The objective of the current study was to develop PpIX-micelles and investigate their photophyscial properties and PDT efficacy in cancer cells. PpIX is a potent photosensitizer and although its precursor, 5-ALA (Levulan®
) has been clinically approved by the FDA, poor membrane permeability was a major limitating factor to achieve the desired clinical efficacy [7
]. To overcome this limitation, we aimed to establish a micellar nanocarrier using a biocompatible and biodegradable PEG-PLA copolymer to directly deliver PpIX to cancer cells. Currently, Genexol™, a PEG-PLA micelle formulation for the delivery of paclitaxel, has already been clinically approved for cancer treatment in S. Korea [30
PpIX is planar molecule with four conjugated pyrrole rings () [31
]. PpIX can easily aggregate in aqueous solution and its aggregation behavior has been extensively studied as a function of pH and ionic strength [32
]. In the pH range of 0-3, PpIX stays as a monomer with a sharp Soret band at 404 nm and four Q-bands; at pH > 8, PpIX exists as a dimer with a sharp Soret band at 388 nm and four weak Q-bands; and in the pH range 3-7, PpIX forms extended aggregates with two splitting weak Soret bands centered at 350 and 460 nm and four weak Q bands. In these larger aggregates, porphyrins preferentially interact axially through π-π interactions and laterally by edge-to-edge hydrophobic interactions. Formation of intermolecular hydrogen bonds between the carboxylic acids was further hypothesized to contribute to the stabilization of the aggregated structures [32
]. At physiological pH (7.4), PpIX has a very low aqueous solubility (~1 μg/mL) and cannot be directly administered intravenously.
Polymeric micelles provide an attractive nanocarrier option for the delivery of PpIX. In this study, we prepared PpIX-micelles by two different strategies: non-covalent encapsulation of PpIX in the hydrophobic cores of micelles and covalent conjugation of PpIX to the core-forming block of the PEG-PLA copolymer (). We systematically examined the UV-Vis absorption, fluorescence emission, 1
yield and PDT efficacy of different PpIX-micelles. At low PpIX loading density (i.e., <0.2%), PpIX mostly stayed in the monomeric state as indicated by the sharp Soret band at 404 nm, strong fluorescence intensity, and high 1
yield regardless of incorporation strategies. At high PpIX loading density (i.e., 4%), however, the incorporation strategy had a dramatic effect on the PpIX state in the micelles. In PpIX-encapsulated micelles, aggregated forms of PpIX were observed as indicated by the split Soret bands and complete quenching of fluorescence and 1
= 0.06). In comparison, the 4% PpIX-conjugated micelles showed that PpIX were present as dimers with retaining of considerable 1
generation capacity (ΦΔ
= 0.48). These results are consistent with the previous reports that only PpIX monomers and dimers can be photoexcited to triplet state thus allowing for the generation of 1
generation efficiency from different PpIX-micelles did not correlate with their phototoxicity in H2009 lung cancer cells. Despite having much higher 1
yields, the two 0.2% PpIX-micelles showed significantly lower phototoxicities compared to the 4% PpIX-micelles at the same micelle dose (). To investigate this discrepancy, we used confocal laser scanning microscopy to examine the micelle uptake and PpIX release in H2009 cells. For pegylated micelle nanoparticles, cell uptake occurs through fluidic phase endocytosis where nanoparticles are internalized via endosomes and distributed to other intracellular organelles (e.g. lysosome, Golgi) [36
]. At 4% drug loading, both micelle formulations had very low fluorescence emissions in cell culture media due to PpIX quenching within the micelle core (). With 4% PpIX-encapsulated micelles, rapid increases in fluorescence in H2009 cells was observed in the first 4 hrs (). We attribute this increase to the release of free PpIX from intact micelles as well as from dissociation of micelles into PEG-PLA unimers and free PpIX, which may further bind to cytosolic proteins or membrane structures leading to further elevated fluorescence. For 4% PpIX-conjugated micelles, higher intracellular fluorescence intensity was also observed over those from 0.2% PpIX-micelles, suggesting the dissociation of some micelles into PEG-PLA-PpIX unimers. Similar micelle dissociation events were also observed following exposure to fluorogenic PEG-b
-poly(ε-caprolactone) micelles under biological conditions [38
]. In comparison, 4% conjugated micelles were more stable and had slower micelle dissociation kinetics compared to 4% encapsulated micelles in cell culture medium (Supplementary Fig. S1
), which is consistent with the fast equilibrium for 4% PpIX-encapsulated micelles and steady increase for 4% conjugated micelles in intracellular fluorescence measurements.
Our data show that 4% PpIX-conjugated micelles provided the largest PDT therapeutic index compared to the other micelle formulations tested (). Several factors may contribute to this enhanced performance. First, conjugation of PpIX to the PLA segment prevented the formation of highly aggregated structures as in the 4% PpIX-encapsulated micelles. Consequently, the intact 4% PpIX-conjugated micelles had much higher 1
= 0.48) than 4% PpIX-encapsulated micelles (ΦΔ
= 0.06). Second, it is known that high concentrations of free PpIX in cells can induce dark toxicity due to the binding of PpIX to mitochondrial membranes [40
]. As such, PpIX conjugation to the PEG-PLA copolymer can effectively prevent the release of free PpIX and avoid dark toxicity. Third, 4% PpIX-conjugated micelles showed pattern of steady increase in intracellular fluorescence, which suggest uptake and the slow release of conjugated PpIX inside the cells for improved phototoxicity over time. The prolonged PpIX accumulation may reflect the slower micelle dissociation kinetics to release PEG-PLA-PpIX. In contrast, in 4% PpIX-encapsulated micelles, after an initial increase no changes in fluorescence intensity was observed from 4 to 24 hrs, which indicates the reaching of equilibrium between PpIX-micelle uptake, PpIX release, and the subsequent clearance of released free PpIX. Although 4% PpIX-encapsulated micelles had higher intracellular fluorescence over 4% PpIX-conjugated micelles in confocal studies, chemical extraction of PpIX from cells showed that PpIX-conjugated micelles had higher intracellular PpIX accumulation than that of encapsulated micelles (Supplementary Fig. S2
). These results further verified the protection of PpIX and its photosensitive properties in conjugated micelle formulations, making 4% PpIX-conjugated micelles the best formulation for PDT efficacy. It is interesting to note that for anticancer drug delivery, it is imperative that intact drug molecules be released from nanocarriers. In this study, release of free PpIX from 4% PpIX-encapsulated micelles caused undesirable dark toxicity, while 4% PpIX-conjugated micelles allowed for an effective dose accumulation and higher phototoxicity with reduced dark toxicity in H2009 cells.