtained dispersion was allowed to cool to room temperature leading to formation of solid lipid nanoparticles by recrystallization of the dispersed lipid. Dispersion was probe sonicated to reduce the size of SLNs and was centrifuged at 9000 rpm for 30 min at 4 °C. Then, the supernatant was separated as SLN dispersion. 2.2.3. Incorporation of SLN in gel base [14] For the preparation of hydrogels, glycerol (7% w/w) was added to SLNs dispersion and mixed. Then a gelling agent (Na CMC high viscosity, HPMC K100M, 2% w/w) was added to the SLNs dispersion and the resulting mixture was stirred to yield gel containing 0.1% (w/w) of Amphotericin B. Methylparaben and propylparaben were used as preservatives. The drug loaded SLNs hydrogel formulation was stored at 2-8 °C until use. 3. Characterizations of SLNs 3.1. Evaluation of physical properties The SLNs were characterized for physical properties such as color, odor and stability and the gel formulations were evaluated for color, odor, and pH. 3.2. Particle size, polydispersity index and zeta potential measurement Particle size and polydispersity index (PDI) of the SLNs were measured by dynamic light scattering technique using Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Dispersions were diluted with double distilled water to ensure that the light scattering intensity was within the instruments sensitivity range. Each sample was analyzed in triplicate and the results were shown as mean ± standard deviation. 3.3. % Entrapment Efficiency [25] The entrapment efficiency (% EE) was determined by measuring the concentration of unentrapped drug in the lipidic dispersion [21]. Briefly, the SLNs dispersion was subjected to centrifugation for 30 min, 4°C at 9,000 rpm (Remi Centrifuge Pvt. Ltd., India) and the amount of Amphotericin B in supernatant was determined by dissolving supernatant in methanol: chloroform (6:4) mixture at 406.5 nm by UV Spectrophotometry. The entrapment efficiency was calculated by the equation (1). % EE
Mi Mf
/ M i *100
……………….Equation (1)
7
Where “Mi” is the mass of initial drug used and “Mf” is the mass of free drug detected in the sediment after centrifugation of the aqueous dispersion. Each value was measured in triplicate. The results are shown as mean ± standard deviation. 3.4. Differential Scanning Calorimetry (DSC) analysis Different components of the formulation, placebo SLNs, drug loaded SLNs and physical mixtures were subjected to DSC (DSC-60, Shimadzu, Japan) analysis. Briefly, samples (4–5 mg) were kept in the standard aluminum pans and sealed. Then the pans were placed under isothermal condition at 25 ± 1 °C for 10 min. DSC analysis was performed at 10 °C/min from 25 to 300 °C under an inert environment. An empty sealed pan was used as a reference. 3.5. FTIR Study A Fourier transform infrared (FTIR) spectrophotometer (Bruker, Germany) was used for infrared analysis of samples. Drug, drug loaded-SLNs, blank SLNs and physical mixtures were analyzed by FTIR. About 1–2 mg of sample was mixed with dry potassium bromide and the samples were examined at transmission mode over wavenumber range of 4,000 to 400 cm−1. 3.6. Powder X-ray diffraction (PXRD) PXRD analysis of Amphotericin B and Amphotericin B loaded SLNs was carried out by a powder X-ray diffractometer (Philips PW 1710, Tokyo, Japan), where CuKα radiation was used as X-ray source. For the analysis, samples were placed in the glass sample holders and scanned from 10º to 40º with a scan angular speed (2θ/min) of 2º/min at 40 kV operating voltage and 30 mA current. The diffraction spectra were recorded. 3.7. Scanning Electrons Microscopy (SEM) SEM was used to characterize the microstructure of Amphotericin B lyophilized SLNs. SEM analysis was performed using JSM-5610LV (JEOL Ltd, Tokyo, Japan). Samples were attached to sample stubs and then viewed using an accelerating voltage at the magnification. In SEM study, electrons are transmitted from specimen surfaces. The picture was taken under inert condition in the electron microscope. 3.8. In vitro antifungal activity test [16] The various formulations SLN4 gel, SLN5 gel, SLN6 gel and plain drug gel were assayed for antifungal activity against the fungal strain Trichophyton rubrum, ATCC Code 28188TM (KWIK-STIK 0444 P), obtained from Microbiologics, Minnesota, USA. This fungus was grown on Sabouraud’s agar plate at 25 ± 2 °C. The plates were first sterilized in hot air oven at 160 °C 8
for 60 min. The fungal culture suspension was made according to the protocol of Microbiologics [17] and was allowed to stand for 20 min before transferring onto the solid agar medium, as the strain was available in lyophilized form. To the sterile petri-dishes in which solidified agar growth medium were taken, the fungal culture suspension was spread with the help of a spreader. The inoculums were spread uniformly over the solid agar surface by spreader glass rod and incubated at 25± 2°C for 7 days to allow fungal growth. Then wells were made in the middle of the plates with the help of a sterile cork borer and were filled with the formulations and then the plates were incubated at 25 ± 2°C in the incubator. The clear rings appeared around the dishes in 48 hrs. The rings are known as the zone of inhibition. The larger the zone of inhibition, the more effectively the formulation works. The antifungal activity was evaluated by measuring zones of inhibition (measured in millimetre) of fungal growth surrounding the formulations. The complete antifungal analysis was carried out under strict aseptic conditions and the mean inhibition zone from three plates was calculated. The results were shown as mean ± standard deviation. 3.9. Ex vivo permeation studies and retention study [18] The full-thickness of abdomen skin of female Albino Wistar rats weighing 200 ± 30 g was used for all the permeation experiments. After removing hair, the skin was excised and examined for integrity using a lamp-inspecting method. The skin was rinsed with physiological saline. The fat tissues below skin were carefully chopped. The thickness of each skin was similar. The skins were clamped between the donor and the receptor chamber of Keshary Chien diffusion cells with an effective diffusion area of 1.54 cm2 and a cell volume of 20 ml. The receptor chambers were filled with freshly prepared mixtures of physiological buffer solution pH 6.8 and methanol (7:3 v/v). Methanol was used to solubilise Amphotericin B and sink condition was maintained. The diffusion cells were maintained at 32 ± 0.5°C using a recirculating water bath and the fluid in the receptor chambers was stirred continuously at 300 rpm. The formulations (0.5 g) were placed in the donor chambers. At 1, 2, 3, 4, 5, 6 and 24 h, 0.5 mL of the fluid in the receptor chambers was withdrawn for UV spectrophotometry analysis and replaced immediately with an equal volume of fresh mixtures of physiological buffer solution pH 6.8 and methanol (7:3 v/v). The cumulative amounts of Amphotericin B permeated through rat skins were plotted as a function of time. The permeation rate (Jss, μg·cm-2˙h-1) of Amphotericin B at a steady-state through rat skin was calculated from the slope of linear portion of the plots of Qt versus time. The experiments were
9
carried out in triplicate for each sample, and the results are presented as mean ± standard deviation. The permeability coefficient (Kp, cm/h) was calculated according to Equation (2). K p Jss / C o
……………….Equation (2)
Where, Kp is the permeability coefficient, Jss is the flux calculated at steady-state and C0 represents the drug concentration which remains constant in the vehicle. At the end of the experiments, the skins were removed and rinsed with distilled water. Then, the skins were soaked in 5 ml of methanol for 24 hrs and were subjected to five sonication cycles for 15 min each in ultrasound bath, followed by centrifugal separation. The skin accumulative amount, namely the total amount of Amphotericin B extracted from skin at the end of the permeation study (at 24 hrs) could be obtained from the concentration of Amphotericin B in supernatant methanol. The extracted Amphotericin B from skins was analyzed by UV spectrophotometry. 3.10. Skin-irritation test [19] The irritation potential of the Amphotericin B SLNs gel formulations were evaluated by carrying out the Draize patch test on the rabbits. Animal care and handling was performed in accordance to the CPCSEA guidelines. White New Zealand rabbits weighing 2.5–3 kg were obtained from Biochemistry Department, The Maharaja Sayajirao University of Baroda, Vadodara, India. Animals were divided into Six groups (n = 3) as follows: Group 1: Conventional plain drug gel. Group 2: Negative control: SLN based gel without Amphotericin B (Placebo gel). Group 3: SLN4 gel formulation containing Amphotericin B (0.1%, w/w). Group 4: SLN5 gel formulation containing Amphotericin B (0.1%, w/w). Group 5: SLN6 gel formulation containing Amphotericin B (0.1%, w/w). Group 6: Positive control: Formalin. The back of the animals was clipped free of hairs 24 hrs prior to the application of the formulations and 0.5 g of formulation was applied on the hair-free skin with uniform spreading within the area of 4 cm2. The skin was observed for any visible change, such as erythema at 24 hrs, 48 hrs and 72 hrs after the application of formulations. Formalin was used as positive control and SLNs based gel without Amphotericin B was used as negative control in the study. The mean erythema scores were recorded (ranging from 0 to 4), depending on the degree of erythema, as follows: no erythema = 0; slight erythema (barely perceptible-light pink) = 1; 10
moderate erythema (dark pink) = 2; moderate to severe erythema (light red) = 3 and severe erythema (extreme redness) = 4 grade. The experimental study was approved by the Institutional Ethics Committee (IAEC, The Maharaja Sayajirao University of Baroda, Vadodara, India). 3.11. Stability study of SLNs [20] The chemical and physical stability of Amphotericin B SLNs were evaluated at 2-8°C and at 25 °C for 3 months for clarity, particle size, zeta potential and drug content. The centrifuge tests were also carried out to assess the physical stability of the Amphotericin B loaded SLN. The Amphotericin B loaded SLNs were centrifuged for 30 min at 2000 rpm in the centrifuge tests. 3.12. Statistical analysis Analysis of variance test (ANOVA) was applied to determine noteworthy results. Data obtained from skin permeation experiments were expressed as mean ± standard error (3 independent samples). The statistical analysis was carried out using Instat 2.1 software (Graph Pad Software Corp., San Diego, CA, USA). The P-value < 0.05 was considered as significant. 4. Results and discussion 4.1. Partitioning behaviour of Amphotericin B Partition coefficients of Amphotericin B were obtained by analysing drug content in aqueous phase. Drug content was found to be 90.2 ± 1.1 %, 96.5 ± 1.4 % and 72.1 ± 2.7 % in compritol ATO 888, Precirol ATO 5 and stearic acid, respectively. Precirol ATO5 in which Amphotericin B exhibited higher partition coefficient was selected for preparation of SLNs gel. 4.2. Evaluation of SLNs gel physical properties HPMC K100M yielded gel with very high viscosity and exhibited very high thickness with tackiness. Na CMC gels loaded with nanoparticulate dispersion were of medium viscosity, yellowish in color, odourless with smooth appearance. The pH of the gels was found in the range of 7.32 to 7.45. On the basis of above observations the Amphotericin B SLN gel (gelled with Na CMC) was used for further evaluations. 4.3. Particle size, Zeta potential and Polydispersity Index (PDI) Particle size, zeta potential and PDI of different Amphotericin B SLN formulations are given in Table 1. The effect of poloxamer F 68, poloxamer F 127 and tween 80 on the particle size is evident from the particle size analysis of samples, in which Poloxamer F127 showed lower particle size whereas Tween 80 showed higher particle size. The average size of all the 11
formulations was found between 111.1±2.2 to 415.8 ± 4.4 nm. Amphotericin B SLN5 has lowest particle size of 111.1±2.2 nm and the narrowest size distribution of 0.1±0.05 compared with the other formulations. In addition, no significant difference of the distribution between Amphotericin B SLNs and drug free SLNs were observed. The incorporation of Amphotericin B into SLNs only resulted in a slight change of average size this may be due to the extremely low amount of drug. The zeta potential of all the formulations was found in the range of –5 to – 23 mV. The incorporation of Amphotericin B into SLNs showed no influence on the zeta potentials of nanoparticles. SLNs prepared by tween 80 showed lower zeta potentials which denote that the SLNs dispersion system was less sterically stabilized in tween 80 surfactant systems. Even though a high zeta potential can provide an electric repulsion, poloxamer F127 provided steric stability for maintaining the stability of SLNs dispersions and produced highly stable dispersion system. 4.4. % Entrapment Efficiency Drug encapsulation efficiency (% EE) was highest and lowest when SLNs were prepared with poloxamer F 127 (>85 %) and tween 80 (<82 %) respectively as given in Table 1. SLNs prepared with poloxamer F 68 also exhibited EE > 80%. % EE decreased as follows: poloxamer F 127 > poloxamer F 68 > tween 80. However, % EE of SLNs significantly increased with increasing surfactant concentration till 0.25%. This might be due to the efficient loading and retention of drug molecules within the nanoparticle matrix or nanoparticle surface at higher surfactant concentration. % EE of SLNs prepared at drug: lipid ratio 1: 10 were not significantly different at 0.25% and 0.50% surfactant concentration. This might be due to the presence of enough lipids to encapsulate the drug molecules. % EE increased with increasing lipid concentration. As the drug: lipid ratio decreases, the lipid concentration increases, so there was an increase in % EE till the ratio of drug: lipid was 1:10 after which there was no significant change. This is reasonable, as higher amount of lipid was available to encapsulate drug molecules at higher lipid concentration. % EE of SLN5 was very high (93.8 ± 1.8%). The high % EE might be beneficial to reduce the skin irritation of drug by avoiding the direct contact between drug and skin surface. 4.5. Differential scanning calorimetry (DSC) analysis
12
DSC analysis was used to characterize the state and degree of crystallinity of lipid dispersions, semi-solid systems, polymers and liposomes. It allows the study of the melting and crystallization behaviour of crystalline materials like solid lipid nanoparticles. As illustrated in Fig.1. The thermogram of Amphotericin B (Fig.1 A, endothermic peak at 211.69°C) showed Thermogram of Precirol ATO 5 (Fig.1 B, endothermic peak of melting at 56.98°C and 61.81°C), Poloxamer F 127 (Fig.1 C, endothermic peak of melting at 54.79°C ), sucrose (Fig.1 D, endothermic peak of melting at 190.31°C and 230.94°C), blank SLNs lyophilized with sucrose (Fig.1 E, endothermic peak of melting at 52.52°C, 187.96°C and 227.36°C), Amphotericin B loaded SLNs lyophilized with sucrose (Fig.1 F, endothermic peak of melting at 56.59°C, 187.13°C and 224.25°C), Amphotericin B loaded SLNs lyophilized without sucrose (Fig.1 G, endothermic peak of melting at 53.33°C and 57.14°C) and physical mixture of drug, Precirol ATO 5 and poloxamer F127 (Fig.1 H, endothermic peak of melting at 55.88°C). Endothermic peak of melting of drug was not seen in Amphotericin B loaded SLNs lyophilized without sucrose and physical mixture, indicating that the physical state of Amphotericin B changed from crystalline to amorphous. Hence it could be concluded that in Amphotericin B SLNs, drug was present in amorphous state and may have been homogeneously dispersed in the lipid matrix [21]. However, when SLNs were lyophilized with sucrose, it showed interference of sucrose in SLNs formulation. So, Fig. 3 F showed the endothermic peak of sucrose which indicates the crystalline state of the formulation. From the Fig. 3 G it can be seen that during the formulation of Amphotericin B SLNs, reduction in the endothermic peak of Precirol ATO 5 was observed. This can be attributed to the change in crystal lattice and increased number of lattice defects in the lipid crystal [22]. 4.6. FTIR study The FTIR spectrum showed characteristic peaks of Amphotericin B such as -OH stretch (strongly H- bond) (3368 cm-1), CH stretch (polyene) (3009 cm-1), N-H2 in plane band (1627 cm1
) and Polyene C=C bond (1554 cm-1) shown in Fig. 2A. In the IR spectrum of lyophilized
SLNs, peaks corresponding to Amphotericin B disappeared or broad peaks of Amphotericin B – OH groups shortened. This indicated drug entrapment in lipid matrix as shown in 2B. 4.7. PXRD study The powder X-ray diffractogram of Amphotericin B exhibits sharp peak at 2θ scattered angle of 13.88º, 21.29º and 21.58º, which indicates crystalline nature of Amphotericin B shown 13
in Fig. 3A [23]. The characteristic peaks for Amphotericin B were absent in the SLNs PXRD pattern suggesting that Amphotericin B was not in crystalline form in SLNs shown in Fig. 3B. Therefore, the study demonstrates that the crystalline structure of the Amphotericin B was changed after formation of SLNs with lipid and surfactant. 4.8. Scanning Electrons Microscope (SEM) SEM image of Amphotericin B lyophilized SLNs shown in Fig. 4. SEM image shows that the Amphotericin B SLNs are discrete, spherical, and regular in shape. There was a large particle due to aggregation of lyophilized SLNs formulation. 4.9. In vitro antifungal test The antifungal activity was measured by Trichophyton rubrum fungal species by growing it on Sabouraud’s agar. The antifungal activity results are given in Table 2. The zone of inhibition after 72 hrs was 1.28 ± 0.07, 1.76 ± 0.09, 2.81 ± 0.13 and 2.0 ± 0.10 mm for plain drug gel, SLN4 gel, SLN5 gel and SLN6 gel, respectively. The zone of inhibition was greater for SLN5 due to the higher concentration of drug as well as higher penetration power of SLNs. The zone of inhibition was according to SLN5 > SLN6 > SLN4 > plain drug gel. This indicated the SLN5 gel was more effective as compared to other formulations. 4.10.Ex vivo drug release and skin retention study To assess the influence of the lipidic nanoparticles on the permeation and accumulation of drug into the skin, ex vivo skin permeation and retention studies were performed using hairless rat skin by Keshary chein diffusion cells. All SLN gel formulations followed Higuchi model indicating that the drug released from SLN gel was by diffusion in sustained manner. In dermatological treatment, improving the efficacy demands high drug levels in the skin. With solid nanoparticle dispersion gels, a greater quantity of drug remained localized in the skin, with more amounts penetrating into the receptor compartment as compared with conventional gel. SLN5 gel had the highest uptake of Amphotericin B in skins when compared with conventional gel, SLN4 gel and SLN6 gel. Thus, drug localizing effect in the skin seems possible with SLNs. The amount of permeated drug was lower in case of conventional gel, while SLNs based formulation represented the more amount of permeated drug (Fig. 5 A). The data indicates that lipidic nanoparticles clearly increased the drug permeation through the skin. It may appear that the structural composition of solid lipid nanoparticles was responsible for rate limiting effect in drug permeation. The cumulative amounts of Amphotericin B from conventional gel, SLN4 gel, 14
SLN5 gel, and SLN6 gel at 24 hrs were 9.24 µg/cm2, 19.84 µg/cm2, 22.34 µg/cm2 and 20.72 µg/cm2 respectively. In other words, conventional gel showed lower drug permeation, in comparison to lipidic nanoparticular systems (Fig. 5A). Permeation parameters are given in Table 2. Data indicates that drug flux were 0.39± 0.01 μg/cm2/hr, 0.83± 0.01 μg/cm2/hr, 0.93± 0.01μg/cm2/hr and 0.86± 0.02 μg/cm2/hr for conventional gel, SLN4 gel, SLN5 gel, and SLN6 gel respectively. Amp B SLN5 gel shown much better drug retention in skin compared Amp B microemulsion [16]. Drug retained in skin was approximately 23 % after 24 hrs in microemulsion whereas in Amp SLN5 gel it was 36.48 after 24 hrs. In fact, lipidic nanoparticles have shown to enhance the penetration (dermal delivery) especially into the upper skin layers [24], hence creating a reservoir which is able to prolong the skin residence time. Amp B SLN5 gel showed more drug deposition as compared Amp B microemulsion. This indicate that the Amp B SLN5 gel was more beneficial for topical fungal diseases because of more amount of drug reserved on skin, so more contact with the fungal species and therefore more effectively treat the fungal species on the skin layer. The increased skin delivery by SLNs include the large surface area due to small particle sizes, an occlusive effect and a penetration enhancer effect due to presence of surfactants. This property of the nanoparticles providing a better permeation condition by maintaining the skin hydration level at higher point. Bouwstra [25] reported that this behavior might be expected, as SLNs system based on lipid composition are more similar to subcutaneous lipids, in contrast to conventional gel. Thus, localized delivery of Amphotericin B from SLNs into skin tissue is affected, leading to an effective treatment of cutaneous fungal infection. The mechanism by which lipids increase skin permeability appears to involve disruption of the densely packed lipids which fill the extracellular spaces of the subcutaneous layer [26]. Their effect as penetration enhancers, after addition to semisolid vehicles, have been studied on a variety of drugs [27], In order to assess the skin uptake and penetration of Amphotericin B from SLNs, the in vitro permeation ability through skins and into skins were performed using diffusion cells [28]. SLNs can increase the uptake of drug in skin [29] and the skin targeting effect was disclosed by fluorescence microscopy [30]. SLNs with small diameters are advantageous to improve the penetration of nanoparticles into skins and the controlled release of SLNs may induce the increase of drug accumulation [31]. 15
4.11. Skin irritation study Conventional gel therapy is associated with noticeable skin irritation, which strongly restricts its applicability and acceptability by the patients. Basically, the Amphotericin B delivery system should be able to reduce irritation. The skin-irritation studies indicated that SLN4 gel, SLN5 gel and SLN6 gel exhibited considerably no irritation as compared to conventional gel, even after 48 hrs of application. Results of skin irritation study are given in Table 3. The primary irritation index (PII) was found to be 0.33 ± 0.33, 0.11 ± 0.19, 0.22 ± 0.38 for SLN4 gel, SLN5 gel and SLN6 gel, respectively showing no irritation. The primary irritation index (PII) was found to be 0.00 ± 0.00, 2.11 ± 1.01 and 2.89 ± 1.71 for control gel (placebo), formalin and conventional gel, respectively. Therefore, the developed SLNs formulation resulted in no erythema or edema on the intact and abraded rabbit skin, as compared to the conventional gel. Thus the formulation can be classified as a non-irritant to the rabbit skin. 4.12. Stability study Amphotericin B SLN exhibited a good stability during the period of 3 months. No significant change of clarity and phase separation was observed. Amphotericin B SLN had a good physical stability. This may imply that the transition of dispersed lipid in SLNs dispersion from β’ form to stable β form might occur extremely slowly. Generally, the SLNs are prepared from solid lipids or blends of solid lipids and, after the preparation by solvent diffusion technique; the particle crystallizes, at least partially, in higher energy modifications α and β’. During storage, these modifications can transform to the low-energy, more-ordered β modification. Due to its high degree of order, the number of imperfections in the crystal lattice is reduced thus leading to drug expulsion. No degradation of Amphotericin B in the formulations was observed. 5. Conclusion Amphotericin B SLNs were formulated for topical application and it was clearly demonstrated that SLNs were effective for topical delivery of Amphotericin B. SLN5 showed higher skin deposition, lower skin irritation and better anti-fungal activity. The developed system may provide better remission from the disease due to localized delivery with minimal side effects and the data from in vitro study were promising but further evaluation is needed to elucidate the clinical efficacy of this topical dosage form.
16
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Figure captions
Fig. 1. DSC analysis of (A) Amphotericin B, (B) Precirol ATO 5, (C) Poloxamer F 127, (D) Sucrose, (E) Drug free SLNs lyophilized with sucrose, (F) Drug loaded SLNs lyophilized with sucrose, (G) Drug loaded SLNs lyophilized without sucrose and (H) Physical mixture
20
Fig. 2. FTIR study of (A) Amphotericin B, (B) Lyophilized SLNs
21
Fig. 3. PXRD study of (A) Amphotericin B and (B) Amphotericin B lyophilized SLNs
22
Fig. 4. SEM images of Amphotericin B SLNs
23
Fig. 5. (A) Drug permeation study (B) Skin retention study of SLN4 gel, SLN5 gel, SLN6 gel and conventional gel (Mean ± SD (Standard Deviation), n=3)
24
Table 1 Different SLN formulations of Amphotericin B with different evaluation parameters. Formulation Drug : Surfactant
Surfactant
Solvent
Purified Particle Size* Zeta
PDI*
%
Code
Lipid
Conc.
Volume
Water
(nm)
Entrapment
Ratio
(% w/v)
(ml)
(nm)
Potential* (mV)
Efficiency*
SLN1
1 : 7.5
Pluronic F 68
0.25 %
10
40
268.3 ± 3.0
-16.02 ± 1.13 0.25 ± 0.05
80.1 ± 1.3
SLN2
1 : 10
Pluronic F 68
0.25 %
10
40
242.0 ± 3.3
-12.64 ± 1.93 0.23 ±0.03
86.4 ± 0.5
SLN3
1 : 12.5
Pluronic F 68
0.25 %
10
40
224.6 ± 2.3
-09.17 ± 1.26 0.27 ± 0.10
83.2 ± 1.0
SLN4
1 : 7.5
Pluronic F 127
0.25 %
10
40
163.1 ± 2.0
-20.95 ± 1.58 0.29 ± 0.05
85.6 ± 2.1
SLN5
1 : 10
Pluronic F 127
0.25 %
10
40
111.1 ± 2.2
-23.98 ± 1.36 0.13 ± 0.04
93.8 ± 1.8
SLN6
1 : 12.5
Pluronic F 127
0.25 %
10
40
133.9 ± 3.0
-15.73 ± 2.72 0.19 ± 0.09
90.0 ± 1.7
SLN7
1 : 7.5
Tween 80
0.25 %
10
40
415.8 ± 4.4
-09.55 ± 2.37 0.31 ± 0.06
72.7 ± 3.8
SLN8
1 : 10
Tween 80
0.25 %
10
40
373.2 ± 4.9
-06.12 ± 1.75 0.34 ± 0.08
81.9 ± 2.4
SLN9
1 : 12.5
Tween 80
0.25 %
10
40
399.2 ± 3.6
-05.69 ± 1.09 0.28 ± 0.04
78.6 ± 3.0
*(Mean ± Standard Deviation, n=3)
18
Table 2 In vitro antifungal activity and percutaneous permeation parameters of various Amphotericin B SLN gel formulations Formulation Code
Zone of Inhibition (diameter in mm)*
Percutaneous permeation parameters*
24 hrs
Jss flux (μg/cm2/hr)
48 hrs
72 hrs
Permeability Co-efficient Kp (cm/hr) × 10-3
Plain Drug Gel
0.53 ± 0.04
0.86 ± 0.06
1.20 ± 0.07
0.39± 0.01
0.78± 0.02
SLN4 Gel
0.61 ± 0.06
1.19 ± 0.05
1.76 ± 0.09
0.83± 0.01
1.65± 0.01
SLN5 Gel
1.22 ± 0.11
1.88 ± 0.09
2.81 ± 0.13
0.93± 0.01
1.86± 0.02
SLN6 Gel
0.94 ± 0.08
1.70 ± 0.06
2.05 ± 0.10
0.86± 0.02
1.73± 0.02
*(Mean ± Standard Deviation, n=3)
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Table 3 Mean erythemal scores and PII observed for various Amphotericin B formulations obtained at the end of 24, 48 and 72 h. Formulation Code
Erythemal Score* 24 hrs
48 hrs
72 hrs
PII
Conventional Gel
1.00 ± 0.00
2.33 ± 0.58
3.00 ± 1.00
2.11 ± 1.01
SLN 4 Gel
0.00 ± 0.00
0.33 ± 0.58
0.66 ± 0.58
0.33 ± 0.33
SLN 5 Gel
0.00 ± 0.00
0.00 ± 0.00
0.33 ± 0.58
0.11 ± 0.19
SLN 6 Gel
0.00 ± 0.00
0.00 ± 0.00
0.66 ± 0.58
0.22 ± 0.38
Control Gel (Placebo)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
Formalin
1.00 ± 0.00
3.33 ± 0.58
4.33 ± 0.58
2.89 ± 1.71
*(Mean ± Standard Deviation, n=3)
20