Defined covalent attachment of three cancer drugs to DNA origami increases cytotoxicity at nanomolar concentration.

DNA nanostructures have captured great interest as drug delivery vehicles for cancer therapy. Despite rapid progress in the field, some hurdles, such as low cellular uptake, low tissue specificity or ambiguous drug loading, remain unsolved. Herein, well-known antitumor drugs (doxorubicin, auristatin, and floxuridine) were site-specifically incorporated into DNA nanostructures, demonstrating the potential advantages of covalently linking drug molecules via structural staples instead of incorporating the drugs by noncovalent binding interactions. The covalent strategy avoids critical issues such as an unknown number of drug-DNA binding events and premature drug release. Moreover, covalently modified origami offers the possibility of precisely incorporating several synergetic antitumor drugs into the DNA nanostructure at a predefined molar ratio and to control the exact spatial orientation of drugs into DNA origami. Additionally, DNA-based nanoscaffolds have been reported to have a low intracellular uptake. Thus, two cellular uptake enhancing mechanisms were studied: the introduction of folate units covalently linked to DNA origami and the transfection of DNA origami with Lipofectamine. Importantly, both methods increased the internalization of DNA origami into HTB38 and HCC2998 colorectal cancer cells and produced greater cytotoxic activity when the DNA origami incorporated antiproliferative drugs. The results here present a successful and conceptually distinct approach for the development of DNA-based nanostructures as drug delivery vehicles, which can be considered an important step towards the development of highly precise nanomedicines.

On the uptake of cationic liposomes by cells: From changes in elasticity to internalization.

In this study, we assessed the capacity of a previously reported engineered liposomal formulation, which had been tested against model membranes mimicking the lipid composition of the HeLa plasma membrane, to fuse and function as a nanocarrier in cells. We used atomic force microscopy to observe physicochemical changes on the cell surface and confocal microscopy to determine how the liposomes interact with cell membranes and released their load. In addition, we performed viability assays using methotrexate as an active drug to obtain proof of concept of the formulation´s capacity to function as a drug delivery-system. The interaction of engineered liposomes with living cells corroborates the information obtained using model membranes and supports the capacity of the engineered liposomal formulation to serve as a potential nanocarrier.

Studying Lipid Membrane Interactions of a Super-Cationic Peptide in Model Membranes and Living Bacteria.

The super-cationic peptide dendrimers (SCPD) family is a valuable class of antimicrobial peptide candidates for the future development of antibacterial agents against multidrug-resistant gram-negative bacteria. The deep knowledge of their mechanism of action is a major challenge in research, since it may be the basis for future modifications/optimizations. In this work we have explored the interaction between SCPD and membranes through biophysical and microbiological approaches in the case of the G1OLO-L2OL2 peptide. Results support the idea that the peptide is not only adsorbed or close to the surface of the membrane but associated/absorbed to some extent to the hydrophobic-hydrophilic region of the phospholipids. The presence of low concentrations of the peptide at the surface level is concomitant with destabilization of the cell integrity and this may contribute to osmotic stress, although other mechanisms of action cannot be ruled out.

Dielectric properties and lamellarity of single liposomes measured by in-liquid scanning dielectric microscopy.

Liposomes are widely used as drug delivery carriers and as cell model systems. Here, we measure the dielectric properties of individual liposomes adsorbed on a metal electrode by in-liquid scanning dielectric microscopy in force detection mode. From the measurements the lamellarity of the liposomes, the separation between the lamellae and the specific capacitance of the lipid bilayer can be obtained. As application we considered the case of non-extruded DOPC liposomes with radii in the range ~ 100-800 nm. Uni-, bi- and tri-lamellar liposomes have been identified, with the largest population corresponding to bi-lamellar liposomes. The interlamellar separation in the bi-lamellar liposomes is found to be below ~ 10 nm in most instances. The specific capacitance of the DOPC lipid bilayer is found to be ~ 0.75 µF/cm2 in excellent agreement with the value determined on solid supported planar lipid bilayers. The lamellarity of the DOPC liposomes shows the usual correlation with the liposome's size. No correlation is found, instead, with the shape of the adsorbed liposomes. The proposed approach offers a powerful label-free and non-invasive method to determine the lamellarity and dielectric properties of single liposomes.

Cholesterol Effect on the Specific Capacitance of Submicrometric DOPC Bilayer Patches Measured by in-Liquid Scanning Dielectric Microscopy.

The specific capacitance of biological membranes is a key physical parameter in bioelectricity that also provides valuable physicochemical information on composition, phase, or hydration properties. Cholesterol is known to modulate the physicochemical properties of biomembranes, but its effect on the specific capacitance has not been fully established yet. Here we use the high spatial resolution capabilities of in-liquid scanning dielectric microscopy in force detection mode to directly demonstrate that DOPC bilayer patches at 50% cholesterol concentration show a strong reduction of their specific capacitance with respect to pure DOPC bilayer patches. The reduction observed (∼35%) cannot be explained by the small increase in bilayer thickness (∼16%). We suggest that the reduction of the specific capacitance might be due to the dehydration of the polar head groups caused by the insertion of cholesterol molecules in the bilayer. The results reported confirm the potential of in-liquid SDM to study the electrical and physicochemical properties of lipid bilayers at very small scales (down to ∼200 nm here), with implications in fields such as biophysics, bioelectricity, biochemistry, and biosensing.

Characterization of monolayers and liposomes that mimic lipid composition of HeLa cells.

In this work, based on several studies, we develop an artificial lipid membrane to mimic the HeLa cell membrane using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and cholesterol (CHOL). This is then a means to further study the fusion process of specific engineered liposomes. To characterize the mimicked HeLa cell membrane, we determined a series of surface pressure-area (π-A) isotherms and the isothermal compression modulus was calculated together with the dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed using a fluorescence technique (Laurdan) and two microscopy techniques: Brewster angle microscopy (BAM) and atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mN m-1. To examine the nature and composition of the observed domains, force spectroscopy (FS) based on AFM was applied to the LBs. Finally, two engineered liposome formulations were tested in a fusion assay against mimicked HeLa cell membrane LBs, showing good results and thereby opening the door to further assays and uses.

Planar lipid bilayers formed from thermodynamically-optimized liposomes as new featured carriers for drug delivery systems through human skin.

The fundamental objective pursued in this work is to investigate how liposomes formed with a thermodynamically optimized molar composition formed by the main components of the stratum corneum matrix behave on the human skin surface when used as drug delivery systems. To this purpose we engineered liposomes using phosphatidylcholines, ceramides and cholesterol. The specific molar ratio of the three components was established after studying the mixing properties of the lipid monolayers of the lipid components formed at the air-water interface. Liposomes loaded and unloaded with ibuprofen and hyaluronic acid were characterized by quasi-elastic light scattering and fluorescence polarization. Optimized liposomes, with and without drugs, were applied onto human skin and the structures formed evaluated using atomic force microscopy. Since penetration enhancers improve the permeation of the drugs encapsulated, we also examined the effects of Tween® 80 on the physical properties of the liposomes and on their extensibility over skin. In the present work we were able to observe the deposition and extension of liposomes in suspension onto human skin demonstrating the potential of liposomes without a secondary vehicle for releasing drugs in transdermal applications.

Effect of cholesterol on monolayer structure of different acyl chained phospholipids.

In this work we have investigated the effect of cholesterol (CHOL) in phospholipid monolayers on a series of phosphatidylcholines differing in acyl chain composition. We have used the CHOL proportion that abolishes the gel (Lß)-to-liquid-crystalline (Lß) transition in bilayers in order to investigate the mixing properties and laterally-segregated domains formed by specific phospholipid-CHOL ratios at the air-water interface. The binary monolayers where formed by mixing CHOL with 1,2-palmitoyl-sn-glycero-3-phos-phatidylcholine (DPPC);1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC); 1-pal-mitoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (PSPC); 1-palmitoyl-2-oleoyl-sn-gly-cero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphatidyl-choline (PLPC), respectively. From surface pressure-area (π-A) isotherms the isothermal compression modulus were calculated, and the mixing properties of the monolayers obtained by performing a basic surface thermodynamic analysis. From the excess Gibbs energy, the interaction parameter and the activity coefficients were also calculated. The study of the monolayers was complemented by determining the molecular dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed by atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mNm-1. To get insight into the nature and composition of the observed domains force spectroscopy (FS) based on AFM was applied to the LBs.

Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Monolayers and Its Possible Biological Relevance.

Because transmembrane proteins (TMPs) can be obtained with sufficient purity for X-ray diffraction studies more frequently than decades ago, their mechanisms of action may now be elucidated. One of the pending issues is the actual interplay between transmembrane proteins and membrane lipids. There is strong evidence of the involvement of specific lipids with some membrane proteins, such as the potassium crystallographically sited activation channel (KcsA) of Streptomyces lividans and the secondary transporter of lactose LacY of Escherichia coli, the activities of which are associated with the presence of anionic phospholipids such as the phosphatidylglycerol (PG) and phosphatidyethanolamine (PE), respectively. Other proteins such as the large conductance mechanosensitive channel (MscL) of E. coli seem to depend on the adaptation of specific phospholipids to the irregular surface of the integral membrane protein. In this work we investigated the lateral compressibility of two homoacid phosphatidylethanolamines (one with both acyl chains unsaturated (DOPE), the other with the acyl chains saturated (DPPE)) and the heteroacid phosphatidyletanolamine (POPE) and their mixtures with POPG. The liquid expanded (LE) to liquid condensed (LC) transition was observed in POPE at a temperature below its critical temperature (Tc = 36 °C). Because Tc lies below the physiological temperature, the occurrence of this phase transition may have something to do with the functioning of LacY. This magnitude is discussed within the context of the experiments carried out at temperatures below the Tc of POPE at which the activity of Lac Y and other TMPs are frequently studied.

Mapping phase diagrams of supported lipid bilayers by atomic force microscopy.

In this work, we present the method followed to construct a pseudophase diagram of two phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol). Two different techniques, DSC and AFM, have been used based in the determination of the onset (Tonset ) and completion (Toffset ) temperatures of the gel-to-liquid crystalline phases (Lß â†’Lα ), the first from the endotherms from liposomes and the second from the topographic images of supported lipid bilayers. The features of both phase diagrams are discussed emphasizing the influence of Ca2+ presence and the substrate (mica) on the transition undergone by the phospholipid mixture. Microsc. Res. Tech. 80:4-10, 2017. © 2016 Wiley Periodicals, Inc.

Enhanced topical delivery of hyaluronic acid encapsulated in liposomes: A surface-dependent phenomenon.

In the present study, we investigated the release and permeation of hyaluronic acid (HA) encapsulated in liposomes when deposited onto two surfaces: cellulose, a model widely used for investigating transport of drugs; and human skin, a natural biointerface used for transdermal drug delivery. We prepared and characterised liposomes loaded with HA and liposomes incorporating two penetration enhancers (PEs): the non-ionic surfactant Tween 80, and Transcutol P, a solubilising agent able to mix with polar and non-polar solvents. In vitro and ex vivo permeation assays showed that PEs indeed enhance HA-release from liposomes. Since one of the possible mechanisms postulated for the action of liposomes on skin is related to its adsorption onto the stratum corneum (SC), we used atomic force microscopy (AFM) topography and force volume (FV) analysis to investigate the structures formed after deposition of liposome formulations onto the investigated surfaces. We explored the possible relationship between the formation of planar lipid structures on the surfaces and the permeation of HA.

Unspecific membrane protein-lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements.

In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca(2+) display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (Lα) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid-to-protein ratio of 40. When the lipid-to-protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self-segregated units of LacY, the lower domain is constituted only by phospholipids in gel (Lß) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid-protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid-LacY interface segregated from the fluid bulk phase where POPG predominates.