Characterization techniques for nanoparticulate carriers

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Characterization techniques for nanoparticulate carriers

Abstract: The scope and commercialization of the therapeutic nanoparticles is severely limited despite their potential for treating several grave diseases at molecular level. This may be partially attributed to the limited availability of well-established, standardized and validated techniques for adequate characterization of these systems. However, a standard set of characterization methods constitutes a primary requirement to expedite the regulatory approval of therapeutic nanoparticles so as to progress to preclinical and clinical trials. A thorough characterization is also necessary for fine tuning of their structural properties, their interand intra-laboratory reproducibility, their therapeutic performance and the evaluation of their safety. This chapter provides a comprehensive summary of the currently available physicochemical methods of characterizing therapeutic nanoparticles. Techniques for characterzing particle size, homogeneity, shape and surface charge are described, with a special focus on the various industrially relevant instruments being employed for this purpose. A brief description has also been included about the in vitro biological assays, such as sterility tests and immunological studies, which are necessary to confirm the bio-compatibility of nanoparticles, before they may be evaluated in in vivo pharmacodynamic models and clinical studies. Key words: size, surface charge, dynamic light scattering, zeta potential, morphology, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, sterility, microbial contamination, mycoplasma testing, toxicity.

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3.1 Need and challenges for characterization techniques of nanoparticulate drug carriers Engineered nanoparticles have established themselves as novel platforms or delivery carriers for otherwise insoluble or poorly soluble drugs, offering tremendous scope for alteration of their pharmacokinetic and disposition profiles, as seen in the previous sections of this book. Additionally, nanoparticles decorated with targeting ligands are now known to bear the potential to limit the drug toxicity by facilitating preferential delivery of drug to the affected areas and hence limiting their pernicious side effects due to non-specific exposure. Despite the promising therapeutic applications of these fascinating delivery vehicles, their transition from laboratory to the market has been a laborious journey; challenges involved in their successful characterization being one of the major responsible factors. Many of the nanoparticles meant for therapeutic and clinical applications comprise a plethora of materials that have unique optical, electronic and structural properties on the sub-micron scale, properties totally unconfronted in bulk materials or isolated molecules. This can adversely affect the development of reproducible and valid assays like colorimetric and enzymatic assays, which rely on measurement of inherent material characteristics, for example, absorbance, surface characteristics, etc. Additionally, therapeutic nanoparticle formulations contain alternative excipients such as stabilizers and surfactants, cryoprotectants employed during lyophilization, which along with the impurities that get adsorbed on particle surfaces may hinder their characterization by the conventional techniques [1, 2]. Multi-functional and multi-component nanoparticles require a more rigorous characterization assessing their individual ‘functional parts’, their stoichiometry and the chemical stability of the connections between them [3]. Some other factors which hinder adequate characterization include particle agglomeration affecting identification of their true size, availability of techniques with slow and cumbersome statistically reliable sampling, a wide variety of conventional techniques which present a difficulty in comparison of the data, lack of appropriate standards and lack of communication between scientists and formulators from various groups. Yet another reason for implementation of standard characterization techniques is the requirement of the mandatory FDA approval for

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establishing a candidate’s therapeutic or diagnostic potential in humans. A definite set of preclinical tests forms an integral aspect of this approval which is processed through either an investigational new drug (IND) or investigative device exemption (IDE) application. Processing of these applications becomes difficult for nanomaterials due to the lack of a standardized set of characterization methods. As a result investigators are forced to design and validate their own novel characterization methods to assess safety, toxicity and quality control. The FDA in turn encounters the difficulty of rational interpretation of data generated through a set of unfamiliar techniques with limited acceptance in the scientific literature. This ultimately results in complications and delay in the preclinical testing, and hence the clinical trials for nanotech-based drugs. Thorough characterization also governs the fine tuning of nanoparticle structures, their inter- and intra-laboratory reproducibility, performance of drug nanoparticles and evaluation of their safety to comply with the policies of the Environment, Health and Safety (EHS) community. The toxicity of nanoparticles in comparison to their micro-counterparts has been a matter of extensive deliberation due to unavailability of standardized methods and materials leading to generation of conflicting research outcomes [1, 4]. Toxicity concerns have also been a major rate limiting factor for the successful commercialization of therapeutic nanoparticles. A sound characterization strategy for biomedical nanoparticles through strategic and concerted interdisciplinary approaches thus forms the need to facilitate their smooth transition from laboratory to the market. The present scenario calls for technology developments in areas of in-line diagnostic facilities to control the particle features at the production stage, improved precision and reproducibility, newer software for statistical data analysis, characterization methods for optically transparent materials and standardized protocols for optimum sample preparation to result in reproducible information [5]. Such a comprehensive understanding of nanoparticle properties will be possible through integration of three different characterization aspects, namely physico-chemical properties, in vitro performance and in vivo activities. Each of these bears particular significance with physicochemical characterization constituting the front-line strategy for predicting the suitability of the nanoparticles for in vitro or in vivo evaluations as well as maintaining the inter-laboratory consistency. In vitro evaluations provide a fair estimation of formulation efficacy and toxicity though complete inference of the formulation pharmacokinetics and efficacy is possible only through animal studies [4]. Published by Woodhead Publishing Limited, 2012

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Several physico-chemical characteristics of the therapeutic nanoparticles, including particle size, homogeneity, shape, surface area, surface charge, aggregation state, diffusivity and surface functional groups including their stability and distribution, can greatly influence their biological interaction and activity [6–11]. A wide variety of techniques have been used to provide information on these parameters with some of them comprising local probes while others with bulk-sensitive probes. These include dynamic light scattering (DLS), mass spectrometry, scanning electron microscopy (SEM), transmission electronic microscopy (TEM), electron diffraction, scanning tunneling microscopy, atomic force microscopy (AFM), as optical absorption spectroscopy, nuclear magnetic resonance (NMR), infrared (IR) spectroscopy (Fourier transform IR), Raman scattering, X-ray diffraction, X-ray photoelectron spectroscopy, and X-ray absorption [1, 4, 12]. Elucidation of particle mechanisms through in vitro biological assays is yet another challenging aspect of characterization of therapeutic nanoparticles. Particle characteristics under conditions mimicking the physiological environment in vivo may vary due to the impact of the physiological pH and ionic strength on the hydrodynamic size and surface charge of the nanoparticles. Measurement of nanoparticle parameters in presence of cell culture medium serves to mimic the nanoparticle protein binding inside the body which is now a known phenomenon affecting the physico-chemical and pharmacokinetic properties of the former. This may offer a big challenge necessitating the use of appropriate controls with known properties to ensure accurate end results. A variety of assays involving immortalized cell lines or primary cells freshly processed from organ and tissue sources or their combinations may be used to evaluate the bio-compatibility of nanoparticles. Complete characterization of nanoparticles also included a battery of toxicity studies, sterility tests, immunological studies to confirm the absence of hemolytic, complement activation, and thrombogenicity potential of the nanoparticles and in vitro phagocytosis studies for prediction of recognition by the immune system and clearance by the reticuloendothelial system (RES) [1]. The subsequent sections of this book present an overview of the current scenario of characterization of therapeutic nanoparticles which have presented significant challenges to formulation scientists and regulatory bodies. Some of the afore-mentioned techniques have been briefly described with discussion regarding the information provided by them. Table 3.1 gives an overview of the methods available for nanoparticle characterization [4]. Focus has been placed on some of the industrially

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Table 3.1

Overview of methods available for characterizing nanoparticles [4]

Parameter assessed

Method available

Presence

Dark field optical microscopy

Size

Dynamic light scattering, Static light scattering, Ultrasonic spectroscopy, Turbidimetry, NMR, Single particle optical sensing, FFF Hydrodynamic fractionation, Filtration

Morphology

TEM, SEM, Atomic force microscopy

Surface charge

Electrophoretic light scattering, U-tube electrophoresis, Electrostatic-FFF

Surface hydrophobicity

Hydrophobic interaction chromatography

Surface adsorbates

Electrophoresis

Density

Isopycnic centrifugation, sedimentation-FFF

Interior structure

Freeze-fracture SEM, DSC, X-ray diffraction, NMR

Abbreviations: DSC, differential scanning calorimetry; FFF, field fractionation; NMR, nuclear magnetic resonance; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

approved methods of characterization of therapeutic nanoparticles. However, an extensive and in-depth survey of all the specific characterization methods is beyond the scope of this book.

3.2 Measuring the size of nanoparticles The size of therapeutic nanoparticles is the most important parameter affecting the kinetics in vivo in terms of cellular internalization, deposition and clearance via the RES [1, 13]. Yet another important factor which accompanies size measurement is the width or shape of the size distribution. Subtle nuances in the experimental measurement may be reflected as large differences in the information conveyed by a complete size distribution and that which can be obtained from the actual experimental signal. Both these parameters are also influenced by the shape of the particle due to difference in the light scattering efficiency of nanoparticles with different facets [4, 14]. The manner in which information can be derived from a sample forms the main basis of classification of different sizing methods. Published by Woodhead Publishing Limited, 2012

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The ensemble methods, including the spectroscopic methods based on light scattering or ultrasonic absorption, estimate the size distribution by appropriate processing of the collective signal generated by the entire particle population. Though these methods have gained much industrial acceptance due to their convenience, they face certain drawbacks such as inability to detect small shifts in distribution which may have a bearing on process of nanoparticle production or their stability. Also these methods require result validation by comparing the distribution widths, shapes, and number of modes generated by various methods. On the other hand, counting methods such as microscopy or singleparticle optical sensing (SPOS) reflect the overall particle distribution by compiling a histogram of the individual particle measurements. Though these methods are more sensitive to small changes in distribution, particular care has to be taken to evaluate a sufficient number of particles lest a few large particles should disturb the fair size determination of a particle population. The third group of methods estimate the data based on physical categorization of particles based on their size. These separation methods like field-flow fractionation or filtration, etc., are valuable in that they analyze all the particles within a population [4].

3.2.1 Dynamic light scattering (DLS) Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS) or quasielastic light scattering (QELS) is the widely available method employed for routine analysis of hydrodynamic size of the particles in solution. This method relies upon the measurement of scattering intensity of nanoparticles in Brownian motion when illuminated by a monochromatic beam of light. This scattering intensity fluctuates on a microsecond timescale, the fluctuations corresponding to the diffusion rate of the particles. The fluctuations are measured by an autocorrelation function which is fit to an exponential, as indicated in Figure 3.1, and the decay of the correlation function is employed to calculate the rate of diffusion [1, 15, 16]. Subsequently using the cumulants method, the least-squares fitting of this correlation function is used to determine the mean decay rate from which the mean size of a particle population is determined. A unitless quantity derived from the cumulants analysis, the polydispersity index (PI), is used to represent the relative variance of the size distribution. This measured diffusion coefficient which is based on the standard assumptions of spherical size, low concentration, and

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Figure 3.1

The averaged (n = 10) intensity distribution plot (a) and correlation curve (b) for PLGA nanoparticles. Samples were prepared in distilled water at concentration of 100 mg/ml and filtered with a 0.45 mm filter. Measurements were conducted at 25 °C, in a 10 mm path length quartz cuvette. The DLS instrument employed a 633 nm laser wavelength and a scattering angle of 173°. The z-average was 29.6 ± 0.2 nm with a PI of 0.162 ± 0.008. A refractive index of 1.332 and a viscosity of 0.890 cP were used for size calculations

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known viscosity of the suspending medium as well as the sample preparation and handling is used to estimate the particle size [1]. DLS has gained popularity in the industrial fraternity due to its capability for rapid analysis, minimum requirement of calibration and sensitivity for detecting and measuring nanometric particles. Furthermore, the method may be applied for measuring long- and short-term pH, and thermal stability of nanoparticles, this stability being correlated to the changes in hydrodynamic particle size with changes in the environment to which the sample is exposed. Temporal stability is determined as longterm stability indicative of the shelf-life of the sample in its native medium monitored periodically and short-term stability measured at different storage temperatures (ambient, 4, and 37 °C) from single to several days. The method is, however, faced with certain shortcomings such as appropriate sample dilution to avoid degradation of signal-to-noise ratio due to low particle concentration or multiple scattering effects or interparticle interaction at too high concentration, care during sample preparation in the use of clean-dried sample cuvettes and filtered dispersion medium to eliminate dust contamination, the inaccuracy of the mathematical procedure used to determine the decay constants and the fact that differences in the weighted averages determined (e.g. number versus intensity) and the physical property actually measured (e.g. hydrodynamic diffusion versus projected area) may affect the comparison of the data obtained with that from other techniques [1, 4, 16, 17]. Some of the particle size measurement instruments based on DLS principle and that have gained popularity for industrial applications are listed below.

The Zetasizer range from Malvern Instruments Ltd., Malvern, Worcestershire, UK [18] This range of instruments is widely used to measure the hydrodynamic size as one of the parameters along with the polydispersity, surface charge and molecular weight of the nanoparticles. In this range of instruments, Zetasizer Nano S and ZS employ the patented Non-Invasive Back-scatter (NIBS) optics where a scattering angle of 173° (back-scattering) is used to measure the size with the optics being non-invasive due to absence of any contact with the sample. Both these instruments are adapted for higher sensitivity with the position of the sample cuvette being suitably adjusted to allow measurements of extremely small particles or the ones at a low concentration as well as the large particles or those at a higher concentration. Back-scattering mechanism minimizes the errors due to

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the presence of dust particles since these are known to scatter light in the forward direction. The instrument allows measurement of particle size with little or no sample dilution.

Delsa™ Nano Series from Beckman Coulter Inc., USA [19] This new generation series of instruments which are based on the patented forward scattering through transparent electrode technology (FST) can measure particles in the range of 0.6 nm to 7 µm. This is accomplished through the utilization of both log-scale and linear-scale correlators. Apart from possessing sensitivity for such a wide range of particle sizes, the instruments are also adapted to handle a wide concentration range from 0.001 to 40%. Precision optics of the instruments prevents light intensity and coherence loss due to fiber optics collection. The instruments operate at three scattering angles of 15, 30 and 160°. Additional benefits include analysis of volumes as low as 30 µl.

Bluewave Particle Size Analyzer from Microtrac Inc., USA [20] This instrument is based on the patented Tri-Laser Diffraction Technolology which performs scattering measurements through an entire 180° angle. This is accomplished through a combination of two short wavelength blue laser diodes along with a high angle red laser and two detector arrays. The instrument provides an excellent resolution for both wet and dry samples over its entire measurement range which varies from 10 nm to 2 µm. Furthermore, proprietary calculations are employed herein for non-spherical particles thus providing ‘realistic’ information.

W130i Dynamic Light Scattering System from Avid Nano Ltd., UK [21] The instrument utilizes a 660 nm fiber-coupled diode laser source in combination with a silicone avalanche photo-diode based photon counter, the detection ability of the latter matching the former. The laser power (30 mW) used herein is potent enough to illuminate the weakest scattering from samples but eliminates any dust particles or heating effects that may disturb the size measurement. The ‘diamond’ geometry of Stabilized Anti-Back Reflection optics of this instrument is the major reason for its compact dimensions. The advantages of the instrument include very low Published by Woodhead Publishing Limited, 2012

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measurement volumes down to 1 µl and the optional but built-in flow mode compatibility allowing the real time measurements of the samples.

The Saturn Digisizer II Particle Size Analyzer from Micromeritics [22] This instrument utilizes an extremely rapid digital detection technology and a CCD (charge-coupled device) detector consisting of over three million detector elements to result in high resolution, accurate, repeatable and reproducible digital representation of light scattering from a sample. The instrument is designed to measure both organic and inorganic particles in the range of 40 nm to 2.5 µm. Yet other attractive features of the instrument include automatic dispersion of sample in the liquid sample handler available both in high and low volumes and optimized analysis program allowing quick and efficient data processing.

Particle size analyzers from Brookhaven Instruments Corp., USA [23] These instruments can measure sizes ranging from 0.5 nm to 6 µm at a wide temperature range of 4 to 90 °C. Some of the instruments can measure sample volumes as low as 2.5 µl. The instruments operate at different scattering angles of 4, 15 and 90°. Some of the instruments are adapted with patented cells to facilitate automatic continuous online monitoring of a wide range of samples. Yet some other instruments are adapted with multi-plate readers, require no sample dilution or chemical modification of substrates and minimize toxic chemical exposure mediated by robotics. These instruments can measure formation of particle aggregrates at initial stages and may be employed for highthroughput handling of biotechnology based nanotherapeutics with extreme sensitivity, precision and accuracy. However, the ultimate choice of the instrument is governed by the size and complexity of the nanotherapeutic requiring a particular range and sensitivity of the instrument to provide accurate and reproducible information.

3.3 Zeta potential measurement [1, 4] The surface charge of the nanoparticles is estimated by the measurement of zeta potential. Charged nanoparticles in an ionic solution bear an 96

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electrical double layer comprising of a layer of oppositely charged ions strongly bound to the particle surface (Stern layer) and a second layer of loosely associated ions comprising the outer diffuse layer. Movement of the nanoparticle leads to a distinct dissimilarity between the ions in the diffuse layer that moves along with it and the ions associated with the bulk dispersant phase. The electrostatic potential at this boundary depends on the surface charge of the nanoparticle and is referred to as the zeta (ζ) potential. Generally nanoparticles with zeta potential values ranging within −10 to +10 mV are considered to have neutral surfaces while those with larger absolute values of 30 mV (+/−) are considered to be strongly cationic or anionic. This importance of the measurement lies in the fact that the surface charge of the individual particles affects the inter-particle interaction, thus affecting their stability in solution. In this regards, nanoparticle formulations with higher absolute zeta potential values are considered to be more stable due to repulsion between them [24]. Biologically, the measurement is relevant since the charge of the particles influences their interaction with the cellular and biological membranes, with cationic particles being more strongly bound to the negatively charged cell surfaces, the latter affecting the cellular internalization of the nanoparticles and hence their efficacy [25]. One of the methods of measuring the zeta potential is from the oscillations in signal resulting from scattering of light by particles in an electric field [26] with most of the instruments achieving this using a Doppler shift. Some of the experimental precautions necessary during the zeta potential measurement is the choice of the appropriate dispersant medium since the value depends on the pH and conductivity of the latter. In most cases it is advisable to note the sample pH along with the zeta potential but working closer to physiological pH is more relevant for therapeutic nanoparticles. In cases where pH of the sample needs to be adjusted, sudden pH changes of the sample should be avoided to maintain the sample stability. One of the approaches to avoid the influence of an external diluent is to employ particle-free supernatant to dilute the sample, the approach lacking application for samples with very small particles. Other approaches that can overcome these dilution problems include electroacoustic methods and phase analysis light scattering based on measurement of the phase delay shift rather than the frequency shift [27, 28]. Care should be taken to assess parameters: the scattering intensity count rate should comply with instrument’s specifications, the phase plots should have alternating slopes with time followed by either a smooth positive or negative peak, the frequency plots should have a smooth baseline and the measurement should not alter with duration or Published by Woodhead Publishing Limited, 2012

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different voltages applied. A typical depiction of these plots has been presented in Figure 3.2. In case of mono-dispersed samples, the zeta potential value arises from a single component and hence the plot is observed as a single peak. However, in case of polydispersed samples, zeta potential values of multiple components can result in multiple peaks in which case the value can be reported as an average of all the values or as value of individual peak. Numerous other factors also require special attention to avoid erroneous zeta potential results. These include absence of air bubbles in the cuvette and at the electrode surface due to the applied voltage, correct orientation of the cuvette in the instrument, employment of optically clear samples, degradation of electrodes, lower number of runs resulting in poor phase plots, too low sample concentration leading to degradation of signal-to-noise ratio and too high sample concentrations

Figure 3.2

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The averaged (n = 5) (a) phase plot, (b) frequency plot, and (c) zeta potential distribution for PLGA nanoparticles. Samples were prepared in distilled water at concentration of 100 mg/ml. Measurements were conducted at 25 °C, in a zeta cell (DTS1060C, Malvern Instruments) using a Malvern ZetaSizer Nano ZS at an applied voltage of 150 V. The zeta potential was +33.0 ± 0.5 mV. A viscosity of 0.891 centiPoise (cP), a dielectric constant of 78.6, and Henry function of 1.5 were used for the calculations Published by Woodhead Publishing Limited, 2012

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leading to inter-particle scattering. Most of these errors can be overcome by taking appropriate experimental care. In cases where the electrodes display signs of degradation, employment of a lower voltage is recommended while adjusting the instrument to an appropriate number of runs. To handle the concentration effects, generally the zeta potential measurements are conducted after the DLS measurements, the sample concentration ranges for the latter being suitable for the former. Additionally, the instruments may be periodically calibrated using an appropriate standard (e.g. zeta potential transfer standard, DTS0050, Malvern Instruments Ltd.). Some of the industrially accepted zeta potential measuring instruments include the following.

3.3.1 The Zetasizer range from Malvern Instruments Ltd., Malvern, Worcestershire, UK [18] The instruments measure zeta potential using laser Doppler electrophoresis (LDE). The Zetasizer Nano series utilizes the principle of Phase Analysis Light Scattering (M3PALS) to measure the particle velocity. Preference of phase analysis over frequency analysis results in a 1000 times enhancement of sensitivity to changes in particle mobility. This bears particular importance with regards to samples at high ionic concentration or those dispersed in low dielectric constant dispersants. Other advantages of the instruments include availability of disposable electrodes and low volume (150–750 µl), disposable sample cuvettes for samples of a wide concentration range (up to 40% w/v) to avoid the problem of contamination due to presence of small amounts of the previous sample. There is no specific practical range for the zeta potential values to be measured with particle size range between 3.8 nm and 100 µm depending on the type of the sample being analyzed.

3.3.2 Zeta potential analyzers from Brookhaven Instruments Corp., USA [23] ZetaPlus from Brookhaven utilizes electrophoretic light scattering and the Laser Doppler Velocimetry (LDV) method to measure the particle velocity of nanoparticles ranging from few nanometers to about 30 µm. Samples in organic or oily dispersants having very low mobility, those in high ionic strength solutions or those near the isoelectric point may be analyzed Published by Woodhead Publishing Limited, 2012

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using Phase Analysis Light Scattering principle of the ZetaPALS instrument bearing about 1000 times greater sensitivity than the other instrument. Disposable sample cuvettes requiring no alignment enable avoidance of sample contamination to facilitate simple, rapid and accurate analysis. Furthermore, the ZetaPALS instrument is adapted to characterize multimodal distributions, if any. Both the instruments can analyze a wide range of samples with zeta potential values varying between −220 and 220 mV with the sample volumes ranging from 180 to 1250 µl. The size range suitable for measurement is between 1 nm and 100 µm.

3.3.3 Delsa™ Nano Series from Beckman Coulter Inc., USA [19] In these instruments the principle of electrophoretic light scattering (ELS) through FST is used to measure the zeta potential of concentrated samples (0.001% up to 40%). FST is the only known technology that works well for the concentrated samples since such samples exhibit Brownian broadening of scattering spectrum at high scattering angles making the application of back scattering difficult for zeta potential measurement. The size of range of the particles that can be measured varied from 0.6 nm to 7 µm with the zeta potential range varying between −200 and 200 mV. The instruments operate with automated titration system over a wide pH range of 1–13 and temperature range of 10–90 °C.

3.4 Characterizing the morphology of the nanoparticles The morphology of therapeutic nanoparticles plays a critical role in their quality control and biodistribution [24]. Detailed morphological characterization of nanoparticles may be possible using a combination of several techniques, some of which have been listed below.

3.4.1 Electron Microscopy [1, 12] Electron Microscopy uses electromagnetic radiation of shorter wavelengths for overcoming the diffraction effects of light to enable efficient resolution and detailed visualization of particles up to 0.1 nm.

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Interaction of electron beam with a particulate sample results in their transmission, back-scattering and diffraction. TEM relies on the electrons which are transmitted through the samples without any significant attenuation of energy, the latter depending upon the thickness and density of the sample. The end result is a 2D projection of sample offering structural information via electron diffraction. SEM analysis provides crystallographic information based on electron beam diffracted by particles with favorable orientation. The method can also measure the additional electrons generated by the collision of primary beam by plotting the former as a function of the position of primary electrons, thus resulting in a 3D projection of sample. A detailed description of both these methods is presented below.

Transmission Electron Microscopy (TEM) [1, 4, 12] In this technique, electrons are accelerated at high voltage potential through a series of electromagnetic field after being emitted from a source. These electrons are subsequently focused on thin sample sections (dried layers in case of nanoparticles) under evaluation after which they move through another magnetic field before finally colliding with fluorescent screen. Those which do not pass through the nanoparticle layers are scattered or diffracted. The collision of the electron converts their kinetic energy to visible light energy which upon exposure to a photographic film or excitation by a CCD camera generates a digital ‘shadow’ image of varying darkness depending on the sample density. The most important experimental precaution while characterizing nanoparticles by this technique are presence of extremely thin sample layers to enable passage of electrons and resistance of the sample to damage by the high energy electron beam. In the event of sample susceptibility to the latter, low electron beam is employed to generate lattice fringe images and electron diffraction [29]. In modern times, the applications of TEM are numerous including high-resolution imaging of biological samples with rigid and dense organelles facilitating scattering of high velocity electrons, visualization of nanoparticles in biological specimens like tissue specimens or cell culture samples including their location in numerous intracellular organelles (mitochondria, endoplasmic reticula, Golgi, centrioles, microtubules, endosomes and ribosomes), and changes in nanoparticle Published by Woodhead Publishing Limited, 2012

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structure resulting from their interactions with gas, liquid or solid substrates [30]. Considering this utility for providing high resolution images (about 1 Å/1 Angstrom, or 1 × 10−10 m) of nanometric structures, the technique has undergone numerous reformations in recent times to adapt it more for nanotechnology based products. Dynamic imaging through in situ microscopy, quantitative chemical mapping, holographic imaging of electric and magnetic fields, and ultrahigh resolution imaging of lesser than even 1 Å are some of the advanced versions of this technique to provide fundamental understanding of nanoparticulate structures [31]. Furthermore, embedding the samples in epoxy resin (plastics) and flash freezing (cryo-TEM) can be used to characterize samples displaying low electron scattering [32]. Examples of such samples may include micellar nanotherapeutics. Yet other methods like variable-pressure or environmental TEM can be used to evaluate ‘wet’ samples where presence of water is essential in the sample to maintain the integrity of its structures. This is of particular relevance in visualizing nanoparticles in fine subcellular organelles. A depiction of TEM and Cryo-TEM images of hard shelled and soft shelled polymeric nanoparticle systems loaded with curcumin formulated by our research group have been depicted in Figure 3.3 A and B. Figure 3.3(a) depicts spherical morphology and homogenous distribution of Eudragit® S100 (Röhm Pharma) loaded with curcumin also depicting their smooth surface [24]. Figure 3.3(b) depicts curcumin loaded hydrogel nanoparticles of a combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone. The soft-shelled nature of the nanoparticles due to the presence of hydrophilic polymers in the latter case call for flash freezing of the sample to enable an enhanced scattering of the electron beam [33]. Despite its numerous advantages the technique has met with a few drawbacks such as requirement of sample staining, employment of a statistically small image area, damaging effects of the vacuum or electron beam irradiation common in high resolution imaging, and overlap of the images of nanoparticles and the matrix which supports them (Polyvinyl formal, Polyvinyl butyral, Nitrocellulose-based polymers, etc.). In certain cases the last drawback is overcome by exploiting the epitaxial relationship between the nanoparticles and their support. A brief description of some TEM instruments which can find industrial application for the analysis of therapeutic nanoparticles is provided below.

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Figure 3.3

A: TEM image of Eudragit® S100 nanoparticles of curcumin; B: Cryo-TEM images of hydrogel nanoparticles of curcumin formulated using hydrophilic polymers. (a) Reproduced with permission from American Scientific Publisher [24]. (b) Reproduced with permission from John Wiley and Sons [33]

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Transmission Electron Microscopes from FEI, USA: Titan™, Tecnai™, Morgagni™ [34] The FEI Titan™ includes a range of commercially available instruments including the Titan™ G2 60-300, Titan3™ G2 60-300, Titan Krios™ and Titan™ ETEM (environmental TEM). This range of instruments comprise of 60–300 kV electron column which provides the mechanical stability required for its probe and aberration correctors responsible for its extremely high resolution imaging. All the instruments are equipped with constant power lenses and advanced power supplies for providing the requisite electronic and thermal stability. Additionally, each of the instruments is empowered with a unique feature to cater to specific applications. The Titan 60–300™ microscope (resolution: 80 pm) incorporates a platform for providing excellent stability and flexibility to its corrector and monochromator. The Titan™ ETEM (resolution: 0.16 nm) is equipped with the facility of using a mixture of up to four different gases which along with suitable pressure settings allow assessment of dynamic behavior of nanoparticles under variable environmental conditions like pressure and temperature. The Titan3 G2 60-300 microscope (resolution: 70 pm) is designed to provide flexibility to high resolution applications such as tomography, cryo-TEM, environmental-TEM and dynamic experiments. Furthermore, feasibility for upgrading the microscope column with additional correctors or a monochromator supports the economic flexibility of the instrument. Finally the fully automated operation of Titan Krios (resolution: 0.204 nm) facilitates complementation of the cellular scale analysis by light microscopy with the atomic scale analysis by NMR and X-ray diffraction to facilitate rapid, easy and high volume imaging. The Tecnai series of transmission electron microscopes includes Tecnai Osiris™ in which combination of a high brightness field emitter and windowless EDX detection using Silicon Drift Detector technology allows for high resolution and rapid analysis to give color-coded elemental information. Tecnai G2 Polara allows cryo-TEM analysis of low contrast samples like micellar structures. The patented technology of Tecnai G2 Spirit provides high-resolution, highcontrast, 2D and 3D images holding special importance for soft matter samples. The Morgagni TEM permits rapid, simple, high-quality image analysis facilitating almost an automated image acquisition once the instrument settings are preset. Moreover the user-friendly instrument features promising operation by users with varying levels of experience.

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Transmission Electron Microscopes from Carl Zeiss NTS, Germany: LIBRA® 120, LIBRA® 200 and LIBRA® range of Energy Filtering Transmission Electron Microscopes (EFTEM) [35] LIBRA®120 PLUS is equipped with features such as in-column OMEGA energy filter, Koehler Illumination System, Patented Automatic Illumination System, open detector and completely dry vacuum operation, all of which permit easy and high throughput sample screening. The Koehler Illumination System in turn offers advantages of reproducible, homogenous beam illumination with measurable dose rates. LIBRA® 200 range offers additional advantages of up-gradation with monochromator to correct aberrations and prevent loss of brightness or incorporating corrector for the objective lens to improvise point resolution of the microscopes. This EFTEM range of instruments selects electrons on the basis of their scattering angles and energy and energy bandwidths which provides a better contrast. They utilize electron optics with unique Koehler illumination which along with the in-column energy filtering offers easy and stable digital imaging. Included in this series of instruments is LIBRA® 120 EFTEM equipped with a LaB6 or tungsten source which provides excellent image contrast from thick or unstained thin specimens. The LIBRA® 200 FE has corrected in-column OMEGA filter and a highly efficient field emission emitter. With both the instruments finding application in life-science and polymer and material science research, they may find use in studying cellular association of different polymeric nanoparticles.

Scanning Electron Microscopy (SEM) [4, 12] SEM is an ideal technique to assess the purity, extent of aggregation and degree of dispersion and homogeneity of nanoparticles. The main advantage of this technique is the prevention of sample destruction in Environmental- or E-SEM mode; the measurements correlate with relative humidity of real atmospheric conditions by suitable variations in the vacuum and temperature inside the sample chamber [36]. Thus the technique is advantageous especially for polymeric nanoparticles since the morphology can be visualized in liquid state since complete drying of polymeric nanoparticles may alter their inherent morphological characteristics. However, the application of SEM is limited since it sometimes fails to distinguish between the nanoparticles and the substrate. The Published by Woodhead Publishing Limited, 2012

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inadequacy of its resolution becomes even more pronounced in systems tending to form agglomerates in which case TEM provides a more feasible alternative to minutely visualize the structural nuances of such nanoparticle clumps. Moreover, in E-SEM the movement of particles in liquid film and lack of conductive coating leads to compromise in the resolution of the final images [36]. Also as with TEM, the sample size is statistically small to reveal an absolute representation of the bulk nanoparticles. A representative SEM image of polymeric nanoparticles of docetaxel formulated using a hydrophobic starch polymer has been depicted in Figure 3.4(a). Imaging was performed after sputter-coating of the sample with gold, under vacuum, to enhance the contrast. Figure 3.4(b) depicts the E-SEM image of the same nanoparticle system. As described earlier the ESEM image exhibits a lower contrast as compared to SEM. However, the images comply with each other in terms of average particle size and sample homogeneity. Also no bridging of the nanoparticles is observed in either case.

Figure 3.4

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(a) SEM and (b) E-SEM image of polymeric nanoparticles of docetaxel formulated using a hydrophobic starch polymer. The SEM image was captured after sputtering the sample with gold to facilitate enhanced resolution. (Reproduced with permission from John Wiley and Sons [36])

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A brief description of some SEM instruments which can find industrial application for the analysis of drug delivery nanoparticles has been presented below.

Scanning Electron Microscopes from FEI, USA: Quanta™ [37] The Quanta series includes a range of instruments which can operate in three modes, viz. high vacuum, low vacuum and environmental mode, allowing analysis of a wide range of nanoparticulate samples. Variable analytical systems including energy dispersive spectrometer, wavelength dispersive X-ray spectroscopy and electron backscatter diffraction provide sufficient versatility and resolution to these microscopes. Additionally, bright-field and dark-field imaging becomes possible with the field emission gun (FEG) systems equipped with S/TEM detector. This becomes specifically beneficial to visualize the morphology of low contrast samples like nucleotide-polymer nanoplexes. The Quanta™ 50 range of microscopes is featured with user-friendly control software, camera for providing colored sample images, beam deceleration option for operation at a low voltage (appropriate for beam sensitive samples) and additional detector options for enhanced resolution. Quanta Morphologi provides both the shape as well as the size information of nanoparticles by a single instrument, making use of the ‘right’ combination of the superior magnification and resolution of FEG-SEM with the well established particle size analysis software of Malvern Instruments. This may overcome the limitations of indirect particle size analysis methods which provide the equivalent spherical diameters of nanoparticles.

Scanning Electron Microscopes from Carl Zeiss NTS, Germany: EVO® [35] The EVO® HD encompasses a novel electron source technology allowing operation at low kV. This provides enhanced resolution even for beam sensitive samples. The EVO® MA and EVO® LS series of microscopes offer some additional image enhancement features including imaging at variable pressures, LaB6 source for enhanced brightness and ability to prevent sample dehydration, all of which may be of particular importance for nanoparticulate samples like polymeric nanoparticles where the stage of sample drying may alter the actual sample morphology. Additionally, Zeiss offers a range of Field Emission Scanning Electron Microscopes (FESEM) again with variable pressure technology in combination with Published by Woodhead Publishing Limited, 2012

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the GEMINI® In-lens SE detector or Energy Selective Backscattered detector or technologies supporting analytical applications like energy and wavelength dispersive X-ray spectroscopy. This enables superior imaging with enhanced resolution and contrast.

3.4.2 Atomic force microscopy (AFM) [4, 38] AFM measurements rely on the deflection of a cantilever with a sharp probe (tip) at one end, which is employed to scan the sample surface. The cantilever is generally made of silicon or silicon nitride with the tip having the radius of curvature on the order of nanometers. The instrument controls a piezoelectric displacement actuator with a built-in cantilever beam that is present at the other end of this cantilever. Attractive or repulsive forces at very close proximity of the tip with the sample causes its deflection which is subsequently measured by laser light, from a solid state diode, reflected from the back of the cantilever onto a position sensitive detector comprising of a split photodiode. The tip displacements are linearly correlated to the deflections, the latter having an extremely small magnitude compared to the cantilever thickness and length. Depending on the nature of tip-surface interactions, the instrument may be operated in contact mode, based on repulsive interactions with the probe actually contacting the sample surface or in non-contact mode, based on long range surface force interactions wherein the probe just hovers over the sample surface. Imaging soft surfaces or particles weakly adhered to a surface is possible via yet another operation form, the intermittent contact or tapping mode, which thus renders it the most suitable for nanoparticle characterization. Here the cantilever is oscillated at amplitude of 100–200 nm, close to its resonating frequency and perpendicular to the sample surface. When very close to the sample surface, various interactive forces between the sample and the tip cause it to intermittently contact the sample surface, thereby reducing the amplitude, which is then maintained at a set magnitude by an electronic servo controlling the piezoelectric actuator. This results in a tapping AFM image due to forces of intermittent contact of the probe with the sample. With reference to qualitative nanoparticle characterization, AFM provides 3D images, with the perpendicular resolution limited to less than 0.1 nm by the vibration element and lateral resolution limited to around 1 nm by the tip diameter. Furthermore, quantitative information can be generated by software based processing of the individual images of nanoparticles or nanoparticulate clusters.

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AFM can be performed both on dry and wet nanoparticulate systems, the former being analyzed in air or controlled gaseous environments (nitrogen/argon) and the latter after distributing on suitable anchoring substrates, silicon or mica being the popular ones. It is important that the nanoparticles have a greater affinity for the substrate than the probe tip to avoid their sticking to the latter with the resulting compromise on the quality of the final images. Additionally, insufficient attachment of the nanoparticles to the surface may lead to a ‘streaking’ effect in the final image when using contact mode, in which case alternative operating means need to be adopted [39]. Some of the advantages of this technique when characterizing nanoparticulate formulations include provision of 3D information about the nanoparticles thus facilitating measurement of their height, operation in air, liquids and vacuums, ease and rapidity of sample preparation, requirement of less working space, economic feasibility with regards to instrument cost and simple operation procedures not requiring highly skilled work force. However, the need to raster the tip makes it a time consuming process with the possibility of the false observations of sizes lower than the actual and possibility of deformation of sample morphology during drying (air or under nitrogen flow) on the silicon or mica substrate [40]. A brief description of atomic force microscopes which can find industrial application for nanoparticle characterization has been included below.

Atomic Force microscopes from Bruker AXS Inc., USA acquired from Veeco Instruments, Inc. [41, 42] Bruker offers a range of high resolution AFM instruments providing superior resolution images in air or in liquids. Additionally, instruments are available which combine the attributes of AFM with high-end inverted and confocal microscopes to facilitate imaging under biologically relevant conditions. This range of instruments offers fast and simple sample operation in various modes and options for advanced research and yet can be operated by personnel with only a preliminary training. Furthermore, these instruments which materialize calibrated measurements of cantilever deflections employing fiber optic interferometry can be used along with techniques like optical/confocal/interferometric microscopy or can be upgraded with Raman or confocal microscopy to provide simultaneous determination of chemical composition or high resolution and quantitative analysis of sample surface. Published by Woodhead Publishing Limited, 2012

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Yet other suppliers of AFM instruments include Agilent Technologies, USA [43], Danish Micro Engineering A/S, Denmark [44], Park Systems Corp., Korea [45], etc., with the latter providing instruments both for research purpose and industrial applications.

3.5 Assessing the sterility and detecting mycoplasma or microbial contamination of nanoparticles [46, 47] As discussed in the earlier chapters of this book, nanoparticles as drug delivery vehicles can be formulated for administration by various routes with intravenous (i.v.) route being one of the primarily exploited ones for improvising therapeutic properties of the encapsulated drug through controlled release, site-specific targeting, prolongation of drug elimination, etc. [48]. These parenteral nanoparticle formulations are required to fulfil the sterility criteria, similar to pharmaceutical injectables, to avoid contamination by bacterial endotoxins. Upon i.v. administration, bacterial endotoxins or lipopolysaccharides can trigger strong inflammatory responses leading to fever, shock or even death [49]. One of the most widely used methods to test the presence of endotoxins in pharmaceutical and clinical sectors is based on the clotting of Limulus amoebocyte lysate (LAL) from the blood of horseshoe crab, Limulus polyphemus. Smaller amounts of endotoxin result in turbidity of LAL while larger amounts lead to its gelation [50]. To ensure safety of the administered products, limits have been imposed by FDA on the number of endotoxin units (EU) [46]. Detection and quantification of these in nanoparticle formulations is carried out either by end-point chromogenic LAL assay or turbidity measurement of LAL extract. The former method relies on the ability of the LAL clotting enzyme to cleave certain synthetic amino acid carriers of a chromogenic p-nitroanilide group into the yellow colored p-nitroaniline (λmax = 405 nm). The intensity of this color is then directly proportional to the amount of endotoxin present. The turbidity assay relies on extrapolating the turbidity generated in test formulation on a standard curve constructed from the turbidity measurements of samples containing known amounts of standard endotoxin [46]. Nanoparticulate formulations, however, may interfere with both these methods leading to enhanced or under-evaluated EU values. This may happen with the formulations absorbing at 405 nm, those triggering the

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LAL proteolytic cascade leading to the formation of colored product (dendrimers), those quenching the absorbance at this wavelength, those which adsorb endotoxins on their surfaces (gold colloids) or those like nanoliposomes and nanoemulsions, with inherent high optical density affecting the turbidity measurements. Such experimental errors can then be avoided following the FDA guidelines for those nonnanoparticulate products which interfere with accurate interpretation of LAL reaction [51]. Some of the special precautions to be followed while conducting this assay for nanoparticles include adjustment of sample pH between 6 and 8 with sterile NaOH or HCl with pH measurement being conducted on a small aliquot of this sample to prevent bulk contamination from the micro-electrode, performing the assay after suitable dilution of the sample not exceeding maximum valid dilution, etc. Generally this dilution is maintained at 1:500 due to the high sensitivity of this assay. It is also important to test the presence of microorganisms like bacteria, yeast and mold in the nanoparticulate formulations to prevent contamination of cell cultures during the various in vitro assays or preventing infections of animals during in vivo efficacy, toxicity and biodistribution studies. The assays make use of Millipore sampler devices and swab test kits to detect the presence of any visible microbial colonies upon incubation with the nanoparticles. For nanoparticles containing bacteriostatic or bactericidal actives, repetition with appropriate dilutions or combination with standard antibiotics is generally recommended to estimate the actual microorganism inhibition capacity of the formulation [46, 52]. Literature also describes the employment of several mycoplasma detection assays which include polymerase chain reaction in combination with restriction fragment length polymorphism, optical biosensors involving fluorescence resonance energy transfer (FRET) and fluorescence microscopy employing appropriate fluorescent dyes to stain the mycoplasma DNA [52].

3.6 Toxicity evaluation of nanoparticles The attractive attributes of the nanoparticulate delivery vehicles as well as their economic and scientific impacts has led to their rampant exploitation in the health-care industry. The tremendous growth in the development of these ‘nano’ drug delivery vehicles has resulted in a corresponding dramatic increase in the levels of their human exposure. Published by Woodhead Publishing Limited, 2012

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However, until recent times the adverse biological effects of these systems were largely overlooked. These included effects of their long-term accumulation and toxicological profiles generated by the very properties of small size, specific shape, large surface area and surface activity, which govern their importance and use in medicine. Since the pioneering article by the Royal Society and Royal Academy of Engineering in 2004 discussing the scarcity of information on effects of engineered nanoparticles on human health and environment, several other research groups and government organizations have attempted to address this issue. Though research in this direction is still not in pace with the one concerning development of such systems, the preliminary reports provide sufficient evidence of the ability of some such systems to induce cytotoxicity and trigger inflammatory cascade and oxidative stress. Thus understanding the toxicological hazards associated with the engineered nanoparticles to prevent danger not only to human health but also to the nanotechnology industry, at large, forms the need of the day [53–55]. Majority of the preliminary investigations, evaluating the toxicity of nanoparticles, focus on cytotoxicity testing usually at high nanoparticle doses. However, exposure at lower doses may lead to subtle alterations at genetic level leading to carcinogenesis instead of cell death. Thus another vital area of investigation is the genotoxicology profiling of these new pharmaceuticals to estimate their carcinogenic or mutagenic potential. Such preclinical characterizations are a necessity before initiating Phase I/ II clinical trials to prevent any potential health hazard not only on an individual level but also upon fertility and the health of subsequent generations [53]. Such toxicological and genotoxicological profiling also holds importance given the fact that it is not only the patients who are vulnerable to moderate to chronic exposure to nanoparticles, for varying time periods, but the workforce of nanotechnology industries also forms a large section of the society to bear this effect. Yet another interesting facet is the correlation of these various toxicological trends with the physico-chemical properties of these drug delivery vehicles. This will facilitate a platform for extrapolating the risks of future nanoparticulate systems with similar characteristics to ensure their safety and biocompatibility. Catering to the important implications of these toxicological evaluations, researchers have attempted to establish the toxicological profile of nanoparticles employing standard pre-clinical tests including short-term and repeated dose toxicity studies. Genotoxicity evaluation is being carried out through a battery of assays including in vivo

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micronucleus assay, in vivo chromosomal aberration assay and in vivo comet assay. The Organisation for Economic Co-operation and Development (OECD) established the OECD Working Party on Manufactured Nanomaterials to focus on human health and environmental safety implications of engineered nanomaterials and establish internationally harmonized standards for hazard, exposure and risk assessment of these. This program published the guidance manual for the testing of manufactured nanomaterials in March 2009 [56]. The details of all these assays for establishing a complete toxicological profile of engineered nanoparticles will be discussed in depth in the subsequent chapters of this manuscript.

3.7 Evaluating immunological potential of nanoparticles Along with toxicity another major concern with regards to nanoparticulate drug delivery systems that requires special attention is their immunological testing. Various regulatory bodies like US FDA, EMEA and MHRA have published documents, which although not regarded as guidance documents, specify various requirements for a safe and effective nanoparticulate drug delivery system. Both USFDA and MHRA reports employ broader terminologies indicating the requirement of a detailed toxicological data in the generation of such a report and further for the generation of regulatory guidelines [57, 58]. The EMEA reflection paper, however, specifically signifies the importance of immunological investigations for the development of therapeutic nanoparticles [59]. Immunological potential of nanoparticles is of particular interest due to the fact that these particles are smaller in size and can penetrate different type of cells and interact with biological barriers. Additionally, these systems are fabricated utilizing numerous polymeric/lipidic materials along with surfactants, stabilizers or antibodies, which in turn may generate immune reactions [60]. On the other hand, various nanoparticles are also under investigation for targeting immune system. Their potential in vaccine delivery has become increasingly important and various such products developed by pharmaceutical industry are currently in different phases of clinical trials [61]. Thus thorough investigation of immunogenic potential becomes important for both these types of nanoparticle systems. Published by Woodhead Publishing Limited, 2012

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The immune system is a tuneable system of the human body and its suppression and stimulation depends on various conditions like environment, diseased state, intake of various materials by different routes, etc. Thus investigation of immunogenic potential should form an integral aspect of the development of various delivery systems, specifically targeted delivery systems [62]. In this regards, our research group has also published the immunostimulatory effect of drug-free chitosan nanoparticles using various immune-markers such as lymphocyte proliferation, nitric-oxide production and IL-6 expression [63]. Studies such as these describe the various methodologies which may be used to investigate interaction of nanoparticles with immune system; with numerous studies and compilations in this direction being published by Marina Dobrovolskaia et al. [64]. Dobrovolskaia and other researchers mainly describe four different methods of characterizing the interaction of nanoparticles with the immune system viz analysis of hemolytic effect of nanoparticles [65], analysis of thrombogenic effect of nanoparticles [66, 67], analysis of complement activation by nanoparticles [68] and analysis of cellular chemotaxis by nanoparticles [69]. Hemolytic properties concern interaction of foreign material with red blood cells (RBCs), and in the present context interaction of nanoparticles with RBCs. Hemolytic activies can be determined using spectrophotometry. In this the absorbance of cyanmethemoglobin, a conversion product of hemoglobin by damaged cells, is measured after incubating nanoparticles with RBCs. Nanoparticle induced hemolysis can then be calculated comparing with suitable control [65, 70]. Though various cytotoxicity assays are available for nanoparticles, determination of hemolytic activity is particularly important for injectable nanoparticles. Various researchers have investigated the effect of the nanoparticles on important hematological properties. Morey et al. studied various hematological functions of microemulsion based nanoparticles with respect to the thromboelastography, platelet contractile force, clot elastic modulus, platelet counting and the structural integrity of erythrocytes [71]. Our group also investigated the hemolytic activity of phospholipid based nanocarriers containing etoposide, an anticancer drug, using spectrophotometry [72]. Neun and Dobrovolskaia developed an in vitro method of analysis of hemolytic properties of nanoparticles. This colorimetric assay determines the concentration of released hemoglobin upon interaction nanoparticles with the blood [73]. Thus spectrophotometry can be regarded as a method of choice for inclusion within regulatory guidelines of regulatory agencies and can be easily adapted by pharmaceutical industry for such evaluations.

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Assessing thrombogenic properties through a battery of tests is yet another method to establish the immunosafety of the nanoparticles. Thrombogenicity can be defined as tendency of a material to generate blood clotting and/or thrombus, when in contact with the blood. A thrombus may lead to the occlusion of blood vessels [74]. Investigating and controlling the circulation time of the nanoparticle by various strategies forms one of the important aspects of their development since nanoparticles have a longer circulation time compared to the conventional drug delivery systems. This property of nanoparticles necessitates investigations concerning their thrombogenic properties to establish a validated method for investigating their thrombogenicity. Various researchers have utilized Lee–White method for testing the thrombogenicity of nanoparticles [75]. This method includes a direct interaction of nanoparticles with human blood and then estimating the duration of its coagulation as a direct indicator of thrombogenic potential of the test nanoparticles. Radomski et al. described a more detailed investigation to understand the interaction of nanoparticles with blood. Here the authors studied vascular thrombosis generated by ferric chloride. Ultrasonic flow probes were utilized for measuring the rate of thrombosis [76]. However, the method was observed to be useful only for in vivo applications. Neun and Dobrovolskaia have described an in vitro method for analysis of thombogenic potential of nanoparticles. In this method the authors describe the interaction of nanoparticles with platelet-rich plasma as against the human blood studied by the earlier researchers. Here the nanoparticles were incubated with platelet-rich plasma followed by particle counting and size analysis to determine the active number of platelets after incubation. The aggregation percentage was calculated after comparing the results with control and the authors claim that this method has potential to meet various regulatory and industrial requirements [66]. In a series of discussions related to the interaction of nanoparticles with blood, complement activation has also been associated with previous two properties as complement activation may lead to a larger reaction by immune system. The complement system supports the antibodies and phagocytic cells to eliminate the pathogen and is a part of immune system. This system comprises of various proteins associated with each other and normally available in bloodstream as a pro-protein. When activated, this system may lead to an amplified immune reaction such as anaphylaxis or allergic reaction [77]. Nanoparticles and nanomedicines may activate this system and hence the complement activation potential Published by Woodhead Publishing Limited, 2012

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of the developed nanoparticles should be determined and validated with various types of nanoparticles. Reddy et al. [68] and Thomas et al. [78] reported an in situ method (C3a sandwich enzyme-linked immunosorbent assay) to determine the complement activation potential of nanoparticles incubated with serum. Some researchers have also employed 2D immune-electrophoresis for this purpose [79]. Since most of the researchers describe a wide array of methods, a quick methodology to screen nanoparticles complement activation potential is desired. Neun and Dobrovolskaia [80] developed a quick qualitative method to determine the complement activation potential. In this method, nanoparticles were incubated with human blood and analysis was carried out by polyacrylamide gel electrophoresis (PAGE), followed by western blot analysis conducted using anti-C3-specific antibodies. Concentration of C3 cleavage products was determined and was compared with control. Cellular chemotaxis is a special phenomenon where cells migrate to their favorable environment, this event normally mediated by certain specific chemicals associated with cells or by chemicals induced from outside. The phenomenon is common during various disease conditions, specifically in cancer. Nanoparticles may act as chemo-attractants and this necessitates the requirement of an assay method to analyze this potential. Skoczen et al. [69] developed a fluorescence based assay where they utilized two part 96-well plate divided by a filter, with the lower part containing the nanoparticles and the upper containing cell line. Fluorescence measurement was then used to estimate the cell migration after specific incubation times followed by their staining. Though various other methods have been proposed to understand the interaction between nanoparticles and immune system, majority of them focus on the determination of hemolytic activity and thrombogenic potential. Thus there is a strict need for assays/methods to determine the complement activation and cellular chemotaxic potential of nanoparticles. This calls for additional research in this direction to promote the faster development of nanoparticle based drug delivery systems. Thus, though appreciable progress has been made in assessing various characteristics of therapeutic nanoparticles, further efforts are still warranted for establishment of validated and standardized techniques as well as their international harmonization to facilitate the regulatory approval and smooth transition of nanoparticulate drug delivery systems into the society.

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3.8 References [1] McNeil SE (2010) Challenges for nanoparticle characterization. In: McNeil SE (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (Methods in Molecular Biology), Humana Press, Springer, New York, pp. 9–15. [2] Rao A, Schoenenberger M, Gnecco M, Glatzel T, Meyer E, et al. (2007) Characterization of nanoparticles using Atomic Force Microscopy. J Phys: Conf Ser, 61: 971–976. [3] Hall JB, Dobrovolskaia MA, Patri AK and McNeil SE (2007) Characterization of nanoparticles for therapeutics. Nanomedicine, 2: 789–803. [4] Haskell RJ (2006) Physical characterization of nanoparticles. In: Gupta RB and Kompella UB (Eds.), Nanoparticle Technology for Drug Delivery, Taylor & Francis Group, New York, pp. 103–132. [5] Saltiel C and Giesche H (2000) Needs and opportunities for nanoparticle characterization. J Nanopart Res, 2: 325–326. [6] Kobayashi H, Kawamoto S, Jo SK, Bryant HL, Jr, Brechbiel MW and Star RA (2003) Macromolecular MRI contrast agents with small dendrimers: pharmacokinetic differences between sizes and cores. Bioconj Chem, 14: 388–394. [7] Oberdorster G, Oberdorster E and Oberdorster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Pers, 113: 823–839. [8] Furumoto K, Nagayama S, Ogawara K, Takakura Y, Hashida M, et al. (2004) Hepatic uptake of negatively charged particles in rats: possible involvement of serum proteins in recognition by scavenger receptor. J Control Rel, 97: 133–141. [9] Oberdorster G (2000) Toxicology of ultrafine particles: in vivo studies. Philos Trans R Soc Lond Ser A Math Phys Eng Sci, 358: 2719–2739. [10] Ogawara K, Yoshida M, Higaki K, Kimura T, Shiraishi K, et al. (1999) Hepatic uptake of polystyrene microspheres in rats: effect of particle size on intrahepatic distribution. J Control Release, 59: 15–22. [11] Ogawara K, Yoshida M, Kubo J, Nishikawa M, Takakura Y, et al. (1999) Mechanisms of hepatic disposition of polystyrene microspheres in rats: effects of serum depend on the sizes of microspheres. J Control Release, 61: 241–250. [12] Herrera JE and Sakulchaicharoen N (2009) Microscopic and spectroscopic characterization of nanoparticles. In: Pathak Y and Thassu D. (Eds.), Drug Delivery Nanoparticles Formulation and Characterization, Informa Healthcare, New Yrok, USA, pp. 239–251. [13] Dong Q, Hurst D, Weinmann H, Chenevert T, Londy F and Prince M (1998) Magnetic resonance angiography with gadomer-17. An animal study original investigation. Invest Radiol, 33: 699–708. [14] Allen T (1997) Particle Size Measurement, 5th ed., Chapman & Hall, London, pp. 1–62. [15] Pecora R (2000) Dynamic light scattering measurement of nanometer particles in liquids. J Nanoparticle Res, 2: 123–131. Published by Woodhead Publishing Limited, 2012

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[37] http://www.fei.com/products/scanning-electron-microscopes/quanta.aspx [38] Grobelny J, DelRio FW, Pradeep N, Kim D-I, Hackley VA and Cook RF (2010) Size measurement of nanoparticles using atomic force microscopy. In: McNeil SE (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (Methods in Molecular Biology), Humana Press, Springer, New York, pp. 71–82. [39] Scalf, J. and West, P. Part I: Introduction to Nanoparticle Characterization with AFM. Pacific Nanotechnology, Inc.; http://www.nanoparticles.org/pdf/ Scalf-West.pdf [40] Montasser I, Fess H and Coleman AW (2002) Atomic force microscopy imaging of novel type of polymeric colloidal nanostructures. Eur J Pharm Biopharm, 54: 281–284. [41] http://www.bruker-axs.com/atomic_force_microscopes.html [42] http://www.bruker-axs.com/news_article.html?&tx_ttnews[tt_news]=213 &cHash=bed68327b1. [43] http://www.home.agilent.com/agilent/product.jspx?nid=-33986.0.00&cc =DE&lc=ger [44] http://www.dme-spm.com/index.html [45] http://www.parkafm.com/product/product_overview_r.php [46] Neun BW and Dobrovolskaia MA (2010) Detection and quantitative evaluation of endotoxin contamination in nanoparticle formulations by LAL-based assays. In: McNeil SE (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (Methods in Molecular Biology), Humana Press, Springer, New York, pp. 121–130. [47] Potter TM and Dobrovolskaia MA (2010) Analysis of microbial contamination in nanoparticle formulations. In: McNeil SE (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (Methods in Molecular Biology), Humana Press, Springer, New York, pp. 131–134. [48] Kreuter J (1994) Nanoparticles. In: Kreuter J. (Ed.), Colloidal Drug Delivery Systems; Marcel Dekker Inc., New York, pp. 219–342. [49] Chan E and Murphy JT (2003) Reactive oxygen species mediate endotoxininduced human dermal endothelial NF-kB activation. J Surg Res, 111: 120–126. [50] Roth RI and Levin J (1992) Purification of Limulus polyphemus proclotting enzyme. J Biol Chem, 267: 24097–24102. [51] FDA (1987) Guideline on validation of the Limulus Amebocyte Lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices. December 1987. [52] Murthy RSR and Pathak Y (2009) In vitro characterization of nanoparticle cellular interaction. In: Pathak Y and Thassu D. (Eds.), Drug Delivery Nanoparticles Formulation and Characterization, Informa Healthcare, New York, pp. 175. [53] Singh N, Manshian B, Jenkins GJS, Griffiths SM, Williams PM, et al. (2009) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials, 30: 3891–3914. [54] Landsiedel R, Kapp MD, Schulz M, Wiench K and Oesch F (2009) Genotoxicity investigations on nanomaterials: methods, preparation and

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