ABSTRACT
Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Cell-Penetrating Peptides/pharmacokinetics , Glioblastoma/drug therapy , Oligopeptides/pharmacokinetics , Tuftsin/pharmacokinetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Carriers/pharmacology , Drug Delivery Systems , Glioblastoma/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Neuropilin-1/metabolism , Oligopeptides/chemistry , Oligopeptides/pharmacology , Rats , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Tuftsin/analogs & derivatives , Tuftsin/pharmacology , Tumor Cells, CulturedABSTRACT
Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.
Subject(s)
Antitubercular Agents/administration & dosage , Cell-Penetrating Peptides/metabolism , Drug Carriers/metabolism , Isoniazid/administration & dosage , Membrane Lipids/metabolism , Amino Acid Sequence , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacokinetics , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Drug Carriers/chemistry , Humans , Isoniazid/chemistry , Isoniazid/pharmacokinetics , Lipid Bilayers/metabolism , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapyABSTRACT
Fluorescent labelling is a common approach to reveal the molecular details of cellular uptake, internalisation, transport, distribution processes in biological systems. The conjugation with a fluorescent moiety might affect relevant physico-chemical and in vitro transport properties of the bioactive component. A representative set of seven cationic peptides-including cell-penetrating peptides as well as antimicrobial peptides and synthetic derivatives-was selected for our comparative study. Membrane affinity of the peptides and their 5(6)-carboxyfluorescein (Cf) derivatives was determined quantitatively and compared applying Langmuir monolayer of zwitterionic (DPPC) and negatively charged (DPPC + DPPG) lipids as cell membrane models. The interaction with neutral lipid layer is mainly governed by the overall hydrophobicity of the molecule which is remarkably increased by Cf-conjugation for the most hydrophobic Magainin, Melittin and Transportan. A significantly enhanced membrane affinity was detected in negatively charged lipid model monolayer for all of the peptides since the combination of electrostatic and hydrophobic interaction is active in that case. The Cf-conjugation improved the penetration ability of Penetratin and Dhvar4 suggesting that both the highly charged character (Z/n) and the increased hydrophobicity by Cf-conjugation present important contribution to membrane interaction. This effect might also responsible for the observed high in vitro internalisation rate of Penetratin and Dhvar4, while according to in vitro studies they did not cause damage of cell membrane. From the experiments with the given seven cationic peptides, it can be concluded that the Cf-conjugation alters the degree of membrane interaction of such peptides which are moderately hydrophobic and highly charged.
