Oil-filled microcapsules of kraft lignin were synthesized by first creating an oil in water emulsion followed by a high-intensity, ultrasound-assisted cross-linking of lignin at the water/oil interface. The rationale behind our approach is based on promoting documented lignin hydrophobic interactions within the oil phase, followed by locking the resulting spherical microsystems by covalent cross-linking using a high intensity ultrasound treatment. As further evidence in support of our rationale, confocal and optical microscopies demonstrated the uniformly spherical morphology of the created lignin microparticles. The detailed elucidation of the cross-linking processes was carried out using gel permeation chromatography (GPC) and quantitative (31)P NMR analyses. The ability of lignin microcapsules to incorporate and release Coumarin-6 was evaluated in detail. In vitro studies and confocal laser scanning microscopy analysis were carried out to assess the internalization of capsules into Chinese hamster ovary (CHO) cells. This part of our work demonstrated that the lignin microcapsules are not cytotoxic and readily incorporated in the CHO cells.

High-intensity ultrasound induces emulsification and cross-linking of protein molecules in aqueous medium. The stability and the functionality of the resultant protein-coated microbubbles are crucial in many of their applications. For example, the stability of drug-loaded microbubbles should be sufficiently long enough, in vivo, so that they can be ruptured only at specific sites for release of the drugs. In this study, we report the synthesis of stable and functional microbubbles, coated with chemically reduced lysozyme, using high-intensity ultrasound in aqueous solution. In the absence of chemical reduction, stable microbubbles were not produced with native lysozyme, indicating the importance of free -SH functional groups for protein cross-linking. The degree of cross-linking between lysozyme molecules was controlled by manipulating both the extent of chemical reduction of the intramolecular disulfide bonds and sonication time. The lysozyme-coated microbubbles are stable for several months and retain the enzymatic (antimicrobial) activity of lysozyme. The layer-by-layer (LbL) deposition of polyelectrolytes onto the protein-shell air-core template has been used as a versatile procedure to modify the surface properties of the microbubbles, indicating the possibility of adsorbing potential drugs and/or biolabels on the surface of these microbubbles for therapeutic and diagnostic applications.

The intracellular delivery of nucleic acids and proteins remains a key challenge in the development of biologic therapeutics. In gene therapy, the inefficient delivery of small interfering RNA (siRNA) to the cytosol by lipoplexes or polyplexes is often ascribed to the entrapment and degradation of siRNA payload in the endosomal compartments. A possible mechanism by which polyplexes rupture the endosomal membrane and release their nucleic acid cargo is commonly defined as the "proton sponge effect". This is an osmosis-driven process triggered by the proton buffering capacities of polyplexes. Herein, we investigate the molecular basis of the "proton sponge effect" through direct visualization of the siRNA trafficking process, including analysis of individual polyplexes and endosomes, using stochastic optical reconstruction microscopy. We probed the sequential siRNA trafficking steps through single molecule superresolution analysis of subcellular structures, polyplexes, and silencing RNA molecules.Specifically, individual intact polyplexes released in the cytosol upon rupture of the endosomes, the damaged endosomal vesicles, and the disassembly of the polyplexes in the cytosol were examined. We found that the architecture of the polyplex and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. We provide evidence that in highly branched and rigid cationic polymers, such as glycogen or polyethylenimine, immobilized on silica nanoparticles, the proton sponge effect is effective in inducing osmotic swelling and rupture of endosomes.KEYWORDS: endosomal escape, polyplexes, proton sponge effect, STORM, glycogenThe complexation of nucleic acids with cationic lipid and polymers to form lipoplexes and polyplexes, respectively, is widely used for intracellular transfection of DNA, siRNA, microRNAs, and lately CRISPR-Cas9. 1,2 To efficiently deliver their cargo and promote alteration 3 of gene expression and transcription, these vectors must overcome multiple biological barriers, including substantial cellular uptake, endosomal escape, and intracellular targeting devoid of offtarget effects. The endolysosomal escape 3-7 and endocytic recycling 8 processes are reported to be the major bottlenecks encountered in the intracellular release of nucleic acid payloads. In particular, the translocation of intact and functional RNA double strands from late endosomes to the cytosol prior to their degradation in the lysosome compartments is usually considered the ratelimiting step in siRNA-mediated knockdown of protein expression (RNAi). 3,6 It has been estimated that the escape of siRNAs from endosomes to the cytosol occurs at a very low efficiency (0.01-2%). 5,7 The delivery of siRNAs, mediated by lipoplexes and polyplexes, has been investigated by time-lapse confocal and electron microscopy. [3][4][5][6] This qualitative and quantitative image-based analysis of lipid and polymer-based nanocarriers, in fixed and live cells, have revealed dif...

Gas filled hollow microparticles, i.e., microbubbles and microballoons, are soft matter devices used in a number of diverse applications ranging from protein separation and purification in food science to drilling technology and ultrasound imaging. Aqueous dispersions of these mesoscopic systems are characterized by the stabilization of the air/water interface by a thin shell of phospholipid bilayer or multilayers or by a denatured and cross-linked proteic matrix. We present a study of a type of microballoons based on modified poly(vinyl alcohol), PVA, a synthetic biocompatible polymer, with new structural features. A cross-linking reaction carried out at the air/water interface provides polymeric air-filled microbubbles with average dimensions depending on the reaction temperature. Characterization of diameters and shell thicknesses for microbubbles obtained at different temperatures has been carried out. Conversion to solvent-filled hollow microcapsules is possible by soaking microbubbles in dimethyl sulfoxide. Microcapsules permeability to fluorescent labeled dextran molecular weight standards was correlated to the mesh size of the polymer network of the shell. Microbubbles were covalently grafted under very mild conditions with beta-cyclodextrin and poly-l-lysine with a view to assay the capability of the device for delivery of hydrophobic drugs or DNA. PVA based microballoons show a remarkable shelf life of several months, their external surface can be decorated with many biologically relevant molecules. These features, together with a tested biocompatibility, make them attractive candidates for use as multifunctional device for diagnosis and therapeutic purposes, i.e., as ultrasound reflectors in ecographic investigation and as drug platforms for in situ sonoporation.

A topologically extended model of a chemically cross-linked hydrogel of poly(vinyl alcohol) (PVA) at high hydration degree has been developed for a molecular dynamics simulation with atomic detail at 323 K. The analysis of the 5 ns trajectory discloses structural and dynamic aspects of polymer solvation and elucidates the water hydrogen bonding and diffusion in the network. The features of local polymer dynamics indicate that PVA mobility is not affected by structural constraints of chemical junctions at the investigated cross-linking density, with a prevailing dumping effect due to water interaction. Simulation results are validated by a favorable comparison with findings of an incoherent quasi-elastic neutron scattering study of the same hydrogel system.

The intracellular delivery of functional nanoparticles (NPs) and the release of therapeutic payloads at a target site are central issues for biomedical applications. However, the endosomal entrapment of NPs typically results in the degradation of active cargo, leading to poor therapeutic outcomes. Current advances to promote the endosomal escape of NPs largely involve the use of polycationic polymers and cell-penetrating peptides (CPPs), which both often suffer from potential toxicity and convoluted synthesis/conjugation processes. Herein, we report the use of metal-phenolic networks (MPNs) as versatile and nontoxic coatings to facilitate the escape of NPs from endo/lysosomal compartments. The MPNs, which were engineered from the polyphenol tannic acid and Fe III or Al III , enabled the endosomal escape of both inorganic (mesoporous silica) and organic (polystyrene and melamine resin) NPs owing to the "proton-sponge effect" arising from the buffering capacity of MPNs. Postfunctionalization of the MPN-coated NPs with low-fouling polymers did not impair the endosomal escape, indicating the modular and generalizable nature of this approach. We envisage that the ease of fabrication, versatility, low cytotoxicity, and promising endosomal escape performance displayed by the MPN coatings offer opportunities for such coatings to be used for the efficient delivery of cytoplasm-targeted therapeutics using NPs.

In this paper, we present some new case examples where the chemical versatility of poly (vinyl alcohol) (PVA) can be used for potential biomedical applications. PVA, the polymeric material used for designing new nanostructured devices, is water soluble, biocompatible and has excellent physical properties. We point out the possibility of obtaining wall-to-wall chemical hydrogels as well as microgels without diminishing the biocompatibility available in the starting PVA material. Injectability is another important factor to take into account in controlled drug delivery for gene therapy. In this respect, in this paper, established and more innovative methods are prospected in order to obtain particles with dimensions suitable for these applications.

In this report, we describe the delivery of small interfering RNA (siRNA) using LbL-assembled microcapsules. The microcapsules are based on negatively charged poly(methacrylic acid) nanometer thin films containing cross-linking disulfide bonds. One system is polycation-free and another contains polylysine for siRNA complexation in the microcapsule void. When microcapsules containing a siRNA targeting survivin were delivered to PC-3 prostate cancer cells, a significant inhibition of the expression of the antiapoptotic protein was observed. However, down-regulation of survivin was also observed in PC-3 cells exposed to microcapsules embedded with a scrambled siRNA as well as in cells treated with empty microcapsules. These findings indicate a capsule-dependent off-target effect, which is supported by a reduction in the expression of other survivin-unrelated proteins. The microcapsules and their polymeric constituents do not affect cell proliferation, as determined by a metabolic assay, even after 4 days of exposure. In addition, in PC-3 cells exposed to microcapsules, we observed a marked accumulation of LC3b, a marker related to autophagy (i.e., self-digestion), a degradation pathway involved in the maintenance of cell homeostasis in response to different stresses. This evidence suggests that empty microcapsules can induce a perturbation of the intracellular environment, which causes the activation of a cell safeguard mechanism that may limit the therapeutic effect of the microcapsules in tumor cells.