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Nano-spray drying technique as a novel approach to formulate stable econazole nitrate nanosuspension formulations for ocular use Amr Maged, Azza Ahmed Mahmoud, and Mahmoud M. Ghorab Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00167 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016
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Molecular Pharmaceutics
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Nano-spray drying technique as a novel approach to formulate stable econazole nitrate nanosuspension formulations for ocular use
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Amr Maged 1,*, Azza A. Mahmoud 1,2, Mahmoud M. Ghorab3 1
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Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt, Cairo, Egypt
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2
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Department of Pharmaceutical Technology, National Research Center, Dokki, Cairo, Egypt 3
Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt
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Corresponding author: Amr Maged
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E-Mail:
[email protected] 15
Tel.: +201005849238
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Address: Department of Pharmaceutics and Pharmaceutical Technology,
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Future University in Egypt, End of 90th St., Fifth Settlement, New Cairo,
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Egypt
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Running Head: Nano-spray dried econazole nanosuspension
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Nano-spray drying technique as a novel approach to formulate stable econazole nitrate nanosuspension formulations for ocular use
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Amr Maged 1,*, Azza A. Mahmoud 1,2, Mahmoud M. Ghorab3 1
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Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt, Cairo, Egypt, 2Department of Pharmaceutical Technology, National Research Center, Dokki, Cairo, Egypt and 3Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt
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Abstract
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The effect of using methyl-β-cyclodextrin and hydroxylpropyl-β-cyclodextrin as
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carriers for econazole nitrate nanoparticles prepared by nano-spray dryer was
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explored
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polyvinylpyrrolidone k30, poloxamer 407, Tween 80 and Cremophor EL were
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used. The nano-spray dried formulations revealed almost spherical particles
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with an average particle size-values ranging from 121 to 1565 nm and zeta
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potential-values ranging from -0.8 to -2.5 mV. The yield-values for the obtained
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formulations reached 80%. The presence of the drug in amorphous state within
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the nanosuspension matrix system significantly improved drug release from
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them compared to that for pure drug. Combination of hydroxylpropyl-β-
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cyclodextrin with Tween 80 achieved important role for preserving the
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econazole nanosuspension from aggregation during storage for one year at room
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temperature as well as improving drug release from the nanosuspension. This
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selected formulation was suspended in chitosan HCl to increase drug release
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and bioavailability. The in-vivo evaluation on Albino rabbit's eyes demonstrated
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distinctly superior bioavailability of the selected formulation suspended in
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chitosan compared to its counterpart formulation suspended in buffer and crude
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drug suspension due to its mucoadhesive properties and nano-size. The nano-
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spray dryer could serve as a one step technique towards formulating stable and
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effective nanosuspensions.
in
this
work.
Stabilizers
namely;
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polyethylene
oxide,
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Molecular Pharmaceutics
Keywords: Nano-spray dryer; nanoparticles; eye drops
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1. Introduction
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The eye, the most sensitive and complicated organ in the body, is easily affected
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by several diseases such as glaucoma, cataract, dry eye and infection 1 . The eye
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may be infected by fungal contamination through wearing contact lenses,
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accident, trauma, surgery or rubbing the eye by infected skin 2.
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Econazole nitrate is classified as antifungal imidazole drug that is used for the
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treatment of topical fungal infections caused by Candida albicans and Tinea.
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Econazole nitrate is a water insoluble drug that create complicated barrier for its
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formulation in ocular drug delivery system 3, 4.
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Introducing the drug into nano-sized drug delivery system have showed many
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advantages
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bioavailability, reducing drug toxicity and increasing drug selectivity to certain
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organ 5. The ideal ocular drug delivery system should be effective in low dose,
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provide drug targeting to certain ocular tissues with minimum side effects,
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prolong drug action by increasing contact time with eye without causing
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irritation and overcome eye clearance and drainage that cause dose loss 6. The
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Nano-ocular drug delivery systems include nanoemulsions, liposomes,
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niosomes, solid lipid nanoparticles (SLN) and nanosuspensions.
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The nanosuspension drug delivery system can be produced by two technologies,
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top down and bottom up methods. In top down method, the reduction in drug
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particle size into smaller particles is done using mechanical forces that may
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resulting in degradation of drug such as milling and high pressure
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homogenization 7, 8, while in bottom up method, the drug is dissolved in organic
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solvent followed by precipitation or drying to get nanoparticles using solvent-
in
improving
aqueous
drug
solubility,
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increasing
drug
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antisolvent or spray drying process. Spray drying process revealed many
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advantages in increasing drug content and encapsulation efficiency as well as
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producing particles with uniform size distribution and high drug yield 9.
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Nano-spray drying process is a new advanced technology to produce
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nanoparticles. The nano-spray dryer made revolution in spray drying technology
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by producing formulations with high yield and particle size in the nano-size
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with very narrow distributions. The nano-spray drying technology depends on
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spraying the liquid formulations through piezoelectric driven actuator that
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vibrate a stainless steel mesh (at ultrasonic frequency of 60 kHz) to create tiny
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aerosol droplets in a smaller size range and with a very narrow distribution
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compared to that obtained from classical spray dryers. A high voltage is applied
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between two electrodes during the spray process to produce dried fine solid
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particles. Those particles are deposited on the inner wall of the electrostatic
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stainless steel collecting cylinder. At the end of the spray drying process, the
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dried fine powders are conveniently collected using a scraper 10.
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The nano-spray dryer was used to prepare gelatine nanoparticles in order to
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provide a sustained release for amoxicillin. The formulated nanoparticles were
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able to release the drug over 12 hours in 0.05 M phosphate buffer solution
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Furthermore, Cyclosporin A and dexamethasone were loaded in poly(d,l-
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lactide-co-glycolide) (PLGA) nanoparticles using the nano-spray dryer. The
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obtained nanoparticles showed sustained drug release from 15 to 30 days in
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.
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phosphate buffer with pH value of 7.4 12.
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The aim of this work was to formulate econazole nitrate nanosuspension using
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spray drying technique aiming to increase its dissolution and bioavailability
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while avoiding the general stability problems encountered during the storage of
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the nanoparticle suspension.
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The resulting nanoparticles were evaluated for their process yield, entrapment
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efficiency, particle size, zeta potential, in-vitro drug release, morphology and
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particle size stability during storage at room temperature. Selected econazole
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nitrate nanoparticles formulation was modified to increase its mucoadhesive
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properties and was subjected to in-vivo bioavailability study in rabbit's eyes and
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compared to that of drug suspension.
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2. Materials and methods
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2.1. Materials
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Econazole nitrate was a kind gift from Minapharm Pharmaceuticals (Egypt).
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Poloxamer 407, poly ethylene oxide (molecular weight of 100,000 g/mole) and
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dialysis tubing cellulose membrane (molecular weight cut-off 14000 g/mole)
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were purchased from Sigma Aldrich (USA). Methyl-β-cyclodextrin (MβCD)
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and hydroxylpropyl-β-cyclodextrin (HPβCD) were a kind gift from Roquette
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(France). Sodium chloride, sodium bicarbonate, calcium chloride dehydrate,
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potassium chloride, Tween 80 and absolute ethyl alcohol were purchased from
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El-Nasr Pharmaceutical Chemicals (Egypt). Cremophor EL was obtained from
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Cisme Chemicals (Italy). Polyvinylpyrrolidone (PVP K 30) was supplied by
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Morgan Chemical Industries Company (Egypt). Acetonitrile (HPLC grade) was
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purchased from Research Lab Company (India). Water (HPLC grade) was
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obtained from Fisher Scientific (UK). Chitosan hydrochloride was a kind gift
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from Zhejiang Chemicals Import and Export Corporation (China).
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2.2. Methods
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2.2.1. Preparation of econazole nitrate nanoparticles
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The econazole nanoparticles were prepared by nano-spray drying technique.
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Exactly, 0.2 g of econazole nitrate and specified amounts of carriers and
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stabilizers (table 1) were dissolved in ethanol (10 ml) followed by spraying
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through the Buchi® nano-spray dryer B-90 (Büchi Labortechnik, Switzerland).
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The nano-spray dryer was operated using inert loop B-295 system (Büchi
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Labortechnik AG, Flawil, Switzerland) operated using nitrogen gas with flow
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rate of 110 L/min. The spray drying processes were operated at an inlet
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temperature of 90°C and an outlet temperature of 35°C with a feeding rate of 40
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mL/hr. The spray mesh with 7.0 µm aperture size was used in our study. The
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obtained nanoparticles were collected from the wall of the cylindrical collecting
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electrode of the nano-spray dryer.
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2.2.2. Evaluation of econazole nitrate nanoparticles
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2.2.2.1. Determination of nano-spray drying process yield
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The econazole nitrate nanoparticles collected from nano-spray dryer were
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weighed and the yield was calculated by the following equation:
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% Yield = (Recovered mass / Mass entered in the spray dryer) × 100 144
2.2.2.2. Entrapment efficiency
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Econazole nitrate nanoparticles were weighed and then dissolved in ethanol to
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determine the amount of drug in them using Shimadzu 1800 UV (Japan)
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measured at 271 nm. The entrapment was calculated using the following
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equation:
% Entrapment efficiency = (Amount of drug in nanoparticles / Weight of nanoparticles) × 100
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2.2.2.3. Determination of particle size and zeta potential
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All formulations of econazole nitrate nanoparticles prepared by nano-spray
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dryer were dispersed in isotonic buffer pH 7.4 and their particle size,
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polydispersity index (PDI) and zeta potential values were measured using
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Malvern Nano Zetasizer ZS (UK).
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2.2.3. Stability of econazole nitrate nanosuspensions
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All the prepared econazole nitrate formulations suspended in isotonic buffer pH
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7.4 were stored for six months at room temperature. The stored nanoparticles
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were characterized for their particle size, PDI and zeta potential values at time
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intervals of 0, 1, 2, 3, 9, 12, 18, 30, 60, 90, 120, 150 and 180 days.
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2.2.4. In-vitro drug release studies
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The prepared econazole nitrate nanoparticles obtained from the nano-spray
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dryer were suspended in isotonic buffer pH 7.4 and were then tested for drug
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release by dialysis method in simulating tear fluid of pH of 7.4 containing 10%
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ethanol. Nanoparticles (equivalent to 2.5 mg of drug) were instilled in dialysis
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bag (molecular weight cut-off 14000 g/mole) containing 1 ml isotonic buffer
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with pH value of 7.4 and was secured by two clamps at each end. The dialysis
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bag was completely soaked in a vessel containing 100 ml of release medium of
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simulated tear fluid
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at 34oC (incubator shaker IKA KS 4000, Germany). The vessel was closed to
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avoid evaporation of the release medium. Samples were taken at specified time
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intervals. The samples were measured spectrophotometrically at 221.5 nm using
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Shimadzu 1800 UV (Japan). Release efficiency (RE) was calculated using the
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following equation 14: RE = (#% y × dt / y100 × t) × 100
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where, y is the amount of drug released at time t and y100 donates 100% release.
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2.2.5. Differential scanning calorimetry (DSC)
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The formulation which gave best drug release and stability was subjected to
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DSC thermogram analysis. DSC analysis was used to study the crystallinity of
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the drug in the selected formulation and physical mixture of the formulation.
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DSC studies were carried out on econazole nitrate powder, the carrier (HPβCD),
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selected formulation and its physical mixture using differential scanning
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with shaking rate of 200 stroke per min and maintaining
$
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calorimeter (DSC-50, Shimadzu, Japan). The procedure involved heating an
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accurately weighed of samples (5 mg) encapsulated in aluminum pan at
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scanning rate of 10⁰C/min and over predetermined temperature range from
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30⁰C to 400⁰C. Dry nitrogen gas was used as carrier gas with flow rate of 25
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mL/min.
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2.2.6. X-ray diffraction studies (XRD)
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X-ray diffraction patterns of econazole nitrate, carrier and selected formulation
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were obtained. The samples were irradiated with Ni filtered Cu Kα radiation
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with an operating voltage of 45kV and current of 40 mA using Diano X-ray
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diffractometer apparatus (USA). The employed scanning rate was 2⁰/min over
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diffraction angle (2θ) range of 3-70⁰.
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2.2.7. Scanning electron microscopy (SEM)
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The shape of selected econazole nanoparticles formulated by spray dryer was
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evaluated using a scanning electron microscope (Quanta FEG250, USA). The
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nanoparticles were coated with gold and were examined at 10 kV.
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2.2.8. Transmission electron microscopy (TEM)
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Selected formulations were examined by transmission electron microscope
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(JEM-HR-2100, Jeol, Japan), where, the samples were stained by 2%
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phosphotungstic acid solution and then dried at room temperature to be
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examined at 160 kV.
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2.2.9. Viscosity determination
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The viscosity of selected nanosuspension formulations was measured by
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Brookfield cone and plate viscometer using spindle CS-40 (DV3T, USA). The
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required volume that was placed between cone and plate was 3 ml and
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temperature was adjusted at room temperature.
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2.2.10. Mucoadhesion study
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Selected formulations suspended in isotonic buffer pH 7.4 or in 0.1% chitosan
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HCl solution were mixed with equal volume of mucin solution (0.4 mg/ml) by
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vortex for 1 min, then the resulted mixtures were evaluated for their zeta
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potential and particle size values 15.
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2.2.11. Ocular irritation study
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The ocular irritation study was performed on 12 Albino rabbits which were
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divided into two groups, each group consisted of 6 rabbits. The first group
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received the selected econazole nitrate nanoparticles that were dispersed in
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isotonic buffer 7.4 (EH-TW2) on the right eye and the second group received
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the selected nanoparticles that were dispersed in 0.1 % chitosan HCl solution
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(EH-TW2C), while the left eye worked as a control for both groups.
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The administration of 50 µL of econazole formulation into rabbit's eyes was
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repeated three times every 2.5 hour per day for the first three days and once on
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the fourth day 16. According to Draize test the rabbit's eye was tested after 1 and
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24 hour from last instillation of the formulation
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adverse reactions and irritation of cornea, conjunctiva, eyelids, anterior chamber
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and iris was performed. The evaluation criteria were classified as follows: no
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irritation (0 – 3.9), slight irritation (4 – 8.9), moderate irritation (9 – 12.9) and
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high irritation (13 – 16) 18.
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2.2.12. In-vivo evaluation of selected econazole nitrate nanoparticles
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2.2.12.1. Study design
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The study was performed on three formulations containing drug concentration
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of 125 µg/ml; prepared nanoparticles suspended in 0.1% chitosan HCl or in
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isotonic buffer pH 7.4 as well as drug suspension. The tested formulations were
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sterilized with gamma radiation using a dose of 10 kGy. This study was
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. The evaluation for any
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approved by the Research Ethics Committee, Faculty of Pharmacy, Cairo
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University, Cairo, Egypt (No: PI 1149). Eighteen healthy Albino rabbits
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(weighing 1.5-2 kg) were randomly divided into three groups, each containing
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six rabbits. A parallel group design was applied, so that each group received
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single ocular dose of tested formulation.
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2.2.12.2. Tear film sampling
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The rabbit's eyes received 50 µL of tested formulation that was instilled in the
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conjunctival sac. Samples of tear films were collected by placing two sterile
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filter paper discs (6 mm in diameter) under the lower eyelid of the rabbit's eye
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for only 1 min at different time points as follows: zero (pre-dose), 0.5, 1, 2, 3, 4,
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5, 6, 7 and 8 hours. Full care was taken during sampling to avoid the irritation
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of the eyelid margin. The collected discs were immersed in 50 µL of acetonitrile
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and stored in Eppendorfs at −20◦C until being analyzed. Depending on the in-
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vivo study design to assess the pharmacokinetic profiles of econazole nitrate, its
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total amount in the collected tears was displayed as a function of time, and the
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following pharmacokinetic parameters were calculated (Cmax, Tmax, AUC(0-24hr),
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AUC(0-∞), AUMC(0-24hr), AUMC(0-∞) and MRT).
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2.2.12.3. Chromatographic conditions
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The concentration of econazole nitrate was analyzed in rabbit's tears applying a
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validated high performance liquid chromatography method described by Ng et
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al
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detector and isocratic pump (LC-20AT, Shimadzu, Japan) was used. A C18
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reversed phase analytical column (250 mm × 4.6 I.D. mm, particle size 5 µm,
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Berlin, Germany) was used.
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The filtered and degassed mobile phase consisted of acetonitrile and 12 mM
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sodium acetate buffer with ratio of 75:25 v/v and its pH value was 8.5. The flow
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. A high performance liquid chromatography system with an ultraviolet
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rate of the mobile phase was set to 1.5 ml/min and the drug was detected at 220
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nm.
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2.2.13. Statistical analysis
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The data were collected and were expressed as mean values ± SD. The data
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were subjected to statistical analysis by applying two-way analysis of variance
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(ANOVA). Statistical analysis was performed using SPSS® software, USA. The
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difference between values was considered to be significant at p
