Fernando Carlos Giacomelli

Living cells exhibit a complex organization comprising numerous compartments, among which are RNA- and protein-rich membraneless, liquidlike organelles known as biomolecular condensates. Energy-consuming processes regulate their formation and dissolution, with (de)phosphorylation by specific enzymes being among the most commonly involved reactions. By employing a model system consisting of a phosphorylatable peptide and homopolymeric RNA, and thanks to time-resolved ultra-small-angle x-ray scattering enabling us to probe a wide range of length and time scales, we elucidate how enzymatic activity remodels the growth kinetics and the internal structure of biomolecular condensates. Under a passive condition, we reveal a nucleation-driven coalescence mechanism maintained over four decades in time, similar to the coarsening of simple binary fluid mixtures. Coarse-grained molecular-dynamics simulations show that peptide-decorated RNA chains assembled immediately after mixing constitute the basic subunits. In contrast, actively formed condensates initially display a networklike internal structure, which gradually matures upon dephosphorylation prior to coalescence. Both pathways lead to a steady state, but fluorescence recovery after photobleaching indicates a peptide diffusivity that is twice as high in actively formed condensates consistent with their loosely packed internal structure. This study contributes to linking effectively cellular active processes such as post-translational modifications to the formation and dissolution pathways of membraneless organelles, impacting other fields including synthetic biology.

Polymersome nanoreactors, vesicular assemblies formed from amphiphilic block copolymers, provide a versatile platform for compartmentalized catalysis and the construction of biomimetic systems. While extensive efforts have focused on the encapsulation of enzymes within such constructs, reports of vesicles displaying intrinsic membrane permeability remain unusual. Typically, selective transport across polymersome membranes requires the incorporation of channel proteins or other porogenic components. Conversely, we herein demonstrate that polymer vesicles comprising poly(butylene oxide) as the hydrophobic segment exhibit inherent permeability to small molecules without the need for artificial machineries, possibly governed by moderate hydrophobicity of this polymer and consequently hydration of the membrane. Synthesis and detailed characterization of diblock and triblock copolymers containing poly(butylene oxide) and poly(glycidol), respectively, as hydrophobic and hydrophilic blocks are first demonstrated; the self-assembly of the chains into polymer vesicles and their inherent permeability to disparate small molecules are subsequently highlighted. Notably, we further reveal that such vesicles can be conveniently loaded with a model enzyme (horseradish peroxidase), which remains entrapped in the aqueous lumen. Using a well-established colorimetric assay, we show that the vesicles are also permeable to small-enzyme substrates and products, and therefore, the reported strategy can be applied to a wide range of enzymes and functional proteins for the design of simple permeable nanoreactors for enzyme-mediated catalysis.

Nanotherapeutic delivery requires particulate systems composed of only a few components yet featuring multiple capabilities to reach the clinic. In this study, we built a platform based on selective reactions (solid-phase peptide synthesis, peptide coupling chemistry, and click chemistry) to enhance cellular uptake of gold nanoparticles (AuNPs) by anchoring cell-penetrating peptides (CPPs) at the outermost layer of the constructs. Model amphipathic peptide (MAP), trans-acting activator of transcription (TAT), hexarginine (R₆), and arginine monomer (Arg) were attached to the surface of AuNPs synthesized directly by using branched polyethylenimine (BPEI) polyelectrolyte. From a different perspective, a water-soluble CGSWQWRR sequence capable of promoting the reduction of auric species and steric stabilization of gold colloids was also synthesized. The structure and dynamics of particles were characterized using imaging, scattering, and spectroscopy techniques, and their biological performance was evaluated by assessing cell viability and cellular uptake. The nanoconstructs were noncytotoxic up to 1.0 ppm in the case of CPP–BPEI@AuNPs (5.2 nm metallic core) or up to 10 ppm for CGSWQWRR@AuNPs (30 nm metallic core). Those featuring CPPs at the surface were internalized faster and to a higher extent (∼40% in 4 h) compared to the precursor (∼20% in 4 h). The highest cellular uptake was found for CGSWQWRR@AuNPs (∼75% in 4 h), which mediated the membrane-wrapping process more effectively and was prepared by the easiest protocol (one-pot, two-reactant, no workup reaction in aqueous media). The findings of this study simplify nanoparticle manufacturing, thereby reducing the gap between chemical synthesis and clinical applications.

Well-defined gold nanoparticles (AuNPs) are accessible via simple synthetic methods, and their surface chemistry stands as a key factor in determining applications in the biomedical field. While macromolecules featuring amino groups are already known to successfully mediate the formation of stable gold colloids in one-pot, two-reactant, no workup reactions in aqueous media, we herein report the discovery that, under mild reaction temperatures, polymers of outstanding biomedical interest not only can play the simultaneous role of reducing and capping agent but also lead to particulate systems with unique features. From a library of samples that included branched polyethylenimine (BPEI), poly­(l-lysine) (PLL), bovine serum albumin (BSA), poly­(2-methyl-2-oxazoline) (PMeOx), poly­(N-(2-hydroxypropyl) methacrylamide) (PHPMA), and amine-functionalized poly­(N-(2-hydroxypropyl)­methacrylamide-co-N-(3-aminopropyl)­methacrylamide) P­(HPMA-co-APMA), we found that PHPMA end-functionalized with nitrile motifs generate spherical and stable AuNPs@PHPMA of very small size (diameter of ∼2.4 nm), as underlined by imaging experiments. Cell viability experiments indicated exceptionally good biocompatibility up to very high numerical particle concentrations as compared to the other systems. The reduced size imparted to the AuNPs@PHPMA outstanding catalytic properties (no induction time and high reaction rate constant for the hydrogenation of p-nitrophenol) and antimicrobial activity (total antibacterial activity against Escherichia coli and dose-dependent antibacterial activity against Staphylococcus aureus). The introduction of primary amine groups (13.4 mol %) of higher nucleophilicity known to work better for AuNP synthesis makes these unique features disappear, as evidenced for P­(HPMA-co-APMA). The other systems yielded 6–28 nm particles whose properties reflected both the size of the metallic core and chemical nature and conformation of the capping agent. These findings point to novel applications of PHPMA polymers worthy of further development, especially in light of their excellent water solubility and biocompatibility.

Sugar-decorated core–shell nanoparticles loaded with ursolic acid and two-fold increase in cell toxicity compared to controls has been produced. Targeted delivery has been shown with enhanced uptake by cancer cells compared to healthy counterparts.

The delivery of nucleic acids requires a gene vector as they are unable to effectively cross cell membranes. Polycations and deoxyribonucleic acid (DNA) can self-assemble into polyplexes enabling intracellular delivery of genetic material. In this framework, we have evaluated the transfection efficiency and cytotoxicity of amino acid-modified branched polyethyleneimine (BPEI). The presence of arginine (Arg), cysteine (Cys) and histidine (His) conjugated to the polymer chains allows for higher transfection capability with lower cytotoxicity compared to the unsubstituted counterpart. BPEI-Arg showed the highest values of transfection efficiency as attributed to its positive charges at nearly neutral pH. The enhanced gene delivery capacity is also linked to the cell-penetrating feature of arginine. The improved efficiency evidenced for BPEI-Cys and BPEI-His is presumably connected to polyplex stabilization due to the formation of bioreducible disulfide bridges, and the protonation of histidine at the intracellular environment. The amino acid modification of branched PEI chains is herein demonstrated to be potentially relevant in ocular gene delivery since human retinal pigment epithelial cells are known to be notoriously difficult to be transfected. This investigation accordingly bridges a gap by combining efficient branched PEI DNA-compacting ability with the superior cell-penetrating properties of arginine, and focused also on potential retinal or corneal therapies.

The permeability and responsiveness of polymer membranes are absolutely relevant in the design of polymersomes for cargo delivery. Accordingly, we herein correlate the structural features, permeability, and responsiveness of doxorubicin-loaded (DOX-loaded) nonresponsive and stimuli-responsive polymersomes with their in vitro and in vivo antitumor performance. Polymer vesicles were produced using amphiphilic block copolymers containing a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) segment linked to poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA, nonresponsive block), poly[4-(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)benzyl methacrylate] [PbAPE, reactive oxygen species (ROS)-responsive block], or poly[2-(diisopropylamino)ethyl methacrylate] (PDPA, pH-responsive block). The PDPA-based polymersomes demonstrated outstanding biological performance with antitumor activity notably enhanced compared to their counterparts. We attribute this behavior to a fast-triggered DOX release in acidic tumor environments as induced by pH-responsive polymersome disassembly at pH < 6.8. Possibly, an insufficient ROS concentration in the selected tumor model attenuates the rate of ROS-responsive vesicle degradation, whereas the nonresponsive nature of the PPPhA block remarkably impacts the performance of such potential nanomedicines.