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Issadore, D.

Publications and source records attributed to Issadore, D..

12 recordsLinked to original sources

Open-source robotic chip-to-plate interface for high-throughput microfluidic generation of materials libraries

Data-driven materials development requires large, well-characterized libraries of precisely defined formulations. While microfluidic platforms excel at generating highly controlled materials, their throughput is often limited by the challenge of efficiently interfacing device outputs with standard well plates. This bottleneck frequently necessitates manual transfer or non-microfluidic workflows, constraining both throughput and reproducibility. Here, we present LMNOP-bot (Libraries of Micro- and Nano-materials, OPen-source bot), an open-source robotic platform for the automated generation and collection of micro- and nanomaterial libraries from serial microfluidic outputs. Using synchronized, pressure-driven flow, LMNOP-bot enables continuous formulation and direct deposition into standard well plates. The system is low-cost (<$700, excluding pressure regulators), constructed from readily available or easily fabricated components, and designed for broad accessibility. LMNOP-bot collects [&ge;]30 {micro}L per formulation at a rate of one sample every four seconds, representing an approximately 50x increase in throughput over existing serial microfluidic workflows, and operates robustly for over 10,000 runs without maintenance. We demonstrate compatibility with both PDMS/glass and commercial polycarbonate devices, with seamless interfacing to 96- and 384-well plates. Repeated sampling confirms high precision and reproducibility. By removing a key bottleneck in microfluidic library generation, LMNOP-bot enables rapid, scalable, and accessible exploration of material design spaces.

bioengineering↗

Scalable flow synthesis of ultrasmall inorganic nanoparticles for biomedical applications via a confined impinging jet mixer

Ultrasmall inorganic nanoparticles (sub-5 nm) have unique biomedical advantages due to rapid clearance, enhanced imaging contrast, and potent therapeutic properties. However, current synthesis methods are limited by low throughput, polydispersity, and reliance on harsh conditions such as organic solvents or high temperatures. We report a scalable, single-step aqueous synthesis using a confined impinging jet mixer (CIJM) that produces size-controlled, clinically relevant nanoparticles, including silver sulfide, silver telluride, cerium oxide, and iron oxide, under ambient conditions. The resulting nanoparticles are homogeneous, stable, and preserve their functional biological properties. We demonstrate consistent performance across scales, establishing the CIJM as a versatile and reproducible method for producing ultrasmall inorganic nanoparticles suitable for clinical translation and high-throughput biomedical applications.

bioengineering↗

Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer's disease

INTRODUCTIONRobust plasma-based biomarkers to distinguish Lewy body disease (LBD) and Alzheimers disease (AD) are currently lacking. We applied track-etch magnetic nanopore (TENPO) sorting for enrichment of brain-derived extracellular vesicle (EV) signatures as potential biomarkers to address this gap. METHODSWe analyzed plasma from 137 autopsy-confirmed patients [30 LBD, 31 AD, 30 AD/LBD, 19 AD with amygdala Lewy bodies (AD/ALB), and 27 controls], sequencing miRNAs from TENPO-isolated GluR2-positive (neuron-enriched) and GLAST-positive (astrocyte-enriched) EVs, and measuring plasma proteins (A{beta}40, A{beta}42, tau, p-Tau181, p-Tau231) via SIMOA. RESULTSWe identified 16 GluR2+, 8 GLAST+, and 4 protein biomarkers with differential expression (false discovery rate-corrected P value < .1) between LBD and AD. A multimodal 15-feature panel classified LBD versus AD with 10-fold cross-validated accuracy = 0.95 and area under the curve (AUC) = 0.96. DISCUSSIONBrain-derived EVs offer accurate and accessible miRNA biomarkers for the differential diagnosis of LBD and AD.

bioengineering↗

Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries

Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and in vivo screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds. Each formulation is defined by varying the reagent flow ratios into one of eight microscale mixers using litho-graphically encoded fluidic resistors and dynamically controlled external pressure supplies. The microfluidic chip is integrated with custom frobotic plate handling for the rapid collection of each distinct formulation. Using this platform, we produce a library of 96 formulations, which we profile physicochemically and evaluate in terms of both in vitro and in vivo transfection.

bioengineering↗

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery

Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how they are influenced by the lipid composition and mixing method. Here we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multi-angle light scattering, and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading, and shape, which depends on both the formulation technique and lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.

bioengineering↗

Multi-stage-mixing to create a core-then-shell structure improves DNA-loaded lipid nanoparticles transfection by orders of magnitude

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilize MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs display a clear core-shell structure, vastly lower frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA-LNPs due to low expression, CTS improved DNA-LNPs protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. These results show that supramolecular arrangement is critical to LNP performance and can be controlled by mixing methodology. Further, MSM/CTS have finally made DNA-LNPs into a practical platform for long-term gene expression.

bioengineering↗

High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level

We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose Bead-based Digital Single Molecule-Single EV Sorting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches. We evaluated the analytical capabilities of BDEVS through head-to-head comparison with gold-standard technologies, and demonstrated a [~]100x improvement in the limit of detection of EV subpopulations. We demonstrate the high throughput ([~]100k beads / minute) profiling of individual EVs for key immune markers PD-L1, CD155, and the melanoma tumor marker TYRP-1, and showed that BDEVS can precisely quantify and sort EVs, offering unprecedented resolution for analyzing tumor-immune interactions and detecting rare EV subpopulations in complex clinical specimens. We demonstrate BDEVSs potential as a transformative tool for EV-based diagnostics and therapeutic monitoring in the context of cancer immunology by analyzing plasma samples from patients with melanoma, where EV heterogeneity plays a critical role in disease progression and response to therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/621423v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1a8a542org.highwire.dtl.DTLVardef@f9b9f0org.highwire.dtl.DTLVardef@11e4758org.highwire.dtl.DTLVardef@de1645_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

BackgroundMitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting normal mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. Results1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. f ConclusionThis study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

neuroscience↗

Towards the Clinical Translation of a Silver Sulfide Nanoparticle Contrast Agent: Large Scale Production with a Highly Parallelized Microfluidic Chip

Ultrasmall silver sulfide nanoparticles (Ag2S-NP) have been identified as promising contrast agents for a number of modalities and in particular for dual-energy mammography. These Ag2S-NP have demonstrated marked advantages over clinically available agents with the ability to generate higher contrast with high biocompatibility. However, current synthesis methods are low-throughput and highly time-intensive, limiting the possibility of large animal studies or eventual clinical use of this potential imaging agent. We herein report the use of a scalable silicon microfluidic system (SSMS) for the large-scale synthesis of Ag2S-NP. Using SSMS chips with 1 channel, 10 parallelized channels, and 256 parallelized channels, we determined that the Ag2S-NP produced were of similar quality as measured by core size, concentration, UV-visible spectrometry, and in vitro contrast generation. Moreover, by combining parallelized chips with increasing reagent concentration, we were able to increase output by an overall factor of 3,400. We also found that in vivo imaging contrast generation was consistent across synthesis methods and confirmed renal clearance of the ultrasmall nanoparticles. Finally, we found best-in-class clearance of the Ag2S-NP occurred within 24 hours. These studies have identified a promising method for the large-scale production of Ag2S-NP, paving the way for eventual clinical translation.

bioengineering↗

Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion

Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres versus anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photocrosslinkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels at low and high particle packing. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore number and size, storage moduli, and extrusion forces influenced by particle shape and packing. Robust in vitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cellular invasion into granular hydrogels when injected subcutaneously in vivo is significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair.

bioengineering↗

Proteomic Profiling and Biological Characterization of Extracellular Vesicles Isolated from Human Alzheimer’s Disease Brain Tissues

IntroductionExtracellular vesicles (EVs) from human Alzheimers disease (AD) biospecimens contain amyloid-{beta} peptide (A{beta}) and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined.\n\nMethodsEVs were isolated from the cortical grey matter of 20 AD and 18 control brains. Tau and A{beta} levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning.\n\nResultsLevels of pS396 tau and A{beta} were significantly elevated in AD EVs. High levels of neuron- and glia- specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy.\n\nDiscussionIn addition to A{beta} and tau, ANXA5, VGF, GPM6A and ACTZ are new signature proteins in AD EVs.

biochemistry↗

Robust Microfabrication of Highly Parallelized Three-Dimensional Microfluidics on Silicon

We present a new, robust three dimensional microfabrication method for highly parallel microfluidics, to improve the throughput of on-chip material synthesis by allowing parallel and simultaneous operation of many replicate devices on a single chip. Recently, parallelized microfluidic chips fabricated in Silicon and glass have been developed to increase the throughput of microfluidic materials synthesis to an industrially relevant scale. These parallelized microfluidic chips require large arrays (> 10,000) of Through Silicon Vias (TSVs) to deliver fluid from delivery channels to the parallelized devices. Ideally, these TSVs should have a small footprint to allow a high density of features to be packed into a single chip, have channels on both sides of the wafer, and at the same time minimize debris generation and wafer warping to enable permanent bonding of the device to glass. Because of these requirements and challenges, previous approaches cannot be easily applied to produce three dimensional microfluidic chips with a large array of TSVs. To address these issues, in this paper we report a fabrication strategy for the robust fabrication of three-dimensional Silicon microfluidic chips consisting of a dense array of TSVs, designed specifically for highly parallelized microfluidics. In particular, we have developed a two-layer TSV design that allows small diameter vias (d < 20 {micro}m) without sacrificing the mechanical stability of the chip and a patterned SiO2 etch-stop layer to replace the use of carrier wafers in Deep Reactive Ion Etching (DRIE). Our microfabrication strategy allows >50,000 (d = 15 {micro}m) TSVs to be fabricated on a single 4\" wafer, using only conventional semiconductor fabrication equipment, with 100% yield (M = 16 chips) compared to 30% using previous approaches. We demonstrated the utility of these fabrication strategies by developing a chip that incorporates 20,160 flow focusing droplet generators onto a single 4\" Silicon wafer, representing a 100% increase in the total number of droplet generators than previously reported. To demonstrate the utility of this chip for generating pharmaceutical microparticle formulations, we generated 5-9 {micro}m polycaprolactone particles with a CV <5% at a rate as high as 60 g/hr (> 1 trillion particles / hour).

bioengineering↗