We have developed an operationally simple, time, and cost-effective protocol to produce a novel tetrazine-based iron oxide nanoparticle using commercially available and inexpensive materials. Our protocol proceeds at room temperature and uses hexafluorophosphate azabenzotriazole tetramethyl uronium, a well-known, widely used reagent for the large-scale industrial production of important pharmaceuticals. The nanoparticles obtained have a diameter range between 16 and 21 nm and showed no toxicity against endothelial cell lines. The tetrazine moiety on the nanoparticle surface could potentially allow further attachment of specific targeting vectors by using so-called copper-free click chemistry. We therefore anticipate that our protocol can represent a significant breakthrough in the future development and commercialization of improved, tissue-specific contrast agents and drug delivery for clinical diagnosis, monitoring and therapy of diseases at an asymptomatic stage

A Kuzmin

AG Kolhatkar

Alexandru Chivu

AM Morawski

BL Oliveira

CS McKay

DMM Jaradat

F Herranz

HL Evans

JB Haun

JM Baskin

JR Dunetz

Juan C. Kaski

M Colombo

M Ohgushi

Marco M. Meloni

MF Debets

MM Meloni

NK Devaraj

RA Trivedi

SA Schmitz

SK Mishra

Stephen Barton

Taigang He

Wenhui Song

Crossref

Facile, productive, and cost-effective synthesis of a novel tetrazine-based iron oxide nanoparticle for targeted image contrast agents and nanomedicines

Meloni, MM

Barton, S

Chivu, A

Kaski, JC

Song, W

He, T

St George's Online Research Archive

  2 Facile, productive and cost-effective synthesis of a novel tetrazine-based iron oxide nanoparticle for targeted image contrast agents and nanomedicines   Marco M. Meloni • Stephen Barton • Alexandru Chivu • 1 Juan C. Kaski • Wenhui Song • Taigang He 2  3 Abstract We have developed an operationally simple, time 4 and cost-effective protocol to produce a novel tetrazine-based 5 iron oxide nanoparticle using commercially available and 6 inexpensive materials. Our protocol proceeds at room 7 temperature and uses Hexafluorophosphate Azabenzotriazole 8 Tetramethyl Uronium, a well-known, widely used reagent for 9 the large-scale industrial production of important 10 pharmaceuticals. The nanoparticles obtained have a diameter 11 range between 16 and 21 nm and showed no toxicity against 12 endothelial cell lines. The tetrazine moiety on the 13 nanoparticle surface could potentially allow further 14 attachment of specific targeting vectors by using so-called 15 copper-free click chemistry. We therefore anticipate that our 16 protocol can represent a significant breakthrough in the future 17 development and commercialization of improved, tissue-18 specific contrast agents and drug delivery for clinical 19 diagnosis, monitoring and therapy of diseases at an 20 asymptomatic stage.  21  22 Keywords Diagnosis • Magnetic Resonance Imaging • Iron 23 Oxide Nanoparticles • Copper-free click Chemistry • 24 Bioconjugation • Synthesis 25  26 Introduction 27  28 Since their discovery (Ohgushi, 1978), Iron Oxide 29 Nanoparticles (IONPs) have emerged as powerful tools for a 30 wide scope of applications such as in the fields of biolabeling, 31 medical diagnostics, and therapy (Colombo et al. 2012). In 32 particular as contrast agents for magnetic resonance imaging 33 (MRI), IONPs have attracted enormous attention. IONPs 34 have a transverse relaxivity which increases at higher 35 magnetic fields, resulting in enhanced signal-to-noise ratio 36 with lower dosages (Mishra et al. 2016). The use of IONPs 37 for diagnostic imaging is therefore expected to increase 38 steadily. Unlike Gd-based contrast agents (CAs), IONPs have 39 proven safe with some systems already approved for clinical 40 use (Schmitz et al. 2001; Trivedi et al. 2004). With the recent 41 advent of molecular imaging (Meloni et al. 2017), IONPs are 42                                                      Marco M. Meloni •  Juan C. Kaski • Taigang He  The Cardiology Academy Group, St George’s University of London, Cranmer Terrace, SW17 0RE, London, UK.  Marco M. Meloni • Stephen Barton  School of Pharmacy and Chemistry, Kingston University, Penhryn Road, KT1 2EE, London, UK.  Alexandru Chivu • Wenhui Song  UCL Centre for Biomaterials, Division of surgery & Interventional Science, University College London, Pond Street, NW3 2PS, London, UK.  increasingly used for the preparation of targeted CAs, 43 allowing non-invasive visualization of biomolecules which 44 are the signatures of diseases in specific body tissues, both in 45 vivo and in real time. The preparation of these targeted CAs 46 firstly involves coating of the IONPs surface with a 47 biocompatible, clinically approved polymer, followed by its 48 functionalization with a targeting vector which allows tissue 49 specificity (A. M. Morawski et al. 2005). 50  51 This process, so-called standard bioconjugation, uses well-52 established synthetic chemistry and involves the formation of 53 stable chemical bonds. Despite being reliable, this method 54 can lead to low yields and produce toxic byproducts, resulting 55 in expensive, time-consuming purifications and toxicological 56 screenings. A number of years may therefore be required for 57 the translation, large scale production and commercialization 58 of targeted, nanoparticle-based CAs into the clinical market. 59  60 Copper-free click reactions recently emerged as a powerful, 61 time-saving bioconjugation strategy to prepare targeted CAs 62 (Devaraj and Weissleder 2011; McKay and Finn 2014). 63 Copper-free click reactions are operationally simpler and 64 proceed at room temperature. They eliminate the use of 65 cytotoxic copper catalysts (Baskin JM et al. 2007) and are 66 inert to both water and oxygen. They also generate minimal, 67 inoffensive byproducts, providing the best yield with the 68 highest reaction rates. Copper-free click reactions can 69 proceed in living organisms, allowing the targeting of 70 biomolecules with high specificity without altering any native 71 biochemical process. Among the arsenal of available copper-72 free click reactions, the Inverse Electron Demand Diels Alder 73 (IEDDA) reaction stands out as the most promising one for 74 its rapid reaction rate and generation of safe, inert nitrogen as 75 the only byproduct (Oliveira et al. 2017). IEDDA reactions 76 also involve tetrazines and cycloalkenes, both of which are 77 easier substrates to prepare if compared to 78 azadibenzocyclooctynes (DIBACs) (Debets et al. 2010) and 79 biarylazacyclooctynones (BARACs) (Kuzmin et al. 2010). 80 Despite such significant advantages, the preparation of 81 targeted, nanoparticle-based CAs using IEDDA is a relatively 82 unexplored field. A possible reason is a shortage of reliable 83 protocols to prepare tetrazine-based IONPs. Therefore, the 84 Taigang He  Royal Brompton Hospital, Imperial College London, London, UK.  Marco M. Meloni () • Taigang He () The Cardiology Academy Group, St George’s University of London, Cranmer Terrace, SW17 0RE, London, UK.  e-mail: mmeloni@sgul.ac.uk e-mail: the@sgul.ac.uk   3 identification of novel, green and efficient ways to prepare 1 these functional nanoparticles can accelerate the development 2 of improved contrast agents for diagnosis and therapy. In this 3 paper we designed and synthesized a novel route to a 4 tetrazine-bound nanoparticle 4 (Scheme 1). 5  6  7 Scheme 1 (a) Oleic acid, oleylamine, phenyl ether (b) BTAC, 8 KMnO4 (c). Tetrazine 5, HATU, DIPEA, CH2Cl2, at room 9 temperature. 10  11 As illustrated above, our strategy involved the loading of a 12 tetrazine moiety to nanoparticles 3 using 13 Hexafluorophosphate Azabenzotriazole Tetramethyl 14 Uronium (HATU), a readily available, inexpensive and well-15 known reagent. Our overall approach to nanoparticles 4 can 16 be exploited for the future production of improved contrast 17 reagents, with potential application also in the fields of 18 nanomedicine. 19  20 Results and discussion 21  22 In this work, we successfully designed and developed an 23 operationally simple, economical and productive approach to 24 produce a novel tetrazine-decorated IONP 4 (Scheme 1, page 25 2). Our protocol is reproducible by taking an advantage of 26 well-established chemistry and usages of building blocks 3 27 and 5, both accessible via established published protocols 28 (Herranz et al. 2008; Evans et al. 2014). Nanoparticles 4 were 29 obtained via coupling between carboxylic acid IONPs 3 and 30 tetrazine-based amine 5 (Scheme 1, page 2). Our method 31 builds upon the reaction between a carboxylic acid and an 32 amine, which produces extremely stable chemical bonds even 33 in a biological environment and is one of the most robust, 34 known reactions in synthetic chemistry (Jaradat, 2018). 35 During this process a challenge also emerged due to the 36 presence of the paramagnetic core both in 3 and 4, which 37 excluded quantitative reaction monitoring via NMR. 38 Therefore, we first decided to validate the chemistry using 39 acetic acid as a surrogate for nanoparticles 3. We wanted a 40 fast, high yielding protocol to obtain model compound 6. 41 After unsuccessful attempts to prepare this latter using N-42 hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-43 dimethylaminopropyl)carbodiimide (EDC) in water (Herranz 44 et al. 2008), we found that the system HATU/DIPEA/CH2Cl2 45 afforded 6 in 92% yield after only 3 hours. 46 We therefore adopted this method for the synthesis of 47 nanoparticles 4. Coupling between 3 and 5 was performed 48 with HATU and DIPEA in CH2Cl2, affording the desired 49 nanoparticles 4. We found this method both safe and 50 operationally simple for many reasons. Firstly, HATU is an 51 established chemical which is widely used during large scale 52 synthesis of pharmaceuticals. Several reports of HATU 53 applications have showcased its use in the synthesis of 54 promising drug candidates (Dunetz et al. 2016 and references 55 cited herein). Secondly, a simple filtration/washing cycle can 56 be used to purify and isolate nanoparticles 4. Thirdly, the 57 reaction also proceeded both at room temperature and in 58 presence of relatively safe chemicals. Finally, our approach is 59 highly reproducible. As a proof, we repeated the synthesis 60 three times, obtaining nanoparticles 4 with the same zeta 61 potentials in all cases (see Table 1). Yields of nanoparticles 4 62 were 91%, 88% and 90% for repetitions 1-3 respectively. We 63 therefore believe that our approach to nanoparticles 4 can be 64 feasible for a scale-up production. The tetrazine grafted on 65 IONP render a nano-platform for conjugating a diverse range 66 of biomolecules or drugs via copper-free click chemistry. 67 This strategy has advantages of simplifying chemical 68 synthesis, avoiding cytotoxic copper catalysts and generating 69 a system to detect biomarkers in live cells with improved 70 sensitivity. One such example is the nanoparticle-antibody 71 conjugate developed to image extracellular receptors in 72 cancer cells (Haun et al. 2010). 73  74 Table 1. Zeta potentials found for nanoparticles precursor 3, 75 tetrazine bound IONPs 4 and yields obtained. 76  77 Sample (repeat) IONPs 3 (mV) IONPs 4 (mV) 1 -37.6 -20.5 2 -41.1 -16.1 3 -42.9 -18.1  78 With nanoparticles 4 successfully prepared, we then decided 79 to assess their shape and average size. These parameters are 80 relevant in assessing IONPs sensitivity for biosensing 81 applications. As a proof, in 2013 Kolhatkar et al. 82 demonstrated that cubic and spherical nanoparticles display 83 different sensitivities due to their different contact areas. With 84 this in mind, we obtained TEM images of IONPs 4 (Figures 85 1a-d). It can be seen that the particles are spherical in shape 86 with a size range between 16 and 21 nm. 87  88  89 Figures 1a-d. TEM Images of nanoparticles 2 90  91 Once that both shape and size of the nanoparticles were 92 assessed, we moved to establish their magnetic properties. 93 (1a) (1b) (1c) (1d)   4 We were particularly interested in the saturation 1 magnetization value because this is another key indicator in 2 assessing IONPs sensitivity for biosensing applications 3 (Colombo et al. 2012). 4 Magnetization curves were performed on a solid sample. 5 IONPs 4 showed a paramagnetic behaviour with a saturation 6 magnetization value of 1.49E-03 emu/g (Figure 2), 7 confirming also that IONPs 4 present a degree of surface 8 functionalisation. 9  10 Fig 2. Magnetization curve of IONPs 4 at 310K 11  12 To further demonstrate the potential applicability of 13 nanoparticles 4 in pre-clinical and clinical fields, we felt 14 mandatory to perform toxicological screenings. After being 15 cultured in the presence of nanoparticles 4 at various 16 concentrations for one to three days, both metabolic activity 17 (Figure 3) and total DNA of HUVECs (Figure 4) were 18 performed, with no cytotoxicity or changes in cell viability 19 and morphology encountered. 20  21 Fig. 3 Metabolic profiles of HUVEC cells treated at different 22 concentrations of IONPs 4. 23  24  25 Fig. 4 Total DNA of HUVEC cells treated at different 26 concentrations of IONPs 4. 27  28  29  30 These results strongly suggest that targeted contrast reagents 31 based on IONPs 4 can be safely used in pre-clinical and 32 clinical imaging or magnetic-nanomedicine.  33 The relaxation rate is another key parameter in the 34 applicability of nanoparticles 4 in preclinical and clinical 35 fields. We plan to obtain this parameter as a part of our future 36 applications in vivo, and the results will be reported in due 37 course. 38  39 Conclusions 40  41 We have developed an operationally simple, high-yielding 42 and cost-effective protocol to produce a novel class of low 43 toxicity, tetrazine-based IONPs. Our protocol is reproducible 44 and the tetrazine motif can be exploited for the preparation of 45 conjugated biomolecules or drug molecules for targeted-46 based contrast agents or nanomedicine by using all the 47 strengths of the well-known copper-free click chemistry. 48 Targeted magnetic nanoparticle-based contrast agents and 49 nanomedicines are rapidly emerging into the preclinical and 50 clinical fields. We therefore anticipate that our method can 51 represent an invaluable breakthrough and provides a multi-52 functional nanoparticle platform for the large-scale 53 production and commercialization of improved, tissue 54 specific contrast agents or magnetic nanomedicine for the 55 clinical diagnosis and therapy of diseases at an asymptomatic 56 stage. 57  58 Conflicts of interest 59  60 None to declare 61  62 Acknowledgements 63  64 This work is supported by the British Heart Foundation 65 [FS/15/17/31411 to M. M. M.]. 66  67 References 68  69 Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, 70 Miller IA, Lo A, Codelli JA, Bertozzi CR (2007). 71 Copper-free click chemistry for dynamic in vivo 72 imaging. Proc Natl Acad Sci USA 104: 16793-73 16797. doi: 10.1073/pnas.0707090104 74 Colombo M, Carregal-Romero S, Casula MF, Gutierrez L, 75 Morales MP, Bohm IB, Heverhagen JT, Prosperi D, 76 Parak WJ (2012) Biological applications of 77 magnetic nanoparticles Chem Soc Rev 41:4306-78 4334 https://doi:10.1039/c2cs15337h 79 Debets MF, van Berkel SS, Schoffelen S, Rutjes FP, van Hest 80 JC, van Delft FL (2010) Aza-dibenzocyclooctynes 81 for fast and efficient enzyme PEGylation via copper-82 free (3+2) cycloaddition ChemComm 46:97-99 83 https://doi:10.1039/b917797c 84 Devaraj NK, Weissleder R (2011) Biomedical Applications 85 of Tetrazine Cycloadditions Acc Chem Res 44:816-86 827 https://doi:10.1021/ar200037t 87 Dunetz JR, Magano J, Weisenburger GA (2016) Large-Scale 88 Applications of Amide Coupling Reagents for the 89 Synthesis of Pharmaceuticals Org Process Res Dev 90 20:140-177 https://doi:10.1021/op500305s 91 Evans HL, Nguyen QD, Carroll LS, Kaliszczak M, Twyman 92 FJ, Spivey AC, Aboagye EO (2014) A 93 bioorthogonal (68) Ga-labelling strategy for rapid in 94 vivo imaging ChemComm 50:9557-9560 95 https://doi:10.1039/c4cc03903c 96 Haun JB, Devaraj NK, Hilderbrand SA, Lee H, Weissleder R 97 (2010) Bioorthogonal chemistry amplifies 98 nanoparticle binding and enhances the sensitivity of 99   5 cell detection Nature Nanotechnonogy 5:660-665 1 https://doi:10.1038/nnano.2010.148 2 Herranz F, Morales MP, Roca AG, Vilar R, Ruiz-Cabello J 3 (2008) A new method for the aqueous 4 functionalization of superparamagnetic Fe2O3 5 nanoparticles Contrast Media Mol Imaging 3:215-6 222 https://doi:10.1002/cmmi.254 7 Jaradat DMM (2018) Thirteen decades of peptide synthesis: 8 key developments in solid phase peptide synthesis 9 and amide bond formation utilized in peptide 10 ligation Amino Acids 50:39-68 11 https://doi:10.1007/s00726-017-2516-0 12 Kolhatkar AG, Nekrashevich I, Litvinov D, Willson RC, Lee 13 TR (2013). Cubic Silica-Coated and Amine-14 Functionalized FeCo Nanoparticles with High 15 Saturation Magnetization. Chem Mater, 25:1092-16 1097 https://doi: 10.1021/cm304111z 17 Kuzmin A, Poloukhtine A, Wolfert MA, Popik VV (2010) 18 Surface Functionalization Using Catalyst-Free 19 Azide−Alkyne Cycloaddition Bioconjugate Chem. 20 21:2076-2085 https://doi:10.1021/bc100306u 21 McKay CS, Finn MG (2014) Click chemistry in complex 22 mixtures: bioorthogonal bioconjugation Chem Biol 23 21:1075-1101 24 https://doi:10.1016/j.chembiol.2014.09.002 25 Meloni MM, Barton S, Xu L, Kaski JC, Song W, He T (2017) 26 Contrast agents for cardiovascular magnetic 27 resonance imaging: an overview J Mater Chem B 28 5:5714-5725 https://doi:10.1039/c7tb01241a 29 Mishra SK, Kumar BS, Khushu S, Tripathi RP, Gangenahalli 30 G (2016) Increased transverse relaxivity in 31 ultrasmall superparamagnetic iron oxide 32 nanoparticles used as MRI contrast agent for 33 biomedical imaging Contrast Media Mol Imaging  34 5:350-361. https://doi: 10.1002/cmmi.1698 35 Morawski AM, Lanza GA, Wickline SA (2005) Targeted 36 contrast agents for magnetic resonance imaging and 37 ultrasound Curr Opin Biotechnol 16:89-92 38 https://doi:10.1016/j.copbio.2004.11.001 39 Ohgushi M, Nagayama K, Wada A (1978) Dextran-40 magnetite: A new relaxation reagent and its 41 application to T2 measurements in gel systems J 42 Magn Reson (1969) 29:599-601 43 https://doi.org/10.1016/0022-2364(78)90018-5 44 Oliveira BL, Guo Z, Bernardes GJL (2017) Inverse electron 45 demand Diels-Alder reactions in chemical biology 46 Chem Soc Rev 46:4895-4950 47 https://doi:10.1039/c7cs00184c 48 Schmitz SA, Taupitz M, Wagner S, Wolf K-J, Beyersdorff D, 49 Hamm B (2001) Magnetic resonance imaging of 50 atherosclerotic plaques using superparamagnetic 51 iron oxide particles J Magn Reson Im 14:355-361 52 https://doi:10.1002/jmri.1194 53 Trivedi RA, U-King-Im JM, Graves MJ, Kirkpatrick PJ, 54 Gillard JH (2004) Noninvasive imaging of carotid 55 plaque inflammation Neurology 63:187-188 56 https://doi:10.1212/01.wnl.0000132962.12841.1d 57  58  59  60  61  62  

We have developed an operationally simple, time, and cost-effective protocol to produce a novel tetrazine-based iron oxide nanoparticle using commercially available and inexpensive materials. Our protocol proceeds at room temperature and uses hexafluorophosphate azabenzotriazole tetramethyl uronium, a well-known, widely used reagent for the large-scale industrial production of important pharmaceuticals. The nanoparticles obtained have a diameter range between 16 and 21 nm and showed no toxicity against endothelial cell lines. The tetrazine moiety on the nanoparticle surface could potentially allow further attachment of specific targeting vectors by using so-called copper-free click chemistry. We therefore anticipate that our protocol can represent a significant breakthrough in the future development and commercialization of improved, tissue-specific contrast agents and drug delivery for clinical diagnosis, monitoring and therapy of diseases at an asymptomatic stage

UCL Discovery

English

Facile, Productive, and Cost-effective Synthesis of a Novel Tetrazine-based Iron Oxide Nanoparticle for Targeted Image Contrast Agents and Nanomedicines

Meloni, Marco M.

Barton, Stephen

Chivu, Alexandru

Kaski, Juan C.

Song, Wenhui

He, Taigang

Kingston University Research Repository

  1 This is a post-peer-review, pre-copyedit version of an article published in Journal of Nanoparticle Research. The final authenticated version is available online at: https://doi.org/10.1007/s11051-018-4370-8.    2 Facile, productive and cost-effective synthesis of a novel tetrazine-based iron oxide nanoparticle for targeted image contrast agents and nanomedicines   Marco M. Meloni • Stephen Barton • Alexandru Chivu • 1 Juan C. Kaski • Wenhui Song • Taigang He 2  3 Abstract We have developed an operationally simple, time 4 and cost-effective protocol to produce a novel tetrazine-based 5 iron oxide nanoparticle using commercially available and 6 inexpensive materials. Our protocol proceeds at room 7 temperature and uses Hexafluorophosphate Azabenzotriazole 8 Tetramethyl Uronium, a well-known, widely used reagent for 9 the large-scale industrial production of important 10 pharmaceuticals. The nanoparticles obtained have a diameter 11 range between 16 and 21 nm and showed no toxicity against 12 endothelial cell lines. The tetrazine moiety on the 13 nanoparticle surface could potentially allow further 14 attachment of specific targeting vectors by using so-called 15 copper-free click chemistry. We therefore anticipate that our 16 protocol can represent a significant breakthrough in the future 17 development and commercialization of improved, tissue-18 specific contrast agents and drug delivery for clinical 19 diagnosis, monitoring and therapy of diseases at an 20 asymptomatic stage.  21  22 Keywords Diagnosis • Magnetic Resonance Imaging • Iron 23 Oxide Nanoparticles • Copper-free click Chemistry • 24 Bioconjugation • Synthesis 25  26 Introduction 27  28 Since their discovery (Ohgushi, 1978), Iron Oxide 29 Nanoparticles (IONPs) have emerged as powerful tools for a 30 wide scope of applications such as in the fields of biolabeling, 31 medical diagnostics, and therapy (Colombo et al. 2012). In 32 particular as contrast agents for magnetic resonance imaging 33 (MRI), IONPs have attracted enormous attention. IONPs 34 have a transverse relaxivity which increases at higher 35 magnetic fields, resulting in enhanced signal-to-noise ratio 36 with lower dosages (Mishra et al. 2016). The use of IONPs 37 for diagnostic imaging is therefore expected to increase 38 steadily. Unlike Gd-based contrast agents (CAs), IONPs have 39 proven safe with some systems already approved for clinical 40 use (Schmitz et al. 2001; Trivedi et al. 2004). With the recent 41 advent of molecular imaging (Meloni et al. 2017), IONPs are 42                                                      Marco M. Meloni •  Juan C. Kaski • Taigang He  The Cardiology Academy Group, St George’s University of London, Cranmer Terrace, SW17 0RE, London, UK.  Marco M. Meloni • Stephen Barton  School of Pharmacy and Chemistry, Kingston University, Penhryn Road, KT1 2EE, London, UK.  Alexandru Chivu • Wenhui Song  UCL Centre for Biomaterials, Division of surgery & Interventional Science, University College London, Pond Street, NW3 2PS, London, UK.  increasingly used for the preparation of targeted CAs, 43 allowing non-invasive visualization of biomolecules which 44 are the signatures of diseases in specific body tissues, both in 45 vivo and in real time. The preparation of these targeted CAs 46 firstly involves coating of the IONPs surface with a 47 biocompatible, clinically approved polymer, followed by its 48 functionalization with a targeting vector which allows tissue 49 specificity (A. M. Morawski et al. 2005). 50  51 This process, so-called standard bioconjugation, uses well-52 established synthetic chemistry and involves the formation of 53 stable chemical bonds. Despite being reliable, this method 54 can lead to low yields and produce toxic byproducts, resulting 55 in expensive, time-consuming purifications and toxicological 56 screenings. A number of years may therefore be required for 57 the translation, large scale production and commercialization 58 of targeted, nanoparticle-based CAs into the clinical market. 59  60 Copper-free click reactions recently emerged as a powerful, 61 time-saving bioconjugation strategy to prepare targeted CAs 62 (Devaraj and Weissleder 2011; McKay and Finn 2014). 63 Copper-free click reactions are operationally simpler and 64 proceed at room temperature. They eliminate the use of 65 cytotoxic copper catalysts (Baskin JM et al. 2007) and are 66 inert to both water and oxygen. They also generate minimal, 67 inoffensive byproducts, providing the best yield with the 68 highest reaction rates. Copper-free click reactions can 69 proceed in living organisms, allowing the targeting of 70 biomolecules with high specificity without altering any native 71 biochemical process. Among the arsenal of available copper-72 free click reactions, the Inverse Electron Demand Diels Alder 73 (IEDDA) reaction stands out as the most promising one for 74 its rapid reaction rate and generation of safe, inert nitrogen as 75 the only byproduct (Oliveira et al. 2017). IEDDA reactions 76 also involve tetrazines and cycloalkenes, both of which are 77 easier substrates to prepare if compared to 78 azadibenzocyclooctynes (DIBACs) (Debets et al. 2010) and 79 biarylazacyclooctynones (BARACs) (Kuzmin et al. 2010). 80 Despite such significant advantages, the preparation of 81 targeted, nanoparticle-based CAs using IEDDA is a relatively 82 unexplored field. A possible reason is a shortage of reliable 83 protocols to prepare tetrazine-based IONPs. Therefore, the 84 Taigang He  Royal Brompton Hospital, Imperial College London, London, UK.  Marco M. Meloni () • Taigang He () The Cardiology Academy Group, St George’s University of London, Cranmer Terrace, SW17 0RE, London, UK.  e-mail: mmeloni@sgul.ac.uk e-mail: the@sgul.ac.uk   3 identification of novel, green and efficient ways to prepare 1 these functional nanoparticles can accelerate the development 2 of improved contrast agents for diagnosis and therapy. In this 3 paper we designed and synthesized a novel route to a 4 tetrazine-bound nanoparticle 4 (Scheme 1). 5  6  7 Scheme 1 (a) Oleic acid, oleylamine, phenyl ether (b) BTAC, 8 KMnO4 (c). Tetrazine 5, HATU, DIPEA, CH2Cl2, at room 9 temperature. 10  11 As illustrated above, our strategy involved the loading of a 12 tetrazine moiety to nanoparticles 3 using 13 Hexafluorophosphate Azabenzotriazole Tetramethyl 14 Uronium (HATU), a readily available, inexpensive and well-15 known reagent. Our overall approach to nanoparticles 4 can 16 be exploited for the future production of improved contrast 17 reagents, with potential application also in the fields of 18 nanomedicine. 19  20 Results and discussion 21  22 In this work, we successfully designed and developed an 23 operationally simple, economical and productive approach to 24 produce a novel tetrazine-decorated IONP 4 (Scheme 1, page 25 2). Our protocol is reproducible by taking an advantage of 26 well-established chemistry and usages of building blocks 3 27 and 5, both accessible via established published protocols 28 (Herranz et al. 2008; Evans et al. 2014). Nanoparticles 4 were 29 obtained via coupling between carboxylic acid IONPs 3 and 30 tetrazine-based amine 5 (Scheme 1, page 2). Our method 31 builds upon the reaction between a carboxylic acid and an 32 amine, which produces extremely stable chemical bonds even 33 in a biological environment and is one of the most robust, 34 known reactions in synthetic chemistry (Jaradat, 2018). 35 During this process a challenge also emerged due to the 36 presence of the paramagnetic core both in 3 and 4, which 37 excluded quantitative reaction monitoring via NMR. 38 Therefore, we first decided to validate the chemistry using 39 acetic acid as a surrogate for nanoparticles 3. We wanted a 40 fast, high yielding protocol to obtain model compound 6. 41 After unsuccessful attempts to prepare this latter using N-42 hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-43 dimethylaminopropyl)carbodiimide (EDC) in water (Herranz 44 et al. 2008), we found that the system HATU/DIPEA/CH2Cl2 45 afforded 6 in 92% yield after only 3 hours. 46 We therefore adopted this method for the synthesis of 47 nanoparticles 4. Coupling between 3 and 5 was performed 48 with HATU and DIPEA in CH2Cl2, affording the desired 49 nanoparticles 4. We found this method both safe and 50 operationally simple for many reasons. Firstly, HATU is an 51 established chemical which is widely used during large scale 52 synthesis of pharmaceuticals. Several reports of HATU 53 applications have showcased its use in the synthesis of 54 promising drug candidates (Dunetz et al. 2016 and references 55 cited herein). Secondly, a simple filtration/washing cycle can 56 be used to purify and isolate nanoparticles 4. Thirdly, the 57 reaction also proceeded both at room temperature and in 58 presence of relatively safe chemicals. Finally, our approach is 59 highly reproducible. As a proof, we repeated the synthesis 60 three times, obtaining nanoparticles 4 with the same zeta 61 potentials in all cases (see Table 1). Yields of nanoparticles 4 62 were 91%, 88% and 90% for repetitions 1-3 respectively. We 63 therefore believe that our approach to nanoparticles 4 can be 64 feasible for a scale-up production. The tetrazine grafted on 65 IONP render a nano-platform for conjugating a diverse range 66 of biomolecules or drugs via copper-free click chemistry. 67 This strategy has advantages of simplifying chemical 68 synthesis, avoiding cytotoxic copper catalysts and generating 69 a system to detect biomarkers in live cells with improved 70 sensitivity. One such example is the nanoparticle-antibody 71 conjugate developed to image extracellular receptors in 72 cancer cells (Haun et al. 2010). 73  74 Table 1. Zeta potentials found for nanoparticles precursor 3, 75 tetrazine bound IONPs 4 and yields obtained. 76  77 Sample (repeat) IONPs 3 (mV) IONPs 4 (mV) 1 -37.6 -20.5 2 -41.1 -16.1 3 -42.9 -18.1  78 With nanoparticles 4 successfully prepared, we then decided 79 to assess their shape and average size. These parameters are 80 relevant in assessing IONPs sensitivity for biosensing 81 applications. As a proof, in 2013 Kolhatkar et al. 82 demonstrated that cubic and spherical nanoparticles display 83 different sensitivities due to their different contact areas. With 84 this in mind, we obtained TEM images of IONPs 4 (Figures 85 1a-d). It can be seen that the particles are spherical in shape 86 with a size range between 16 and 21 nm. 87  88  89 Figures 1a-d. TEM Images of nanoparticles 2 90  91 Once that both shape and size of the nanoparticles were 92 assessed, we moved to establish their magnetic properties. 93 (1a) (1b) (1c) (1d)   4 We were particularly interested in the saturation 1 magnetization value because this is another key indicator in 2 assessing IONPs sensitivity for biosensing applications 3 (Colombo et al. 2012). 4 Magnetization curves were performed on a solid sample. 5 IONPs 4 showed a paramagnetic behaviour with a saturation 6 magnetization value of 1.49E-03 emu/g (Figure 2), 7 confirming also that IONPs 4 present a degree of surface 8 functionalisation. 9  10 Fig 2. Magnetization curve of IONPs 4 at 310K 11  12 To further demonstrate the potential applicability of 13 nanoparticles 4 in pre-clinical and clinical fields, we felt 14 mandatory to perform toxicological screenings. After being 15 cultured in the presence of nanoparticles 4 at various 16 concentrations for one to three days, both metabolic activity 17 (Figure 3) and total DNA of HUVECs (Figure 4) were 18 performed, with no cytotoxicity or changes in cell viability 19 and morphology encountered. 20  21 Fig. 3 Metabolic profiles of HUVEC cells treated at different 22 concentrations of IONPs 4. 23  24  25 Fig. 4 Total DNA of HUVEC cells treated at different 26 concentrations of IONPs 4. 27  28  29  30 These results strongly suggest that targeted contrast reagents 31 based on IONPs 4 can be safely used in pre-clinical and 32 clinical imaging or magnetic-nanomedicine.  33 The relaxation rate is another key parameter in the 34 applicability of nanoparticles 4 in preclinical and clinical 35 fields. We plan to obtain this parameter as a part of our future 36 applications in vivo, and the results will be reported in due 37 course. 38  39 Conclusions 40  41 We have developed an operationally simple, high-yielding 42 and cost-effective protocol to produce a novel class of low 43 toxicity, tetrazine-based IONPs. Our protocol is reproducible 44 and the tetrazine motif can be exploited for the preparation of 45 conjugated biomolecules or drug molecules for targeted-46 based contrast agents or nanomedicine by using all the 47 strengths of the well-known copper-free click chemistry. 48 Targeted magnetic nanoparticle-based contrast agents and 49 nanomedicines are rapidly emerging into the preclinical and 50 clinical fields. We therefore anticipate that our method can 51 represent an invaluable breakthrough and provides a multi-52 functional nanoparticle platform for the large-scale 53 production and commercialization of improved, tissue 54 specific contrast agents or magnetic nanomedicine for the 55 clinical diagnosis and therapy of diseases at an asymptomatic 56 stage. 57  58 Conflicts of interest 59  60 None to declare 61  62 Acknowledgements 63  64 This work is supported by the British Heart Foundation 65 [FS/15/17/31411 to M. M. M.]. 66  67 References 68  69 Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, 70 Miller IA, Lo A, Codelli JA, Bertozzi CR (2007). 71 Copper-free click chemistry for dynamic in vivo 72 imaging. Proc Natl Acad Sci USA 104: 16793-73 16797. doi: 10.1073/pnas.0707090104 74 Colombo M, Carregal-Romero S, Casula MF, Gutierrez L, 75 Morales MP, Bohm IB, Heverhagen JT, Prosperi D, 76 Parak WJ (2012) Biological applications of 77 magnetic nanoparticles Chem Soc Rev 41:4306-78 4334 https://doi:10.1039/c2cs15337h 79 Debets MF, van Berkel SS, Schoffelen S, Rutjes FP, van Hest 80 JC, van Delft FL (2010) Aza-dibenzocyclooctynes 81 for fast and efficient enzyme PEGylation via copper-82 free (3+2) cycloaddition ChemComm 46:97-99 83 https://doi:10.1039/b917797c 84 Devaraj NK, Weissleder R (2011) Biomedical Applications 85 of Tetrazine Cycloadditions Acc Chem Res 44:816-86 827 https://doi:10.1021/ar200037t 87 Dunetz JR, Magano J, Weisenburger GA (2016) Large-Scale 88 Applications of Amide Coupling Reagents for the 89 Synthesis of Pharmaceuticals Org Process Res Dev 90 20:140-177 https://doi:10.1021/op500305s 91 Evans HL, Nguyen QD, Carroll LS, Kaliszczak M, Twyman 92 FJ, Spivey AC, Aboagye EO (2014) A 93 bioorthogonal (68) Ga-labelling strategy for rapid in 94 vivo imaging ChemComm 50:9557-9560 95 https://doi:10.1039/c4cc03903c 96 Haun JB, Devaraj NK, Hilderbrand SA, Lee H, Weissleder R 97 (2010) Bioorthogonal chemistry amplifies 98 nanoparticle binding and enhances the sensitivity of 99   5 cell detection Nature Nanotechnonogy 5:660-665 1 https://doi:10.1038/nnano.2010.148 2 Herranz F, Morales MP, Roca AG, Vilar R, Ruiz-Cabello J 3 (2008) A new method for the aqueous 4 functionalization of superparamagnetic Fe2O3 5 nanoparticles Contrast Media Mol Imaging 3:215-6 222 https://doi:10.1002/cmmi.254 7 Jaradat DMM (2018) Thirteen decades of peptide synthesis: 8 key developments in solid phase peptide synthesis 9 and amide bond formation utilized in peptide 10 ligation Amino Acids 50:39-68 11 https://doi:10.1007/s00726-017-2516-0 12 Kolhatkar AG, Nekrashevich I, Litvinov D, Willson RC, Lee 13 TR (2013). Cubic Silica-Coated and Amine-14 Functionalized FeCo Nanoparticles with High 15 Saturation Magnetization. Chem Mater, 25:1092-16 1097 https://doi: 10.1021/cm304111z 17 Kuzmin A, Poloukhtine A, Wolfert MA, Popik VV (2010) 18 Surface Functionalization Using Catalyst-Free 19 Azide−Alkyne Cycloaddition Bioconjugate Chem. 20 21:2076-2085 https://doi:10.1021/bc100306u 21 McKay CS, Finn MG (2014) Click chemistry in complex 22 mixtures: bioorthogonal bioconjugation Chem Biol 23 21:1075-1101 24 https://doi:10.1016/j.chembiol.2014.09.002 25 Meloni MM, Barton S, Xu L, Kaski JC, Song W, He T (2017) 26 Contrast agents for cardiovascular magnetic 27 resonance imaging: an overview J Mater Chem B 28 5:5714-5725 https://doi:10.1039/c7tb01241a 29 Mishra SK, Kumar BS, Khushu S, Tripathi RP, Gangenahalli 30 G (2016) Increased transverse relaxivity in 31 ultrasmall superparamagnetic iron oxide 32 nanoparticles used as MRI contrast agent for 33 biomedical imaging Contrast Media Mol Imaging  34 5:350-361. https://doi: 10.1002/cmmi.1698 35 Morawski AM, Lanza GA, Wickline SA (2005) Targeted 36 contrast agents for magnetic resonance imaging and 37 ultrasound Curr Opin Biotechnol 16:89-92 38 https://doi:10.1016/j.copbio.2004.11.001 39 Ohgushi M, Nagayama K, Wada A (1978) Dextran-40 magnetite: A new relaxation reagent and its 41 application to T2 measurements in gel systems J 42 Magn Reson (1969) 29:599-601 43 https://doi.org/10.1016/0022-2364(78)90018-5 44 Oliveira BL, Guo Z, Bernardes GJL (2017) Inverse electron 45 demand Diels-Alder reactions in chemical biology 46 Chem Soc Rev 46:4895-4950 47 https://doi:10.1039/c7cs00184c 48 Schmitz SA, Taupitz M, Wagner S, Wolf K-J, Beyersdorff D, 49 Hamm B (2001) Magnetic resonance imaging of 50 atherosclerotic plaques using superparamagnetic 51 iron oxide particles J Magn Reson Im 14:355-361 52 https://doi:10.1002/jmri.1194 53 Trivedi RA, U-King-Im JM, Graves MJ, Kirkpatrick PJ, 54 Gillard JH (2004) Noninvasive imaging of carotid 55 plaque inflammation Neurology 63:187-188 56 https://doi:10.1212/01.wnl.0000132962.12841.1d 57  58  59  60  61  62  

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Facile, productive, and cost-effective synthesis of a novel tetrazine-based iron oxide nanoparticle for targeted image contrast agents and nanomedicines

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