Photodynamic Killing of Human Cancer Cells with Smart Photosensitizer Materials and An Endoscopic Implement for Singlet Oxygen Delivery

The thesis describes progress on probe tips for a microoptic device for the precise delivery of the components necessary for photodynamic therapy (PDT) in a highly localized and controllable fashion. The thesis also summarizes results of a photosensitized oxidation study. The work focused on i) developing a photoactive fluoropolymer surface that will release sensitizer drug molecule for use in PDT, ii) designing new probe tips surfaces for use as sensitizer support for a microoptic PDT device, iii) exploring strategies for covalent attachment of sensitizers and model compounds to Teflon/PVA surfaces with the aim of being coupled with our microoptic device, and iv) initiating photosensitized dissociation of peroxides at silica surface by triplet sensitizer energy transfer as a strategy to break peroxide O–O bonds for RO∙ release to study the oxidative process. Development of a photoactive fluoropolymer surface that releases sensitizer drug molecule was achieved. The surface is a Teflon/poly(vinyl alcohol) (PVA) nanocomposite bearing a photoreleasable PEGylated photosensitizer that generates 1O2 (1Δg) [chlorin e6 methoxy tri(ethylene glycol) trimester]. We observed that the Teflon-like fluorinated surface showed resistance to drug adsorption and that there is also an increase in ground state oxygen by the material. The relative surface efficiency to photorelease the PEGylated sensitizer was slightly higher for the nanocomposite when compared with fluorinated PVG surface. We also found that the presence of C−F bonds in the polymers was beneficial for high O2 solubility, repelling action, and low physical quenching of 1O2. The fluoropolymer could be shaped into device tips to discharge controlled sensitizer and 1O2 quantities for tissue repair or pointsource photodynamic therapy in vivo. Two different types of probe tips material for a micooptic PDT device were successfully synthesized and their mechanical strength, stability and efficiency for their used as sensitizer support was determined. The surfaces were Teflon-like nanocomposite, made of polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) and a silica monolith, prepared using an acid based catalyzed sol-gel method. Two types of sensitizer-silica monolith surfaces were synthesized: sensitizer coated silica monolith and sensitizer doped silica monolith. Both xerogel surfaces, in presence of light and oxygen, successfully generated singlet oxygen in water which was detected by chemical trapping with alkene, trans-2-methyl-2-pentenoic acid, and anthracene. PDT sensitizers and model compounds were covalently attached to the plain or succinic acid functionalized Teflon/PVA surface. We were successful in (1) synthesizing 9-bromomethylanthracene-Teflon/PVA heterogeneous surfaces via an alkylation reaction (2) covalently attaching chlorin e6 onto the plain Teflon/PVA surface via bromopropanol linker (3) synthesizing a hybrid Teflon/PVA-9-anthracenemethanol surface by covalently attaching 9-anthracenemethanol via an esterification reaction to the Teflon/PVA surface, previously modified with succinic acid and (4) synthesizing Teflon/PVA probe tip with photo detachable pheophorbide molecules. The heterogeneous surface when placed in solution, in presence of light and oxygen, was observed to show coloration of butanol solution where 99 % sensitizer detached from the probe tip. Lastly, we explored photochemical reactions at silica surface with the focus on photosensitized dissociation of peroxides. Triplet sensitizer energy transfer as a strategy to break peroxide O–O bonds for RO∙ release was confirmed. We successfully observed the photosensitized dissociation of dicumyl peroxide, upon irradiation of 4,4’-dimethyl benzyl sensitizer adsorbed on fume silica particles. The highest amount of cleaved peroxide (25.3 %) was detected in solution when particles were loaded with dicumyl peroxide: 4,4’-dimethyl benzyl in 1:10 ratio

Photosensitizer Drug Delivery via an Optical Fiber

: An optical fiber has been developed with a maneuverable miniprobe tip that sparges O2 gas and photodetaches pheophorbide (sensitizer) molecules. Singlet oxygen is produced at the probe tip surface which reacts with an alkene spacer group releasing sensitizer upon fragmentation of a dioxetane intermediate. Optimal sensitizer photorelease occurred when the probe tip was loaded with 60 nmol sensitizer, where crowding of the pheophorbide molecules and self-quenching were kept to a minimum. The fiber optic tip delivered pheophorbide molecules and singlet oxygen to discrete locations. The 60 nmol sensitizer was delivered into petrolatum; however, sensitizer release was less efficient in toluene-d8 (3.6 nmol) where most had remained adsorbed on the probe tip, even after the covalent alkene spacer bond had been broken. The results open the door to a new area of fiber optic-guided sensitizer delivery for the potential photodynamic therapy of hypoxic structures requiring cytotoxic control

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose

Autocatalytic-Assisted Photorelease of a Sensitizer Drug Bound to a Silica Support

The photorelease of a sensitizer from a fluorinated silica surface occurs by a reaction of singlet oxygen with the vinyl ether bond linker with scission of a dioxetane intermediate. Irradiation of the released sensitizer generates singlet oxygen, which accelerates the release of more sensitizer via an autocatalytic reaction. Sigmoidal behavior of sensitizer release in <i>n-</i>butanol and <i>n-</i>octanol occurs at an optimal temperature of 20 °C. The photorelease efficiency was reduced at low temperatures, where the sensitizer was retained on the surface due to a long-lived dioxetane with inefficient scission, and also reduced at high temperatures, due to a slower reaction of ¹O₂ with the vinyl ether bond. Immediate acceleration is a result of released sensitizer being used as a dopant to eliminate the induction step, further implicating an autocatalytic mechanism. However, the sigmoidal sensitizer release was not correlated to solvent viscosity, heat, or light from the dioxetane decomposition or to minor O₂ solubility enhancements caused by the fluorinated silica. The mechanistic information collected here can be used to help control the pace of drug release; however, it remains to be seen whether an autocatalytic-based drug delivery system has an advantage to those with non-sigmoidal kinetics

A Hand-held Fiber-optic Implement for the Site-specific Delivery of Photosensitizer and Singlet Oxygen

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)-1,2-dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first-order photocleavage rate constant of 1.13 h−1 was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide-a derivative that carries a p-formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide-a, otherwise a pH effect was observed in a binary methanol-water system (9:1) at pH below 2 or above 8

Photoactive Fluoropolymer Surfaces That Release Sensitizer Drug Molecules

We describe a physical–organic study of two fluoropolymers bearing a photoreleasable PEGylated photosensitizer that generates ¹O₂(¹Δ_g) [chlorin e₆ methoxy tri­(ethylene glycol) triester]. The surfaces are Teflon/poly­(vinyl alcohol) (PVA) nanocomposite and fluorinated silica. The relative efficiency of these surfaces to photorelease the PEGylated sensitizer [shown previously to be phototoxic to ovarian cancer cells (Kimani, S. et al. <i>J. Org. Chem</i> <b>2012</b>, <i>77</i>, 10638)] was slightly higher for the nanocomposite. In the presence of red light and O₂, ¹O₂ is formed, which cleaves an ethene linkage to liberate the sensitizer in 68–92% yield. The fluoropolymers were designed to deal with multiple problems. Namely, their success relied not only on high O₂ solubility and drug repellency but also on the C–F bonds, which physically quench little ¹O₂, for singlet oxygen’s productive use away from the surface. The results obtained here indicate that Teflon-like surfaces have potential uses in delivering sensitizer and singlet oxygen for applications in tissue repair and photodynamic therapy (PDT)