Luca Digiacomo

Stealth Effect of Biomolecular Corona on Nanoparticle Uptake by Immune Cells

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 nanomaterials 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 NPs 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.

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

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.

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

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.

The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells.

In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

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

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.

In vivo protein corona patterns of lipid nanoparticles

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 NPs 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.

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

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.

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

UNLABELLED 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 systems 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. STATEMENT OF SIGNIFICANCE Recent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low backgrounds sensitivity. This study overcomes some of the approximations adopted so far, by decoupling the flow terms of the investigated dynamics and thus recovering ensembles information from specific single particle behaviors. Finally, relevant implications for nanoparticle-based drug delivery are shown.

Time-lapse confocal imaging datasets to assess structural and dynamic properties of subcellular nanostructures

Time-lapse optical microscopy datasets from living cells can potentially afford an enormous amount of quantitative information on the relevant structural and dynamic properties of sub-cellular organelles/structures, provided that both the spatial and temporal dimensions are properly sampled during the experiment. Here we provide exemplary live-cell, time-lapse confocal imaging datasets corresponding to three sub-cellular structures of the endo-lysosomal pathway, i.e. early endosomes, late endosomes and lysosomes, along with detailed guidelines to produce analogous experiments. Validation of the datasets is conducted by means of established analytical tools to extract the structural and dynamic properties at the sub-cellular scale, such as Single Particle Tracking (SPT) and imaging derived Mean Square Displacement (iMSD) analyses. In our aim, the present work would help other researchers in the field to reuse the provided datasets for their own scopes, and to combine their creative approaches/analyses to similar acquisitions.

Brain targeting by liposome-biomolecular corona boosts anti-cancer efficacy of Temozolomide in glioblastoma cells

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.