No abstract

BackgroundRecently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.ResultsIn total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B) six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance.ConclusionsA FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Δ1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.

The general aim of our work is to build a set of engineered protein channels to be used in liposome and polymersome technology with a special emphasis in delivery applications. The channel proteins FhuA and OmpF are modified to answer to chemical (Angew. Chem. Int. Ed., 2008), pH (Soft Matter, 2011), and light stimuli. In this study a first light triggered release system is developed by employing the photo‐cleavable label 6‐nitroveratryloxycarbonyl chloride (NVOC‐Cl) and FhuA variants with six, five, and only one lysine in the barrel. Kinetic studies on liposome inserted FhuA variants, using 3,3′,5,5′‐tetramethylbenzidine (TMB)/horseradish peroxidase (HRP) as detection system led to the discovery of a single labeled amino acid position, K556, that is sufficient to act as a gate and that controls TMB translocation through the FhuA Δ1‐160 pore. Background conversion of TMB in the absence of FhuA Δ1‐160 ranges from 13 (non‐photo‐irradiated) to 27 (photo‐irradiated) n · s−1. A “fully” open FhuA Δ1‐160 channel reaches TMB conversions up to 113 × 10−9 M · s−1; a “fully” labeled FhuA Δ1‐160 shows a TMB conversion of 29 × 10−9 M · s−1 which is close to background levels. The engineered FhuA Δ1‐160 with only one lysine in the barrel interior (K556) shows a TMB conversion of 33 × 10−9 M · s−1 after labeling and after NVOC photo‐cleavage a conversion of 94 × 10−9 M · s−1. The latter proves the gate keeping role of position 556 in sterically modulating TMB fluxes. CD spectra, cryogenic TEM, and DLS experiments were performed to characterize the employed liposomes with embedded FhuA Δ1‐160 variants.

Liposomes are colloidal structures formed by the self-assembly of lipid molecules in solution into spherical, self-closed structures through their amphiphilic properties. All liposome preparation protocols reported consist of several steps of preparation, homogenization, and purification, which are labor-intensive, arduous, and lengthy to execute. In this work, a new procedure has been developed to reduce the time of the postrehydration sizing of liposomes from multilamellar vesicles, while improving the uniformity of the resulting liposomes produced and achieving high encapsulation efficiencies. For the homogenization step, the typically used method of filter extrusion was substituted by centrifugation. Purification of liposomes to eliminate nonencapsulated molecules and lipids is routinely carried out via gel permeation chromatography, an extremely lengthy procedure, and in the method we report, this lengthy step was replaced by the use of molecular-weight cut-off filters. Using this novel method, large unilamellar vesicles were produced and the time required, postrehydration, was dramatically reduced from almost 48 to less than 2 hours, with a highly uniformly sized population of liposomes being produced-the homogeneity of the liposome population achieved using our method was 99%, as compared to 88% attained by using the traditional method of production. We have used this approach to encapsulate fluorescein isothiocyanate (FITC), and 160,000 FITC molecules were encapsulated and the liposomes were demonstrated to be stable for at least 10 weeks at 4 degrees C.