The effect on the cell proliferation of compounds Man-Tb and 149 in HeLa cells was evaluated via an Alamar Blue assay in a range of concentrations (100 – 1 μM). The cytotoxicy of Man-Nap was already reported in Chapter 2 and it was found that the compound was not toxic in HeLa cells after 24 h incubation (IC50 >100 μM), which was not surprising since the cells did not take up the compound. Since compounds Man-Tb and 149 were found inside the cells at 24 h, their anti-proliferative effect after longer times (72 h) was also assessed. Table 5.2 summarises the IC50 values obtained after 24 and 72 h incubation, respectively. Even though cell internalization was occurring for compounds Man-Tb and 149, no effect on the cell proliferation (i.e. IC50 >100 μM) was observed even after long incubation times of 72 h.
Table 5.2. IC50 (µM) values of compound Man-Tb and 149 in HeLa cells measured at different times.
Compound IC50 (μM)
t (h) 24 72
Man-Tb >100 >100
149 >100 >100
The fact that these compounds proved to be non-toxic towards HeLa cells was a gratifying result as it highlighted the potential for these probes to be used as non-toxic probes for the selective detection of lectin in vitro.
5.5 Conclusions
A novel Tröger’s base naphthalimide was successfully synthesised and fully characterised spectroscopically. Both of the compounds, Man-Nap and Man-Tb, were shown to successfully bind with Con A, as demonstrated spectroscopically by the significant changes in their fluorescence intensity upon binding. This was demonstrated at low
Chapter 5. Glycosylated Naphthalimides for Lectin Detection
171 concentrations of 1 × 10-5 M, with a significant increase in the fluorescence emission intensity of Man-Nap being observed. However, this was not the case for the Man-Tb as it showed scattering even at very low concentrations of Con A (0.02 equiv). This scattering is a consequence of the formation of a less soluble complex. Due to the poor resolution observed in the fluorescence emission spectra of Man-Tb, a direct comparison between the two ligans cannot be drawn. However, the fact that more scattering was observer in the case of Man-Tb may suggest a better binding due to the presence of the two -D-mannose.
Nonetheless, better results were obtained with Man-Nap, as the binding with Con A invoked a noticeable increase in the fluorescence emission. Importantly, both compounds showed to be able to bind to Con A at low and high concentrations and the Con A binding was measured with both compounds up to 1 μM concentrations of Con A.
The compounds demonstrated the ability to bind selectively with Con A even in the presence of other macromolecules such as BSA or PNA. Similar compounds were used as negative controls to provide further evidence that the changes in the fluorescence only occur when the compounds bind to Con A.
The binding occurring was supported by SEM studies, where it was observed that notable changes in the morphologies of both compounds were evident upon binding with Con A.
In vitro studies of both compounds in HeLa cells showed that they were non-toxic, even though Man-Tb was internalised by the cells. Compound 149 was also evaluated in vitro due to its higher lipophilicity. Uptake studies showed large aggregates being dissolved and taken up by the cells over time.
In conclusion, these findings offer a successful proof-of-principle study that demonstrates the potential of glycosylated naphthalimide derivatives to be used as molecular probes for lectin detection. Their simple structures could pave the design of more complex probes in the future.
Chapter 6
Supramolecular Self-Assembly of Glycosylated
Naphthalimides
Chapter 6. Supramolecular Self-Assembly of Glycosylated Naphthalimides
173 6.1 Introduction
Various supramolecular structures have been formed by the glycosylated naphthalimides described in the previous chapters. Taking into account that the spectroscopic and biological properties of the compounds (as free molecules) had already been explored, it was decided to examine their supramolecular structures to investigate if they could be of interest as novel biomaterials. The use of biomaterials for biological applications has been expanded in the recent years.231,232 Biomaterials applications range from drug delivery, antibacterial, or the improvement or replacement of natural functions.233-235 The main objective in this field is to create materials that respond to artificial stimuli such as pH, temperature, light or magnetic irradiation, etc. Recently, examples of biomaterials inducing cancer apoptosis or a drug-delivery mechanism have emerged using hyperthermia.236,237 Photothermal therapy (PTT) uses nano-materials that absorb in the near infrared (tissue penetrating) and transform this energy into heat, thus causing apoptosis in the cancerous cells. For instance, Leiyin et al.236 developed polyethylene glycol (PEG) nanoparticles of compound 150 (Figure 6.1.1), which absorbs in the NIR and transforms this energy into heat. Assays carried out using λexc = 808 nm for only 8 min irradiation showed a temperature increase from 25 °C to 47 °C, leading to a significant size tumour reduction in mice bearing HSC tumour.
Figure 6.1.1. Compound 150 developed by Leiyin et al. forming PEG-nanoparticles that induces apoptosis
via PTT.236
However, preventing the accumulation of nanoparticles within the body is still challenging and therefore the development of degradable materials is still needed.238,239
Chapter 6. Supramolecular Self-Assembly of Glycosylated Naphthalimides
174
Therefore, many groups have focussed their research in the development of biodegradable materials.240-243 Chenand co-worker240 developed silica nanoparticles bearing Doxorubicin (Dox) coated with collagen and hyaluronic acid, which will improve biocompatibility and cancerous cell delivery, as the CD44 receptor for hyaluronic acid is overexpressed in certain tumours. More importantly, hyaluronidase (HAase) would degrade the nano particles thus releasing the drug (Dox) inside the cells and inducing cell death. Similarly, gels have also been used to encapsulate drugs,244,245 and DeForest and co-workers,243 have recently developed hydrogels that can be selectively released by a combination of stimuli, such as enzymatic, light irradiation or reducing conditions (Scheme 6.1.1). These hydrogels can be engineered depending on the desired combinations of stimuli required to reach the degradation, as certain diseases-states present overexpressed enzymes or reducing environments.243
Scheme 6.1.1. Different stimuli that trigger hydrogels degradation designed by DeForest group.243
As it was previously discussed, naphthalimides can interact with each other via - interaction of their aromatic cores. This aggregation can lead to the formation of supramolecular structures in solution such as spherical aggregates or gels. The following section describes recent examples of self-assembled naphthalimides and their applications.