Macrophage-targeted photodynamic therapy

Abstract

Photodynamic therapy (PDT) uses light activatable molecules that after illumination produce reactive oxygen species and unwanted tissue destruction. PDT has dual selectivity due to control of light delivery and to some extent selective photosensitizer (PS) accumulation in tumors or other diseased tissue, additional targeted selectivity of PS for disease is necessary. The delivery of drugs to selected lesions can be enhanced by the preparation of targeted macromolecular conjugates that employ cell type specific targeting by ligand-receptor recognition. Macrophages and monocytes express a scavenger-receptor that is a high-capacity route for delivering molecules into endocytic compartments in a cell-type specific manner. We have shown that by attaching PS to scavenger-receptor ligands it is possible to get three logs of selective cell killing in macrophages while leaving non-macrophage cells unharmed. The capability to selectively kill macrophages has applications in treating cancer and in the detection and therapy of vulnerable atherosclerotic plaque and possibly for autoimmune disease and some infections.

Fig. 1

Photophysical/photochemical mechanisms underlying PDT. Light is absorbed by the ground state PS that then moves to the first singlet excited state, from where it can lose energy by fluorescence or internal conversion and return to the ground state. Alternatively the excited singlet state PS can undergo intersystem crossing to the excited triplet state, which in addition to losing energy by phosphorescence, can transfer its energy to the ground state of molecular oxygen, which is also a triplet. This results in the PS returning to the ground singlet state, and the oxygen rising to the excited singlet state (Type II). Alternatively the triplet PS may undergo reactions with substrates leading to free radicals (Type I). Both these pathways lead to highly cytotoxic species by reacting with proteins, nucleic acids and lipids in cells.

Fig. 2

Cells were incubated with conjugate (5 µM for J774 cells, 2 µM for other cells) for 24 hours and uptake was determined by flow cytometry (Fig 2A); or cells were illuminated with 660-nm light at a fluence rate of 50 mW/cm2 and survival fractions were calculated with respect to non-illuminated cells by the MTT assay 24 hours later (Fig 2B).

Fig. 3

In vivo tumoricidal effects of macrophage-targeted PDT compared to non-targeted free c_e6. A) subcutaneous J774 tumors were grown in Balb/c mice and 14 days later injected IV with BSA-c_e6-mal or free c_e6 (2 mg c_e6 equivalent/kg) and 24 hours later illuminated with 200 J/cm² 660 nm light. B) Subcutaneous EMT-6 tumors were grown in Balb/c mice and treated exactly as described for J774 tumors in Fig 3A.

Fig. 4

Intimal fluorescence signal from different sections of aortas from atherosclerotic and normal rabbits. Values are means of 4–8 spectra obtained from sections taken from aortas from 6 atherosclerotic and 6 normal rabbits and bars are SEM.

Fig. 5

Trichrome (left) and reverse DAPI (right) staining of aortic sections (upper is non-illuminated and lower is illuminated) taken from a rabbit injected with BSA-ce6-mal and receiving intravascular light 24 hours later.