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Wnorowski, A.

Publications and source records attributed to Wnorowski, A..

2 recordsLinked to original sources

Systemic delivery of cationic liposome-mediated siRNA EGFR enhances therapeutic efficacy in a human colorectal cancer model

The clinical translation of RNA interference (RNAi) therapeutics remains limited by inefficient delivery and cancer-target accumulation. Here, we report the development of a new cationic liposome (CLP) nanocarrier engineered for delivery and controlled-release of small interfering RNA (siRNA) targeting the epidermal growth factor receptor (EGFR) in human colorectal cancer. CLPs were synthesized from ethylphosphocholine-based lipids and PEGylated components, with folic acid (FA) tissue-specific ligand and fluorophore labelling. These nanocarriers exhibited robust physicochemical stability across a broad pH and temperature range, efficient siRNA complexation, and nuclease-protection of siRNA. Functional studies revealed that CLP-siEGFR achieved effective cytosolic siRNA cargo release and EGFR silencing in vitro, proving to be more effective than conventional lipid-based transfection systems. In human xenograft models, intravenously administered CLP-siEGFR showed enhanced tumor localization, prolonged siRNA retention, and significant tumor growth suppression, accompanied by marked downregulation of EGFR. Importantly, systemic dosing was well-tolerated, with no evidence of hepatotoxicity, nephrotoxicity, or hematological abnormalities. These results position CLP nanocarriers as an effective platform for targeted RNAi therapeutics, offering translational potential for precision oncology applications.

cancer biology↗

Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

The ability of styrene-maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene-maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human {beta}2-adrenergic receptor (rh{beta}2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rh{beta}2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems. HighlightsO_LIThe SMALP technology enables a direct solubilization of GPCRs from cell membranes. C_LIO_LIThe isolation of GPCRs from mammalian cells is usually a low-yield procedure. C_LIO_LISMALPs embedding the {beta}2-adrenergic receptor were prepared from a HEK293T cell line. C_LIO_LIThe ionic strength of buffers plays a key role in SMALP formation and isolation. C_LIO_LIStandard SMALP protocols can be finely optimized to increase purification yields. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/428305v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14a3bdcorg.highwire.dtl.DTLVardef@a00d14org.highwire.dtl.DTLVardef@1baca87org.highwire.dtl.DTLVardef@4686c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗