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Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol–DOPE/DNA lipoplexes in serum

Abstract

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.

Published in Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010

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Evolution of the protein corona of lipid gene vectors as a function of plasma concentration

Abstract

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.

Published in Langmuir, 2011

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Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells

Abstract

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.

Published in Molecular pharmaceutics, 2012

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Cationic liposome/DNA complexes: from structure to interactions with cellular membranes

Abstract

Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.

Published in European biophysics journal, 2012

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Time evolution of nanoparticle–protein corona in human plasma: relevance for targeted drug delivery

Abstract

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as “protein corona”. This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle’s lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol–DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol–DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C–II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol–DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

Published in Langmuir, 2013

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Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems

Abstract

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Published in Molecular pharmaceutics, 2013

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Selective targeting capability acquired with a protein corona adsorbed on the surface of 1, 2-dioleoyl-3-trimethylammonium propane/DNA nanoparticles

Abstract

A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers in vivo, can be used for targeting. Here, we show that nanoparticles can acquire a selective targeting capability with a protein corona adsorbed on the surface. We demonstrate that lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction with human plasma components, spontaneously become coated with vitronectin that promotes efficient uptake in cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor.

Published in ACS applied materials & interfaces, 2013

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Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition

Abstract

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.

Published in Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014

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Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells

Abstract

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.

Published in Nanoscale, 2014

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The liposome–protein corona in mice and humans and its implications for in vivo delivery

Abstract

As soon as nanomaterials, such as nanoparticles (NPs), are injected into a physiological environment a rich coating of biomolecules known as the “protein corona” rapidly covers them. This protein dress is the main factor, which affects the interaction of NPs with living systems. While the relationship between NP features and the biomolecule corona has been extensively investigated, whether and how changes in the physiological environment affect the NP–protein corona remains under-investigated. This is one of the most important steps in translating results in animal models to the clinic. Here we investigated thoroughly the biological identity of lipid NPs (size, charge, aggregation state and composition of the corona) after incubation with human plasma (HP) and mouse plasma (MP) by dynamic light scattering, micro-electrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC/MS-MS). Specifically, we used two different liposomal formulations: the first one was made of polyethyleneglycol (PEG)-coated 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), while the second one was made of 30% of DOTAP, 50% of neutral saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 20% cholesterol. The temporal evolution and complexity of the NP–protein corona was found to be strongly dependent on the biological environment. In MP, liposomes were more negatively charged, less enriched in opsonins and appreciably more enriched in apolipoproteins than their counterparts in HP. Collectively, our results suggest that the biological identities of NPs in mice and humans can be markedly different from each other. Relevance of results to in vivo applications is discussed.

Published in Journal of Materials Chemistry B, 2014

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Size and charge of nanoparticles following incubation with human plasma of healthy and pancreatic cancer patients

Abstract

When nanoparticles (NPs) enter a biological environment, proteins bind to their surface forming a protein coating, which alters NP features giving it a biological identity, which controls its physiological response. The NP biological identity (size, charge and aggregation state) does strictly correlate with its physicochemical properties and the nature of the biological environment. While the former relationship has been extensively investigated, whether and how alterations in the physiological environment affect the biological identity of the NPs remains unclear. In this work we enrolled healthy and histologically proven pancreatic cancer patients. A statistically significant reduction in the level of clinically relevant proteins in cancer patients occurred. Positively and negatively charged lipid nanoparticles with two different surface chemistries (plain and PEGylated) were incubated with human plasma from both groups and characterized thoroughly by dynamic light scattering and zeta potential measurements. Only when plain positively charged NPs were tested, significant difference in zeta-potential between healthy and pancreatic cancer groups was found. This result implies that pooling human plasma from healthy volunteers might lead to a bias and thus unpredictable consequences in regard to previously optimized targeting profile.

Published in Colloids and Surfaces B: Biointerfaces, 2014

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Liposome–protein corona in a physiological environment: challenges and opportunities for targeted delivery of nanomedicines

Abstract

Active targeting that exploits the (over)expression of surface receptors in target cells by ligand incorporation is a central concept in nanomedicine research. Despite unprecedented efforts, no targeted liposome-based therapeutics is commercially available for clinical practice. What is inhibiting the efficient translation of targeted liposome technology from bench to bedside? After introduction in the bloodstream, the lipid surface is immediately modified by the adsorption of a “protein corona” and preserving the surface functionality appears to be challenging. On the other hand, a long-standing corona with receptor-binding sites could associate with the target cell long enough to activate the cell's uptake machinery, triggering liposome endocytosis and intracellular cargo delivery. This opens the intriguing possibility to manipulate the corona composition by liposome design. This review will focus on the emerging field of liposome–protein corona research from basic, descriptive research to readily applicable knowledge and technologies for implementation in drug improvement and development.

Published in Nanomedicine: Nanotechnology, Biology and Medicine, 2015

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Surface chemistry and serum type both determine the nanoparticle–protein corona

Abstract

The protein corona that forms around nanoparticles in vivo is a critical factor that affects their physiological response. The potential to manipulate nanoparticle characteristics such that either proteins advantageous for delivery are recruited and/or detrimental proteins are avoided offers exciting possibilities for improving drug delivery. In this work, we used nanoliquid chromatography tandem mass spectrometry to characterize the corona of five lipid formulations after incubation in mouse and human plasma with the hope of providing data that may contribute to a better understanding of the role played by both the nanoparticle properties and the physiological environment in recruiting specific proteins to the corona. Notably, we showed that minor changes in the lipid composition might critically affect the protein corona composition demonstrating that the surface chemistry and arrangement of lipid functional groups are key players that regulate the liposome–protein interactions. Notably, we provided evidence that the protein corona that forms around liposomes is strongly affected by the physiological environment, i.e., the serum type. These results are likely to suggest that the translation of novel pharmaceutical formulations from animal models to the clinic must be evaluated on a case-by-case basis.

Published in Journal of proteomics, 2015

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Lipid composition: a ‘key factor’ for the rational manipulation of the liposome–protein corona by liposome design

Abstract

When liposomes are exposed to biological fluids, a dynamic coating of proteins immediately covers them. Similarly to the aura of plasma surrounding the Sun, plasma proteins are thought of as establishing an aura that surrounds each liposome, hence the phenomenon was dubbed ‘protein corona’. This natural functionalization includes proteins engaged from the blood that can interact with receptors (over)expressed on the plasma membrane of target cells, thus targeting the liposomes to their final destination. Exploiting the liposome–protein corona for targeting has the potential to revolutionize the treatment of many disorders and requires a deep understanding of the factors shaping the corona. Following incubation with human plasma (HP), here we manipulated this corona by using six liposomal formulations with systematic changes in lipid composition. The lipids we employed are among the most frequently used lipid species for drug and gene delivery applications in vitro and in vivo. The six liposome–protein coronas were thoroughly characterized by synchrotron small angle X-ray scattering, dynamic light scattering, zeta-potential and nanoliquid-chromatography tandem mass spectrometry experiments. We identified general principles shaping the liposome–protein corona and established clear-cut relationships between lipid species and classes of plasma proteins. This knowledge sets the basis for a rational manipulation of the protein corona for targeted drug delivery by liposome design.

Published in RSC advances, 2015

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The biomolecular corona of nanoparticles in circulating biological media

Abstract

When nanoparticles come into contact with biological media, they are covered by a biomolecular ‘corona’, which confers a new identity to the particles. In all the studies reported so far nanoparticles are incubated with isolated plasma or serum that are used as a model for protein adsorption. Anyway, bodily fluids are dynamic in nature so the question arises on whether the incubation protocol, i.e. dynamic vs. static incubation, could affect the composition and structure of the biomolecular corona. Here we let multicomponent liposomes interact with fetal bovine serum (FBS) both statically and dynamically, i.e. in contact with circulating FBS (≈40 cm s−1). The structure and composition of the liposome–protein corona, as determined by dynamic light scattering, electrophoretic light scattering and liquid chromatography tandem mass spectrometry, were found to be dependent on the incubation protocol. Specifically, following dynamic exposure to FBS, multicomponent liposomes were less enriched in complement proteins and appreciably more enriched in apolipoproteins and acute phase proteins (e.g. alpha-1-antitrypsin and inter-alpha-trypsin inhibitor heavy chain H3) that are involved in relevant interactions between nanoparticles and living systems. Supported by our results, we speculate that efficient predictive modeling of nanoparticle behavior in vivo will require accurate knowledge of nanoparticle-specific protein fingerprints in circulating biological media.

Published in Nanoscale, 2015

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Stealth effect of biomolecular corona on nanoparticle uptake by immune cells

Abstract

When injected in a biological milieu, a nanomaterial rapidly adsorbs biomolecules forming a biomolecular corona. The biomolecular corona changes the interfacial composition of a nanomaterial giving it a biological identity that determines the physiological response. Characterization of the biomolecular structure and composition has received increasing attention mostly due to its detrimental impact on the nanomaterial’s metabolism in vivo. It is generally accepted that an opsonin-enriched biomolecular corona promotes immune system recognition and rapid clearance from circulation. Here we applied dynamic light scattering and nanoliquid chromatography tandem mass spectrometry to thoroughly characterize the biomolecular corona formed around lipid and silica nanoparticles (NPs). Incubation with human plasma resulted in the formation of NP-biomolecular coronas enriched with immunoglobulins, complement factors, and coagulation proteins that bind to surface receptors on immune cells and elicit phagocytosis. Conversely, we found that protein-coated NPs were protected from uptake by macrophage RAW 264.7 cells. This implies that the biomolecular corona formation provides a stealth effect on macrophage recognition. Our results suggest that correct prediction of the NP’s fate in vivo will require more than just the knowledge of the biomolecular corona composition. Validation of efficient methods for mapping protein binding sites on the biomolecular corona of NPs is an urgent task for future research.

Published in Langmuir, 2015

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The protein corona of circulating PEGylated liposomes

Abstract

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.

Published in Biochimica et Biophysica Acta (BBA) - Biomembranes, 2016

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Exploring cellular interactions of liposomes using protein corona fingerprints and physicochemical properties

Abstract

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.

Published in ACS nano, 2016

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The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

Abstract

Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

Published in Scientific reports, 2016

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Exploitation of nanoparticle–protein corona for emerging therapeutic and diagnostic applications

Abstract

Exposure of nanoparticles (NPs) to biological fluids (e.g., plasma, interstitial fluid, and cytoplasm) leads to the absorption of proteins on the NP surface, forming a protein corona (PC) that drastically influences the NP physicochemical properties. Herein, we highlight the emerging applications of PC towards its use in therapeutics and diagnostics. In particular, special emphasis is given to the exploitation of PC for targeted delivery of nanomaterials and early cancer detection. By highlighting such recent applications of PC, we hope to demonstrate that this bio-entity has the potential to determine the success of NPs in biomedicine beyond their currently envisioned purposes.

Published in Journal of Materials Chemistry B, 2016

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Personalized liposome–protein corona in the blood of breast, gastric and pancreatic cancer patients

Abstract

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.

Published in The international journal of biochemistry & cell biology, 2016

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Nanoparticles-cell association predicted by protein corona fingerprints

Abstract

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a “protein corona” layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ≈ 100–250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs’ physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.

Published in Nanoscale, 2016

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Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles

Abstract

Fluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system’s symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations.

Published in Acta biomaterialia, 2016

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A protein corona-enabled blood test for early cancer detection

Abstract

Pancreatic cancer is a very aggressive malignancy that is often diagnosed in the advanced stages, with the implication that long-term survivors are extremely rare. Thus, developing new methods for the early detection of pancreatic cancer is an urgent task for current research. To date, nanotechnology offers unprecedented opportunities for cancer therapeutics and diagnosis. The aim of this study is the development of a new pancreatic cancer diagnostic technology based on the exploitation of the nano-bio-interactions between nanoparticles and blood samples. In this study, blood samples from 20 pancreatic cancer patients and 5 patients without malignancy were allowed to interact with designed lipid nanoparticles, leading to the formation of a hard “protein corona” at the nanoparticle surface. After isolation, the protein patterns were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). We found that the protein corona of pancreatic cancer patients was much more enriched than that of healthy individuals. Statistical analysis of SDS-PAGE results allowed us to discriminate between healthy and pancreatic cancer patients with a total discriminate correctness rate of 88%.

Published in Nanoscale, 2017

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In vivo protein corona patterns of lipid nanoparticles

Abstract

In physiological environments (e.g. the blood), nanoparticles (NPs) are surrounded by a layer of biomolecules referred to as a ‘protein corona’ (PC). The most tightly NP-bound proteins form the so-called hard corona (HC), the key bio-entity that determines the NP's biological identity and physiological response. To date, NP-HC has been almost exclusively characterized in vitro, while NP–protein interactions under realistic in vivo conditions remain largely unexplored. In this study, we thoroughly characterized the in vivo HC of a NP formulation that forms around lipid nanoparticles with a lipid composition equal to that of clinically used liposomal amphotericin B (AmBisome®) after the recovery of the NPs from the blood circulation of FVB/N mice 10 minutes post intravenous administration. In vitro HC formed by 10 minutes incubation of NPs in FVB/N mouse plasma was used for comparison. Here we show that the biological identity (i.e. size, zeta-potential and aggregation state) of NPs in vivo is significantly different from that acquired in vitro. Furthermore, the variety of protein species in the in vivo HC was considerably larger. The present work has demonstrated that characterization of the in vivo HC is essential to provide an accurate molecular description of the biological identity of NPs in physiological environments.

Published in RSC advances, 2017

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Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells

Abstract

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.

Published in Nanomedicine: Nanotechnology, Biology and Medicine, 2017

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Biological identity of nanoparticles in vivo: clinical implications of the protein corona

Abstract

Despite the advances in biomedical applications of nanoparticle (NP) and numerous publications, few NPs have made it to clinical trials and even fewer have reached clinical practice. This wide gap between bench discoveries and clinical applications is mainly because of our limited understanding of the biological identity of NPs. In physiological environments, NPs are coated by a ‘protein corona’ (PC), critically affecting physiological and therapeutic responses. To date, nearly all studies attempting to characterize the PC have been conducted in vitro. Here, we review recent advances in our understanding of the in vivo PC. We also discuss recent developments of quantitative models to predict biological interactions and how they offer new opportunities for the clinical translation of NPs.

Published in Trends in Biotechnology, 2017

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Influence of dynamic flow environment on nanoparticle-protein corona: From protein patterns to uptake in cancer cells

Abstract

The fast growing use of nanoparticles (NPs) in biotechnology and biomedicine raises concerns about human health and the environment. When introduced in physiological milieus, NPs adsorb biomolecules (especially proteins) forming the so-called protein corona (PC). As it is the PC that mostly interacts with biological systems, it represents a major element of the NPs’ biological identity with impact on nanotoxicology, nanosafety and targeted delivery of nanomedicines. To date, NP-protein interactions have been largely investigated in vitro, but this condition is far from mimicking the dynamic nature of physiological environments. Here we investigate the effect of shear stress on PC by exposing lipid NPs with different surface chemistry (either unmodified and PEGylated) to circulating fetal bovine serum (FBS). PC formed upon in vitro incubation was used as a reference. We demonstrate that PC is significantly influenced by exposure to dynamic flow and that changes in PC composition are dependent on both exposure time and NP’s surface chemistry.

Published in Colloids and Surfaces B: Biointerfaces, 2017

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Impact of the biomolecular corona on the structure of PEGylated liposomes

Abstract

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.

Published in Biomaterials science, 2017

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Clinically approved PEGylated nanoparticles are covered by a protein corona that boosts the uptake by cancer cells

Abstract

Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been ‘opsonization’, i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not “stealth” in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.

Published in Nanoscale, 2017

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An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles

Abstract

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP–cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Published in Nanoscale, 2017

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Dynamic fingerprinting of sub-cellular nanostructures by image mean square displacement analysis

Abstract

Here we provide demonstration that image mean square displacement (iMSD) analysis is a fast and robust platform to address living matter dynamic organization at the level of sub-cellular nanostructures (e.g. endocytic vesicles, early/late endosomes, lysosomes), with no a-priori knowledge of the system, and no need to extract single trajectories. From each iMSD, a unique triplet of average parameters (namely: diffusivity, anomalous coefficient, size) are extracted and represented in a 3D parametric space, where clustering of single-cell points readily defines the structure “dynamic fingerprint”, at the whole-cell-population level. We demonstrate that different sub-cellular structures segregate into separate regions of the parametric space. The potency of this approach is further proved through application to two exemplary, still controversial, cases: i) the intracellular trafficking of lysosomes, comprising both free diffusion and directed motion along cytoskeletal components, and ii) the evolving dynamic properties of macropinosomes, passing from early to late stages of intracellular trafficking. We strongly believe this strategy may represent a flexible, multiplexed platform to address the dynamic properties of living matter at the sub-cellular level, both in the physiological and pathological state.

Published in Scientific reports, 2017

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Clinically approved liposomal nanomedicines: lessons learned from the biomolecular corona

Abstract

Nowadays, liposomes are the most successful drug delivery systems with a dozen drug products available in the clinic. Grafting poly-(ethylene glycol) (PEG) onto the liposome surface prevents protein binding thus prolonging blood circulation, while synthetic modification of the terminal PEG molecule with ligands (e.g. monoclonal antibodies and peptides) should promote selective accumulation in the tumor region with respect to healthy tissues. However, despite big efforts, advances have not outgrown the development stage and just a few targeted liposomal drugs are commercially available. The latest studies have clarified that following exposure to physiological environments liposomes are covered by a biomolecular corona (BC). Main factors shaping the BC are the liposomes’ physicochemical properties (i.e. size, surface charge and lipid composition), the biological fluid (e.g. plasma of healthy volunteers vs. plasma of cancer patients) and environmental factors (e.g. temperature). Combining the most recent evidence reported in the literature, herein we suggest that the liposome–BC could act as a personalized “endogenous trigger” affecting off-target interactions and controlling the indication for disease of clinically approved formulations. In this Opinion paper, we suggest that a better understanding of the liposome–BC together with improvements in mapping corona proteins will open the fascinating possibility to manipulate the BC by liposome design. This is not an easy task, but it could represent a turning point in the development of novel liposome-based targeting strategies for personalized nanomedicines.

Published in Nanoscale, 2018

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Human biomolecular corona of liposomal doxorubicin: The overlooked factor in anticancer drug delivery

Abstract

More than 20 years after its approval by the Food and Drug Administration (FDA), liposomal doxorubicin (DOX) is still the drug of choice for the treatment of breast cancer and other conditions such as ovarian cancer and multiple myeloma. Yet, despite the efforts, liposomal DOX did not satisfy expectations at the clinical level. When liposomal drugs enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BC). The BC changes liposome’s synthetic identity, providing it with a new one, referred to as “biological identity” (size, aggregation state, and BC composition). Today, the concept is emerging that specific BCs may determine either success (e.g., stealth effect and accumulation at the target site) or failure (e.g., rapid blood clearance and off-target interactions) of liposomal drugs. To get a comprehensive investigation of liposome synthetic identity, biological identity, and cellular response as a function of human plasma (HP) concentration, here we used a straightforward combination of quantitative analytical and imaging tools, including dynamic light scattering, microelectrophoresis, synchrotron small-angle X-ray scattering, transmission electron microscopy (TEM), fluorescence lifetime imaging microscopy (FLIM), nano-liquid chromatography tandem mass spectrometry/mass spectrometry (nano-LC–MS/MS), confocal microscopy, flow cytometry, and cell viability assays. Doxoves was selected as a reference. Following exposure to HP, Doxoves was surrounded by a complex BC that changed liposome’s synthetic identity. Observations made with nano-LC–MS/MS revealed that the BC of Doxoves did not evolve as a function of HP concentration and was poorly enriched of typical “opsonins” (complement proteins, immunoglobulins, etc.). This provides a possible explanation for the prolonged blood circulation of liposomal DOX. On the other hand, flow cytometry showed that protein binding reduced the internalization of DOX in MCF7 and MDA-MB-435S human breast carcinoma. Combining FLIM and TEM experiments, we clarified that reduction in DOX intracellular content was likely due to the frequent rupture of the liposome membrane and consequent leakage of the cargo. In light of reported results, we are prompted to speculate that a detailed understanding of BC formation, composition, and effects on liposome stability and uptake is an indispensable task of future research in the field, especially along the way to clinical translation of liposomal drugs.

Published in ACS applied materials & interfaces, 2018

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Brain targeting by liposome–biomolecular corona boosts anticancer efficacy of temozolomide in glioblastoma cells

Abstract

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.

Published in ACS Chemical Neuroscience, 2018

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Principal component analysis of personalized biomolecular corona data for early disease detection

Abstract

Today, early disease detection (EDD) is a matter of more importance than ever in medicine. Upon interaction with human plasma, nanoparticles are covered by proteins leading to formation of a biomolecular corona (BC). As the protein patterns of patients with conditions differ from those of healthy subjects, current research into technologies based on the exploitation of personalized BC patterns could be a turning point for early disease detection. Here, we present a framework based on principal component analysis of large personalized BC datasets. We comment on how principal component analysis of personalized BC data is a fundamental step towards turning the output of basic research into fast, safe and inexpensive technologies with superior prediction ability than current methods.

Published in Nano Today, 2018

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Improving the accuracy of pancreatic cancer clinical staging by exploitation of nanoparticle-blood interactions: a pilot study

Abstract

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.

Published in Pancreatology, 2018

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Cationic lipid/DNA complexes manufactured by microfluidics and bulk self-assembly exhibit different transfection behavior

Abstract

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.

Published in Biochemical and biophysical research communications, 2018

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Microfluidic manufacturing of surface-functionalized graphene oxide nanoflakes for gene delivery

Abstract

Graphene oxide (GO) is a single-atomic-layered material made of a sheet of oxidized carbon atoms arranged in a honeycomb structure. Thanks to the notable physical and chemical properties of GO, GO-based nanomaterials have applications in many fields of research, including gene delivery. It has been reported that pristine GO can absorb single-stranded DNA and RNA through π–π stacking, which cannot be used as a gene carrier because it is hard to load double-stranded DNA (dsDNA). To tackle this issue, this work was aimed at developing a hybrid nanoparticle (NP) system made of GO coated with cationic lipids (hereafter referred to as GOCL) with suitable physical–chemical properties for gene delivery applications. To this end, nanosized GO flakes (nGO) were coated with the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) by microfluidic mixing. Comprehensive characterization of GOCL NPs was performed by a combination of dynamic light scattering (DLS), micro-electrophoresis and atom force microscopy (AFM). Our results show that GOCL NPs exhibit adequate size (<150 nm) and surface charge (ξ = +15 mV) for gene delivery purposes. Complexes made of GOCL NPs and plasmid DNA (pDNA) were used to transfect human cervical cancer cells (HeLa) and human embryonic kidney (HEK-293) cells. Pristine nGO and DOTAP cationic liposomes were used as a reference. GOCL NPs exhibited a similar TE but a much higher cell viability compared with DOTAP cationic liposomes. Confocal fluorescence microscopy provided a reasonable explanation for the superior performance of GOCL/DNA complexes showing that they are much more numerous, regular in size and homogeneously distributed than DOTAP/DNA complexes, thus splitting their gene payload over the entire cell population. Because of the imperative demand for efficient and safe nanocarriers, this study will contribute to the development of novel surface-functionalized GO-based hybrid gene vectors.

Published in Nanoscale, 2019

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Protein corona fingerprints of liposomes: New opportunities for targeted drug delivery and early detection in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of cancer-related mortality in the Western world and is envisaged to become the second cause by 2030. Although our knowledge about the molecular biology of PDAC is continuously increasing, this progress has not been translated into better patients’ outcome. Liposomes have been used to circumvent concerns associated with the low efficiency of anticancer drugs such as severe side effects and damage of healthy tissues, but they have not resulted in improved efficacy as yet. Recently, the concept is emerging that the limited success of liposomal drugs in clinical practice is due to our poor knowledge of the nano–bio interactions experienced by liposomes in vivo. After systemic administration, lipid vesicles are covered by plasma proteins forming a biomolecular coating, referred to as the protein corona (PC). Recent studies have clarified that just a minor fraction of the hundreds of bound plasma proteins, referred to as “PC fingerprints” (PCFs), enhance liposome association with cancer cells, triggering efficient particle internalization. In this study, we synthesized a library of 10 liposomal formulations with systematic changes in lipid composition and exposed them to human plasma (HP). Size, zeta-potential, and corona composition of the resulting liposome–protein complexes were thoroughly characterized by dynamic light scattering (DLS), micro-electrophoresis, and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS). According to the recent literature, enrichment in PCFs was used to predict the targeting ability of synthesized liposomal formulations. Here we show that the predicted targeting capability of liposome–protein complexes clearly correlate with cellular uptake in pancreatic adenocarcinoma (PANC-1) and insulinoma (INS-1) cells as quantified by flow-assisted cell sorting (FACS). Of note, cellular uptake of the liposomal formulation with the highest abundance of PCFs was much larger than that of Onivyde®, an Irinotecan liposomal drug approved by the Food and Drug Administration in 2015 for the treatment of metastatic PDAC. Given the urgent need of efficient nanocarriers for the treatment of PDAC, we envision that our results will pave the way for the development of more efficient PC-based targeted nanomaterials. Here we also show that some BCs are enriched with plasma proteins that are associated with the onset and progression of PDAC (e.g., sex hormone-binding globulin, Ficolin-3, plasma protease C1 inhibitor, etc.). This could open the intriguing possibility to identify novel biomarkers.

Published in Pharmaceutics, 2019

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Exploitation of nanoparticle-protein interactions for early disease detection

Abstract

The main diagnostic tools for primary and metastatic central nervous system (CNS) tumors are the anamnestic neurological examination and the imaging tests, which are expensive and lack specificity. In recent years, the shell of macromolecules which forms on nanoparticles (NPs) when they are exposed to human blood, also known as hard corona (HC), became a powerful tool in diagnostics. Indeed, HC can act as a “nano-concentrator” of serum proteins and can detect minor changes in the protein concentration at the very early stages of disease development. In this paper, we characterized lipid NP HC formed in blood samples from patients affected by meningeal tumors. We found that the HCs of meningeal tumor patients could be discriminated from those of healthy subjects. Our results show that emerging HC-based technologies will pave the way for early diagnosis of CNS cancer.

Published in Applied Physics Letters, 2019

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Effect of molecular crowding on the biological identity of liposomes: an overlooked factor at the bio-nano interface

Abstract

Once embedded in a physiological environment, the surface of nanoparticles (NPs) gets covered with a biomolecular corona (BC) that alters their synthetic characteristics and subsequently gives them a peculiar biological identity. Despite recent studies having clarified the role of NP composition, surface chemistry and biological source (e.g., human/animal serum or plasma) in the formation of the BC, little is known about the possible impact of molecular crowding. To fill this gap, we used a cationic liposomal formulation as a model system and studied its biological identity upon incubation with human plasma, at a fixed liposome-to-plasma volume ratio and different concentrations. We carried out dynamic light scattering measurements to quantify the size and zeta potential of the investigated systems and gel electrophoresis to evaluate the composition of the corresponding coronas. Our findings suggest that NP stability may be compromised by molecular crowding, but the corona composition is stable over a wide range of concentrations, which extend over more than two orders of magnitude. As the biological identity of NPs eventually determines their final fate in vivo, we predict that this study could contribute to the development of a safe and effective nanosystem for the targeted delivery of therapeutic agents.

Published in Nanoscale Advances, 2019

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Microfluidic-generated lipid-graphene oxide nanoparticles for gene delivery

Abstract

Graphene oxide (GO) is employed in a broad range of biomedical applications including antimicrobial therapies, scaffolds for tissue engineering, and drug delivery, among others. However, the inability to load it efficiently with double-stranded DNA impairs its use as a gene delivery system. To overcome this limitation, in this work, the functionalization of GO with cationic lipids (CL) is proficiently accomplished by microfluidic manufacturing. To this end, we use CLs 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and {3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]} cholesterol (DC-Chol) and zwitterionic dioleoylphosphatidylethanolamine and cholesterol to generate a library of 9 CL formulations with systematic changes in lipid composition. Combined dynamic light scattering, microelectrophoresis, and atomic force microscopy reveal that graphene oxide/cationic lipid (GOCL) nanoparticles (NPs) are positively charged and uniformly coated by one lipid bilayer. GOCL NPs are able to condense plasmid DNA into stable, nanosized complexes whose size and zeta-potential can be finely tuned by adjusting the DNA/GOCL weight ratio, Rw. Luciferase assay results show that positively charged GOCL/DNA complexes (Rw = 0.2) efficiently transfect HeLa cells with no appreciable cytotoxicity. In particular, the ternary GOCL formulation made of DOTAP, DC-Chol, and Cholesterol (GOCL8) is as efficient as Lipofectamine® 3000 in transfecting cells, but much less cytotoxic. Confocal microscopy clarifies that the high transfection efficiency of GOCL8 is due to its massive cellular uptake and cytosolic DNA release. Implications for nonviral gene delivery applications are discussed.

Published in Applied Physics Letters, 2019

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Disease-specific protein corona sensor arrays may have disease detection capacity

Abstract

The earlier any catastrophic disease (e.g., cancer) is diagnosed, the more likely it can be treated, providing improved patient prognosis, extended survival and better quality of life. In early 2014, we revealed that various types of disease can substantially affect the composition/profile of protein corona (i.e., a layer of biomolecules that forms at the surface of nanoparticles upon their interactions with biological fluids). Here, by combining the concepts of disease-specific protein corona and sensor array technology we developed a platform with disease detection capacity using blood plasma. Our sensor array consists of three cross-reactive liposomes, with distinct lipid composition and surface charge. Rather than detecting a specific biomarker, the sensor array provides pattern recognition of the corona protein composition adsorbed on the liposomes. As a feasibility study, sensor array validation was performed using plasma samples obtained from patients diagnosed with five different cancer types (i.e. lung cancer, glioblastoma, meningioma, myeloma, and pancreatic cancer) and a control group of healthy donors. Although no single corona composition is specific for any one cancer type, overlapping but distinct patterns of the corona composition constitutes a unique “fingerprint” for each type of cancer (with a high classification accuracy, i.e. 99.4%). To finally probe the capacity of this sensor array for early detection of cancers, we used cohort plasma obtained from healthy people who were subsequently diagnosed several years after plasma collection with lung, brain, and pancreatic cancers. Our results suggest that the disease-specific protein corona sensor array will not only be instrumental in the screening, detection, and identification of diseases, but may also help identify novel protein pattern markers whose role in disease development and/or disease biology has not been appreciated so far.

Published in Nanoscale Horizons, 2019

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The biomolecular corona of gold nanoparticles in a controlled microfluidic environment

Abstract

Nanoparticles (NPs) exposed to biological media are coated by proteins and other biomolecules forming a biomolecular corona (BC) on the particle surface. Recent studies have shown that shear stress as that created by laminar fluid flow generates more complex coronas with systematic changes in composition with respect to counterparts formed under static incubation. However, in most studies reported so far, dynamic environments have been produced by peristaltic pumps and comparing experimental results appears challenging. On the other side, generating shear stress by microfluidic devices could help to remove user variability and ensure better reproducibility of experimental data. This study was therefore aimed at exploring formation of NP-BC in a microfluidic environment. To this end, 100 nm gold nanoparticles and human plasma (HP) were used as models for nano-formulation and biological medium. We injected gold nanoparticles and HP in each of the islets of a remote-controlled microfluidic cartridge. Static incubation was used as a reference. BC-decorated NPs were thoroughly characterized by dynamic light scattering (DLS), micro-electrophoresis (ME), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS). By varying the incubation time from 30 s to 2.5 min we demonstrate that BC is already determined by the earliest exposure time point and does not appreciably evolve in time. DLS and ME results demonstrate that the BC formed in a microfluidic chip is thicker and more negatively charged than its counterpart formed under static incubation. SDS-PAGE and nano-LC MS/MS revealed that the incubation procedure had a major effect on BC composition. As an example, immunoglobulins are the most abundant plasma proteins of the BC generated in a microfluidic environment (relative protein abundance ∼30%), while tissue leakage proteins (relative protein abundance ∼26%) are the most enriched proteins when the BC is formed upon static incubation. Potential implications in emerging biomedical research arenas are discussed.

Published in Lab on a Chip, 2019

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Converting the personalized biomolecular corona of graphene oxide nanoflakes into a high-throughput diagnostic test for early cancer detection

Abstract

Advances in nanotechnology are introducing the exciting possibility of cancer identification at early stages via analysis of the personalized biomolecular corona (BC), i.e. the dynamic “halo” of proteins that adsorbs onto NPs following exposure to patients' plasma. In this study, we develop a blood test for early cancer detection based on the characterization of the BC that forms around Graphene Oxide (GO) nanoflakes. Among its elective properties, GO binds low amounts of albumin, the most abundant protein in the blood and one of the most enriched proteins in the BC of many nanomaterials. This unique property of GO allows strong adsorption of poorly concentrated plasma proteins without abundant protein depletion. In our study, GO nanometric flakes have been used to analyze BCs from 50 subjects, half of them diagnosed with pancreatic cancer and half of them being healthy volunteers. Pancreatic cancer was chosen as the model of a high mortality disease with poor survival rates due to its delayed diagnosis. The receiver operating characteristic (ROC) curve analysis was applied to measure the diagnostic accuracy of the BC-based test. We obtained an area under the curve (AUC) of 0.96 and the test discriminated cancer patients from healthy subjects with a sensitivity of 92%. Finally, a double-blind validation was made using a second test dataset (10 healthy subjects + 10 pancreatic cancer patients) and it confirmed the results obtained on the first training dataset. Being highly accurate, fast, inexpensive and easy to perform, we believe that the BC-enabled blood test has the potential to become a turning point in early detection of cancer and other diseases.

Published in Nanoscale, 2019

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Interplay of protein corona and immune cells controls blood residency of liposomes

Abstract

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.

Published in Nature communications, 2019

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Challenges in molecular diagnostic research in cancer nanotechnology

Abstract

Development of robust cancer prevention strategies have the capacity to reduce the need for therapeutic products, relieve patient’s suffering and alleviate the crushing financial burden for both patients and their families. Therefore, the obvious choice that would benefit the largest number of people is to realign the importance and prioritization of diagnostics alongside therapeutics. However, entrepreneurs and policy-makers are more inclined to invest in manufacturers of therapeutics, which produce much higher revenues compared to diagnostics companies. The central aim of this opinion article is i) to identify the reasons for this chasm gap between therapeutic and diagnostic approaches in cancer research; and ii) to draw the attention of researchers, entrepreneurs, and policy-makers to the importance and potential promises of diagnostic approaches in lowering the social burden and cost of cancer.

Published in Nano Today, 2019

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Impact of the protein corona on nanomaterial immune response and targeting ability

Abstract

Over the last decade nanomaterials have had a major impact on human health for the early detection and treatment of many diseases. The future success of clinically translatable nanomaterials lies in the combination of several functionalities to realize a personalized medical experience for patients. To maintain promises, concerns arising from toxic potential and off‐target accumulation of nanomaterials must be addressed first. Upon introduction to a complex biological system (e.g., following systemic administration), nanomaterials interact with all the encountered biomolecules and form the protein corona, a complex coating of plasma proteins that provides them with a totally new biological identity. As the protein corona controls the nanomaterial behavior in vivo, a precise knowledge of the relationship between biological identity and physiological response is needed but not yet achieved. Based on impressive progress made thus far, this review critically discusses how the protein corona activates immune response and influences the targeted delivery of nanomaterials. Furthermore, we comment on emerging strategies to manipulate protein binding in order to promote formation of designer artificial coronas and achieve a desired therapeutic outcome. We conclude by debating challenges that must be overcome to obtain widespread clinical adoption of nanomaterials.

Published in Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 2020

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Nanoparticle-enabled blood tests for early detection of pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is often detected too late to allow adequate treatments with the result that patients are condemned to sufferings and early death. Most efforts have been therefore aimed at identifying sensitive PDAC biomarkers. Although biomarkers have numerous advantages, sample size, intra-individual variability, existence of several biases and confounding variables and cost of investigation make their clinical application challenging. In recent years, nanotechnology is providing new options for early cancer detection. Among recent discoveries, the concept is emerging that the protein corona, i.e. the layer of plasma proteins that surrounds nanomaterials in bodily fluids, is personalized. In particular, the protein corona of cancer patients is significantly different from that of healthy individuals. Herein, we review this concept with a particular focus on clinical relevance. We also discuss the recently developed nanoparticle-enabled blood (NEB) tests that demonstrated to be promising in discriminating PDAC patients from healthy volunteers by global change of the nanoparticle-protein corona. We conclude with a critical discussion of research perspectives aimed at further improving the prediction ability of the test.

Published in Cancer Letters, 2020

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Effect of Protein Corona on The Transfection Efficiency of Lipid-Coated Graphene Oxide-Based Cell Transfection Reagents

Abstract

Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to explore the bionano interactions between GOCL/DNA complexes (hereafter referred to as ”grapholipoplexes”) and human plasma. When exposed to increasing protein concentrations, grapholipoplexes get covered by a protein corona that evolves with protein concentration, leading to biocoronated complexes with modified physicochemical properties. Here, we show that the formation of a protein corona dramatically changes the interactions of grapholipoplexes with four cancer cell lines: two breast cancer cell lines (MDA-MB and MCF-7 cells), a malignant glioma cell line (U-87 MG) and an epithelial colorectal adenocarcinoma cell line (CACO-2). Luciferase assay clearly indicates a monotonous reduction of the transfection efficiency of biocoronated grapholipoplexes as a function of protein concentration. Finally, we report evidence that a protein corona formed at high protein concentrations (as those present in in vivo studies) promotes a higher capture of biocoronated grapholipoplexes within degradative intracellular compartments (e.g., lysosomes), with respect to their pristine counterparts. On the other hand, coronas formed at low protein concentrations (human plasma = 2.5%) lead to high transfection efficiency with no appreciable cytotoxicity. We conclude with a critical assessment of relevant perspectives for the development of novel biocoronated gene delivery systems.

Published in Pharmaceutics, 2020

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A mechanistic explanation of the inhibitory role of the protein corona on liposomal gene expression

Abstract

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.

Published in Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020

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A comprehensive analysis of liposomal biomolecular corona upon human plasma incubation: The evolution towards the lipid corona

Abstract

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.

Published in Talanta, 2020

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Personalized Graphene Oxide-Protein Corona in the Human Plasma of Pancreatic Cancer Patients

Abstract

The protein corona (PC) that forms around nanomaterials upon exposure to human biofluids (e.g., serum, plasma, cerebral spinal fluid etc.) is personalized, i.e., it depends on alterations of the human proteome as those occurring in several cancer types. This may relevant for early cancer detection when changes in concentration of typical biomarkers are often too low to be detected by blood tests. Among nanomaterials under development for in vitro diagnostic (IVD) testing, Graphene Oxide (GO) is regarded as one of the most promising ones due to its intrinsic properties and peculiar behavior in biological environments. While recent studies have explored the binding of single proteins to GO nanoflakes, unexplored variables (e.g., GO lateral size and protein concentration) leading to formation of GO-PC in human plasma (HP) have only marginally addressed so far. In this work, we studied the PC that forms around GO nanoflakes of different lateral sizes (100, 300, and 750 nm) upon exposure to HP at several dilution factors which extend over three orders of magnitude from 1 (i.e., undiluted HP) to 103. HP was collected from 20 subjects, half of them being healthy donors and half of them diagnosed with pancreatic ductal adenocarcinoma (PDAC) a lethal malignancy with poor prognosis and very low 5-year survival rate after diagnosis. By dynamic light scattering (DLS), electrophoretic light scattering (ELS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nano liquid chromatography tandem mass spectrometry (nano-LC MS/MS) experiments we show that the lateral size of GO has a minor impact, if any, on PC composition. On the other side, protein concentration strongly affects PC of GO nanoflakes. In particular, we were able to set dilution factor of HP in a way that maximizes the personalization of PC, i.e., the alteration in the protein profile of GO nanoflakes between cancer vs. non-cancer patients. We believe that this study shall contribute to a deeper understanding of the interactions among GO and HP, thus paving the way for the development of IVD tools to be used at every step of the patient pathway, from prognosis, screening, diagnosis to monitoring the progression of disease.

Published in Frontiers in Bioengineering and Biotechnology, 2020

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Protein corona-enabled serological tests for early stage cancer detection

Abstract

Early stage cancer detection is a major issue in current medicine. In recent years, nanotechnology is providing new alternatives for early diagnosis. Upon exposure to human plasma, several nanoparticle types (e.g. gold nanoparticles) are surrounded by a protein layer referred to as protein corona (PC). The PC changes the original identity of the nanoparticle conferring a new biological character. It is now accepted that slight variations in the composition of a protein source significantly varies the PC composition. Thus, nanomaterials incubated with plasma proteins of individuals with different physiological conditions generate PCs with different compositions. This gives rise to the new concept of personalized PC. Therefore, since protein patterns of subjects affected by certain pathologies differ from those of healthy ones, diagnostic technologies based on the evaluation of personalized PC could represent a fascinating opportunity for early disease detection. Herein, we review the concept of personalized PC along with recent advances on the topic, giving an overview of some innovative analytical approaches for early stage cancer detection.

Published in Sensors International, 2020

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Mechanistic Insights into the Release of Doxorubicin from Graphene Oxide in Cancer Cells

Abstract

Liposomal doxorubicin (L-DOX) is a popular drug formulation for the treatment of several cancer types (e.g., recurrent ovarian cancer, metastatic breast cancer, multiple myeloma, etc.), but poor nuclear internalization has hampered its clinical applicability so far. Therefore, novel drug-delivery nanosystems are actively researched in cancer chemotherapy. Here we demonstrate that DOX-loaded graphene oxide (GO), GO-DOX, exhibits much higher anticancer efficacy as compared to its L-DOX counterpart if administered to cellular models of breast cancer. Then, by a combination of live-cell confocal imaging and fluorescence lifetime imaging microscopy (FLIM), we suggest that GO-DOX may realize its superior performances by inducing massive intracellular DOX release (and its subsequent nuclear accumulation) upon binding to the cell plasma membrane. Reported results lay the foundation for future exploitation of these new adducts as high-performance nanochemotherapeutic agents.

Published in Nanomaterials, 2020

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Optimal centrifugal isolating of liposome–protein complexes from human plasma

Abstract

In the past few years, characterization of the protein corona (PC) that forms around liposomal systems has gained increasing interest for the development of novel therapeutic and diagnostic technologies. At the crossroads of fast-moving research fields, the interdisciplinarity of protein corona investigations poses challenges for experimental design and reporting. Isolation of liposome–protein complexes from biological fluids has been identified as a fundamental step of the entire workflow of PC characterization but exact specifications for conditions to optimize pelleting remain elusive. In the present work, key factors affecting precipitation of liposome–protein complexes by centrifugation, including time of centrifugation, total sample volume, lipid[thin space (1/6-em)]:[thin space (1/6-em)]protein ratio and contamination from biological NPs were comprehensively evaluated. Here we show that the total amount of isolated liposome–protein complexes and the extent of contamination from biological NPs may vary with influence factors. Our results provide protein corona researchers with precise indications to separate liposome–protein complexes from protein-rich fluids and include proper controls, thus they are anticipated to catalyze improved consistency of data mining and computational modelling of protein corona composition.

Published in Nanoscale Advances, 2021

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Protein corona profile of graphene oxide allows detection of glioblastoma multiforme using a simple one-dimensional gel electrophoresis technique: a proof-of-concept study

Abstract

Glioblastoma multiforme (GBM) is the most aggressive form of gliomas. The development of supplementary approaches for glioblastoma diagnosis, limited to imaging techniques and tissue biopsies so far, is a necessity of clinical relevance. In this context, nanotechnology might afford tools to enable early diagnosis. Upon exposure to biological media, nanoparticles are coated with a layer of proteins, the protein corona (PC), whose composition is individual and personalized. Here we show that the PC of graphene oxide nanosheets has a capacity to detect GBM using a simple one-dimensional gel electrophoresis technique. In a range of molecular weights between 100 and 120 kDa, the personalized PC from GBM patients is completely discernible from that of healthy donors and that of cancer patients affected by pancreatic adenocarcinoma and colorectal cancer. Using tandem mass spectrometry, we found that inter-alpha-trypsin inhibitor (ITI) heavy chain H4 is enriched in the PC of all tested individuals but not in the GBM patients. Overall, if confirmed on a larger cohort series, this approach could be advantageous at the first level of investigation to decide whether to carry out more invasive analyses and/or to follow up patients after surgery and/or pharmacological treatment.

Published in Biomaterials science, 2021

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A Proteomic Study on the Personalized Protein Corona of Liposomes. Relevance for Early Diagnosis of Pancreatic DUCTAL Adenocarcinoma and Biomarker Detection

Abstract

Due to late diagnosis, high incidence of metastasis, and poor survival rate, pancreatic cancer is one of the most leading cause of cancer-related death. Although manifold recent efforts have been done to achieve an early diagnosis of pancreatic cancer, CA-19.9 is currently the unique biomarker that is adopted for the detection, despite its limits in terms of sensitivity and specificity. To identify potential protein biomarkers for pancreatic ductal adenocarcinoma (PDAC), we used three model liposomes as nanoplatforms that accumulate proteins from human plasma and studied the composition of this biomolecular layer, which is known as protein corona. Indeed, plasma proteins adsorb on nanoparticle surface according to their abundance and affinity to the employed nanomaterial, thus even small differences between healthy and PDAC protein expression levels can be, in principle, detected. By mass spectrometry experiments, we quantified such differences and identified possible biomarkers for PDAC. Some of them are already known to exhibit different expressions in PDAC proteomes, whereas the role of other relevant proteins is still not clear. Therefore, we predict that the employment of nanomaterials and their protein corona may represent a useful tool to amplify the detection sensitivity of cancer biomarkers, which may be used for the early diagnosis of PDAC, with clinical implication for the subsequent therapy in the context of personalized medicine.

Published in Journal of Nanotheranostics, 2021

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Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery

Abstract

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Published in Pharmaceutics, 2021

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Undergraduate course, University 1, Department, 2014

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