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The present study investigates the physicochemical properties and stability of a novel lipid-based formulation-surfactant-enriched oil marbles containing abiraterone acetate. While the biopharmaceutical performance of this formulation has been reported recently, this study aims to fill the gap between a promising in vivo performance and industrial applicability. A series of techniques were employed to assess the solid-state characteristics of oil marble cores along with their physicochemical properties upon stability testing. The chemical stability of abiraterone acetate in the formulation was also investigated. The core of the formulation was found to be stable both physically and chemically over 12 months of storage. The in vitro performance of stressed samples was evaluated using a dissolution experiment. The formulation has successfully self-emulsified upon incubation in bio-relevant media, resulting in a fast and complete API release. An important issue connected with the excipient used as a covering material of oil marbles has been identified. The seemingly insignificant water sorption caused agglomeration of the oil marbles and consequently compromised the dissolution rate in some of the stressed samples. Replacing HPMC with lactose as a covering material resulted in more favorable properties upon storage. Overall, it has been shown that oil marbles are an industrially applicable concept of the solidified lipid-based formulation.
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Produtos Biológicos , Excipientes , Acetato de Abiraterona , Carbonato de Cálcio , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Excipientes/química , Lactose , Lipídeos/química , Solubilidade , Tensoativos/química , ÁguaRESUMO
Colloid deposition in granular media is relevant to numerous environmental problems. Classic filtration models assume a homogeneous pore space and largely ignore colloid aggregation. However, substantial evidence exists on the ubiquity of aggregation within porous media, suggesting that deposition is enhanced by it. This work studies the deposition process in relation to aggregate size and structure. We demonstrate that aggregation is induced at typical groundwater velocities by comparing the repulsive DLVO force between particle pairs to the hydrodynamic shear force opposing it. Column experiments imaged with high-resolution X-ray computed tomography are used to measure aggregate structure and describe their morphology probability distribution and spatial distribution. Aggregate volume and surface area are found to be power-law distributed, while Feret diameter is exponentially distributed with some flow rate dependencies caused by erosion and restructuring by the fluid shear. Furthermore, size and shape of aggregates are heterogeneous in depth, where a small number of large aggregates control the concentration versus depth profile shape. The range of aggregate fractal dimensions found (2.22-2.42) implies a high potential for restructuring or breaking during transport. Shear-induced aggregation is not currently considered in macroscopic models for particle filtration, yet is critical to consider in the processes that control deposition.
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Coloides , Filtração , Fractais , Tamanho da Partícula , PorosidadeRESUMO
Long-term continuous protein production can be reached by perfusion operation. Through the continuous removal of waste metabolites and supply of nutrients, steady-state (SS) conditions are achieved after a certain transient period, where the conditions inside the reactor are not only uniform in space but also constant in time. Such stable conditions may have beneficial influences on the reduction of product heterogeneities. In this study, we investigated the impact of perfusion cultivation on the intracellular physiological state of a CHO cell line producing a monoclonal antibody (mAb) by global transcriptomics and proteomics. Despite stable viable cell density was maintained right from the beginning of the cultivation time, productivity decrease, and a transition phase for metabolites and product quality was observed before reaching SS conditions. These were traced back to three sources of transient behaviors being hydrodynamic flow rates, intracellular dynamics of gene expression as well as metabolism and cell line instability, superimposing each other. However, 99.4% of all transcripts and proteins reached SS during the first week or were at SS from the beginning. These results demonstrate that the stable extracellular conditions of perfusion lead to SS also of the cellular level.
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Anticorpos Monoclonais/genética , Proteoma/genética , Transcriptoma , Animais , Anticorpos Monoclonais/análise , Células CHO , Técnicas de Cultura de Células/métodos , Cricetulus , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Perfusão/métodos , Proteoma/análise , Proteômica/métodosRESUMO
The rheological behavior of particle suspensions is a challenging problem because its description depends on the interaction of two phases with different material properties. This interaction can lead to complex behavior because of acting forces at the solid-liquid interface such as lubrication. The goal of this work is to propose a method for the modeling of fluids viscoelasticity in the presence of spherical particles including fluid-particle interactions. To accomplish this, we employed a simplified approach using the discrete element method (DEM) coupled with computational fluid dynamics (CFD) to simulate a suspension of particles under oscillatory flow in a three-dimensional computational domain. The choice of DEM provides versatility to customize the constitutive relations of particle-particle and fluid-particle interactions. Particularly, we focused on studying the effect of solid-liquid interaction (lubrication forces) on the viscoelasticity of the particulate system. To analyze the effect of this interfacial force, we simplified the particle-particle interaction to a nonadhesive elastic contact, thus avoiding aggregation of the particles. The work consists of two parts: the first one is a pure CFD model of the oscillatory motion applied to a Newtonian fluid (without particles), and the second is an extended version including DEM to simulate the viscoelasticity of the particle suspension. In this way, we can isolate the effect of fluid inertia on the viscoelasticity of the particulate system. The obtained results show that the model is capable to reproduce qualitatively the increase of the storage modulus as a function of the solid volume fraction and the dependence of dynamic moduli on the applied shear strain. The presented methodology provides a new insight into modeling of rheology by customizing interactions at the particle level based purely on first-principles with model parameters including solely material properties and physically identifiable quantities.
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The mechanical behavior of nanoparticle assemblies depends on complex particle interactions that are difficult to study experimentally. Depending on the nanoparticle morphology, these interactions could lead to adhesive and elastic-plastic behavior during contact deformation. The aim of this research is to study the effect of contact interactions between polymer nanoparticles and their impact on the macroscopic properties of formed aggregates. For this purpose, the discrete element method (DEM) was used to develop an interaction model combining elastic-plastic deformation and adhesion to study the behavior of spherical polymeric nanoparticles. Initially, a pair of particles interacting in the normal direction was simulated to evaluate the effect of adhesion and plastic deformation in the pull-off force of the contact. Based on these results, the simulations were extended to a dispersed system of nanoparticles, in which multibody interactions become dominant. Considering the aggregation between the nanoparticles induced by a shear flow, we performed an analysis of the number of aggregates and aggregates size in time to characterize the strength of clusters formed during the process. The simulation results showed that the interaction strength upon breakage of the clusters, correlating with the aggregates size, depends on the nanoparticle's softness. In this way, we verified that the type of contact interaction directly influences the macroscopic mechanical response of nanoparticle assemblies. Therefore, our model represents a new way of predicting the mechanical behavior of polymer nanoparticle systems and of optimizing it by adjusting primary particle properties.
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N-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this analysis to CHO fed-batch immunoglobulin G (IgG) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modeling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalysed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the IgG production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, galactosylation fluxes decreased with the cultivation time more significantly than the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.
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Galactosiltransferases/metabolismo , Imunoglobulina G/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Galactosiltransferases/genética , Glicosilação , Imunoglobulina G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 106 cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307. © 2016 Wiley Periodicals, Inc.
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Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células/métodos , Perfusão/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/normas , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Agregados Proteicos , Isoformas de Proteínas/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.
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Anticorpos Monoclonais/metabolismo , Reatores Biológicos , Modelos Biológicos , Perfusão/instrumentação , Perfusão/métodos , Polissacarídeos/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Simulação por Computador , Desenho Assistido por Computador , Cricetulus , Desenho de Equipamento , Análise de Falha de Equipamento , GlicosilaçãoRESUMO
Shearing lyophobic colloidal suspensions can lead to aggregation, followed by gelation, if the formed clusters grow to sizes large enough to percolate. If the temperature is set over the glass transition temperature of the suspended material, the particles embedded in the same aggregate start to coalesce with one another. Coalescence occurs to the finite viscosity of the particles' material, which leads to material diffusion from particle to particle. The driving force of this process is the reduction of the particle-dispersant interface and, as a consequence, the decrease the center-to-center separation of the particles. This leads to decreased cluster size, and hence a delayed gelation. Simultaneously, coalescence reinforces the particle-particle bonds formed upon aggregation, leading to clusters that are able to resist higher hydrodynamic forces before breaking up, hence leading to faster gelation. These two competing effects, combined with the natural complexity of colloidal aggregation makes it rather difficult to understand and predict which trend becomes dominant. In the present work, the shear-induced gelation of model polymeric colloidal systems with different glass transition temperatures has been studied. Starting with their interaction potential we investigate the impact of temperature on the gel time in concentrated suspensions (φ = 5%) under steady shear, followed by the effect of temperature on the stress-resistance of fully destabilized clusters under agitation. The results of the present work allow for a systematic view and deepened understanding of the factors governing shear-induced gelation in the presence of coalescence.
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Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.
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Anticorpos Monoclonais/isolamento & purificação , Técnicas de Cultura de Células/métodos , Análise Serial de Proteínas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anticorpos Monoclonais/química , Formação de Anticorpos , Peso MolecularRESUMO
Concentrated suspensions of non-Brownian particles exhibit a decrease in their viscosity with the increasing shear rate, a phenomenon called shear thinning. We present a possible explanation for this long-standing problem based on recent advances in the connection between the slip on the surface of the particles and the suspension viscosity. By expressing the energy dissipation between a pair of particles as a function of the local shear rate, it is possible to directly link the decrease of the viscosity with the shear rate to the slip of solvent molecules on the particle surface. Good agreement with various experimental data suggests that the surface slip might be important for the rheology of suspensions. The implications of this idea are relevant for a broad spectrum of applications as they show that not only the bulk properties, but also the properties of the solid-liquid interface are crucial for the flow in crowded systems.
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The rheological behavior of concentrated suspensions is a complicated problem because it originates in the collective motion of particles and their interaction with the surrounding fluid. For this reason, it is difficult to accurately model the effect of various system parameters on the viscosity even for highly simplified systems. We model the viscosity of a hard-sphere suspension subjected to high shear rates using the dynamic discrete element method (DEM) in three spatial dimensions. The contact interaction between particles was described by the Hertz model of elastic spheres (soft-sphere model), and the interaction of particles with flow was accounted for by the two-way coupling approach. The hydrodynamic interaction between particles was described by the lubrication theory accounting for the slip on particle surfaces. The viscosity in a simple-shear model was evaluated from the force balance on the wall. The obtained results are in close agreement with literature data for systems with hard spheres. Namely, the viscosity is shown to be independent of shear rate and primary particle size for monodisperse suspensions. In accordance with theory and experimental data, the viscosity grows rapidly with particle volume fraction. We show that this rheological behavior is predominantly caused by the lubrication forces. A novel approach based on the slip of water on a particle surface was developed to overcome the divergent behavior of lubrication forces. This approach was qualitatively validated with literature data from AFM measurements using a colloidal probe. The model presented in this work represents a new, robust, and versatile approach to the modeling of viscosity in suspensions with the possibility to include various interaction models and study their effect on viscosity.
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Aggregates grown in mild shear flow are released, one at a time, into homogeneous isotropic turbulence, where their motion and intermittent breakup is recorded by three-dimensional particle tracking velocimetry (3D-PTV). The aggregates have an open structure with a fractal dimension of â¼2.2, and their size is 1.4 ± 0.4 mm, which is large, compared to the Kolmogorov length scale (η = 0.15 mm). 3D-PTV of flow tracers allows for the simultaneous measurement of aggregate trajectories and the full velocity gradient tensor along their pathlines, which enables us to access the Lagrangian stress history of individual breakup events. From this data, we found no consistent pattern that relates breakup to the local flow properties at the point of breakup. Also, the correlation between the aggregate size and both shear stress and normal stress at the location of breakage is found to be weaker, when compared with the correlation between size and drag stress. The analysis suggests that the aggregates are mostly broken due to the accumulation of the drag stress over a time lag on the order of the Kolmogorov time scale. This finding is explained by the fact that the aggregates are large, which gives their motion inertia and increases the time for stress propagation inside the aggregate. Furthermore, it is found that the scaling of the largest fragment and the accumulated stress at breakup follows an earlier established power law, i.e., dfrag â¼ σ(-0.6) obtained from laminar nozzle experiments. This indicates that, despite the large size and the different type of hydrodynamic stress, the microscopic mechanism causing breakup is consistent over a wide range of aggregate size and stress magnitude.
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Although several scaling bioreactor models of mammalian cell cultures are suggested and described in the literature, they mostly lack a significant validation at pilot or manufacturing scale. The aim of this study is to validate an oscillating hydrodynamic stress loop system developed earlier by our group for the evaluation of the maximum operating range for stirring, based on a maximum tolerable hydrodynamic stress. A 300-L pilot-scale bioreactor for cultivation of a Sp2/0 cell line was used for this purpose. Prior to cultivations, a stress-sensitive particulate system was applied to determine the stress values generated by stirring and sparging. Pilot-scale data, collected from 7- to 28-Pa maximum stress conditions, were compared with data from classical 3-L cultivations and cultivations from the oscillating stress loop system. Results for the growth behavior, analyzed metabolites, productivity, and product quality showed a dependency on the different environmental stress conditions but not on reactor size. Pilot-scale conditions were very similar to those generated in the oscillating stress loop model confirming its predictive capability, including conditions at the edge of failure.
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Técnicas de Cultura de Células/instrumentação , Animais , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Proliferação de Células , Humanos , Hidrodinâmica , Projetos PilotoRESUMO
The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing.
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Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Glicosilação , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , SpodopteraRESUMO
In this work, we describe a method for the production of porous polymer materials in the form of particles characterized by narrow pore size distribution using the principle of shear-induced reactive gelation. Poly(styrene-co-divinylbenzene) primary particles with diameter ranging from 80 to 200 nm are used as building blocks, which are assembled into fractal-like clusters when exposed to high shear rates generated in a microchannel. It was found that independent of the primary particle size, it is possible to modulate the internal structure of formed fractal-like aggregates having fractal dimension ranging from 2.4 to 2.7 by varying the residence time in the microchannel. Thermally induced postpolymerization was used to increase the mechanical resilience of such formed clusters. Primary particle interpenetration was observed by SEM and confirmed by light scattering resulting in an increase of fractal dimension. Nitrogen sorption measurements and mercury porosimetry confirmed formation of a porous material with surface area ranging from 20 to 40 m(2)/g characterized by porosity of 70% and narrow pore size distribution with an average diameter around 700 nm without the presence of any micropores. The strong perfusive character of the synthesized material was confirmed by the existence of a plateau of the height equivalent to a theoretical plate measured at high reduced velocities using a chromatographic column packed with the synthesized microclusters.
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Fenômenos Mecânicos , Polímeros/química , Géis , Tamanho da Partícula , Polimerização , PorosidadeRESUMO
The coagulation process has a dramatic impact on the properties of dispersions of colloidal particles including the change of optical, rheological, as well as texture properties. We model the behavior of a colloidal dispersion with moderate particle volume fraction, that is, 5 wt %, subjected to high shear rates employing the time-dependent Discrete Element Method (DEM) in three spatial dimensions. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was used to model noncontact interparticle interactions, while contact mechanics was described by the Johnson-Kendall-Roberts (JKR) theory of adhesion. The obtained results demonstrate that the steady-state size of the produced clusters is a strong function of the applied shear rate, primary particle size, and the surface energy of the particles. Furthermore, it was found that the cluster size is determined by the maximum adhesion force between the primary particles and not the adhesion energy. This observation is in agreement with several simulation studies and is valid for the case when the particle-particle contact is elastic and no plastic deformation occurs. These results are of major importance, especially for the emulsion polymerization process, during which the fouling of reactors and piping causes significant financial losses.
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By combining elements from colloidal and polymer reaction engineering a new approach toward macroporous, mechanically robust polymer particles is presented, which does not require any porogenic additives. Specifically, aggregation and breakage in turbulent conditions of aggregates originating from fully destabilized primary latex particles is applied to produce compact, micrometer-sized clusters. Post-polymerization of monomer introduced initially to swell the primary particles is imparting mechanical rigidity and permanence to the internal structure. The resulting microclusters exhibit an internal porosity on the order of 70% and relatively broad pore size distribution, with exceptionally large pores, ranging from about 50 nm to 10 µm in diameter. These particulate microclusters, produced via reactive gelation under shear, are fractal objects with fractal dimension around 2.7, as opposed to the more open fractal structure of a monolith produced via stagnant reactive gelation, with fractal dimension of 1.9. Such macroporous particles are thought to be useful in applications requiring pores on the micrometer scale, e.g., in the chromatography of biomolecules or for packing beds perfusive to convective flow.
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Nanoclusters (NCs) composed of nanoparticles (NPs) with different functionalities and having final size in the sub-micrometer range are of great interest for biomedical imaging, drug delivery, sensors, etc. Because some of the functionalities cannot be incorporated into a single NP, e.g., high drug loading combined with strong magnetic properties, here, we present a proof of the concept using an alternative way to combine these properties using different NPs. In particular, starting from polymer and magnetite nanoparticles (MNPs), we produce NCs made out of a statistical distribution of the two components through a process based on aggregation and breakup. The effect of all involved operating parameters, i.e., primary NP size and composition, surfactant type and concentration, and applied hydrodynamic stress on the NC size and internal structure, was systematically investigated using dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM) analyses. It was found that, by properly tuning the balance between attractive and steric repulsive forces on one side and hydrodynamic stress on the other, NCs as small as 100 nm can be produced. In all cases, the produced NCs have a very compact internal structure characterized by fractal dimension around 2.6. The proposed production strategy to synthesize hetero-NCs composed of mixtures of various primary particles is suitable for the production of multifunctional devices of nanometer size (i.e., approximately 100 nm) for material and biomedical applications.
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Aggregates prepared under fully destabilized conditions by the action of Brownian motion were exposed to an extensional flow generated at the entrance of a sudden contraction. Two noninvasive techniques were used to monitor their breakup process [i.e. light scattering and three-dimensional (3D) particle tracking velocimetry (3D-PTV)]. While the first one can be used to measure the size and the morphology of formed fragments after the breakage event, the latter is capable of resolving trajectories of individual aggregates up to the breakage point as well as the trajectories of formed fragments. Furthermore, measured velocity gradients were used to determine the local hydrodynamic conditions at the breakage point. All this information was combined to experimentally determine for the first time the breakage rate of individual aggregates, given in the form of a size reduction rate K(R), as a function of the applied strain rate, as well as the properties of the formed fragments (i.e., the number of formed fragments and the size ratio between the largest fragment and the original aggregate). It was found that K(R) scales with the applied strain rate according to a power law with the slope being dependent on the initial fractal dimension only, while the obtained data indicates a linear dependency of K(R) with the initial aggregate size. Furthermore, the probability distribution function (PDF) of the number of formed fragments and the PDF of the size ratio between the largest fragment and the original aggregate indicate that breakage will result with high probability (75%) in the formation of two to three fragments with a rather asymmetric ratio of sizes of about 0.8. The obtained results are well in agreement with the results from the numerical simulations published in the literature.