Effect of surface functionalization and loading on the mechanical properties of soft polymeric nanoparticles prepared by nano-emulsion templating.

Drug and gene delivery systems based on polymeric nanoparticles offer a greater efficacy and a reduced toxicity compared to traditional formulations. Recent studies have evidenced that their internalization, biodistribution and efficacy can be affected, among other factors, by their mechanical properties. Here, we analyze by means of Atomic Force Microscopy force spectroscopy how composition, surface functionalization and loading affect the mechanics of nanoparticles. For this purpose, nanoparticles made of Poly(lactic-co-glycolic) (PLGA) and Ethyl cellulose (EC) with different functionalizations and loading were prepared by nano-emulsion templating using the Phase Inversion Composition method (PIC) to form the nano-emulsions. A multiparametric nanomechanical study involving the determination of the Young's modulus, maximum deformation and breakthrough force was carried out. The obtained results showed that composition, surface functionalization and loading affect the nanomechanical properties in a different way, thus requiring, in general, to consider the overall mechanical properties after the addition of a functionalization or loading. A graphical representation method has been proposed enabling to easily identify mechanically equivalent formulations, which is expected to be useful in the development of soft polymeric nanoparticles for pre-clinical and clinical use.

A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density.

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

Complex pBAE Nanoparticle Cell Trafficking: Tracking Both Position and Composition Using Super Resolution Microscopy.

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(ß-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Versatile Vessel-on-a-Chip Platform for Studying Key Features of Blood Vascular Tumors.

Tumor vessel-on-a-chip systems have attracted the interest of the cancer research community due to their ability to accurately recapitulate the multiple dynamic events of the metastatic cascade. Vessel-on-a-chip microfluidic platforms have been less utilized for investigating the distinctive features and functional heterogeneities of tumor-derived vascular networks. In particular, vascular tumors are characterized by the massive formation of thrombi and severe bleeding, a rare and life-threatening situation for which there are yet no clear therapeutic guidelines. This is mainly due to the lack of technological platforms capable of reproducing these characteristic traits of the pathology in a simple and well-controlled manner. Herein, we report the fabrication of a versatile tumor vessel-on-a-chip platform to reproduce, investigate, and characterize the massive formation of thrombi and hemorrhage on-chip in a fast and easy manner. Despite its simplicity, this method offers multiple advantages to recapitulate the pathophysiological events of vascular tumors, and therefore, may find useful applications in the field of vascular-related diseases, while at the same time being an alternative to more complex approaches.

Real-Time Ratiometric Imaging of Micelles Assembly State in a Microfluidic Cancer-on-a-Chip.

The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy.

The future of gene therapy relies on the development of efficient and safe delivery vectors. Poly(ß-amino ester)s are promising cationic polymers capable of condensing oligonucleotides into nanoparticles - polyplexes - and deliver them into the cell nucleus, where the gene material would be expressed. The complexation state during the crossing of biological barriers is crucial: polymers should tightly complex DNA before internalization and then release to allow free DNA to reach the nucleus. However, measuring the complexation state in cells is challenging due to the nanometric size of polyplexes and the difficulties to study the two components (polymer and DNA) independently. Here we propose a method to visualize and quantify the two components of a polyplex inside cells, with nanometre scale resolution, using two-colour direct stochastic reconstruction super-resolution microscopy (dSTORM). With our approach, we tracked the complexation state of pBAE polyplexes from cell binding to DNA release and nuclear entry revealing time evolution and the final fate of DNA and pBAE polymers in mammalian cells.

Super-resolution Imaging of Structure, Molecular Composition, and Stability of Single Oligonucleotide Polyplexes.

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

Influence of Enzyme Quantity and Distribution on the Self-Propulsion of Non-Janus Urease-Powered Micromotors.

The use of enzyme catalysis to power micro- and nanomachines offers unique features such as biocompatibility, versatility, and fuel bioavailability. Yet, the key parameters underlying the motion behavior of enzyme-powered motors are not completely understood. Here, we investigate the role of enzyme distribution and quantity on the generation of active motion. Two different micromotor architectures based on either polystyrene (PS) or polystyrene coated with a rough silicon dioxide shell (PS@SiO2) were explored. A directional propulsion with higher speed was observed for PS@SiO2 motors when compared to their PS counterparts. We made use of stochastically optical reconstruction microscopy (STORM) to precisely detect single urease molecules conjugated to the micromotors surface with a high spatial resolution. An asymmetric distribution of enzymes around the micromotor surface was observed for both PS and PS@SiO2 architectures, indicating that the enzyme distribution was not the only parameter affecting the motion behavior. We quantified the number of enzymes present on the micromotor surface and observed a 10-fold increase in the number of urease molecules for PS@SiO2 motors compared to PS-based micromotors. To further investigate the number of enzymes required to generate a self-propulsion, PS@SiO2 particles were functionalized with varying amounts of urease molecules and the resulting speed and propulsive force were measured by optical tracking and optical tweezers, respectively. Surprisingly, both speed and force depended in a nonlinear fashion on the enzyme coverage. To break symmetry for active propulsion, we found that a certain threshold number of enzymes molecules per micromotor was necessary, indicating that activity may be due to a critical phenomenon. Taken together, these results provide new insights into the design features of micro/nanomotors to ensure an efficient development.

Amphiphilic poly(α)glutamate polymeric micelles for systemic administration of siRNA to tumors.

RNAi therapeutics carried a great promise to the area of personalized medicine: the ability to target "undruggable" oncogenic pathways. Nevertheless, their efficient tumor targeting via systemic administration had not been resolved yet. Amphiphilic alkylated poly(α)glutamate amine (APA) can serve as a cationic carrier to the negatively-charged oligonucleotides. APA polymers complexed with siRNA to form round-shaped, homogenous and reproducible nano-sized polyplexes bearing ~50 nm size and slightly negative charge. In addition, APA:siRNA polyplexes were shown to be potent gene regulators in vitro. In light of these preferred physico-chemical characteristics, their performance as systemically-administered siRNA nanocarriers was investigated. Intravenously-injected APA:siRNA polyplexes accumulated selectively in tumors and did not accumulate in the lungs, heart, liver or spleen. Nevertheless, the polyplexes failed to induce specific mRNA degradation, hence neither reduction in tumor volume nor prolonged mice survival was seen.

Micellar Stability in Biological Media Dictates Internalization in Living Cells.

The dynamic nature of polymeric assemblies makes their stability in biological media a crucial parameter for their potential use as drug delivery systems in vivo. Therefore, it is essential to study and understand the behavior of self-assembled nanocarriers under conditions that will be encountered in vivo such as extreme dilutions and interactions with blood proteins and cells. Herein, using a combination of fluorescence spectroscopy and microscopy, we studied four amphiphilic PEG-dendron hybrids and their self-assembled micelles in order to determine their structure-stability relations. The high molecular precision of the dendritic block enabled us to systematically tune the hydrophobicity and stability of the assembled micelles. Using micelles that change their fluorescent properties upon disassembly, we observed that serum proteins bind to and interact with the polymeric amphiphiles in both their assembled and monomeric states. These interactions strongly affected the stability and enzymatic degradation of the micelles. Finally, using spectrally resolved confocal imaging, we determined the relations between the stability of the polymeric assemblies in biological media and their cell entry. Our results highlight the important interplay between molecular structure, micellar stability, and cell internalization pathways, pinpointing the high sensitivity of stability-activity relations to minor structural changes and the crucial role that these relations play in designing effective polymeric nanostructures for biomedical applications.

Super-Resolution Microscopy Unveils Dynamic Heterogeneities in Nanoparticle Protein Corona.

The adsorption of serum proteins, leading to the formation of a biomolecular corona, is a key determinant of the biological identity of nanoparticles in vivo. Therefore, gaining knowledge on the formation, composition, and temporal evolution of the corona is of utmost importance for the development of nanoparticle-based therapies. Here, it is shown that the use of super-resolution optical microscopy enables the imaging of the protein corona on mesoporous silica nanoparticles with single protein sensitivity. Particle-by-particle quantification reveals a significant heterogeneity in protein absorption under native conditions. Moreover, the diversity of the corona evolves over time depending on the surface chemistry and degradability of the particles. This paper investigates the consequences of protein adsorption for specific cell targeting by antibody-functionalized nanoparticles providing a detailed understanding of corona-activity relations. The methodology is widely applicable to a variety of nanostructures and complements the existing ensemble approaches for protein corona study.

Capturing "Extraordinary" Soft-Assembled Charge-Like Polypeptides as a Strategy for Nanocarrier Design.

The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary-extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.

Versatile Methodology to Encapsulate Gold Nanoparticles in PLGA Nanoparticles Obtained by Nano-Emulsion Templating.

PURPOSE: Gold nanoparticles have been proved useful for many biomedical applications, specifically, for their use as advanced imaging systems. However, they usually present problems related with stability and toxicity. METHODS: In the present work, gold-nanoparticles have been encapsulated in polymeric nanoparticles using a novel methodology based on nano-emulsion templating. Firstly, gold nanoparticles have been transferred from water to ethyl acetate, a solvent classified as class III by the NIH guidelines (low toxic potential). Next, the formation of nano-emulsions loaded with gold nanoparticles has been performed using a low-energy, the phase inversion composition (PIC) emulsification method, followed by solvent evaporation giving rise to polymeric nanoparticles. RESULTS: Using this methodology, high concentrations of gold nanoparticles (>100 pM) have been encapsulated. Increasing gold nanoparticle concentration, nano-emulsion and nanoparticle sizes increase, resulting in a decrease on the stability. It is noteworthy that the designed nanoparticles did not produce cytotoxicity neither hemolysis at the required concentration. CONCLUSIONS: Therefore, it can be concluded that a novel and very versatile methodology has been developed for the production of polymeric nanoparticles loaded with gold nanoparticles. Graphical Abstract Schematic representation of AuNP-loaded polymeric nanoparticles preparation from nano-emulsion templating.

Spontaneous Protein Adsorption on Graphene Oxide Nanosheets Allowing Efficient Intracellular Vaccine Protein Delivery.

Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer.