335 nm (Wilhelm et al., 1991). Pyrene fluorescence intensity (I335/332) values remain nearly unchanged at low concentrations of mPEG-PA but with further increase of concentration, intensity begin to increase significantly, implying the onset of self aggregation and formation of micelle nanostructures (Figure 2.3). No change in pyrene fluorescence was observed with mPEG which clearly indicated that mPEG cannot aggregate to form micelle like structure. It implies that the presence of hydrophobic palmitate chain was necessary for the micelle formation. The concentration of the mPEG-PA at which the micelles were started to form is known as CMC which is the most important parameter regarding the thermodynamic stability of the micelles. The micelle formation is a thermodynamic process and the relationship between CMC and the free energy of micellization (ǻG°M) of an amphiphile in aqueous solution can be written as:
Where, R is universal gas constant and T is temperature.
So, the lower is the CMC value of a given amphiphilic polymer, the more stable are the micelles. This is specially important from the pharmacological point of view, since upon dilution with a large volume of the blood only micelles with low CMC value will able to exist, while micelles with high CMC value may dissociate into unimers and their content may precipitate in blood. The value of CMC obtained for mPEG-PA was much lower than low molecular weight surfactant (e.g. 2.3 g/L for sodium dodecyl sulfate in water). It indicates micelles formed from mPEG-PA conjugate were thermodynamically stable and could preserve stability without dissociation after dilution caused by intravenous injection.
The average size of the drug loaded micelles was found to be less than 50 nm, which was comparable to the other nanoformulations of curcumin. This size range of the nanocarriers is encouraging with regards to passive targeting of tumor tissues through EPR effect.
0M ln( )
G RT CMC
'
To investigate whether these micelle nano-carriers would be advantageously used for encapsulating highly hydrophobic drug curcumin for delivery to cancer cells, we studied the encapsulation of curcumin by the novel mPEG-PA conjugate. The encapsulation efficiency of curcumin increased as the amount of conjugate increased. This was because of the fact that, with the increase of conjugate, more micelles formed which solubilized more amount of curcumin in their core. The solubilization of curcumin in mPEG-PA made a clear yellowish solution whereas without the nanocarriers free curcumin was not able to solubilize in water (Figure 2.6). This clear solution was obtained due to the encapsulation of curcumin in core of the micelle nanocarriers (Scheme 2.2).
Curcumin has intrinsic fluorescence properties. The photophysical properties and fluorescence spectra of curcumin are highly sensitive to the polarity of the environment and showed significant solvent dependent shifts in emission maxima (Chignell et al., 1994). We found that, after encapsulation in micelles the absorption and fluorescence intensity of curcumin increased significantly. The fluorescence peak maxima of micellar curcumin exhibited a large blue shift. This increase of absorption and fluorescence intensity and blue shift of peak maxima suggests that curcumin in mPEG-PA is encapsulated in the hydrophobic core of the micelle where the environment is nonpolar.
For effective passive targeting of cancerous tissues drug loaded micelles should be able to retain the drug for prolonged time in circulation. We observed that mPEG-PA micelles were able to retain more than 80% of drug after 48 hours. Thus the micellar formulation was very stable under broad physiological pH conditions.
The mPEG-PA conjugate is biodegradable as evidenced by enzyme degradation studies.
When lipase acts on mPEG-PA micelle we anticipated release of free mPEG in the solution and palmitic acid as an insoluble precipitate (Scheme 2.3). 1H NMR of the precipitate
(Figure 2.10) confirmed that it was palmitic acid and micelle degradation occurred due to the cleavage of the ester bond of the conjugate as per proposed scheme (Scheme 2.3).
To verify that the ester linkage of mPEG-PA can be cleaved inside the cells to release the entrapped drug, we performed an in vitro experiment with HeLa cell lysate. The cell lysate successfully degraded the micelles to release curcumin (Figure 2.12). So, the conjugate is biodegradable and can be degraded in situ by the cells. Taking advantage of intrinsic fluorescence properties of curcumin we observed that cells can internalize micelle encapsulated curcumin as effectively as free curcumin. But the mPEG-PA encapsulated curcumin has the added advantage of enhanced solubility and can therefore be directly used without any organic solvent.
Since, mPEG is nontoxic and approved by FDA for pharmaceutical applications and palmitic acid is a natural fatty acid found in human body, we expect the mPEG-PA conjugate should be biocompatible, biodegradable and potentially safe as a drug carrier for clinical use. In vitro cytotoxicity assay proved that empty nanocarriers showed only minimal cytotoxicity and nanocarriers encapsulated drug was as effective as free curcumin. Also under the microscope we observed significant change in morphology of the cells after treatment with nanocarrier encapsulated curcumin. The green fluorescence of curcumin was observed all over the cells, earlier which were mainly in the cytoplasm (Figure 2.14). So, in the cell cytoplasm, the nanocarriers were degraded by the action of enzymes and released the drug which ultimately diffuses slowly into the nucleus and exerted its effect. It has been reported that curcumin induce cytotoxic effects in HeLa cells through apoptosis. Although we have not done any molecular assay to find out the mechanism of cell death but our microscopic study suggests that, nanocarrier entrapped curcumin also induced cell death
through apoptosis. These results demonstrated that this delivery system could be a promising choice for administering curcumin without loss of its therapeutic efficacy.
Scheme 2.2: Self-assembly of mPEG-PA in aqueous solution to form micelle nanocarriers and simultaneous encapsulation of curcumin in the core of nanocarriers.
Scheme 2.3: Lipase catalyzed degradation of mPEG-PA. Lipase hydrolyzes the ester bond of mPEG-PA to yield mPEG and palmitic acid in physiological condition (37ºC, pH 7.4).