Field-flow Fractionation Research Articles

Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Flow field-flow fractionation and single particle inductively coupled plasma mass spectrometry as a powerful tool for tracking and understanding the sensing mechanism of Ag–Au bimetallic nanoparticles toward cobalt ions

BackgroundAg–Au bimetallic nanoparticles (BNPs), synthesized by using citrate reduction of Ag and Au ions, were used as sensor for detection of Co2+. In order to optimize sensing performance, it is necessary to control the particle size and size distribution of the original Ag–Au BNPs. Therefore, analytical methods based on the use of single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) and flow-field flow fractionation (FlFFF)-ICP-MS were developed to track the signal of Ag and Au in bimetallic nanoparticles at each step of the procedure: BNP synthesis, aggregation and sensing in order to understand the sensing mechanism. To better understand colorimetric sensing of Co2+ using Ag–Au BNPs, various solution mixtures were analyzed by using SP-ICP-MS and FlFFF-ICP-MS. ResultsSP-ICP-MS provided the information on the core size, size distribution and particle number concentration, as well as the heterogeneity of the particles synthesized by using various citrate concentrations and metal ratios. FlFFF-ICP-MS offered the information on hydrodynamic size as well as the signal intensity ratio of Ag and Au in BNPs and for the understanding of the aggregation of BNPs arising from the [Co(II)(en)3]2+ complex surrounding the surface of the BNPs. Under optimum sensing condition, the use of SP-ICP-MS for BNPs assisted detection of Co2+ improved the sensitivity of Co2+ determination by 20-fold in comparison with the conventional spectrophotometric analysis. SignificanceThe information obtained from SP-ICP-MS and FlFFF-ICP-MS can be combinedly used to understand sensing mechanism and to select the best condition for synthesis of BNPs used as sensor. This study illustrates the usefulness of SP-ICP-MS and FlFFF-ICP-MS in the nanoparticle-based sensor development research area.

Focusing Mechanisms in Micro-thermal Field-Flow Fractionation

Focusing Mechanisms in Micro-thermal Field-Flow Fractionation

Colloids facilitate transport of cadmium and uranium in arable soils, which is undetected by suction cups in the field

AbstractColloids can facilitate the transport of trace elements in soil, but earlier studies fell short either because of sampling artefacts or because no contrasting soils were used; both factors hamper a better understanding of where this occurs. Here, leaching in undisturbed and unsaturated soil columns was observed to identify colloidal transport of cadmium (Cd) and uranium (U) in soils with contrasting properties. Crucially, it was examined how such column data relate to more traditional in situ data obtained with porous suction cups. Soils were collected from three agricultural fields in Switzerland (soil pH 5.1–7.3), leached during 36 days (0.6 pore volumes, ~1.6 mm day−1), and elements trapped in the PTFE suction plates were recovered and were ascribed to colloids. Of the total mobile mass (sum percolate and plate), 18%–53% (Cd) or 8–89% (U) was retained by plates, the range depending on the field where soil was sampled and that fraction depends on soil pH and for U also on dissolved inorganic carbon. The total mobile Cd or U mass divided by the percolated volume indicates the metal concentration in the average leachate and was logically highest for Cd in the most acidic soil and for U in the most alkaline soil. The concentrations of Cd and U in these column leachates were >10‐fold higher than corresponding concentrations in the suction cups in the field for those soils and elements where colloids dominated leaching. In contrast, they were within a factor of two where colloids are <20% of the mobile mass. Flow Field‐Flow Fractionation (FlFFF) analyses showed an association of colloidal U with oxyhydroxides, clays and organic matter and, for colloidal Cd, an association with organic matter. These data show that suction cups or filtered percolates can underestimate the leaching of Cd and U by up to an order of magnitude.

Pilot plant approach combining photocatalysis and adsorption for antibiotics removal from slaughterhouse and urban wastewater treatment plant effluents

The main objective of this research is to perform a real case-study of antibiotic decontamination from real slaughterhouse and wastewater effluents by applying a sequential treatment of TiO2/UV–vis photocatalysis and adsorption. More precisely, the removal of five classes of antibiotics: enrofloxacin, sulfadiazine, trimethoprim, azithromycin, amoxicillin and amoxicillin degradation products was studied in an 80 L/h photocatalytic and adsorption pilot-scale plant over multiple cycles, operating in semi-continuous mode. The results exhibited significant removal rates, ranging from 77% to 100% for the slaughterhouse effluent, and 61–89% for the wastewater treatment plant effluent, thus demonstrating that the treatment is more effective when it is applied directly in the emission source, previous to antibiotic dilution in the municipal collector. Using the two processes sequentially results in greater efficiency than when they are used in isolation. After photocatalysis, antibiotic degradation products are easily adsorbed, since they have more affinity for the adsorbent, and the presence of competing compounds decreases considerably. After applying several cycles of treatment, the adsorption performance remains almost constant. By contrast, the photocatalysis performance decreased, which was attributed to catalyst agglomeration determined by Asymmetric Flow Field-Flow Fractionation (AF4) coupled with Dynamic Light Scattering (DLS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts

ABSTRACT The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

Soil colloids as binding agents in the formation of soil microaggregates in wet-dry cycles: A case study for arable Luvisols under different management

In the hierarchical model of soil aggregates, small soil microaggregates (small SMA; <20 μm) are often considered to be fundamental building units at the micron scale. Below which, soil colloids (<1 µm) have recently been proposed as binding agents of (micro)aggregates. However, the way in which soil colloids contribute to the formation and stability of soil micro- and macroaggregates remains largely unknown. For clarification, we evaluated potential impacts of the colloidal content, particularly the <450 nm colloids, on the aggregation of small SMA. Free water stable small SMA and <450 nm colloids were isolated from Ap-horizons of Stagnic Luvisols under different management (cropped and bare fallow). The size-resolved elemental composition of the <450 nm colloids was analyzed by asymmetric flow field-flow fractionation in combination with an inductively coupled plasma mass spectrometer and an organic carbon detector. To vary the colloidal content in small SMA, (1) suspensions containing different amounts of <450 nm colloids were added in small SMA, or (2) <1 µm colloids were removed from small SMA by centrifugation. In the maximum colloidal addition treatment, the mass ratios of added colloids to small SMA were 3.0 and 5.1 wt% for the cropped and bare fallow soil samples, respectively. Aggregation of small SMA with different colloidal amounts was performed in three successive wet-dry cycles. Afterwards, the size distribution of the resulting aggregates was measured by laser diffraction. Our results indicated that, in wet-dry cycles, colloids were important binding agents for the formation of SMA. Their presence, especially those <450 nm, was likely to support the formation of solid bridges during drying at particle contacts of 1–10 µm small SMA, favoring hereby SMA build-up in a relatively small size range of 1–40 µm. In contrast, the absence of <1 μm colloids in small SMA led to a preferential generation of relatively large aggregates in wet-dry cycles, i.e., typically with sizes >40 μm up to 1700 μm in maximum. Our study on aggregation in wet-dry cycles revealed that the colloidal content has a controlling effect on the size distribution of resulting aggregates by acting as a binding agent and provides hereby new insights into the evolvement of aggregate hierarchy in soils.

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Characterization of molar mass and conformation of relevant (non-)starch polysaccharides in cereal-based beverages

Arabinoxylans, β-glucans, and dextrins influence the brewing industry's filtration process and product quality. Despite their relevance, only a maximum concentration of β-glucans is recommended. Nevertheless, filtration problems are still present, indicating that although the chemical concentration is essential, other parameters should be investigated. Molar mass and conformation are important polymer physical characteristics often neglected in this industry. Therefore, this research proposes an approach to physically characterize enzymatically isolated beer polysaccharides by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering and differential refractive index detector. Based on the obtained molar masses, root-mean-square radius (rrms from MALS), and hydrodynamic radius (rhyd), conformational properties such as apparent density (ρapp) and rrms/rhyd can be calculated based on their molar mass and size. Consequently, the ρapp and rrms/rhyd behavior hints at the different structures within each polysaccharide. The rrms/rhyd 1.2 and high ρapp values on low molar mass dextrins (1–2·105 g/mol) indicate branches, while aggregated structures at high molar masses on arabinoxylans and β-glucans (2·105 –6·106 g/mol) are due to an increase of ρapp and a rrms/rhyd (0.6–1). This methodology provides a new perspective to analyze starch and non-starch polysaccharides in cereal-based beverages since different physical characteristics could influence beer's filtration and sensory characteristics.

Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Copper Speciation in Wine Growing-Drain Waters: Mobilization, Transport, and Environmental Diffusion

Copper (Cu) has been used to treat vines for a long time, which has led to its accumulation in vineyard soils. In the present work, the mobilization of copper from these soils and its transport, and diffusion outside the plots by drain water were investigated. For this, the distribution of copper between the dissolved and colloidal phases, and within the colloidal phase, of these waters was determined using an investigation strategy based on the coupling between a size separation technique, asymmetric flow field-flow fractionation, and several detectors. First, the total copper concentrations in water from different drains were monitored over a period of 2 years: Cu was mainly found in the fraction of &lt; 450 nm. Then, the distribution of copper on the size continuum was more closely studied in water from one of the drains, sampled over a winter period. Between 45 and 75% of Cu was found in the 2–450 nm colloidal fraction. The &lt;450 nm colloidal phase of the drain waters was found to be mainly composed of humic acids (~15 to 60 mg L−1) and clay-rich particles (~100 to 650 mg (Al) L−1). These particles also contained (hydr)oxides of iron and manganese. The concentrations of Fe and Mn were approximately 100 to 200 times lower than those of Al. The majority of humic acids had an apparent molar mass of ≤ 10 kDa. They were distributed along the size continuum: (i) in a population with an average size of ~20 nm, probably consisting of supramolecular entities, and (ii) associated with clay-rich particles with a size of ~120–200 nm. Copper was found to be complexed with humic acids and associated with clays via clay-humic complexes. Copper mobilization from the soil to the water and its transport to the drain water appeared governed by the soil humidity level and the rainfall.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10nm SiNPs significantly contributed to the production of reactive oxygen species.

Immunopotentiating Polyphosphazene Delivery Systems: Supramolecular Self-Assembly and Stability in the Presence of Plasma Proteins.

Studies on the biological performance of nanomedicines have been increasingly focused on the paradigm shifting role of the protein corona, which is imminently formed once the formulation is placed in a complex physiological environment. This phenomenon is predominantly studied in the context of protein adsorption science, while such interactions for water-soluble systems remain virtually unexplored. In particular, the importance of plasma protein binding is yet to be understood for pharmaceuticals designed on the basis of supramolecular architectures, which generally lack well-defined surfaces. Water-soluble ionic polyphosphazenes, clinically proven immunoadjuvants and vaccine delivery vehicles, represent an example of a system that requires supramolecular coassembly with antigenic proteins to attain an optimal immunopotentiating effect. Herein, the self-assembly behavior and stability of noncovalently bound complexes on the basis of a model antigen─hen egg lysozyme─and polyphosphazene adjuvant are studied in the presence of plasma proteins utilizing isothermal calorimetry, asymmetric flow field flow fractionation, dynamic light scattering, and size exclusion chromatography methods. The results demonstrate that although plasma proteins, such as human serum albumin (HSA), show detectable avidity to polyphosphazene, the strength of such interactions is significantly lower than that for the model antigen. Furthermore, thermodynamic parameters indicate different models of binding: entropy driven, which is consistent with the counterion release mechanism for albumin versus electrostatic interactions for lysozyme, which are characterized by beneficial enthalpy changes. In vitro protein release experiments conducted in Franz diffusion cells using enzyme-linked immunoassay detection suggest that the antigen-adjuvant complex stability is not adversely affected by the presence of the most physiologically abundant protein, which confirms the importance of the delivery modality for this immunoadjuvant. Moreover, HSA shows an unexpected stabilizing effect on complexes with high antigen load─an important consideration for further development of polyphosphazene adjuvanted vaccine formulations and their functional assessment.

Determination and Characterization of Gold Nanoparticles in Liquor Using Asymmetric Flow Field-Flow Fractionation Hyphenated with Inductively Coupled Plasma Mass Spectrometry.

The EU has approved the usage of gold as a food additive (E175) and it has been applied in numerous foods for coloring and decoration purposes. Different from the general assumption that edible gold is mainly present in the form of flakes or external coating in foods, this work demonstrated that gold nanoparticles (Au NPs) can be released from gold flakes and extracted under optimized conditions. To support future risk assessment associated with the exposure of Au NPs to human health, an effective approach was established in this study for both size characterization and mass determination of Au NPs released in a commercial gold-containing liquor using Asymmetric Flow Field-flow Fractionation (AF4) hyphenated with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Our results showed that no Au NPs were detected in the original liquor product and only after ultrasonication for several minutes did Au NPs occur in the ultrasound-treated liquor. Particularly, Au NPs released in the liquor can be well extracted after 100-fold enrichment of gold flakes and the subsequent ultrasonication for 25 min. Size characterization of Au NPs was conducted by AF4-ICP-MS under calibration with Au NP standards. The gold particle sizes detected ranged from 8.3-398.0 nm and the dominant size of the released Au NPs was around 123.7 nm in the processed liquor. The mass concentration of gold particles determined in the liquor sample with gold flakes concentrated and subsequently sonicated was 48.1 μg L-1 by pre-channel calibration and the overall detection recoveries ranged over 82-95%. For the comparison control samples without ultrasonication, there was no detection of Au NPs. The established method was demonstrated to be useful for monitoring Au NPs in liquor and is possibly applied to other similar foodstuffs.

The dual nature of biomimetic melanin.

Melanin-inspired nanomaterials offer unique photophysical, electronic and radical scavenging properties that are widely explored for health and environmental preservation, or energy conversion and storage. The incorporation of functional melanin building blocks in more complex nanostructures or surfaces is typically achieved via a bottom-up approach starting from a molecular precursor, in most cases dopamine. Here we demonstrate that indeed, the oxidative polymerization of dopamine, for the synthesis of melanin-like polydopamine (PDA), leads to the simultaneous formation of more than one nanosized species with different compositions, morphologies and properties. In particular, a low-density polymeric structure and dense nanoparticles (NP) are simultaneously formed. The two populations could be separated and analyzed in real time using a chromatographic technique free of any stationary phase (flow field fractionation, FFF). The results following the synthesis of melanin-like PDA showed that the NP are formed only during the first 6 hours as a result of a supramolecular self-assembly-driven polymerization, while the formation of the polymer continues for about 36 hours. The two populations were also separated and characterized using TEM, UV-vis absorption spectroscopy, fluorescence and light scattering spectroscopy, DLS, FTIR, ζ-potential measurements, gel electrophoresis and pH titrations. Interestingly, very different properties between the two populations were observed: in particular the polymer contains a higher number of catechol units (8 mmol g-1 -OH) with respect to the NP (1 mmol g-1 -OH) and presents a much higher antioxidant activity. The attenuation of light by NP is more efficient than that by the polymer especially in the Vis-NIR region. Moreover, while the NP scatter light with an efficiency up to 27% they are not fluorescent, and the polymer does not scatter light but shows an excitation wavelength-dependent fluorescence typical of multi-fluorophoric uncoupled systems.

Evaluation of Drug Release from Polymeric Nanoparticles in Simulated Saliva and Gastric Media by Asymmetric Flow Field–Flow Fractionation (AF4)

Nanocarriers for oral drug delivery will encounter various biochemical environments throughout the digestive tract, which could induce different drug release behaviors. Conventional drug release assays can provide total drug release...

An in-depth physicochemical investigation of drug-loaded core-shell UiO66 nanoMOFs.

Nanosized UiO66 are among the most studied MOF materials. They have been extensively applied in various areas, such as catalysis, gas absorption, electrochemistry, chemical sensing, and biomedical applications. However, the preparation of stable nano-sized UiO66 for drug delivery applications is challenging because of the high tendency of UiO66 to aggregate during storage. To address this issue, we coated UiO66 with oligomers made of crosslinked cyclodextrins. The coated UiO66 exhibited a good stability upon storage for more than three weeks, even for low quantities of coating materials. The resulting core-shell UiO66 were characterized using a set of complementary methods including microscopies, spectroscopies, X-ray diffraction, and thermogravimetric investigations. Size distribution was assessed by orthogonal methods. Cisplatin was loaded in the core-shell nanoparticles, followed by an in-depth analysis by asymmetric flow field-flow fractionation (AF4) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS). This method combines the extremely high elemental selectivity and ultratrace detection limits of mass spectrometry with the capacity of AF4 to differentiate the diverse populations present in the sample. Free cisplatin and UiO66-associated cisplatin could be well separated by AF4. AF4-ICP-MS/MS analysis provided the exact drug loading, without the need of separating the nanoparticles from their suspension media. These data suggest the potential of AF4-ICP-MS/MS in the optimization of drug delivery systems.

Correction to: Nanoparticles separation by different conditions at asymmetric flow field-flow fractionation

Correction to: Nanoparticles separation by different conditions at asymmetric flow field-flow fractionation

Assessment of Protein Binding Using Asymmetric-Flow Field-Flow Fractionation Combined with Multi-angle Light Scattering and Dynamic Light Scattering.

Asymmetric-flow field-flow fractionation (AF4) is a valuable tool to separate and assess different size populations in nanotherapeutics. When coupled with both static light scattering and dynamic light scattering, it can be used to qualitatively assess protein binding to nanoparticles by comparing the shape factors for both non-plasma-incubated samples and plasma-incubated samples. The shape factor is defined as the ratio of the derived root mean square radius (bystatic light scattering) to the measured hydrodynamic radius (bydynamic light scattering). The shape factor gives an idea of where the center of mass lies in a nanoparticle, and any shift in the shape factor to larger values is indicative of a mass addition to the periphery of the nanoparticle and suggests the presence of protein binding. This protocol will discuss how to set up an experiment to assess protein binding in nanoparticles using AF4, multi-angle light scattering (MALS), and dynamic light scattering (DLS).

Nanoparticle Size Distribution and Stability Assessment Using Asymmetric-Flow Field-Flow Fractionation.

Nanomaterials are inherently polydisperse. Traditional techniques, such as the widely used batch-mode dynamic light-scattering (DLS) analysis, are not ideal nor thoroughly descriptive enough to define the full complexity of these materials. Asymmetric-flow field-flow fractionation (AF4) with various in-line detectors, such as ultraviolet-visible (UV-vis), multi-angle light scattering (MALS), refractive index (RI), and DLS, is an alternative technique that can provide flow-mode analysis of not only size distribution but also shape, drug release/stability, and protein binding.