- Email: [email protected]
Fatty alcohol containing nanostructured lipid carrier (NLC) for progesterone oral delivery: In vitro and ex vivo studies
Accepted Manuscript Fatty alcohol containing nanostructured lipid carrier (NLC); Strategy to fade away progesterone oral delivery drawbacks Mohammed Elmowafy, Khaled Shalaby, Mohamed M. Badran, Hazim M. Ali, Mohamed S. Abdel-Bakky, Ibrahim El-Bagory PII:
S1773-2247(17)30891-2
DOI:
10.1016/j.jddst.2018.03.007
Reference:
JDDST 600
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 14 October 2017 Revised Date:
23 January 2018
Accepted Date: 5 March 2018
Please cite this article as: M. Elmowafy, K. Shalaby, M.M. Badran, H.M. Ali, M.S. Abdel-Bakky, I. ElBagory, Fatty alcohol containing nanostructured lipid carrier (NLC); Strategy to fade away progesterone oral delivery drawbacks, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.03.007. 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.
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Fatty alcohol containing nanostructured lipid carrier (NLC); strategy to fade away progesterone oral delivery drawbacks Alternative title:
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Fatty alcohol containing nanostructured lipid carrier(NLC) for progesterone oral delivery: In vitro and ex vivo studies
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Abdel-Bakky5, Ibrahim El-Bagory1
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Department of Pharmaceutics, College of Pharmacy, Aljouf University, Skaka, P.O. Box 2014,
KSA 2
Department of Pharmaceutics and Ind. Pharmacy, Faculty of Pharmacy (Boys), Al-Azhar
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University, Nasr City, Cairo, Egypt 3
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Mohammed Elmowafy1,2*, Khaled Shalaby1,2, Mohamed M. Badran2,3,Hazim M. Ali4, Mohamed S.
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, P.O.
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Box 2457, KSA
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Department of Chemistry,, College of Sciences, Aljouf University, Skaka, P.O. Box 2014, KSA
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Department of Pharmacology and Toxicology, Faculty of Pharmacy (Boys), Al-Azhar University,
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Nasr City, Cairo, Egypt
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*Corresponding author
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Tel.: +966541869569
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Email:[email protected], [email protected]
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Abstract
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progesterone. Fatty alcohols (cetyl, cetostearyl and 1:1 mixture) containing NLCs were thought to
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be one of the most relevant systems based on progesterone drawbacks as acid sensitivity and poor
29
bioavailability. Formulations were prepared characterized for physicochemical evaluation, in vitro
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release, short term stability, interaction possibility and ex vivo permeation studies. The results
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showed nanorange particle size with major influence of fatty alcohol concentration. In vitro release
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results suggested protection of formulation from gastric environment with controlling the drug
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This study aims to overcome most of the obstacles that that hinder oral bioavailability of
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interaction among fatty alcohol, stearic acid and progesterone. Ex vivo results showed that
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formulations significantly (p˂0.05) permeated through duodenum when compared with suspension
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after 8h. The results collectively indicated that the cetyl alcohol containing NLC was a potential
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drug delivery system for oral administration of progesterone.
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release as a function of fatty alcohol concentration. Infrared and thermal analyses showed
Key words: Progesterone; NLC; Fatty alcohol; Controlled release; Ex vivo
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1. Introduction
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mammals particularly in female reproduction. Physiologically, PG is essential for maintenance
51
during menstrual cycle and all stages of normal pregnancy for development of the foetus. Irregular
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PG levels produces many disturbances as palpitations, headaches, hotness, vaginal dryness and
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breast tenderness [1]. In addition PG deficiency may be a predisposing factor in endometrial
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hyperplasia and endometrial cancer [2]. PG is indicated in the prevention of preterm labor in
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women at risk, especially women with history of previous spontaneous preterm labor [3]. Oral
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Progesterone (PG; Fig. 1), is a natural steroid hormone with multifunctional roles in
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water solubility (log P of 3.87; class II according to Biopharmaceutical Classification System),
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short half life due to extensive hepatic first pass effect [4] and enzymatic degradation within the
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gastrointestinal tract [5].Administration of high PG doses is not tolerated by patients and side
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effects (as somnolence and dizziness) appear. To address these biopharmaceutical challenges
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associated with PG, many approaches were developed such as nanosphere [6], chitosan [7]
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administration of PG is accompanied by many drawbacks including poor bioavailability due to poor
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into another route of administration as sublingual [5] and transdermal delivery using nano-
64
architecture liquid crystalline particles [9].
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incorporation and self-nanoemulsifying drug delivery systems (SNEDDS) [8] or even by shifting
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(NLC), is consisted of solid lipid and liquid lipid blend which in turn leads to imperfections
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between the lipids and creates spaces to incorporate drugs in the matrix, the gate to provide higher
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drug payload than the first generation (solid lipid nanoparticles; SLN) and prohibit drug expulsion
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during storage [10, 11]. In addition, NLC surpass many other colloidal carriers in terms of
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inexpensive required materials, easiness of manufacture, lipid biocompatibility, controlling drug
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release and stability. Particularly, NLC preponderate polymeric nanoparticles in terms of safety and
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biocompatibility (considered the lowest toxic nanoparticles when administrated in vivo [12]) of raw
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The second generation of lipid nanoparticles, the so-called nanostructured lipid carriers
ACCEPTED MANUSCRIPT materials and liposomes in term of better ability to control the release of encapsulated drug. Fatty
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alcohols were found to control the release of some drugs such as theophylline [13] and felodipine
75
[14].
76 77
fatty alcohol containing NLCs in order to (1) improve oral bioavailability by dissolution
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enhancement, (2) control the release and (3) protect the drug from gastrointestinal degradation.
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Stearic acid was selected as long chain fatty acid solid lipid while sesame oil was the liquid lipid.
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The PG loaded NLCs were formulated and characterized according to physicochemical evaluation.
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Based on the aforementioned data, the aim of the current work was to incorporate PG into
2. Materials and Methods 2.1. Materials
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The chosen formulations were subjected to ex-vivo permeation studies.
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cetyl alcohol, cetostearyl alcohol and polyethylene glycol (PEG) were purchased from Lobal
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PG was purchased from BDH chemicals (England). Sesame oil, stearic acid, Tween 80,
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Techno Pharmchem (India). Sodium azide was purchased from Sigma Aldrich (Germany). All the
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other chemicals, reagents and solvents used were of analytical reagent grade.
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Chemie (India). PEG1500 was purchased from MERK (Germany). PEG4000was purchased from
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2.2. Preparation of PG loaded NLCs
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The NLCs were prepared by high shear homogenization followed by sonication. Briefly, the
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lipid phase (10% w/v from total formulation) consisted of 2% stearic acid (solid lipid) and 8%
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sesame oil (liquid lipid) content. PG was firstly dissolved in sesame oil, heated at 65°C and mixed
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with preheated solid lipid. Different amounts of fatty alcohols (cetyl alcohol, cetostearyl alcohol or
96
1:1 mixture; Table1) were added to molten lipid phase along with PEG mixture
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(PEG400:PEG1500:PEG400; 1:1:1). Aqueous phase (containing 3% Tween 80) was heated at 65°C
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(Ultra Turrax, IKA Eurostar 40 digital, GmbH, Germany) at 1300 rpm for 15 min in water bath.
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Hence, the formulations were subjected to homogenization using high shear homogenizer (Yellow
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line DI25 basic, Germany) at 12,000 rpm for 10 min to get smaller particle size. The formulations
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were subjected to bath sonication (BranSonic, 5510E-DTH, Mexico) for 5 min at 60 W to get
103
uniform size distribution. The obtained dispersions were cooled at room temperature and stored at
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4°C till use.
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for 5min and slowly added to the melted lipid under continuous stirring by a high-speed stirrer
2.3. Particle size and surface charge
106 107 108
loaded NLCs were carried out using dynamic light scattering technique (Zetasizer Nano ZS,
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Malvern Instruments, UK). All samples were properly diluted with double distilled water at room
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temperature. All the measurements were performed in triplicate.
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Determination of average particle size, PDI and zeta potential (surface charge) of the PG
2.4. Chromatographic conditions
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Dionex UltiMate 3000UHPLC+ ) focused standard systems equipped with quaternary analytical
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Analysis of PG concentration was carried out using UHPLC system (Thermo Scientific
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autosampler,TCC-3000SD thermostatted column compartment, SR-3000 solvent rack without
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degasser and DAD-3000 diode array detector. The apparatus was interfaced to a DELL PC
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compatible computer with a Chromeleon™ 7.2 Chromatography Data System. The separation was
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carried out on an ACCLAIM™ 120 C18 column, 4.6 mm×150 mm, 5µm with a mobile phase
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consisting of acetonitrile, methanol and 20mM citric acid in volume ratio of 45:45:10 at a flow rate
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of 1 ml/min. The wavelength for PG detection was set at 245 nm, with an injection volume of 10 µl
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at 25°C.
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pump LPG-3400SD with degasser,WPS-3000TSL analytical split-loop thermostatted well plate
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ACCEPTED MANUSCRIPT 126 2.5. Validation of assay method
127 128
accuracy, the limit of detection (LOD), limit of quantification (LOQ) and system suitability. The
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linearity of the method was studied using different concentrations covering the range from 0.05 to
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Validation of UHPLC method for determination of PG included linearity, precision,
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30 µg/ml) by comparing the values for the relative standard deviation: RSD= (SD/ mean measured
132
concentration) × 100. Accuracy was expressed as the percentage recovery and was determined by
133
the following equation:
× 100,
(1)
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Recovery (%) =
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90 µg/ml at five replicates for each level. Precision was estimated at three levels of PG (5, 15 and
134 135 136
LOD and LOQ values were estimated based on the standard deviation of intercept (SDb) and slope
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(a) of calibration curve, according to the following formulas;
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LOQ =
! ×"."
,
! ×%&
(2)
, (3)
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LOD =
139 140 141
and peak asymmetry for 50 µg/ml of PG.
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The system suitability was tested by determining peak retention time, peak area, theoretical plates
2.6. Drug encapsulation efficiency
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The entrapment efficiency (EE) is of major importance as the drug release from system is
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greatly influenced by amount of drug encapsulated inside that system. EE% was determined by
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indirect method basing upon precipitation of NLC upon adjusting pH value of the NLC dispersion
148
to 1.2 (by adding 0.1N HCL) followed by centrifugation at 11,000 rpm for 45 min at 16°C [15]. The
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supernatant and pellets wash out were filtered through 0.45 µm filter and diluted appropriately with
150
ACCEPTED MANUSCRIPT methanol and analyzed by above mentioned UHPLC method. The EE% of PG loaded NLCs was
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then calculated according to the following equation (4):
152 153
)(
* +
,
+-
. * +
* +
,
,
+- /+0
+-
)
× 100,
(4)
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EE% =
2.7. In vitro PG release
154 155 156 157 158
described by Yang et al. [16] with some modifications. The receptor compartment consisted 500ml
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In vitro release of PG from NLCs was evaluated by the dialysis bag diffusion technique
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bags was soaked in PBS overnight prior experiment. PG loaded NLCs (equivalent to 2mg of PG)
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were placed dialysis bag (molecular weight cut off: 12–14 kDa, Livingstone, NSW, Australia),
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containing 0.1N HCl to simulate gastric pH (1.2) and then the bags were sealed at both sides. The
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bags were immersed in receptor compartment placed in USP apparatus I vessels automatically
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adjusted at 37± 2°C and 100 rpm. At predetermined intervals of time (15, 30, 60 and 120 min),
165
aliquots of 2 ml were withdrawn from the diffusion media and HPLC analyzed. Equal amounts of
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phosphate buffered saline (PBS) of pH-7.4 to maintain sink conditions. Membrane of dialyzing
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constant receptor volume. After 2 h, the bags were opened and the internal pH was adjusted into 6.8
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fresh PBS were added to diffusion media immediately after withdrawal time point to maintain
to simulate intestinal pH. Samples of 2ml were withdrawn at regular time intervals (30, 60, 120, 180
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and 240 min) to be analyzed and the same volume was replaced by fresh release medium. All the
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experiments were done in triplicate, and the average values were taken.
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At the end of experiment, the kinetic parameters for the in vitro data were determined in
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order to estimate the best fit to distinct kinetic model (zero, first or Higuchi order) to determine the
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PG release mechanism from NLC formulations. Stating the proper mode of release is based on the
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ACCEPTED MANUSCRIPT Correlation Coefficient Parameter (r) for parameters involved, where the highest correlation
175
coefficient represents the actual mode of the release [17].
176 177
2.8. Stability
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In order to assess the physicochemical stability of the prepared systems, particle size, PDI,
179 180
months when formulations stored at 4°C.
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zeta potential and EE% were considered as stability criteria. They were monthly measured for 3
2.9. Morphology
182 183 184
6360 LV, JEOL, Tokyo, Japan). Formulations were freeze-dried (Alpha 1-4LDPlus,GmbH,
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Osterode am Harz, Germany) and fixed on carbon tape and sputter-coated with a thin gold layer.
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The formulations were then scanned and photographed.
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2.10. Thermal analysis
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The shape of PG loaded NLCs was observed by scanning electron microscopy (SEM; JSM-
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interaction possibility using differential scanning calorimeter (DSC) tool. Accurately weighed 8mg
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In addition to individual components, freeze-dried NLC2 formulations were examined for
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(Shimadzu Corporation, Tokyo, Japan). Standard empty aluminum pan was used as reference and
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the thermograms were recorded between 30°C and 250°C at a scan rate of 10°C/min under nitrogen
194
gas.
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of samples were placed in aluminum pans, crimped and analyzed using a Shimadzu DSC-60
196 2.11. FTIR
197
Interaction between PG and NLC components (stearic acid and fatty alcohol) was studied by 198 Fourier transform infrared (FT-IR) in the transmission mode. FT-IR analysis was performed between 199 4400 and 350 cm−1 at a resolution of 4 cm−1.
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2.12. Ex vivo permeability studies
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NLCs and conventional suspension. Adult female albino rabbits, weighing 1.75–2kg, were used in
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This study was carried out in order to investigate the intestinal permeability of PG from
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Aljouf University (Skaka, Northern region, KSA) and housed under conventional laboratory
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conditions with free access to food and water till sacrifice. The study was conducted in accordance
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with ethical procedures and policies approved by the Local Committee for Research Bioethics,
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Aljouf University (Ethical code; LCBE8/3/37/38).
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the experiment. They were obtained from the Animal House Colony of College of Pharmacy,
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PBS to get rid of the mucous and gut wastes. Parts of 4cm long were cut and one side was tied up.
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Besides PG suspension (PG was dispersed in 2% carboxymethylcellulose sodium), formulations of
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NLC1 and NLC2 (equivalent to 2 mg of PG) were placed in intestinal parts and the remaining side
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was tied with cotton thread. The sacs were placed in USP apparatus vessels containing 500 ml
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Tyrode solution (pH 7.4) of 0.05% sodium azide and maintained at 37± 2°C and stirred at
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100rpm.Samples of 2mlwere withdrawn at regular time intervals (0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6,7
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Animals were sacrificed by ether inhalation and the duodenal part was excised and flushed with
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compartment. All the experiments were done in triplicate, and the average values were taken.
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and 8 h) and UHPLC analyzed. At each time point 2 ml of fresh solution were added to receptor
Cumulative amount of PG permeating through intestinal wall per unit area was plotted against time.
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Steady state flux (Jss) was calculated from the slope of the linear portion of the plot.
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Permeability coefficient was also calculated using the following equation (2):
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Kp =
344 , (5) Ci 223
ACCEPTED MANUSCRIPT Where Kp was the permeability coefficient, and Ci was the initial concentration of PG in donor
224
compartment. The efficacy of NLCs to permeate PG through intestinal wall was also determined by
225
enhancement ratio (ER). ER was also calculated using the following equation (3) [18]:
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Jss of NLC , (6) Jss of suspension
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ER =
2.13. Statistical Analysis
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analysis of obtained results was evaluated by one-way ANOVA followed by Tukey-Kramer test
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All results in this work are expressed as a mean ± standard deviation (SD). The statistical
using the StatPlus software (Analyst Soft Inc., USA). Difference at P < 0.05 was considered to be
232
significant.
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3. Results and discussion
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3.1. Rationale behind components and formulations
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of PG. Of course and as previously mentioned, NLCs themselves behave as controlled release
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system, but their constituents largely affect that behavior. Stearic acid, a long chain fatty acid, was
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chosen as solid lipid which was reported as the degree of metformin HCl release was stearic acid
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The components of NLCs were selected on the basis of the purpose; controlling the release
concentration dependent [19]. Sesame oil, plant oil containing long chain triglycerides, was selected
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as liquid lipid based upon PG solubility, slow dissolution [20] and safety terms. The addition of
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PEG to the formulation aimed to increase NLC stabilization and enhance bioavailability and not to
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improve PG solubilization as PG completely dissolved in sesame oil. It was also reported that PG
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showed a declined solubility in PEG400 [21]. The wisdom of using of PEG in a mixture of different
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grades was firstly to control the release rate [22] of PG. Secondly to minimize the gastrointestinal
246
absorption of PEG as well as prevent the induced toxicity. Oral absorption and toxicity were
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type and concentration, NLC formulations were designed. Two fatty alcohols were selected (cetyl
249
alcohol and cetostearyl alcohol) with 0.125, 0.25 and 0.5% concentrations to study the effect of
250
different concentrations on physicochemical characteristics. It should be noted that higher
251
concentration of fatty alcohol than 0.5% produced very viscous and hardly flowable formulations
252
simultaneously upon cooling.
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reviewed to be inversely proportional to molecular weight of PEG [23]. As function of fatty alcohol
3.2.1.UHPLC method development and optimization
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3.2. Chromatography
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Separation of PG was achieved on ACCLAIM™ 120 C18 column (maintained at 25 ºC) and
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compositions of acetonitrile, methanol and citric acid were studied. 20 mM citric acid, methanol
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and acetonitrile (45:45: 10% v/v) resulted in the good chromatographic separation of progesterone.
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The chromatogram of separation of PG measured at the optimized UHPLC conditions is shown in
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isocratic elution with a mobile phase consisting of acetonitrile, methanol and citric acid. Several
262
3.2.2. Method validation
263
3.2.2.1. Linearity
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Fig. 2A. PG peak was observed at the retention time of 3.41 min.
264 265 266
µg/ml. The calibration curve was created by plotting the peak area against the concentration (Fig.
267
2B), and the average value of linear regression equation for five replicates was y=0.567(±0.0012) x
268
+0.153(±0.0077) with an average correlation coefficient (r2) value of 0.9995(± 4.4x10-5).
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3.2.2.2. Precision and accuracy
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The linearity of calibration curve of progesterone was obtained over a range of 0.05-90
Intra-day accuracy and precision were performed by analyzing five replicates at three
271
concentration levels: 5, 15 and 30 µg/ml in the same day as listed in Table 2. Inter-day accuracy and
272
precision were evaluated on three successive days at the same concentration levels of PG as shown
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ACCEPTED MANUSCRIPT in Table 2. From the listed data, we can see that the established method is accurate and precise.
274 275
3.2.2.3. LOD and LOQ
276 277
and 0.136 µg/ml respectively. The low values of LOD and LOQ refer to the good sensitivity of
278
developed method.
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LOD) and LOQ of the proposed method for PG determination were found to be 0.045 µg/ml
3.2.2.4. System suitability
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System suitability was performed by calculating the peak retention time, peak area, peak asymmetry
280 281 282
time, peak area, peak asymmetry and number of theoretical plates were found to be 3.41± 0.004,
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28.68± 0.35, 1.1± 0.016 and 9251.33± 30.23 respectively. These results confirm the suitability of
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the proposed method and ensuring system performance.
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3.3. Characterization of PG loaded NLCs
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and number of theoretical plates for 50 µg/ml of PG (six replicate). The value of peak retention
286 287 288
formulations are depicted in Table 1. The values of particle size lied between 137.2±4.1 and
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The results of particle size, PDI, zeta potential and EE% of freshly prepared NLC
290
that addition of fatty alcohols to NLC enlarged the particle size except NLC 2 which contained the
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228.2±5.6 nm which confirmed nanometric range of all investigated formulations. It was noticed
smallest amount of cetyl alcohol. Cetyl alcohol containing NLCs exhibited significant smaller
292
particle size than cetostearyl alcohol containing NLCs (p ˂ 0.05) while formulations containing
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cetyl and cetostearyl alcohols mixture showed insignificant (p > 0.05) particle size values
294
differences. Cetostearyl alcohol consists of mixture of 50-70% stearyl alcohol (C-18) and 20-35%
295
cetyl alcohol (C-16) as well as small amount of myristyl alcohol. Longer carbon chain of
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cetostearyl alcohol might be the cause of larger particle size [24].
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Bandyopadhyay and coworker concluded that stearic acid (fatty acid) based system
298
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These results do not contradict with our study as the other study formulated the systems completely
300
stearic acid or cetyl alcohol whereas our study formulations based upon addition of fatty alcohols
301
(with different amounts) to stearic acid based system. PDI results reveal homogeneity of particle
302
distribution (˂0.35) as typical of monodispersed systems [26].
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produced higher particle size when compared with cetyl alcohol (fatty alcohol) based system [25].
304
via electrostatic repulsion which in turn prohibits their aggregation. All investigated samples
305
showed markedly negative charge and ranged from -22.1±1.47 to -28.1±3.4 mV without significant
306
difference among formulations. This means that addition of fatty alcohols to NLC did not influence
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Zeta potential determines surface charge of the particles which greatly affects their stability
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Tween 80 and PEG. The EE% of PG when loaded in nanostructured formulations was found to
309
vary between 93.7±2.7 and 97.2±1.5% (Table 1).These satisfying values of EE% resulted from high
310
solubility of PG in sesame oil which aided to entrap the large amount of PG and decreased the drug
311
crystallinity which helped better stability and suitability for the controlled release [27]. It should be
312
mentioned that the effect of fatty alcohols on EE% was negligible.
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surface charge the resulting formulations. The negative charge may be attributed to presence of
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3.4. In vitro PG release and release kinetics
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In vitro release of PG from the NLC formulations was assessed using a USP dissolution apparatus and are graphically represented in Fig. 3 (A and B). The sink conditions were maintained using 500 ml PBS (pH 7.4) as the solubility of PG was predetermined in receptor compartment of 0.0198±0.003 mg/ml. The method used in this article differs from both direct placing of nanoparticles in receptor media and conventional dialysis bag method. Sampling from the former results in loss of nanoparticles and decreasing the concentration of the donor while in the later the donor compartment is not diluted which does not correlate the in vivo behavior. In the current method, two drawbacks were overcome by placing nanoparticles formulations in dialysis bag and
314 315
ACCEPTED MANUSCRIPT diluting them by in vivo simulating media (without enzymes). The rationale behind that was to consider the media inside bag simulating gastric (0.1N HCl) and intestinal (pH=6.8) environments whereas the media outside simulating circulation (pH=7.4). Therefore release pattern by this way can largely reflect in vivo release behavior. From Fig. 3, we can propose that at the end of first stage
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(in acidic environment), the highest amount of PG released was less than 19% (NLC1 and NLC2) without initial burst release pattern which confirmed the results of EE% (above 93%). These results also were promising in protecting most of encapsulating PG from gastric acidic media during the
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proposed time (2h). By the beginning of the second stage, initial burst of all formulation was pronounced. NLC1 showed the highest release by the end (>77%) while NLC7 exhibited the lowest
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one (˂22%). These results imparted release of higher amount of PG in intestinal environment (the optimum site for drug absorption) with controlled release behavior. Opposite pattern might be attributed to ionization degree of stearic acid in both media. In the first stage the majority of stearic acid was non-ionized and its hydrophobicity greatly retained which in the second stage degree of
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ionization increased and hence the leakage of PG was improved. Quadir and coworkers studied the release pattern theophylline from different hydrophobic matrices. They attributed the faster release from stearic acid to the formation of hydrogen bond between free carboxylic acid and dissolution
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media [13]. The feasibility of our explanation (ionization degree) lies in that the release pattern completely varied in different pH values. It was also of worth to mention that the release of PG was
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dependent upon fatty alcohol type and concentration. Cetostearyl alcohol showed the highest degree to release retardation while cetyl alcohol showed the lowest degree. Cetostearyl alcohol has longer aliphatic carbon chain which increases its hydrophobicity and hampers wetting of the system by dissolution media [13]. Shorter length of aliphatic chain in cetyl alcohol than cetostearyl alcohol decreased the hydrophobicity and promoted PG release. Stearic acid is of higher hydrophilicity (due to carboxylic group) and molecular dimension than cetyl alcohol which in turn produces higher release from stearic acid containing NLC (NLC1). As expected, the release pattern of cetyl alcohol
ACCEPTED MANUSCRIPT and cetostearyl alcohol mixture lied in between them. The higher the concentration of fatty alcohol, lower percent of PG released after 6h. The controlled release behavior of fatty alcohols is attributed to participating in structure of lipidic particles and stabilizing the system [25]. Different kinetic models were applied to the mechanism by which PG was released from NLCs. Kinetic fits (data not
coefficient (r) than both zero and Higuchi diffusion models.
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3.5. Stability of NLCs
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shown) revealed that PG released from NLC by first order kinetic model with higher correlation
As the major storage stability problems facing lipid nanocarriers are gelation phenomena,
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particle size growth and drug leakage [28], stability estimation was performed on basis of particle size, zeta potential, PDI and EE%. Results are shown in Table 3. It is obvious that the particle size growth increased with fatty alcohol increase after 3 months. This phenomenon might be attributed to formation of hydrogen bond between hydroxyl group of fatty alcohol molecules impeded in lipid
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particles which in turn improved aggregation and enlarged particles. PDI results show higher values after 3 months than 0.3 except NLC2 and NLC3 indicating unimodal size distribution. There were no noticeable variations in zeta potential and EE% results after 3months. Collectively, NLC2
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showed minimal particle size growth, uniform particle size distribution, absence of visible particulate matter as well as reasonable zeta potential value. For that, NLC2 (as selected fatty
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alcohol containing NLC) as well as NLC1 (as non-fatty alcohol containing NLC) were selected for next studies.
3.6. Morphology Fig. 4 shows the surface morphology of PG loaded NLC2 determined by SEM after freeze drying. The SEM images showed homogeneous and rounded particles with smooth surface.
ACCEPTED MANUSCRIPT 3.7. The crystal form of PG loaded NLC To investigate the effect of incorporation of PG on its melting and crystalline state DSC analysis of pure components and lyophilized NLC2 were performed (Fig. 5). PG exhibited sharp endothermic peak at 131°C indicating the crystalline nature while cetyl alcohol and stearic acid
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showed main peaks at 51.4°C and 56.8°C respectively. Lyophilized NLC2 exhibited two apexes (at 43.5°C and 50.1°C) peak. This shift of cetyl alcohol and stearic acid peaks into lower temperature
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depressed broad peaks confirmed the concurrent melting and consequent interaction between them in lipid matrix of NLC. Similar finding was reported by Sanna and coworkers who studied the
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effect of econazole nitrate lipid nanoparticles of glycerol palmitostearate (Precirol® ATO 5) and different chain length fatty alcohols [29]. They attributed the peak depression to lattice defect which resulted from interaction between econazole nitrate and lipid. Hu and coworkers correlated the peak depression of monostearin nanoparticles to amount of added liquid lipid (Caprylic/capric
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triglycerides); the higher concentration of liquid lipid the wider solid lipid peak. This in turn augmented the less ordered mechanism which allowed more vacancy to encapsulate the drug with high loading [30]. The decrease in melting temperature of the lipids in nanoparticles when
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compared to bulk lipids might be attributed to decrease in particle size when formulated in nanorange particles. Absence of main endothermic peak of PG confirmed the presence in
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amorphous state which in turn improved PG solubility.
3.8. FTIR analysis
Interaction between drug and other NLC components was studied using FTIR tool. The FTIR spectra of pure PG, stearic acid, cetyl alcohol and NLC2 are shown in Fig. 6. In pure PG spectrum, the region below 1500cm-1 represents the finger print of PG polymorph 1 [31]. After 1500cm-1 there were four main sharp peaks; 1612cm-1 (represents aromatic C=C stretch), 1661cm-1 (represents conjugated ketone at C3 in PG structure), 1699cm-1(represents non-conjugated ketone at
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features; the first is the absence conjugated C=O at C3 in PG. The second is shifting of both nonconjugated ketone (1703cm-1) and OH (3388cm-1) peaks which collectively confirmed stable dimeric intermolecular hydrogen bonding (in the condensed phase) between PG and NLC
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components and in turn can enhance PG solubility by NLC formulation. The third is the appearance of new peak at 1744cm-1 (represents ester) which could explain the interaction between stearic acid
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and cetyl alcohol.
3.9. Ex vivo studies
As PG is class II drug, its absorption is dissolution rate limited; the first step in absorption
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process. Therefore, ex vivo studies was performed to have an insight on the capability of NLCs to enhance PG absorption (Fig. 7). Permeation profiles of PG from NLC1 and NLC2 relative to the suspension through duodenal wall are shown in Fig. 6. It was observed that the NLC1 and NLC2
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formulations significantly (p˂0.05) permeated through intestinal wall (53.4±17.5% and 67.3±8.4% respectively) when compared with suspension (34.8±6.2%) after 8h. This indicated the absorption
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superiority of NLC formulations over conventional suspension due to nanometric particles as well as the presence of lipids and surface active agent. It is also clear that incorporation of cetyl alcohol in NLC2 produced slight enhancement in extent of PG permeation when compared to NLC1 after 8h. Partitioning of cetyl alcohol between stearic acid (partly explained in DSC and FTIR sections) and biological membrane lipid affinity might be also a proposed mechanism. However, lower flux and hence Kp of NLC2 (0.00128±0.0002 and6.4±0.34 respectively), when compared to NLC1 (0.0016±0.0003 and 8.05±0.27 respectively), augmented the controlled release behavior of cetyl
ACCEPTED MANUSCRIPT alcohol containing formulation during ex vivo experiment (Table 4).However, lack of blood supply resulted in absence of drug clearance and accumulation of PG in receptor compartment. This in turn fainted controlled release behavior of NLC2. Another point of concern is that cumulative amount of PG released from cellulose membrane was much higher than that of the rabbit intestine. This might
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be attributed to difference in thickness of both barriers (100 µm of the cellulose membrane) and structural complexity of biological membranes. It is worth to mention that linearity was more pronounced (r equals 0.969 and 0.984 for NLC1 and NLC2 respectively) when the plot was
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constructed between the percentages of PG permeated against square root of time (h1/2). This
described the Higuchi diffusion model.
4. Conclusion
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indicated that PG release was controlled by a passive (Fickian) diffusion mechanism, which was
In conclusion, NLCs based on stearic acid and cetyl and /or cetostearyl alcohols were
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successfully prepared by high shear homogenization and in vitro characterized. The developed NLC formulations showed particles in the nanometric size range, low PI values, good physical stability as well as efficient PG encapsulation. The higher amount of fatty alcohols the slower release and
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higher instability. Cetyl alcohol (1.25% of total lipid content) showed controlled release pattern, good stability and efficient duodenal permeation. Hence, it seems to be a promising system for oral
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delivery of PG.
Conflict of interest The authors declare that they have no conflicts of interest.
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Al-Safi, Z.A. and N. Santoro, Menopausal hormone therapy and menopausal symptoms. Fertility and sterility, 2014. 101(4): p. 905-915. Schindler, A.E., Progestogen deficiency and endometrial cancer risk. Maturitas, 2009. 62(4): p. 334-337. Cetingoz, E., et al., Progesterone effects on preterm birth in high-risk pregnancies: a randomized placebo-controlled trial. Archives of gynecology and obstetrics, 2011. 283(3): p. 423-429. Biruss, B. and C. Valenta, Skin permeation of different steroid hormones from polymeric coated liposomal formulations. European journal of pharmaceutics and biopharmaceutics, 2006. 62(2): p. 210-219. Vaugelade, C., et al., Progesterone freeze-dried systems in sublingual dosage form. International journal of pharmaceutics, 2001. 229(1): p. 67-73. Memişoğlu, E., et al., Non-surfactant nanospheres of progesterone inclusion complexes with amphiphilic β-cyclodextrins. International journal of pharmaceutics, 2003. 251(1): p. 143153. Cerchiara, T., et al., Effect of chitosan on progesterone release from hydroxypropyl-βcyclodextrin complexes. International journal of pharmaceutics, 2003. 258(1): p. 209-215. Hassan, T.H. and K. Mäder, Novel semisolid SNEDDS based on PEG-30-di(polyhydroxystearate): Progesterone incorporation and in vitro digestion. International journal of pharmaceutics, 2015. 486(1): p. 77-87. Elgindy, N.A., M.M. Mehanna, and S.M. Mohyeldin, Self-assembled nano-architecture liquid crystalline particles as a promising carrier for progesterone transdermal delivery. International journal of pharmaceutics, 2016. 501(1): p. 167-179. Müller, R., et al., Nanostructured lipid carriers (NLC) in cosmetic dermal products. Advanced drug delivery reviews, 2007. 59(6): p. 522-530. Saupe, A., et al., Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)– structural investigations on two different carrier systems. Bio-medical materials and engineering, 2005. 15(5): p. 393-402. Severino, P., et al., Crystallinity of Dynasan® 114 and Dynasan® 118 matrices for the production of stable Miglyol®-loaded nanoparticles. Journal of thermal analysis and calorimetry, 2011. 108(1): p. 101-108. Quadir, M.A., et al., Evaluation of hydrophobic materials as matrices for controlled-release drug delivery. Pak J Pharm Sci, 2003. 16(2): p. 17-28. Savolainen, M., et al., Evaluation of controlled-release polar lipid microparticles. International journal of pharmaceutics, 2002. 244(1): p. 151-161. Tan, S., et al., Surfactant effects on the physical characteristics of Amphotericin Bcontaining nanostructured lipid carriers. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2010. 372(1): p. 73-79. Yang, S., et al., Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharmaceutical research, 1999. 16(5): p. 751-757. Guinedi, A.S., et al., Preparation and evaluation of reverse-phase evaporation and multilamellar niosomes as ophthalmic carriers of acetazolamide. International Journal of Pharmaceutics, 2005. 306(1): p. 71-82. Sanka, K., et al., Improved oral delivery of clonazepam through liquisolid powder compact formulations: in-vitro and ex-vivo characterization. Powder Technology, 2014. 256: p. 336344.
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Nanjwade, B.K., S.R. Mhase, and F. Manvi, Formulation of extended-release metformin hydrochloride matrix tablets. Tropical journal of pharmaceutical research, 2011. 10(4): p. 375-383. Windbergs, M., C.J. Strachan, and P. Kleinebudde, Understanding the solid-state behaviour of triglyceride solid lipid extrudates and its influence on dissolution. European Journal of Pharmaceutics and Biopharmaceutics, 2009. 71(1): p. 80-87. Nandi, I., et al., Synergistic effect of PEG-400 and cyclodextrin to enhance solubility of progesterone. AAPS PharmSciTech, 2003. 4(1): p. 1-5. Sukuru, K., Hydrophilic vehicle-based dual controlled release matrix system. 2012, Google Patents. Gullapalli, R.P. and C.L. Mazzitelli, Polyethylene glycols in oral and parenteral formulations—A critical review. International journal of pharmaceutics, 2015. 496(2): p. 219-239. Yoo, J.W., D.S. Yun, and H.J. Kim, Influence of reaction parameters on size and shape of silica nanoparticles. Journal of nanoscience and nanotechnology, 2006. 6(11): p. 33433346. Bandyopadhyay, P., Fatty alcohols or fatty acids as niosomal hybrid carrier: effect on vesicle size, encapsulation efficiency and in vitro dye release. Colloids and Surfaces B: Biointerfaces, 2007. 58(1): p. 68-71. Sanna, V., et al., Preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration. Aaps Pharmscitech, 2004. 5(2): p. 17-23. Müller, R.H., M. Radtke, and S.A. Wissing, Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Advanced drug delivery reviews, 2002. 54: p. S131-S155. Mehnert, W. and K. Mäder, Solid lipid nanoparticles: production, characterization and applications. Advanced drug delivery reviews, 2001. 47(2): p. 165-196. Sanna, V., G. Caria, and A. Mariani, Effect of lipid nanoparticles containing fatty alcohols having different chain length on the ex vivo skin permeability of Econazole nitrate. Powder Technology, 2010. 201(1): p. 32-36. Hu, F.-Q., et al., Preparation and characteristics of monostearin nanostructured lipid carriers. International journal of pharmaceutics, 2006. 314(1): p. 83-89. Araya-Sibaja, A.M., et al., Dissolution properties, solid-state transformation and polymorphic crystallization: progesterone case study. Pharmaceutical development and technology, 2014. 19(7): p. 779-788.
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Table captions
Table 1. Compositions, particle size, PDI, zeta potential and EE% of different formulations (n=3,
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±SD).
Table 2. Accuracy and precision of the proposed method for the determination of PG (n=5).
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Table 3. Particle size, PDI, zeta potential and EE% of PG loaded NLC formulations within 3
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months (n=3, ±SD).
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Table 4. Permeation parameters of PG from PG suspension, NLC1 and NLC2 across rabbit duodenum (mean ±S.D., n = 3).
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Tables Table1 Cetyl Alc.%
Cetostearyl alc.%
PEG mix%
NLC 1 NLC 2 NLC 3 NLC 4 NLC 5 NLC 6 NLC 7 NLC 8 NLC 9 NLC 10
2 2 2 2 2 2 2 2 2 2
8 8 8 8 8 8 8 8 8 8
------0.125 0.25 0.5 ------------------0.0625 0.125 0.25
------------------------0.125 0.25 0.5 0.0625 0.125 0.25
3 3 3 3 3 3 3 3 3 3
Aqueous surfactant % (Tween 80) 3 3 3 3 3 3 3 3 3 3
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Particle size (nm)
PDI
Zeta potential (mV) -28.1±3.4 -22.6±0.7 -24.1±1.65 -25.4±0.75 -24.3±1.9 -23.5±1.2 -23.9±0.2 -25.65±0.9 -25.2±0.3 -22.1±1.47
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Sesame Oil%
142.3±6.7 137.2±4.1 194.3±9.7 165.4±3.6 210.1±7 228.2±5.6 219.0±2.7 201.6±1.9 191.3±5.1 226.7±2.8
0.142±0.002 0.235±0.009 0.341±0.007 0.215±0.075 0.129±0.014 0.201±0.004 0.294±0.005 0.27±0.008 0.210±0.05 0.323±0.004
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Stearic acid%
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EE%
97.2±1.5 95.3±3.6 94.8±4.1 96.1±7.18 93.8±5.8 95.1±7.9 96.6±6.2 93.7±2.7 95.4±4.8 94.7±4.1
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Table2
Intra-day
Inter-day
Concentration added (µg/ml)
Average concentration found (µg/ml)
Recovery (%)
RSD (%)
Average concentration found (µg/ml)
Recovery (%)
5
4.97±0.023
99.4
0.463
4.97±0.012
99.4
0.241
15
14.99±0.014
99.93
0.095
14.81±0.136
98.73
0.918
30
29.91±0.005
99.7
0.017
29.66±0.197
98.87
0.664
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RSD (%)
Table 3.
1st month
Code
2nd month
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3rd month
PDI
Zeta potential (mV)
EE%
Particle size (nm)
PDI
Zeta potential (mV)
EE%
Particle size (nm)
PDI
Zeta potential (mV)
EE%
NLC 1
150.7±2.9
0.161±0.003
-27.6±1.8
96.5±3.4
177.6±4.5
0.212±0.007
-28.2±1.65
94.1±5.9
199.4±5.2
0.341±0.024
-28.3±2.23
92.3±4.9
NLC 2
144.9±3.4
0.262±0.006
-16.5±0.5
95.5±7.9
160.3±5.6
0.269±0.012
-23.5±1.44
94.2±6.1
168.6±4.7
0.250±0.012
-18.1±1.48
92.4±5.3
NLC 3
210.6±7.1
0.356±0.039
-23.4±1.7
92.3±6.7
249.1±6.3
0.427±0.062
-24.2±2.22
92.6±2.4
266.9±7.2
0.515±0.092
-22.7±1.78
90.1±1.6
NLC 4
272.0±2.8
0.227±0.032
-22.6±1.87
94.3±5.3
322.2±2.8
0.286±0.022
-26.6±2.77
92.9±3.9
332.5±2.8
0.299±0.031
-21.5±1.65
91.3±1.1
NLC 5
213.6±4.4
0.133±0.027
-23.9±0.23
92.3±3.8
252.8±7.4
0.280±0.036
-24.6±1.12
91.3±6.9
276.0±3.5
0.304±0.062
-20.1±1.56
90.6±2.6
NLC 6
232.3±11.3
0.299±0.042
-22.8±1.6
95.7±6.8
293.8±5.8
0.380±0.054
-21.1±0.98
94.9±2.5
446.4±7.1
0.431±0.039
-24.5±1.91
93.7±1.8
NLC 7
224.6±5.4
0.377±0.035
-24.7±0.34
95.1±1.7
255.8±8.7
0.384±0.055
-22.4±1.74
93.4±2.3
297.1±6.6
0.402±0.073
-20.2±1.57
92.8±1.7
NLC 8
202.2±9.2
0.299±0.066
-26.7±0.1
94.1±8.2
214.3±6.4
0.331±0.066
-25.8±3.22
93.6±1.2
243.3±5.2
0.379±0.055
-27.8±3.18
92.8±2.5
NLC 9
197.1±5.3
0.275±0.020
-24.1±0.4
94.3±1.8
249.7±6.9
0.364±0.071
-23.6±2.34
91.6±3.5
277.1±8.4
0.393±0.062
-20.1±3.25
90.5±1.5
NLC 10
234.9±4.5
0.479±0.053
-20.8±0.6
93.1±6.7
-20.7±2.85
90.4±3.1
311.2±4.8
0.533±0.072
-18.8±1.95
88.8±1.3
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Particle size (nm)
0.535±0.038
Table 4.
Jss (mg.cm-2.h-1) 0.0016±0.0003 0.00128±0.0002 0.00079
Kp (cm.h-1) 8.05±0.27 6.4±0.34 3.96±0.15
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NLC1 NLC2 Suspension
Cumulative amount permeated (mg) 1.068±0.35 1.347±0.169 0.696±0.124
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Formulation
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2.03 1.62 --------
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Figure captions
Figure 1. Chemical structure of PG.
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Figure 2. PG analysis method; (A) Chromatogram of PG at the optimized UHPLC conditions and (B) Linearity of PG calibration curve.
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Figure 3. In vitro release profiles of PG by two stages experiments; the first in 1.2 pH and the second in 6.8 pH; (A) NLC1-4 and (B) NLC5-10
and NLC1-5 and (B) NLC6-10
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Figure 4. In vitro stability of PG in simulated gastric fluid (without enzymes); (A) PG suspension
Figure 5. Transmission electron microscope image of NLC2.
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Figure 6. DSC thermogram of (A) stearic acid, (B) cetyl alcohol, (C) PG and (D) NLC2.
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Figure 7. FTIR spectra of PG, stearic acid, cetyl alcohol and NLC2.
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Figure 8. Ex vivo permeation studies of PG across rabbit duodenum from PG suspension, NLC1 and NLC2.
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Figures
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Figure 4.
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Figure 8.
S1773-2247(17)30891-2
DOI:
10.1016/j.jddst.2018.03.007
Reference:
JDDST 600
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 14 October 2017 Revised Date:
23 January 2018
Accepted Date: 5 March 2018
Please cite this article as: M. Elmowafy, K. Shalaby, M.M. Badran, H.M. Ali, M.S. Abdel-Bakky, I. ElBagory, Fatty alcohol containing nanostructured lipid carrier (NLC); Strategy to fade away progesterone oral delivery drawbacks, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.03.007. 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.
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Fatty alcohol containing nanostructured lipid carrier (NLC); strategy to fade away progesterone oral delivery drawbacks Alternative title:
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Fatty alcohol containing nanostructured lipid carrier(NLC) for progesterone oral delivery: In vitro and ex vivo studies
1 2 3 4 5 6 7 8 9
Abdel-Bakky5, Ibrahim El-Bagory1
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Department of Pharmaceutics, College of Pharmacy, Aljouf University, Skaka, P.O. Box 2014,
KSA 2
Department of Pharmaceutics and Ind. Pharmacy, Faculty of Pharmacy (Boys), Al-Azhar
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University, Nasr City, Cairo, Egypt 3
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Mohammed Elmowafy1,2*, Khaled Shalaby1,2, Mohamed M. Badran2,3,Hazim M. Ali4, Mohamed S.
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, P.O.
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Box 2457, KSA
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Department of Chemistry,, College of Sciences, Aljouf University, Skaka, P.O. Box 2014, KSA
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5
Department of Pharmacology and Toxicology, Faculty of Pharmacy (Boys), Al-Azhar University,
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Nasr City, Cairo, Egypt
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*Corresponding author
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Tel.: +966541869569
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Email:[email protected], [email protected]
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Abstract
26 27
progesterone. Fatty alcohols (cetyl, cetostearyl and 1:1 mixture) containing NLCs were thought to
28
be one of the most relevant systems based on progesterone drawbacks as acid sensitivity and poor
29
bioavailability. Formulations were prepared characterized for physicochemical evaluation, in vitro
30
release, short term stability, interaction possibility and ex vivo permeation studies. The results
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showed nanorange particle size with major influence of fatty alcohol concentration. In vitro release
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results suggested protection of formulation from gastric environment with controlling the drug
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This study aims to overcome most of the obstacles that that hinder oral bioavailability of
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interaction among fatty alcohol, stearic acid and progesterone. Ex vivo results showed that
35
formulations significantly (p˂0.05) permeated through duodenum when compared with suspension
36
after 8h. The results collectively indicated that the cetyl alcohol containing NLC was a potential
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drug delivery system for oral administration of progesterone.
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release as a function of fatty alcohol concentration. Infrared and thermal analyses showed
Key words: Progesterone; NLC; Fatty alcohol; Controlled release; Ex vivo
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1. Introduction
49 50
mammals particularly in female reproduction. Physiologically, PG is essential for maintenance
51
during menstrual cycle and all stages of normal pregnancy for development of the foetus. Irregular
52
PG levels produces many disturbances as palpitations, headaches, hotness, vaginal dryness and
53
breast tenderness [1]. In addition PG deficiency may be a predisposing factor in endometrial
54
hyperplasia and endometrial cancer [2]. PG is indicated in the prevention of preterm labor in
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women at risk, especially women with history of previous spontaneous preterm labor [3]. Oral
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Progesterone (PG; Fig. 1), is a natural steroid hormone with multifunctional roles in
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water solubility (log P of 3.87; class II according to Biopharmaceutical Classification System),
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short half life due to extensive hepatic first pass effect [4] and enzymatic degradation within the
59
gastrointestinal tract [5].Administration of high PG doses is not tolerated by patients and side
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effects (as somnolence and dizziness) appear. To address these biopharmaceutical challenges
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associated with PG, many approaches were developed such as nanosphere [6], chitosan [7]
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administration of PG is accompanied by many drawbacks including poor bioavailability due to poor
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into another route of administration as sublingual [5] and transdermal delivery using nano-
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architecture liquid crystalline particles [9].
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incorporation and self-nanoemulsifying drug delivery systems (SNEDDS) [8] or even by shifting
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(NLC), is consisted of solid lipid and liquid lipid blend which in turn leads to imperfections
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between the lipids and creates spaces to incorporate drugs in the matrix, the gate to provide higher
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drug payload than the first generation (solid lipid nanoparticles; SLN) and prohibit drug expulsion
69
during storage [10, 11]. In addition, NLC surpass many other colloidal carriers in terms of
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inexpensive required materials, easiness of manufacture, lipid biocompatibility, controlling drug
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release and stability. Particularly, NLC preponderate polymeric nanoparticles in terms of safety and
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biocompatibility (considered the lowest toxic nanoparticles when administrated in vivo [12]) of raw
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The second generation of lipid nanoparticles, the so-called nanostructured lipid carriers
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alcohols were found to control the release of some drugs such as theophylline [13] and felodipine
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[14].
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fatty alcohol containing NLCs in order to (1) improve oral bioavailability by dissolution
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enhancement, (2) control the release and (3) protect the drug from gastrointestinal degradation.
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Stearic acid was selected as long chain fatty acid solid lipid while sesame oil was the liquid lipid.
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The PG loaded NLCs were formulated and characterized according to physicochemical evaluation.
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Based on the aforementioned data, the aim of the current work was to incorporate PG into
2. Materials and Methods 2.1. Materials
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The chosen formulations were subjected to ex-vivo permeation studies.
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cetyl alcohol, cetostearyl alcohol and polyethylene glycol (PEG) were purchased from Lobal
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PG was purchased from BDH chemicals (England). Sesame oil, stearic acid, Tween 80,
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Techno Pharmchem (India). Sodium azide was purchased from Sigma Aldrich (Germany). All the
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other chemicals, reagents and solvents used were of analytical reagent grade.
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Chemie (India). PEG1500 was purchased from MERK (Germany). PEG4000was purchased from
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2.2. Preparation of PG loaded NLCs
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The NLCs were prepared by high shear homogenization followed by sonication. Briefly, the
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lipid phase (10% w/v from total formulation) consisted of 2% stearic acid (solid lipid) and 8%
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sesame oil (liquid lipid) content. PG was firstly dissolved in sesame oil, heated at 65°C and mixed
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with preheated solid lipid. Different amounts of fatty alcohols (cetyl alcohol, cetostearyl alcohol or
96
1:1 mixture; Table1) were added to molten lipid phase along with PEG mixture
97
(PEG400:PEG1500:PEG400; 1:1:1). Aqueous phase (containing 3% Tween 80) was heated at 65°C
98
ACCEPTED MANUSCRIPT 99
(Ultra Turrax, IKA Eurostar 40 digital, GmbH, Germany) at 1300 rpm for 15 min in water bath.
100
Hence, the formulations were subjected to homogenization using high shear homogenizer (Yellow
101
line DI25 basic, Germany) at 12,000 rpm for 10 min to get smaller particle size. The formulations
102
were subjected to bath sonication (BranSonic, 5510E-DTH, Mexico) for 5 min at 60 W to get
103
uniform size distribution. The obtained dispersions were cooled at room temperature and stored at
104
4°C till use.
105
SC
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for 5min and slowly added to the melted lipid under continuous stirring by a high-speed stirrer
2.3. Particle size and surface charge
106 107 108
loaded NLCs were carried out using dynamic light scattering technique (Zetasizer Nano ZS,
109
Malvern Instruments, UK). All samples were properly diluted with double distilled water at room
110
temperature. All the measurements were performed in triplicate.
111
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Determination of average particle size, PDI and zeta potential (surface charge) of the PG
2.4. Chromatographic conditions
112 113 114 115
Dionex UltiMate 3000UHPLC+ ) focused standard systems equipped with quaternary analytical
116
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Analysis of PG concentration was carried out using UHPLC system (Thermo Scientific
117
autosampler,TCC-3000SD thermostatted column compartment, SR-3000 solvent rack without
118
degasser and DAD-3000 diode array detector. The apparatus was interfaced to a DELL PC
119
compatible computer with a Chromeleon™ 7.2 Chromatography Data System. The separation was
120
carried out on an ACCLAIM™ 120 C18 column, 4.6 mm×150 mm, 5µm with a mobile phase
121
consisting of acetonitrile, methanol and 20mM citric acid in volume ratio of 45:45:10 at a flow rate
122
of 1 ml/min. The wavelength for PG detection was set at 245 nm, with an injection volume of 10 µl
123
at 25°C.
124
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pump LPG-3400SD with degasser,WPS-3000TSL analytical split-loop thermostatted well plate
125
ACCEPTED MANUSCRIPT 126 2.5. Validation of assay method
127 128
accuracy, the limit of detection (LOD), limit of quantification (LOQ) and system suitability. The
129
linearity of the method was studied using different concentrations covering the range from 0.05 to
130
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Validation of UHPLC method for determination of PG included linearity, precision,
131
30 µg/ml) by comparing the values for the relative standard deviation: RSD= (SD/ mean measured
132
concentration) × 100. Accuracy was expressed as the percentage recovery and was determined by
133
the following equation:
× 100,
(1)
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Recovery (%) =
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90 µg/ml at five replicates for each level. Precision was estimated at three levels of PG (5, 15 and
134 135 136
LOD and LOQ values were estimated based on the standard deviation of intercept (SDb) and slope
137
(a) of calibration curve, according to the following formulas;
138
LOQ =
! ×"."
,
! ×%&
(2)
, (3)
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LOD =
139 140 141
and peak asymmetry for 50 µg/ml of PG.
142
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The system suitability was tested by determining peak retention time, peak area, theoretical plates
2.6. Drug encapsulation efficiency
143 144 145
The entrapment efficiency (EE) is of major importance as the drug release from system is
146
greatly influenced by amount of drug encapsulated inside that system. EE% was determined by
147
indirect method basing upon precipitation of NLC upon adjusting pH value of the NLC dispersion
148
to 1.2 (by adding 0.1N HCL) followed by centrifugation at 11,000 rpm for 45 min at 16°C [15]. The
149
supernatant and pellets wash out were filtered through 0.45 µm filter and diluted appropriately with
150
ACCEPTED MANUSCRIPT methanol and analyzed by above mentioned UHPLC method. The EE% of PG loaded NLCs was
151
then calculated according to the following equation (4):
152 153
)(
* +
,
+-
. * +
* +
,
,
+- /+0
+-
)
× 100,
(4)
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EE% =
2.7. In vitro PG release
154 155 156 157 158
described by Yang et al. [16] with some modifications. The receptor compartment consisted 500ml
159
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In vitro release of PG from NLCs was evaluated by the dialysis bag diffusion technique
160
bags was soaked in PBS overnight prior experiment. PG loaded NLCs (equivalent to 2mg of PG)
161
were placed dialysis bag (molecular weight cut off: 12–14 kDa, Livingstone, NSW, Australia),
162
containing 0.1N HCl to simulate gastric pH (1.2) and then the bags were sealed at both sides. The
163
bags were immersed in receptor compartment placed in USP apparatus I vessels automatically
164
adjusted at 37± 2°C and 100 rpm. At predetermined intervals of time (15, 30, 60 and 120 min),
165
aliquots of 2 ml were withdrawn from the diffusion media and HPLC analyzed. Equal amounts of
166
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phosphate buffered saline (PBS) of pH-7.4 to maintain sink conditions. Membrane of dialyzing
167
constant receptor volume. After 2 h, the bags were opened and the internal pH was adjusted into 6.8
168
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fresh PBS were added to diffusion media immediately after withdrawal time point to maintain
to simulate intestinal pH. Samples of 2ml were withdrawn at regular time intervals (30, 60, 120, 180
169
and 240 min) to be analyzed and the same volume was replaced by fresh release medium. All the
170
experiments were done in triplicate, and the average values were taken.
171
At the end of experiment, the kinetic parameters for the in vitro data were determined in
172
order to estimate the best fit to distinct kinetic model (zero, first or Higuchi order) to determine the
173
PG release mechanism from NLC formulations. Stating the proper mode of release is based on the
174
ACCEPTED MANUSCRIPT Correlation Coefficient Parameter (r) for parameters involved, where the highest correlation
175
coefficient represents the actual mode of the release [17].
176 177
2.8. Stability
178
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In order to assess the physicochemical stability of the prepared systems, particle size, PDI,
179 180
months when formulations stored at 4°C.
181
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zeta potential and EE% were considered as stability criteria. They were monthly measured for 3
2.9. Morphology
182 183 184
6360 LV, JEOL, Tokyo, Japan). Formulations were freeze-dried (Alpha 1-4LDPlus,GmbH,
185
Osterode am Harz, Germany) and fixed on carbon tape and sputter-coated with a thin gold layer.
186
The formulations were then scanned and photographed.
187
2.10. Thermal analysis
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The shape of PG loaded NLCs was observed by scanning electron microscopy (SEM; JSM-
188 189 190
interaction possibility using differential scanning calorimeter (DSC) tool. Accurately weighed 8mg
191
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In addition to individual components, freeze-dried NLC2 formulations were examined for
192
(Shimadzu Corporation, Tokyo, Japan). Standard empty aluminum pan was used as reference and
193
the thermograms were recorded between 30°C and 250°C at a scan rate of 10°C/min under nitrogen
194
gas.
195
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of samples were placed in aluminum pans, crimped and analyzed using a Shimadzu DSC-60
196 2.11. FTIR
197
Interaction between PG and NLC components (stearic acid and fatty alcohol) was studied by 198 Fourier transform infrared (FT-IR) in the transmission mode. FT-IR analysis was performed between 199 4400 and 350 cm−1 at a resolution of 4 cm−1.
200
ACCEPTED MANUSCRIPT 201 202 203
2.12. Ex vivo permeability studies
204
NLCs and conventional suspension. Adult female albino rabbits, weighing 1.75–2kg, were used in
205
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This study was carried out in order to investigate the intestinal permeability of PG from
206
Aljouf University (Skaka, Northern region, KSA) and housed under conventional laboratory
207
conditions with free access to food and water till sacrifice. The study was conducted in accordance
208
with ethical procedures and policies approved by the Local Committee for Research Bioethics,
209
Aljouf University (Ethical code; LCBE8/3/37/38).
210
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the experiment. They were obtained from the Animal House Colony of College of Pharmacy,
211
PBS to get rid of the mucous and gut wastes. Parts of 4cm long were cut and one side was tied up.
212
Besides PG suspension (PG was dispersed in 2% carboxymethylcellulose sodium), formulations of
213
NLC1 and NLC2 (equivalent to 2 mg of PG) were placed in intestinal parts and the remaining side
214
was tied with cotton thread. The sacs were placed in USP apparatus vessels containing 500 ml
215
Tyrode solution (pH 7.4) of 0.05% sodium azide and maintained at 37± 2°C and stirred at
216
100rpm.Samples of 2mlwere withdrawn at regular time intervals (0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6,7
217
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Animals were sacrificed by ether inhalation and the duodenal part was excised and flushed with
218
compartment. All the experiments were done in triplicate, and the average values were taken.
219
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and 8 h) and UHPLC analyzed. At each time point 2 ml of fresh solution were added to receptor
Cumulative amount of PG permeating through intestinal wall per unit area was plotted against time.
220
Steady state flux (Jss) was calculated from the slope of the linear portion of the plot.
221
Permeability coefficient was also calculated using the following equation (2):
222
Kp =
344 , (5) Ci 223
ACCEPTED MANUSCRIPT Where Kp was the permeability coefficient, and Ci was the initial concentration of PG in donor
224
compartment. The efficacy of NLCs to permeate PG through intestinal wall was also determined by
225
enhancement ratio (ER). ER was also calculated using the following equation (3) [18]:
226
Jss of NLC , (6) Jss of suspension
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ER =
2.13. Statistical Analysis
227 228 229 230
analysis of obtained results was evaluated by one-way ANOVA followed by Tukey-Kramer test
231
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All results in this work are expressed as a mean ± standard deviation (SD). The statistical
using the StatPlus software (Analyst Soft Inc., USA). Difference at P < 0.05 was considered to be
232
significant.
233
3. Results and discussion
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3.1. Rationale behind components and formulations
234 235 236 237
of PG. Of course and as previously mentioned, NLCs themselves behave as controlled release
238
system, but their constituents largely affect that behavior. Stearic acid, a long chain fatty acid, was
239
chosen as solid lipid which was reported as the degree of metformin HCl release was stearic acid
240
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The components of NLCs were selected on the basis of the purpose; controlling the release
concentration dependent [19]. Sesame oil, plant oil containing long chain triglycerides, was selected
241
as liquid lipid based upon PG solubility, slow dissolution [20] and safety terms. The addition of
242
PEG to the formulation aimed to increase NLC stabilization and enhance bioavailability and not to
243
improve PG solubilization as PG completely dissolved in sesame oil. It was also reported that PG
244
showed a declined solubility in PEG400 [21]. The wisdom of using of PEG in a mixture of different
245
grades was firstly to control the release rate [22] of PG. Secondly to minimize the gastrointestinal
246
absorption of PEG as well as prevent the induced toxicity. Oral absorption and toxicity were
247
ACCEPTED MANUSCRIPT 248
type and concentration, NLC formulations were designed. Two fatty alcohols were selected (cetyl
249
alcohol and cetostearyl alcohol) with 0.125, 0.25 and 0.5% concentrations to study the effect of
250
different concentrations on physicochemical characteristics. It should be noted that higher
251
concentration of fatty alcohol than 0.5% produced very viscous and hardly flowable formulations
252
simultaneously upon cooling.
253
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reviewed to be inversely proportional to molecular weight of PEG [23]. As function of fatty alcohol
3.2.1.UHPLC method development and optimization
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3.2. Chromatography
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Separation of PG was achieved on ACCLAIM™ 120 C18 column (maintained at 25 ºC) and
254 255 256 257 258
compositions of acetonitrile, methanol and citric acid were studied. 20 mM citric acid, methanol
259
and acetonitrile (45:45: 10% v/v) resulted in the good chromatographic separation of progesterone.
260
The chromatogram of separation of PG measured at the optimized UHPLC conditions is shown in
261
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isocratic elution with a mobile phase consisting of acetonitrile, methanol and citric acid. Several
262
3.2.2. Method validation
263
3.2.2.1. Linearity
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Fig. 2A. PG peak was observed at the retention time of 3.41 min.
264 265 266
µg/ml. The calibration curve was created by plotting the peak area against the concentration (Fig.
267
2B), and the average value of linear regression equation for five replicates was y=0.567(±0.0012) x
268
+0.153(±0.0077) with an average correlation coefficient (r2) value of 0.9995(± 4.4x10-5).
269
3.2.2.2. Precision and accuracy
270
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The linearity of calibration curve of progesterone was obtained over a range of 0.05-90
Intra-day accuracy and precision were performed by analyzing five replicates at three
271
concentration levels: 5, 15 and 30 µg/ml in the same day as listed in Table 2. Inter-day accuracy and
272
precision were evaluated on three successive days at the same concentration levels of PG as shown
273
ACCEPTED MANUSCRIPT in Table 2. From the listed data, we can see that the established method is accurate and precise.
274 275
3.2.2.3. LOD and LOQ
276 277
and 0.136 µg/ml respectively. The low values of LOD and LOQ refer to the good sensitivity of
278
developed method.
279
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LOD) and LOQ of the proposed method for PG determination were found to be 0.045 µg/ml
3.2.2.4. System suitability
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System suitability was performed by calculating the peak retention time, peak area, peak asymmetry
280 281 282
time, peak area, peak asymmetry and number of theoretical plates were found to be 3.41± 0.004,
283
28.68± 0.35, 1.1± 0.016 and 9251.33± 30.23 respectively. These results confirm the suitability of
284
the proposed method and ensuring system performance.
285
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3.3. Characterization of PG loaded NLCs
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and number of theoretical plates for 50 µg/ml of PG (six replicate). The value of peak retention
286 287 288
formulations are depicted in Table 1. The values of particle size lied between 137.2±4.1 and
289
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The results of particle size, PDI, zeta potential and EE% of freshly prepared NLC
290
that addition of fatty alcohols to NLC enlarged the particle size except NLC 2 which contained the
291
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228.2±5.6 nm which confirmed nanometric range of all investigated formulations. It was noticed
smallest amount of cetyl alcohol. Cetyl alcohol containing NLCs exhibited significant smaller
292
particle size than cetostearyl alcohol containing NLCs (p ˂ 0.05) while formulations containing
293
cetyl and cetostearyl alcohols mixture showed insignificant (p > 0.05) particle size values
294
differences. Cetostearyl alcohol consists of mixture of 50-70% stearyl alcohol (C-18) and 20-35%
295
cetyl alcohol (C-16) as well as small amount of myristyl alcohol. Longer carbon chain of
296
cetostearyl alcohol might be the cause of larger particle size [24].
297
Bandyopadhyay and coworker concluded that stearic acid (fatty acid) based system
298
ACCEPTED MANUSCRIPT 299
These results do not contradict with our study as the other study formulated the systems completely
300
stearic acid or cetyl alcohol whereas our study formulations based upon addition of fatty alcohols
301
(with different amounts) to stearic acid based system. PDI results reveal homogeneity of particle
302
distribution (˂0.35) as typical of monodispersed systems [26].
303
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produced higher particle size when compared with cetyl alcohol (fatty alcohol) based system [25].
304
via electrostatic repulsion which in turn prohibits their aggregation. All investigated samples
305
showed markedly negative charge and ranged from -22.1±1.47 to -28.1±3.4 mV without significant
306
difference among formulations. This means that addition of fatty alcohols to NLC did not influence
307
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Zeta potential determines surface charge of the particles which greatly affects their stability
308
Tween 80 and PEG. The EE% of PG when loaded in nanostructured formulations was found to
309
vary between 93.7±2.7 and 97.2±1.5% (Table 1).These satisfying values of EE% resulted from high
310
solubility of PG in sesame oil which aided to entrap the large amount of PG and decreased the drug
311
crystallinity which helped better stability and suitability for the controlled release [27]. It should be
312
mentioned that the effect of fatty alcohols on EE% was negligible.
313
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surface charge the resulting formulations. The negative charge may be attributed to presence of
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3.4. In vitro PG release and release kinetics
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In vitro release of PG from the NLC formulations was assessed using a USP dissolution apparatus and are graphically represented in Fig. 3 (A and B). The sink conditions were maintained using 500 ml PBS (pH 7.4) as the solubility of PG was predetermined in receptor compartment of 0.0198±0.003 mg/ml. The method used in this article differs from both direct placing of nanoparticles in receptor media and conventional dialysis bag method. Sampling from the former results in loss of nanoparticles and decreasing the concentration of the donor while in the later the donor compartment is not diluted which does not correlate the in vivo behavior. In the current method, two drawbacks were overcome by placing nanoparticles formulations in dialysis bag and
314 315
ACCEPTED MANUSCRIPT diluting them by in vivo simulating media (without enzymes). The rationale behind that was to consider the media inside bag simulating gastric (0.1N HCl) and intestinal (pH=6.8) environments whereas the media outside simulating circulation (pH=7.4). Therefore release pattern by this way can largely reflect in vivo release behavior. From Fig. 3, we can propose that at the end of first stage
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(in acidic environment), the highest amount of PG released was less than 19% (NLC1 and NLC2) without initial burst release pattern which confirmed the results of EE% (above 93%). These results also were promising in protecting most of encapsulating PG from gastric acidic media during the
SC
proposed time (2h). By the beginning of the second stage, initial burst of all formulation was pronounced. NLC1 showed the highest release by the end (>77%) while NLC7 exhibited the lowest
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one (˂22%). These results imparted release of higher amount of PG in intestinal environment (the optimum site for drug absorption) with controlled release behavior. Opposite pattern might be attributed to ionization degree of stearic acid in both media. In the first stage the majority of stearic acid was non-ionized and its hydrophobicity greatly retained which in the second stage degree of
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ionization increased and hence the leakage of PG was improved. Quadir and coworkers studied the release pattern theophylline from different hydrophobic matrices. They attributed the faster release from stearic acid to the formation of hydrogen bond between free carboxylic acid and dissolution
EP
media [13]. The feasibility of our explanation (ionization degree) lies in that the release pattern completely varied in different pH values. It was also of worth to mention that the release of PG was
AC C
dependent upon fatty alcohol type and concentration. Cetostearyl alcohol showed the highest degree to release retardation while cetyl alcohol showed the lowest degree. Cetostearyl alcohol has longer aliphatic carbon chain which increases its hydrophobicity and hampers wetting of the system by dissolution media [13]. Shorter length of aliphatic chain in cetyl alcohol than cetostearyl alcohol decreased the hydrophobicity and promoted PG release. Stearic acid is of higher hydrophilicity (due to carboxylic group) and molecular dimension than cetyl alcohol which in turn produces higher release from stearic acid containing NLC (NLC1). As expected, the release pattern of cetyl alcohol
ACCEPTED MANUSCRIPT and cetostearyl alcohol mixture lied in between them. The higher the concentration of fatty alcohol, lower percent of PG released after 6h. The controlled release behavior of fatty alcohols is attributed to participating in structure of lipidic particles and stabilizing the system [25]. Different kinetic models were applied to the mechanism by which PG was released from NLCs. Kinetic fits (data not
coefficient (r) than both zero and Higuchi diffusion models.
SC
3.5. Stability of NLCs
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shown) revealed that PG released from NLC by first order kinetic model with higher correlation
As the major storage stability problems facing lipid nanocarriers are gelation phenomena,
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particle size growth and drug leakage [28], stability estimation was performed on basis of particle size, zeta potential, PDI and EE%. Results are shown in Table 3. It is obvious that the particle size growth increased with fatty alcohol increase after 3 months. This phenomenon might be attributed to formation of hydrogen bond between hydroxyl group of fatty alcohol molecules impeded in lipid
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particles which in turn improved aggregation and enlarged particles. PDI results show higher values after 3 months than 0.3 except NLC2 and NLC3 indicating unimodal size distribution. There were no noticeable variations in zeta potential and EE% results after 3months. Collectively, NLC2
EP
showed minimal particle size growth, uniform particle size distribution, absence of visible particulate matter as well as reasonable zeta potential value. For that, NLC2 (as selected fatty
AC C
alcohol containing NLC) as well as NLC1 (as non-fatty alcohol containing NLC) were selected for next studies.
3.6. Morphology Fig. 4 shows the surface morphology of PG loaded NLC2 determined by SEM after freeze drying. The SEM images showed homogeneous and rounded particles with smooth surface.
ACCEPTED MANUSCRIPT 3.7. The crystal form of PG loaded NLC To investigate the effect of incorporation of PG on its melting and crystalline state DSC analysis of pure components and lyophilized NLC2 were performed (Fig. 5). PG exhibited sharp endothermic peak at 131°C indicating the crystalline nature while cetyl alcohol and stearic acid
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showed main peaks at 51.4°C and 56.8°C respectively. Lyophilized NLC2 exhibited two apexes (at 43.5°C and 50.1°C) peak. This shift of cetyl alcohol and stearic acid peaks into lower temperature
SC
depressed broad peaks confirmed the concurrent melting and consequent interaction between them in lipid matrix of NLC. Similar finding was reported by Sanna and coworkers who studied the
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effect of econazole nitrate lipid nanoparticles of glycerol palmitostearate (Precirol® ATO 5) and different chain length fatty alcohols [29]. They attributed the peak depression to lattice defect which resulted from interaction between econazole nitrate and lipid. Hu and coworkers correlated the peak depression of monostearin nanoparticles to amount of added liquid lipid (Caprylic/capric
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triglycerides); the higher concentration of liquid lipid the wider solid lipid peak. This in turn augmented the less ordered mechanism which allowed more vacancy to encapsulate the drug with high loading [30]. The decrease in melting temperature of the lipids in nanoparticles when
EP
compared to bulk lipids might be attributed to decrease in particle size when formulated in nanorange particles. Absence of main endothermic peak of PG confirmed the presence in
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amorphous state which in turn improved PG solubility.
3.8. FTIR analysis
Interaction between drug and other NLC components was studied using FTIR tool. The FTIR spectra of pure PG, stearic acid, cetyl alcohol and NLC2 are shown in Fig. 6. In pure PG spectrum, the region below 1500cm-1 represents the finger print of PG polymorph 1 [31]. After 1500cm-1 there were four main sharp peaks; 1612cm-1 (represents aromatic C=C stretch), 1661cm-1 (represents conjugated ketone at C3 in PG structure), 1699cm-1(represents non-conjugated ketone at
ACCEPTED MANUSCRIPT C20 in PG structure) and 2849-2923cm-1 (represents methyl C-H stretch). Stearic acid spectrum shows main peaks at 1701cm-1 and 2849cm-1 (represent carboxylic acid and C-H stretch respectively). Cetyl alcohol exhibits spectrum of two main peaks at 2881cm-1 and 3374cm-1 (C-H stretch and normal OH stretch respectively). In NLC2 spectrum, there are three characteristic
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features; the first is the absence conjugated C=O at C3 in PG. The second is shifting of both nonconjugated ketone (1703cm-1) and OH (3388cm-1) peaks which collectively confirmed stable dimeric intermolecular hydrogen bonding (in the condensed phase) between PG and NLC
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components and in turn can enhance PG solubility by NLC formulation. The third is the appearance of new peak at 1744cm-1 (represents ester) which could explain the interaction between stearic acid
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and cetyl alcohol.
3.9. Ex vivo studies
As PG is class II drug, its absorption is dissolution rate limited; the first step in absorption
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process. Therefore, ex vivo studies was performed to have an insight on the capability of NLCs to enhance PG absorption (Fig. 7). Permeation profiles of PG from NLC1 and NLC2 relative to the suspension through duodenal wall are shown in Fig. 6. It was observed that the NLC1 and NLC2
EP
formulations significantly (p˂0.05) permeated through intestinal wall (53.4±17.5% and 67.3±8.4% respectively) when compared with suspension (34.8±6.2%) after 8h. This indicated the absorption
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superiority of NLC formulations over conventional suspension due to nanometric particles as well as the presence of lipids and surface active agent. It is also clear that incorporation of cetyl alcohol in NLC2 produced slight enhancement in extent of PG permeation when compared to NLC1 after 8h. Partitioning of cetyl alcohol between stearic acid (partly explained in DSC and FTIR sections) and biological membrane lipid affinity might be also a proposed mechanism. However, lower flux and hence Kp of NLC2 (0.00128±0.0002 and6.4±0.34 respectively), when compared to NLC1 (0.0016±0.0003 and 8.05±0.27 respectively), augmented the controlled release behavior of cetyl
ACCEPTED MANUSCRIPT alcohol containing formulation during ex vivo experiment (Table 4).However, lack of blood supply resulted in absence of drug clearance and accumulation of PG in receptor compartment. This in turn fainted controlled release behavior of NLC2. Another point of concern is that cumulative amount of PG released from cellulose membrane was much higher than that of the rabbit intestine. This might
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be attributed to difference in thickness of both barriers (100 µm of the cellulose membrane) and structural complexity of biological membranes. It is worth to mention that linearity was more pronounced (r equals 0.969 and 0.984 for NLC1 and NLC2 respectively) when the plot was
SC
constructed between the percentages of PG permeated against square root of time (h1/2). This
described the Higuchi diffusion model.
4. Conclusion
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indicated that PG release was controlled by a passive (Fickian) diffusion mechanism, which was
In conclusion, NLCs based on stearic acid and cetyl and /or cetostearyl alcohols were
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successfully prepared by high shear homogenization and in vitro characterized. The developed NLC formulations showed particles in the nanometric size range, low PI values, good physical stability as well as efficient PG encapsulation. The higher amount of fatty alcohols the slower release and
EP
higher instability. Cetyl alcohol (1.25% of total lipid content) showed controlled release pattern, good stability and efficient duodenal permeation. Hence, it seems to be a promising system for oral
AC C
delivery of PG.
Conflict of interest The authors declare that they have no conflicts of interest.
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References
6.
7. 8.
9.
10. 11.
12.
13. 14. 15.
16. 17.
18.
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5.
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4.
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3.
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Al-Safi, Z.A. and N. Santoro, Menopausal hormone therapy and menopausal symptoms. Fertility and sterility, 2014. 101(4): p. 905-915. Schindler, A.E., Progestogen deficiency and endometrial cancer risk. Maturitas, 2009. 62(4): p. 334-337. Cetingoz, E., et al., Progesterone effects on preterm birth in high-risk pregnancies: a randomized placebo-controlled trial. Archives of gynecology and obstetrics, 2011. 283(3): p. 423-429. Biruss, B. and C. Valenta, Skin permeation of different steroid hormones from polymeric coated liposomal formulations. European journal of pharmaceutics and biopharmaceutics, 2006. 62(2): p. 210-219. Vaugelade, C., et al., Progesterone freeze-dried systems in sublingual dosage form. International journal of pharmaceutics, 2001. 229(1): p. 67-73. Memişoğlu, E., et al., Non-surfactant nanospheres of progesterone inclusion complexes with amphiphilic β-cyclodextrins. International journal of pharmaceutics, 2003. 251(1): p. 143153. Cerchiara, T., et al., Effect of chitosan on progesterone release from hydroxypropyl-βcyclodextrin complexes. International journal of pharmaceutics, 2003. 258(1): p. 209-215. Hassan, T.H. and K. Mäder, Novel semisolid SNEDDS based on PEG-30-di(polyhydroxystearate): Progesterone incorporation and in vitro digestion. International journal of pharmaceutics, 2015. 486(1): p. 77-87. Elgindy, N.A., M.M. Mehanna, and S.M. Mohyeldin, Self-assembled nano-architecture liquid crystalline particles as a promising carrier for progesterone transdermal delivery. International journal of pharmaceutics, 2016. 501(1): p. 167-179. Müller, R., et al., Nanostructured lipid carriers (NLC) in cosmetic dermal products. Advanced drug delivery reviews, 2007. 59(6): p. 522-530. Saupe, A., et al., Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)– structural investigations on two different carrier systems. Bio-medical materials and engineering, 2005. 15(5): p. 393-402. Severino, P., et al., Crystallinity of Dynasan® 114 and Dynasan® 118 matrices for the production of stable Miglyol®-loaded nanoparticles. Journal of thermal analysis and calorimetry, 2011. 108(1): p. 101-108. Quadir, M.A., et al., Evaluation of hydrophobic materials as matrices for controlled-release drug delivery. Pak J Pharm Sci, 2003. 16(2): p. 17-28. Savolainen, M., et al., Evaluation of controlled-release polar lipid microparticles. International journal of pharmaceutics, 2002. 244(1): p. 151-161. Tan, S., et al., Surfactant effects on the physical characteristics of Amphotericin Bcontaining nanostructured lipid carriers. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2010. 372(1): p. 73-79. Yang, S., et al., Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharmaceutical research, 1999. 16(5): p. 751-757. Guinedi, A.S., et al., Preparation and evaluation of reverse-phase evaporation and multilamellar niosomes as ophthalmic carriers of acetazolamide. International Journal of Pharmaceutics, 2005. 306(1): p. 71-82. Sanka, K., et al., Improved oral delivery of clonazepam through liquisolid powder compact formulations: in-vitro and ex-vivo characterization. Powder Technology, 2014. 256: p. 336344.
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Table captions
Table 1. Compositions, particle size, PDI, zeta potential and EE% of different formulations (n=3,
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Table 2. Accuracy and precision of the proposed method for the determination of PG (n=5).
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Table 3. Particle size, PDI, zeta potential and EE% of PG loaded NLC formulations within 3
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Table 4. Permeation parameters of PG from PG suspension, NLC1 and NLC2 across rabbit duodenum (mean ±S.D., n = 3).
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Tables Table1 Cetyl Alc.%
Cetostearyl alc.%
PEG mix%
NLC 1 NLC 2 NLC 3 NLC 4 NLC 5 NLC 6 NLC 7 NLC 8 NLC 9 NLC 10
2 2 2 2 2 2 2 2 2 2
8 8 8 8 8 8 8 8 8 8
------0.125 0.25 0.5 ------------------0.0625 0.125 0.25
------------------------0.125 0.25 0.5 0.0625 0.125 0.25
3 3 3 3 3 3 3 3 3 3
Aqueous surfactant % (Tween 80) 3 3 3 3 3 3 3 3 3 3
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Particle size (nm)
PDI
Zeta potential (mV) -28.1±3.4 -22.6±0.7 -24.1±1.65 -25.4±0.75 -24.3±1.9 -23.5±1.2 -23.9±0.2 -25.65±0.9 -25.2±0.3 -22.1±1.47
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Sesame Oil%
142.3±6.7 137.2±4.1 194.3±9.7 165.4±3.6 210.1±7 228.2±5.6 219.0±2.7 201.6±1.9 191.3±5.1 226.7±2.8
0.142±0.002 0.235±0.009 0.341±0.007 0.215±0.075 0.129±0.014 0.201±0.004 0.294±0.005 0.27±0.008 0.210±0.05 0.323±0.004
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Stearic acid%
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EE%
97.2±1.5 95.3±3.6 94.8±4.1 96.1±7.18 93.8±5.8 95.1±7.9 96.6±6.2 93.7±2.7 95.4±4.8 94.7±4.1
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Table2
Intra-day
Inter-day
Concentration added (µg/ml)
Average concentration found (µg/ml)
Recovery (%)
RSD (%)
Average concentration found (µg/ml)
Recovery (%)
5
4.97±0.023
99.4
0.463
4.97±0.012
99.4
0.241
15
14.99±0.014
99.93
0.095
14.81±0.136
98.73
0.918
30
29.91±0.005
99.7
0.017
29.66±0.197
98.87
0.664
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RSD (%)
Table 3.
1st month
Code
2nd month
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3rd month
PDI
Zeta potential (mV)
EE%
Particle size (nm)
PDI
Zeta potential (mV)
EE%
Particle size (nm)
PDI
Zeta potential (mV)
EE%
NLC 1
150.7±2.9
0.161±0.003
-27.6±1.8
96.5±3.4
177.6±4.5
0.212±0.007
-28.2±1.65
94.1±5.9
199.4±5.2
0.341±0.024
-28.3±2.23
92.3±4.9
NLC 2
144.9±3.4
0.262±0.006
-16.5±0.5
95.5±7.9
160.3±5.6
0.269±0.012
-23.5±1.44
94.2±6.1
168.6±4.7
0.250±0.012
-18.1±1.48
92.4±5.3
NLC 3
210.6±7.1
0.356±0.039
-23.4±1.7
92.3±6.7
249.1±6.3
0.427±0.062
-24.2±2.22
92.6±2.4
266.9±7.2
0.515±0.092
-22.7±1.78
90.1±1.6
NLC 4
272.0±2.8
0.227±0.032
-22.6±1.87
94.3±5.3
322.2±2.8
0.286±0.022
-26.6±2.77
92.9±3.9
332.5±2.8
0.299±0.031
-21.5±1.65
91.3±1.1
NLC 5
213.6±4.4
0.133±0.027
-23.9±0.23
92.3±3.8
252.8±7.4
0.280±0.036
-24.6±1.12
91.3±6.9
276.0±3.5
0.304±0.062
-20.1±1.56
90.6±2.6
NLC 6
232.3±11.3
0.299±0.042
-22.8±1.6
95.7±6.8
293.8±5.8
0.380±0.054
-21.1±0.98
94.9±2.5
446.4±7.1
0.431±0.039
-24.5±1.91
93.7±1.8
NLC 7
224.6±5.4
0.377±0.035
-24.7±0.34
95.1±1.7
255.8±8.7
0.384±0.055
-22.4±1.74
93.4±2.3
297.1±6.6
0.402±0.073
-20.2±1.57
92.8±1.7
NLC 8
202.2±9.2
0.299±0.066
-26.7±0.1
94.1±8.2
214.3±6.4
0.331±0.066
-25.8±3.22
93.6±1.2
243.3±5.2
0.379±0.055
-27.8±3.18
92.8±2.5
NLC 9
197.1±5.3
0.275±0.020
-24.1±0.4
94.3±1.8
249.7±6.9
0.364±0.071
-23.6±2.34
91.6±3.5
277.1±8.4
0.393±0.062
-20.1±3.25
90.5±1.5
NLC 10
234.9±4.5
0.479±0.053
-20.8±0.6
93.1±6.7
-20.7±2.85
90.4±3.1
311.2±4.8
0.533±0.072
-18.8±1.95
88.8±1.3
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Particle size (nm)
0.535±0.038
Table 4.
Jss (mg.cm-2.h-1) 0.0016±0.0003 0.00128±0.0002 0.00079
Kp (cm.h-1) 8.05±0.27 6.4±0.34 3.96±0.15
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Cumulative amount permeated (mg) 1.068±0.35 1.347±0.169 0.696±0.124
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Formulation
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Figure captions
Figure 1. Chemical structure of PG.
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Figure 2. PG analysis method; (A) Chromatogram of PG at the optimized UHPLC conditions and (B) Linearity of PG calibration curve.
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Figure 3. In vitro release profiles of PG by two stages experiments; the first in 1.2 pH and the second in 6.8 pH; (A) NLC1-4 and (B) NLC5-10
and NLC1-5 and (B) NLC6-10
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Figure 4. In vitro stability of PG in simulated gastric fluid (without enzymes); (A) PG suspension
Figure 5. Transmission electron microscope image of NLC2.
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Figure 6. DSC thermogram of (A) stearic acid, (B) cetyl alcohol, (C) PG and (D) NLC2.
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Figure 7. FTIR spectra of PG, stearic acid, cetyl alcohol and NLC2.
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Figure 8. Ex vivo permeation studies of PG across rabbit duodenum from PG suspension, NLC1 and NLC2.
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