Date Published: January 17, 2019
Publisher: Public Library of Science
Author(s): Camille E. Pinguet, Esther Ryll, Alexander A. Steinschulte, Jón M. Hoffmann, Monia Brugnoni, Andrey Sybachin, Dominik Wöll, Alexander Yaroslavov, Walter Richtering, Felix A. Plamper, Tushar Jana.
We propose a strategy to counteract the salt-driven disassembly of multiliposomal complexes made by electrostatic co-assembly of anionic small unilamellar liposomes and cationic star-shaped polyelectrolytes (made of quaternized poly(dimethylaminoethyl methacrylate) (qPDMAEMA100)3.1). The combined action of (qPDMAEMA100)3.1 and a nonionic star-shaped polymer (PEO12-b-PPO45)4, which comprises diblock copolymer arms uniting a poly(ethylene oxide) PEO inner block and a poly(propylene oxide) PPO terminal block, leads to a stabilization of these complexes against disintegration in saline solutions. Hereby, the anchoring of the PPO terminal blocks to the lipid bilayer and the bridging between several liposomes are at the origin of the promoted structural stability. Two-focus fluorescence correlation spectroscopy verifies the formation of multiliposomal complexes with (PEO12-b-PPO45)4. The polyelectrolyte and the amphiphilic polymer work synergistically, as the joint action still assures some membrane integrity, which is not seen for the mere (PEO12-b-PPO45)4—liposome interaction alone.
Small unilamellar liposomes (spherical bilayer lipids vesicles) are used in many disciplines for various applications: cosmetics , food industry , pharmaceutics … The function of liposomes as carriers has been extensively exploited particularly for cancer therapy . Liposomes have the advantage to encapsulate hydrophobic substances in the lipidic membrane and hydrophilic substances in the cavity [5, 6]. Then, stimuli like temperature or pH variation can cause a triggered release [7, 8]. To increase their specific performance, liposomes can be modified. As an example, coatings can increase the stability of the encapsulated compound  and maintain its activity [10, 11]. More specifically, addition of poly(ethylene oxide) PEO chains as a liposome coating improves the circulation time . However, increasing the chain length of PEO enhances the permeability of the membrane and, thus, causes leakage due to the phase separation of the liposomal bilayer membrane . In contrast, PEO-dendron phospholipids owing to their increase in the liposomal stability make the liposomes stealthier .
Small unilamellar anionic liposomes (50 nm in diameter) were prepared by evaporation and sonification method [37, 38]. DOPC (zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine), purchased by Avanti lipids USA, was dissolved in chloroform:ethanol (1:1 v:v) and POPS (anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine), purchased by Avanti Lipids USA was dissolved in chloroform (Fig 2). The molar fraction of anionic lipids can be described as ν = [POPS]/([POPS]+[DOPC]) = 0.1. Subsequently, the organic solvent was removed on a rotary evaporator (typically by drying 2 mL of a 10 mg/mL organic lipid solution). The resulting lipid film was dispersed usually in 2 mL Tris buffer (pH 7, 10−2 mol/L) by sonication during 2 times 5 minutes (with a break between both) with the ultrasonic homogenizer Bandelin Sonopuls HD 60 (at 80% power during continuous sonication), yielding a stock solution of liposomes (10 mg/mL), which was further diluted (0.01 mL of a stock solution filled up to total sample volume of 2 mL with the respective buffer/salt/polymer solutions) to reach a final lipid concentration of 0.05 mg/mL (except where otherwise stated; 0.05 mg/mL corresponds to 6.37 10−6 mol/L of negative charges). For the conductivity measurements, the liposome preparation was altered: the lipid film was resolvated and dispersed in Tris buffer (pH 7, 10−2 mol/L) containing sodium chloride (4 mol/L). Subsequent dialysis against pure buffer led to a dispersion of NaCl-loaded liposomes. For two-focus fluorescence correlation spectroscopy (2fFCS) measurements, a fluorescence-labelled lipid is added during the preparation of the liposomes, the 1-palmitoyl-2-(6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-phosphoethanolamine (NBD PE from Avanti; Fig 2). 20 μL of NBD PE lipids (1mg/mL) is added to the DOPC and POPS. Approximately 0.1% of the lipids are labelled with the dye being incorporated into the membrane of the liposomes. The liposomes were stored always only for a short time (max. 2 weeks) to avoid the fusion of their membranes, which could reduce the reproducibility of the experiments.
In this part, different aspects are examined: 1. The size evolution of multiliposomal complexes, which are formed by addition of (PPO45-PEO12)4 and incorporation of PPO residues into different liposome membranes. 2. The retention or the loss of the integrity of the complexes. 3. The effects of salt on size/aggregative stability of the complexes.
We prepared aggregates of liposomes linked together by an amphiphilic star-shaped polymer and in some cases additionally by an oppositely charged polyelectrolyte. The hydrophobic stickers at the end of the arms of the amphiphilic polymer interact with the inner membrane (i.e. lipid tails) of several liposomes, while the oppositely charged polyelectrolyte leads to an additional electrostatic “gluing” of lipid head groups of various liposomes. It was demonstrated that the size of the liposome aggregates increases due to a fluffy interconnection of the liposomes at rather low concentrations of the amphiphilic polymer “glue” . However, the structure of the liposome aggregates is compacted at higher concentrations of the amphiphilic polymer. Though the presence of amphiphilic polymer is detrimental to the membrane integrity, the liposome aggregates do not disassemble at high salt concentrations (which is the case for mere electrostatic binding). Even more, the combined action of polyelectrolyte and amphiphilic polymer leads synergistically to salt-resistant aggregates with a still considerable integrity of the membrane. By using the described approach, a multi-liposomal container with especially high capacity to hydrophobic substances (like drugs ) could be established.