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For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes' synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or "proteoDNAsome", which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

Nuclear breakdown was found to be the dominant route for DNA entry into the nucleus in actively dividing cells. The possibility that alternative routes contribute to DNA entry into the nucleus, however, cannot be ruled out. Here we address the process of lipofection by monitoring the localization of fluorescently-labelled DNA plasmids at the single-cell level by confocal imaging in living interphase cells. As test formulation we choose the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE) with plasmidic DNA pre-condensed by means of protamine. By exploiting the spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) we monitor the position of the nuclear envelope (NE), while concomitantly imaging the whole nucleus (by Hoechst) and the DNA (by Cy3 fluorophore) in a multi-channel 3D confocal imaging experiment. Reported results show that DNA clusters are typically associated with the NE membrane in the form of tubular invaginations spanning the nuclear environment, but not completely trapped within the NE invaginations, i.e. the DNA may use these NE regions as entry-points towards the nucleus. These observations pave the way to investigating the molecular details of the postulated processes for a better exploitation of gene-delivery vectors, particularly for applications in non-dividing cells., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

When drug nanocarriers enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BMC) mainly constituted by proteins. Although a deep investigation has been performed on the composition of BMC in terms of proteins, scarce attention has been posed to low molecular weight metabolites present in human plasma. In this work, for the first time, the investigation of the BMC of liposomal nanoparticles (NPs) constituted by 1,2-dioleoyl-3-trimethylammonium-propane polar lipid has been carried out by an ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry based untargeted metabolomics approach. Compounds were tentatively identified based on matches with online databases and comparison of MS/MS spectra with available spectral libraries. Moreover, a comparison of three metabolite extraction strategies, including an ultrafiltration membrane based protocol, a methanol extraction based protocol, and Wessel & Flügge protocol, was performed. Methanol extraction procedure resulted in the widest metabolic coverage of liposomal NP BMC. A total of 193 metabolites has been tentatively identified, 166 of which belonged to the class of lipids including phospholipids, steroids, carnitines, fatty alcohols, diglycerides and fatty acids. The high abundance of lipids in the BMC can be explained by the adsorption of plasma lipoproteins onto liposome surface, confirming previous works on other kinds of NPs. Lipids are important bioactive molecules, which could impact NP circulation and uptake by cells. Extending the investigation of BMC beyond the protein corona and towards the "lipid corona" may be the keystone of a better understanding and control of NP fate in human body., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The past three decades have witnessed fast advances in the use of cationic liposome-DNA complexes (lipoplexes) for gene delivery applications. However, no lipoplex formulation has reached into the clinical practice so far. The primary drawback limiting clinical use of lipoplexes is the lack of mechanistic understanding of their low transfection efficiency (TE) in vivo. In physiological environments, lipoplexes are coated by a protein corona (PC) that mediates the interactions with the cell machinery. Here we show that the formation of PC can change the interactions of multicomponent (MC) lipoplexes with our cell model (i.e., HeLa). At the highest lipoplex concentration, the formation of PC can reduce the TE of MC lipoplexes from 60% to <5%. Combining dynamic light scattering and synchrotron small-angle X-ray scattering (SAXS), we clarify that the formation of PC modifies physical-chemical properties of MC lipoplexes so as to affect their TE. Moreover, we examined single transfection barriers by a combination of fluorescence-activated cell sorting, single-cell real-time fluorescence confocal microscopy, and synchrotron SAXS. We demonstrate that PC formation has the ability to modify the relative contribution of caveolae-mediated endocytosis and macropinocytosis in lipoplexes uptake, in favor of the latter, increasing accumulation of PC-decorated lipoplexes into degradative lysosomal compartments. Finally, we report evidences that PC reduces the structural stability of lipoplexes against solubilization by cellular lipids, likely favoring premature DNA release and cytosolic digestion by DNAase. These combined effects revealed here offer a comprehensive mechanistic explanation on the reason behind reduction in gene expression of MC lipoplexes., (Copyright © 2019. Published by Elsevier B.V.)

In vivo liposomes, like other types of nanoparticles, acquire a totally new 'biological identity' due to the formation of a biomolecular coating known as the protein corona that depends on and modifies the liposomes' synthetic identity. The liposome-protein corona is a dynamic interface that regulates the interaction of liposomes with the physiological environment. Here we show that the biological identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a critical assessment of the key concepts of liposome technology that need to be reviewed for its definitive clinical translation.

Recently, the concept is emerging that the reduced success of nanoparticles in clinical practice is due to the adsorption of the "biomolecular corona (BC)," which alters their biological identity. Apart from protein variations, alterations in the human metabolome may change the BC decoration, which has poorly been addressed so far. Here, glucose is used as a model metabolite and how the interactions between liposomes (as a model nanoparticle) and plasma proteins are influenced by normal and diabetic sugar blood levels is explored. As model liposomes, Doxoves and Onivyde are used that are used for the treatment of breast and metastatic pancreatic cancer, respectively. It is shown that glucose does affect the structure and composition of BC. The biological effects of liposome-BC complexes are investigated in MCF 7 and MDA-MB-231 breast cancer cells for Doxoves and in pancreatic adenocarcinoma (PANC-1) and insulinoma (INS-1) cells for Onivyde. In the presence of glucose, the cellular toxicity of liposome-protein complexes and uptake by human monocytic THP1 cell line increases. These results demonstrate that alterations in glucose concentration, and more generally changes in the human metabolome, may play a fundamental role in the biological identity of liposomes and, consequently, on their in vivo physiological readouts including therapeutic efficacy., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Temozolomide (TMZ) is the current first-line chemotherapy for treatment of glioblastoma multiforme (GBM). However, similar to other brain therapeutic compounds, access of TMZ to brain tumors is impaired by the blood-brain barrier (BBB) leading to poor response for GBM patients. To overcome this major hurdle, we have synthesized a set of TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical-chemical properties. The targeting nature of this nanomedicine is provided by the recruitment of proteins, with natural targeting capacity, in the biomolecular corona (BC) layer that forms around CLs after exposure to human plasma (HP). TMZ-loaded CL-BC complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS). BCs were found to be enriched of typical BC fingerprints (BCFs) (e.g., Apolipoproteins, Vitronectin, and vitamin K-dependent protein), which have a substantial capacity in binding to receptors that are overexpressed at the BBB (e.g., scavenger receptor class B, type I and low-density lipoprotein receptor). We found that the CL formulation exhibiting the highest levels of targeting BCFs had larger uptake in human umbilical vein endothelial cells (HUVECs) that are commonly used as an in vitro model of the BBB. This formulation could also deliver TMZ to the human glioblastoma U-87 MG cell line and thus substantially enhance their antitumor efficacy compared to corona free CLs. Thus, we propose that the BC-based nanomedicines may pave a more effective way for efficient treatment of GBM.

Recent advances in biochemical and biophysical research have been achieved through the employment of microfluidic devices. Microfluidic mixing of therapeutic agents with biomaterials yields systems with finely tuned physical-chemical properties for applications in drug and gene delivery. Here, we investigate the role of preparation technology (microfluidic mixing vs. bulk self-assembly) on the transfection efficiency (TE) and cytotoxicity of multicomponent cationic liposome/DNA complexes (lipoplexes) in live Chinese hamster ovarian (CHO) cells. Decoupling TE and cytotoxicity allowed us to combine them in a unique coherent vision. While bulk self-assembly produces highly efficient and highly toxic MC lipoplexes, microfluidics manufacture leads to less efficient, but less cytotoxic complexes. This discrepancy is ascribed to two main factors controlling lipid-mediated cell transfection, i.e. the lipoplex concentration at the cell surface and the lipoplex arrangement at the nanoscale. Further research is required to optimize microfluidic manufacturing of lipoplexes to obtain highly efficient and not cytotoxic gene delivery systems., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Pancreatic ductal adenocarcinoma (PDAC) early diagnosis is  crucial  and new, cheap and user-friendly techniques for biomarker identification  are  needed. "Protein corona" (PC) is emerging a new bio-interface potentially useful in tumor early diagnosis. In a previous investigation, we showed that relevant differences between the  protein patterns of  PCs formed on lipid NPs after exposure to PDAC and non-cancer plasma  samples exist. To extend that research, We performed this pilot study to investigate the effect of PDAC tumor size and distant metastases on PC composition., Methods: Twenty PDACs were clinically staged according to the UICC TNM staging system 8 t h Edition. Collected plasma samples were let to interact with lipid NPs; resulting PCs were characterized by SDS-PAGE. To properly evaluate changes in the PC, the protein intensity profiles were reduced to four regions of molecular weight: < 25 kDa, 25-50 kDa, 50-120 kDa, > 120 kDa.  RESULTS: Data analysis allowed to distinguish T1-T2 cases from T3 and above all from metastatic ones (p < 0.05). Discrimination power was particularly due to a subset of plasma proteins with molecular  weight comprised between 25-50 kDa  and 50-120 kDa., Conclusions: PC composition is critically influenced by tumor size and presence of distant metastases in PDAC. If our findings will be further confirmed, we envision that future developments of cheap and user-friendly PC-based tools will allow to improve the accuracy of PDAC clinical staging, identifying among resectable  PDACs with potentially better prognosis (i.e. T1 and T2) those at higher risk of occult distant metastases., (Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Driven by the promises of gene therapy, PEGylated cationic liposomes (CLs) have been investigated for decades, but their use in the clinical setting is far from established. Such a dichotomy is due to several factors that have been ignored over the last two decades. The hardest challenge seems to occur when PEGylated CLs come into contact with a physiological environment (e.g. the blood). Recent evidence has demonstrated that PEGylation does not completely prevent protein binding (as believed so far), but a biomolecular shell, termed "biomolecular corona" (BC), covers the liposome surface. Here we show that the formation of a BC not only affects the surface properties of PEGylated CLs, but also, and significantly, their bilayer structure thus impairing their ability to safely deliver their cargo to the target site. Therefore, a mechanistic understanding of the structures emerging from liposome-protein interactions may represent a truly new paradigm for the clinical translation of PEGylated CLs.

To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results., (Copyright © 2016 Elsevier Inc. All rights reserved.)

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome-protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To control liposomes fate and transport upon contact with biofluids, it is essential to consider several parameters affecting the synthetic and biological identity of liposomes, as well as liposome-protein corona (PC) aspects. As a powerful tool in this data mining adventure, quantitative structure-activity relationship (QSAR) approach is used to correlate physicochemical properties of liposomes and their PC fingerprints to multiple quantified biological responses. In the present study, the relationship between cellular interactions of a set of structurally diverse liposomal formulations and their physicochemical and PC properties has been investigated via linear and nonlinear QSAR models. Significant parameters affecting cellular uptake and cell viability of liposomes in two important cancer cell lines (PC3 and HeLa) have been identified. The developed QSARs have the capacity to be implemented in advanced targeted delivery of liposomal drugs.

Following systemic administration, liposomes are covered by a 'corona' of proteins, and preserving the surface functionality is challenging. Coating the liposome surface with polyethylene glycol (PEG) is the most widely used anti-opsonization strategy, but it cannot fully preclude protein adsorption. To date, protein binding has been studied following in vitro incubation to predict the fate of liposomes in vivo, while dynamic incubation mimicking in vivo conditions remains largely unexplored. The main aim of this investigation was to determine whether shear stress, produced by physiologically relevant dynamic flow, could influence the liposome-protein corona. The corona of circulating PEGylated liposome was thoroughly compared with that formed by incubation in vitro. Systematic comparison in terms of size, surface charge and quantitative composition was made by dynamic light scattering, microelectrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). Size of coronas formed under static vs. dynamic incubation did not appreciably differ from each other. On the other side, the corona of circulating liposomes was more negatively charged than its static counterpart. Of note, the variety of protein species in the corona formed in a dynamic flow was significantly wider. Collectively, these results demonstrated that the corona of circulating PEGylated liposomes can be considerably different from that formed in a static fluid. This seems to be a key factor to predict the biological activity of a liposomal formulation in a physiological environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Here we introduce a proteomics methodology based on nanoliquid-chromatography tandem mass spectrometry (nanoLC/MS-MS) to investigate the "protein corona effect for targeted drug delivery", an innovative strategy, which exploits the "protein corona" that forms around nanoparticles in a physiological environment to target cells.

When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.

Surfactant nanocarriers have received considerable attention in the last several years as interesting alternative to classic liposomes. Different pH-sensitive vesicular colloidal carriers based on Tween 20 derivatives, obtained after functionalization of the head groups of the surfactant with natural, or simply modified, amino acids, were proposed as drug nanocarriers. Dynamic light scattering, Small Angle X-ray Scattering, Trasmission Electron Microscopy and fluorescence studies were used for the physico-chemical characterization of vesicles and mean size, size distribution, zeta potential, vesicle morphology and bilayer properties were evaluated. The pH-sensitivity and the stability of formulations, in absence and in presence of foetal bovine serum, were also evaluated. Moreover, the contact between surfactant vesicles and liposomes designed to model the cellular membrane was investigated by fluorescence studies to preliminary explore the potential interaction between vesicle and cell membranes. Experimental findings showed that physico-chemical and technological features of pH-sensitive vesicles were influenced by the composition of the carriers. Furthermore, proposed carriers are able to interact with mimetic cell membrane and it is reasonable to attribute the observed differences in interaction to the architectural/structural properties of Tween 20 derivatives. The findings reported in this investigation showed that a deep and extensive physico-chemical characterization of the carrier is a fundamental step, according to the evidence that the knowledge of nanocarrier properties is necessary to translate its potentiality to in vitro/in vivo applications.

Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a "protein corona" onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.

The concept that the effective unit of interest in the cell-nanomaterial interaction is the particle and its corona of associated proteins is emerging. Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concentrations (2.5-80%). The composition of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. We show that the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes with the plasma concentration. This result may have deep biological implications for the application of lipid-based gene vectors both in vitro and in vivo., (© 2011 American Chemical Society)

The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids.

The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

The self-assembling of cationic liposomes (CLs) and DNA can give rise to a variety of nanostructures and morphologies. Multilamellar complexes are made of DNA intercalated between opposing lipid bilayers, while clusters formed by intact vesicles may exist as 'beads-on-a-string' (i.e. CLs attached to a string of DNA). Precise knowledge of the structure and morphology of complexes is relevant in many biological important processes such as gene delivery. Here dynamic light scattering, zeta potential, synchrotron small angle X-ray scattering and one-dimensional polyacrylamide gel electrophoresis were performed to investigate the equilibrium structure, morphology and interactions with plasma proteins of lipoplexes made of the cationic lipid (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl])-cholesterol (DC-Chol), the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE) and DNA. Results show that DC-Chol-DOPE/DNA complexes are multilamellar systems with DNA protected by cationic lipids. On the other hand, the 'protein corona' associated to lipoplexes after interaction with human plasma was found to be much richer in basic immunoglobulins gamma proteins (Ig-Gs) than that of pure lipid vesicles in the absence of DNA. According to the most recent evidences reported in the literature, this finding would suggest the existence of hybrid structures made of multilamellar complexes either stuck together by DNA or coexisting with DNA-loaded intact vesicles. Because surface properties of lipoplexes may determine their interaction with cells and tissues, these results may be important for predicting biological responses in vivo., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids.

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a 'protein corona'. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications., (Copyright 2009 Elsevier B.V. All rights reserved.)

We have investigated the effect of serum on nanometric structure, size, surface potential, DNA-binding capacity, and transfection efficiency of DDAB-DOPE/DNA and DC-Chol-DOPE/DNA lipoplexes as a function of membrane charge density and cationic lipid/DNA charge ratio. In the absence of serum, the nanometric structure and DNA binding capacity of lipoplexes determined the transfection efficiency. When serum was added, the transfection efficiency of all lipoplex formulations was found to increase. We identified structural stability and an increase in size in serum as major parameters regulating the efficiency of lipofection. By extrapolation, we propose that serum, regulating the size of resistant lipid-DNA complexes, can control the mechanism of internalization of lipoplexes and, in turn, their efficiency.

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.

Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (lipoplexes) and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent lipoplexes, incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than binary lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge density of lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes induced by anionic lipids strictly depended on the lipid composition of lipoplexes. We found evidence of the existence of three different regimes of stability related to the interaction between complexes and anionic membranes. Both unstable (with low membrane charge density, sigmaM) and highly stable lipoplexes (with high sigmaM) exhibited low transfection efficiency whereas highly efficient multicomponent lipoplexes exhibited an "optimal stability". This intermediate regime reflects a compromise between two opposing constraints: protection of DNA in the cytosol and endosomal escape. Here we advance the concept that structural stability, upon interaction with cellular anionic lipids, is a key factor governing the transfection efficiency of lipoplexes. Possible molecular mechanisms underlying experimental observations are also discussed.

The mechanism of formation of multicomponent lipoplexes was investigated by means of synchrotron Small-Angle X-ray Diffraction (SAXD). Mixed lipid dispersions were prepared by mixing different populations of binary cationic liposomes. When adding DNA to mixed lipid dispersions, multicomponent lipoplexes spontaneously formed exhibiting structural properties, i.e., membrane thickness, surface charge density, and one-dimensional DNA packing density, intermediate between those of binary lipoplexes. These results suggested that DNA lets liposomes come into contact and fuse and that a complete lipid mixing at the molecular level occurs. The equilibrium structure of multicomponent lipoplexes was found to be unique and did not depend on the number and kind of populations composing lipid dispersion but only on the lipid species involved and on their relative molar ratio. According to recent theoretical models we identified two-dimensional lipid mixing entropy as the key factor regulating the existence of only multicomponent lipoplexes with ideally mixed lipid species.

The formation of lipid-DNA (CL-DNA) complexes called lipoplexes, proposed as DNA vectors in gene therapy, is obtained by adding DNA to a solution containing liposomes composed of cationic and neutral lipids. The structural and dynamic properties of such lipoplexes are determined by a coupling between the electrostatic interactions and the elastic parameters of the lipid mixture. An attempt to achieve a better understanding of the structure-dynamics relationship is reported herein. In particular, an elastic neutron scattering investigation of DOTAP-DOPC (dioleoyl trimethylammonium propane-dioleoyl phosphatidylcoline) complexed with DNA is described. Proton dynamics in this oriented CL-DNA lipoplex is found to be strongly dependent upon DNA concentration. Our results show that a substantial modification of the membrane dynamics is accompanied by the balancing of the total net charge inside the complex, together with the consequent displacement of interlayer water molecules.

The supramolecular structural nature of some cationic liposomes-DNA complexes, currently used as vehicles in non-viral gene delivery, has been elucidated by recent X-ray diffraction experiments. The relationship between the chemico-physical properties of these self-assembled structures and their transfection efficiency is extensively studied. Here we report a first comprehensive structural study by using energy dispersive X-ray diffraction, of the complex DC-Chol--DOPE/DNA (3beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol dioleoylphosphatidylethanolamine/DNA), which has been classified as one of the most effective in in-vivo experiments. Our results show that DC-Chol--DOPE/DNA lipoplexes have a columnar inverted hexagonal structure, which is not influenced by the cationic liposome/DNA charge ratio. The transfection efficiency of C6 rat glioma cells by DC-Chol--DOPE/DNA lipoplexes and the toxicity of lipoplexes to cells are dramatically affected by cationic liposome/DNA weight ratio. It seems therefore that the lipoplex structures have not any influence on transfection efficiency and toxicity in our experimental system.

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

In order to utilize virosomes or proteoliposomes for the delivery of drugs or macromolecules to specific pathologic target cells we elaborated a system to shuttle drugs to solid tissue (liver) as well as to the macrophages, a crucial cellular compartment of the immune system. Using virosomes prepared from the P3HR1 strain of Epstein-Barr virus, we demonstrated that these particles fused with human hepatocarcinoma cell line Li7A and therefore might be used as drug vectors. Furthermore, we report that proteoliposomes prepared by reconstituting in a cocktail of phosphatidylserine-phosphatidylcholine the anion transporter band 3 protein markedly increased the phagocytic activity of macrophages in culture. This could represent a new device to be used as a drug delivery system to enhance specific macrophagic functions.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

We report a comprehensive study of the lamellar dioleoyl trimethylammonium propane and DNA complexes (DOTAP–DNA) as a function of the lipid/DNA weight ratio ρ in order to determine their structure and thermodynamic stability using an energy dispersive X-ray diffraction technique (EDXD). These self-assemblies consist of a periodic multilayer structure with DNA chains adsorbed between cationic membranes. Furthermore, above and below the isoelectric point, the DNA packing density significantly varies as a function of ρ. Our results confirm that EDXD is a powerful technique to characterize self-assembled ordered structures of oppositely charged macromolecules. [Copyright &y& Elsevier]