Research Archive Citation for published version: Martinho N., Silva L. C., Florindo H. F., Brocchini S., Zloh M., and Barata T. S., ‘Rational design of novel, fluorescent, tagged glutamic acid dendrimers with different terminal groups and in silico analysis of their properties’, International Journal of Nanomedicine, Vol. 12: 7053-7073, September 2017. DOI: https://doi.org/10.2147/IJN.S135475 Document Version: This is the Published version. Copyright and Reuse: This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Enquiries If you believe this document infringes copyright, please contact the Research & Scholarly Communications Team at [email protected] Journal name: International Journal of Nanomedicine Article Designation: Original Research Year: 2017 Volume: 12 International Journal of Nanomedicine Dovepress Running head verso: Martinho et al Running head recto: Design of glutamic acid dendrimers with different terminal groups open access to scientific and medical research DOI: http://dx.doi.org/10.2147/IJN.S135475 Open Access Full Text Article ORIGINAL RESEARCH Rational design of novel, fluorescent, tagged glutamic acid dendrimers with different terminal groups and in silico analysis of their properties Nuno Martinho1–3 Abstract: Dendrimers are hyperbranched polymers with a multifunctional architecture that can Liana C Silva1,4 be tailored for the use in various biomedical applications. Peptide dendrimers are particularly Helena F Florindo1 relevant for drug delivery applications due to their versatility and safety profile. The overall lack Steve Brocchini2 of knowledge of their three-dimensional structure, conformational behavior and structure–activity Mire Zloh3 relationship has slowed down their development. Fluorophores are often conjugated to dendrimers Teresa S Barata2 to study their interaction with biomolecules and provide information about their mechanism of action at the molecular level. However, these probes can change dendrimer surface properties and 1 Research Institute for Medicines have a direct impact on their interactions with biomolecules and with lipid membranes. In this study, (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, we have used computer-aided molecular design and molecular dynamics simulations to identify 2 For personal use only. Lisbon, Portugal; Department of optimal topology of a poly(L-glutamic acid) (PG) backbone dendrimer that allows incorporation Pharmaceutics, UCL School of of fluorophores in the core with minimal availability for undesired interactions. Extensive all- Pharmacy, London, 3School of Life and Medical Sciences, University of atom molecular dynamic simulations with the CHARMM force field were carried out for different Hertfordshire, Hatfield, UK; 4Centro generations of PG dendrimers with the core modified with a fluorophore (nitrobenzoxadiazole and de Química-Física Molecular and IN – Institute of Nanoscience and Oregon Green 488) and various surface groups (glutamic acid, lysine and tryptophan). Analysis Nanotechnology, Instituto Superior of structural and topological features of all designed dendrimers provided information about their Técnico, Universidade de Lisboa, size, shape, internal distribution and dynamic behavior. We have found that four generations of a Lisboa, Portugal PG dendrimer are needed to ensure minimal exposure of a core-conjugated fluorophore to exter- nal environment and absence of undesired interactions regardless of the surface terminal groups. Our findings suggest that NBD-PG-G4 can provide a suitable scaffold to be used for biophysical studies of surface-modified dendrimers to provide a deeper understanding of their intermolecular interactions, mechanisms of action and trafficking in a biological system. Keywords: dendrimers, peptide dendrimers, molecular dynamics, fluorescence, CHARMM, International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 147.197.30.88 on 02-Feb-2018 structure-activity, surface properties Introduction Dendrimers belong to a class of hyperbranched polymers with well-defined radial architecture that has been extensively studied for biomedical applications.1 The topology Correspondence: Mire Zloh of dendrimers can be described by their core, internal branches (or spacers) and terminal School of Life and Medical Sciences, groups. By using various monomers to build different topological architectures, it is University of Hertfordshire, College Lane, Hatfield AL10 9AB, UK possible to design their size, their shape, the surface groups available for interaction Email [email protected] and overall physicochemical properties.2–4 Similar to other drug delivery systems, one of the key requirements is to have an adequate safety profile.5 In this regard, Teresa S Barata Department of Pharmaceutics, UCL peptide dendrimers consisting of natural amino acids are attractive candidates for use School of Pharmacy, 29/39 Brunswick in biological systems due to their potential biodegradability and biocompatibility.6 Square, London WC1N 1AX, UK Email [email protected] Peptide dendrimers can be synthesized either by linear combinations of amino acids submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2017:12 7053–7073 7053 Dovepress © 2017 Martinho et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/IJN.S135475 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Powered by TCPDF (www.tcpdf.org) 1 / 1 Martinho et al Dovepress assembled at a branching point7,8 or by using amino acids as to incorporate a fluorophore (of different size) into its core. focal branching points themselves (eg, glutamic acid [GLU],9 The goal of this approach was to determine the generation lysine [LYS]10,11 and aspartic acid12). Owing to side chain size at which the fluorophore can be protected from the external and the combination of amino acids, these peptide dendrimers environment, thus ensuring that the probe does not interfere might have an asymmetric arrangement that will create dif- with the interaction of PG dendrimers with other biomol- ferent local microenvironments within the dendrimer and be ecules. Furthermore, different surface modifications were suitable to different surface modifications.13–15 studied to determine their overall effect on the structural Although extensively studied, the mechanism of properties of the modified core dendrimers. We have used action of bioactive dendrimers is still difficult to probe all-atom MD simulations using the CHARMM FF as an experimentally.16 One way to track dendrimers during their FF suitable to study structural properties of dendrimers.23 interaction with biomolecules and cells is to conjugate a fluo- These simulations were used to describe the structure and rescent probe at the surface of these carriers. However, there conformational behavior of PG dendrimers, as well as the are two main drawbacks of using this approach. First, fluores- effects of modifying the dendrimer core by attaching different cent probes may randomly be distributed at the surface. This size fluorophores, namely, nitrobenzoxadiazole (NBD) would result in a heterogeneous population of dendrimers, or Oregon Green 488 (ORG). NBD was chosen due to its ranging from non-labelled dendrimers to dendrimers with small size and optical properties that are suitable to monitor multiple fluorophores, where the proximity between the polarity changes in the surrounding environment, whereas probes might lead to possible quenching effects.17 Second, the ORG was selected due to its bigger size and suitable the fluorophore probes can themselves interact with cells spectroscopic properties to be used in monitoring cell events. and interfere or mask the real interaction of dendrimers with We further evaluated the effects of diverse terminal groups these biological systems.18,19 Furthermore, the targeted drug with different charges on the structure of the dendrimers by delivery achieved by conjugation of different monomers to using GLU, LYS or tryptophan (TRP; Figure 1). This study surface groups13,14 may be affected by the introduction of a demonstrates the use of MD simulation to provide an insight For personal use only. fluorophore on the surface. into the versatility of the PG dendrimers that can be further In order to avoid this kind of interferences, phosphorus explored for different biomedical applications. dendriplexes with a fluorophore attached to the core have been synthesized to study their interaction with cells.20 Methods Thus, designing dendrimers that have a defined number of Monomer parameterization probes that are hindered from the external environment