CONCLUSIONS AND FUTURE WORK

This project was successfully able to determine the wound healing potential of the MNPs and ZnO NPs on the inflammatory and proliferative phases of repair, using in vitro and in vivo models. We have found that the MNPs tested exhibit limited wound healing potential, whilst the ZnO NPs enhanced an in vitro model of re-epithelialisation and accelerated wound closure in mice by promoting epidermal and collagen remodelling. The NP’s wound repair enhancing properties were shown experimentally to be dependent upon the characteristics of the particle. We found that a particle’s dose, size and agglomeration state are essential parameters responsible for its wound healing activity, whereby a low dose of a ZnO NP, with a small primary particle size, was more effective than its bulk and monodispersed variant. Although the mechansim of action is still poorly understood, evaluation of the keratinocytes exposed to the ZnO NPs in culture has shown that the NPs do not appear to enhance cell proliferation, the rate of cell adhesion, or the expression of integrin β1. This suggests that other cellular proteins involved in motor function, such as the cytoskeletal proteins, require investigation. In addition, the identification of a mechanism of action provides the potential to manipulate the scratch repair or the in vivo model systems, and by specifically targeting an identified protein or process, we may also discover how to make ZnO NP’s effects greater.

This project has reinforced the beneficial use of the ZnO NPs for the management of acute wounds. In support of its utility, ZnO is inexpensive, easily accessible and safe for topical application. While this thesis has predominantly focused on evaluating the NPs on the basis of their physiochemical characteristics, several avenues are available to further assess their mechanism of action and biological properties. Furthermore, there are some methods which may enhance its efficacy

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As shown in Chapter 4, the ZnO 30nm NPs had the greatest impact on keratinocyte re-epithelialisation. It was also the only particle to accelerate wound closure in vivo. As mentioned above, ZnO’s mechanism of action requires further investigation. Although the ZnO NPs did not mediate expression of the integrin β1 protein, which is involved in keratinocyte adhesion, there are several other families of adhesion proteins, such as selectins, CAMs and cadherins which are expressed in keratinocytes (358). Furthermore, cytoskeletal proteins also have a vital role in cell movement. The cytoskeleton is principally composed of actin microfilaments, intermediate filaments and microtubules. Whilst intermediate filaments primarily have an important structural role and do not participate in cell motility, actin microfilaments and microtubules possess motor function. Cell movement is fundamentally driven by the continuous rearrangement of the actin cytoskeleton. Cell movement is achieved via actin polymerisation and de-polymerisation and via its ability to contract upon interaction with myosin. Microtubules are also highly dynamic polymers which regulate cell contractility and migration (386). Both actin and tubulin contain several zinc binding sites (387), which may modulate cell migration when bound. Therefore, it is reasonable to assess the effects of low dose ZnO 30nm NPs on the expression and function of these cytoskeletal proteins.

In Chapter 4, we also observed the keratinocyte re-epithelialisation enhancing ability of the TiO2 25nm NPs. Since TiO2 is typically considered chemically inert, poorly soluble and of low toxicity, it was suggested that the ZnO 30nm and TiO2 25nm NPs promoted re-epithelialisation on the basis of their similar primary particle size. To investigate this further, a thorough study assessing the effects of other metal oxides on HaCaT and HEK cell re-epithelialisation is warranted. This study may also assist in the identification of the mechanism of action of these NPs.

To further assess the biological properties and the wound healing benefit of the ZnO NPs, evaluating their effect on the growth of common bacterium found in acute and chronic wounds (as listed in Table 1.6) may strengthen their routine use for wound management. Although the literature has demonstrated ZnO’s inhibitory effect on the growth of some of these species, such as S.

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effects in a dose- and particle-size dependent manner is lacking, and may reveal its potential in the management of chronic wounds, for which microbial colonisation is problematic.

Finally, as we demonstrated that the ZnO 30nm NPs were able to enhance wound closure by the single application of a 20 µL aliquot of a 0.5 µg/mL dose, future studies could employ a dosing regimen that evaluates its wound healing benefit when dosed daily or on alternating days. An evaluation by this method has the potential to see a further enhancement in ZnO’s wound healing ability, as an increased dosing frequency can reduce both a particle wash-out effect and dilution by wound exudate. Moreover, incorporation of the NPs into a hydrogel-based dressing enables greater control of the wound microenvironment and the potential for a sophisticated NP delivery mechanism. The dressing can be designed to regulate moisture content of the wound, inhibit microbial colonisation, and maintain parameters such as pH and temperature, whereby an acidic wound environment (pH 4-6), kept within a temperature range of 33°C to 35°C, has been shown to favour wound resolution (392). Furthermore, with several types of hydrogels available, such as those based on collagen, hyaluronic acid, chitosan or alginate, the physiochemical properties of the dressing, such as the hydrophilicity, porosity, swelling ratio and degradation can be controlled. These properties can be exploited to control the rate of fluid passage from the wound, as well as the rate of NP release from the hydrogel, and its ability to diffuse throughout the tissue (393).

Although this project has highlighted ZnO’s wound healing potential in a dose- and particle-size dependent manner, further research as outlined above, is required to both identify its mechanism of action and to determine whether greater healing can be achieved with modification of the NP delivery method.

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