Conclusion on the biological experiments

The research presented and discussed in Chapter III covered the in vitro and ex vivo testing of the most interesting nanogels described in Chapter II.

After applying the Zein protein test to a variety of nanogels, it was found that nanogels are, in general, quite mild and would not induce skin irritation. The values found for the fluorescent nanogels were generally twice those found with the non-fluorescent, although this was still below the level normally considered to be mild irritants.

The toxicity of the nanogels was tested with Alamar Blue to determine the cell viability of keratinocytes incubated for 24 and 48 hours with the nanogels. The results show an excellent percentage of viable cells after 48h for non-fluorescent nanogels with concentrations of up to 10 mg/mL. Introducing comonomers like AEA and fluorescent monomers decreased the viability to 1mg/mL or lower. The experiments on cell viability elucidated the influence of the cell media on the LCST. A study of the LCST in EpiLife established the drop in temperature to be around 1.5-2°C in fluorescent, neutral and negatively charged nanogels. The decrease in the LCST for fluorescent, positively charged nanogels was around 8°C, bringing the response temperature too close to room temperature for this nanogels to be suitable for the final application. The drop in the LCST was shown to be caused by the ionic strength of the cell media.

The fluorescent, neutral and negatively-charged nanogels were used to study the ability of keratinocyte cells to internalise them at skin temperature (35°C).

The former ones were internalised after 48 hours and caused aggregates; the latter were internalised after 24 hours. In both cases the nanogels were localised in the cytoplasm and nucleus.

Finally, the experiments with human excised skin showed that the fluorescent, neutral nanogels were able to diffuse through the stratum corneum and reach the viable epidermis without the help of any enhancer. Furthermore, the presence of benzyl alcohol or DMSO did not improve the penetration of the nanogels. In the other hand, fluorescent negatively-charged nanogels were unable to penetrate the skin without the help of an enhancer, the most efficient being benzyl alcohol.

4.4 Future work

Chapter III focused on testing the nanogels in biological systems as drug delivery vehicles. There are several areas for possible future work.

The first area of study would be the viability of keratinocyte cells treated with the drug FA in its free form, compared to cells treated with FA-loaded nanogels, containing the equivalent amount of the drug. This study would show the potential of the drug delivery system for sustaining the drug release and delivering it inside the cell. In addition, if the nanogels are able to release the drug more slowly, the LD50 should be higher than for the free drug alone, making it safer to use. (LD50 is a measure of a drug’s lethal dose and it refers to the concentration of drug required to kill half the cells after an specified test duration.)

The efficacy of non-steroidal, anti-inflammatory drugs (NSAIDs) such as FA is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. As COX is the rate-limiting enzyme in prostaglandin biosynthesis, it can be inferred that the beneficial effect of NSAIDs may be achieved through the inhibition of prostaglandin biosynthesis. To assess the efficacy of the loaded nanogels, compared to the free drug, cells should be incubated with FA and FA-containing nanogels, in order to check the protein expression after the exposition time. If the nanogels uploaded with FA performed better at inhibiting the effect of COX, it would show a successful internalisation of the drug in to the skin cells.

The cell viability experiments, measured the percentage of cells that were alive after incubation with nanogels but did not give any information about the type of cell death when the nanoparticles were cytotoxic. A flow cytometer could give information on the type of cellular death: apoptosis (programed cell death, a regulated process, during an cells’ lifecycle) or necrosis (non-physiological process that occurs as a result of external factors such as trauma or infection). Such a study would be a logical continuation of the work on the viability of the keratinocyte cells

The second area of interest is how long it takes to be able to detect the nanogels in the cells. The cell uptake experiments on keratinocyte cells were

carried out at the same time interval as the cell viability experiments, 24 and 48 hours. It would be of interest to perform more experiments at more time points, specifically to assess how long it takes to be able to detect the nanogels in the cells. For this experiment high concentrations of fluorescent polymer should be used, probably at 1mg/ml or higher. It would be interesting to see if the nanogels are located in specific organelles during the early part of the experiment.

Linked to these experiments, it would also be interesting to study the cell-uptake pathways, which are either by passive diffusion, endocytosis, phagocytosis, etc. Organelle location gives clues to the type of uptake pathway; for example, early location of the nanogels in the lysosomes indicates endocytosis.

The third issue is the easy availability of fresh human skin from the operating theatre. It would be an appropriate additional protocol to screen potential formulations with a Franz Cell, using porcine skin from the abattoir or synthetic skin. This would allow a first pre-selection of candidates for later testing in human skin.

Fourthly, because of the reduced amount of skin available for experiments, some were never performed. It would therefore be of interest to perform sensitivity studies of nanogel penetration, using skins from different genders, ages, races and parts of the body.

Finally, if more human excised skin were to be available, penetration experiments with FA-containing nanogels could be performed, to assess the ability of this carrier to deliver the drug. After the penetration experiments, the skin should be washed, ground and extracted with chloroform (CHCl3), in order to remove the drug and quantify how much of it had been able to penetrate the cells.