Investigating 3R in vivo approaches for bio-distribution and efficacy evaluation of nucleic acid nanocarriers: studies on peptide-mimicking ionizable lipid

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.Drug Delivery Technolog

Characterization of the interactions between various hexadecylmannoside-phospholipid model membranes with the lectin Concanavalin A

The specific interaction of Concanavalin A (ConA) with glycolipid-containing model membranes was investigated using (a) surface pressure-time (Pi-t) curves, (b) epifluorescence microscopy connected to a film balance, (c) atomic force microscopy (AFM) of the monofilms after Langmuir-Blodgett (LB) transfer and (d) quartz crystal microbalance (QCMB) weight-quantification of the adhered protein on the glycolipid model membrane. The adsorption of ConA on a model membrane was mannose-specific and concentration-dependent in the range 1-50% (1% was the lower detection limit, whereas above 30% saturation began). Adsorption kinetics was followed by QCMB and Pi-t measurements. Saturation was reached after 1 h. Hydrophilic spacers were introduced between the alkyl chain and the mannose headgroup of the ConA ligands. The quantity of specific ConA-adhesion increased with spacer length and also the adhesion kinetics was accelerated using protruding ligands. With AFM it was possible to detect morphological differences of mixed hexadecylmannoside-1,2-distearyl-sn-glycero-3-phosphocholine (DSPC) films in dependence on spacer length of the glycolipid before and after molecular contact with ConA

Characterization of the interactions between various hexadecylmannoside-phospholipid model membranes with the lectin Concanavalin A

The specific interaction of Concanavalin A (ConA) with glycolipid-containing model membranes was investigated using (a) surface pressure-time (Pi-t) curves, (b) epifluorescence microscopy connected to a film balance, (c) atomic force microscopy (AFM) of the monofilms after Langmuir-Blodgett (LB) transfer and (d) quartz crystal microbalance (QCMB) weight-quantification of the adhered protein on the glycolipid model membrane. The adsorption of ConA on a model membrane was mannose-specific and concentration-dependent in the range 1-50% (1% was the lower detection limit, whereas above 30% saturation began). Adsorption kinetics was followed by QCMB and Pi-t measurements. Saturation was reached after 1 h. Hydrophilic spacers were introduced between the alkyl chain and the mannose headgroup of the ConA ligands. The quantity of specific ConA-adhesion increased with spacer length and also the adhesion kinetics was accelerated using protruding ligands. With AFM it was possible to detect morphological differences of mixed hexadecylmannoside-1,2-distearyl-sn-glycero-3-phosphocholine (DSPC) films in dependence on spacer length of the glycolipid before and after molecular contact with ConA

Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA

The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (NN-di-(beta-steaorylethyl)-N,N-dimethylammonium chloride) or cetylpyridinium chloride as charge carrier. The resulting particles were approximately 100 nm in size and showed zeta potentials around +40 mV at pH 7.4. DNA binding was tested by agarose gel electrophoresis. The resulting SLN-DNA complexes were further characterised by AFM and zeta potential measurements. Only the SLN batch SII-13, composed of 4% Compritol, 4% Tween/Span and 1% EQ1, was able to form stable complexes with DNA. Typical complexes were 300 to 800 nm in size. Cytotoxicity and transfection efficiency was tested in vitro on Cos-1 cells. Cationic SLN produced by modification with EQ1 were well tolerated, with LD50 values >3 mg/ml in the LDH release assay and >0.6 mg/ml in the WST-1 assay. Further, SLN-DNA complexes containing between 10 and 200 weight equivalents of SII-13 (matrix lipid) efficiently transfected the galactosidase expression plasmid pCMVbeta in the absence and presence of the endosomolytic agent chloroquine. (C) 2001 Published by Elsevier Science B.V