Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze–thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10⁸ to 10¹³ ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation

Donor systems that can be used in Copper(II)-complex loading.

Copper is able to form complexes with compounds containing S-Donor, O-Donor and N,O-Donor systems as well as other mixed donor systems. Examples of drugs that are described here, in addition to DDC (an S-Donor), include Quercetin (Qu) (an O-Donor), Clioquinol (CQ) (an N,O donor) as well as CX-5461, previously not identified as a copper complexing agent. Each was loaded into DSPC/Chol (55:45 mol ratio) liposomes prepared with 300 mM CuSO4. The loading temperature used in these examples was 25, 50, 40 and 60°C, respectively.</p

The anticancer activity of diethyldithiocarbmamate (DDC) is dependent on copper.

<p><b>(A)</b> Disulfiram is metabolized to diethyldithiocarbamate (DDC) and DDC complexes with Copper (Cu) (II). <b>(B)</b> Cytotoxicity curves for DSF (●) and DSF + CuSO₄ (■) were obtained with the IN CELL Analyzer using U87 glioblastoma cells where cell viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. <b>(C)</b> Cytotoxicity curves for DDC (●) and DDC + CuSO₄ (■); where cytotoxicity was measured as described above. <b>(D)</b> DDC and Cu(DDC)₂ IC₅₀ for U251, MDA-231-BR, and A549 cancer cell lines as well as HBEcP a normal cell line; averages (±SEM) are reported from three separate experiments each done in triplicate. <b>(E)</b> Photograph of DDC, CuSO₄ and Cu(DDC)₂ solutions in water.</p

Characterization of copper-complex loading method.

<p><b>(A)</b> Measured (AAS) copper to liposomal lipid ratio (black bars) compared to measured Cu(DDC)₂ (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bars) after DDC was added to CuSO₄-containing DSPC/Chol liposomes prepared with different amounts of DSPE-PEG₂₀₀₀ (ranging from 0 to 5 mole%). <b>(B)</b> Formation of Cu(DDC)₂ inside CuSO₄-containing DSPC/Chol liposomes as a function of the CuSO₄ concentration used to prepare the liposomes (ranging from 0 to 300 mM); where the measured copper (AAS) to liposomal lipid ratio (black bar) is compared to the measured Cu(DDC)₂ (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bar). <b>(C)</b> Linear regression analysis comparing measured (AAS) copper concentration (assuming encapsulated copper was free in solution) to measured Cu(DDC)₂ (UV-Vis spectrophotometer) concentration (assuming encapsulated Cu(DDC)₂ was free in solution); R² = 0.9754; each data point represents a mean ± SEM determined from at least three separate experiments done in duplicate.</p

Preliminary tolerability and plasma elimination profiles for liposomal formulations of Cu(DDC)₂, Cu(CQ)₂, CuQu and CuCX-5461 after intravenous injection into CD-1 mice.

<p>Mice were injected with a single dose of 15 mg/kg Cu(DDC)₂ (-●-), 30mg/kg Cu(CQ)₂ (-■-), 70mg/kg CuQu (-▲-) and 50 mg/kg Cu-CX-5461 (-▼-). <b>(A)</b> Changes in body weight following administration of the indicated liposomal formulation where body weights were measured over 14 days after injection (n = 3). <b>(B)</b> Preliminary plasma elimination profiles of the indicated liposomal formulations where the copper-complexed compound was measured at 1, 4, 8 and 24 hrs after administration (n = 4); concentrations were measured using HPLC or AAS as described in the Methods.</p

Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO₄.

<p><b>(A)</b> Photograph of solutions consisting of DDC (5mg/mL) and added to CuSO₄-containing DSPC/Chol (55:45) liposomes (20 mM liposomal lipid) over a 1 hour at 25°C. <b>(B)</b> Formation of Cu(DDC)₂ inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hour at 4(●), 25(■) and 40(▲)°C following addition of DDC at a final DDC concentration of (5 mM); Cu(DDC)₂ was measured using a UV-Vis spectrophotometer and lipid was measured using scintillation counting. <b>(C)</b> Cu(DDC)₂ formation inside DSPC/Chol (55:45) liposomes over time where the external pH was 7.4 (▲) and 3.5 (▼). <b>(D)</b> Measured Cu(DDC)₂ as a function of increasing DDC added, represented as the theoretical Cu(DDC)₂ to total liposomal lipid ratio; where the lipid concentration was fixed at 20 mM and final DDC concentration was varied. <b>(E)</b> Cryo-electron microscopy photomicrograph of CuSO₄- containing DSPC/Chol (55:45) liposomes and the same liposomes after formation of encapsulated Cu(DDC)₂. <b>(F)</b> Size of the CuSO₄- containing liposomes and liposomes with encapsulated Cu(DDC)₂ as determined by quasi-electric light scattering and cryo-electron microscopy; data points are given as mean ± SD.</p

Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named “EV Fingerprinting” that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5′-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities

Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named “EV Fingerprinting” that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity

Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named “EV Fingerprinting” that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity