Photo-Triggered Polymeric Nitric Oxide (NO) Donors: Synthesis and Biomedical Applications

Paul, Soumya (2024) Photo-Triggered Polymeric Nitric Oxide (NO) Donors: Synthesis and Biomedical Applications. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Soumya Paul (20RS085)) 20RS085.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Gasotransmitters belong to the subfamily of endogenous gaseous signalling molecules, which finds wide range of biomedical applications. Among the various gasotransmitters, nitric oxide (NO) has enormous effect on cardiovascular system. Apart from this, NO showed pivotal role in neurological, respiratory and immunological systems. Moreover, the paradoxical concentration-dependent activities make this gaseous signalling molecule more interesting. The gaseous NO has negligible stability in physiological conditions (37 oC, pH 7.4), which restrict their potential therapeutic applications. To overcome this issue, various NO delivering carriers were reported so far. Unfortunately, most of these NO donors have low stability, short half-life or low NO payload. Herein, we review the synthesis of NO delivering motifs, development of macromolecular NO donors, their advantages-disadvantages, and biological applications. Various NO detection analytical techniques are discussed briefly and finally a viewpoint about the design of polymeric NO donors with improved physico-chemical characteristics is predicted. To develop nitric oxide (NO)-releasing donors, we synthesized N-nitrosamine functionalized water-soluble alternating copolymer as the macromolecular scaffold with a high half-life. Reversible addition-fragmentation chain transfer (RAFT) polymerization of maleic anhydride (MA) and styrene (S) using poly(ethylene glycol) methyl ether (mPEG) containing macro-chain transfer agent (mPEG2K-CDP) produced the mPEG-b-P(MA-alt-S) copolymer, which was further modified with NO binding motifs. NO detection performed by Griess reagent and 1,2-diaminoanthraquinone demonstrated controlled release of NO from the polymer in acidic pH or with 254 nm UV light. N-nitrosamines are well established motifs to release the gasotransmitter, nitric oxide (NO), by homolytic cleavage of the N‒NO bond. For a particular application, since controlled NO release is the decisive factor under biological milieu, a series of amphiphilic N-nitrosamine-based block copolymers (BCPx-NO) are developed that release NO under photoirradiation (365 nm, 3.71 mW/cm², total 33.39 J/cm²). The BCPx-NO are water-soluble, formed micellar architecture in aqueous medium, and exhibited sustained 92‒160 μM of NO release up to 11.5 h, which is 36.8‒64.0% of the calculated value. To understand the mechanism of NO release, the small molecular NO donor (NOD) resembling the NO releasing functional motif of BCPx-NO is synthesized, which display a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar BCPx-NO enhances the antibacterial activity over NOD and exhibits superior bactericidal effect on Gram-positive Staphylococcus aureus compared to Gram-negative Pseudomonas aeruginosa. In relation to biomedical applications, this work offers a comprehensive insight on tuning light-triggered NO release to improve antibacterial activity. N-nitrosamine-derived nitric oxide (NO) delivery agents find widespread use in diverse biomedical applications including cancer therapy. To understand how NO is released from these compounds and acts within cells, herein we report a facile approach to synthesize N-nitrosamine bound water-soluble napthalimide-based block copolymers with an improved regulation over their molecular weight and aqueous solution self-assembly. The polymer exhibits fluorescence “turn-on” response upon photo-stimulated (365 nm, 3.71 mW/cm²) NO release, delivering 47‒53 μM of NO within 10 h. This accounts for approximately 67‒75% of the theoretically bound NO within the polymer. The fluorescence “turn-on” response is characteristic of the small molecule NO donor (NOD), albeit the emission time has a longer half-life in the polymers. The type of NO released from the NOD is nitric oxide radical (NO), as per the electron paramagnetic resonance (EPR) spectroscopy results. The in vitro NO release in response to the photoirradiation and the consequent acquired fluorescence is corroborated using flow cytometry and confocal imaging studies. It was also manifested that these NO conjugated polymers can physically encapsulate doxorubicin (DOX) in aqueous environment, and the synergistic effect of DOX and NO is reflected in the exhibited cytotoxicity against the MCF-7 (human breast adenocarcinoma) cells. The spatiotemporally modulated NO release steered fluorescence “turn-on” may find abundant biomedical applications. We report a facile stimuli-responsive strategy to generate reactive oxygen and nitrogen species (ROS and RNS) in the biological milieu from a photcleavable water-soluble block copolymer under photoirradiation. Herein, an anthraquinone-based water-soluble polymeric nitric oxide (NO) donor (BCPx-NO) is synthesized, which exhibits NO release in the range of 40-65 μM within 10 h of photoirradiation with half-life of 30-103 min. Moreover, the BCPx-NO produces peroxynitrite (ONOO⁻) and and singlet oxygen (¹O₂) under photoirradiation (427 nm, 2.25 mW/cm²). To explore the mechanism of NO release and photolysis of the functional group under blue light, a small molecular anthraquinone-based N-nitrosamine (NOD) is prepared. Furthermore, the cytotoxicity and in vitro ROS and RNS generation by the polymeric nanoparticles are evidenced a triple negative breast adenocarcinoma (MDA-MB-231) under visible light irradiation (white light, 5.83 mW/cm2; total 31.5 J/cm²). The spatiotemporally controlled ONOO⁻ and ¹O₂ generation from the polymeric NO donor holds significant potential for applications in cancer therapy.

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