Brocorens, Patrick

Gerbaux, Pascal

Saintmont, Fabrice

ORBi UMONS

Structural characterization of PAMAM dendrimers:Molecular modeling and ion mobility mass spectrometry investigation Fabrice Saintmont 1,2, Emilie Halin 1, Patrick Brocorens 2 and Pascal Gerbaux 11) Organic Synthesis & Mass Spectrometry Laboratory, Interdisciplinary Center for Mass Spectrometry (CISMa), Center of Innovation and Research in Materials and Polymers (CIRMAP), University of Mons - UMONS, 23 Place du Parc, 7000 Mons, Belgium; 2) Laboratory for Chemistry of Novel Materials, Center of Innovation and Research in Materials and Polymers (CIRMAP), Research Institute for Science and Engineering of Materials, University of Mons - UMONS, 23 Place du Parc, 7000 Mons, BelgiumIntroductionExperimental sectionResults and discussionConclusion1. A.-M. Caminade, C.-O. Anderson; J. Mater. Chem. B, 2, 4055-4066 (2014)2. A. Buczkowski, D. Waliszewski, et al.; Int. J. Pharm., 505, 1-13 (2016)3. D. A. Tomalia, B. Huang, et al.; Tretrahedron, 59, 3799-3813 (2003)4. Q. Duez, F. Chirot, et al.; J. Am. Soc. Mass Spectrom., 28, 2483-2491 (2017)Ref:AcknowlegdmentsThe MS laboratory acknowledges the “Fonds National de la Recherche Scientifique (FRS-FNRS)” for its contribution to the acquisition of the Synapt G2-Si mass spectrometer. F. S. thanks UMONS for its grant.Dendrimers are monodisperse hyperbranched polymers with a well-defined structure (Figure 1). Their step-by-step synthesis allows a control of the molecular structure, going from a generation (G) tothe following increasing the number of surface groups. One of the most studied dendrimer is poly(amidoamine) (PAMAM), especially in the context of drug delivery. PAMAM dendrimers arebiocompatible, water soluble and have a nanometric size, which are interesting properties for nanocarriers of drug molecules. Dendrimers can be used to transport drug molecules by covalently linkingthem to the surface groups of the dendrimer or by the formation of non-covalent complexes. Two possibilities exist for the later: encapsulation inside the dendrimer or electrostatic interaction at thesurface [1]. The type of non-covalent complex will have an impact on the nanodrug properties and it is important to distinguish them. Up to now, the techniques usually used (solubility, equilibriumdialysis, NMR) cannot clearly identify the structure [2]. A way to fully describe the structure of the complexes would be to use Ion Mobility Mass Spectrometry (IMMS) and molecular modeling.But first, it is necessary to characterize the structure of the dendrimers alone to validate the method.Theoretical sectionPAMAM with cystamine core (CYS) (Figure 2) G0 to G3 andwith ethylene diamine core (EDA) (Figure 3) G0 and G1 weresynthesized by a two step divergent method: alkylation of theamine with methyl acrylate, then amidation with ethylenediamine [3]. EDA PAMAM G2 and G3 are commercialproducts.Ion mobility MS experiments were performed on a WatersSynapt G2-Si (Figure 4), equiped with Travelling Wave IonMobility (TWIM). Experimental collision cross sections(CCSexp) were obtained via a polymer calibration (PEG 600,1000, 2000 and 3350, and PLA 3300 and 5500) [4]. Dendrimersolutions were prepared at a concentration of 50 micromolar ina 1:1:1 mixture of H2O:MeOH:ACN and infused directly in theESI source.Figure 4: Waters Synapt G2-Si mass spectrometer PAMAM ions observed in IMMS were also studied bymolecular mechanics and dynamics (MM/MD) withpartially reparametrized DREIDING force field andCOMPASS charges.The different charge states of PAMAM were protonatedat terminal amine, maximizing the distance between theprotons. The structures were optimized by twoconsecutive annealing from 5 to 800K for 50 cycles. Themost stable structure for each charge state and generationwas then submitted to an equilibration MD at 298K for20 ns followed by a second identical dynamic.200 frames from the second dynamic were injected intoCollidoscope program to calculate the theoretical CCS(CCSth) by the Trajectory Method (TM)coreG0 G1Figure 1: general structure of dendrimersFigure 2: cystamineFigure 3: ethylenediamneFigure 5: experimental CCS evolution of different generations of EDA PAMAM with the charge state.Colors are the same as in figure 6100300500700900110013000 1 2 3 4 5 6 7 8 9CCS (Å²)Charge (H+) EDA G0 EDA G1 EDA G2 EDA G3Figure 5 shows that the CCSexpincreases with the number of charges atthe surface of the dendrimer, for each ofthe 4 generations studied. This increasein CCS, and so the expansion of the 3Dstructure with the charges can beexplained by the tendency of the ions tominimize the coulombic repulsionbetween the charged branches, goingfrom globular structure to fullyextended.Structure of these ions were calculatedby MM/MD, along with thecorresponding CCSth in order tocharacterize them.Figure 8: comparison between experimental and theoretical CCS for EDA PAMAM G2. Errors bars represent 5% of the experimental value, which is the usually admitted error.The structures come from the 2nd dynamic with the solvent accessible surface area (SASA) with He as solvent in blue.4505005506006507007508001 2 3 4 5 6 7CCS (Å²)Charge (H+) CCS exp CCS thThe good agreement between CCSexp and CCSth (Figure 8) allows to determine the 3D structure of the ions inthe gas phase. As supposed based on the experimental values, the ions with a low number of charges canstabilize them and stay globular, but the structure expends when the coulombic repulsion became too high andseparates the branches of the dendrimer.As the CCsth are calculated in He, the solvent accessible surface Area (SASA) with He as solvent wascalculated. This surface can be seen as the surface on which the gas can collide and would be linked to the CCS.Indeed, figure 7 shows that the SASA increases with the expansion of the structure, and so with the increase ofthe number of charges.200022002400260028003000320034005005506006507007508001 2 3 4 5 6 7SASA (Å²)CCS (Å²)Charge (H+) CCS th SASAFigure 9: comparison between theoretical CCS and calculated SASA for EDA PAMAM G2There is a correlation between thetwo theoretical parameters (Figure9), so the SASA could be use for aquick qualitative comparison betweentheoretical structures andexperimental CCS.Note that the CCSth were averaged on200 frames of the 2nd dynamic andthe SASA was calculated only on thefirst frame of the same dynamic.IMMS and molecular modeling allow to investigate the 3D structure of PAMAM ions in the gas phase; especially with a special attention paid to the charge state influence.The combination of theoretical and experimental data appears useful to characterize dendrimers and could also be used for investigating their non covalent complexes, if those are stable enough to survive the IMMS.Figure 7: Experimental CCS of CYS and EDA PAMAM from G0 to G3100300500700900110013000 1 2 3 4 5 6 7 8 9 10CCS (Å²)Charge (H+)G0G1G2G3CYSEDAComparison between CYSand EDA PAMAM showsthat the core have a weakinfluence on the structureand its modification withcharges (Figure 7). Thecomparison betweenexperiment and theory alsoreveals a good agreementfor CYS PAMAM.G0G1G2Figure 6: EDA PAMAM G2 with the different generations highlightedG2:sali 1:10 10% MeOH dans H2Om/z550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300%0100QTOFUS_FASA_170320_G2-SALI_02 247 (4.683) Cm (1:378) TOF MS ES+ 1.24e41086E2 + 3H+EDA G2 = E2Salicylic acid= SA815E2 + 4H+652E2 + 5H+544E2 + 6H+680E2 + 1SA+5H+ 849E2 + 1SA+ 4H+1132E2 + 1SA + 3H+884E2 + 2SA+ 4H+1178E2 + 2SA + 3H+1224E2 + 3SA + 3H+m/z600 700 800 900 1000 1100 1200 1300Figure 10: ESI-MS spectra of a solution 1:10 EDA G2 : salicylic acid in H2OFigure 10 shows non covalentcomplexes between EDA PAMAMG2 and salicylic acid. Thestoichiometry changes with thecharge state and complexes arefragile. They are non-specific andcreated by the interaction betweenprotonated amine and carboxylicacid at the surface. No inclusioncomplexes were observedirrespective of the experimentalconditions, the generation ofPAMAM and the guest.

Structural characterization of PAMAM dendrimers: molecular modeling and ion mobility investigation

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Structural characterization of PAMAM dendrimers: molecular modeling and ion mobility investigation

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