Although selective tumor delivery of anticancer drugs has been sought by exploiting either passive targeting or by ligand-mediated targeting, a selective anticancer therapy remains an unmet medical need. Despite the advances which have been achieved by nanomedicines, nanosystems such as polymer-drug conjugates still miss the goal of clinical efficacy. In this study, we demonstrated that polymer-drug conjugates require a thoroughly chemical design and the right targeting agent/polymer ratio to be selective and effective towards cancer cells. In particular, two PEG conjugates carrying paclitaxel and targeted with different folic acid (FA)/PEG ratios (one or three) were investigated. The cytotoxicity study in positive (HT-29) and negative (HCT-15) FA receptor (FR)-cell lines demonstrated that the conjugates with one or three FAs were 4- or 28-fold more active in HT-29 cells, respectively. The higher activity of the 3-FA conjugate was confirmed by its strong impact on cell cycle arrest. Furthermore, FA targeting had a clear effect on migration and invasiveness of HT-29 cells, which were significantly reduced by both conjugates. Interestingly, the 3-FA conjugate showed also an improved pharmacokinetic profile in mice. The results of this study indicate that thorough investigations are needed to optimize and tune drug delivery and achieve the desired selectivity and activity towards cancer cells.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

The structural dispersity of poly­[oligo­(ethylene glycol) methacrylate] (POEGMA) brushes critically influences the stabilization of Au nanoparticles (NPs) and their interactions with anti-PEG antibodies (APAs). Commercial oligo­(ethylene glycol) methacrylate (OEG _p MA) macromonomers are intrinsically polydisperse, featuring a distribution of ethylene glycol (EG) units spanning from n = 2 to n = 15. Flash chromatography was applied to isolate OEG ₈ MA with discrete length (n = 8)the most abundant species in commercial mixtures. Controlled radical polymerization of polydisperse and discrete monomer sources was subsequently applied to generate POEG _p MA and POEG ₈ MA, which feature heterogeneous and homogeneous structures, respectively, while displaying an overall identical composition. When used to form shells on Au NPs, uniform POEG ₈ MA brushes provided enhanced colloidal stability across a wide temperature range compared to their polydisperse counterparts. While serum protein corona formation was largely determined by polymer composition, APA binding was promoted by longer OEG segments present in polydisperse POEG _p MA shells, which acted as epitopes for antibody recognition. These findings highlight how controlling polymer architecture and dispersity in the design of PEG-based shells for NPs could give access to nonimmunogenic formulations. More broadly, polymer dispersity emerges as an additional tool for modulating the behavior of nanomaterials within biological systems.

In this study, a circular conjugate of granulocyte colony-stimulating factor (G-CSF) was prepared by conjugating the two end-chains of poly(ethylene glycol) (PEG) to two different sites of the protein. For the orthogonal conjugation, a heterobifunctional PEG chain was designed and synthesized, bearing the dipeptide ZGln-Gly (ZQG) at one end-chain, for transglutaminase (TGase) enzymatic selective conjugation at Lys41 of G-CSF, and an aldehyde group at the opposite end-chain, for N-terminal selective reductive alkylation of the protein. The cPEG-Nter/K41-G-CSF circular conjugate was characterized by physicochemical methods and compared with native G-CSF and the corresponding linear monoconjugates of G-CSF, PEG-Nter-G-CSF, and PEG-K41-G-CSF. The results demonstrated that the circular conjugate had improved physicochemical and thermal stability, prolonged pharmacokinetic interaction, and retained the biological activity of G-CSF. The PEGylation strategy employed in this study has potential applications in the design of novel protein-based therapeutics.

Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.

RhDNase I (recombinant human deoxyribonuclease I, known as Pulmozyme®) has mucolytic action, which is clinically exploited in cystic fibrosis (CF). In the lungs, its efficacy is hampered by both limited stability and presence of inhibitors such as G-actin. Here we synthesized three PEGylated rhDNase I conjugates differing in PEG length and conjugation chemistry, the latter affecting the overall charge of the conjugate. The aim was to preserve as much as possible the activity of the enzyme and to protect it from inhibition and degradation. Of note, derivatization with PEG24mer, the shortest chain, is accomplished through amide bonds, thus reducing the overall positive charge of the conjugate. The activity of these conjugates was tested on supercoiled DNA plasmids, and then on the rheological properties of DNA-supplemented artificial mucus and of CF patients' mucus. The use of PEG24mer produced the largest loss in DNase activity in an in vitro assay (33.7% vs. 83.8% with mPEG20kDa and 71.4% with mPEG2kDa) but in mucus showed the highest reduction in viscosity (complex viscosity at 1.0 Hz: ≈1 Pa*s vs. 2.5-5.0 Pa*s with native enzyme or other conjugates), which is likely to be due to less intense inhibitory interactions with G-actin, both with artificial and CF patients' mucus. In nebulization, mPEG24mer-rhDNase I also showed a more promising aerodynamic profile than rhDNase I (a mass median droplet aerodynamic diameter of 3.69 μm vs. 4.12 μm; fraction of fine particles of 72.3% vs. 64.1%), supporting its potential as an enhanced treatment option.