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

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

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

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

Efficient pancreatic cancer detection through personalized protein corona of gold nanoparticles

Characterization of the personalized protein corona (PC) that forms around nanomaterials upon exposure to human plasma is emerging as powerful technology for early cancer detection. However, low material stability and interbatch variability have limited its clinical application so far. Here, we present a nanoparticle-enabled blood (NEB) test that uses 120 nm gold nanoparticles (NPs) as the accumulator of blood plasma proteins. In the test, the personalized PC of gold NPs is characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. As a paradigmatic case study, pancreatic ductal adenocarcinoma (PDAC) was chosen due to the lack of effective detection strategies that lead to poor survival rate after diagnosis (<1 year) and extremely low 5-years survival rate (15-20%). Densitometric analysis of 75 protein patterns (28 from healthy subjects and 47 from PDAC patients) allowed us to distinguish nononcological and PDAC patients with good sensitivity (78.6%) and specificity (85.3%). The gold NEB test is completely aligned to affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria stated by the World Health Organization for cancer screening and detection. Thus, it could be very useful in clinical practice at the first level of investigation to decide whether to carry out more invasive analyses or not

Reproducibility of biomolecular corona experiments: a primer for reliable results

The biomolecular corona is a key component controlling the identity of nanomaterials in physiological environments. Studies aimed at identifying factors shaping the biomolecular corona have proliferated in the last decade but have been performed by research groups with different backgrounds. Efforts made within the scientific community to guarantee the reproducibility of experimental data have identified protocol standardization as an indispensable step for advancing knowledge in this arena. To contribute to fulfill this gap, here the relevance of interoperator variability in biomolecular corona studies and the benefits arising from automated systems usage are explored. Moreover, the role of molecular crowding during nanoparticle-biofluid incubation and the effect of washing the pellet during corona isolation are thoroughly investigated. It is believed that the findings will help researchers enhance the accuracy of experimental design and reporting

The biomolecular corona of gold nanoparticles in a controlled microfluidic environment

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

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

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

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

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

Microfluidic formulation of dna-loaded multicomponent lipid nanoparticles for gene delivery

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

Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo

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