Pleiotropic effects of trisomy and pharmacologic modulation on structural, functional, molecular, and genetic systems in a Down syndrome mouse model.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments.

Microcephaly with a disproportionate hippocampal reduction, stem cell loss and neuronal lipid droplet symptoms in Trappc9 KO mice.

Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.

Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release.

The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.

Metabolic changes in glioblastomas in response to choline kinase inhibition: In vivo MRS in rodent models.

Changes in glioblastoma (GBM) metabolism was investigated in response to JAS239, a choline kinase inhibitor, using MRS. In addition to the inhibition of phosphocholine synthesis, we investigated changes in other key metabolic pathways associated with GBM progression and treatment response. Three syngeneic rodent models of GBM were used: F98 (N = 12) and 9L (N = 8) models in rats and GL261 (N = 10) in mice. Rodents were intracranially injected with GBM cells in the right cortex and tumor growth was monitored using T2 -weighted images. Animals were treated once daily with intraperitoneal injections of 4 mg/kg JAS239 (F98 rats, n = 6; 9L rats, n = 6; GL261 mice, n = 5) or saline (control group, F98 rats, n = 6; 9L rats, n = 2; GL261 mice, n = 5) for five consecutive days. Single voxel spectra were acquired on Days 0 (T0, baseline) and 6 (T6, end of treatment) from the tumor as well as the contralateral normal brain using a PRESS sequence. Changes in metabolite ratios (tCho/tCr, tCho/NAA, mI/tCr, Glx/tCr and (Lip + Lac)/Cr) were used to assess metabolic pathway alterations in response to JAS239. Tumor growth arrest was noted in all models in response to JAS239 treatment compared with saline-treated animals, with a significant reduction (p < 0.05) in the F98 model. A reduction in tCho/tCr was observed with JAS239 treatment in all GBM models, indicating reduced phospholipid metabolism, with the highest reduction in 9L followed by GL261 and F98 tumors. A significant reduction (p < 0.05) in the tCho/NAA ratio was observed in the 9L model. A significant reduction in mI/tCr (p < 0.05) was found in JAS239-treated F98 tumors compared with the saline-treated animals. A non-significant trend of reduction in Glx/tCr was observed only in F98 and 9L tumors. JAS239-treated F98 tumors also showed a significant increase in Lip + Lac (p < 0.05), indicating increased cell death. This study demonstrated the utility of MRS in assessing metabolic changes in GBM in response to choline kinase inhibition.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

Multi-resistive pulse sensor microfluidic device.

Resistive pulse sensors have been used to characterise everything from whole cells to small molecules. Their integration into microfluidic devices has simplified sample handling whilst increasing throughput. Typically, these devices measure a limited size range, making them prone to blockages in complex sample matrixes. To prolong their life and facilitate their use, samples are often filtered or prepared to match the sample with the sensor diameter. Here, we advance our tuneable flow resistive pulse sensor which utilises additively manufactured parts. The sensor allows parts to be easily changed, washed and cleaned, its simplicity and versatility allow components from existing nanopore fabrication techniques such as glass pipettes to be integrated into a single device. This creates a multi-nanopore sensor that can simultaneously measure particles from 0.1 to 30 µm in diameter. The orientation and controlled fluid flow in the device allow the sensors to be placed in series, whereby smaller particles can be measured in the presence of larger ones without the risk of being blocked. We illustrate the concept of a multi-pore flow resistive pulse sensor, by combining an additively manufactured tuneable sensor, termed sensor 1, with a fixed nanopore sensor, termed sensor 2. Sensor 1 measures particles as small as 10 µm in diameter, whilst sensor 2 can be used to characterise particles as small as 100 nm, depending upon its dimensions. We illustrate the dual pore sensor by measuring 1 and 10 µm particles simultaneously.

A simple displacement aptamer assay on resistive pulse sensor for small molecule detection.

A universal aptamer-based sensing strategy is proposed using DNA modified nanocarriers and Resistive Pulse Sensing (RPS) for the rapid (≤20 min) and label free detection of small molecules. The surface of a magnetic nanocarrier was first modified with a ssDNA (anchor) which is designed to be partially complimentary in sequence to the ssDNA aptamer. The aptamer and anchor form a stable dsDNA complex on the nanocarriers surface. Upon the addition of the target molecule, a conformational change takes place where the aptamer preferentially binds to the target over the anchor; causing the aptamer to be released into solution. The RPS measures the change in velocity of the nanocarrier as its surface changes from dsDNA to ssDNA, and its velocity is used as a proxy for the concentration of the target. The length of the aptamer and the ability to extract and preconcentrate the nanocarriers using a magnet, is shown to affect the sensitivity. We illustrate the versatility of the assay using the same anchor sequence and Aptamers to the antibiotic Moxifloxacin, and chemotherapeutics Imatinib and Irinotecan. In addition, the proposed assay can be easily extended to detect multiple analytes simultaneously, by utilizing nanocarriers with different diameters. Each sized particle is functionalised with a the same anchor but a unique aptamer. We illustrate this with the simultaneous detection of Imatinib and Moxifloxacin. The strategy could be easily adapted to a range of targets and unlike previous strategies that use aptamer modified nanocarriers, the signal is not dependent upon the tertiary structure of the aptamer-target interaction.

DNAzyme Sensor for the Detection of Ca2+ Using Resistive Pulse Sensing.

DNAzymes are DNA oligonucleotides that can undergo a specific chemical reaction in the presence of a cofactor. Ribonucleases are a specific form of DNAzymes where a tertiary structure undergoes cleavage at a single ribonuclease site. The cleavage is highly specificity to co-factors, which makes them excellent sensor recognition elements. Monitoring the change in structure upon cleavage has given rise to many sensing strategies; here we present a simple and rapid method of following the reaction using resistive pulse sensors, RPS. To demonstrate this methodology, we present a sensor for Ca2+ ions in solution. A nanoparticle was functionalised with a Ca2+ DNAzyme, and it was possible to follow the cleavage and rearrangement of the DNA as the particles translocate the RPS. The binding of Ca2+ caused a conformation change in the DNAzyme, which was monitored as a change in translocation speed. A 30 min assay produced a linear response for Ca2+ between 1-9 µm, and extending the incubation time to 60 min allowed for a concentration as low as 0.3 µm. We demonstrate that the signal is specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.

Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling.

Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1ß involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.

A Tunable Three-Dimensional Printed Microfluidic Resistive Pulse Sensor for the Characterization of Algae and Microplastics.

Technologies that can detect and characterize particulates in liquids have applications in health, food, and environmental monitoring. Simply counting the numbers of cells or particles is not sufficient for most applications; other physical properties must also be measured. Typically, it is necessary to compromise between the speed of a sensor and its chemical and biological specificity. Here, we present a low-cost and high-throughput multiuse counter that classifies a particle's size, concentration, and shape. We also report how the porosity/conductivity or the particle can influence the signal. Using an additive manufacturing process, we have assembled a reusable flow resistive pulse sensor capable of being tuned in real time to measure particles from 2 to 30 µm across a range of salt concentrations, i.e., 2.5 × 10-4 to 0.1 M. The device remains stable for several days with repeat measurements. We demonstrate its use for characterizing algae with spherical and rod structures as well as microplastics shed from tea bags. We present a methodology that results in a specific signal for microplastics, namely, a conductive pulse, in contrast to particles with smooth surfaces such as calibration particles or algae, allowing the presence of microplastics to be easily confirmed and quantified. In addition, the shapes of the signal and of the particle are correlated, giving an extra physical property to characterize suspended particulates. The technology can rapidly screen volumes of liquid, 1 mL/min, for the presence of microplastics and algae.

Droplet factories: Synthesis and assembly of metal nanoparticles on magnetic supports.

The interface between two immiscible liquids represent an ideal substrate for the assembly of nanomaterials. The defect free surface provides a reproducible support for creating densely packed ordered materials. Here a droplet flow reactor is presented for the synthesis and/or assembly of nanomaterials at the interface of the emulsion. Each droplet acts as a microreactor for a reaction between decamethylferrocene (DmFc) within the hexane and metal salts (Ag+/Pd2+) in the aqueous phase. The hypothesis was that a spontaneous, interfacial reaction would lead to the assembly of nanomaterials creating a Pickering emulsion. The subsequent removal of the solvents showed how the Ag nanoparticles remain trapped at the interface and retain the shape of the droplet, however the Pd nanoparticles were dispersed with no tertiary structure. To further exploit this, a one-step process where the particles are synthesised and then assembled into core-shell materials was proposed. The same reactions were performed in the presence of oleic acid stabilised iron oxide nanoparticles dispersed within the hexane. It was shown that by changing the reaction rate and ratio between metal and iron oxide a continuous coating of metal nanoparticles can be formed on top of an iron oxide microsphere, or form a uniform composite. These insights offer a new method and chemistry within flow reactors for the creation of palladium and silver nanoparticles. We use the technique to create metal coated iron oxide nanomaterials but the methodology could be easily transferred to the assembly of other materials.

Rapid quantification of prion proteins using resistive pulse sensing.

Prion diseases are a group of fatal transmissible neurological conditions caused by the change in conformation of intrinsic cellular prion protein (PrPC). We present a rapid assay using aptamers and resistive pulse sensing, RPS, to extract and quantify PrPC from complex sample matrices. We functionalise the surface of superparamagnetic beads, SPBs, with a DNA aptamer. First SPB's termed P-beads, are used to pre-concentrate the analyte from a large sample volume. The PrPC protein is then eluted from the P-beads before aptamer modified sensing beads, S-beads, are added. The velocity of the S-beads through the nanopore reveals the concentration of the PrPC protein. The process is done in under an hour and allows the detection of picomol's of protein.

Peptide Nanocarriers for Detection of Heavy Metal Ions Using Resistive Pulse Sensing.

The use of nanocarriers within resistive pulse sensing facilitates the detection and quantification of analytes. To date the field has been dominated by polyionic carriers or nanomaterials. Together they combine the recognition elements of a ligand with a stable support, facilitating the sample handling, analysis times, and multiplex detection. Here we develop the use of peptide-functionalized superparamagnetic nanocarriers to extract and quantify metal ions in solution. The interaction between nickel and the peptide ligand is measured as a change in translocation velocity of the carrier. The magnitude of change is proportional to the concentration of the metal ions in solution. Unlike DNA aptamers where a change in the tertiary structure and the folding of the polyanionic backbone influences the carrier velocity, the peptides here had a lower net charge under the assay conditions. To try and enhance the signal we engineered charged groups within the peptide to explore the effects on the signal. In all cases the metal ion binding dominated the velocity of the carrier. The assay was shown to work across 3 orders of magnitude and can detect Ni2+ in the presence of some other heavy metal ions. We demonstrate this by quantifying Ni2+ in both tap and pond water. The work allows for future multiplexed sensing strategies using both peptides and DNA aptamers in resistive pulse sensors.

Raman spectral signatures of urinary extracellular vesicles from diabetic patients and hyperglycemic endothelial cells as potential biomarkers in diabetes.

Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.

Additively Manufactured Flow-Resistive Pulse Sensors.

Resistive pulse sensors (RPSs) provide detailed characterization of materials from the nanoparticle up to large biological cells on a particle-to-particle basis. During the RPS experiment, particles pass through a channel or pore that conducts ions, and the change in the ionic current versus time is monitored. The change in current during each translocation, also known as a "pulse", is dependent on the ratio of the particle and channel dimensions. Here we present a facile and rapid method for producing flow-RPSs that do not require lithographic processes. The additively manufactured sensor has channel dimensions that can be easily controlled. In addition, the fabrication process allows the sensor to be quickly assembled, disassembled, cleaned, and reused. Furthermore, the RPS can be created with a direct interface for fluidic pumps or imaging window for complementary optical microscopy. We present experiments and simulations of the RPS, showing how the pulse shapes are dependent on the channel morphology and how the device can count and size particles across a range of flow rates and ionic strengths. The use of pressure-driven fluid flow through the device allowed a rapid characterization of particles down to concentrations as low as 1 × 10-3 particles per mL, which equated to one event per second.

Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a†.

Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging.

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20-30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.

The Design and Characterization of Multifunctional Aptamer Nanopore Sensors.

Aptamer-modified nanomaterials provide a simple, yet powerful sensing platform when combined with resistive pulse sensing technologies. Aptamers adopt a more stable tertiary structure in the presence of a target analyte, which results in a change in charge density and velocity of the carrier particle. In practice the tertiary structure is specific for each aptamer and target, and the strength of the signal varies with different applications and experimental conditions. Resistive pulse sensors (RPS) have single particle resolution, allowing for the detailed characterization of the sample. Measuring the velocity of aptamer-modified nanomaterials as they traverse the RPS provides information on their charge state and densities. To help understand how the aptamer structure and charge density effects the sensitivity of aptamer-RPS assays, here we study two metal binding aptamers. This creates a sensor for mercury and lead ions that is capable of being run in a range of electrolyte concentrations, equivalent to river to seawater conditions. The observed results are in excellent agreement with our proposed model. Building on this we combine two aptamers together in an attempt to form a dual sensing strand of DNA for the simultaneous detection of two metal ions. We show experimental and theoretical responses for the aptamer which creates layers of differing charge densities around the nanomaterial. The density and diameter of these zones effects both the viability and sensitivity of the assay. While this approach allows the interrogation of the DNA structure, the data also highlight the limitations and considerations for future assays.

Rapid Assessment of Site Specific DNA Methylation through Resistive Pulse Sensing.

Many diseases are defined by patterns of DNA methylation which result in aberrant gene expression. We present a rapid assay based upon resistive pulse sensing, RPS, to characterize sequence specific DNA methylation sites in genomic DNA. We modify the surface of superparamagnetic beads, SPBs, with DNA (capture probe). The particles are added to solution where they bind to and extract sequence specific DNA (target DNA). The target loaded SPBs are then incubated with antibodies which bind to the methylation sites, and the velocity of the SPBs through the nanopore reveals the number and location of the epigenetic markers within the target. The approach is capable of distinguishing between different methylation sites within a DNA promoter region. Crucially the approach is not dependent on accurate sequencing of assayed DNA, with genomic regions targeted through complementary probes. As such the number of stages and reagents costs are low and the assay is complete in under 60 min which includes the incubation and run times. The format also allows simultaneous quantification of number of copies of methylated DNA, and we illustrate this with a dose response curve.

Synthesis of Gold Nanoparticles Using the Interface of an Emulsion Droplet.

A facile and rapid method for synthesizing single crystal gold spherical or platelet (nonspherical) particles is reported. The reaction takes place at the interface of two immiscible liquids where the reducing agent decamethylferrocene (DmFc) was initially added to hexane and gold chloride (AuCl4-) to an aqueous phase. The reaction is spontaneous at room temperature, leading to the creation of Au nanoparticles (AuNP). A flow focusing microfluidic chip was used to create emulsion droplets, allowing the same reaction to take place within a series of microreactors. The technique allows the number of droplets, their diameter, and even the concentration of reactants in both phases to be controlled. The size and shape of the AuNP are dependent upon the concentration of the reactants and the size of the droplets. By tuning the reaction parameters, the synthesized nanoparticles vary from nanometer to micrometer sized spheres or platelets. The surfactant used to stabilize the emulsion was also shown to influence the particle shape. Finally, the addition of other nanoparticles within the droplet allows for core@shell particles to be readily formed, and we believe this could be a versatile platform for the large scale production of core@shell particles.