In-silico method development and optimization of on-line comprehensive two-dimensional liquid chromatography via a shortcut model.

Comprehensive two-dimensional liquid chromatography (LCxLC) represents a valuable alternative to conventional single column, or one-dimensional, liquid chromatography (1D-LC) for resolving multiple components in a complex mixture in a short time. However, developing LCxLC methods with trial-and-error experiments is challenging and time-consuming, which is why the technique is not dominant despite its significant potential. This work presents a novel shortcut model to in-silico predicting retention time and peak width within an RPLCxRPLC separation system (i.e., LCxLC systems that use reversed-phase columns (RPLC) in both separation dimensions). Our computationally effective model uses the hydrophobic-subtraction model (HSM) to predict retention and considers limitations due to the sample volume, undersampling and the maximum pressure drop. The shortcut model is used in a two-step strategy for sample-dependent optimization of RPLCxRPLC separation systems. In the first step, the Kendall's correlation coefficient of all possible combinations of available columns is evaluated, and the best column pair is selected accordingly. In the second step, the optimal values of design variables, flow rate, pH and sample loop volume, are obtained via multi-objective stochastic optimization. The strategy is applied to method development for the separation of 8, 12 and 16 component mixtures. It is shown that the proposed strategy provides an easy way to accelerate method development for full-comprehensive 2D-LC systems as it does not require any experimental campaign and an entire optimization run can take less than two minutes.

Predicting sample injection profiles in liquid chromatography: A modelling approach based on residence time distributions.

The pharmaceutical and bio-pharmaceutical industries rely on simulations of liquid chromatographic processes for method development and to reduce experimental cost. The use of incorrect injection profiles as inlet boundary condition for these simulations may, however, lead to inaccurate results. This study presents a novel modelling approach for accurate prediction of injection profiles for liquid chromatographic columns. The model uses the residence time distribution theory and accounts for the residence time of the sample through the injection loop, connecting tubes and heat exchangers that exist upstream of the actual chromatographic column, between the injection point and the column inlet. To validate the model, we compare simulation results with experimental injection profiles taken from the literature for 20 operating conditions. The average errors in the predictions of the mean and variance of the injection profiles result to be 8.98% and 8.52%, respectively. The model, which is based on fundamental equations and actual hardware details, accurately predicts the injection profile for a range of sample volumes and sample loop-filling levels without the need of calibration. The proposed modelling approach can help to improve the quality of in-silico simulation and optimization for analytical chromatography.

A versatile non-fouling multi-step flow reactor platform: demonstration for partial oxidation synthesis of iron oxide nanoparticles.

In the last decade flow reactors for material synthesis were firmly established, demonstrating advantageous operating conditions, reproducible and scalable production via continuous operation, as well as high-throughput screening of synthetic conditions. Reactor fouling, however, often restricts flow chemistry and the common fouling prevention via segmented flow comes at the cost of inflexibility. Often, the difficulty of feeding reagents into liquid segments (droplets or slugs) constrains flow syntheses using segmented flow to simple synthetic protocols with a single reagent addition step prior or during segmentation. Hence, the translation of fouling prone syntheses requiring multiple reagent addition steps into flow remains challenging. This work presents a modular flow reactor platform overcoming this bottleneck by fully exploiting the potential of three-phase (gas-liquid-liquid) segmented flow to supply reagents after segmentation, hence facilitating fouling free multi-step flow syntheses. The reactor design and materials selection address the operation challenges inherent to gas-liquid-liquid flow and reagent addition into segments allowing for a wide range of flow rates, flow ratios, temperatures, and use of continuous phases (no perfluorinated solvents needed). This "Lego®-like" reactor platform comprises elements for three-phase segmentation and sequential reagent addition into fluid segments, as well as temperature-controlled residence time modules that offer the flexibility required to translate even complex nanomaterial synthesis protocols to flow. To demonstrate the platform's versatility, we chose a fouling prone multi-step synthesis, i.e., a water-based partial oxidation synthesis of iron oxide nanoparticles. This synthesis required I) the precipitation of ferrous hydroxides, II) the addition of an oxidation agent, III) a temperature treatment to initiate magnetite/maghemite formation, and IV) the addition of citric acid to increase the colloidal stability. The platform facilitated the synthesis of colloidally stable magnetic nanoparticles reproducibly at well-controlled synthetic conditions and prevented fouling using heptane as continuous phase. The biocompatible particles showed excellent heating abilities in alternating magnetic fields (ILP values >3 nH m2 kgFe-1), hence, their potential for magnetic hyperthermia cancer treatment. The platform allowed for long term operation, as well as screening of synthetic conditions to tune particle properties. This was demonstrated via the addition of tetraethylenepentamine, confirming its potential to control particle morphology. Such a versatile reactor platform makes it possible to translate even complex syntheses into flow, opening up new opportunities for material synthesis.

In-Silico Conceptualisation of Continuous Millifluidic Separators for Magnetic Nanoparticles.

Magnetic nanoparticles are researched intensively not only for biomedical applications, but also for industrial applications including wastewater treatment and catalytic processes. Although these particles have been shown to have interesting surface properties in their bare form, their magnetisation remains a key feature, as it allows for magnetic separation. This makes them a promising carrier for precious materials and enables recovery via magnetic fields that can be turned on and off on demand, rather than using complex (nano)filtration strategies. However, designing a magnetic separator is by no means trivial, as the magnetic field and its gradient, the separator dimensions, the particle properties (such as size and susceptibility), and the throughput must be coordinated. This is showcased here for a simple continuous electromagnetic separator design requiring no expensive materials or equipment and facilitating continuous operation. The continuous electromagnetic separator chosen was based on a current-carrying wire in the centre of a capillary, which generated a radially symmetric magnetic field that could be described using cylindrical coordinates. The electromagnetic separator design was tested in-silico using a Lagrangian particle-tracking model accounting for hydrodynamics, magnetophoresis, as well as particle diffusion. This computational approach enabled the determination of separation efficiencies for varying particle sizes, magnetic field strengths, separator geometries, and flow rates, which provided insights into the complex interplay between these design parameters. In addition, the model identified the separator design allowing for the highest separation efficiency and determined the retention potential in both single and multiple separators in series. The work demonstrated that throughputs of ~1/4 L/h could be achieved for 250-500 nm iron oxide nanoparticle solutions, using less than 10 separator units in series.

Development of an in-line magnetometer for flow chemistry and its demonstration for magnetic nanoparticle synthesis.

Despite the wide usage of magnetic nanoparticles, it remains challenging to synthesise particles with properties that exploit each application's full potential. Time consuming experimental procedures and particle analysis hinder process development, which is commonly constrained to a handful of experiments without considering particle formation kinetics, reproducibility and scalability. Flow reactors are known for their potential of large-scale production and high-throughput screening of process parameters. These advantages, however, have not been utilised for magnetic nanoparticle synthesis where particle characterisation is performed, with a few exceptions, post-synthesis. To overcome this bottleneck, we developed a highly sensitive magnetometer for flow reactors to characterise magnetic nanoparticles in solution in-line and in real-time using alternating current susceptometry. This flow magnetometer enriches the flow-chemistry toolbox by facilitating continuous quality control and high-throughput screening of magnetic nanoparticle syntheses. The sensitivity required to monitor magnetic nanoparticle syntheses at the typically low concentrations (<100 mM of Fe) was achieved by comparing the signals induced in the sample and reference cell, each of which contained near-identical pairs of induction and pick-up coils. The reference cell was filled only with air, whereas the sample cell was a flow cell allowing sample solution to pass through. Balancing the flow and reference cell impedance with a newly developed electronic circuit was pivotal for the magnetometer's sensitivity. To showcase its potential, the flow magnetometer was used to monitor two iron oxide nanoparticle syntheses with well-known particle formation kinetics, i.e., co-precipitation syntheses with sodium carbonate and sodium hydroxide as base, which have been previously studied via synchrotron X-ray diffraction. The flow magnetometer facilitated batch (on-line) and flow (in-line) synthesis monitoring, providing new insights into the particle formation kinetics as well as, effect of temperature and pH. The compact lab-scale flow device presented here, opens up new possibilities for magnetic nanoparticle synthesis and manufacturing, including 1) early stage reaction characterisation 2) process monitoring and control and 3) high-throughput screening in combination with flow reactors.

Stable Iron Oxide Nanoflowers with Exceptional Magnetic Heating Efficiency: Simple and Fast Polyol Synthesis.

Magnetically induced hyperthermia has reached a milestone in medical nanoscience and in phase III clinical trials for cancer treatment. As it relies on the heat generated by magnetic nanoparticles (NPs) when exposed to an external alternating magnetic field, the heating ability of these NPs is of paramount importance, so is their synthesis. We present a simple and fast method to produce iron oxide nanostructures with excellent heating ability that are colloidally stable in water. A polyol process yielded biocompatible single core nanoparticles and nanoflowers. The effect of parameters such as the precursor concentration, polyol molecular weight as well as reaction time was studied, aiming to produce NPs with the highest possible heating rates. Polyacrylic acid facilitated the formation of excellent nanoheating agents iron oxide nanoflowers (IONFs) within 30 min. The progressive increase of the size of the NFs through applying a seeded growth approach resulted in outstanding enhancement of their heating efficiency with intrinsic loss parameter up to 8.49 nH m2 kgFe-1. The colloidal stability of the NFs was maintained when transferring to an aqueous solution via a simple ligand exchange protocol, replacing polyol ligands with biocompatible sodium tripolyphosphate to secure the IONPs long-term colloidal stabilization.

Small iron oxide nanoparticles as MRI T1 contrast agent: scalable inexpensive water-based synthesis using a flow reactor.

Small iron oxide nanoparticles (IONPs) were synthesised in water via co-precipitation by quenching particle growth after the desired magnetic iron oxide phase formed. This was achieved in a millifluidic multistage flow reactor by precisely timed addition of an acidic solution. IONPs (≤5 nm), a suitable size for positive T1 magnetic resonance imaging (MRI) contrast agents, were obtained and stabilised continuously. This novel flow chemistry approach facilitates a reproducible and scalable production, which is a crucial paradigm shift to utilise IONPs as contrast agents and replace currently used Gd complexes. Acid addition had to be timed carefully, as the inverse spinel structure formed within seconds after initiating the co-precipitation. Late quenching allowed IONPs to grow larger than 5 nm, whereas premature acid addition yielded undesired oxide phases. Use of a flow reactor was not only essential for scalability, but also to synthesise monodisperse and non-agglomerated small IONPs as (i) co-precipitation and acid addition occurred at homogenous environment due to accurate temperature control and rapid mixing and (ii) quenching of particle growth was possible at the optimum time, i.e., a few seconds after initiating co-precipitation. In addition to the timing of growth quenching, the effect of temperature and dextran present during co-precipitation on the final particle size was investigated. This approach differs from small IONP syntheses in batch utilising either growth inhibitors (which likely leads to impurities) or high temperature methods in organic solvents. Furthermore, this continuous synthesis enables the low-cost (<£10 per g) and large-scale production of highly stable small IONPs without the use of toxic reagents. The flow-synthesised small IONPs showed high T1 contrast enhancement, with transversal relaxivity (r2) reduced to 20.5 mM-1 s-1 and longitudinal relaxivity (r1) higher than 10 mM-1 s-1, which is among the highest values reported for water-based IONP synthesis.

Whither Magnetic Hyperthermia? A Tentative Roadmap.

The scientific community has made great efforts in advancing magnetic hyperthermia for the last two decades after going through a sizeable research lapse from its establishment. All the progress made in various topics ranging from nanoparticle synthesis to biocompatibilization and in vivo testing have been seeking to push the forefront towards some new clinical trials. As many, they did not go at the expected pace. Today, fruitful international cooperation and the wisdom gain after a careful analysis of the lessons learned from seminal clinical trials allow us to have a future with better guarantees for a more definitive takeoff of this genuine nanotherapy against cancer. Deliberately giving prominence to a number of critical aspects, this opinion review offers a blend of state-of-the-art hints and glimpses into the future of the therapy, considering the expected evolution of science and technology behind magnetic hyperthermia.

A Modular Millifluidic Platform for the Synthesis of Iron Oxide Nanoparticles with Control over Dissolved Gas and Flow Configuration.

Gas-liquid reactions are poorly explored in the context of nanomaterials synthesis, despite evidence of significant effects of dissolved gas on nanoparticle properties. This applies to the aqueous synthesis of iron oxide nanoparticles, where gaseous reactants can influence reaction rate, particle size and crystal structure. Conventional batch reactors offer poor control of gas-liquid mass transfer due to lack of control on the gas-liquid interface and are often unsafe when used at high pressure. This work describes the design of a modular flow platform for the water-based synthesis of iron oxide nanoparticles through the oxidative hydrolysis of Fe2+ salts, targeting magnetic hyperthermia applications. Four different reactor systems were designed through the assembly of two modular units, allowing control over the type of gas dissolved in the solution, as well as the flow pattern within the reactor (single-phase and liquid-liquid two-phase flow). The two modular units consisted of a coiled millireactor and a tube-in-tube gas-liquid contactor. The straightforward pressurization of the system allows control over the concentration of gas dissolved in the reactive solution and the ability to operate the reactor at a temperature above the solvent boiling point. The variables controlled in the flow system (temperature, flow pattern and dissolved gaseous reactants) allowed full conversion of the iron precursor to magnetite/maghemite nanocrystals in just 3 min, as compared to several hours normally employed in batch. The single-phase configuration of the flow platform allowed the synthesis of particles with sizes between 26.5 nm (in the presence of carbon monoxide) and 34 nm. On the other hand, the liquid-liquid two-phase flow reactor showed possible evidence of interfacial absorption, leading to particles with different morphology compared to their batch counterpart. When exposed to an alternating magnetic field, the particles produced by the four flow systems showed ILP (intrinsic loss parameter) values between 1.2 and 2.7 nHm2/kg. Scale up by a factor of 5 of one of the configurations was also demonstrated. The scaled-up system led to the synthesis of nanoparticles of equivalent quality to those produced with the small-scale reactor system. The equivalence between the two systems is supported by a simple analysis of the transport phenomena in the small and large-scale setups.

Unravelling the growth mechanism of the co-precipitation of iron oxide nanoparticles with the aid of synchrotron X-Ray diffraction in solution.

Co-precipitation is the most ubiquitous method for forming iron oxide nanoparticles. For a typical co-precipitation synthesis, the pH of a ferrous and/or ferric ion solution is increased via the addition of a base. The latter can be added either slowly (a steady addition over either minutes or hours) or fast (a one-time addition) resulting in an abrupt increase in the pH. However, understanding the mechanism of particle formation is still lacking, which limits the reproducibility of the co-precipitation reaction due to intermediate phases still being present in the final product. In this work, we study in detail a co-precipitation synthesis with an abrupt increase in pH via the addition of sodium carbonate. Fast and reproducible mixing at defined precursor and base solution temperatures was achieved utilising a flow reactor. Transmission electron microscopy, electron diffraction and room temperature 57Fe Mössbauer spectroscopy showed a distinct transition from an amorphous ferrihydrite phase to a mixture of magnetite-maghemite (Fe3O4/γ-Fe2O3). Synchrotron X-ray diffraction revealed the initial formation of crystalline iron hydroxide carbonate (green rust) plates occurring before the Fe3O4/γ-Fe2O3 appeared. The ferrihydrite particles increase in size over time as the proportion of iron hydroxide carbonate plates are re-dissolved into solution, until the ferrihydrite particles crystallise into Fe3O4/γ-Fe2O3.

Crystal Shape Modification via Cycles of Growth and Dissolution in a Tubular Crystallizer.

Besides size and polymorphic form, crystal shape takes a central role in engineering advanced solid materials for the pharmaceutical and chemical industries. This work demonstrates how multiple cycles of growth and dissolution can manipulate the habit of an acetylsalicylic acid crystal population. Considerable changes of the crystal habit could be achieved within minutes due to rapid cycling, i.e., up to 25 cycles within <10 min. The required fast heating and cooling rates were facilitated using a tubular reactor design allowing for superior temperature control. The face-specific interactions between solvent and the crystals' surface result in face-specific growth and dissolution rates and hence alterations of the final shape of the crystals in solution. Accurate quantification of the crystal shapes was essential for this work, but is everything except simple. A commercial size and shape analyzer had to be adapted to achieve the required accuracy. Online size, and most important shape, analysis was achieved using an automated microscope equipped with a flow-through cell, in combination with a dedicated image analysis routine for particle tracking and shape analysis. Due to the implementation of this analyzer, capable of obtaining statistics on the crystals' shape while still in solution (no sampling and manipulation required), the dynamic behavior of the size shape distribution could be studied. This enabled a detailed analysis of the solvent's effect on the change in crystal habit.

Crystal Engineering in Continuous Plug-Flow Crystallizers.

Size, shape, and polymorphic form are the critical attributes of crystalline particles and represent the major focus of today's crystallization process design. This work demonstrates how crystal properties can be tuned efficiently in solution via a tubular crystallizer that facilitates rapid temperature cycling. Controlled crystal growth, dissolution, and secondary nucleation allow a precise control of the crystal size and shape distribution, as well as polymorphic composition. Tubular crystallizers utilizing segmented flow such as the one presented in our work can provide plug flow characteristics, fast heating and cooling, allowing for rapid changes of the supersaturation. This makes them superior for crystal engineering over common crystallizers. Characterization of particle transport, however, revealed that careful selection of process parameters, such as tubing diameter, flow rates, solvents, etc., is crucial to achieve the full benefits of such reactors.

Modelling tumour cell proliferation from vascular structure using tissue decomposition into avascular elements.

Computer models allow the mechanistically detailed study of tumour proliferation and its dependency on nutrients. However, the computational study of large vascular tumours requires detailed information on the 3-dimensional vessel network and rather high computation times due to complex geometries. This study puts forward the idea of partitioning vascularised tissue into connected avascular elements that can exchange cells and nutrients between each other. Our method is able to rapidly calculate the evolution of proliferating as well as dead and quiescent cells, and hence a proliferative index, from a given amount and distribution of vascularisation of arbitrary complexity. Applying our model, we found that a heterogeneous vessel distribution provoked a higher proliferative index, suggesting increased malignancy, and increased the amount of dead cells compared to a more static tumour environment when a homogenous vessel distribution was assumed. We subsequently demonstrated that under certain amounts of vascularisation, cell proliferation may even increase when vessel density decreases, followed by a subsequent decrease of proliferation. This effect was due to a trade-off between an increase in compensatory proliferation for replacing dead cells and a decrease of cell population due to lack of oxygen supply in lowly vascularised tumours. Findings were illustrated by an ectopic colorectal cancer mouse xenograft model. Our presented approach can be in the future applied to study the effect of cytostatic, cytotoxic and anti-angiogenic chemotherapy and is ideally suited for translational systems biology, where rapid interaction between theory and experiment is essential.