Structural, dynamical and optical properties of gold nanorod/DNA complexes for gene delivery applications

Structural, dynamical and optical properties of gold nanorod/DNA complexes for gene delivery applications Luca Digiacomo Supervisor: Giulio Caracciolo Internal supervisor: Stefano Luin Co-supervisor: Stefano Coppola Enhancement of the optical response by nanometric metallic systems (e.g. gold and silver nanoparticles) has raised large fundamental and technological interest during the past decade, due to their localized surface plasmon resonances (LSPRs). Such enhancement is associated with the collective resonant electron oscillation, that is driven by an external electromagnetic field. Gold nanorods (AuNRs) have broadly tunable LSPRs, which can be adjusted in the visible and near-infrared (NIR) range by acting on their shape, size, and composition. The geometrical aspects of gold nanoparticles (AuNPs) can be modulated in the synthesis procedure. Generally for spherical objects, with the increase of the particle size the surface plasmon absorption maximum redshifts, when the shape of the particle changes from sphere-like to rod-like, a degree of anisotropy is introduced. As a consequence, the LSPR spectrum splits into two bands and the absorption spectrum strongly depends on the aspect ratio (i.e. the length-to-diameter ratio). Due to their interesting and versatile features, the high tolerability in living organisms and the ease of the functionalization, noble metal nanoparticles have several applications in different fields of nanotechnology, including surface-enhanced Raman spectroscopy, luminescence and fluorescence correlation spectroscopy, biological imaging and biomedicine. The biosensing and biomedical potential of these metallic nanoparticles depends on how they interact with the NIR light and the biological environment. The size affects cellular uptake, diffusion and cytotoxicity. The shape mainly rules the plasmon resonance wavelengths, therefore the optical response. They can both be modulated in the synthesis procedure. Hence, an intriguing perspective is to gain a substantial control over the principal properties of these nanoparticles, efficiently and systematically, in order to optimize them for biotechnological applications. Our work is focused on the structural and optical analysis of the systems under study, involved in gold-nanoparticle-mediated gene delivery applications: the NR/DNA complexes represent a non-viral vector, the nucleic acid (DNA) within them is their cargo, which has to be protected during the cellular uptake, while the complexes overcome several extra- and intra-cellular barriers. Once efficiently internalized, the complexes have to traverse the cytoplasm and only in the proximity of the nucleus the DNA should be released. One of the most interesting aspects of gold nanorods is the controlled gene-payload release. It can be mediated and driven by the optical response of NRs to a laser beam, tuned at the wavelength of the localized surface plasmon resonance. The electronic excitations and the subsequent nonradiative decays induce an increase of the kinetic energy, a local overheating and a rapid and deep alteration of the structure. The bonds between nanoparticles and nucleic acid are broken and, as a consequence, the DNA is released. The formation of the complexes is a self-assembling process, driven by the Coulomb interaction between the nucleic acid and a shell of cationic surfactant, namely cetyltrimethylammonium bromide (CTAB), which coats the gold nanorods. The nanoparticle functionalization with the surfactant is performed in the synthesis procedure. The size, charge, symmetry, geometrical and dynamical properties strongly depend on the molar ratio between their constituents, while the nanorod uniaxial anisotropy affects the optical response and defines the way that particles and DNA can be organized in three dimensions. In this work we investigate CTAB-capped gold nanorods and nanorod/DNA complexes, through the following experimental techniques. The trasmission electron microscopy represents the most direct approach to estimate size and shape of NRs. It provides high accuracy and allowed us to evaluate the CTAB-capped AuNR dimensions, as well as the slight deviation from the cylindrical geometry. The system azimuthal symmetry is peculiar in further analysis, i.e. synchrotron small angle X-ray scattering (SAXS) and dynamic light scattering (DLS) experiments. The former provides an estimation of the NR optical element structure. Indeed, because of its intrinsic features, the synchrotron SAXS technique allowed us to investigate the gold core of the larger CTAB-gold unit. Then, NR length and radius have been related to the localized surface plasmon resonance (LSPR) wavelengths, which have been measured by UV-Vis absorption spectroscopy. Further, we studied the NR dynamics through DLS, by measuring the translational and rotational brownian diffusion coefficients. The DLS approach represents also a relevant starting point for the NR/DNA complexes study. More precisely, the system symmetry affects the response to an incident light beam, thus it can be considered as a discriminating element for our analysis. We evaluated NR/DNA complexes symmetry, size and dynamics as a function of the component molar ratio and coupled them to the corresponding zeta potentials, which have been measured through electrophoretic light scattering (ELS). The most suitable complexes as drug delivery systems can be selected through this structural analysis, which allowed us to determine a specific molar ratio that corresponds to a class of potential gene delivery systems. Finally, we investigated the nucleic acid thermal stability through the UV-Vis absorption spectroscopy, both for naked DNA and within the complexes. The Coulomb interaction between DNA and CTAB is responsible for the self-assembling process, but it slightly affects the nucleic acid secondary structure. This binding effect is however negligible in a temperature range wide enough to ensure the DNA stability for the expected application perspectives. The thesis is divided into 4 chapters. Chapter 1 underlines some physical aspects of gold nanoparticles and how the optical properties of noble metal systems are related to the nanobiotechnology. After a brief historical introduction, the application in gene delivery is presented, then the physical background of the surface plasmon resonance, the approximations adopted, the theories developed and the validity ranges are discussed. In Chapter 2 a presentation of the materials involved precedes the sections about the techniques adopted, within which the theoretical and experimental aspects are explained. All the results obtained are discussed in Chapter 3 and, finally, Chapter 4 gives a work overview, focused on the conclusions related to the experimental results and the consequent perspectives of application

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

The possible role of sex as an important factor in development and administration of lipid nanomedicine-based COVID-19 vaccine

Nanomedicine has demonstrated a substantial role in vaccine development against severe acute respiratory syndrome coronavirus (SARS-CoV-2 and COVID-19). Although nanomedicine-based vaccines have now been validated in millions of individuals worldwide in phase 4 and tracking of sex-disaggregated data on COVID-19 is ongoing, immune responses that underlie COVID-19 disease outcomes have not been clarified yet. A full understanding of sex-role effects on the response to nanomedicine products is essential to building an effective and unbiased response to the pandemic. Here, we exposed model lipid nanoparticles (LNPs) to whole blood of 18 healthy donors (10 females and 8 males) and used flow cytometry to measure cellular uptake by circulating leukocytes. Our results demonstrated significant differences in the uptake of LNP between male and female natural killer (NK) cells. The results of this proof-of-concept study show the importance of recipient sex as a critical factor which enables researchers to better consider sex in the development and administration of vaccines for safer and more-efficient sex-specific outcomes

Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles

PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development

Inhibiting the growth of 3D brain cancer models with bio-coronated liposomal temozolomide

Nanoparticles (NPs) have emerged as an effective means to deliver anticancer drugs into the brain. Among various forms of NPs, liposomal temozolomide (TMZ) is the drug-of-choice for the treatment and management of brain tumours, but its therapeutic benefit is suboptimal. Although many possible reasons may account for the compromised therapeutic efficacy, the inefficient tumour penetration of liposomal TMZ can be a vital obstacle. Recently, the protein corona, i.e., the layer of plasma proteins that surround NPs after exposure to human plasma, has emerged as an endogenous trigger that mostly controls their anticancer efficacy. Exposition of particular biomolecules from the corona referred to as protein corona fingerprints (PCFs) may facilitate interactions with specific receptors of target cells, thus, promoting efficient internalization. In this work, we have synthesized a set of four TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical−chemical properties. We have demonstrated that precoating liposomal TMZ with a protein corona made of human plasma proteins can increase drug penetration in a 3D brain cancer model derived from U87 human glioblastoma multiforme cell line leading to marked inhibition of tumour growth. On the other side, by fine-tuning corona composition we have also provided experimental evidence of a non-unique effect of the corona on the tumour growth for all the complexes investigated, thus, clarifying that certain PCFs (i.e., APO-B and APO-E) enable favoured interactions with specific receptors of brain cancer cells. Reported results open new perspectives into the development of corona-coated liposomal drugs with enhanced tumour penetration and antitumour efficacy

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.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the National Council of Science and Technology (CONACYT), Mexico, Reg. # 217101, the Spanish Ministry of Education (Grants CTQ2010-20541, CTQ2010- 14897), the Basque Government (Department of Education, University and Research, predoctoral BFI- 2011-2226 grant) and by Spanish grants MAT2012-39290-C02-01 and IPT-2012-0574- 300000. Technical and human support provided by SGIker (UPV/ EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU). GC acknowledges support by the Italian Minister for University and Research (MIUR) (Futuro in Ricerca, Grant No. RBFR08TLPO).Peer reviewe

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

CFBM - A Framework for Data Driven Approach in Agent-Based Modeling and Simulation

Recently, there has been a shift from modeling driven approach to data driven approach in Agent Based Modeling and Simulation (ABMS). This trend towards the use of data-driven approaches in simulation aims at using more and more data available from the observation systems into simulation models [1, 2]. In a data driven approach, the empirical data collected from the target system are used not only for the design of the simulation models but also in initialization, evaluation of the output of the simulation platform. That raises the question how to manage empirical data, simulation data and compare those data in such agent-based simulation platform. In this paper, we first introduce a logical framework for data driven approach in agent-based modeling and simulation. The introduced framework is based on the combination of Business Intelligence solution and a multi-agent based platform called CFBM (Combination Framework of Business intelligence and Multi-agent based platform). Secondly, we demonstrate the application of CFBM for data driven approach via the development of a Brown Plant Hopper Surveillance Models (BSMs), where CFBM is used not only to manage and integrate the whole empirical data collected from the target system and the data produced by the simulation model, but also to initialize and validate the models. The successful development of the CFBM consists not only in remedying the limitation of agent-based modeling and simulation with regard to data management but also in dealing with the development of complex simulation systems with large amount of input and output data supporting a data driven approach

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

Combined Inhibition of CDK4/6 and PI3K/AKT/mTOR Pathways Induces a Synergistic Anti-Tumor Effect in Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16INK4a and p14ARF, deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM