Kulkarni, J. A. | bioRxiv Science

bioRxiv2020

EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

Extracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

bioengineering↗

bioRxiv2020

Modular lipid nanoparticle platform technology for siRNA and lipophilic prodrug delivery

Successfully employing therapeutic nucleic acids, such as small interfering RNA (siRNA), requires chemical modifications or the use of nanocarrier technology to prevent their degradation in the circulation and to facilitate intracellular delivery. Lipid nanoparticles (LNP) are among the most advanced nanocarriers culminating in the first siRNA therapeutics clinical translation and approval. At the same time, their applicability as modular platform technology due to the interchangeable building blocks and siRNA payload hallmarks one of LNPs major advantages. In addition, drug derivatization approaches to synthesize lipophilic small molecule prodrugs enable stable incorporation in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, we describe how the modular LNP platform can be applied for combined gene silencing and chemotherapy to induce additive anti-cancer effects. We show that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs. In addition, we demonstrate that prodrug incorporation does not affect LNPs gene silencing ability and that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, we quantitively determined the LNPs and prodrugs pharmacokinetic properties and biodistribution following systemic administration in tumor-bearing mice. Our results indicate that co-encapsulation of siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward approach for combination therapy development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/907394v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b61be4org.highwire.dtl.DTLVardef@1e130c5org.highwire.dtl.DTLVardef@148e5dcorg.highwire.dtl.DTLVardef@eb0cb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗