Atomic force microscopy to elucidate lipid membranes enhanced by engineered liposomes

[eng] The research in this thesis aimed to study and generate an engineered formulation that can fuse with cell membranes and carry drugs or other compounds into cells. HeLa cells were chosen as the target cells and prior to their use, a model membrane mimicking the lipid membrane of HeLa cells was developed. Starting from the basic components and using a bottom-up approach, different phospholipids were studied and compared to identify the construction blocks of liposomes and assess the effects of cholesterol on these phospholipids. After selecting the desired composition, a membrane model mimicking the HeLa cell membrane was developed to test its fusion with the engineered liposomes and to understand the fusion process before starting in vitro assays with living HeLa cells. In the in vitro assays, the engineered liposomes were able to fuse with the cell membrane as well as carry and liberate a model drug (methotrexate) into the cells, demonstrating that the engineered liposomes can work efficiently as nanocarriers. Across the entire thesis, one technique was constantly used, atomic force microscopy (AFM). This technique enables the study of the smallest samples, such as lipid monolayers, as well as larger samples, like HeLa cells. AFM can also be used to obtain the physicochemical properties of samples using the force spectroscopy mode, allowing the analysis of samples and providing insight into the nanomechanics of the samples studied. Several techniques were used in this thesis, including the application of a Langmuir-Blodgett trough to study the physicochemical properties of lipids, fluorescence resonance energy transfer (FRET) to determine the fusion of the engineered liposomes, visualization techniques like AFM and confocal microscopy, as well as viability assays to test the toxicity of the engineered liposomes to HeLa cells. Finally, we demonstrated the ability of the engineered liposomes to fuse with cells, acting as nanocarriers based on their physicochemical properties. The ability of the membrane model to mimic the HeLa cell lipid membrane was also validated.[cat] Aquesta tesi té com a objectiu l'estudi i el disseny d'una formulació capaç de fusionar i transportar fàrmacs o altres molècules a les cèl·lules. Per a l'estudi, les cèl·lules objectiu seleccionades han estat cèl·lules HeLa i abans del seu ús, s'ha desenvolupat un model de membrana que imita la membrana lipídica de les cèl·lules HeLa. Partint dels components bàsics, des d'un punt de vista del desenvolupament “bottom-up”, s'han estudiat i comparat diferents fosfolípids per trobar els blocs de construcció adequats per als liposomes, també s'han estudiat els efectes del colesterol sobre aquests fosfolípids. Després de seleccionar la composició desitjada, s'ha desenvolupat una formulació que imita en composició la membrana cel·lular de les cèl·lules HeLa per provar la fusió dels liposomes dissenyats i per intentar entendre el procés de fusió abans d'iniciar els assajos in vitro amb cèl·lules HeLa. Pel que fa als assajos in vitro, els liposomes han demostrat ser capaços de fusionar-se a la membrana, així com transportar i alliberar un fàrmac model (metotrexat) a les cèl·lules, demostrant que els liposomes dissenyats en aquesta tesi són capaços de funcionar de manera eficient com a “nanocarriers”. Al llarg d’aquesta tesi, una tècnica ha estat constantment present, la microscòpia de força atòmica (AFM), ja que ofereix la possibilitat de realitzar estudis des de les mostres més petites, com l'estudi de monocapes lipídiques, fins a mostres més grans com les cèl·lules HeLa. Aquesta tècnica també permet fer observacions fisicoquímiques de qualsevol d'aquestes mostres mitjançant el mode d'espectroscòpia de força que permet sondejar les mostres i obtenir informació sobre la nanomecànica de les mostres estudiades. Amb aquesta finalitat s'han utilitzat diverses tècniques, tant les que han ajudat a estudiar les propietats fisicoquímiques dels lípids, com el de Langmuir-Blodgett, com altres per determinar els efectes de fusió dels liposomes com la transferència d'energia per ressonància fluorescent (FRET) o tècniques de visualització com l’AFM o microscòpia confocal i fins i tot tècniques de viabilitat per provar la viabilitat de la formulació a les cèl·lules HeLa. Finalment, hem desenvolupat i demostrat les capacitats dels liposomes per fusionar-se amb les cèl·lules, podent, en funció de les seves propietats fisicoquímiques, actuar com a “nanocarriers”. El model de membrana que imita les cèl·lules HeLa s'ha validat corroborant la capacitat per imitar la membrana lipídica de les cèl·lules HeLa reals

Engineering and development of model lipid membranes mimicking the HeLa cell membrane

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug deliver

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.

Engineering and development of model lipid membranes mimicking the HeLa cell membrane

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug delivery.This study was supported by the Spanish Ministry of Economy and Competitiveness (PID2019-110210GB-I00), the Catalan Government (Generalitat de Catalunya) (214SGR 1442), the Basque Government (grants No. IT1264-19 and IT1270-19), the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC).Peer reviewe

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.This study was carried out with the support of grants ART2017 from the IN2UB, TEC2016-79156-P from the Spanish Ministry of Economy and Competitiveness and 214SGR 1442 from the Catalan authorities (Generalitat de Catalunya). J.S. thanks Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government.Peer reviewe

Improving the genistein oral bioavailability via its formulation into the metal–organic framework MIL-100(Fe)

International audienceDespite the interesting chemopreventive, antioxidant and antiangiogenic effects of the natural bioflavonoid genistein (GEN), its low aqueous solubility and bioavailability make it necessary to administer it using a suitable drug carrier system. Nanometric porous Metal-Organic Frameworks (nanoMOFs) are appealing systems for drug delivery. Particularly, the mesoporous MIL-100(Fe) possesses a variety of interesting features related to its composition and structure, which make it an excellent candidate to be used as a drug nanocarrier (highly porous, biocompatible, can be synthetized as homogenous and stable nanoparticles (NPs), etc.). In this study, GEN was entrapped by simple impregnation in MIL-100 NPs achieving a remarkable drug loading (27.1 wt%). A combination of experimental and computing techniques was used to achieve a deep understanding of the encapsulation of GEN in MIL-100 nanoMOF. Subsequently, GEN delivery studies were carried out under simulated physiological conditions, showing on the whole a sustained GEN release for 3 days. Initial pharmacokinetics and biodistribution studies were also carried out upon the oral administration of the GEN@MIL-100 NPs in a mouse model, evidencing a higher bioavailability and showing that this oral nanoformulation appears very promising. To the best of our knowledge, the GEN-loaded MIL-100 will be the first antitumor oral formulation based on nanoMOFs studied in vivo, and paves the way to efficiently deliver nontoxic antitumorals by a convinient oral route.