New polymeric nanoparticles to interrupt the ros and rns derived misregulation of cells

New polymeric nanoparticles to interrupt the ROS- and RNSderived misregulation of cells Van Nam DAO BPharm (Hons), MPharmSc A thesis submitted for the degree of Doctor of Philosophy at Monash University in 2021 Faculty of Pharmacy and Pharmaceutical Sciences New polymeric nanoparticles to interrupt the ROS- and RNS- derived misregulation of cells Van Nam DAO BPharm (Hons), MPharmSc Supervisors: Dr John Quinn Dr Michael Whittaker A/ Prof Erica Sloan Panel members: Dr Angus Johnston Dr Betty Exintaris Dr Daniel Poole A thesis submitted for the degree of Doctor of Philosophy at Monash University in 2021 Faculty of Pharmacy and Pharmaceutical Sciences Copyright notice © Van Nam DAO (2021) I certify that I have made all reasonable efforts to secure copyright permissions for thirdparty content included in this thesis and have not knowingly added copyright content to my work without the owner's permission Abstract Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have critical roles in various biological processes and signaling pathways Moreover, perturbations in ROS and RNS levels often occur with changes in metabolism and health disorders The application of scavengers, which are capable of reducing ROS and RNS levels, could potentially protect cells from being damaged by these reactive molecules Further, manipulating ROS and RNS levels can also interrupt downstream communication between impaired cells and other surrounding tissue, as seen in the tumour microenvironment (TME) wherein cancer cells use these molecules to communicate with supporting cells The delivery of H2S and persulfide compounds that mimic cellular defense pathways and offer inherent ROS scavenging activities is another potential approach to alleviate ROS levels Further, the recent development of nanomedicine has enabled researchers to address issues associated with small molecular drugs, such as nonspecific binding, low tissue permeation and short retention time In this thesis, polymer-based scavengers for ROS and RNS have been developed which i) exploit advanced polymer design, ii) contain novel reactive moieties, iii) scavenge ROS and/or RNS intracellularly, and iv) confer downstream effects in the biological milieu Firstly, in Chapter 1, the background of ROS and RNS production is discussed, including how these species are regulated in biological systems Recent advances in development of hydrogen sulfide and persulfide donors, as well as macromolecular ROS/RNS scavengers, are also described Subsequently, in a series of experimental chapters, data are presented about four main categories of materials that have been developed: linear and brush polymers bearing trisulfide moieties for releasing hydrogen sulfide and persulfides (Chapter and Chapter 3), polymer carriers for delivering N-acetyl L-cysteine intracellularly (Chapter 4), star polymers for conjugating a small molecule antioxidant (TEMPO) (Chapter 5) and nitric oxide scavenging polymers (Chapter 6) The synthesised compounds were characterised using a range of analytical techniques, including H 2S-specific amperometry Further, the impact of the synthesised materials on cell viability, and their biological performance, i.e ROS RNS scavenging ability, were evaluated on several models, including a naturally elevated ROS level model which can simulate TME communication (a co-culture of fibroblasts and breast cancer cells, Chapters 2, and 5), an externally stimulated oxidative stress model (HEK293 treated with H2O2, Chapter 3), and a naturally elevated RNS model (activated primary and cell line macrophages, Chapter 6) Where appropriate, other intracellular behaviour of the synthesised compounds was evaluated, including cell association, co-localisation with subcellular organelles and intracellular H2S release Certain key secondary effects associated with polymer treatment were also examined, including ROS-associated cellular changes (collagen-1 and F-actin expression, Chapter 2), NOlinked phagocytosis activity (Chapter 6), or mitochondrial functions (superoxide anion production and co-localisation, Chapter 5) The materials developed in the thesis represent novel entities for manipulating cellular signaling pathways, and are potentially applicable to diverse fields including the pharmaceutical, polymer and material sciences, as well as oncology and pharmacology Publications during enrolment Urquhart MC, Dao NV, Ercole F, Boyd BJ, Davis TP, Whittaker MR, et al Polymers with dithiobenzoate end groups constitutively release hydrogen sulfide upon exposure to cysteine and homocysteine ACS Macro Lett 2020;9(4):553-7 Dao NV, Ercole F, Kaminskas LM, Davis TP, Sloan EK, Whittaker MR, et al Trisulfide-bearing PEG brush polymers donate hydrogen sulfide and ameliorate cellular oxidative stress Biomacromolecules 2020: 21(12):5292-305 Dao NV, Ercole F, Urquhart MC, Kaminskas LM, Nowell CJ, Davis TP, et al Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer co-culture model Biomaterials Science 2021 DOI: 10.1039/D0BM01544J Thesis including published works declaration I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma at any university or equivalent institution and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference is made in the text of the thesis This thesis includes 02 original papers published in peer reviewed journals and submitted publications The core theme of the thesis is the development of antioxidant polymers and nanomedicine The ideas, development and writing up of all the papers in the thesis were the principal responsibility of myself, the student, working within the Drug Delivery, Disposition and Dynamics Theme, Monash Institute of Pharmaceutical Sciences under the supervision of Dr John Quinn, Dr Michael Whittaker and Associate Professor Erica Sloan The inclusion of co-authors reflects the fact that the work came from active collaboration between researchers and acknowledges input into team-based research In the case of Chapter and 3, my contribution to the work involved the following: (If this is a laboratory-based discipline, a paragraph outlining the assistance given during the experiments, the nature of the experiments and an attribution to the contributors could follow.) Thesis Chapter Publication Title Status Nature and % of student contribution the polymers and revise manuscripts cholesteryl 80% PEG conjugate design, intracellular and Published collagen-1 cancer co-culture C Urquhart helped J Nowell provided collecting expertise in imaging and analysis and data revised manuscript and Yes synthesise the polymers 3) Cameron analysis, and production in a breast 2) Matthew Research attenuates ROS Coauthor(s), Monash student 1) Francesca Ercole helped synthesise Trisulfide linked Co-author name(s), Nature and % of Co-author’s contribution* 4) Thomas P Davis, Lisa M Kaminskas, preparing Erica K Sloan, Michael R Whittaker manuscript and John F Quinn formulated the idea of the project, provided supervision, and model had input into manuscript preparation 1) Francesca Ercole provided expertise Trisulfidebearing PEG brush polymers hydrogen sulfide Published and ameliorate cellular oxidative stress on synthesis and amperometric sensing Research of H2S and revised manuscript design, donate 80% 2) Thomas P Davis, Lisa M Kaminskas, collecting Erica K Sloan formulated the idea of the data project and revise manuscript and analysis, and No 3) Michael R Whittaker and John F preparing Quinn formulated the idea of the project, manuscript provided supervision, and had input into manuscript preparation I have renumbered sections of published papers in order to generate a consistent presentation within the thesis Student name: Van Nam Dao Date: 22nd April 2021 I hereby certify that the above declaration correctly reflects the nature and extent of the student’s and co-authors’ contributions to this work In instances where I am not the responsible author I have consulted with the responsible author to agree on the respective contributions of the authors Main Supervisor name: Dr John F Quinn Date: 22nd April 2021 Acknowledgements Doing a PhD is truly a journey, and to get to the end of the journey with this thesis in hand, I have received enormous support from all the people around me First and foremost, I would like to acknowledge Dr John Quinn and Dr Michael Whittaker for being my main supervisors, who have deep knowledge of not only polymer chemistry but also other disciplines, ranging from organic chemistry to analytical chemistry and biology They have spent a great amount of time and effort in supervising me, and have given me valuable advice whenever I am in trouble Thanks for always encouraging me to explore new knowledge without hesitation I also would like to thank Associate Professor Erica Sloan for the supervision and useful advice she gave me while I was working with cancer models Erica also gave me the opportunity to think critically when preparing manuscripts for publication I would not have been able to finish my PhD without the great support of Dr Francesca Ercole Fran has provided me with amazing expertise in hydrogen sulfide, sulfur and polymer chemistry She also taught me chemistry techniques that were essential for my PhD and advised me in my personal life I would like to express my deepest appreciation to Fran I would like to acknowledge Dr Lisa Kaminskas for her advice during the project and for raising many important points in preparing the manuscript for the co-culture work, Mr Cameron Nowell for the impressive expertise in imaging and analysis, and Dr Jason Dang for always being nice and providing great assistance in analysing NMR data and HRMS data without any hesitation I also want to acknowledge Dr Jibriil Ibrahim for the assistance in doing lung alveolar macrophage experiments and Dr Yuhuan Li for guiding me with the very first steps in cell experiments I would like to thank my Milestone Review Panel, Dr Angus Johnston, Dr Betty Exintaris and Dr Daniel Poole for the inputs and advice during my entire PhD At CBNS and MIPS, I was also assisted by Professor Thomas Davis, the CBNS centre and D4 Theme Dr Nghia Truong, Dr Asuntha Munasinghe and Ms Karen Drakatos were really helpful during my PhD enrolment Thank you all for creating an excellent working and research environment I also want to acknowledge the Funding from the Vietnamese Government (in the form of VIED scholarship), the Faculty Sponsorship from MIPS, the support from the Department of Physical Chemistry and Physics, Hanoi University of Pharmacy and from the Ministry of Education and Training, Vietnam Without the funding and support, I would have not been able to pursue my PhD at Monash University Friends are the people who are willing to help whenever I seek assistance, and I would like to express my acknowledgement to Aadarash for being a very nice neighbour, who also helped me with many aspects of my life Thank you Matt, Alex, Ken, John F for all the social chats, lab work support and your generosity and kindness I also want to thank Scott for advising me in imaging and your kindness in lending me chemicals You all have made my PhD journey better than I expected I also got help from all the members in level 4, the Nanomedine team and the CBNS administration team, May, Aykut, James, Kristian, Meike, Ayaat, Cheng, Erny, Paulina, Jeffiri, Stefan, Nikos, Ximo, Adrian, Jason W, Loshini, Anne, Sam, Natalie, Charlotte, Moore, Daniel, Rob, Joanne, Carlos, Emily, Stephanie, Xiaotong, Inin, Song, Elly, Lars, Kwan, Nil Mrs Oanh Nguyen and Mr Cuong Nguyen are my closest friends in Australia, who aided me when I first arrived in Melbourne and always welcome me as a family member I really appreciate their kindness and assistance It had been good experience in sharing house with Gerard, Artur, Yoni and Lauren, who made my life much easier and meaningful Thank you Mai Vu, my colleague in both Vietnam and Australia and other Vietnamese friends, for your great support and encouragement Finally and most importantly, I would like to acknowledge my Mother and my Father, who have always stayed beside me and supported me with their love My great-grandmother, grandparents, sister and all members in my beloved family are also so important to me, thanks for their care and support! Nam Dao Figure SB33 1H-NMR (400 MHz, CDCl3) spectrum of M2-10 polymer with peak assignments Figure SB34 1H-NMR (400 MHz, CDCl3) spectrum of M2-20 polymer with peak assignments 293 Figure SB35 1H-NMR (400 MHz, CDCl3) spectrum of M0 with peak assignments 294 Table SB1 Specific polymerisation characteristics and conditions % Mx M1 M2 Feed Ratios Mx OEGMA950 RAFT Agent AIBN (transferred as an aliquot from stock solution) ×10-4 mg mmol DMSO [Mx + OEGMA950] ml (mol/ g solvent) [Mx] : [OEGMA950] : [RAFT] : AIBN mg ×10-2 mmol mg ×10-1 mmol mg ×10-3 mmol 10 : 54 : : 0.1 20.0 4.09 350 3.68 2.75 6.82 0.112 6.82 0.308 1.2 20 12 : 48 : : 0.1 45.0 9.21 350 3.68 3.10 7.67 0.126 7.67 0.342 1.2 30 18 : 42 : : 0.1 55.1 11.28 250 2.66 2.53 6.27 0.103 6.27 0.288 1.2 10 : 54 : : 0.1 21.1 4.09 350 3.68 2.75 6.82 0.112 6.82 0.313 1.2 20 12 : 48 : : 0.1 42.1 7.89 300 3.16 2.66 6.58 0.108 6.58 0.302 1.2 30 18 : 42 : : 0.1 64.1 12.18 270 2.84 2.73 6.77 0.111 6.76 0.310 1.2 0 : 60 : : 0.1 0 300 3.16 2.13 5.26 0.086 5.24 0.245 1.2 Figure SB36 FT-IR NIR spectra of polymerising reaction mixture of P[OEGMA950] with 10% of trisulfide based monomer 1, M1 (MPEGESMA) at t=0h and t=36h The peak corresponding to the double bond (CH2=C(CH3)-) has been marked with an oval 295 Figure SB37 Total monomer conversion as a function of time for the polymerisation of P[OEGMA 950] without any trisulfide monomer for 3h (A); with trisulfide monomer M1 for 14h (B); with trisulfide monomer M2 for 16h (C) 296 Figure SB38 Electrochemical detection of H2S evolution from co-polymers of P[OEGMA950] with 30% trisulfide monomer M1 (M1-30) after exposing to GSH at different levels, i.e 5.0 µM or 5.0 mM The experiments were conducted in deoxygenated DBPS containing 0.2 mg/ml of polymer Data shows that low amounts of H2S was detected when GSH was used at 5.0 μM, while subsequently 5.0 mM led to more substantial H2S liberation The insert illustrates the intensity over three hours after 5.0 µM of GSH was used Notably, all the voltage values were presented in relative to the intensity right before adding 5.0 mM of GSH Figure SB39 Size distribution by volume (A) and by number (B) of polymer with M1-30 and M2-30 in DPBS at 0.2 mg/ml Data were obtained using DLS technique 297 B Trigger/ pHa Polymer tmax ~ (mins)b [H2S]max (µM)c [Sulfides]tot (µM)d [trisulfide] (µM)e Sulfides released (%)f GSH 6.1 M1-30 40 36.6 41.0 73.6 56 M2-30 25 24.7 27.7 70.0 40 CYS 7.0 M1-30 12 28.1 55.0 73.6 75 M2-30 15 30.4 59.5 70.0 85 Figure SB40 (A) Total sulfides released from M1-30 or M2-30 using glutathione (GSH) or cysteine (CYS) to trigger H2S release The experiments were conducted in deoxygenated Dulbecco’s phosphate buffer saline (DBPS) at 0.2 mg/ml of polymer; glutathione or cysteine was added to a final concentration of mM at t=0 N=3 Error bar: S.E.M (B) H2S releasing characteristics: a pH was measured when a fresh solution was prepared at a concentration of mM in DPBS and was found to be ~ 6.1 for GSH and 7.0 for CYS; b determined when H2S start to fall into equilibrium state; [Sulfides]tot = [𝐻2 𝑆]𝑚𝑎𝑥 × (10pH−p𝐾1 + 1), where pK1 = 7.02 at RT; e c determined at tmax; d [trisulfide] (µM) = 0.2/ Mn x 106 x m, where 0.2 is the concentration of polymer (mg/mL), M n is the molecular weight of polymer by NMR (g/mol) and m is the average number of trisulfide monomer (DP) in polymer chain as achieved from table 1; f Sulfides released at tmax (%) = [Sulfides]tot/ [trisulfide].2 298 Figure SB41 Cell viability (%) of HEK293 cells treated with co-polymers of P[OEGMA950] incorporating M1 (A) or M2 (B) at different co-polymer compositions, compared to the non-treated control group Homo-polymer M0 was used as a control polymer Data were presented for three independent experiments and were plotted as mean ± S.E.M Cell viability was assessed after 24 hour treatment using AlamarBlue assay 299 Figure SB42 ROS levels of cells pre-treated with polymers containing no trisulfide moiety (M0) or polymers with 30% of H2S releasing group (M1-30 or M2-30), compared to the value for the control group without any treatment All the polymers were used at 0.1 mg/ml OxiSelect (DCFHDA) was employed for ROS staining Data were obtained one hour after treatment using an Operetta High content imaging system with a 20X magnification objective Results presented are for three independent experiments (n=3) Matched one-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis; ns: not significant; error bar: S.E.M 300 REFERENCES Ercole F, Li Y, Whittaker MR, Davis TP, Quinn JF H 2S-Donating trisulfide linkers confer unexpected biological behaviour to poly(ethylene glycol)–cholesteryl conjugates J Mater Chem B 2020;8:3896-907 Ercole F, Whittaker MR, Halls ML, Boyd BJ, Davis TP, Quinn JF Garlic-inspired trisulfide linkers for thiol-stimulated H2S release Chem Commun 2017;53(57):8030-3 301 Appendix C SUPPORTING INFORMATION FOR CHAPTER TEMPO-conjugated star polymers: old compound, new applications 302 Figure SC1 1H-NMR (A) and 19F-NMR (B) spectra of the reaction mixture of star P2 and amino-TEMPO at different time points: 15 minutes (a), hours (b), 12 hours (c) and 24h (d) Solvent: CDCl Table SC1 Size distribution by volume and by number of S1-S3 in water 1.0 mg/ml Data were obtained using DLS technique Star Z-average (d.nm) PDI Volume mean (d.nm) Number mean (d.nm) S1 35 0.43 16 11 S2 57 0.30 18 10 S3 56 0.24 27 17 303 Figure SC2 CellROX Deep Red channels for ROS detection in co-cultured breast cancer cells and fibroblasts when challenged with TBHP: cells were treated with S1, S2, S3 (0.125 mg/mL, D, E, C) or TEMPO (0.38 mM, F) for one hour followed by exposure to TBHP (200 µM for 20 hours) before ROS staining Control cells without treatment (A) or treated with S3 only (B) are also presented Scale bar: 100 µm Images were obtained from an Operetta High content imaging system with a 20X magnification objective Figure SC3 Mean fluorescence intensity per cell (MFI) when TEMPO-Cy5-polymer S4 was introduced to mono-cultured or co-cultured cells of BJ-5ta and MCF-7 for hours, hours and 24 hours Each graph represents data from one experiment from three technical replicates 304 Figure SC4 Fluorescence intensity from cell media containing AlamarBlue with added S3 (0.125 mg/mL) or free TEMPO (0.38 mM) Media (Alamar Blue diluted at 10% in DMEM - 10% FBS - 1% pen/strep) were collected after introducing for two hours to co-cultured wells without any polymer treatment and were analysed using a Clariostar plate reader For each run, media were collected from different wells Figure SC5 Fluorescence spectrum of S4 when excited by a wavelength 650 nm, with and without the presence of ascorbic acid [S4] = 0.25 mg/mL; [ascorbic acid] = 0.5 mM (normal tissue concentration of ascorbic acid is 0.03-10 mM).1, 305 Figure SC6 Merged channels of Hoechst 33342 (blue) and caveolin-1 (green) (a, b, c) and the corresponding MitoSOX channels (d, e, f) of BJ-5ta - MCF-7 co-cultured cells when treated with TEMPO-star S3 (b, e) or its ascorbic acid-reduced analogue S5 (c, f) or left untreated for 24 hours (a, d) Polymers were employed at 0.125 mg/mL MitoSOX Red was employed for staining mitochondrial superoxide anion before the images (d, e, f) were captured Subsequently, cells were fixed and stained for visualisation of caveolin-1 (a fibroblast marker3) and nuclei and the images were captured at the same position (a, b, c) Images were obtained from an Operetta High content imaging system with a 20X magnification objective Scale bar 20 µm 306 REFERENCES Kojo S Vitamin C: basic metabolism and its function as an index of oxidative stress Curr Med Chem 2004;11(8):1041-64 Zhang X, Kim WS, Hatcher N, Potgieter K, Moroz LL, Gillette R, et al Interfering with nitric oxide measurements 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid J Biol Chem 2002;277(50):48472-8 Martinez-Outschoorn UE, Pavlides S, Whitaker-Menezes D, Daumer KM, Milliman JN, Chiavarina B, et al Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors Cell Cycle 2010;9(12):2423-33 307 ...Monash University in 2021 Faculty of Pharmacy and Pharmaceutical Sciences New polymeric nanoparticles to interrupt the ROS- and RNS- derived misregulation of cells Van Nam DAO BPharm (Hons), MPharmSc... theme of the thesis is the development of antioxidant polymers and nanomedicine The ideas, development and writing up of all the papers in the thesis were the principal responsibility of myself, the. .. through the activity of NOX complex systems; the expression of the NOXs and their assembly; the phosphorylation, expression and translocation to the membrane, and the conformational changes and binding