Effective SARS-CoV-2 antiviral activity of hyperbranched polylysine nanopolymers.

The coronavirus pandemic (COVID-19) had spread rapidly since December 2019, when it was first identified in Wuhan, China. As of April 2021, more than 130 million cases have been confirmed, with more than 3 million deaths, making it one of the deadliest pandemics in history. Different approaches must be put in place to confront a new pandemic: community-based behaviours (i.e., isolation and social distancing), antiviral treatments, and vaccines. Although behaviour-based actions have produced significant benefits and several efficacious vaccines are now available, there is still an urgent need for treatment options. Remdesivir represents the first antiviral drug approved by the Food and Drug Administration for COVID-19 but has several limitations in terms of safety and treatment benefits. There is still a strong request for other effective, safe, and broad-spectrum antiviral systems in light of future emergent coronaviruses. Here, we describe a polymeric nanomaterial derived from L-lysine, with an antiviral activity against SARS-CoV-2 associated with a good safety profile in vitro. Nanoparticles of hyperbranched polylysine, synthesized by L-lysine's thermal polymerization catalyzed by boric acid, effectively inhibit the SARS-CoV-2 replication. The virucidal activity is associated with the charge and dimension of the nanomaterial, favouring the electrostatic interaction with the viral surface being only slightly larger than the virions' dimensions. Low-cost production and easiness of synthesis strongly support the further development of such innovative nanomaterials as a tool for potential treatments of COVID-19 and, in general, as broad-spectrum antivirals.

Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor.

Poly(ethylene glycol) (PEG) is a widely used polymer employed to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. PEG attaches to free amines, typically at lysine residues or at the N-terminal amino acid. This lack of selectivity can present problems when a PEGylated protein therapeutic is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic modification of proteins is one route to overcome this limitation. Bacterial transglutaminases are enzyme candidates for site-specific modification, but they also have rather broad specificity. The need arises to be able to predict a priori potential PEGylation sites on the protein of interest and, especially, to be able to design mutants where unique PEGylation sites can be introduced when needed. We investigated the feasibility of a computational approach to the problem, using human granulocyte colony-stimulating factor as a test case. The selected protein is therapeutically relevant and represents a challenging problem, as it contains 17 potential PEGylation sites. Our results show that a combination of computational methods allows the identification of the specific glutamines that are substrates for enzymatic PEGylation by a microbial transglutaminase, and that it is possible to rationally modify the protein and introduce PEG moieties at desired sites, thus allowing the selection of regions that are unlikely to interfere with the biological activity of a therapeutic protein.

A new PEG-beta-alanine active derivative for releasable protein conjugation.

A new PEGylating agent, PEG-betaAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines. Furthermore, the conjugates obtained with PEG-betaAla-NHCO-OSu showed an interesting slow release of polymer chains upon incubation under physiological conditions. Further investigations determined that the PEG chains released are those coupled to histidine residues, and this finally yields less PEGylated species as well as free protein. This release allows a partial recovery of protein activity that is often remarkably and permanently reduced after stable PEGylation, and it occurs in water or blood without the involvement of enzymes. On the other hand, the rate of PEG release, tuned by the chemical structure of this new PEGylating agent, is not too high, and therefore, the achievement of a desired prolongation of protein half-life in vivo is still feasible. The pharmacokinetics of hGH-PEG6k-betaAla conjugate was compared to that of native hGH in rats and monkeys, and the blood residence times were increased by 10- and 7-fold, respectively. The conjugate potency was evaluated in hypophysectomized rats demonstrating a superior pharmacodynamic profile with respect to native hGH.

Supramolecular association of recombinant human growth hormone with hydrophobized polyhydroxyethylaspartamides.

The protein delivery properties of polymer supramolecular assemblies were investigated by using recombinant human growth hormone (rh-GH) and two polyhydroxyethylaspartamide (PHEA) derivatives: (a) PHEA-C16 obtained by PHEA random grafting with hexadecylalkylamine; (b) PHEA-PEG 5000-C16 obtained by PHEA random co-grafting with hexadecylalkylamine and 5 kDa poly(ethylene glycol). The two polymers possessed similar self-assembling properties: critical micelle concentration (CMC) and particle size. The protein loading (protein/polymer, w/w, %) was 12.1+/-1.3% and 8.5+/-0.4% with PHEA-C16 and PHEA-PEG 5000-C16, respectively. The rh-GH/polymer association constant calculated by Scatchard analysis was 1.87 x 10(5)M(-1) with PHEA-C(16) and 0.27 x 10(5)M(-1) with PHEA-PEG 5000-C16. The Klotz analysis showed that 5 PHEA-C16 and 9 PHEA-PEG 5000-C16 polymer chains associated with one protein molecule. The protein dissociation from the PHEA-C16 and PHEA-PEG 5000-C16 supramolecular complexes was complete in about 350-450 and 450-550 h, respectively. With both polymers, the protein release was faster as the protein/polymer ratio increased. Pharmacokinetic studies were performed by subcutaneous administration to rats of protein/polymer solutions at different w/w ratios (1:75 and 1:150). Both polymer formulations slowed the protein absorption. The protein bioavailability increased as the protein/polymer complex stability decreased and the protein/polymer w/w ratio increased indicating that efficient protein delivery can be achieved by proper polymer choice and formulation composition.

Site-specific pegylation of G-CSF by reversible denaturation.

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.

Effect of 1-butanol on the microstructure of lecithin/water/tripalmitin system.

Warm microemulsions based on lipids characterized by a melting point over 50 degrees C have been successfully used as starting matrix in a quenching process to obtain solid lipid nanoparticles (SLN). In this work, we have investigated the effect of 1-butanol (B) on the phase behavior of the lecithin (LCT)/water (W)/tripalmitin (TP) system at 70 degrees C. The study has been carried out at LCT/B=1 (weight ratio). Emulsion and liquid crystalline phase regions have been observed in the ternary phase diagram, while the presence of 1-butanol in the LCT/W/B/TP system allows the formation of a wide area of liquid isotropic phase from the whole (LCT+B)/TP binary axis up to 37 wt% of water. The microstructure of this isotropic phase has been investigated by means of 1H NMR PGSE technique. The self-diffusion coefficients of the different components along oil and water dilution lines indicate a microstructural organization characterized by a highly connected water in oil domains.