Acoustic spectroscopy: A powerful analytical method for the pharmaceutical field?

Accepted Manuscript Title: Acoustic spectroscopy: a powerful analytical method for the pharmaceutical field? Author: Giulia Bonacucina Diego R. Perinelli Marco Cespi Luca Casettari Riccardo Cossi Paolo Blasi Giovanni F. Palmieri PII: DOI: Reference:

S0378-5173(16)30208-3 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.03.009 IJP 15607

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

3-12-2015 13-2-2016 9-3-2016

Please cite this article as: Bonacucina, Giulia, Perinelli, Diego R., Cespi, Marco, Casettari, Luca, Cossi, Riccardo, Blasi, Paolo, Palmieri, Giovanni F., Acoustic spectroscopy: a powerful analytical method for the pharmaceutical field?.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.03.009 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.

Acoustic spectroscopy: a powerful analytical method for the pharmaceutical field?

Giulia Bonacucina1, Diego R. Perinelli1, Marco Cespi1, Luca Casettari2, Riccardo Cossi3, Paolo Blasi1, Giovanni F. Palmieri1*

1

University of Camerino, School of Pharmacy, 62032 Camerino (MC), Italy

2

University of Urbino, Department of Biomolecular Sciences, 61029 Urbino (PU), Italy 3

QI, Via Monte D‟Oro 2/A, 00040 Pomezia (RM), Italy

*Corresponding author: [email protected], Tel: +39 737 402233. Fax: +39 737 637345

Graphical abstract

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ABSTRACT Acoustics is one of the emerging technologies developed to minimize processing, maximize quality and ensure the safety of pharmaceutical, food and chemical products. The operating principle of acoustic spectroscopy is the measurement of the ultrasound pulse intensity and phase after its propagation through a sample. The main goal of this technique is to characterise concentrated colloidal dispersions without dilution, in such a way as to be able to analyse non-transparent and even highly structured systems. This review presents the state of the art of ultrasound-based techniques in pharmaceutical preformulation and formulation steps, showing their potential, applicability and limits. It reports in a simplified version the theory behind acoustic spectroscopy, describes the most common equipment on the market, and finally overviews different studies performed on systems and materials used in the pharmaceutical or related fields.

Keywords: Ultrasound, sound speed, sound attenuation, nanoparticles, soft matter, disperse systems

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INTRODUCTION Although studies of inaudible acoustic waves started in the 19th century, the modern science of ultrasonics began to around 1917 (Graff, 1981). Ultrasound is defined as sound pressure waves whose frequency exceeds the audible range for the human ear (~20 kHz). Some animals use ultrasound for navigation (dolphins) or hunting (bats), exploiting the information carried by backscattered sound waves. Ultrasound is one of the technologies developed to minimize processing, maximize quality and ensure the safety of pharmaceutical, food and chemical products (Knorr et al., 2011). An important application of this technique for the pharmaceutical, food and chemical industries is the characterisation of dispersed systems, which requires identification of the fraction of the dispersed phase (volume or weight) and the size distribution of the droplets or particles. Both can be obtained from acoustic measurements with the advantages that ultrasonic techniques are fast, nondestructive, reliable and can be applied to opaque media, making it possible to study dispersions as they are. Ultrasound is used at different power levels and frequencies for different purposes, low-intensity ultrasound at high frequency (> 1MHz) and high-intensity ultrasound at low frequency (20-100 kHz) being the main combinations (Povey, 1998) . When waves of low-intensity ultrasound (< 1 W/cm2) travel through a material, they do not alter its physical or chemical properties. Since the speed and efficiency of the transmission depend on the nature of the bonds and masses of the molecules present and therefore on the composition of the material (Coupland and McClements, 2001), low-intensity ultrasound can be used to obtain information about the physicochemical properties of the materials. By comparing the incident and resultant ultrasonic wave, the structure of the sample can be defined (McClements, 1995). On the contrary, high-intensity ultrasound uses a markedly greater amount of power (typically 10-1000 W/cm2), with consequent physical and chemical changes to the material to which they are applied (McClements, 1995). For this reason, high intensity ultrasound is used to homogenize or decompose samples, or to promote such chemical reactions as oxidation. The creation of ultrasonic pulses is based on the ability of piezoelectric elements to convert electric energy to mechanical energy and vice versa. The piezoelectric elements do not have a symmetric centre; the application of an alternating voltage (AC) on the piezoelectric element causes the element to oscillate (compress and expand) at very high frequencies, producing high frequency mechanical sound waves (Povey, 2013). Waves can be differentiated into two main forms, the longitudinal (compressional) ultrasound wave, where the propagation direction is identical with the oscillation direction (the medium is 3

locally compressed and dilated), and the transversal (shear) ultrasound wave, where the direction of propagation is vertical to that of the oscillation plane (the medium is exposed to shear stress) (Povey, 2013). Sound propagates with certain velocity (sound speed), intensity and intensity decay (attenuation). The intensity decreases and the phase changes as the ultrasound propagates through and interacts with a heterogeneous system. The decrease in fluctuation amplitude is usually referred to as “ultrasound attenuation.” The phase is related to the speed of ultrasound propagation through the particular system. The presence of a dispersed phase strongly influences the total attenuation and causes a change of phase in the sound speed parameter, in comparison to that of the pure liquid medium. Thus, if we measure the variation of these ultrasound properties, we can also extract some information about the properties of the whole system ( Dukhin et al, 2005). While measurements at a single frequency are usually sufficient to obtain such information as the volume (or weight) fraction of the dispersed phases, measurements at varied frequencies offer even more information, as these frequency changes cause large changes in the acoustic attenuation, a particularly important parameter for identifying particle size (Hipp et al., 1999). Thus, in recent years ultrasonic spectroscopy has attracted considerable attention as a promising particle size measurement technique. Indeed, it offers a unique possibility for characterizing concentrated colloidal dispersions, since it avoids sample dilution and can be used to analyze non-transparent and even highly structured systems (Dukhin, 2002). Hence, issues like the difficulty of analyzing optically opaque, highly or non-electrically conducting media, and highly concentrated dispersed systems (up to 40% of dispersed phase) can be resolved (Dukhin and Goetz, 1996). Furthermore, acoustic spectroscopy provides a suitable technique for characterizing colloidal systems and also for studying their stability, with the additional possibility of distinguishing between aggregation and flocculation phenomena for both solid rigid particles and soft particles (lattices, emulsions and microemulsions). However, the success of a measurement technique is also determined by the quality of the analysis algorithm, thus, many researchers have examined the applicability of ultrasonic spectroscopy to various material systems, the validity of the different models, the performance at high particle concentration and the sensitivity to particle shape (Babick et al., 2000). Though acoustic spectroscopy is widely used to analyze several systems as paints, cosmetics, ceramics, minerals, pigments, oxides, chemical-mechanical polishing materials and foods, its use is still limited in the pharmaceutical field. However, in recent years some progress has been made on the use of this technology in this field as well, and an overview of these developments should be very helpful for those doing pharmaceutical research and development, as well as production and quality control. 4

Thus, this review describes the state of the art of ultrasound techniques in the pre-formulation, formulation and quality control of different types of pharmaceuticals, highlights the potential, applicability and limits of this technique, and provides an overview of different studies performed on systems and materials with implications for the pharmaceutical field.

2. Theory and Instrumentation 2.1 Theory The operating principle of acoustic spectroscopy is based on the measurement of the ultrasound pulse amplitude and phase after its propagation through the sample. During the passage through the sample, sound is attenuated by the presence of the liquid medium and any particles in dispersion; by applying the appropriate theories, we can exploit sound attenuation to obtain information about particle properties. There are six loss mechanisms of sound interaction with a dispersed system:  Viscous: related to the shear waves generated by the dispersed particles oscillating in the acoustic pressure field due to the difference in the densities between particle and medium,  Thermal: related to the temperature gradients generated near the particle surfaces,  Scattering: similar phenomenon occurring in light scattering,  Intrinsic: involving losses of acoustic energy due to due to dissipation within the particles and within the continuous phase during sound propagation,  Structural: caused by the oscillation of a network of particles; this mechanism is specific for structured systems such as concentrated colloid dispersions,  Electrokinetic: related to ultrasound/electric double layer interactions. Electrokinetic losses are negligible in terms of the total attenuation, making it possible to separate electroacoustic spectroscopy from acoustic spectroscopy. Electroacoustic spectroscopy deals with the interaction between electric and acoustic fields. Thus, it is possible to apply a sound field and measure the resultant electric current, which is referred to as the colloid vibration current (CVI), or, on the contrary, one can apply an alternating electric field and measure the resultant acoustic field, which is referred to as the electronic sonic amplitude

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(ESA). Thus, the sixth mechanism of particle interaction with sound leads to the transformation of part of the acoustic energy to electric energy. Total attenuation is mainly the sum of the first five contributions:

Different acoustic theories have been developed to characterise particulate systems in order to find a relationship between the acoustic properties (sound speed, attenuation, etc.) and the microscopic characteristics of a heterogeneous system, such as composition, structure, and particle size. Among them, the ECAH theory, whose acronym comes from the names of its creators (Epstein, Carhart, Allegra and Hawley), (Epstein and Carhart, 1953, Allegra and Hawley, 1972) is probably the most well-known. This theory describes the interaction of single elastic or viscous particles in viscous media with sound waves for all combinations of size and frequency. This means it addresses compressional, viscous and thermal waves, but it does not address visco-elastic particles, visco-elastic or elastic dispersion media, effects of surface tension or surface rheology, any kind of interaction among neighbouring particles, electroacoustic effects etc. The primary results are the shapes of the compressional, viscous and thermal waves generated around the particle (analogy to Mie theory in optics). Then, extrapolation to suspensions is done by assuming a random configuration of particles in the suspension and large interparticles spacing (i.e. relative to wavelengths of the compressional, viscous or thermal wave); this extrapolation delivers attenuation and phase velocity of sound within the dispersed system and also the scattering from a suspension (analogy to Lambert-Beer in optics). It is constructed in two stages. The first, the „„single particle theory,‟‟ attempts to account for ultrasound disturbances surrounding one single particle. This stage relates the microscopic properties of both the fluid and the particle to the system properties at a single particle level. The second stage, referred to as the „„macroscopic theory,‟‟ relates this single particle level to the macroscopic level at which the experimental raw data are obtained. At this stage, the total attenuation is regarded as a superposition of the contributions from each particle, and particle– particle interactions are neglected. Thus this theory presents some limitations, but can surely be considered valid for moderately concentrated samples when „„thermal losses‟‟ are the dominant mechanism of attenuation, as in emulsion or latex systems. Two different parameters play an important role, “viscous depth” and “thermal depth.” These two parameters are a measure of the decay of the shear and thermal waves in the liquid. They are actually involved in the differences observed between soft and solid particle dispersions, since 6

particles oscillating in the sound wave generate these shear and thermal waves, which damp in the vicinity of the particle. In practice „„viscous depth‟‟ (dv) is the characteristic distance for the shear wave amplitude to decay, while „„thermal depth‟‟ (dt) is the corresponding distance for the thermal wave: (2)

where  is the kinematic viscosity of the medium,  the sound frequency, m the medium density, m the medium heat conductance and Cm p is the heat capacity at constant pressure of the medium. Since for most liquids such as water, „„thermal losses‟‟ are much less sensitive than „„viscous losses‟‟ to „„particle–particle‟‟ interactions, the ECAH theory is valid for emulsions or other soft dispersed systems characterised by a considerable volume fraction of the dispersed phase. (Dukhin and Goetz, 2001) In practice, in the measuring cell, a transducer converts an electrical input to an ultrasound pulse of a certain frequency and intensity and launches it into the sample. The intensity of this pulse decreases as it passes through the sample due to the interaction with the fluid. A second transducer converts this weakened acoustic pulse back to an electric signal and sends it to the electronics for comparison with the initial one. The total loss and time delay from the input to output transducer for each frequency can be considered the „„raw data‟‟ from which further interpretation is done. It is convenient to present these raw data in terms of an attenuation coefficient  defined as (4)

where f is the frequency of the pulse, L the distance between transmitter and receiver and Iin and Iout are the intensities of the emitted and received pulse, respectively. The ECAH theory is the most common approach to get particle size from sound attenuation data. However, it requires a set of physical parameters describing solid and liquid phases (i.e. density, 7

heat capacity, shear rigidity, etc.). Li et al (Li et al, 2004) and Babick and coworkers (Babick et al., 2006) developed alternative strategies able to overcome the issue of partially unknown material properties. In any case both these approaches are quite complex, require a certain measurement experience and are not applicable for all the systems. The sound speed c is obtained from the following equation, t being the delay time between emitting and receiving the pulse: c = L/t

(5)

Such experimental data can be used either for empirical correlations with other properties of the system under investigation, or for further theoretical treatment. This second step is particularly interesting, as noted above, for the determination of the particle size distribution. In fact, starting from the ECAH, several other theories, rather complex but demonstrated and experimentally verified, allow the calculation of each single contribution to the whole acoustic attenuation and the transformation of these data into particle size. For further insight into these complex theories, the reader is invited to consult specific treatises on this subject. (Dukhin and Goetz, 2002) The acoustic spectrometer can also act as an extensional, or longitudinal microrheometer, taking into account the fact that in this case „„longitudinal‟‟ viscoelastic properties are measured because the stress is not tangential (as for an oscillation experiment in a rotational rheometer), but normal. Thus, it is possible to relate measurements of ultrasonic absorption and velocity to the real and imaginary part of the complex modulus. (Litovitz and Davis, 1964) It is demonstrated that:

(6)

(7)

2.2 Instrumentation Instruments can be classified as continuous, quasi-continuous and pulsed, according to the way the sound wave is generated. While the most common type of sound transducer employs a piezoelectric ceramic called lead zirconium titanate (PZT), there are many other types of construction, including electrically polarized plastics and single crystal materials such as quartz or lithium niobate. Single 8

frequency, frequency scanning or broad-band (simultaneous multiple frequencies) generation of sound wave are possible, typically over frequencies between a few Hz and 100 MHz. Sample volumes range from a few hundred microliter, in resonant cell systems, to 500 ml in a quasicontinuous, frequency sweeping spectrometer. (Povey, 2006) A brief description of four of the most common devices on the market follows.

2.2.1 DT 1200 series Combined acoustic and electroacoustic spectrometers developed during the last decade by Dispersion Technology Inc. (DT-1200, 1201 and 1202) allow for separate or combined use of the sensors, both of which have two piezo crystal transducers and measure signals using a pulse technique. The Acoustic sensor has identical transmitting and receiving piezoelectric transducers separated by a variable motor-controlled gap. The Electroacoustic sensor consists of a third piezoelectric transducer that transmits ultrasound pulses and an electroacoustic antenna receiver separated by a fixed gap. The combined sensor has a single piezoelectric transducer serving as both the transmitter and receiver of the ultrasound pulses and an electroacoustic antenna receiver separated by a variable motor controlled gap. A single transducer can be used for the acoustic measurement by exploiting the reflected signal from the face of the antenna and measuring the round trip attenuation. The Acoustic spectrometer measures the attenuation spectra using a pulse technique with a variable gap between the transducers. The instrument can work in a single gap mode or in variable gap mode (from 0.15 to 0.20 mm in 21 steps). The principle advantage of the variable gap mode is that it offers the possibility to obtain attenuation data over a wider range of frequencies, since only larger gaps provide useful information at low frequencies, while for higher frequencies, only the smaller gaps give reliable data. The instrument also provides data on the sound speed by measuring the phase of the sound wave. The measured sound speed serves to verify the suitability of the model dispersion, which is used for the calculation of both the particle size distribution and the ζ-potential. The Acoustic attenuation spectra are used for calculating the particle size distribution when relevant sample properties are known, as highlighted in the theory section. Such physical and chemical properties as density, viscosity and heat capacity should be determined before performing any acoustic measurement. They must be input into a database containing different substances or materials that constitute the dispersed and the dispersing phase of the system to test. The theoretical attenuation is calculated using the equation, and the PSD is derived using three different model distributions (monodisperse, 9

modified lognormal and bimodal). The best fit is determined for each PSD model by minimizing the "error" between the theoretical attenuation and the experimental data. The final PSD is then selected according to the fitting errors calculated. (Dukhin and Goetz, 2000) The calculation of zeta potential from CVI requires information about particle size distribution, acoustic impedance, and sound attenuation, parameters that may be known a priori or that can be derived from the acoustic attenuation spectra when acoustic and electro-acoustic spectrometers are combined together. (Dukhin and Goetz, 2002b) The frequency used in the analyses changes from 1 to 100 MHz in an adjustable number of steps. The total volume of sample required for the analysis is about 50ml. The cell is equipped with a magnetic stirrer to prevent sedimentation and assure correct mixing during titration experiments. Indeed, the instrument is outfitted with two burettes for automatic titration and also has conductivity and temperature probes. ( Dukhin and Goetz, 1998) Five different US patents describe aspects of this instrument. (Dukhin and Goetz, 2000; Dukhin and Goetz, 2002a; Dukhin and Goetz, 2002b, 2005a; Dukhin. and Goetz, 2005b) Potentially, the instrument could be used in different fields for the determination of particle sizing and zeta potential, (Dukhin and Goetz, 1998; Wines et al, 1999a), in nanotechnology, (Comba and Sethi, 2009; Dukhin, 2007; Sun et al., 2006), bio-sciences (Bonacucina et al., 2008; Dukhin et al., 2006; Mueller and Mann, 2007; Rezwan et al., 2004), food sciences (Dukhin et al, 2005), cosmetics (Dukhin et al., 2010) and, as reviewed in this article, for the characterization of pharmaceutical systems (Dukhin et al., 1996; Richter et al., 2007; Wines et al., 1999a).

2.2.2 Opus Sympatech This instrument is designed as a probe for in-line particle size analysis and can be adapted to nearly all kinds of process pipes or vessels using a DN 100 flange or process adapters. The new ultrasonic extinction sensor is a “one flange solution,” which allows measurements to be carried out in-line inside a pipe or a vessel. The part of the sensor inside the sample is made of stainless steel, while the gaskets are made of Kalrezez®. The sensor can stand a maximum temperature of 120 °C and is designed for a pressure range of 0–40 bar (Geers and Witt, 2003). It offers an automatic change of the gap width over the complete, acceptable pressure range, and the actual gap width can be measured by an integrated gap width sensor, so the sensor can be adapted during the measurement to the demands of the suspension (e.g. concentration) to be analyzed. In this instrument, an electrical high frequency generator is connected to a piezoelectric ultrasonic transducer that transmits ultrasonic waves into 10

the sample to interact with the dispersed particles. Thus particles that are very much smaller than the wavelength of the ultrasonic wave are dragged and do not attenuate the signal, whereas particles larger than the wavelength scatter and attenuate the signal. Anyway, viscous and/or thermal losses of very small particles is not always completely neglectable and could give a contribute to attenuation and also to acoustic dispersion. After passing the measuring zone, the ultrasonic waves are received by an ultrasonic detector and converted into an electrical signal. The extinction of the ultrasonic waves is calculated from the ratio of the signal amplitudes on the generator and detector side, and the frequency of the ultrasonic wave may be varied between 100 KHz and 200 MHz. In order to measure particle size distribution, the attenuation at 31 frequencies is recorded as one measurement lasting 60 seconds, which leads to an attenuation spectrum. The particle size distribution and solid concentration is calculated from the effective signal from the complete attenuation spectra. The entire measuring range of OPUS covers 0.01 to 3,000 µm. The most important size parameters , x50 and x90 and x10, are shown as a trend diagram. (Andrew Smith, 2010). The primary measurement provided by this instrument, the frequency-dependent Ultrasonic Extinction, can be converted by a mathematical algorithm into a particle size distribution.The device is especially designed for in- and on-line particle size distributions in highly concentrated suspensions and emulsions (up to 70% Vol. typ.) (H. Geers 2003) without any need for dilution. (J. Jordan, 2007)

2.2.3 HR-US High-resolution ultrasonic spectroscopy (HR-US Sonas Technologies, Dublin, Ireland) is an ultrasonic device based on the phenomenon of resonance. A mechanical longitudinal ultrasonic wave of a certain frequency is generated by the transmitter, travels through the sample cell, and then is reflected back by the receiver. In a stationary medium, the positive interference between two waves travelling in opposite directions generates a wave which is called a stationary or standing wave. This wave will be characterised by the same frequency as the incident wave but with greater amplitude. The speed of a standing wave is determined from the wavelength and the frequency. The ultrasonic tester graphs a peak of energy whenever the transducer receives a reflected wave. Sound is reflected any time a wave changes mediums, generating a peak. The half-bandwidth (broadness) of the resonant peaks of the ultrasonic wave is determined by ultrasonic attenuation, and thus the

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measurements of the profile of the resonant peaks allow determination of the ultrasonic velocity and attenuation in the systems. The measurements of different peaks of different frequencies makes it possible to analyse the frequency dependence of velocity and attenuation parameters. In order to measure the ultrasonic parameters, it is usually necessary to select one or several resonance peaks, which is equivalent to choosing one or more frequencies. In fact, the ultrasonic spectra are usually wide, and so only a few frequency peaks are required to cover the frequency range typical of the instrument (1-18MHz). In this device, the ultrasonic wave is generated and received by two piezotransducers positioned one in front of the other. The HR-US spectrometers employ a principle by which the path length of the ultrasonic wave in the sample exceeds the size of the sample. This instrument allows the attainment of ultrasonic measurements with record resolution (down to ±0.2mm/s for ultrasonic velocity) in a broad range of sample volumes, down to a single droplet. The standard version is equipped with built-in 1-mL ultrasonic cells that can be thermostated between -20ºC and 120ºC, and the ultrasonic wave is generated and received by two piezotransducers in contact with the cell itself. The cell construction allows stirring of the sample. Measurements can be made using different programs, such as temperature ramp, kinetic mode, titration mode and multi-frequency mode in the temperature range of -20ºC to 120ºC. In addition, the HR-US can be equipped with specialized cells (external moduli) such as flow through cells, reduced-volume cells (down to 0.03 mL), cells for non-liquid samples and cells with extendedfrequency ranges (the frequency range achievable is 0.1–20 MHz). This instrument allows the attainment of ultrasonic measurements with very high resolution (down to ±0.2mm/s for ultrasonic velocity and 0.2% for attenuation) in a broad range of sample volumes. Its high resolution makes it possible to perform measurements in low concentration solutions, typically on the scale of about 1 mg/mL scale, and in some cases down to the scale of µg/mL. (Buckin et al., 2002).

2.2. 4 Resoscan® The ResoScan® URT System is a compact benchtop instrument enabling high resolution URT measurement in small liquid samples. The ultrasonic resonator cell, made of titanium, contains two identical cavities of 180µl for convenient measurements of independent samples or a reference sample. The instrument is equipped with an ultra-high precision Peltier thermostating system that reaches temperature constancy better than 1mK (0.001°). This is very important, since ultrasonic measurements are sensitive to temperature changes. Moreover, the device features a fast heating, 12

cooling and equilibration time between 5° and 85°C with a rate of 100–350 m/C/min-1. The resolution of the ultrasonic velocity is 0.001 m/s-1. Repeatability of absolute velocity after automatic re-initialization is ±0.01 m/s-1. The cavity material is titanium and gold, and the ultrasonic transducers are fabricated from high quality lithium niobate. The ResoScan® has two closed sample cells, one sample cell and one reference cell, each with a path length of 7.0 mm. They are equipped with closed lids to avoid evaporation of the samples. The ResoScan® system measures ultrasonic velocity and the attenuation through the resonance technique. The sample cells are constructed as ultrasonic resonators in which a standing compressional wave is generated. During the initialization, a frequency range of about 7–9 MHz is scanned. The system locates the resonance frequencies within this range, calculates the order of the resonance peaks, and automatically selects an optimal one (the master peak) for the measurement. From the frequency and the shape of this peak, it then calculates ultrasonic velocity and attenuation. The deviations of the observed frequencies from the ideal resonator model and the fit of these deviations to a special transfer function of the ultrasonic resonator are then used to calculate the parameters required. The system evaluates the resonance orders and selects one resonance as the pilot-resonance, and the measurements are carried out for a well-known resonance peak ( Lautscham et al., 2000).

3. CHARACTERISATION OF PHARMACEUTICAL SYSTEMS 3.1 SOLID NANOPARTICLES Particle size and size distribution of solid nanoparticles systems may affect the final stability, bioavailability and even toxicity of drugs. Ultrasound represents a particle sizing technique which is receiving considerable attention from the industry and the academic community (Dukhin and Goetz, 1996, Povey, 1997, Babick et al, 1998, Challis et al, 2005, Shuklaand Prakash, 2006). An example of the use of ultrasound to study solid nanoparticles is provided by Horák and coworkers, who characterised surface-modified iron oxide nanoparticles for stem cell labeling by investigating and controlling their surface and morphological characteristics. In fact, surface modification of iron oxide nanoparticles improved their permeability through the cell membrane. Ultrasounds (Ultrasound Spectrometer HR-US 102) were utilized in this project to monitor differences in ultrasonic velocity between reference (water) and iron oxide colloid under the slow addition of various concentrations of D-mannose and to characterise both uncoated and D-mannosemodified iron oxide nanoparticles. At low D-mannose/γ-Fe2O3 ratios, the ultrasonic velocity 13

increased steeply with addition of D-mannose. The authors understood this to mean that there was the formation of a more ordered structure than that in solution. After exceeding the critical Dmannose concentration, namely, at a D-mannose/γ-Fe2O3 ratio > 0.065, the ultrasonic velocity barely changed, proving that the excess of D-mannose remained in solution. This indicated that until the critical D-mannose/γ-Fe2O3 ratio is reached, D-mannose molecules preferentially adsorb on the surface of iron oxide nanoparticles, forming a coat. Thus, this technique made it possible to verify that D-mannose molecules adsorb on the surface of iron oxide nanoparticles, forming the desired coat, and to identify the critical D-mannose/γ-Fe2O3 ratio necessary to achieve this goal. (Hora´k et al., 2007) In another study, ultrasonic spectroscopy (HR-US) was used as a novel tool for studying differences in the compressibility of hyaluronic acid (HA) nanoparticles with different features, in order to modulate the viscosity of model commercially available viscosupplements (Synvisc, Orthovisc, and Hyalgan) used to reduce pain and improve joint function. These supplements made with high molecular-weight HA suffer from poor injectability. Nanoparticles were synthesized from 17 and 1500 kDa HA on the basis of cross-linking polymer chains through their carboxyl groups via carbodiimide chemistry. The nanoparticles were freeze-dried, and dry powder was stored at −20 °C. Nanoparticle suspensions of HA were studied at different concentrations and in blends with the model viscosupplement. Nanoparticles made from 1500 kDa HA reduced the viscosupplement viscosity and elasticity to a much greater degree than nanoparticles made from 17 kDa HA. The authors reasoned that the difference in the nanoparticle effect on viscoelasticity suggests that nanoparticles made from 17 kDa HA may have dangling surface polymers that facilitated interactions with HA in solution, a hypothesis that was supported by ultrasonic vibrational spectroscopy analysis. In fact, data from ultrasonic spectroscopy was effective in the characterisation of this kind of system. HA nanoparticles made using 17 kDa HA were significantly more compressible, showing a significantly lower differential wave velocity compared to the 1500 kDa HA nanoparticle. This result is indicative of a lower degree of HA chain packing compared to that of 1500 kDa HA nanoparticles, which were expected to have a greater degree of intramolecularity because of their long polymer chain length, higher coiled structure, and propensity for self-association. Thus, increasing nanoparticle concentration in polymer/nanoparticle mixtures (overall HA concentration

of 1.4% w/v) reduced the viscosity and viscoelasticity of the samples, but the type of nanoparticle (17 or 1500 kDa) influenced the polymer−nanoparticle interactions, affecting the bulk rheological properties of the mixtures, because the surface structure of nanoparticles was implicated in the different rheological properties of the polymer/nanoparticle mixtures. ( Fakhari et al, 2013) 14

Juhnke et al. evaluated the stabilization of drug suspensions (proprietary crystalline drug provided by Novartis Pharma AG) by using a nonionic polymer (hydroxypropylcellulose, type Klucel LF), in presence of charged nanoparticles as a complementary method compared to conventional stabilization mechanisms. In this study, CVI (DT-1200) was used to characterise the ultrasoundinduced dipole formation within the electrical double layer of the charged particles. The colloidal drug suspension without addition of charged nanoparticles exhibited negative surface charge with low CVI amplitude. The addition of positively charged Ludox CL and negatively charged Ludox AM resulted in slightly increased CVI amplitudes and in positive and negative electric surface potentials, according to the charges added. The addition of positively charged Latex AL resulted in a decrease of CVI amplitude with positive surface potential. Interestingly, the increased CVI amplitude could be correlated to the pronounced colloidal stabilization shown by the liquid structure and minor thixotropy. This study demonstrated the successful evaluation of the stabilization of drug colloids by the presence of charged nanoparticles. This result opens a number of possibilities, such as the creation of a technology platform without the need for drug surfacespecific anchor segments of molecularly dissolved stabilizers, and the prevention of particle growth of drug colloids by surfactant-induced micellar solubilisation, due to surfactant-free colloidal drug suspension compositions. Furthermore, considering the transformation of colloidal drug suspensions into solid dosage forms, (tablets and capsules), charged nanoparticles can potentially be utilized as matrix-forming agent providing appropriate re-dispersibility of drug colloids (Juhnke M., 2012) Awad and co-workers (Awad et al., 2008) examined the possibility of using ultrasonic velocity measurements while scanning temperature, to check thermal transitions in solid lipid nanoparticle (SLN) suspensions. These suspensions, which consist of fully or partially crystalline lipid nanoparticles suspended in an aqueous continuous phase, offer a number of advantages as delivery systems, among them high encapsulation capacity, good chemical and physical stability, good oral bioavailability, and potential for large scale production (Mehnert and Mäder, 2001). The Awad study monitored the complex thermal transitions occurring during the melting and crystallization of the triglyceride used for the formulation of the solid lipid nanoparticles themselves (SLNs). A Resonic Instruments GmbH (Ditzingen, Germany) was used to measure the ultrasonic velocity of tripalmitin O/W nanoemulsions at temperatures ranging from 5 to 80 °C and the results were compared with those obtained by differential scanning calorimetry (DSC) (figure 1). Both techniques were sensitive to the complex melting behavior of the solidified tripalmitin, which was attributed to the dependency of the SLNs melting point on the particle size. Moreover, results obtained with both techniques matched perfectly. 15

In a more recent study, the authors found ultrasound technology to be dissatisfactory for their purposes. They tested ultrasonic resonance technology (ResoScan®) together with other techniques (dialysis, turbidity measurement methods, fibre optics, asymmetrical flow-field-flow fractionation, ion-selective electrode and syringe filters) in assessing the dissolution kinetics from nanosized drug crystals, such as fenofibrate, aprepitant, sirolimus and megestrole acetate. (Jünemann and Dressman., 2012). The authors initially chose to use ultrasound because its velocity is dependent on solute concentration and structure, charge of the analyte and sample homogeneity, and thus, if the environment in the dissolution medium remains constant, the only changing parameter would be the concentration of dissolved drug. They also were motivated by the advantage that with ultrasound, a minimal quantity of sample solution (ca 200 ml) would suffice. However, they found that this technique was not effective for this kind of study, nor was it suitable for creating a working analytical system for fast and reliable analysis of drug content. Using the highest concentration of a standard solution (FaSSIF, saturated with fenofibrate), the equilibration of temperature was slow (>5 min). After equilibration, this ResoScan® analysis failed to detect fenofibrate in FaSSIF. Thus, the ResoScan® ultrasound detection system failed to yield the expected results; no improvement of analysis at lower drug concentrations was achieved and the time needed for temperature equilibration was too long to analyse a system containing undissolved nanoparticles. Instead, a 2011 study found ultrasound spectroscopy (Resoscan®) useful for studying water suspension of magnetic nanoparticles characterised by a core–shell structure with a magnetic core (Fe3O4) and a surfactant shell (Jo´zefczakn and Skumiel., 2011). The authors explained that magnetic nanoparticles have attracted considerable interest for their potential application in biological and medical fields, for example as drug delivery systems (Alexiou et al, 2002a; Alexiou et al, 2002b) and in a treatment to kill cancer cells, magnetic hyperthermia, which uses an alternating magnetic field to heat nanoparticles. The use of nanoparticles in this treatment is advantageous because their size is compatible with that of viruses, proteins and genes (Brusentsov et al., 2002; Jordan et al, 2001), and also because nanoparticles can be manipulated under this magnetic field. The preparation took place as follows: the surface of magnetic particles was coated with sodium oleate as the primary layer and polyethylene glycol (PEG) as the secondary layer to improve the stability of water-based magnetic fluids. The preparation of the aqueous magnetic fluids was based on co-precipitation of Fe2+ and Fe3+ salts in alkali aqueous medium (NH4OH). Sodium oleate 16

(C17H33COONa) as a first surfactant was used to modify prepared magnetic particles to prevent their agglomeration. The polyethylene glycol was added to the magnetite-oleate system and stirred over 3h to improve stability and increase the half time circulation of the particles. Having prepared the suspension of magnetic nanoparticles the authors used ultrasound technique (ResoScan®) to examine their acoustic properties, specifically through measurement of the velocity and attenuation of ultrasonic waves sent through the suspension. They first examined the ultrasonic wave propagation velocity as a function of temperature, demonstrating that ultrasonic propagation velocity in magnetic fluids is smaller than in pure water. Moreover, their results showed that the polyethylene glycol molecular weight had a clear influence on the acoustic properties of the magnetic fluids studied. A decrease of ultrasonic velocity was observed when the concentration of magnetic particles increased, at constant polyethylene glycol molecular weight, while this effect was significantly reduced by the increase of polyethylene glycol molecular weight. By plotting the ultrasonic wave velocity as a function of magnetite (Fe3O4) volume concentration, it was observed that the velocity increased when polyethylene glycol (PEG) molecular weight increased as well. In fact, the velocity of ultrasound was higher in the system containing nanoparticles coated with PEG10000 than in that containing nanoparticles coated with PEG1000. This difference increased with the concentration of nanoparticles. Using the density values of the nanoparticle systems and ultrasonic velocity data, the authors also calculated the adiabatic compressibility βs of nanoparticle water suspension, observing a decrease of this parameter with the increase of temperature. The authors also studied ultrasonic wave attenuation, and found that the presence of nanoparticles in the suspension caused an increase in the attenuation. In fact, it is known that the attenuation of the ultrasonic wave in magnetic fluids is determined mainly by size, shape, concentration and distribution of nanoparticles dispersed in liquids. Based on the changes of ultrasonic wave attenuation under the influence of the external magnetic field, the authors concluded that the magnetic material was inclined to aggregate, notwithstanding the presence of two surfactant shells. This work provides a good example of the use of ultrasound spectroscopy for characterizing nanoparticle suspension. Another application of ultrasound technology (ResoScan®) served to evaluate nanosystems based on gelatin nanoparticles for the delivery of oligodeoxynucleotide (ODN). (Fuchs, 2010) In this work, the authors compared Ultrasonic Resonator Technology (URT) with more common techniques, such as photon correlation spectroscopy (PCS), in the characterization of GNPs in various sizes and concentrations and ODN-loaded GNPs, and found that URT can be used in 17

formulation development to monitor the size, concentration and ODN loading of gelatin nanoparticles. Bhosale and Berg (Bhosale and Berg, 2010) investigated the possibility of using the ultrasound technique (DT 1201) to determine particle size distribution in gel media, using dispersions of silica particles of varying size (Ludox SM-30, Ludox HS-30, and Ludox TM-40) in aqueous hydroxypropylcellulose (HPC) gels of varying cross-link density. This experimental work explored the limits of the technique and the current theory for the determination of particle size distribution in gel media. The samples for acoustic analysis were prepared by mixing the desired amount of Ludox particles with a 0.5 wt % solution of hydroxylpropyl cellulose (HPC, 1000 kDa) in a 20 mM NaOH solution; the HPC solutions were cross linked to varying degrees using divinyl sulfone (DVS). This study demonstrated that ultrasounds can be used in dense dispersions confined in gels, providing that the particle size is smaller than the mesh size of the gel, according to the simple rubber elasticity theory. This type of system can be encountered in the pharmaceutical field, specifically in drug targeting, but is often difficult to study with other techniques because of the semisolid texture. On the other hand, the authors concluded, if the particles are larger than the mesh size, the resulting viscoelastic response from the gel matrix cannot be interpreted using the existing theoretical framework. They suggested that current models be extended to predict the PSD for geltrapped colloids, since acoustic attenuation can be used to probe the viscoelastic properties (microrheology) of the polymer gel matrix at the MHz scale of frequency.

3.2 SOFT NANOPARTICLES Though acoustics and electroacoustics can be extremely helpful in characterizing particle size, zpotential and several other properties of dispersed systems, the literature offers few examples of the characterisation of soft nanoparticles systems, particularly for pharmaceutical applications. As seen thus far, acoustics and electroacoustics can be extremely helpful in characterizing particle size, zpotential and several other properties of dispersed systems. Along these lines, the use of ultrasound technology to characterise soft nanoparticles systems, particularly for pharmaceutical applications, has been the described in a number of studies in the literature. Ultrasound technique (ResoScan®) was proposed by Cavegn (Cavegn et al., 2011) as a Process Analytical Technology (PAT) tool in homogenization of nanoparticulate gels. Cavegn and coworkers studied homogenization kinetics of aqueous dispersions of colloidal microcrystalline cellulose (MCC) and a mixture of clay particles with xanthan gum and compared them with 18

colloidal silicon dioxide in oil, using ultrasonic resonator technology as a monitoring tool. They wanted to address the problem of the variable thixotropic behavior of nanoparticulate viscosity enhancers, caused by inconstant aggregation of the primary nanoparticles due to differences in their microstructure. They noted that the addition of polymers increases the complexity of the structure and the possibility for interaction with the nanoparticles and their aggregates, and indicated that to ensure robust drug product quality, it is important to standardize the structure in a nanoparticulate vehicle, with a real-time monitoring. They pointed out that in-line analysis is challenging because it is difficult to measure particles of colloidal size and characterise interactions with other components such as polymers. The authors analysed the changes of ultrasound velocity in these systems as a function of the homogenization time and described the kinetics thus obtained using a new heuristic equation, assuming that changes of ultrasound speed are approximated by first-order kinetics. In addition, they identified the rheological properties offline to evaluate the structure of the resulting gels. Their results demonstrated the appropriateness of ultrasound velocimetry to set times needed for complete homogenization. In particular, this technique demonstrated remarkable sensitivity in detecting production differences, for example, those derived from different suppliers of colloidal MCC and subtle effects of concentration, temperature, and homogenization speed. They concluded that this ability to detect changes in nanoparticulate vehicles makes the novel ultrasound technology highly interesting for the monitoring of pharmaceutical homogenization processes. Stillhart et al. (Stillhart et al., 2013) used ultrasound technology in their study of Pouton type IV systems, which are typically mixtures of a hydrophilic surfactant and co-solvent without the presence of an oily phase. (Pouton, 2006) This kind of formulation can give rise to supersaturation upon aqueous dilution, accompanied by undesirable drug precipitation. In particular, Stillhart and co-workers studied the characteristics of aqueous dispersions obtained from two hydrophilic surfactants (Cremophor® RH 40 and polysorbate 80) mixed with ethanol as co-solvent and fenofibrate as model drug. They investigated how the drug solubilisation capacity of the two surfactant/co-solvent systems changed upon aqueous dispersion. The knowledge of such changes in drug solubilisation and supersaturation is important for predicting the destiny of a lipid-based system after dispersion. Thus, the precipitated model drug fenofibrate was characterised by focused beam reflectance, X-ray diffraction, and Raman spectroscopy, while vehicle phase changes upon aqueous dilution were studied using dynamic light scattering and ultrasound analysis. Ultrasound technique (ResoScan®) monitored structural changes as a function of dilution level, since a change in ultrasound speed is attributed to a difference in apparent density or compressibility of the medium. The authors measured the difference in ultrasound velocity (DU) between water and the 19

formulations at different dilution ratios, and observed a qualitative change of the acoustic response. The plot of the acoustic response as a function of drug concentration indicated that for most samples there was an approximately linear dependency between DU and the formulation concentration. At low dilution levels, the presence of high DU values indicated elevated stiffness (corresponding to low medium compressibility), due to the existence of coherent liquid-crystalline structures. Further dilution led to a pronounced decrease in DU, which can be associated with the presence of isolated micelles and a loss of mechanical stiffness. Wang et al. (Wang et al, 2005) compared measurements of the ultrasonic velocity and attenuation (Resoscan®) during heating and cooling with those obtained from rheological oscillatory measurements to study the sol/gel and gel/sol transition in carrageenan solutions (figure 2). Carrageenans were chosen because these polysaccharides are widely used in the food industry as gelling and stabilizing agents. It should also be noted that because of their biocompatibility, nontoxicity and low cost, and their ability to form gel, these biopolymers are highly useful in pharmaceutical applications such as controlled release systems. Wang et al. used ultrasound at 7.8 MHz to investigate the gelation of different carrageenans in aqueous solutions by following the increase in ultrasonic attenuation and decrease in ultrasonic velocity. They noted that the decrease in ultrasonic velocity is assumed to correlate with the aggregation of carrageenan molecules in ordered conformation, and that the increase in attenuation may be related to the friction between the gel network and water molecules. They found that a mixture of and carrageenans showed very different ultrasonic characteristics compared to a natural /hybrid-carrageenan, indicating that the molecular structure of carrageenans strongly influences their gelation properties. Their acoustic results matched with those obtained by oscillatory rheological analysis; acoustic spectroscopy was more sensitive in differentiating carrageenan types, while they found rheology preferable for detecting gel formation. In some cases, an electroacoustic technique has been used to characterise the potential of hydrogel systems. In particular, Piai et al (Piai et al, 2009) synthesized and characterised a hydrogel constituted of chitosan (CT) and chondroitin sulfate (CS), and used a DT 1200 electro-acoustic (CVI) spectrometer (Dispersion Technology) to determine the dependence of the -potential of chitosan/chondroitin sulfate hydrogel on pH, in order to better understand the hydrogel structure, chain interactions and reorganization in the hydrogel-forming 3-D matrix, which seemed to be particularly significant between pH 6 and 12.

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Ultrasound techniques have also been used to study the self-assembling behavior of amphiphilic block copolymer systems. Bonacucina et al. sought to demonstrate the potential offered by the use of an electroacustic spectrometer (DT 1200) for characterising the self-assembling behaviour of Poloxamer 407 systems (3–25%, w/v), both alone or after the addition of various amounts of hydroxypropyl-cyclodextrin (5–20%, w/v). Because of its self-assembling and thermogelling properties, Poloxamer 407 is widely used in the pharmaceutical field. They identified particle size and the microrheological extensional moduli (G‟and G‟‟) of the systems based on sound attenuation and speed, and studied Poloxamer 407 phase transitions and how the HP

-CD affected them, by examining the variation

of the particle size and the rheological extensional moduli at increasing temperatures. (Bonacucina et al, 2008) By analysing the evolution of the particle size with temperature, they were able to identify the different Poloxamer transitions in water. Ultrasound analysis revealed very small mean diameter values at low temperatures, which they associated with the presence of unimer species in solution. Temperature increase led to the formation of aggregates (micelle structures) that corresponded to higher values of the size. In addition, they analysed the influence of the presence of 10% of HP -CD and found that the final particle size was about half that of the corresponding Poloxamer 407 samples alone. These results led them to suppose that HP -CD interacts with micelles, reducing the formation of clusters and shifting the gelation temperature (characterised by micelle–micelle interactions). The same transitions were confirmed by the determination of the rheological moduli. Both G‟ and G‟‟ moduli allowed the identification of the system phase behaviour, showing a different trend depending on the temperature of analysis. They observed that the G‟ modulus for the Poloxamer 407 decreased during micellization and gelation until reaching a plateau value, indicating that probably a decrease of the micelle/water interactions caused an increased amount of „„free‟‟ water in the system. They reasoned that desolvation gave rise to a reduction of „„sound speed,‟‟ since sound speed in free water is lower than that of water involved in the interaction with micelles, and indicated that the sound speed decrease could be related to an increasing micelle rigidity and a decrease in their compressibility. In presence of the 10% of HP b-CD, the phase transitions appeared less defined, as can be observed by the less marked change of the analysed parameters in (figure 3). The ultrasonic parameters of velocity and attenuation measured using an HR-US 102 were used to study the micellization process of Poloxamer 407 by Cespi and coworkers (Cespi et al, 2010a). Six 21

different concentrations of copolymer water dispersions were prepared and analysed between 5° and 35°C using high resolution ultrasonic spectroscopy. For comparison, the same samples were also analysed by the DSC technique. The authors sought not only to demonstrate the potential of ultrasound technique as a valid alternative to standard methods of analysis, but also to show how the transition observed in ultrasound parameters could be easily interpreted, particularly when the aggregation process for self-assembling polymers is analysed. Sound speed and attenuation were used successfully to monitor the micellization process, although with different sensitivity. In particular, sound speed proved to be very effective for studying the aggregation–deaggregation process, but failed to detect the gelation process. Instead, sound attenuation data allowed identification of both transitions (micellization and gelation). Both parameters gave a very good correlation with DSC data in terms of characteristic transition temperatures and also in terms of micellization kinetics and related parameters, though small differences were observed in the identification of the exact temperature range of the analysed processes (figure 4). In sum, the authors concluded that high-resolution ultrasound spectroscopy can be a very effective and accurate technique for monitoring temperature-induced polymer-assembling processes (micellisation), thus providing an interesting alternative to DSC measurement. In another study, Farrugia et al. (Farrugia et al., 2014) assembled ultrasound equipment to assess the temperature-induced phase changes in Pluronic F127 hydrogels. Pluronic F127 solutions under investigation were injected into a 7.3 mm long cylindrical, 25 mm diameter sample chamber consisting of a hollow polymer tube enclosed by 0.175 mm thick Mylar sheets at each end to act as acoustic windows. The sample chamber was placed in a temperature controlled water tank and clamped in position. Two 13 mm diameter, 2.25 MHz ultrasound unfocussed immersion transducers (Olympus) were placed in the water tank on opposite sides of the sample chamber. One transducer was used as a transmitter and the other as a receiver. Over the temperature range studied, the transducer nearfield distance was between 63 mm and 66 mm. The spacing between the transducers was set to 250 mm, with the sample sitting at a distance of 125 mm from each transducer, placing it well within the acoustic farfield. The transducers were then aligned to produce a maximum transmitted signal through the sample chamber. Next, a model hydrophobic molecule, pyrene, was loaded into the system. This fluorescent probe was used to validate micelle formation and to study the uptake of the drug for controlled release applications, as fluorescence is very sensitive to subtle changes in the microenvironment around the probe and microstructural changes in the micelles at a molecular level. 22

Through validation with a fluorescence spectroscopy technique, this study showed that the temperature dependent phase transitions in Pluronic solutions could be identified through analysis of the relative changes in ultrasound velocity and attenuation as a function of temperature. This phase transition was more clearly detected through the first and the second derivatives of both ultrasound parameters with respect to temperature. Moreover, when the authors compared ultrasound and the fluorescence results, they observed that a sudden change in the ultrasound parameters occurred in the temperature region corresponding to the onset of the fluorescence peak. Thus, this work demonstrated that ultrasound techniques may have a key role to play in the characterisation and design of hydrogel systems used for therapeutic purposes. High resolution ultrasonic spectroscopy (HR-US) was also used by Perinelli et al (Perinelli et al., 2013) to monitor the influence of inorganic salts, in particular potassium phosphate (KH2PO4/K2HPO4), commonly used as buffer system, on the micellisation of Poloxamer 407. They then compared the results obtained by ultrasonic spectroscopy with the microcalorimetry and dynamic light scattering data in order to better understand the role of ions on the dynamic of micellization. Next, they compared equilibrium thermal parameters derived from DSC with two other sets of data, namely the results obtained with DLS, which represented the process of micelles formation at microscopic level, and those obtained with HR-US for variations in sound speed and attenuation (figure 5). When they merged data generated with the different methodologies, it was evident that the main factors driving the self-aggregation process were the interactions between water and poloxamer, and especially water and water, and that the increase of free water moved the whole system towards a more chaotic structure. In fact, ions increased the water–water interactions and decreased the water–poloxamer ones, modifying the general order of the systems and entropically promoting the micellisation process. Comparing the performance of the single techniques, the authors concluded that they were sensitive enough to identify any influence of additives on the self-assembling behavior of copolymer systems. However, they found HR-US and mDSC to be more suitable techniques for general characterisation and determination of the CMT or CMC because they afforded easier analysis and data interpretation; instead, DLS offered the possibility of monitoring the contemporary presence of unimers and micelles during the transition. A similar study but on a different polymeric system was performed by Van Durme et al. (Van Durme et al., 2005), who used high-resolution ultrasonic spectroscopy (HR-US) and modulated temperature differential scanning calorimetry (MTDSC) to evaluate the effect of salt additives 23

(NaCl, CaCl2,orNa2SO4) and surfactants (sodium dodecyl sulfate, SDS) on the lower critical

solution temperature (LCST) phase behavior of aqueous solutions of either poly(Nisopropylacrylamide) (PNIPAM) or poly(vinyl methyl ether) (PVME). This very interesting study highlighted how the addition of surfactants can influence the phase separation properties of polymer/water mixtures, a problem frequently encountered in the formulation of pharmaceutical systems. In particular, the authors studied the influence of both salts and surfactants on the water solubility of PNIPAM (type II LCST) and PVME (type III LCST) for mixtures spanning the entire composition range, in contrast to previous works that only investigated dilute polymer solutions. The results obtained showed that the evolutions of the ultrasonic signals (velocity and attenuation) and of the apparent heat capacity signal followed the ongoing compositional changes with temperature, according to the specific shape (type II or III) of the LCST demixing curve. Furthermore, HR-US provided additional information concerning the solute-solute and water-solute interactions in the temperature range studied, showing that both types of additives affected the polymer-water hydration structure in different ways. Salt ions mostly dislocated the structured water molecules, while surfactants interacted mainly with the polymer. Both techniques revealed that the water-structuring capacity of salt ions caused a decrease in demixing temperature, with a more pronounced effect noted at high polymer concentration. The presence of surfactant resulted in an increase of Tdemix due to the increased solubilisation of the polymer chains. Ultrasound (HR-US) was also used to study the kinetics of solubilisation of the hydrophobic compounds carvacrol and eugenol in micelles solutions of two nonionic surfactants containing an acetylenic group and two polyoxyethylene chains in the molecule (Surfynol® 485W and Surfynol® 465) (Gaysinsky et al., 2008). Surfactant dispersions were prepared in the concentrations of 1, 2, 3.5, 5, 7.5 and 10% (w/w). Eugenol and carvacrol were added to surfactant systems at concentrations ranging from 0.025 to 3% (w/w). Ultrasound allowed the determination of the uptake of carvacrol and eugenol in surfactant micelles, showing that below the maximum surfactant concentration, the phytophenols were completely solubilized in the micelles in less than 30 min. In particular, the increment in ultrasonic velocity decreased with increasing time, due to the uptake of carvacrol or eugenol by surfactant micelles. The decrease of ultrasonic velocity increment with time was fitted to an exponential decay mode Microemulsions are another type of system of interest in the pharmaceutical field. Characterised by a complex inner structure, they have a thermodynamic stability that allows for self-emulsification at a wide range of temperatures and affords easy preparation. The presence of a considerable amount 24

of surfactants and cosurfactants makes microemulsions „„supersolvents‟‟ for different kinds of drugs, even those that are relatively insoluble in both aqueous and hydrophobic solvents. (Bonacucina et al., 2009a). Ultrasonic spectroscopy has considerable potential in the characterisation of these systems (Ballaró et al., 1980; Cao et al., 1997; Lang et al., 1980; Letamendia et al., 2001; Mehta and Kawaljit, 1998; Wines et al, 1999b; Zana et al, 1982) even in fields other than pharmaceuticals. In general, the formulation of microemulsions requires a deep knowledge of their microstructures, droplet sizes, phase diagrams, and the state of solutes loaded in microemulsion droplets. The use of ultrasonic spectroscopy may offer the possibility to monitor transitions between different phases, as well as to characterise the state of water in the emulsion droplets and their size (Hickey et al, 2006, 2010). Buckin and Hallone ( Buckin and Hallone, 2012) used HR-US to study the relationships between compressibility and physical characteristics of microemulsions, such as their microstructure and the state of their components. In particular, they investigated the state of water in w/o microemulsion droplets composed of isopropyl myiristate, n-propanol, ethyl oleate, lecithin, tween 80, Span 20, and AOT. Ultrasonic phase diagrams were obtained from titration profiles of ultrasonic velocity and attenuation by measuring ultrasonic parameters at various frequencies. Furthermore, phase transitions were identified by abrupt modifications of the profiles of ultrasonic velocity and attenuation and the dependence of these parameters on frequency. Furthermore, they observed different transitions within the microemulsion phase, which they identified as additional „sub phases‟ within this phase that could be related to the state of water and surfactant. Thus, their results demonstrated that the ultrasonic technique enabled efficient mapping of the areas of microemulsion phase diagrams with different states of water, including water in nano-droplets.

Smyth and coworkers (Smyth et al., 2005) used ultrasonic spectroscopy (HR-US) to characterise a microemulsion formulated with isopropyl myristate, lecithin, n-propanol as a cosurfactant in a w/w ratio of 6:1:1, and water. They observed a steady increase in ultrasonic velocity as a consequence of the molecular dissolving of water in the mixture at low concentrations (up to 2 w/w%), and posited that it could be associated with the hydration of lipids and cosurfactants, and thus with a microstructural reorganization, which was shown by an additional increase in ultrasonic velocity when the water concentration ranged between 2 and 6%. Formation of the microemulsion could be seen from approximately 8 w/w% of water with ultrasonic attenuation quickly increasing, which was caused by the scattering of the ultrasonic wave on the particles. Thus this study as well showed

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the potential of ultrasonic spectroscopy in providing information previously unattainable by existing methods of analysis. In another work a year later (Hickey et al., 2006), the same system was again characterised using ultrasonic spectroscopy (HR-US), but with the aim of building an “ultrasonic” phase diagram. Thus, the oil/surfactant/cosurfactant mixture was titrated with water at 25°C and modifications in the ultrasonic velocity and attenuation in the megahertz frequency range were measured. Quantitative analysis of the ultrasonic parameters allowed the characterisation of various phases (swollen micelles, microemulsion, coarse emulsion, and pseudo-bicontinuous) as well as the evaluation of the state of the water and the particle size, which in turn made possible the construction of a phase diagram. The particle size obtained for the microemulsion region ranged from 5 to 14 nm over the measured concentrations of water/isopropyl myristate/Epikuron 200 and n-propanol, in agreement with previous data in the literature. The ability to analyse these systems in titration mode is an important advantage of this technique for microemulsion characterisation. (Hickey et al, 2006) High-resolution ultrasonic spectroscopy was applied by Hickey et al. (Hickey et al., 2010) to analyse a pseudo-ternary phase diagram of a system composed of water/ethyl oleate/Tween 80 and Span 20 by measuring the changes of ultrasonic velocity and attenuation when the oil and surfactants were titrated with water. This system did not contain co-surfactant, but included two different surfactants, Span 20 and Tween 80. Changes in the ultrasonic velocity and attenuation with concentration of water in oil/surfactant mixtures showed several well defined patterns, which were related to the microstructural state of the samples. An „ultrasonic‟ phase diagram was built from the ultrasonic titration profiles and the particle size in the microemulsion region was estimated using the thermo-physical properties of the dispersed water phase and that of the continuous oil and surfactants phase. Thus, in this study, ultrasonic spectroscopy enabled the construction of a pseudoternary phase diagram and highlighted transitions occurring in it. These results matched with those obtained in a previous work using other techniques (Hickey et al., 2010). Bonacucina and co-workers (Bonacucina et al., 2013) reported the use of electroacoustic spectroscopy (DT-1200, Dispersion Technology) for the characterisation of a ternary system based on isopropylmiristate (IPM)/polysorbate80(T)/water (W). This study dealt with the determination of particle size and microrheological extensional moduli (i.e. G‟and G‟‟) of the different tested systems by means of acoustic parameters such as sound attenuation and speed (figure 6). Electric conductivity was also measured using the same instrument. This work highlighted some types of

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information that could be gained with this technique for a deeper understanding of the systems containing different surfactant/oil/water ratios. In brief, polysorbate 80 and isopropylmiristate were mixed in order to obtain different surfactant/oil ratios: 10/90, 20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, and 90/10. An increasing amount of water was then added to the same portion of each of the above S/O ratios, in order to obtain systems having a final water content of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Ultrasonic analyses were performed in cell lengths between 0.325 and 20 mm, in the frequency range of 3-100 MHz, at 20◦C. Particle size and rheological G‟ and G‟‟ moduli were calculated from sound attenuation and phase variation raw data. At the same time, electric conductivity was measured using the appropriate probe provided with the instrument. Different zones of the ternary diagram were identified, corresponding to the presence of microemulsion, emulsion and gel zones. Data obtained in this study confirmed that acoustic spectroscopy is a powerful technique for acquiring accurate information about these kinds of systems. In particular, the ability to determine particle size was important for following the different phase transitions and identifying which kind of system was being formed, while G‟ and G‟‟ moduli gave useful information about the compressibility of the system and consequently about the state of the water present in the system itself. Stillhart and Kuentz (Stillhart and Kuentz, 2012) compared Raman and ultrasonic (ResoScan®) spectroscopy for the quantification of three model drugs (fenofibrate, indomethacin and probucol) in different self-emulsifying formulations. Their goal was to compare the two methods in terms of their analytical performance in the different systems. Moreover, two optical Raman configurations were considered: one for in-line measurement in the bulk solution and the other, a multi-fiber sensor, for quantification of the drug in hard-gelatin capsules filled with the self-emulsifying formulations. The ultrasound results indicated that there was a linear relationship between acoustic parameters and API concentration, and that the ability of attenuation (A) of being more sensitive to drug concentration than sound speed (U). The authors also studied how the density of the formulations was affected by different drug amounts by testing five drug concentrations (range 0.5–4%, w/w). The results indicated that density in the mixture increased with drug load. A different result was found for indomethacine, as this drug strongly affected the structure of the lipid mixture, reducing compressibility by a perturbation of the liquidcrystalline structure. It can be stated that drugs with a strong effect on the structuring of SEDDS are suitable candidates for being studied by ultrasound velocity.

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This work demonstrated that both methods can be used for rapid and non-destructive drug quantification. Moreover, the authors noted that not only are they useful in process analysis, but they can also be adopted when simple and rapid calibration is required, for example, during formulation development, when various compositions of the formulations are tried and changed. Consequently, they argued, both methods have strong potential for implementation as a PAT tool in real-time monitoring of the production of SEDDS or other lipid mixtures. In addition, this study highlighted that Raman spectroscopy was more advantageous than the ResoScan® URT System because it enabled detection of impurities and degradation products. However, the ultrasound analysis was simpler, faster and provided excellent calibration performance. It also showed high sensitivity and good predictability and provided interesting analytical performance in cases in which the drug strongly affected the density or compressibility of the self-emulsifying formulations. In another study, atomic force microscopy (AFM) was used together with acoustic spectroscopy (ResoScan®) for the characterisation of oily core nanocapsules by investigating the mechanical properties at different stages of the nanocapsule preparation (Preetz et al, 2010). The authors sought to study the nanocapsule shell in detail since it is the barrier for drug release and degradation of the particle itself. They used AFM to investigate the mechanical properties at different stages of the nanocapsule preparation process, in order to improve understanding of the nanocapsule formation and to follow the transition of nanoemulsions into nanocapsules. They used acoustic spectroscopy to observe the physical phenomenon of ultrasound absorption by polyelectrolyte nanocapsules featuring different capsule wall compositions. After having prepared an O/W nanoemulsion, oily core nanocapsules were obtained by sequential addition of positively and negatively charged polyelectrolytes (octenyl succinic anhydride modified (OSA) starch, carrageenan and chitosan) and transformation thereof into a core–shell structure (figure 7). Different propagation velocities of ultrasound, could be explained by the different shell composition and properties. Both methods were for the first time applied on nanocapsules and demonstrated the change of an emulsion into a solid capsule wall with an increasing number of layers, thus contributing to the understanding of the preparation process for oily core nanocapsules. In particular, ultrasound results allowed the comparison of the absolute velocity values, showing that the ultrasound propagation increased with the increase in the number of layers around the oil core. The difference between three and five polyelectrolyte layers was greater than that between three and one layer. Both AFM and URT made it possible to observe that when emulsion droplets were transformed into 28

polyelectrolyte nanocapsules with a shell composed of three or five layers, the wall became stiffer as the number of shell layers increased. Niederquell and Kuentz (Niederquell and Kuentz, 2013) studied 20 pharmaceutical nanoemulsifying formulations with respect to the physical stability of the aqueous dispersions. In pharmaceutical practice, it was often unclear whether a micro- or nano-emulsion evolves upon dilution of a pre-concentrate, and thus this study sought to propose stability categories for dispersions obtained from self-emulsifying drug delivery systems. The dispersion was subjected to a thermal stress test, then analysed using dynamic laser light scattering, ultrasonic technology (URT) and near-infrared (NIR) analytical centrifugation. Specifically, a ResoScan® system was used to measure the acoustic responses of the formulations in terms of the ultrasonic velocity and attenuation. The authors considered acoustic attenuation an interesting parameter for evaluating the stability of the dispersions. This study demonstrated that there was not a strict correlation between the changes in the light scattering and the ultrasonic attenuation data. The authors posited that the thermal stress test may have accelerated several changes in the more unstable formulations, and reasoned that possible compositional changes in the microstructure would have affected the ultrasound signal, since it is a response of the sum of microstructural changes. They concluded that droplet coalescence was the only possible mechanism that explained their results, and noted that even microemulsions can theoretically exhibit subtle microstructural changes, given the droplet polydispersity. However, the result was in line with the expectation that unstable emulsions would reveal more pronounced changes in the light scattering and acoustic attenuation data than the assumed microemulsions. Ultrasound technology (ResoScan® System) has been also used to characterise nano-emulsions as a part of the overall Quality by Design (QbD) goal. (Shah et al, 2007) The self-nano emulsified drug delivery systems (SNEDDs) examined in this study consisted of various ratios of oil/surfactant/cosurfactant (sweet orange oil, polyethoxylated castor oil, surfactant), and mono and diglycerides of caprylic acid (co-surfactant), with the Cyclosporine A loaded in the oily phase. Ultrasonic measurements of velocity and absorption at a single resonator frequency served to study the compressibility of the individual components in the droplets, the hydration of the droplets and the stability of the mixed phase of surfactant and co-surfactant. ResoScan® was used for measuring the ultrasonic velocity and absorption. The results obtained showed that the compressibility of the 29

systems increased with the addition of the oily component, thus reducing the sound velocity. Furthermore, the ultrasonic attenuation increased as the droplet diameter increased. The authors indicated that this moderate relationship between the ultrasonic absorption data and the droplet size of these systems could be due to the structuring influences of the non-polar part of the surfactant, which prevents a significant bending of polar surface groups. Thus attenuation data contained information on the droplet size, demonstrating that this parameter could be measured by ultrasonic methods. Regarding sound velocity, this parameter diminished as a function of the amount of oil in the formulation. Ultrasounds might provide an alternative method for determining the physical properties of nanoemulsions and estimating the diameter of the nano-droplets, which is an important indication of the stability of these types of systems.

Ultrasound spectroscopy has been also applied to the characterisation of liposomal formulations. Even though not all of these studies regarded the pharmaceutical field, they offer important information that can be transferred to pharmaceutical applications. For example, Taylor et al. (Taylor et al., 2005) used ultrasonic spectroscopy (HR-US 102) and thermal analysis to investigate the thermal stability of liposomes composed of synthetic phospholipids with different acyl chain lengths (C16:0 and C18:0) and containing either an aqueous buffer or a low concentration of nisin, in order to determine the gel-liquid crystalline phase transition temperature (TM) of liposomes and the influence of nisin on this parameter. The resulting gel-to-liquid crystalline phase transition temperatures (TM) determined by DSC agreed perfectly with those determined by ultrasonic velocity and attenuation measured at 5 MHz.

In another study, Krivanek et al (Krivanek et al., 2008) analysed the isothermal compressibility of dipalmitoylphosphocholine (DPPC) and DPPC-containing cholesterol and ergosterol vesicles by using molecular acoustics, differential scanning and pressure perturbation calorimetric techniques. In particular, the authors focused on differences in structural properties of sterols in lipid bilayers, by studying new thermodynamic properties of dispersions of DPPC with cholesterol and ergosterol. Thus, in addition to differential scanning calorimetric (DSC) and pressure perturbation calorimetric (PPC) analyses, measurements of ultrasonic velocity were performed to define the adiabatic and isothermal compressibilities, and to obtain information about the volume fluctuations of the dispersions of DPPC with cholesterol and ergosterol. The ultrasound velocity studies were carried out with a ResoScan®, using a frequency range of 7.2–8.5 MHz. The authors found no marked 30

difference in the effect of the different sterols (cholesterol and ergosterol) on the various parameters studied (partial specific volume, adiabatic and isothermal compressibility, and volume fluctuations). Ergosterol was more effective than cholesterol in promoting lo-phase domains in DPPC bilayers, that is, it was less effective in promoting lateral packing order in the liquid-like phase. Ultrasound analysis highlighted an important difference in the adiabatic and isothermal compressibility of fluid and solid phases of lipidic bilayer, one that became dramatic in the gel-to-liquid transition region, indicating a significant degree of slow relaxational processes in the microsecond time range of the transition region. Maximum values of 15% for relative volume fluctuations were reached for DPPC at the main transition, but these values were strongly dampened upon addition of both sterols. Within the accuracy of the measurements, no significant differences were observed for the two sterols. The work demonstrated that all three techniques of analysis provided a contribution to understanding the influence of the two sterols on the thermodynamic properties of lipid bilayers and on the nature of the critical point region by determining thermodynamic fluctuation parameters.

A similar study was carried out by Schultz and Levin (Schultz and Levin, 2008). The authors performed a structural characterisation of model lipid microdomain complexes using both vibrational infrared spectroscopy and ultrasonic velocimetry. Vibrational infrared spectroscopy estimated lipid microdomain sizes at the nanometer level in a model ternary bilayer assembly, composed of non-hydroxy galactocerebroside (GalCer), cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC). Adiabatic compressibility was obtained from ultrasonic spectroscopy (ResoScan®) in order to assess microcluster effects on the overall bilayer properties. The multilamellar binary GalCer/DPPC and ternary GalCer/Chol/DPPC mixtures were prepared by combining the appropriate amounts of lipids in chloroform. Solvent was evaporated under a stream of N2 gas and then left under vacuum overnight. The mixtures were dispersed in H2O. To ensure complete hydration and mixing, the dispersed mixtures were heated above the GalCer Tm at about 95°C and then rapidly (within 1–2 min) cooled below the gel to liquid crystalline transition temperature (typically to 0°C). This heating-cooling cycle was repeated at least five times. The authors noted that one challenge in describing microdomain complexes is the rapid diffusion of lipids in the liquid crystalline phase. For this reason, they prepared the assemblies in the liquid crystalline phase and then rapidly cooled the dispersions to induce the gel phase, where lateral diffusion is dramatically inhibited. This method provided a representation of the lipid distributions and organization in the liquid crystalline phase so that the role of cholesterol in microdomain behavior could be studied. Regarding acoustic spectroscopy study, sound velocity measurement allowed the determination of different parameters such as velocity number, partial specific volume 31

and partial specific adiabatic compressibility of the DPPC-sterol mixtures. Upon the addition of cholesterol, the authors observed a modest increase in the compressibility of the system but a substantial change occurred at higher temperature, indicating a disruption of the GalCer domain. The changes in the total compressibility of the system provided further insight into the role of lipids in modulating membrane activity.

In another study, Okoro (Okoro, 2010) used acoustic spectroscopy and densimetry studies to investigate the influence of melittin at mole fractions of up to 3.75mol% on the dynamic and mechanical

properties

of

1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine

(DPPC).

The

combination of the two techniques was very effective in the determination of the considerable influence of melittin on the thermodynamic, mechanical, and compressibility properties of DPPC bilayers, demonstrating substantially disruptive effects of melittin on membrane bilayers. In another work, ultrasound velocity measurements (ResoScan®) were complemented by those afforded by six other techniques (Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), atomic force microscopy (AFM), Laurdan fluorescence spectroscopy and fluorescence microscopy) to study the temperature and pressure dependent phase behaviour of the five-component anionic model raft lipid mixture phospholipids

1,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC),

1,2-dioleoyl-sn-glycero-3-

phospho-(1'-rac-glycerol) sodium salt (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-racglycerol)

sodium

salt

(DPPG)

and

1,2-dipalmitoyl-sn-glycero-3-phosphocholin

(DPPC)

(20:5:45:5:25 mol%). (Kapoor et al., 2011) According to the authors, the chosen phospholipid model membrane system acted as a realistic model of biomembranes and allowed study of protein interactions with anionic lipid bilayers. They noted that anionic lipid bilayers are characterised by liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature–pressure plane, and observed that ultrasound velocimetry data yielded additional information about the compressibility of the membrane system as a function of temperature. The measure of sound velocity highlighted the presence of a kink at ~35 °C, in good agreement with the peak temperature obtained from DSC and PPC measurements and with the adiabatic compressibility data. They explained that this sharp curve was related to the conversion of solid-ordered lipid chains to fluid chains in the phase region of the lipid membrane. On the other hand, they reported that the fluidordered to fluid-disordered transition was not visible by acoustic measurements, and thus concluded that for this kind of test, FT-IR spectroscopy is more sensitive to local conformational changes.

32

3.3 Miscellanea The changes in ultrasound intensity and speed when propagating through a liquid or a suspension were used to study blood cells and protein (bovine serum albumin, BSA) solutions (Dukhin et al., 2006). This study showed the potential and efficacy of acoustic and electroacoustic measurement techniques (DT 1200) for testing biological systems. The authors collected plasma and erythrocytes from initial blood samples of a human donor, performed multiple measurements of the plasma, and a measure of an erythrocyte sample at 95% weight fraction. Next, they diluted the erythrocyte sample with varying amounts of plasma, and measured the resulting erythrocyte dispersions at the different concentrations. It was demonstrated that the difference between water and plasma was 150 times greater than the measurement precision. This opened the possibility of characterising plasma proteins using ultrasound attenuation. The results showed that blood cells were characterised by a strong increase in acoustic attenuation, particularly when the concentration of erythrocytes increased, which indicated that the differences observed could be attributed to the erythrocytes. Differences were most significant at high frequency (100 MHz and higher). Sound speed increased linearly with the increase of the erythrocyte volume fraction. In the same study, electroacoustic measurements on BSA demonstrated that BSA molecules carried a (negative) charge equivalent to seven monovalent charge groups. The electroacoustic signal (CVI) measured for several different volume fractions of protein indicated that the CVI was proportional to the volume fraction. This experiment allowed calculation of the electric charge of the particles, which indicated that the surface charge should be independent of the volume fraction of particles, because it is related to the electrochemical equilibrium between protein and the bulk liquid. Moreover, it was found that each BSA molecule carries a (negative) charge equivalent to seven monovalent charge groups. Thus this work showed that ultrasound-based techniques can provide information on acoustic and electroacoustic properties of biomolecular solutions and dispersions of biological cells (Dukhin et al, 2006).

Bonacucina et al. (Bonacucina et al., 2009b) used the DT1200 acoustic spectrometer to study the dissolution kinetics of hydrophilic polymers, in particular, three different model polysaccharides: lambda carrageenan, gellan gum, and xanthan gum. The main characteristic of the chosen 33

polysaccharides and in general of all hydrocolloids is to interact with water medium, making them suitable for manifold applications in the biomedical, food, and pharmaceutical fields. Fundamental for the use of a water-soluble hydrocolloid is an understanding of the mechanisms governing hydrocolloid/water interactions. In fact, a progressive polymer/solvent interaction that necessarily leads to a change in system structure is responsible for a certain dissolution kinetics. The authors evaluated the influence of particle size and temperature of analysis (25 or 45◦C) through observation of the evolution over time of the microrheological acoustic parameters G‟ and G‟‟ and comparison of the ultrasound results with those obtained by classical rheological analysis (shear viscosity measurements over time). In particular, acoustic analysis was used to investigate the mechanisms underlying the dissolution process. The rheological data obtained with both techniques confirmed that acoustic spectroscopy is a powerful tool for monitoring and quantifying dissolution kinetics and for following over time the physical changes involved in the progressive polymer/solvent interaction that necessarily leads to a modification in system structure. The dissolution process led to an increase in the G‟ modulus and a reduction of the G‟‟ modulus, which the authors explained in terms of a gradual decrease in the “free” water in the systems. This work demonstrated that acoustic spectroscopy data made it possible to effectively distinguish the tested polysaccharides and fractions, in some cases with greater accuracy than rheology. Moreover, this technique could prove particularly useful for polymers that cannot be analysed with common methods such as UV analysis due to their poor UV absorption.

Bonacucina and co-workers (Bonacucina et al, 2009c) also used the acoustic spectrometer DT 1200 to characterise gels and o/w emulsion gel formulations based on Sepineo P 600, a concentrated dispersion of acrylamide/sodium acryloyldimethyl taurate copolymer in isohexadecane, which possesses self-gelling and thickening properties and the ability to emulsify oily phases. In this paper, gels were obtained using the 0.5% and 5% (w/w) Sepineo P 600 concentration, while emulsion gel was prepared from a 3% Sepineo gel by adding a specific amount of almond oil. All the prepared samples were analysed and characterised by oscillation rheology to assess the stability of the systems and by acoustic spectroscopy to determine the particle size of the oil droplets and the microrheological extensional moduli (G′ and G″) of the systems. Both techniques revealed that Sepineo P 600 thickened and gelled well, a property that depended strongly on polymer concentration, and that its addition caused a change from the typical behavior of a concentrated non-entangled solution to that of a “gel-like” sample. What is interesting is that ultrasounds made it possible to analyse the inner structure of the gel samples, revealing that the physical interactions forming this gel-like structure were not particularly strong. On the other hand, the addition of an 34

oily phase increased system consistency only minimally, and the viscoelastic characteristics depended exclusively on the gel structure (figure 8). Furthermore, ultrasound technique was used to monitor emulsion microrheological behavior and stability by considering the variation of both their rheological parameters and particle size (mean diameter). The frequency spectra used to analyse system stability confirmed that the emulsion remained stable over three months. In fact, the G′ and G″ plots obtained after 1 day, 1 month, and 3 months were very similar except for the G″ modulus at low frequencies, confirming the oscillatory analysis results. The results obtained from the analysis of the particle size (mean diameter values in micrometer) confirmed the previous statements concerning the stability of the emulsion. Thus, in this case as well, ultrasound analysis proved to be an excellent tool for investigating the structural properties and stability of dispersed systems at any conditions of concentration and consistency. Moreover, with the aid of ultrasounds, this study demonstrated that Sepineo P 600 is a good candidate for use in the formulation of gels and emulsion gels with rheological properties suitable for topical administration.

In another application, the acoustic spectrometer DT-1200 was used to measure particle size and size distribution of sunscreen formulations developed as nano and macroemulsions, composed of the same raw materials (BHT, disodium EDTA, phenoxyethanol, different kinds of parabens, sodium chloride, glycerin, cetearyl alcohol, water, ethylhexyl methoxycinnamate, benzophenone-3, caprylic/capric triglyceride, oleth-3, and oleth-20) and prepared using the phase inversion temperature method (PIT). A sample of each emulsion (nano and macro) was prepared, stored 24 hours and then placed separately into the DT-1200 without dilution. Analyses were performed three times, in the gap interval of 0.325-20 mm and in the frequency range of 3-100 MHz. Sound attenuation and speed were monitored over time. The nano emulsions differed from the macroemulsions in their appearance and particle size distributions (PSD), and these characteristics in the different formulations coincided with measured differences in efficacy as a sunscreen in in vitro tests. Thus in this case as well, ultrasounds showed great potential as a support technique that can help in optimizing formulations (Granemann e and Silva et al., 2013).

Acoustic spectroscopy by HR-US also proved to be a powerful tool able to provide a microstructural fingerprint of emulsions and suspensions, with the advantage of using small sample volume (typically 1 mL) and controlled temperature conditions. Smyth and co-workers (Smyth et al., 2004) provided different examples of the application of HR-US spectroscopy to study the binding of ligands to the surface of colloid particles and the thermal stability of emulsions and 35

dispersions, and to determine particle size in dilute and concentrated emulsions. They pointed out that dispersed systems such as emulsions and suspensions play an important role in various fields, particularly in the pharmaceutical one, and that the determination of their stability is a key factor in assessing the quality of pharmaceutical formulations. Thus, they asserted, design and manufacture of these products require analytical tools able to characterise the microstructural behaviour of these systems. They explained that absorption of ligands (metal ions) on the surface of particles is an important phenomenon with regards to the colloidal stability of suspensions, and that when the concentration of cations on the particle surface increases, it results in the electrostatic neutralisation of particle charge followed by their aggregation and precipitation, as indicated by a decrease in ultrasonic attenuation, allowing calculation of the quantity of ions bound to the particle surface and the binding affinity. Ultrasound measurements (HR-US) were also used in the same work by Smyth (Smyth et al., 2004) to evaluate the thermal stability of two emulsions by testing the samples under the temperature ramp regime. Measurements were performed while temperature was changing with a programmed speed. Results highlighted reversible changes of ultrasonic parameters in one of the emulsions during several cycles of heating and cooling, while in the second sample, a sharp drop in ultrasonic velocity and attenuation was observed at the first heating circle, which did not recover during the next ramps, thus indicating irreversible changes in the emulsion. The author explained that the decrease in ultrasonic parameters could be due to the temperature-induced coagulation of the oil particles and the aggregation of the original particles into larger particles following the phase separation between oil and water phases. The same technique was used to determine particle size in dilute and concentrated emulsions, in particular, to analyse the particle size in a concentrated (undiluted) water-in-oil emulsion and its change in the course of dilution. This study demonstrated that as the internal phase was diluted, the droplet size decreased according to in accordance with the ultrasonic measurements, confirming that measurements of particle size obtained using traditional techniques, which require dilution of original and concentrated emulsion, would not provide correct information (Smyth et al, 2004). Smyth et al. also used acoustic spectroscopy (HR-US) to monitor crystallization of lysozyme being the crystalline form of this protein. Data showed three stages in the crystallization process. At the end of stage (I), the ultrasonic velocity and attenuation started to increase due to the formation of crystals. This increase continued through stage (II), as the concentration and size of the crystals grew. The rise in the ultrasonic velocity was caused by the increase in rigidity (decrease in compressibility) of the sample as a result of the formation of crystals. The rise of the ultrasonic attenuation could be attributed to the scattering of the ultrasonic wave on the solid crystals formed. 36

During stage (II), the ultrasonic velocity started to decrease and in Stage (III), the ultrasonic attenuation nearly leveled off, indicating the end of crystal formation in the micro range size (Smyth et al, 2004).

In another application, acoustic spectroscopy (the HR-US apparatus) was used for simultaneous measurements of the concentrations of HPMCAS, selected as a model excipient (hypromellose acetate succinate), and fenofibrate, as a model drug in acetone solution. (Chen et al, 2005) They used the measured values of velocity and attenuation from the sample cell directly for analysis. Since all solutions were prepared in acetone, all measurements were performed with acetone in the reference cell. An experimental design was used to assess the viability of simultaneous determination of HPMCAS polymer and fenofibrate concentrations in test solution by ultrasonic spectrometry. Nine runs were designed to probe two factors (concentrations of HPMCAS polymer and fenofibrate) with three levels (high, normal, and low) for each factor. Their analysis of the results revealed a linear relationship of increasing velocity in response to increasing HPMCAS polymer concentration. Similarly, there was also a linear relationship between the change in velocity and the change in fenofibrate concentration. Moreover, a linear relationship of increasing attenuation in response to increasing polymer concentration was observed in solutions containing 4% of fenofibrate, and the slopes became progressively larger as the frequency increased. It was also evident that the attenuation was a strong function of frequency with larger attenuation at higher frequency, which, the authors explained, was an expected behavior of long polymer chains dissolved in solution. Similarly, they reasoned that the absence of a significant linear correlation between the measured attenuation and the fenofibrate concentration was due to the fact that the attenuation for small molecules is usually much less than that for polymeric chains. In conclusion, they found that the ultrasonic spectrometer gave accurate measurements of both velocity and attenuation of acoustic waves in acetone solutions of a model drug (fenofibrate) and a model excipient (HPMCAS polymer). By establishing linear relationships of measured velocity and attenuation to the concentrations of HPMCAS polymer and the Fenofibrate in a series of standard solutions, it was feasible to simultaneously analyse both concentrations of HPMCAS polymer and Fenofibrate.

Cespi et al. used sound speed measurements from acoustic spectroscopy (HR-US apparatus) (Cespi et al, 2010b) to develop an alternative method for evaluating the mucoadhesiveness of polymers. Water solutions of each polymer, alone (0.3–1.0% w/w) or in mixture with mucin (mucin fixed at 1.0% w/w), were studied at two different frequencies (5.2 and 8.2 MHz). Polymer–mucin 37

interaction was evaluated by comparing experimental sound speed values of polymer–mucin samples with values calculated by simple addition of sound speeds recovered from each component alone, analysed at the same concentration. Thus, in this work the interactions occurring between polymer and mucin were evaluated through the comparison of experimental and theoretical sound speed values of their water dispersions. The mucoadhesion degree (MD) was calculated using the rule of additivity: in a system containing one or more polymers dispersed in water, no interaction between the polymeric chains means that the relative sound speed of the whole system is equal to the sum of the relative sound speed of each polymer dispersion. On the other hand, if an interaction occurs, the total relative sound speed measured is lower than the theoretical one. The MD can be considered a quantitative index related to the total number of bonds between mucin and polymers. Results showed that sound speed was very effective in the evaluation of mucoadhesiveness and discriminated between mucoadhesive and non-mucoadhesive polymers (figure 9).

In a different work, Konrad and colleagues (Konrad et al., 1998) measured backscattered ultrasound signals to characterise advancement of the eroding front of the HPMC matrix tablets using a homemade apparatus, composed of an ultrasound transducer (Panametrics V 311, 10 MHz) positioned 8 cm from the tablet, and a Donoson 2 (Minhorst) receiver system. The swelling behavior result for the HPMC matrix tablets obtained using the ultrasound method did not differ from that obtained with the penetrometer. The advantage of the ultrasound method over the penetrometer method is that the tablet need not be removed from the dissolving medium, and thus it is possible to have continuous measurements.

Another example along these lines was offered by Leskinen and coworkers (Leskinen et al., 2011), who used the ultrasound technique to explore movement of the eroding front of HPMC and PEO tablets. All acoustic measurements were conducted using an UltraPACsystem (Physical Acoustics Corporation, Princetown, NJ, USA) which consisted of a tank and 3D-scanning drives, a high frequency A/D-board (PAC-AD-500) and focused broadband ultrasound transducers (Panametrics V307, Panametrics Inc., Waltham, MA, USA; center frequency=5MHz, frequency range (−6 dB) = 3.3–6.7 MHz, focal length = 50 mm, beam diameter of 0.6 mm). The measurements were done using the pulse-echo (PE) geometry with 62.5 MHz sampling frequency. Six identical samples with similar structural characteristics were measured simultaneously in each test. Samples were set into a line with a distance of 30 mm between the center points. In addition, there were two polished stainless steel reference plates at each end of the sample line. The sample surfaces were adjusted to a distance similar to the focal length of the ultrasound transducer with a custom-made sample 38

holder made of polymethylmetacrylate (PMMA, a transparent material). Acoustic measurements were conducted at room temperature 24±1◦C and the same measurement time of 8 h was used in all tests. The sound speed was measured in 0.1 M phosphate buffer (PB, pH 6.8), as specified in the European Pharmacopeia. This work demonstrated that ultrasound could be used to follow the swelling process of hydrophilic polymer tablets. However, one limitation is that in order for the medium-polymer interface to be detectable, the polymers must possess certain acoustic properties, depending on the ultrasound frequency in use. It was found that the ultrasound window technique introduced in this study was a promising method for simultaneous multifront detection (swelling and erosion). The result showed that the sensitivity of ultrasonic monitoring for following hydrogel formation and thickening varied according to the polymer under study. Thus, multifront detection proved challenging, as the hydrogels formed by different polymers might have totally different acoustic properties.

Yuan and Li (Yuan et al, 1999) demonstrated that acoustic spectroscopy is a very powerful tool for investigating materials such as polymer thin films and gels, in particular the behavior of polymer gels that are able to swell or shrink in response to a variety of external stimuli such as temperature, pH, electrical fields and light. In particular, the authors used an MBS-8000 (Matec Instruments) to study the ultrasonic attenuation of N-isopropylacrylamide (NIPA) gel samples as a function of temperature at various frequencies. (Alba, 1992; Yuan K) N-isopropylacrylamide (NIPA) gel showed a temperature-dependent behavior (phase transition temperature equal to 34 °C), thus it belongs to the class of smart material, widely used in controlled drug release. It was found that, at room temperature, the attenuation of the ultrasonic wave in the gel was small and similar to that of pure water. However, as the temperature increased above the sol/gel transition point, the attenuation increased drastically due to the microdomains formed in the gel. This change of the attenuation was completely reversible.

Medendorp and his group (Medendorp and Lodder, 2006) demonstrated that acoustic resonance spectrometry (ARS) can be used as an alternative tool to near infrared spectroscopy (NIR) in Process Analytical Technology, for the quantification of active pharmaceutical ingredients (API) in semi-solid formulations such as creams, gels, ointments, and lotions. The ARS used in this work was constructed from readily available parts. It was built in the near-field configuration, where the wavelength of the excitation signal was much larger than the quartz rod or the sample that was applied to the rod. An acoustic signal was applied to one of the piezoelectric transducers (PZTs) and received at the other. The sample, in mechanical contact with the vertex of the quartz rod, 39

constituted a load on the resonant system. When a sample was placed in contact with the vertex of the waveguide, acoustic waves escaped and propagated through the lotion/quartz interface and into the sample holder. The added mass effect caused a shift in the resonant frequency of the system, while the frictional or viscous drag force caused a reduction in peak amplitude. This pattern gave rise to the characteristic AR spectrum for any given analyte. The authors used this technique to quantify colloidal oatmeal (CO), used as an ingredient in several pharmaceutical lotions, cosmetics, and toiletries, but which is one of many examples in cosmetics and other household products that fall into a gray area for the US Food and Drug Administration (FDA), because it is listed as inert in some lotions but as an active ingredient in others. The FDA Process Analytical Technology (PAT) initiative called for the development and implementation of manufacturing processes to guarantee a predefined quality of pharmaceutical materials as warranted by risk analysis. These processes included multivariate data acquisition, analysis tools, and in-process and end point monitoring tools. The development of acoustic resonance spectrometry as an in-process and end point monitoring device was within the scope of the PAT initiative. Since colloidal oatmeal is a lyophilic colloid, readily hydrated and dispersed evenly through a solution, the resonant acoustic signal received at the detector was taken as an approximate representation of the bulk of the sample, regardless of microscopic differences between individual colloids. With this paper, the authors reported that ARS was faster, less expensive, and outperformed NIR spectroscopy for the quantification of CO in lotions.

CONCLUSIONS This paper has reviewed the use of ultrasound technique in the pharmaceutical field as an aid in preformulation and formulation steps. Although many more studies of ultrasound applications have been done for the food and chemical fields than for pharmaceutics, the works described here highlight the potential of ultrasound for obtaining deeper insights into the physical and chemical structure of pharmaceutical systems as summarized in Table 1. This technique makes it possible to adjust formulation parameters in order to achieve the desired quality and specification of the final systems. In particular, the studies presented in this review demonstrated that there are some physical parameters that can be easily monitored or calculated while the technique fails in some others. 40

Monitoring of sound speed and attenuation parameters in pharmaceutical systems open a wide range of properties and transitions to be studied without the limit of dispersed phase concentration despite the thermal behavior of the systems analyzed represents a very important factor that can be have a certain impact on acoustic properties particularly for soft matter systems. Concerning the size of the dispersed particles only few papers have been published on this subject for pharmaceutical systems (suspension, emulsion, microemulsion and nanoparticels). The main issue is that the mathematical model (ECAH theory) involves several physical properties of materials that affect the particle size and a correct setting of parameters is crucial to obtain reliable results. Thus, despite good results have been obtained, ultrasound is not the election technique in measuring particle size due to the fact that different systems parameters need to be known a priori to set instrument database. Moreover, the results are not directly comparable with those obtained with more common technique used for size analysis (DLS) probably due to the different range of concentration of dispersed phase. The different acoustic equipments have been more utilized to characterize transitions involving attenuation or sound speed variation such as polymers gelation, temperature induced phase changes, self-assembling properties, thermal behavior and stability of liposomes and hydrogel structure. Some other minor but not less important applications focused on the determination of polymers mucoadhesive properties, dissolution kinetics studies of API and polysaccharides, rheological properties of liquid and semisolid systems. This technique revealed also very effective in the characterization of ternary systems in terms of formulation, determination of the physical characteristics, microstructure and state of the different components. In addition, ultrasound can be used to check ongoing processes in-line, making it a valid option for Process Analytical Technology. The authors hope this review has successfully demonstrated the great potential of this technology, and that researchers and pharmaceutical firms will be encouraged to explore its applications in their development and quality control work.

41

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Yuan K, Hu, Z., Li Y., 1999. Polymer gel as thermally responsive attenuator for ultrasonic waves. Appl. Phys. Lett. 74, 2233 - 2235. Zana, R., Lang, J., Sorba, O., Cazabat , A.M., Langevin, D., 1982. Ultrasonic investigation of critical behaviour and percolation phenomena in microemulsions. J. Phys. Lett-Pariss 43, 829-837.

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Legends to figures Figure 1. Plots of the ultrasonic velocity gradient versus temperature for 100% tripalmitin (top) and 75% tripalmitin/25% fish oil (bottom) in oil-in-water emulsions during cooling (a) and heating (b) at 0.3 °C min-1. According to Awad et al., 2008. Figure 2. Ultrasonic and rheological measurement of gelling and melting temperature Tg and Tm depending on K+-concentration in 0.5% -carrageenan. (Wang et al, 2005) Figure 3. A) Particle size versus temperature plots of Poloxamer 407 samples at different concentrations (6–20%, w/v). B) Particle size versus temperature plots of Poloxamer 407 samples at different concentrations (6–20%, w/v) containing the 10% (w/v) of HP b-CD. (Bonacucina et al., 2008). Figure 4. Comparison of characteristic micellization transition and gelation (inset) temperatures (DSC data (black), sound speed data (red), attenuation data (blue)). (Cespi et al, 2010a) Figure 5. Comparison among mDSC, DLS and HR-US (sound speed) data related to the micellisation transition of Poloxamer 407 2.5% (w/w). (Perinelli et al., 2013) Figure 6. Sound attenuation as function of frequency (Bonacucina et al., 2013) Figure 7. Schematic design of primary nanoemulsion (PE), three- and five-layered nanocapsules (Preetz et al, 2010). Figure 8. Mean curves of G″ and G′ moduli from the acoustic spectroscopy of Sepineo/almond oil emulsion (Bonacucina et al., 2009c). Figure 9. Mucoadhesion degree (MD %) of polymer–mucin (1% w/w) dispersions as function of their concentrations (% w/w) calculated at the frequencies of a 5.2 MHz and b 8.2 MHz. Blue, green, red and gray colors refer to PEG, pectin, HPMC, and Carbopol, respectively. (Cespi et al., 2010b)

49

Tables Table1. Summary of the main ultrasound technique applications and equipments.

Applications

REFERENCES

Equipment

Fuchs et al, 2010

Resoscan®

Bohasale and Berg, 2010,

Dt-1200

Bonacucina et al., 2009c DT-1200

Size determination Granemann e Silva et al, 2013

DT-1200 Smyth et al., 2004 HR-US Horá‟K et al., 2007

HR-US

Fakhari et al., 2013

HR-US

Characterization of

Junke et al., 2012

DT-1200

Nanoparticle , SLN,

Awad et al, 2008

Resonic Instruments

Nanocapsules

Jo´zefczakn and Skumiel, 2011

Resoscan®

Fuchs, 2010 Preetz et al, 2010

Characterization of self

Resoscan® Resoscan®

Bonacucina et al, 2008

DT-1200

Cespi et al, 2010a

HR-US

assembling behaviour or

Farrugia et al, 2014

temperature phase changes

Perinelli et al, 2013

HR-US

Van Durme et al, 2005

HR-US

Smyth et al, 2005

HR-US

Buckin and Hallone, 2012

HR-US

systems,

Hickey et al., 2006

HR-US

micro-nanoemulsions

Hickey et al, 2010

HR-US

Stillhart et al., 2013

ResoScan®

Characterization of ternary

50

Bonacucina et al, 2013

DT-1200

Stillhart and Kuentz, 2012

ResoScan®

Niederquell and Kuentz, 2013

ResoScan®

Shah et al, 2007

ResoScan®

Gaysinsky et al, 2008

HR-US

Jünemann and Dressman., 2011

Resoscan®

Bonacucina et al., 2009b

DT-1200

Chen et al, 2005

HR-US

Smyth et al, 2004

HR-US

Dukhin et al., 2006

DT-1200

Bonacucina et al, 2009c

DT-1200

Granemann e Silva et al, 2013

DT-1200

Cespi et al, 2010b

HR-US

Leskinen et al, 2011

UltraPAC system

Dissolution kinetics

Characterization of emulsion, suspension

Mucoadhesion

Tablets erosion Konrad et al., 1998 Cavegn et al., 2011

Resoscan®

PAT Medendorp and Lodder, 2006

Characterization of gel systems

Characterization of lipidic systems

Wang et al,2005

ResoScan®

Piai et al, 2009

DT-1200

Yuan et al, 1999

MATEC Instrument

Taylor et al., 2005

HR-US

Krivanek et al., 2008

ResoScan®

Schultz and Levin, 2008

ResoScan®

Okoro, 2010 Kapoor et al., 2011

ResoScan®

51