Surface Modification of Pollen-Shape Carriers for Dry Powder

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Surface modification of pollen-shape carriers for dry powder inhalation through surface etching Thi Quynh Ngoc Nguyen, Hung Loong Giam, Yabo Wang, Adam Pac#awski, Jakub Szl#k, Aleksander Mendyk, Yu-Hsuan Shao, and Raymond Lau Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/ie502980k • Publication Date (Web): 04 Dec 2014 Downloaded from http://pubs.acs.org on December 10, 2014

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Industrial & Engineering Chemistry Research

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Surface modification of pollen-shape carriers for dry powder inhalation through surface

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etching

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Thi Quynh Ngoc Nguyen,† Hung Loong Giam,† Yabo Wang,† Adam Pacławski,‡ Jakub Szlęk,‡

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Aleksander Mendyk,‡ Yu-Hsuan Shao,*,§ and Raymond Lau*,†

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†

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Nanyang Drive, Singapore 637459

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‡

School of Chemical and Biomedical Engineering, Nanyang Technological University, 62

Department of Pharmaceutical Technology and Biopharmaceutics, Jagiellonian University

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Medical College, Medyczna 9 St, 30-688 Krakow, Poland

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§

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Taipei City, Taiwan 110

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Abstract

Graduate Institute of Biomedical Informatics, Taipei Medical University, 250 Wu-Hsing Street,

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Surface modification of pollen-shape hydroxyapatite (HA) carriers is achieved using

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surface etching technique. Characterization of HA carriers before and after etching are

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performed by scanning electron microscopy (SEM), Carr’s compressibility index (CI), X-ray

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diffraction (XRD), and thermogravimetric analysis (TGA). Four surface etching temperatures are

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tested but only the particles etched at 40ºC and 50ºC are found satisfactory. Proper surface

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etching allows a reduction of the petal-like structure on the particle surface and improves the

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crystallinity of the particles. While the reduction of petal-like structure decreases the emitted

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dose (ED) of the drug particles, an increase in fine particle fraction (FPF) can be attained owing

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to the improvement in drug liberation. An increase in the air flow rate, however decreases the *

Corresponding author. Tel.: +65 6316 8830 (R. Lau); +886 2-2736-1661-3356 (Y.-H. Shao) Email addresses: [email protected] (R. Lau); [email protected] (Y.-H. Shao)

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ACS Paragon Plus Environment


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significance of particle surface morphology and the difference in FPF among the used of

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different carrier particles diminishes. Surface etching technique is found to have good potential

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as an economical process to improve dry powder inhalation efficiency through surface

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modification.

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Keywords: dry powder inhalation, drug carrier, surface etching, in vitro deposition, emitted

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dose, fine particle fraction

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Introduction

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The use of pollen-shape drug carriers in dry powder inhalation (DPI) has been found to

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have a great potential in the delivery of powdered drugs through inhalation1-6. The pollen-shape

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carriers are capable of achieving a higher emitted dose (ED) and fine particle fraction (FPF)

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compared to irregular-shape lactose carriers1,2. The pollen-shape surface allows a long time of

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flight for the detachment of drug to take place. In addition, pollen-shaped carriers have large

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surface areas and binding sites that allow high drug attachment capacity. Both factors contribute

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to the high ED and FPF achieved1,2. Based on the regional deposition results, it is found that the

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high drug attachment characteristics of the pollen-shape carriers also restrict the drug liberation

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during inhalation. It is anticipated that the FPF can be further improved by reducing the surface

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interaction between the carriers and the drugs. However, reduction of particle adhesion can also

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lower the ED4,7-9. It is important to strive for a balance between the attachment and the liberation

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of drugs at the carrier surfaces.

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Several approaches have been proposed to reduce the interaction between particles, such as

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the use of spacer particles10, addition of nano/micro-particles11-13, surface coating14,15, etc. While

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these approaches have shown satisfactory results in a number of studies, the effect is specific to

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the carrier particles applied. The use of spacer particles and nanoparticles was found to give

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negligible improvement on the performance pollen-shape carriers. It is also a great challenge to

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apply surface coating on the pollen-shape surfaces. Thus, surface etching is proposed in this

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study as an alternative surface modification method to reduce the surface interactions.

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The surface etching technique involves the dissolution of the carrier surface partially in an

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etching solution. The etching solution can be an acid or a saturated solution of the same or

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similar composition as the carrier particles. Acid has the ability to dissolve many types of

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organic or inorganic compounds. The degree of dissolution is dependent on the dissolution time

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and the concentration of the acid16. It has been reported in the literature that the dissolution

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mechanism of hydroxyapatite starts with the formation of dissolution nuclei17. The nuclei appear

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most likely on inhomogeneous surfaces and both polynuclear dissolution and etch pits formation

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mechanisms can happen simultaneously17,18. Alternatively, the carrier particles can be mixed

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with a saturated solution of the same composition19. A subsequent increase in temperature can

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increase the solubility of the solution. Certain parts of the carrier surface can be dissolved and

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hence achieved surface etching. The use of surface etching causes minimal change in the

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material properties of the original particles. The simple nature of the process allows a good

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potential in scale-up and utilization in the pharmaceutical industry.

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In this study, the modification of pollen-shape carrier surfaces by surface etching through

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temperature control is explored. Pollen-shape hydroxyapatite (HA) particles are used the carriers.

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HA has the chemical formula of Ca5(PO4)3(OH). Saturated solution of phosphate salt is used as

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the etching solution. The HA particles before and after surface etching are characterized by

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scanning electron microscopy (SEM), Carr’s compressibility index (CI), X-ray diffraction

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(XRD), and thermogravimetric analysis (TGA). The in vitro aerosolization and deposition

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properties of the modified and original HA carriers are also determined using Budesonide (Bd)

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as the model drug using Andersen cascade impactor (ACI).

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Experimental

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Preparation of HA

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Hydroxyapatite (HA, Ca5(PO4)3(OH)) particles are synthesized by hydrothermal reaction

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of potassium dihydrogen phosphate (KH2PO4, Sigma-Aldrich) and calcium nitrate tetrahydrate

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(Ca(NO3)2.4H2O, Sigma-Aldrich). The resulting pollen-shape morphology is governed by the

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synthesis temperature and the concentrations of poly (sodium-4-styrene sulfonate) (PSS, Sigma-

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Aldrich) and urea (Sigma-Aldrich) used. The synthesis condition used in this study is following

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the one used in a previous study2. 60 mL of KH2PO4 (0.02 M) solution is mixed with 100 mL of

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Ca(NO3)2.4H2O (0.02 M) solution. Subsequently, sufficient PSS and urea are added to obtain a

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respective concentration of 40 g/L and 0.5 M. The final mixture is stirred for 30 minutes to allow

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complete dissolution of the urea. The solution mixture is then kept in an autoclave and put into

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an oven. A reaction temperature of 120ºC is maintained for 6 hours. The final precipitated

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product is then filtered, washed, and dried at 80ºC.

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Surface Etching

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A saturated solution of potassium dihydrogen phosphate, KH2PO4 is used as the etching

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solution. 20 g of KH2PO4 is mixed with 100 mL of distilled water. The amount of KH2PO4 added

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exceeds the solubility limit of KH2PO4 in distilled water. Upon constant stirring of 30 minutes,

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the undissolved KH2PO4 is filtered. Afterwards, HA carriers are introduced to the saturated

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KH2PO4 solution. A constant stirring is maintained to suspend the carriers in the solution. The

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suspension temperature is then raised to a desired point slowly by a heater. The carriers are

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allowed to re-dissolve into the solution at the desired temperature for 15–30 minutes and hence

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the carrier surface is etched. The resulting HA carriers are finally filtered, washed, and dried at

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80ºC. Four surface etching temperatures, 30ºC, 40ºC, 50ºC, and 60ºC are being explored in this

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study.

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Shape and Surface Morphology

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The physical properties such as the shape and surface morphology of the HA carriers are

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characterized by the scanning electron microscope (SEM) (JSM-5600, JEOL, Tokyo, Japan).

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Dry HA particles are placed uniformly on a carbon tape attached on a stub. The particles are then

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coated with platinum under an argon atmosphere (JFC-1600, JEOL, Auto Fine Coater, Tokyo,

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Japan) for 60 seconds with a current of 20 mA. SEM images are taken at random locations over

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the coated particles to obtain an overall indication of the particle physical properties.

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Particle Densities and Flowability

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The bulk density (ρbulk) and tap density (ρtap) of the particles can be used to give an

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indication of the particle flowability. 100 mg of a sample is placed in a 1 mL micro-syringe. The

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syringe tube is then tapped against a tabletop by hand at a rate of about 4 times per second for

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2,000–2,500 times until no reduction in the sample volume is noticed. The volumes of the

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sample before and after tapping are subsequently used to compute ρbulk and ρtap, respectively2.

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Triplicate measurements were performed.

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Particle flowability can be characterized by Carr’s Compressibility Index (CI) which relates to ρbulk and ρtap based on the equation:

ρ −ρ  bulk  ×100% CI =  tap ρ   tap

(1)

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Crystalline structure

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The crystalline structure of the HA carriers are determined using X-ray diffraction (XRD).

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A thinner layer of powder samples are placed into a sample holder. It is then inserted into a

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LabX-Shimadzu XRD6000 diffractometer (Shimadzu Corporation, Kyoto, Japan) with Cu Kα as 6


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X-ray source (λ = 1.5406 Å). The parameters are fixed at a scan rate of 0.5/min, voltage of 40

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kV, current of 40 mA and step size of 0.02 for all measurements.

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Moisture content

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Thermogravimetric analysis (TGA, Simultaneous TGA/DSC SDT Q600, TA Instrument,

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Instrument Research) is conducted to measure the moisture absorbed on the surface of particles.

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TGA measurements are performed from 30ºC to 130ºC at a heating rate of 10ºC/min under

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nitrogen atmosphere with 8 – 10 mg of each sample.

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Specific surface area

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The BET surface area of each HA carrier is determined. The Autosorb® Degasser

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(Quantachrome Instruments, FL, USA) is used to dry the samples at 90ºC for 24h under nitrogen.

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The surface area of the each sample is then measured in an Autosorb® 6B (Quantachrome

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Instruments, FL, USA) surface analyzer using nitrogen adsorption method.

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Blending of carriers with Bd

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Budesonide (Bd) (Sigma, Singapore) is used as the model drug in the current study. The

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Bd drug particles had a D50 of 2.8 µm and were used as received. A 100 mg mixture of carrier

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and drug in a ratio of 10:1 by weight is mixed in a REAX top mixer (Heidolph, Kelheim,

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Germany). Each blend is blended under 1000 rpm for 15 min.

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Drug content and content uniformity

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Average drug content and content uniformity are examined by measuring the quantity of

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Bd in the blend. 5 mg of each blend is randomly taken and dissolved into a fixed amount of

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solvent, comprises of a mixture of 2 % nitric acid (Fluka, Singapore) with ethanol (Merck,

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Singapore) in a volume ratio of 3:1. The solution is then analyzed using a UV spectrophotometer

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(Shimadzu, Japan, Nottingham, UK) with a wavelength of 250 nm. The measurement is

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performed in triplicate for all blends. The content uniformity for the mixture is assessed from the

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coefficient of variance (CV). The CV is defined as the ratio of the standard deviation of the drug

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content to the average drug content in the samples as percentage.

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In Vitro Aerosolization and Deposition Properties

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An eight–stage Anderson Cascade Impactor (ACI) (Copley Scientific, Nottingham, UK) is

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used to characterize the in vitro aerosolization and deposition properties of Budesonide (Bd,

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Sigma, Singapore) blended with the pollen-shape carriers. Rotahaler® is used as the inhaler

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device. A schematic diagram of the ACI setup is shown in Fig. 1. For each run of the experiment,

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8 mL of solvent containing ethanol and nitric acid is poured into the pre-separator. The impactor

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plate of each stage is coated with 1% (w/v) solution of silicon oil in hexane to prevent bouncing

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and re-entrainment of particles. 10 ± 0.5 mg of each blend is loaded into a hard gelatin capsule

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(Gelatin Embedding Capsules, size 4, 0.25 cm3, Polysciences, Inc., PA) before putting into the

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inhaler. An actuation time of 8 seconds and 4 seconds is used for air flow rate of 30 L/min and

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60 L/min, respectively to completely disperse all the particles in the capsule. Each experimental

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condition is repeated a minimum of three times to ensure repeatability.

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At the end of each run, particles remained in the capsule, the inhaler and different stages of

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the ACI are extracted. A solvent mixture of 2% nitric acid (Fluka, Singapore) with ethanol

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(Merck, Singapore) in a volume ratio of 3:1 is used to extract the Bd. The concentration of the

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Bd in the solvent is measured using a UV spectrophotometer (Shimadzu, Japan, Nottingham,

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UK) with a wavelength of 250 nm. Each solution is analyzed three times and the average is

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computed.

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The in vitro aerosolization and deposition properties of the blend can be characterized by

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the emitted dose (ED) and fine particle fraction (FPF). ED is the mass percentage of the drug that

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is delivered from the inhaler and FPF is the mass percentage of the drug particles whose

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aerodynamic diameter is smaller than 5.8 µm classified by the different stages of the ACI.

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Results and discussions

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Physical characteristics of surface modified pollen-shape HA carriers

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For simplicity, the original unmodified HA carriers is denoted as “HA-original”. The HA

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carriers modified by the surface etching technique is denoted as “HA-30”, “HA-40”, etc, where

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the number represents the surface etching temperature. The SEM images of the original and the

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surface modified HA carriers are shown in Fig. 2. It can be seen from Fig. 2(a) that HA-original

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particles are spherical and have pollen-shape surface with dense petal-like structures. A

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comparison of Figs. 2(a) and 1(b) indicates that a surface etching temperature of 30ºC does not

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provide any noticeable change in the pollen-shape surface. On the other hand, Fig. 2(e) shows

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that a surface etching temperature of 60ºC destroyed the pollen-shape surface and the particle

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becomes irregularly shaped. As shown in Fig. 2(c), a surface etching temperature of 40ºC retains

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the pollen-shape morphology but the petal-like structure becomes less dense. This is attributed to

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a successful surface etching of the phosphate content present in the HA-original particles and

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leads to a reduction in the petal-like structure of the resulting HA-40 particles. At a higher

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surface etching temperature of 50ºC, a number of fibrous structures are formed on top of the

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petal-like structures of HA-50 particles shown in Fig. 2(d). However, the high degree of surface

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etching causes a certain degree of deformation from the round overall particle structure. Since

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surface etching temperatures of 40ºC and 50ºC are able to give sufficient structural change to the

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pollen-shape surface and still maintain the overall shape of the particles. Therefore, only HA-40

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and HA-50 carriers are used for further investigations. It is also important to note that there is no

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obvious change in the particle size before and after surface modification. The differences

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observed for the carrier performance can be considered a result of surface morphology alone.

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One important particle characteristic for DPI is the particle flowability. It directly impacts

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the dose uniformity. A poor particle flowability leads to irregular flow and causes a large

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variation in the amount of particles released from the capsule20. The CI index can be used to

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characterize the particle flowability and can be calculated from ρtap and ρbulk. A high CI index

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indicates poor flowability based on the concept that compressible and poorly flowable particles

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are more cohesive and thus a large difference between ρtap and ρbulk. ρtap, ρbulk and CI of the HA

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carriers before and after surface modification and their respective particle size distribution and

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specific surface areas are shown in Table 1. It can be seen that the surface etching treatment

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increases both ρtap and ρbulk. Through surface etching, there is a reduction in the surface density

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of the petal-like structure. It allows the particles to be more tightly packed even in a loose

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packing condition. Therefore, an increase in ρbulk and ρtap is observed. For HA-50, the particles

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deviate from a round overall structure and further increases ρbulk and ρtap. However, the degree of

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increase in ρtap and ρbulk are not at all the same. As the surface density of the petal-like structure

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decreases, there is a reduction in the particle-particle contact and subsequently reduces the

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cohesive nature of the particles. As shown in Table 1, HA-40 has a CI value of 39.6% and HA-

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50 has a CI value of 32.9%. This indicates that surface etching is effective in reducing the

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particle cohesiveness and improves the particle flowability.

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Moisture can affect particle dispersion and flowability by enhancing the particle

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aggregation tendency. TGA is therefore carried out to measure the moisture content. The

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thermogram showed a negligible amount of free water in all samples by expressing the marginal

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amount of weight loss (

Surface Modification of Pollen-Shape Carriers for Dry Powder

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