Pegylation of charged polymer-photosensitiser conjugates: effects on photodynamic efficacy

Abstract

Conjugates between photosensitisers (PS) and charged polymeric carriers are under investigation for photodynamic therapy of cancer and may allow targeting to certain cell types or compartments in tumours. Covalent attachment of polyethylene glycol to macromolecules (pegylation) may alter their pharmacokinetics, cell type targeting, and photophysical properties. Macrophages may take up large amounts of aggregated PS, thus lessening the selectivity for cancer cells in tumours. We investigated the effect of pegylation on the uptake and phototoxicity of poly-L-lysine chlorin(e6) conjugates with either cationic or anionic charges in two cell lines, human ovarian cancer cells and mouse macrophages. The cationic conjugate after pegylation became less aggregated, consumed less oxygen and had reduced cellular uptake. However, the phototoxicity corrected for cellular uptake increased three- to five-fold. In contrast, the anionic succinylated conjugate on pegylation became more aggregated, consumed similar amounts of oxygen, and had higher cellular uptake. The anionic conjugate showed the highest relative phototoxicity towards both the cell lines (compared to the other three conjugates) and it decreased most towards the macrophages after pegylation. Pegylation reduced the amount of oxygen consumed per chlorin(e6) molecule when photosensitised cells were illuminated. These in vitro studies suggest that pegylation alters the phototoxicity of PS conjugates depending on the effect produced on the aggregation state.

Figure 1

Effect of pegylation on the extent to which conjugates are aggregated at varying concentrations in serum-containing medium. (A) cationic pL–c_e6 and pL–c_e6–PEG, (B) anionic pL–c_e6–succ, and pL–c_e6–PEG–succ. Points are derived from the difference in fluorescence in 0.1 M NaOH/1% SDS between aliquots taken from samples centrifuged at 16 000 g at 4°C and from those agitated at room temperature. Each point is the mean of fluorescence determinations from three aliquots and error bars are s.d.

Figure 2

Phototoxicity curves comparing the light dose responses of the survival fractions of OVCAR-5 (A, B) and J774 (C, D,) cells incubated with (A, C) cationic pL–c_e6 and pL–c_e6–PEG, (B, D) anionic pL–c_e6–succ and pL–c_e6–PEG–succ. Cells were incubated for 3 h in serum-containing medium with conjugates added at 1 μM c_e6 equivalent concentrations. After illumination, cells were given fresh medium and 24 h later mitochondrial activity was determined by the MTT test. Survival fraction was calculated as the ratio of the 480 nm absorption from PDT-treated cells, to that from those given conjugate and kept at room temperature in the dark for the duration of the illumination. Points are the means from four separate experiments each containing six wells and bars are s.d.

Figure 3

Relative phototoxicity curves comparing the light dose responses of the phototoxicities per nanomoles of c_e6 taken up by the two cell lines (J774 and OVCAR-5) with (A) cationic pL–c_e6 and pL–c_e6–PEG, (B) succinylated pL–c_e6–succ and pL–c_e6–PEG–succ. Points were calculated from the reciprocal of the survival fraction (from Figures 2A to D) divided by the uptake in nanomoles c_e6 equivalent per milligram cell protein (from Table 2). Error bars are the s.d. of the ratios calculated in quadrature.