Dual mechanism drug-eluting hydrogels with potential synergistic antitumour and antibacterial properties

Abstract

Multifunctional hydrogels can coat medical devices and provide properties inhibiting bacterial adherence and biofilm formation. Hydrogel surfaces can be treated with bactericidal nanoparticles or act as antimicrobial coatings to reduce bacterial colonisation, lowering the risk of device-related illnesses and improving patient outcomes. Antibacterial substances, such as antibiotics, can be delivered locally and continuously to infected areas by being included in multifunctional hydrogels. The antibacterial technique known as photodynamic therapy (aPDT) is also getting much attention due to the unlikely development of bacterial resistance to this therapy. PDT's antibacterial activities are principally controlled by two molecular pathways: types I and II. A series of strategies on the rational combination of PDT with other therapeutic approaches to enhance antimicrobial and antitumor efficacy has been reported. Few researchers investigated cationic porphyrin and antibiotic combinations' antibacterial, cancer-treating, and wound-healing properties. Therefore, creating a hydrogel system that can address bacterial and tumour killing, and wound healing is crucial.

This thesis aims to synthesize and characterize a hydrogel loaded with ciprofloxacin (CIP) (a broad-spectrum antibiotic) and cationic tetrakis(4-N-methylpyridyl)porphyrin (TMPyP). In Chapter 2, singlet oxygen generation from a poly(2-hydroxyethyl methacrylate -co-methyl methacrylate) p(HEMA-co-MMA) copolymer loaded with TMPyP. The effect of varying concentrations of TMPyP on singlet oxygen generation rates under irradiation with LED light was examined. Four different concentrations of TMPyP were loaded into the hydrogel, and it was found that hydrogel loaded with a higher amount of TMPyP generated relatively high rates of singlet oxygen production. A linear relationship between TMPyP concentration and singlet oxygen production was found. Furthermore, the microbiological assays and adherence tests demonstrated that the samples loaded with the highest amount of TMPyP (22.72 ± 0.11 μg/dm3) significantly reduced S. aureus and E. coli after 120 min illumination with white LED light (>3 log10 reductions), suggesting the suitability of this candidate for further studies.

Chapters 3 and 4 aim to prepare a dual drug-loaded biomaterial containing two therapeutic agents. The first agent is CIP, a second-generation fluoroquinolone with a broad spectrum of action. The second agent is TMPyP, a cationic photosensitiser that can generate reactive oxygen species to kill pathogens when activated using light. This innovation is promising in the design of antibacterial surfaces and in advancing the solution to medical device-associated infections. The loading assessment of CIP and TMPyP into a p(HEMA-co-MMA) copolymer was confirmed using Attenuated Total Reflectance - Fourier Transform Infrared spectroscopy (ATR-FTIR). Equilibrium water content percentage (EWC%) and contact angle analysis were also assessed. The drug release of CIP from the p(HEMA-co-MMA) copolymers was measured using UV-visible spectroscopy. CIP was released slowly in the first 4 hours (30%) and then increased quickly after 8 hr (60%). 85% of CIP was released from the hydrogel after 24 h. Loading TMPyP combined with CIP did not have a negative effect on the cumulative release of CIP. In vitro microbiological assessments were carried out in Chapter 4 to examine the antibacterial efficacy of the dual drug-loaded material against S. aureus and E. coli.

In Chapter 4, checkerboard assays were performed to determine the phenotypic effect of the combination of TMPyP and CIP (synergism, additive, or antagonism effect). The time-kill assay was performed to assess the antimicrobial activity of CIP/TMPyP loaded into a p(HEMA-co-MMA) hydrogel to help determine the mechanism and the kinetic release of TMPyP and CIP (either bactericidal or bacteriostatic). In comparison, adherence assays evaluate the ability of the developed materials to eradicate S. aureus and E. coli in both darkness and under LED light irradiation after 4 h and 24 h of incubation. Promising microbiological results were demonstrated; combining CIP/TMPyP into p(HEMA-co-MMA) copolymers with irradiation using LED light resulted in > 3 log10 reductions in the survival of gram-positive and gram-negative bacteria. The checkerboard assay revealed the synergism between CIP/TMPyP with a Fractional Inhibitory Concentration Index (FICI) value < 0.5.

Skin cancer is of particular concern; it still poses a severe threat to health, and metastasis is frequently the cause of death. Surgical resection is critical to the current clinical strategy for treating melanoma. However, significant tissue defects in the resection site and potential bacterial infection after surgical resection may exacerbate skin wounds, which are challenging to self-heal. For this, Chapter 5 mainly proposed CIP/TMPyP-loaded p(HEMA-co-MMA) as a novel coating material for wound dressing with promising antibacterial and anticancer properties. In this chapter, cell viability analysis was done using a human melanoma cell line (LOX-IMVI), and the cell viability of melanoma cells exposed to hydrogels loaded with dual therapy CIP and TMPyP decreased to < 70% compared to control. The wound-healing results demonstrated that TMPyP and CIP increased the wound-healing capacity of fibroblasts. The migration of fibroblasts was increased compared to control by 51.9% after 24 h exposure to CIP/TMPyP treatment.

Finally, the cytotoxicity of the materials was examined using the MTT assay. A CIP, TMPyP, and CIP/TMPyP-incorporated p(HEMA-co-MMA) copolymer and untreated p(HEMA-co-MMA) copolymer were found to have low cytotoxicity (cell viability > 70%) after 24 hours direct contact and indirect contact with fibroblast L929 cells.

In conclusion, incorporating antibacterial and anticancer properties in a single drug delivery platform demonstrates potential synergistic therapeutic effects, providing a promising approach for simultaneously addressing two critical healthcare issues. The developed hydrogels have favorable properties to minimize the adverse effects and complications associated with conventional drugs, providing targeted and localized treatment approaches. The findings in this study pave the way for further investigations in the future to optimize these preliminary hydrogels into practical clinical tools.

Thesis is embargoed until 31 December 2028.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Jadara University
Supervisor	Colin McCoy (Supervisor), Louise Carson (Supervisor) & Jessica Moore (Supervisor)