Can graphene oxide be a suitable platform for the complexation with nucleic acids

Université de Strasbourg ÉCOLE DOCTORALE DES SCIENCES CHIMIQUES Can graphene oxide be a suitable platform for the complexation with nucleic acids? by Ngoc Do Quyen CHAU Thesis submitted for the degree of Doctor of Philosophy in Chemistry 24th November 2017 Supervisor: Dr BIANCO Alberto Members of the jury: Dr PALERMO Vincenzo Dr CAMPIDELLI Stéphane Dr BAATI Rachid UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES CHIMIQUES UPR 3572 THÈSE présentée par : Ngoc Do Quyen CHAU soutenue le : 24 Novembre 2017 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Chimie L’oxyde de graphène peut-il devenir une plateforme appropriée pour la complexation d'acides nucléiques ? THÈSE dirigée par : M BIANCO Alberto Directeur de recherche, CNRS RAPPORTEURS : M PALERMO Vincenzo Senior Researcher, CNR, Italie M CAMPIDELLI Stéphane Chercheur, CEA AUTRES MEMBRES DU JURY : M BAATI Rachid Directeur de recherche, CNRS INDEX Index Abstract Acronyms and Abbreviations RESUME DE THESE 16 INTRODUCTION 36 1.1 Graphene oxide 36 1.2 Promise, facts and challenges of graphene oxide in biomedical applications 40 1.2.1 Non-covalent and covalent approaches 40 1.2.1.1 Non-covalent interactions and their driving forces 40 1.2.1.2 Covalent functionalization 43 1.2.2 Graphene oxide based therapy 47 1.2.2.1 Drug and gene delivery 47 1.2.2.2 Photothermal therapy and photodynamic therapy 52 1.2.2.3 Biomedical imaging 55 1.2.3 Biocompatibility and toxicity 58 1.3 Graphene oxide and nucleic acids interactions 61 1.3.1 Graphene oxide bio-interfacing with nucleic acids and biological effects of the graphene/nucleic acid complexes 61 1.3.2 Bioapplication in gene delivery 65 1.4 Future perspective of graphene oxide in cancer therapy 72 1.5 Thesis objectives and outline 73 1.6 Bibliography 76 ELUCIDATION OF siRNA COMPLEXATION EFFICIENCY BY GRAPHENE OXIDE AND REDUCED GRAPHENE OXIDE 101 Abstract 102 Introduction 103 Experimental section 106 Results and discussions 111 Conclusion 131 Bibliography 133 ABSTRACT Doctoral Thesis By Ngoc Do Quyen CHAU In the last decades, graphene oxide (GO) has been predicted as a wonderful nanomaterial in myriad applications for its unique and outstanding properties Intensive research is ongoing to scrutinize its potential role as a prominent vector in gene delivery, especially in gene silencing The main aim of my Thesis is to design a graphene-based hybrid material as a non-viral vector for delivery of small interfering RNA (siRNA) Hence, control of the oxygenated groups on the surface of GO can lead to different behavior in term functionalization ability and interaction with biomolecules In this context, one of the first approach has been to develop various green and facile reduction and reepoxidation methods to obtain GO with different levels of oxygenated moieties In the next step, the introduction of different amines and polymers on these prepared graphene materials via the epoxy ring opening reaction allowed to obtain a novel platform for better complexation with siRNA I have figured out that the driving forces of the ability of complexing with siRNA are dependent on the functional groups conjugated to GO, either due to electrostatic interaction or to hydrogen bond interaction on the other hand, several works demonstrated the ability of GO to efficiently adsorb siRNA on its surface and to transport it into the cells However, studies whether and how siRNA interacts with GO are still inconclusive For this reason, the interactions between GO and siRNA molecules have been then systematically investigated I have found that the siRNA secondary structure is clearly altered by the interaction with GO flakes Interestingly, GO functionalized with low molecular weight polyethyleneimine is able to protect siRNA from structural modifications and to improve the complexing with siRNA Various techniques have been explored to characterize GO with various oxygen percentages, conjugation of cationic molecules with graphene materials, and the interaction of GO with siRNA Besides, the preliminary biological tests proved the efficiency of our graphene derivatives as a vehicle for delivery of siRNA into the cells I believed that this research effort will improve our understanding of the behavior of the GO/siRNA complexes, and thus facilitate the design of new appropriate and efficient gene silencing systems Acronyms and abbreviations ACRONYMS AND ABBREVIATIONS 5-FU 5- fluorouracil 6-FAM 6-carboxyfluorescein A549 cells human lung carcinoma cells AA siRNA with one strand labeled at 5’ position with Alexa Fluor(donor) and another strand labeled at 5’ position with Alexa Fluor647 (acceptor) ADR Adriamycin AFM atomic force microscopy Boc2O di-tert-butyl dicarbonate BPEI branched PEI CC siRNA with one strand labeled at 5’ position with Cy(donor) and another strand labeled at 3’ position with Cy5 (acceptor) CD circular dichroism Ce6 chlorin e6 CMG magnetic rGO CNTs carbon nanotubes Acronyms and abbreviations CS chitosan D1 dendron first generation D2 dendron second generation DAPI fluorescent stain, 4',6-Diamidino-2-phenylindole DCM dichloromethane DIEA N,N-Diisopropylethylamine DLS dynamic light scattering DMA/c N, N-dimethylacetamide DMDO dimethyldioxirane DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DNA deoxyribonucleic acid DOX doxorubicin dsDNA double strand DNA dsRNA double strand RNA E FRET efficiency EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide FA folic acid 10 Figure Direct polarization 13C ssNMR of rGOA-O3-PEI (a); rGOA-O3 (b); mechanical mixture of rGOA-O3 and PEI (c); and PEI alone (d) The spectrum of PEI was obtained with a smaller spectral window and processed with a smaller line broadening (150Hz) Although the resulting spectra appear rather featureless with broad lines and relatively low S/N ratio they remain informative as far as we may separate a minimal set of peaks related to chemical functions Clearest trends may be also extracted from the experimental data by fitting with a pure Chemical Shift Anisotropy model (Figure 7, red lines) The NMR spectrum of rGOA-O3 shows two peaks centered at 160 and 121 ppm attributed to C=O and C=C conjugated bonds, respectively (Figure 6b) [50] The presence of the C=O peak is in agreement with the XPS data (Figure 4d) Comparing this spectrum with that of rGOA-O3-PEI (Figure 7a) we can observe one additional (although weak) peak at 35 ppm assigned to the methylene groups of the PEI chain To 125 understand if the covalent functionalization with PEI occurred, a simple mechanical mixture of rGOA-O3 and PEI was analyzed by ssNMR (Figure 7c) In this case, the methylene PEI groups appear at 45 ppm very closed to the peak obtained from the polymer alone (Figure 7d), although larger (ca ppm) This peak broadening can be attributed to the highly heterogeneous magnetic susceptibility surrounding due to the aromatic ring currents on the graphene sheets For rGOA-O3-PEI the CH2 signal is rather wide (ca 30 ppm) and exhibits a ppm upfield shift relative to the equivalent signal (45 ppm) appearing in the physical mixture of PEI and rGOA-O3 The spectral differences measured among the three samples support further the covalent bonding of PEI to the graphene surface 3.3 Complexation with siRNA The final aim of this study was to investigate the capacity of the different GOs to interact and complex siRNA For this purpose, we used double-stranded siRNA that is a small molecule of 19 nucleobases Complexation was performed in aqueous solutions without adding any complementary salts or solvent GO binding performances have been investigated by gel electrophoresis In each run, the various GOs have been mixed in water with siRNA at 0, 50, 100, 150, 200 and 300 GO/siRNA mass ratios Each complexation experiment was repeated at least three times Free siRNA molecules were then estimated comparing the fluorescence signal between the blank (GO/siRNA at mass ratio 0) and the other complexation ratios as shown in Figure 126 Figure Complexation of GO-TEG Top: image of the electrophoresis gel; Bottom: histograms showing the free siRNA signal at different GO/siRNA mass ratios All different electrophoresis gels are shown in SI (Figure S3) As an example, free siRNA values at mass ratio GO/siRNA of 200 are reported in Table Table Percentage of free siRNA signal compared to blank of non-functionalized and amino-functionalized GOs at 200 GO/siRNA mass ratio Type of functionalization GO rGOA rGOB rGOA-O3 Non-functionalized 67±9 81±7 85±9 86±8 TEG functionalization 43±6 98±2 81±8 62±9 PEI functionalization 56±9 8±6 23±6 3±2* *at mass ratio GO/siRNA of 100 First, we were interested in investigating the complexation abilities of the nonfunctionalized materials As reported in Table 4, GO exerts the best complexing 127 capacity with 67±9% siRNA released, while the hydrothermally (rGOA) and vitamin Ctreated (rGOB) GOs have a lower interaction capacity with 81±7% and 85±9% of free siRNA migrating in the gel, respectively Ozone treatment (rGOA-O3) also shows negligible complexation ability with a release of 86±8% of siRNA molecules Since reduction and epoxidation treatments mainly affect the graphene surface, the complexation trend may be explained by comparing the amount of oxygenated groups present on each GO sheet The interaction between GO and nucleotide molecules has not been yet totally understood It was stated that GO can bind to single stranded DNA oligonucleotides via π-π stacking interactions between the nucleobases and the π system on the surface of GO [51,52] Besides, other experiments displayed that siRNA complexation is trigged by polar interaction between the GO oxygenated groups and the polar groups on the nucleotide strand[53] In particular, GO surface is rich in Hbond donors (alcohols and carboxylic acids) and acceptors (epoxides) On the other hand, oxygenated groups induce a negative charge on the GO surface [48] that may cause the coulombic repulsion of the negatively charged siRNA, thus hampering the complexation The oxidation level of each of the graphene sample was investigated above by XPS (Figure S2 and Table 2) The complexation trend GO>> rGOA  rGOB appears correlated to the C/O ratio (Table and Figure S4a), and in particular it seems to increase with the oxidation levels of the graphene sheets For this reason, the interaction between GOs and siRNA strands is likely driven mostly by the H-bond interactions The re-epoxidation of rGOA is not able to restore the affinity of the complexation ability of GO Most probably, the introduced epoxy groups than can act 128 only as H-bond acceptors are not able to efficiently increase the binding of siRNA molecules About the complexation ability of TEG functionalized GOs, we can observe the most remarkable difference on GO-TEG with a free siRNA of 43±6% Comparing the TEG functionalization of two reduced GOs, the one reduced with vitamin C exerts a higher complexation ability than the hydrothermally reduced GO (81±8% and 98±2% of free siRNA respectively) More interestingly, a stronger ability of complexation was observed when rGO underwent the ozone treatment (rGOA-O3-TEG) (62±9% of free siRNA) TEG functionalized graphene materials show similar complexation trend to non-functionalized materials GO-TEG exhibits the best complexing ability followed by rGOA-O3-TEG The two reduced rGOA and rGOB, also in this case, displayed very low complexation levels TEG functionalization allows the introduction of primary amines on GO surface Those protonable groups can bind siRNA via coulombic interactions (in case they are protonated) or simply via H-bond interactions There is generally a positive effect of the TEG functionalization on the complexation in comparison to the corresponding non-functionalized materials Besides, the complexation with the different TEG-modified GOs cannot be attributed to the amount of TEG (Table 3) Most probably, the interaction between GOs-TEG and siRNA molecules is still driven by the oxygenated groups present on the GO, as in the case of non-functionalized GOs The trend in the complexation is matching the trend of C/O ratio (Figure S4b) Compared to rGOA-O3, the remarkable enhancement on rGOA-O3TEG complexing ability can be explained by the amino functionalization Indeed, TEG functionalization converts epoxide groups in more polar - amino alcohols These H- 129 bond donor groups, together with the primary amine, may enhance the complexation degree of rGOA-O3-TEG for siRNA PEI functionalized graphene materials (Table 4) exhibit a significant increase in complexation with siRNA molecules, especially in the case of ozone treated GO, showing a complete complexation even at GO/siRNA mass ratio 100 (3±2%) The PEI derivatives of GO, rGOA, rGOB showed a value of 56±9%, 8±6%, 23±6% of free siRNA, respectively In the case of PEI functionalized GOs, the trend is nearly inverted compared to non-functionalized GO with rGOA-O3> rGOA>rGOB>GO As it was mentioned before, PEI can efficiently complex oligonucleotides because this polymer is a protonable amine and can interact with siRNA by ionic forces [54] In addition, PEI has a higher molecular weight than TEG diamine, and a higher amount of amines leads to a stronger siRNA complexation with the polymer chain As for TEG functionalized GOs, comparing GO, rGOA and rGOB PEI derivatives on the basis of XPS data (Table and Figure S4c), there is a small difference on polymer functionalization ratio between the three graphenes that however would not directly explain the huge change on GO complexation capacities The difference in the C/O ratio of these three materials should be attributed to the presence of other oxygenated functions such as hydroxyl or carboxylic groups These groups may negatively affect the ability of GO in term of complexation with siRNA Indeed, the presence of proximal negatively charged oxygenated groups on the surface of GO may disfavor the interactions between the different GO-PEI and the negatively charged siRNA molecules This fact would explain the higher performance in complexation of the two rGO-PEI compared to GO-PEI In case of the epoxidation, we were able to introduce 130 selectively the epoxy groups to the rGOA The very high complexation capacity of rGOA-O3 for siRNA can be explained by the high amount of PEI functionalization (Table and Figure S4c) and by the low presence of proximal oxygenated species on the graphene sheets Conclusions In summary, the interaction of siRNA with a series of graphene materials has been explored We have designed and tested different green and facile approaches to obtain GO with different oxygen percentages by reduction via a hydrothermal treatment, vitamin C-induced reduction, and ozonation We were able to functionalize these materials with two different amines through epoxy ring opening The reactions were performed in water at room temperature This strategy could be extended to more complex amino-containing bioactive molecules We confirmed that nitrogen was successfully introduced on the surface of these graphene materials More interestingly, the complexation of siRNA with the non-functionalized graphene materials and the amino conjugated GOs have been thoroughly investigated By gel electrophoresis, we proved that the rGOs and ozonated rGO functionalized with low molecular weight PEI were the most efficient in complexing siRNA molecules Finally, the behavior of GO complexation could be attributed to two main contributions corresponding to polar interactions by H-bonds and ionic interactions H-bond interactions seem to prevail in the case of non-functionalized GOs, while ionic forces have a main role for PEI-functionalized GOs To the best of our knowledge this is the first work aiming to understand the interactions between graphene surface groups and 131 double stranded RNA molecules (i.e siRNA) We believe that the identification and the rationalization of the supramolecular forces that affect complexation is the key point for the preparation of efficient GO nano-carrier Our systems proved to be good candidates for gene silencing and will be furtherly tested in vitro and in vivo Acknowledgements The authors gratefully acknowledge financial support from EU H2020-Adhoc-2014-20 GrapheneCore1 (no 696656) and from the Agence Nationale de la Recherche (ANR) through the LabEx project Chemistry of Complex Systems (ANR-10-LABX0026_CSC) This work was partly supported by the Centre National de la Recherche Scientifique (CNRS), by the International Center for Frontier Research in Chemistry (icFRC), by JST PRESTO, and by JSPS KAKENHI (Science of Atomic Layers (SATL), Grant Number 16H00915) N.D.Q.C 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S Rheiner, Y Bae, Increased poly(ethylene glycol) density decreases transfection efficacy of siRNA/poly(ethylene imine) complexes, AIMS Bioeng (2016) 454–467 139 ... cells AA siRNA with one strand labeled at 5’ position with Alexa Fluor(donor) and another strand labeled at 5’ position with Alexa Fluor647 (acceptor) ADR Adriamycin AFM atomic force microscopy... and structure as well as its favorable physicochemical properties and remarkable features, GO has shown a great potential in biomedical applications and has become a suitable carrier for a variety... these prepared graphene materials via the epoxy ring opening reaction allowed to obtain a novel platform for better complexation with siRNA I have figured out that the driving forces of the ability