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...

CONSPECTUS: Biocompatible hydrogels are materials that hold great promise in medicine and biology since the porous structure, the ability to entrap a large amount of water, and the tunability of their mechanical and tissue adhesive properties make them suitable for several applications, including wound healing, drug and cell delivery, cancer treatment, bioelectronics, and tissue regeneration. Among the possible developed systems, injectable hydrogels, owing to their properties, are optimal candidates for in vivo minimally invasive procedures. To be injectable, a hydrogel must be liquid before and during the injection, but it must quickly jellify after injection to form a soft, self-standing, solid material. The possibility to work with a liquid precursor encoding the functions that will be available after gelation allows the development of biocompatible materials that can be employed in surgery and, in particular, in noninvasive procedures. The underlying idea is to reach the target tissue by using just a needle, or by exploiting the natural body orifices, reducing surgery procedure time, induced pain, and risk of infections. Hydrogels with different properties can be obtained by changing the type of cross-linking, the cross-linking density or the molecular weight of the polymer, or by introducing pending functional groups. The introduction of a nanofiller in the hydrogel network allows for expanding the suite of the structural and functional properties and for better mimicking native tissues. In this Account, we discuss how to provide a hydrogel network with designed properties by playing with both the polymeric chains and the fillers. We present selected examples from the literature that show how to introduce stiffness, stretchability, adhesiveness, self-healing, anisotropy, antimicrobial activity, biodegradability, and conductivity in injectable hydrogels. We further describe how the chemical composition, the mechanical properties, and the microarchitecture of the hydrogel influence cell adhesion, proliferation, and differentiation. Examples of injectable hydrogels for innovative minimally invasive procedures are then discussed in detail; in particular, we showcase the use of hydrogels for tumor resection and as vascular chemoembolization agents. We further discuss how one can improve the rheological properties of injectable hydrogels to exploit them in osteochondral tissue engineering. The effect of the introduction of a conductive filler is then presented in relation to the development of electroactive scaffolds for cardiac-tissue engineering and neural and nerve repair. We believe that the rational design of biocompatible, injectable hybrid hydrogels with tunable properties will likely play a crucial role in reducing the invasiveness and improving the outcome of several clinical and surgical setups.

The direct delivery of specific proteins to live cells promises a tremendous impact for biological and medical applications, from therapeutics to genetic engineering. However, the process mostly involves tedious techniques and often requires extensive alteration of the protein itself. Herein we report a straightforward approach to encapsulate native proteins by using breakable organosilica matrices that disintegrate upon exposure to a chemical stimulus. The biomolecule-containing capsules were tested for the intracellular delivery of highly cytotoxic proteins into C6 glioma cells. We demonstrate that the shell is broken, the release of the active proteins occurs, and therefore our hybrid architecture is a promising strategy to deliver fragile biomacromolecules into living organisms.