Biology subjects

Whitehead, K. A.

Publications and source records attributed to Whitehead, K. A..

7 recordsLinked to original sources

bioRxiv2025

Hydrophobic Ion Pairing for Simple, Non-Toxic Transfection

Although biomacromolecules require intracellular delivery for therapeutic effect, existing transfection agents are often characterized by high cost, low efficiency, and/or cytotoxicity. Here, we describe a new transfection approach based on hydrophobic ion pairing (HIP), which involves the simple mixing of a hydrophobic counterion with charged biomacromolecules. Among tested cargoes (proteins, siRNA, and pDNA), the HIP siRNA system performed especially well, achieving silencing in fibroblasts (80%), T cells (90%), and neurons (70%). HIP siRNA was also highly potent in mice, with tropism dependent on the route of administration. Most notably, intraperitoneal administration enabled [~]40% LAMP-1 knockdown in the pancreas, and intravenous delivery resulted in a remarkable 80% silencing in the heart. Heart delivery was also highly selectively, with no significant knockdown in the liver. Together, these data demonstrate a new, inexpensive approach to biomacromolecular delivery with the potential to target difficult-to-transfect organs, thus expanding the therapeutic potential of nucleic acids.

bioengineering↗

bioRxiv2025

Enhanced epigenetic modulation via mRNA-encapsulated lipid nanoparticles enables targeted anti-inflammatory control

Temporal transcriptional modulation of immune-related genes offers powerful therapeutic potential for treating inflammatory diseases. Here, we introduce an enhanced zinc finger (ZF)-based transcriptional repressor delivered via lipid nanoparticles for controlling immune signaling pathways in vivo. By targeting Myd88, an essential adaptor molecule involved in immunity, our system demonstrates therapeutic efficacy against septicemia in C57BL/6J mice and improves repeated AAV administration by reducing antibody responses. This epigenetic engineering approach provides a platform for safe and efficient immunomodulation applicable across diseases caused by imbalanced inflammatory responses.

immunology↗

bioRxiv2025

Protein corona formed on lipid nanoparticles compromises delivery efficiency of mRNA cargo

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems. In vivo, proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/633942v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@2fef29org.highwire.dtl.DTLVardef@fe7613org.highwire.dtl.DTLVardef@1f9de01org.highwire.dtl.DTLVardef@15e5237_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape without Side Effects

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo. We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

bioengineering↗

bioRxiv2023

Lipid nanoparticle structure and delivery route during pregnancy dictates mRNA potency, immunogenicity, and health in the mother and offspring

Treating pregnancy-related disorders is exceptionally challenging because many small molecule drugs on the market may cause maternal and fetal toxicity. This potential danger has hindered the development and clinical evaluation of new drugs for several decades. Lipid nanoparticle (LNP)-based RNA therapies with high delivery efficacy, favorable immune response, and minimal transplacental transport can quell maternal-fetal toxicity concerns and propel the development of pregnancy-safe drugs. To this extent, we report potent LNP structures that robustly deliver mRNA to maternal organs and placenta. Using structure-function analysis, we show that LNP efficacy is influenced by the polyamine headgroup, and toxicity is governed by the acrylate tail. Our lead nanoparticle shows robust protein expression via multiple clinically relevant administration routes in pregnant mice. In the placenta, it transfects trophoblasts, endothelial cells, and immune cells. Further, by varying ionizable lipid structure, we demonstrate that LNP immunogenicity affects organ expression and pup health during pregnancy. Immunogenic LNPs show lower efficacy in lymphoid organs in an IL-1{beta} dependent manner in pregnant mice. Further, pro-inflammatory immune responses provoke the infiltration of adaptive immune cells in the placenta and restrict pup growth after birth. Together, our results provide a mechanistic basis for designing safe and potent LNPs that can be administered during pregnancy.

bioengineering↗

bioRxiv2021

Profiling of mature stage human breastmilk cells identifies host-defense lactocyte sub-populations

Breastmilk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breastmilk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature stage breastmilk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, qPCR, and single cell RNA sequencing data identified the predominant cell population as epithelial with smaller populations of macrophages and T cells. Two percent of epithelial cells expressed four stem cell markers: SOX2, TRA-1-60, NANOG, and SSEA4. Furthermore, milk contained six distinct epithelial lactocyte sub-populations, including three previously unidentified sub-populations programmed towards host-defense and intestinal development. Pseudotime analysis delineated the differentiation pathways of epithelial progenitors. Together, these data define healthy human maternal breastmilk cells and provide a basis for their application in maternal and infant medicine.

bioengineering↗

bioRxiv2020

From Farm to Pharmacy: Strawberry-Enabled Oral Delivery of Protein Drugs

Although oral drug delivery is preferred by patients, it is not possible for proteins because the gastrointestinal tract is not sufficiently permeable. To enable the non-toxic oral uptake of protein drugs, we investigated plant-based foods as intestinal permeation enhancers, hypothesizing that compounds found in food would be well-tolerated by the gastrointestinal tract. Following a screen of over 100 fruits, vegetables, herbs, and fungi, we identified strawberry as a potent enhancer of macromolecular permeability in vitro and in mice. Natural product chemistry techniques identified pelargonidin, an anthocyanidin, as the active compound. In mice, insulin was orally administered with pelargonidin to induce sustained pharmacodynamic effects with doses as low as 1 U/kg and bioactivity of over 100% relative to the current gold standard of subcutaneous injection. Pelargonidin-induced permeability was reversible within two hours of treatment, and one month of daily dosing did not adversely affect mice as determined by weight tracking, serum concentrations of inflammatory markers, and tight junction gene expression. Results underscore the utility of plant-based foods in biomedical applications and demonstrate pelargonidin as an especially potent enhancer for the oral delivery of biologics.

bioengineering↗