Biological timing as a design principle: enzyme-triggered and clock-regulated uptake mechanisms for chronotherapeutic nanomedicines

Almuthri, Ghaida Khalid K. 2026. Biological timing as a design principle: enzyme-triggered and clock-regulated uptake mechanisms for chronotherapeutic nanomedicines. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Introduction: Rheumatoid arthritis (RA) displays pronounced circadian variation, with peak pain, stiffness, and inflammatory cytokine activity occurring in the early morning. Although these rhythms are well-recognised, current drug delivery systems (DDS) rarely align therapeutic release with endogenous biological rhythms. Circadian rhythms are endogenous near-24-hour oscillations that regulate physiology, metabolism, immune activity and enzyme secretion. These predictable rhythms offer an opportunity to engineer DDS that synchronise therapeutic availability with predictable cycles of disease activity. This thesis investigates whether circadian oscillations in enzyme activity can function as internal triggers for timecontrolled drug release from polymer-drug conjugates, using RA as a model disease. A secondary aim was to determine whether circadian rhythms modulate cellular internalisation of polymeric nanocarriers, revealing an additional temporal layer for optimising nanomedicine design. The central hypothesis was that dextrin-dexamethasone conjugates could exploit daily fluctuations in α-amylase activity to achieve rhythm-aligned glucocorticoid release, and that cellular uptake of polymeric carriers is itself subject to circadian regulation- together forming a dual-level chronotherapeutic platform. Results: Dextrin-dexamethasone conjugates were synthesised using two coupling chemistries (EDC and DCC, both carbodiimide-based reagents that activate carboxylic acid groups to form covalent ester bonds with the drug) at two degrees of succinoylation (30 and 60 mol%). EDC-derived conjugates demonstrated higher drug loading (0.8-2.6 μg/mg) than DCC-derived conjugates (0.4-1.3 μg/mg). The 30 mol% EDC conjugate (DD30E) showed the strongest enzyme-triggered release, exhibiting consistent and statistically significant differences between simulated circadian peak and trough enzyme levels (p<0.0001-0.05). The 60 mol% EDC conjugate (DD60E) displayed moderate responsiveness, whereas the DCClinked conjugates (DD30D and DD60D) showed minimal or no enzyme-dependent release. These findings identify 30 mol% succinoylation with EDC coupling as the most effective structural combination for α-amylase-responsive chronotherapy. To establish the biological plausibility of enzyme-triggered chronotherapy, α-amylase rhythmicity was characterised in synchronised AR42J pancreatic acinar cells. Serum shock synchronisation induced rhythmic gene expression of CLOCK and Cry1, whilst dexamethasone synchronisation induced rhythmic gene expression in CLOCK and Cry2. The Phadebas assay confirmed that both synchronisation methods generated statistically significant circadian oscillations in α-amylase activity, whereas ELISA showed no rhythmicity in protein levels. Unsynchronised cells displayed no rhythmic patterns, confirming that rhythmic α-amylase secretion is clock-driven. The influence of circadian rhythms on polymer internalisation was evaluated in synchronised NIH-3T3 fibroblasts. Serum shock induced rhythmicity in core clock genes (CLOCK, Cry1, and Per1), and flow cytometry revealed significant time-of-day-dependent uptake for macromolecules ³10 kDa, dextrin-51 kDa, linear PEG-10 kDa and 4-arm PEG-10 kDa. Smaller polymers (£2 kDa) and free fluorophore showed no rhythmicity. Polymer architecture also shaped circadian gating: at equivalent molecular weight, 4-arm PEG exhibited ~3.8-fold higher rhythmic amplitude than linear PEG. No rhythmic uptake was observed in unsynchronised cells, confirming dependence on a functional circadian clock. Conclusion: This work provides the first demonstration that both enzyme-triggered drug release and cellular uptake of polymer nanocarriers can be governed by endogenous circadian biology. The identification of DD30E as the optimal α-amylase-responsive conjugate, together with the demonstration of robust α-amylase rhythmicity and clock-dependent, size- and architecture-specific rhythmic cellular uptake, establishes a dual-level chronotherapeutic framework. These findings highlight the potential of polymer-based systems to self-synchronise with the body’s internal timekeeping, offering a rational foundation for developing circadian-aligned drug delivery strategies for RA and other diseases characterised by rhythmic symptomatology.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Dentistry
Subjects:	R Medicine > RS Pharmacy and materia medica
Date of First Compliant Deposit:	11 August 2026
Last Modified:	12 Aug 2026 08:33
URI:	https://orca.cardiff.ac.uk/id/eprint/188865

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