Improving the Therapeutic Potential of G-CSF through Compact Circular PEGylation Based on Orthogonal Conjugations.

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.

Anti-HER2 Super Stealth Immunoliposomes for Targeted-Chemotherapy.

Liposomes play an important role in the field of drug delivery by virtue of their biocompatibility and versatility as carriers. Stealth liposomes, obtained by surface decoration with hydrophilic polyethylene glycol (PEG) molecules, represent an important turning point in liposome technology, leading to significant improvements in the pharmacokinetic profile compared to naked liposomes. Nevertheless, the generation of effective targeted liposomes-a central issue for cancer therapy-has faced several difficulties and clinical phase failures. Active targeting remains a challenge for liposomes. In this direction, a new Super Stealth Immunoliposomes (SSIL2) composed of a PEG-bi-phospholipids derivative is designed that stabilizes the polymer shielding over the liposomes. Furthermore, its counterpart, conjugated to the fragment antigen-binding of trastuzumab (Fab'TRZ -PEG-bi-phospholipids), is firmly anchored on the liposomes surface and correctly orients outward the targeting moiety. Throughout this study, the performances of SSIL2 are evaluated and compared to classic stealth liposomes and stealth immunoliposomes in vitro in a panel of cell lines and in vivo studies in zebrafish larvae and rodent models. Overall, SSIL2 shows superior in vitro and in vivo outcomes, both in terms of safety and anticancer efficacy, thus representing a step forward in targeted cancer therapy, and valuable for future development.

Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice.

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.

The evolution of polymer conjugation and drug targeting for the delivery of proteins and bioactive molecules.

Polymer conjugation can be considered one of the leading approaches within the vast field of nanotechnology-based drug delivery systems. In fact, such technology can be exploited for delivering an active molecule, such as a small drug, a protein, or genetic material, or it can be applied to other drug delivery systems as a strategy to improve their in vivo behavior or pharmacokinetic activities such as prolonging the half-life of a drug, conferring stealth properties, providing external stimuli responsiveness, and so on. If on the one hand, polymer conjugation with biotech drug is considered the linchpin of the protein delivery field boasting several products in clinical use, on the other, despite dedicated research, conjugation with low molecular weight drugs has not yet achieved the milestone of the first clinical approval. Some of the primary reasons for this debacle are the difficulties connected to achieving selective targeting to diseased tissue, organs, or cells, which is the main goal not only of polymer conjugation but of all delivery systems of small drugs. In light of the need to achieve better drug targeting, researchers are striving to identify more sophisticated, biocompatible delivery approaches and to open new horizons for drug targeting methodologies leading to successful clinical applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Folic Acid-Targeted Paclitaxel-Polymer Conjugates Exert Selective Cytotoxicity and Modulate Invasiveness of Colon Cancer Cells.

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.