Jol of Contd Release 235 (2016 237-351 Contents lists avaiable at ScienceDirect Journal of Controlled Release ELSEVIER journal homepage: www-else r.com/locate/jconre! Review article DLS and zeta potential - What they are and what they are not? Oe Sourav Bhattacharjee Sch Wray Mein, Uneaten (UCD), ai Dn. and ARTICLE INFO ABSTRACT ‘rl ey ‘Adequate characterization of NPs (nanoparticles) is f paramount importance to develop well defined Received 13 Ape2016 ‘nanoformulations of therapeutic relevance. Determination of particle size and surface charge of NPs ae ince eve in evs form 8 Jue 2016 [Pensable for proper characterization of NPs. DLS (dynamic ight scattering) and 2 (zeta potential) measure ‘ecepied 9 june 2016 ee ‘ments have gained popularity as simple, easy and reproducibe tools to ascertain particle size and surface charge Unfortunately on practical grounds plenty of challenges exis regarding these two techniques including Inadequate understanding of the operating prinples and dealing with critical sues ike sample preparation and = Sr -C—rt—“—i——s—T—CisSCs=Ctcs Bl Hees eee, | ita ake ag een sacech als roars essere othe ces eer Tsk ssa tour wc pang tc eet pencpset seis sae maalane ‘ons he hens pcr an oeig pce sieson ting cman ened oes tres wc uming 1S and eaucments ay. he ee stom lane ee See fae ermene CLL _ mtn Pa DiS ramet) . io. 3 aed = gee = 3b paola 2 seein : haar xa _ 3 at ter a Tek Cpe ti its [ 23% thangs an aah : 2s ome xe Toe 2 222 Site cmettas x oo a ‘Atbevrons te cleerophoctic mobility: 2M, lpha-2 macroglobulin: AC autocrat unctn: APD Avalanche photo da; apa, Aolipprtia A: BSA, bovine serum albu: CCD, charge-coupled device; Penis TAB cetsietyarmoniabroie: OS, eres centefzal sedimentation: DOS dug every systems: DL deonized DS. dmameliph stern DMEM, Dibee' ae apes meds: DS, dimethyl sll: Dy eatin son cece: Dy asians fson cee: DI. ‘ete dbl er: EM, ton miosepy EU, ropa Une FC, tl al ser FT, ois nae petro: GS ld manoparties HB,Nemoslb Hehe: HS Helmfolez-Smolchowsk; HSA, hunan serum abun i mmunezlbulit IC. soteral ato alormety ky Batzrann conta ep, lo aunts Det second; KNO, paso state, ills mW milinat: NaC sodium chloride; Re eon; NBS, non-invasive backscatter systems; NST, National Institue of Standards ae “Techoogy (USA): NP. nanoparticle: NTA. nanoparte tacking nays: PALS, phase analysts Hat catering: POL poles inex: PE plylctraye complex: EC ‘eine gal: PLA poly tic ct PMMA ply ethylene corto ion; Rath rato: yr as eteacivende: RPM, sel ark Memorial nse media: 5) nano; SAS, sal angle Xray sealer SL, sa ight seating: SPS, anni bi partie er SOP, standard opeating prota, absolute temperate TEM ans lect moscopy UV abratigh acu; 2, acta potent fave permis alse ntl ee sora Bache uci nog 10.1019} nore201605017 IGE 18910 201 Elser BY. Al gts esexe ‘hack oul of Controle Ree 25 (206) 27-251 234 Effect of agglomeration 8 235. Shape of Ns 0 236, Rotational fusion of NPs 30 237, Issues related tocuvette 0 238, Maintenance ofthe instrument 33 24, Practialites 0 241, — Resolution of DIS. 30, 242 Expressing the Ry base on intensity volume or number 8 243. Importance of feeding ght information to the software 38 244 Proper reporting o DIS data. a 245, Comparison of DIS with ther techniques to measure parce size a 246, DLS incellcuture medium 245 2:47, DLS on aetosls and foams 34s, 3. Zeta potential (ZP) 345, 3A. Principles of 345, 3.1. Understanding the EDL and sipping plane 45 312. Fundamental mathematical operators whle measuring 2? 246 32, _Insiumentation in ZP measurements, 346 33. Interpretation of ZP data 7 331. Factors influencing 2P 7 332. ZPand colloid staiity v7 333, ZPand surface charge of NPS oar 34, Practialites 348 B41, Reference materials 348 342, Reusing samples ater measuring 2? 248 33. Using bufers with metalic ons 248 344 Measuring? in cell colture medium, 348 4. Discussion 348 Conic interest 248 ‘Acknowledgements 49 Appendix A. Supplementary data 39 References, 49 1. Introduction integrated, compact and affordable instruments offer user-friendly dig- Physicochemical properties of nanomaterials contribute towards their behavior within biological milieu [1.2} Hence, adequate characterization ofthe nanoparticles (NPs) isessentia in order to obtain reliable data with high translatory output This also becomes relevant due tothe safety con- cerns which are often attributed tothe physical (eg, particle size [3], sur- face charge [4, shape [5]) and chemical (eg, surface functionalization with efferent ligands including PEGylation [], impurity (7). crystalinity [8) properties of NPs. Sufficient characterization helps to explain the NPS a chemical species which are highly reactive and exhibit unprecedented characteristics compared to bulk materials (ea. conductivity [9] fuores- «cence | 10), magnetism {11)). To broaden the scope for applications of NPs, a research intensive framework within the academia and industry has femerged and attracted significant funding plus media attention in ast few years [12] Surface charge and particle size are the two most com- ‘monly mentioned factors that are responsible fora range f biological ef fects of NPs including celular uptake (13), toxicity [14] and dissolution [15] Emerging data indicate the influence ofthese two factors in release profile from NPs designed to cary drug payloads (eg, macromolecules 16), peptides [17)) and release at target sites (eg. small intestine [18] for orl drug delivery purposes) I is important co investigate these two parameters duting development of nanoparticulate DDS (drug delivery systems) especially given the fact that biological matrices are known t0 alter these two features of NPs with diferent mechanisms (eg. protein adsorption causing the characteristic corona (19.20). DDS (dynamic light scatering) also known as photon correlation spectroscopy [21] or quasi-elastic ight scattering [22] - and ZP (zeta po- tential) have emerged as simple table-top techniques executable under ordinary lab environments to investigate the (hydrodynamic) size and surface charge of NPS, respectively. From techniques that were exclusively available to colloid chemists, both DLS and 2 have evolved into popular tools within pharmacy community. The ital interfaces along with possibility for comprehensive data analysis. ‘Additionally, the techniques are non-invasive, require minimal sample preparation and no pre-experimental calibration. The modern instru- ‘ments are able to guide the users on the quality f the generated data with possibilty for time-dependent measurements and ability to export the data traces a les compatible wit various plotting softwares. Unfortunately, due to frequent use with lack of caution and proper training, the quality of the reported data with DLS and ZP in nanomedicine research is not always excellent. Dispersions of NPs in colloidal systems show dual phases (dispersed and dispersant), do not settle overtime [23] and are characterized by Brownian motion of the Particles 24].For charged NPs the system becomes even more complex ‘due tothe interactions between surfaces, molecules and ions leading to the creation of adsotbed layers on NPs [25] Both DLS and ZP utilize these properties of colloid dispersions in order to deduce the hydrody- namic radius (R,) [26] and potential difference at the characteristic sipping plane of electrophoretcally mobile particles [27 In this review, ‘an effort is made to offer a simple account on these two techniques ‘while referring only to essential mathematical priniples in order to un- ‘derstand their strengths and weaknesses. Succinct discussions are of- fered on why and how different factors influence these measurements ‘which cumulatively determine the quality ofthe data. The review also tries to deliver realistic examples while touching practicalities of these techniques which are relevant for drug delivery, 2.DLS (dynamic light scattering) 2.1. Background 2.11 Particle size in defining nanomaterials ‘Size isan important factor to define NPs although considerable de- bate exists on the size threshold to distinguish NPs rom bulk materialsDhak Jura of Conroe Rese 25 (2016) 357-351 Inorder to address this issue, the FU (European Union) released a dtec- tive (2011/696/EU) which offered specific guidelines on how to define a ‘nanomacerial. Some relevant excerpts ftom the document ae: (1) [Paragraph A defined size range would facilitate a uniform nter- pretation, The lower limit was proposed at 1 rm. An upper li of| 100 nm scommoniy used by general consensus, ut theres no sci- “entfic evidence to support the appropriateness of this value. The use ‘fa single upper limit might be too Kiting forthe classification of nanomaterials and a differentiate approach might be more “appropriate: (2) [Paragraph 9} "The International Organisation for Standardisation defines the term nanomateria? as ‘material with any external di- ‘mensions inthe nanoscale or having internal structure or surface “structure in the nanascale. The term ‘nanoscale is defined as size ange of approximately 1m and 100 nm." (3) [Paragraph 11] A nanomateral as defined in this recommendation should consis for 50% or more of particles having a size between 1 ‘am-100 nm" Apart from providing legally binding definition of nanomaterials the document also gave clear guidelines on how to characterize them: (4) [Paragraph 8] “For regulatory purposes, he number size distribu tion should ako be considered using the mean size and the standard deviation of the size to refine the definition The sizecstibution ofa ‘material shouldbe presented as size distribution based onthe num- ber concentration (ie the number of objects within a given size range divided by the number of objects in toral) and not on the mass fraction of nanoscale particles inthe nanomateral as a small mass fraction may contain the largest number of particles There are few interesting propositions made inthe document and it ‘would be appropriate to understand DLS asa nanoparticulate size mea- suring tool from that perspective. The document relaxed the widely popular size threshold of 1-100 nm to be considered as nanomaterials although still defined nanoscale materials t have at least one external «dimension <100 nm, Even a mixture of particles with different sizes can be considered as nanomaterials as long as =50% of the particles (by number) present are $100 nm. As a result it now becomes impor- {ant to know the size distribution ofthe different population of particles within a mixture. Thirdly, not only there isa requirement to know pre- ‘number distribution of NPs in samples. These findings will become im- portant while discussing DLS in subsequent sections. 21.2. Principles of DIS 2112.1, Seatteringof light by particles. Dispersed NPs scatter incident light ‘proportional tothe 6 power of their radii [2]. When the particles are 1/10th of the wavelength ofthe incident light (ie. 3/10) in siz, the scattered light caries same energy (elastic scattering) to the incident light and is not angle-dependent (Rayleigh scattering) [29]. However, when the size ofthe particles exceeds this threshold of \/10 then Ray- leigh scattering is replaced by anisotropic Mie scattering where the scattered lights unequal in energy (inelastic scattering) to the incident light and angle-depenclent (Fg. 1) [30] where the scattered lightis most Intense towards the direction ofthe incident light [31] Thissize thesh- cold (X/10) is due to the way electromagnetic waves (eg light) interact with a particle and falls beyond the scope of this review although excel- lent textbooks and reference literature are available for consultation (3233), 2.122, Fundamental mathematical operators Partilesin a colloid disper Sion scatter an incident laser and the intensity of the scattered light is detected in DLS. The continuously mobile particles within dispersion cause constructive and destructive interferences and hence the intensi- ty of scattered light fluctuates overtime (Fig. 2A) [34 n DLS the fuctu- ation of intensity in scattered light is correlated against short decay intervals (7) andthe intensity ACF (autocorrelation function) is obtain- ed [25] through the following mono-exponential equation (Fig. 2B) for samples with purely monodisperse particles (Eq, (1)): Giz) = 1+ beer a Here, b = constant dependent upon the instrument and setings of ‘optics, D, = translational dfusion coefficient andl q = scattering vector ‘which can further be expressed as (Eg, (2) anny He @ a] where, a = refractive index (RI) ofthe solvent, Ay = wavelength in, vacuum and 8 = scattering angle The intensity ACF (G(n)) is often written as G2(7) and is expressed asa function of field correlation function G1(r) as mentioned in the fol lowing Ea. (3): cise size distribution but it is now equally important to know the 2(7) = 1+ GI 8 Rayleigh scattering Mie scattering Mie scattering Particle size-/10 angle-dependence increases = Notangle-dependent ‘+ Angle-dependent ‘with bigger particles = Eastic scattering *Inclatic scattering KS eS Incident laser wavelengthh Fig 1. Senora showing the erences etcenRaygh an Mle stern“0 ‘hack of Controle Ree 25 (206) 27-251 A Laser A= 633 nm 3 Tinete) Fe. 2.(A) Facuston nen ofthe catered ih by Ns during DLS due to cnsrctive an destructive aefrences (2) Te cael generated by the software node to éstnate they, Te rests are om LS measuretnts period ar 25 Co bighly mendlspese 100 an carboxylate latex beads (PHI) cspetsed in wate (100) a vera Manag instrument wig pst vets (DTSIO0) and aj by Zeta vers 710) sla In DLS instruments (eg, Malvern Zetasizer®) a cortelogtam is gen- hydrodynamic radius (Ry) of sold spherical particles can be derived erated where RCF (raw correlation function) s plotted (Fig. 2B) against as shown in Eq (5) (Stokes-Einstein equation) delay time (2) as shown in Eg (4): kat 7 orm 6 er) cure a 2D ‘which shows that the RCFis dependent onthe field correlation func- tion Gtr) Where ky = Boltzmann constant (1.38064852 x 10-3 J/K),T The autocorrelation functions (G2(=) or G2(z)~ 1] in DIS arecaleu- temperature, 17 = absolute viscosity and Ry, = hydrodynamic lated by data fitting and then the D, is calculated using Eq. (1).The radius. Soft Corona (low affnityhveaky bound) Hard Corona (high afnty/strongly bound) Fi 3. Schematic of sot and hard cron forme onthe suri of aNP. Protein sorption iz kinetic (kan thesmedynaic(K faton of bth te india roti nd NP properties sch sufice modification composton. an dae. italy hig abundance ano high-protein othe naneprce suace. Over ne, hese protes ae replaced lower mbity proteins with ihr bing afin. Serm proteins commonly sere NP rons ae shown aa representative orn erm ami ing C(g phe? margin (ADM), and appre A (apo) Figs gre legend reproduced er elec [4 under AUS open aes aly.Dhak Jura of Conroe Rese 25 (2016) 357-351 an ‘The mathematical equations described indicate that the DLS results {depend on few variables induding viscosity of solvent [36] instrument [37], temperature [3], R (refractive index) of the material [39] ete. 2.1.2.3, Particles dispersed in a colloidal system. While the movernent of te particles in colloidal dispersion is random and translational, the par- ticles also rotate very fast [40] Te inter-particular interactions are also {important as with increasing concentration, the number of colisions within particles increases while the average pathlength traversed by the particles between successive collisions falls [41]. The surfaces of te dispersed particles ae altered depending on the adsorbed layer. A ‘common example to this phenomenon is the adsorption of the proteins ‘nthe surface of the NPs creating the characteristic protein corona {42,43}. The dispersed particles exhibit a hydrated surface wrapped within a cloak of molecules which are not the ingredients of the part- cles itself. The corona is often found to be composed of hard’ and ‘soft components (Fig. 3) [44]. The hard corona refers to the inner stable layer tightly bound tothe particles [45]. The soft corona isthe compar- atively loose layer on top ofthe hard corona composed of molecules of lferent charges and sizes [46]. In colloidal system what scatters Hight are these constructs composed ofthe NP-cores wrapped within the co- ‘ona of hydrated/solvated surfaces with altered compositions. There- fore, in DLS the particles that are assayed are diferent in composition and surface chemistry than those originally synthesized, Ina recent study, DLS was successfully employed in order to deter- ‘mine stabilities of PLA (poly-latic acid) and PMMA (poly-methyl meth- acrylate) NPs in buffers, simulated biological fluids (saliva, gastric juice, intestinal luid and lysosomal uid), serum and tissue homogenates (mice brain, spleen, liver) (47) While the PLA NPs showed reasonable stability in such biologically relevant conditions, the PMMA NPs were ‘unstable and aggregated over time. Such systematicDLS studies provide ‘with an in vitro tool to investigate NPS or stability befare in vivo studies, Inanother study Khan etl. (2015) [48] have used DIS effectively to in vestigate the adsorption of proteins eg, HSA (human serum albumin), SA (bovine serum albumin) and HE (hemoglobin) on CTAB-stabilized GGNPS (gold NPs) of diferent sizes (2-40 nm). Furthermore they corre- lated the data with mathematical modeling in order ta deduce the ad- sorption kinetics and subsequent development of protein corona on GGNPs. Additionally, they provided insights on how the structure of pro- tein and surface chemistry of NPs influenced the development of coro- na, Understanding the dynamics of the growth of protein corana onto [NP-susfaces is important from the perspective of how the NPs behave in vivo 49}. Salvat etal. (2013) has shown that growth of protein coro- ‘na on top of transferrin-functionalized silica NPs (-50 nm) eliminated their receptor-targeting capabilites [50], Hence, detailed investigation {0 understand the chemistry of protein corona on different NPs will enhance their translatory potential and DLS can be an effective tool in such studies along with techniques lke ITC (isothermal titration calorimetry), FT-IR (Fourier transform infrared spectroscopy) and fluo- rescence spectroscopy. 2.1.24. Ry (hydrodynamic radius) and R, (radius of gyration). The Ry, (hydrodynamic radius) is the radius ofthe hypothetical hard sphere ‘that diffuses with the same speed asthe particles assayed under DLS [51]. Hence, Ry isa hypothetical measurement as such hard spheres rarely exist in colloidal dispersions. In reality the dispersed particles are hydrated solvated which along with its corona are often not spher- ‘cal, The composition of the corona ~ especially the soft corona — is dy- ‘namic and fluctuates over time depending on the ionic strength, types ‘ofsmaller and bigger molecules present inthe environment and nature of solvents 52,53} Therefore DLS provides only an indicativesize ofthe colloid. The R, (radius of gyration) isthe massaverage distance from the center of mass to every atom within the molecule (protein) or NP. For smaller NPs exhibiting Rayleigh scattering the R, is measured by SSAXS (small angle X-ray scattering) [54] while static light scattering, (SIS) is used for bigger particles showing anisotropic Mie scattering Digtalsignel Fle 4 Schema stowing the nsrameatin of IS [55], The Ryu ratio provides insights into the compactness and shape of the dispersed particles (-0.78 for spherical NPs, 15-21 for cols and >2 for nanotubes) [56:57 22, Instrumentation and technical aspects of DLS ‘Arange of ligt scattering instruments [e.g, Malvern (Zetasizer® se- res), Brookhaven (NanoDLS® series), Microtrac (Wave I1® series)] hhave appeated in recent years. The Malvern Zetasizen® series of instru- ‘ments are widely popular within university graduates and has emerged as gradual evolution ofthe original Malvern Correlator® marketed in 1970, Overall these instruments have three major components ~ laser, sample and light detector (Fig. 4). 221. Laser ‘The laser used in Malvern Zetasizers® is 4 mW He—Ne laser cof 633 nm wavelength with exceptions of Zetasizer® APS and Zetasizer® WV where 60 mW diode lasers (830 rim) [58] are used. ‘Malvern also supplies DLS instruments with other wavelengths eg, 532 nm (green). The NanoDLS® series cartes laser of 638 nm wave- length [58]. The laser sources provide witha stable beam of coherent ‘monochromatic light. There is an attenuator available to alter the power of laser. 222.Sample ‘lean and square cuvettes made of scratch-fee glass or optically translucent disposable plastic (3 = 3 mm, 5 x 5 mm or 10 x 10 mm) are used, Plastic cuvettes with inbuilt electrodes capable of both DLS and 2? measurements (DTS1070 compatible with Zetasizer® 25, Zand 590) are also available. The sample should be clean, homogeneous and transparent without any precipitation. The minimal volume ofsam- ple required varies with the model (12 pl for Zetasizer® S, 2 i for Zetasizer® pv), However, at least 1-2 ml of sample should be prepared inorder to obtain good quality data 223. Detector ‘Modern DLS instruments are equipped with APD (avalanche photo, diode) detectors which have -65% quantum efficiency in red wave- lengths and thus, lasers of 633 nm are used. In latest instruments the de- tectors are placed at 173° angle to detect backscattering although in some olde versions (Nano $90, 2590) the angle is stil 90". Placing de- tectors at 173” enables detection of backscattering and excludes excess scattered light, This helps to unmask seattered light signals of lowEr ‘hack oul of Controle Ree 25 (206) 27-251 Intensity originating from smaller particles. It also increases (-8 folds) the area of the illuminated sample within cuvette compared to 90° arrangement (60), In such NIBS (non-invasive backscatter system) at- rangements focusing ens is available to alter the path length of the scattered light before reaching the detector by selecting the illuminated area ofthe sample to be either in the middle (fr dilute samples) or close tothe wall (for highly concentrated samples) of the cuvettes, iF necessary. 224. Operating software interface The current softwares (eg, Zetasizer®) provide users with possbi ity 1o design custom SOPs (standard operating protacols) by offering a seties of options. The interface allows the usr to insert data or both sl- vents (eg. solvent name, viscosity) and the materials (es Rl. absorp- tion). The data for most commonly used dispersants (e.g. water, toluene) and materials (eg, polystyrene, protein) are present by default inthe latest versions while online resourcesare available (61 | for details ‘on other solvents and materials. This information becomes particularly important during anisotropic Mie scattering. Inputs on experimental conditions (eg. temperature, equilibration time) are also required. Once generated, the SOPs can then be run on further samples without ‘modifications. The Zetasizer® shows the size distribution data in the ‘main tab while the second tab shows the ACF (Gi(st)-1) andthe inten- sity of fluctuating scattered light (ps) overtime. The third tab is most- ly for guidance which systematically tabulates all currently running experiments and shows the quality of data. Upon completion of exper- ments the data are stored in the retrievable database. Typically, both 2- average size and siz distribution with PDI (polydispersity index) over intensity, volume and number are provided. The size distribution data is available both as line plots or histograms and can be exported with softwares feely available to download from Malvern online resources 225. Data fting algorithms and analysis In DIS the ACF of the scattered light is fitted with two different ‘mathematical algorithms: 4) In cumulant method the intial part (up to 10%) of the ACF s fitted into a single exponential decay where the frst and the second ‘cumulant term provide wit the 2-average size and PDI, respectively {62}. Therefore, z-average size always provides with single value for every sample. The cumulant method is less vulnerable to noise than other algorithms. However, it is unsuitable for heteroge- neous polydisperse samples and in cases may be misleading (Suppl ‘Matetial 1), ) CONTIN algorithm is preferred for polydisperse and heterogeneous samples where cumulant fitting is unsuitable [63] Here, the correla tion function is fitted against longer periods of time and provides, size distribution analysis with average size and width for every peak. For perfectly monodisperse samples both these algorithms should, produce same results, However, in realty samples are rarely monodis: perse and hence the results obtained through these two algorithms di fer. The PD for DLS typically depicts the intensity of light scattered by various fractions of the particles differing in ther sizes and is calculated by (widthvmean)? for each peak. While PDI of <0.1 i considered to be highly monodisperse values of 01-0.4 and >0.4 are considered to be ‘moderately and highly poiydisperse, respectively. 23, Factor that inluence DLS results 23.1. Sample preparation ‘Sample preparation is crucial in DLS measurements. The samples are prepared either solvents (eg. water, methanol, ethanol, toluene) [64] or diluents eg. 10% methanol in water) [65].Some solvents (eg. tolu- ene) scatter ight [65] which interferes as background noise while some (eg. DMSO) shows considerable changes in viscosity with variation in {emperature [67] The samples for DLS measurements should be clea, hhomogencous and without haze. Checking the bottom ofthe cuvette for precipitation is useful. Any precipitation confirms the presence of bigger particles which can be due to poor dispersion, wrong pH, inadequate sonication and compromises the experiment. Using DI (deionized) water is usually not recommended as the absence of ions fails to shield the long-distance interactions between particles. Hence, the size obtained in DI water is always 2-10 nm larger than actual size [68 In few circumstances (eg, PEC) using dilute saline water gives bet- ter data asthe ions shield the particles from long-distance interactions. Using 10 mMKNO; isbetter compared to NaCl as choride ions are high- ly reactive. Filtering samples to exclude dust particles or lumpsis helpful although can precuce artificial narrow size distribution. Using properly ‘washed flters of pore sizes three times bigger than (eg. 5 wm) the ex- pected largest particles within samples can be done. Large particles of ow density may float on top of the solvent layer (creaming) [69] and ‘an render DLS ineffective. For powder formulations (eg, freeze dried procicts) string vigorously can dissolve the NPs. Sonication deserves ‘aution especially when proteins are involved. For polymeric NPs it ‘am take up to 24h by sonication to obtain a stable and homogeneous dispersion 2.32. Sample concentration Higher concentration of NPs results in multi-scatering where the scattered light from one particle interacts with other particles before reaching detector an oses intensity [70] Asa result the obtained size isartificialy smaller, Unpredictable agglomeration happensin high con- ‘centrations unless surfactants are used [71] On the contrary, using (00 dliluce samples may not generate enough scattered light to analyze. ‘Therefore, finding optimal sample concentration is essential It is difi- ‘ult to provide a general guideline on the ideal concentration for DLS sit varies and depends upon factors related to both the instrument (eg. scattering volume, angle of scattering, laser power, detector sensi- tivity) [72] and material properties (e.g, molecular weight, compact- ness) [73] ofthe particles. For example. NPs that are spherical and ‘more compact scatter more light than NPs that are less compact [74 ‘The Malvern Zetasizer® manual states that atleast one milion (10°) re sidual photons should be detected during the experiment to acquire ‘good quality data with high signal-to-noise (S/N) ratio where residual photons mean the difference between scattered photons from the sam- ple and solvent. The user manual of Brookhaven Instruments suggests a sample concentration able to generate counts maximum up to 600 keps although counts within 500-600 kcps work fine for most samples, The noise in DLS experiments varies inversely proportional to the square root of photons counted and hence, there is a minimal threshold for photon counts required to achieve good S/N ratio. In practice, it may bbe necessary to run DLS experiments on seval dilutions to determine the optimal concentration 23.3. Colored and fluorescent samples Ideally colored and fluorescent samples should be avoided in DLS. 175], The use of fluorescent NPs has increased as they are often coupled ‘with microscopy. It is essential to run control experiments to exclude the possibilty of absorption of ight by the fluorophores at the wave- length of laser. in case of absorption, the intensity of the scattered light will be lower and hence, artifical smaller size for the particles ‘will be estimated [76]. Fluorescent ligt is non-coherent and recorded as noise. The APD detectors are often not capable to differentiate be- tween various intensities. As a result in case af fluorescence there can be heightened noise which deteriorates S/N ratio resulting in low qual- ity data and broadening of peaks. Unfortunately, alot ofthe popular flyorophores absorb and emit within 600-700 1am whic interferes with DLS, To minimize such interferences, in some instruments a narrow band filter to sereen out wavelengths different from the laser‘ Dhauachre /Jural of Conrolel Rese 25 (2016) 357-351 28 (eg, 633 + 25 nm) is present. However, it also reduces the number of photons detected and hence, higher concentrations may be required. 234. Effect of agglomeration NPs tend to agglomerate [77] and while some of these agglomera- tions are reversible, often itis not the case, Therefore, diferent surf {ants are frequently used to produce stable dispersions [78]. It is lieu to obtain high quality data from dispersions with agglomerated [NPs asthe bigger agglomerated lumps scatter too much ight which may damage the detector, Excessive scattering also masks low intensity scattered light from smaller particles. Hence, broadened! peaks emerge hile confidence in the data is reduced. Agglomeration is enhanced by increasing concentrations and thus DLSisreliable ony at dilute concen- trations (typically 50-100 pg/ml) which makes it unsuitable for many ‘therapeutic formulations where much higher (up to few mg/ml) con- centrations are used [79] 2315. Shape of NPs Often NPs are not spherical eg..nanostars {80}, nanotubes (81). For such NPs the DLS provide a Ry which by definition isthe radius fa hy- pothetical hard sphere moving a the same speed to that ofthe aspher- ical NPs within dispersion. In a recent paper Nair eral. 82) have ‘modified the Stoks-Einstein equation to fit the data obtained from cy- lindrical structures (eg. nanotubes): 0-8 [in +03] ° ‘Where ke = Boltzmann constant (1.38064852 x 10-** ik), T= temperature, 1) = absolute viscosity, L = length of ylinder andl d = di- ameter of cylinder. The aspect ratio (L/A) is known for nanotubes and hence, the D, for such cylindrical nanomaterials can also be determined byDIs. 236, Rotational difsion of NPS In DLS typically the translational difusion coeiient (D,) is deter- ‘mined while the rotational diffusion coefficient (D,) often goes unde- tected asthe dispersed particles rotate extremely fast. However, for some particles (eg, colloidal gold, larger and cxystalline NPs) intensity peaks at smaller sizes (0-10 nm) may appear de to rotational fusion ‘of particles [83], The easiest way to identify these peaks is to compare the DIS spectra on the same sample at 90° and 173° scattering angles. Unlike translational diffusion the scatering of light due to rotation of particles is not angle-dependent and hence, no shift for peaks due to ro- {ational difusion wil be observed. On the contrary peaks due to trans- Jational diffusion shift at different scattering angles. 237. Issues related to cuvete Plastic cuvettes should be avoided for samples with organic solvents, ‘or experiments requiring temperature of 50 °C. Especially for glass cu- vettes, when the area of illumination is lose to its wall ~a part of the selected laser from the wall may be recorded by the detector as high in- tensity light. Ths is known as flaring and typically shows up as sharp ppeak at 1-10 um, To avoid flaring the area of illumination should be moved towards the middle ofthe cuvette using the focusing lens. 238. Maintenance ofthe instrament Proper maintenance of the instrument is required to obtain cons tent high quality data from DLS, The instrument should be left und {urbed for at least 30 min ater switching it on to give sufficient time {or the laser to stabilize. As per the guidelines published by the NIST (National institute of Standards and Technology) (84), the instruments should be regularly checked with reference NPs with precisely known size and very low PDI. Cytochrome C or BSA (bovine serum albumin) and latex beads of different sizes (eg, 100 nm) can be used as refer- fences fr sizes <20 nmand 220 nm, respectively. The instrument should bee able to show sizes within 2% deviation for reference samples Colloidal god ofcifferent sizes can also be used as reference mateta 24, Practicalities 2A]. Resolution of DIS ‘An inherent weakness of DLS is its low resolution [85]. For example DIS is unable to distinguish between particles of 90 and 110 am and a broad peak with high PD! will appear. In order to offer peak resolution, DIS requites particles diferent in size by atleast a factor of 3 (eg. 10 and! 30 nm, 50 and 150 nm) [86}, This i a limitation of DLS especially for polydisperse samples. To increase resolution prior size-separation ‘of paticles can be done [87]. Latest DLS instruments ean be coupled with analytical instruments which enable DLS to determine size of every Faction precisely 242. Expressing the Ry based on intensity, volume or number ‘Acommon dilemma faced by researchers is how to express the par- ticle size as most of the current DLS softwares offer options to express Particle size distribution based on intensity, number and volume. Ex- pressing the data on these three parameters often produce three differ- ent Ry and size distributions. The principles of DLS are based on ‘measuring intensity of scattered ight and hence, the representative Ry value should always be deduced on intensity measurements while the other two parameters (volume and number) should be used as supporting information only 243. Importance of feeding right information tothe software Softwares like Zetasizer® needs tobe fed with data regarding both the dispersant (e., Rl viscosity) and dispersed (e.. Rl, absorption) phases. For Rayleigh scatterers (<100 nm) this information is often ‘ot required. However, for particles >100 nim - which i often the case for nanoparticulate DDS ~ this information becomes crucial. Knowing the accurate RI and viscosity of the sample i essential in such cases and can be obtained by use of refractometer and rheometer espective- ly. [tis dificult to predict hw NPs alter the viscosity ofthe ample. Asa ‘Working guideline it can be stated that ifthe numberof NPs per uit vol- ‘ume of dispersant increases with increasing concentration then itusual- ly also increases the viscesity ofthe dispersion [88 Its crucial to know these parameters about the dispersant an instruct the software accord- ingly to avoid erroneous data. An example where DIS was run on a dis- persion of stable and highly monodisperse carboxylated latex beads (100 nm) in water (100 g/ml) at 25 °Cbut analyzed on setups for cif ferent dispersant (40% sucrose, wate, methanol and toluene) is shown in Fig. 5. Iis surprising that the z-average size ofthe particles in the same sample varied drastically from ~15 nm (40% sucrose) to =155 nm (toluene) showing how dependent DLS data are on these inputs. 244. Proper reporting of DIS data The NIST has issued the following instructions on reporting DIS data, [sh “Ata minimum, the mean z-average diameter (or radius) and mean, polydispersity inex shouldbe reported, along with thei standard devi- ‘ations based on at 3 to 10 replicate measurements. The number of rep- licate measurements should also be reported. Ifa size distribution analysis algorithm is applied, then It should be identified along with, any key parameter values used in the analysis. Other critical informa tion tha should be reported includes: particle concentration (mass or volume based), dispersion medi composition, refractive index values, {forthe particles and the dispersion medium, viscosity value for the me- dum, measurement temperature, filtration or other procedure used to remove extraneous paticulates/dust prior to analysis (including pore size and filter type), cuvette type and size (pathlength), instrument, ‘make and model, scattering angl(s), an aser wavelength. Adtional‘hack oul of Controle Ree 25 (206) 27-251 40% sucrose —water Methanol Toluene |. of 7 ‘ os my Toi seven) Solvent | Viscosity Ri zaverage | PDI (e) (om) 40% sucrose | 5.1178 14 3 0.008 water | 0.8872 1.33 7 0.008 methanol | 0.5476 1326 322 012 toluene | 0.5568 1.496 153 (0.005 Figs neosity based IS data on same 10 ym dispersion commercial aval carboxy arexbeads 100m mean sein water at 25"Cwith2etaser stare whe set ps wa ou Sens at dierent cess and (A surose water, ethanol andes) werecose,Thez-veauesue vated om ISamta 153. depending onthe save {information that can be helpful to include ina report: measured auto correlation y-intercet (amplitude), mean count rate during measure- ‘ments, duration ofa single measurement, and mean signal-to-noise ratio: ‘Therefore, extensive information on how the DLS measurements were done needs to be provided to ensure both quality and reproduc- ibility. Unfortunately, the DLS data reported in drug, delivery literature rarely meets such high standards and this issue needs to be addressed urgentiy. 25. Comparison of DLS with other techniques to measure particle size 245.1. TEM (transmission electron microscopy). Different formats of EM, (lectron microscopy) [85] eg, TEM [90] are quite popular for imaging, NP, With image analysis softwares (eg, Image}®) itis now possible to ‘obtain size distributions of NPs from TEM images with information on ‘mean size, standard deviation and overall estimation of PDL. However, such information from TEM images often do not corroborate well with data obtained from DLS as the latter isan intensity-based technique [91] whereasTEM is amumber-based one [92] making them fundamen- tally different. While the samples for DLS are solvated, TEM works on dry samples under UHV (ultrahigh vacuum) conditions [93]. DLS mea- sures the Ry, of the dispersed particles whereas TEM provides the projected surface area based on how much of the incident electrons Were transmitted through the sample. Hence, the size obtained by DLS is usually bigger than TEM. An advantage with DLS is its capability to ‘measute bigger number of particles (in millions) compared to TEM (few hundreds). Therefore, DLS provides more robust data on size dis- tribution and PDI 24.52. NTA® (nanoparticle tracking analysis). With the launch of [Nanosight® series of instruments from Malvern the use of NTA® soft- ware has increased rapidly for determining particle size [94] Both these techniques determine particle size from the D, (Section 2.12.2 ofthis review) with Stokes-Einstei equation. However, the way they determine the D; is different and hence, the sizes obtained by these two techniques are often not same, While NTA® detects the D; by re- cording the mobility ofthe NPs from scattered light captured as videos through CCD cameras, DLS detects it by correlating the fluctuation ia intensity ofthe scattered light over time. To compare these two tech- niques the following point are made [39 (i) The particle density with- in samples required for DLS (10%-10""/ml) is usually higher than NTA® (107-10 ml; (i) For same sample usually the size obtained from DLs is smaller but with more error compared to NTA®; (il) NTA@ is more ‘effective than DLS in analyzing polydisperse samples. The data analysis report in NTA@ plots the sizes of the particles on 3D space compared to the 2D report in DLS. As a result, NTAW offers better resolution nd is less vulnerable than DLS to be influenced by high intensity seat- tering from bigger particles; (iv) While both techniques are capable in analyzing submicron particles, DLS is more capable to detect sizes <30 nm. Overall NTA@ offers better analytical suite for therapeutic nanoformulations dealing especially with peptides and macromole- ‘cules, However, it lacks the simplicity of DLS, is mote expensive, re- ‘quires extensive sample preparation and training. 2453, AFM (atomic force microscopy). AFM has emerged as.an effective tool to image NPs especially due tots ability to work on biological sam- ples rich in water [95:96] AFM provides precise information on particle size a shape while also able co recognize paticls of diferent sizes it 2 mixtute, However, the numberof particles analyzed by AFM is much smaller and thus DLS provides better size distribution and PDI 245 Particle sie determination by sedimentation (X-ray disc centrdtuge ‘and DCS ferential centrifugal sedimentation), Recently, determination ‘of NP size based on sedimentation technique has gained popularity (eg. encapsulated drugs, viruses, iposomes, emulsions), although the principles of such techniques were already known before. A detailed ‘liscussion and in-lepth analysis ofthese techniques fall out of the scope for this review although excellent literature material and techni- ‘al notes are available [97-99], In short, these techniques utilize high ‘centrifugal force to deposit NPs infractions based on density. The sizing ‘of the NPs is done by monitoring the deposition ofthe particles ona ro- tating isc either by X-ray absorbance (X-ray disc centrifuge) or mono- ‘chromatic light A = 400-500 nm (DCS). The mathematical operator for these techniques s the Stoke's aw (Eq. (7): yf 8l%-P1) 181) a‘ Dhauachre /Jural of Conrolel Rese 25 (2016) 357-351 a6 ‘where V = velocity (settling rate), d = diameter of particle (cm), _gravitation acceleration (981 crs) fp = particle density (g/m)). fluid ispersant density (g/ml) 7 = viscosity (poise). ‘Such particle sizing based on sedimentation techniques offer certain advantages over DLS: (i) They yield accurate and highly reproducible data with excellent (~2%) peak resolution which is not achievable ‘with DLS; (ji) These tools can operate on particles with avery broad size range (2 nm-80 jan) whereas the operational size-detection win- «dow for DLS is only 10-200 nm: i) The sizes of particles measured with these techniques are comparable to SEM/TEM data whereas for DIS itis almost always bigger than SEM/TEM: (iv) These techniques ‘offer multi-modal size determination with high-throughput modes ‘where -40 samples of 100 yd volume each can be run simultaneously, Itis crucial to realize that the sedimentation techniques determine size of NPs based on their density. Hence, DCS is unable to differentiate ‘between two different NPs as longs their density is same. For example, DCS may yield same size fr smaller solid and larger porous particles {rom same material given the particles are of same density. DCS also ‘may be difficult to perform for non-spherical particles and usvally pro- vides smaller size (Stoke's equivalent) than actual measurements. For ‘example, rods with aspect ratios of 2 and 3 are known to generate sizes which are 5% and 10% lesser than actual measurement, respec- tively. DCS i dfficlt for particles with density lower than the disper- sant as they tend to float. However, with current instrumental advancements such challenges are usually resolved. DCS however re- ‘uires bigger sample volumes (100 pl) whereas modern DLS instru- ‘ments can operate on. as low as 12 yl. Additionally, with current ‘compact instruments lke Malvern Zetasizer® both particle (hydrody- namie) size and zeta potential can be measured while DLS which is not possible in DCS. Pr 245.5, Particle size determination by laser diffraction Laser difaction isa ‘apable too to determine thesize of NPs and its core principles, ike DLS, are also based on scattering of light | 100]. Particles scatter lightin a size- ‘dependent way where bigger particles scater more intensely at smaller angles and smaller particles scater with lesser intensity at wider angles, In laser dliraction technique, the scattering of light is expressed as a function of scattering angle which in turn is used to measure particle The main differences with DLS are: (i) Laser diffraction technique determines the size ofa particle which scatters light ina similar way to that of the particles under investigation. On the contrary DS mea- sures the (hydrodynamic) radius ofa hypathetical solid particle scatter- ing light with same intensity as the particles under investigation while diffusing in the dispersion. Therefore, the results obtained through these techniques are not same and often der by 10-20% depending, (nthe experimental conditions and type of NPs (i) The lower limit of detection is smaller for DLS whereas the upper limit of detection is big- ger in laser diffraction. Therefore for smaller NPs (e., $50 nm) DLS provides better data whereas for bigger patices (eg..> 1pm) laser if faction is more suitable; (i) Much lesser volume of sample is required {or DLS (ing) whereas bigger volumes are required for laser diffaction (in mi); (iv) Laser diffraction is more suitable in samples with particu Jate impurities of larger sizes. Overall laser diffraction offer a better sizing tool for nanoformulations meant for drug delivery as they are ‘often > 100 nm. However. it doesnot offer the compact size and zeta po- tential determining experimental suite and hence, the simplicity of DLS is absent with ase diffraction technique. 2446, DIS in cel culture medkum Performing DLS in cell culture medium (eg, DMEM, RPMI) can be dificult as they contain a wide range of smaller (e.g, ions) and larger (ee. vitamins) molecules, Asa result of adsorption of these molecules, the surface properties and the sizes of the NPs change. Tis becomes particulary evident for medium containing FCS (Fetal calf serum) enriched with proteins. Adsorption of larger protein molecules can show drastic and rapid increase of Ry for the dispersed NPs 101,102}. ‘The adsorbed layers on NPs are dynamic in composition and hence, the fluctuates overtime before stabilizing. Cellculture medium with- ‘ut pH indicators (eg. phenol red) should be used as the colored mate- sialcan absorb the laser and interfere. 247. DIS on aerosols and foams "Newer colloid formulations eg. aerosols, foams are becoming popu lari drug delivery. Iti difficult to perform DLS measurements on such samples and SMPS (scanning mobility particle sizer) is the technique of choice for such preparations, However, there are reports available in i= erature where DLS was used to measure particle size for aerosols and foams. As example [103], the sizeof aerosolized carbon soot originating from burning cigarete tips was measured in a custom-built light scat- tering instrument at 90° scattering angle. Similar attempts were made by Durianet al. (1991) where light scattering technique was used to as- certain particle size as well as internal dynamics of foams [104] 3.Zeta potential (ZP) 31. Principles of The 2, also termed as electrokinetic potential, isthe potential atthe slipping/shear plane of a colloid particle moving under electric field [105]. Electric potential ofa surface isthe amount of work that needs to be done to bring a unit positive charge from infinity to the surface without any acceleration The ZP reflects the potential difference be- tween the EDL (electric double layer) of electrophoretically mobile par- ticles and the layer of dispersant around them at the sipping plane. 3.14. Understanding the EDL and sipping plane ‘When a charged particle is dispersed, an adsorbed double layer — often referred as EDL [106] - develops on its surface (Fig 6). The inner layer consists predominantly of ions/molecules with opposite charge to that ofthe particle (Ster layer. Beyond Stern layer the electrostatic effects due to the surface charge onthe particles decrease as per Debye's lay which states thac withthe distance of each Debye length the feld decreases by a factor of 1 [107] ‘Although mathematically this electrostatic effect extends til infinty, experimentally its only present till ew nm from particle surface. Due to the electrostatic field ofthe charged NPs, a diffuse layer consisting ‘of both same and opposite charged ions/molecules grow beyond the Stern layer which along with the Stern layer forms the EDL. The compo- sition ofthis difuse layer is dynamic and varies depending on a variety of factors eg. pH. ionic strength, concentration te, When an electric field is applied to such dispersion the charged particles move towards the opposite electrode (electrophoresis). Within tis difuse layer there is a hypothetical plane which acts as the interface between the moving, particles and the layer of dispersant around it while electrophoresis, ‘This plane is the characteristic slippng/shear plane and ZP is the poten- tial at this particle fluid interface. Greek letter ¢ (zeta) was used origi- nally in mathematical equations while describing it and hence, the rname zeta potential. The potential on the particle surface itself is known asthe Nernst potential (p) [108] and cannot be measured. The electrostatic field decreases in dispersion with distance from the part- cle surface as per Eg (8) w= We 6 where y= surface potential at distance x from the stem layer, surface potential at stern layer, = Debye-Huckel parameter, x distance. ‘When the slipping plane is in close proximity tothe stern layer~ the ‘a= and hence, Eq, (8) can be modified as Ea. (9): wate o6 ‘hack oul of Controle Ree 25 (206) 27-251 Electrophoresis -ve Electrode +ve Electrode Sippingplane (¥,) Particle surface (a) Stefn layer Fig. 6 Caton showing the EDL on negatively caged patie. mediately op othe partie surface there i seg adhered ae (Sem ayer) competing ons of oppaie ‘ove tonal the laces pov ectode ns as) with he ppg plane bcos neat ete the ae pares and pest. he ZP the eeeetintc tenth siping pine: ‘The « (Debye-Huckel parameter) depends on the ionic strength. Hence, with increased ionic presence (e.g. addition of electrolytes) the double layer gets compressed and the ZP decreases. 3.12. Fundamental mathematical operators wile measuring ZP 7P cannot be measured directly and is deduced from electrophoretic mobility of charged particles under an applied electric field. The electrophoretic mobility (jt) of the particles is first calculated as (&q,(10)) (Volt/em) ~ both known quantities. The ZP is then calculated from the obtained by the Henry's equation (Ea, (11)): 2e,s0if ka) a) where z= relative permittivty/dielectric constant, g = permi tivity of vacuum, ¢ = 2P,f{ka) = Henry's function and 9) = viscosity at experimental temperature ‘When the thickness of the EDLis much smaller compared tothe par- ticle radius - which can be due to bigger particles (up to 1 ym) within aqueous solutions of high sat concentration (10? M) - the value of ka) is taken as 15 and the Henry's equation then modifies into the Heimholtz-Smotuchowsh (HS) equation (Eq, (12)); feat (2) 7 aH ‘The HS equation aplies to most of the pharmaceutical preparations and hence, very important for developing nano-DDS [109 (On the contrary when the thickness of EDLs much bigger than the particle itself due to smaller («100 nm) particles dispersed in low salt ‘concentration (10M) the value of fa) istaken as 1 and the Henry's ‘equation can be modified as the Hickel equation (Eq, (13)): Devtod 5 03) “The Hickel equations usually not relevant fr pharmaceutical prep- arationsasit isnot applicable for aqueous dispersions although itis pop- ‘lar in ceramic industry. 22, Instrumentation in ZP measurements {An electric field is applied and the electrophoretic mobility of the particles is measured inthe following two ways: 4) Electrophoretic light scattering: The mobile particles during electro- phoresis scatter an incident laser. As the particles are mobile the catered light has diferent frequency than the orginal laser and the frequency shift is proportional tothe speed ofthe particles (Doppler shift), The instrumentation used for this technique is shown in Fig 7. n short the laser beam is split into two and while one beam, is directed towards the sample the other one is used as reference ‘beam, The scattered light From the sample is combined or optically ‘mixed with the reference beam to determine the Doppler shift. The ‘magnitude of particle velocity (V) is deduced from the Doppler shift, and then the 2P is measured through the series of mathematical equa- tions enlisted as Eqs (10)-(13). This technique is often used in con- junction with DLS and hence, a range of instruments (e., Malvern Zetasizer®) providing integrated measurement suite for both DLS, and 2P have emerged which are popular within university graduates, and nanoformulation groups. The Zetasizer® series of instruments, Use sophisticated laser interferomettic M3-PALS (phase analysis, light scattering) technique {110] for such applications. Disposable plastic (polycarbonate) cuvetes with inbuilt gold plated copper ele ‘wodes and bent capillary tube to hold 0.75 ral of sample are available to conduct both DLS and ZP measurements a a single run Like DLS. the Zetsizer® software interface allows the user to develop custom- {zed SOPs an insert relevant information,Dhak Jura of Conroe Rese 25 (2016) 357-351 Beam Attenuator Sample Digital signal processor (correlator) | Combining optics Compensation optics 6 ig 2. Schematic showing the trenton 2 meus by ectophoeic ght etering b) Electroacoustic phenomenon: In this technique an electric field of high frequency is applied which makes the particles in sample oscillate ‘while the oscillation depends on their size and ZP. The oscillation is analyeed on magnitude and phase angle to determine both the pa le size and ZP 111] This technique is less popular in drug delivery research, 33. Interpretation of ZP data 3.1 Factors influencing 2P 33.11 pH pis perhaps the most influential parameter in ZP measure- ‘ments especially in aqueous dispersions which makes it relevant for pharmaceutical formulations. The ZP varies with pH and becomes ‘more positive and negative in magnitude with acidic and basic pH, re- spectively [112]. Therefore, titration curve of ZP agains different pH values is often generated which helps to determine the isoelectric point ie. the pH where the ZP becomes zea [113]. For aqueous disper- sions - where H' and OH are major ionic constituent - the isoelectric point also denotes the P2C (point of zero charge) [114]. Colloids lose sta- bility and agglomerate/Macculate when the pH is close to the isoelectric point. 33.12. lonie strength. With increasing ionic strength the EDL becomes _more compressed while the ZP decreases and vice versa, The valency ‘ofthe ions is also important while measuring ZP. For ions with higher valency (eg. Ca, AP" having higher valency than monovalent Na 1H", OF) the EDL becomes more compact and the ZP decreases in magnitude, 43.1.3. Concentration. The relation between ZP and particle concent tion is complex and usually determined by both surface adsorption and the effect of EDL Ii difficult to provide with a general guideline ‘n effect of concentration on ZP. However, it can be stated that overall in dilute conditions the surface adsorption phenomenon dominates and hence, the ZP increases with concentration, However, at higher concentration range the thickness of EDL dominates and then by in- ‘teasing concentration an opposite effect ie. dectease in ZP with lesser stability ofthe dispersion is observed [115). 332, ZP and colloid stability ‘One ofthe most popular uses ofP data isto relat it with colloid sta- bility. Guidelines classifying NP-dispersions with ZP values of 10 mV, + 10-20 mV and +20-30 mV and" + 30 mV as highly unstable, relatively stable, moderately stable and highly stable, respectively are common in drug delivery literature [116]. Unfortunately, the reality is ‘more complex than that. Although ZP does provide indications on col- loid stability it does not reflect the entire picture. As per the most widely accepted DLVO (named after inventors Derjagun, Landau, Verwey and Overbeek) theory colloid stability depends on the sum of van der Waals attractive forces and electrostatic repulsive forces due to the EDL| 1171 While 2? provides information on the electrostatic repulsive forces it does not provide any insight onthe attractive van der Waals forces. Therefore, itis nt uncommon to come across stable colloids with low 2P and vice versa. There is plenty of theory involved in undes- standing such attractive forces in nature lke van det Waals which alls beyond the scope of this review. One important point to note is that the van der Waals attractive force is dependent on the Hamaker constant [118] which indirect corresponds to the difference between the RI of the particle and the dispersant. Therefore, ifthe Hamaker constant is low the van der Waals attractive forces also become weak and then ‘mild electrostatic repulsion reflected by low ZP (eg. 10-15 mV) may be enough to ensure colloid stability. Materials like colloidal silica shows exceptional stability at very low ZP (119. It should be noted that steric interactions can also contribute to colloid stability. Fr exam- ple some water-in-oil emulsions are highly stable despite having low 2? [120], PEGylation is also known to facilitate stability of NPs while de- (creasing the ZP [121]. 3.33. ZP and surface charge of NPs ‘Another widely popular use of 2s to use it in assessing the surface charge of NPs. The positive or negative dimensions ofZP are determined by identifying which electrode the particles are moving towards during electrophoresis. It should be noted that ZP never measures charge oFue ‘hack oul of Controle Ree 25 (206) 27-251 charge density and rather deals with surface potential, Thevefore, only tHe magnitude of2P is important while the positive/negative finding as- sociated with it not robust and should not be related with surface charge or charge density or making comparisons between different nnanoformulations. A stated in previous sections ofthis review, a wide varity of factors (e., pH which is relevant for nanoformulations) can change it from +ve t -ve and vice versa. P only provides with indic- ative evidence towards the nature of surlace charge (positive/negative) assuming that the predominant ions inthe EDL up tothe slipping plane ate similar (positivenegative) compared tothe surface ofthe particle it- self Unfortunately, there are too many exceptions to such assumption. ‘The practical way to confirm the nature (positive/negative) as well a, to determine charge density on NPs ito titrate it with known armounts of fons. A detailed description of such titration technique falls beyond the scope ofthis article although excellent reference literature is avail able (122,123), CCharge on particle-clispersant interface (eg. slipping plane) is a complicated and less understood phenomenon. Usually, almost all of the naturally occurring surfaces and molecules exhibit negative charge (eg, cell membrane, proteins lipids, mucus etc). On the contrary cat- ‘onic surfaces and molecules are often synthetic Iti also inappropriate to claim having “neutral” NPs based on 2 as there are never neutral NPs in dispersion due to inevitable charge build up on their surfaces An in- teresting factis that surface charge on NPs can actualy vary depending, ‘onthe dlfferent phases within the colloid. As per Cohen's rule ifboth sol- ‘ute and solvent are insulators then the one with higher relative permit- tivity (e, becomes positiveat the interface. Hence, in room temperature silica (c; = 39) NPs are negatively charged in water (e; = 80) but pos- itvely charged in benzene (¢, = 2.27) 34, Practicalities 3.1 Reference materials Unlike DLS there are as such no reference or standard material for 2 whic is inconvenient in practical terms, The NIST has suggested the use of Goethite (ct-FeO(OH)) which upon preparation under speci- fied conditions should deliver ZPof (32.5012) mV [124]. Howev- ex, the samples need tobe made fresh every time and it may foul the cuvette. Sight variation in data based on instruments may lo occur. 3.42. Reusing samples after measuring ZP Electrophoresis may degrade some NPs and hence, may render the samples unsuitable for reuse after measuring ZP. Asa common guide- line it can be stated that if possible reusing samples for experiments after measuring 2P should be avoided, IFthat is not the case (eg. due to small volume of sample) then adequate re-characterization of the particles including DLS and gel electrophoresis should be done after ‘measuring ZP to exclude any degradation ofthe particles under applied voltage 3.43. Using bufes with metalic ions ‘The electrodes i cells for 2P measurements are prone to react espe cially wth metallic fons (eg. Fe®*) [125]. Such reactions can destroy the electrode and compromise the quality of data. Therefore, regular checking ofthe electrode is advisable. In case the contact between the electrodes and the ions cannot be prevented in spite of deleterious reac- tions then diusion barrier method [126] can be used where the electro- phoretic mobility ofthe particles can stil be measured while preventing, any contact between electrode and the buffer. However, it requires ad- Aitional expertise. 3.4.4, Measuring P in cel culture medium can be challenging to measure Pin cell culture medium. Enriched with plenty of ions, the cel culture mediums have very high conductiv- ity and interfere with ZP measurements, Such high conductivity can ‘generate enough heat under constant voltage which may degrade the sample. Using higher concentrations of NPs (5-10 mg/ml) under low ‘voltage can be helpful to obtain a stable 2P reading. Unfortunately, ic becomes much more complex in cel culture medium carrying FCS ‘which contains plenty of proteins. The available protein molecules ‘get adsorbed on the NP-suffaces and influence both the dispersion ‘and ZP [127], The protein molecules also sometimes make small rnanoagglomerates which interfere with the readings and can generate ‘additional aberrant peaks. 4. Discussion ‘The fundamental principles of both DLS and ZP are rooted within the realms of physical colloid chemistry and its essential to have strong ‘rip over the care physical and mathematical principles in order to un- ‘derstand their applied aspects. The basics of DLS and ZP are taught al- ready to the undergraduate students in physical chemistry in most universities. Unfortunately similar training and structured grooming process for young researchers performing DLS and 2P are often missing in drug delivery research groups. This gap in knowledge as well as lack ‘of proper training needs to be addressed. Both DLS and ZP measurements are based on light scattering and hhence, only clear samples can be subjected to these two techniques. Ad- 200 nm present in it Asa result the prepared nanoformulation samples are often neither ‘lear nor dilute enough to be fit for DLS and ZP measurements. I¢ reeds to be emphasized that DLS and ZP have their own shares of lini- tations and their inability to handle high concentrations is a major ‘weakness of both these techniques. Surface chemistry is important in ‘measuring the DLS and ZP anc! any change in surfaces ofthe particles will alter the results, n therapeutically relevant samples with high con- ‘centration containing complex engineered nanoconstrucs - plenty of parameters (eg. viscosity, pH dielectric constant, RI etc.) change and hence, the dilute samples used for DLS and ZP measurements are never an adequate representation ofthe therapeutic formulations to be used in vivo. Hence, results from DIS and ZP should not provide ‘grounds fr rushing nanoformulations fr in vivo studies. The DLS and ZP are run mostly on aqueous dispersants with known ionic strength, {and pil under controlled laboratory environments that are hardly com= parable to in vivo circumstances where the dispersion medium often is blood with a complex matrix. The DIS and ZP neither operate in blood ‘hor can predict the behavior of NPs in blood. These two techniques ‘were originally developed for protein dispersions and although they ‘work fine with engineered NPs within certain operational conditions, the scope of them in characterizing nanoformulations in vivo is limited. ‘Unfortunately, at this stage the lack of adequate analytical tools ca- pable to handle complex biological matrices (eg, blood) is missing ‘and the focus of ongoing research work should try to address this issue urgently in order to facilitate translation In ast few years a wide range of nanoformulations have emerged although thei translational impact overall has been disappointing with poor in vitro ~ in vivo corre- lation [128-130]. There are challenges in characterization of NPs under physiologically relevant concitions. DIS and ZP are excellent tools to ‘characterize NPs at ther intial stages of development, However, the Scopes for these two techiiques become increasingly limited in futher phases of nanoformulation preparation orto provide sound data for in vivo correlation, Confit of interest ‘The author would like to declare no conflict of interest.Dhak Jura of Conroe Rese 25 (2016) 357-351 20 Acknowledgements Punding and acces to scientific literature was provided by UCD. The author would lke to thank his colleagues and students for innumerable discussions which laid the foundation of this review. Dr. Dermot Brougham and Dr. Delyan Hrstov from UCD Schoo! of Chemistry are ‘thanked for stimulating discussions and providing latex beads, respectively Appendix A. Supplementary data ‘Supplementary daa to this article can be found online at hitp//dx. doiorg/10.1016)}jconrel 201606017. References (0) tree, 1 sthion D. Dots, T-Lang. 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