In this work, a pegylated poly(L
-glutamic acid) bifunctional conjugate containing an MRI contrast agent and a photosensitizer was prepared to reduce non-specific uptake, particularly the liver uptake of conjugate and to improve tumor targeting in MRI-guided photodynamic therapy. Covalent conjugation of PEG onto polymers prevents their recognition by macrophages and increases their blood circulation time (28
). In addition, pegylated macromolecular photosensitizer conjugates have shown increased tumor retention and improved therapeutic profile (29
Three-dimensional high-resolution dynamic contrast enhanced MRI is effective for non-invasive visualization of the real-time pharmacokinetics and biodistribution of the polymer conjugates in mouse tumor models (31
). The MRI study revealed that PGA-(Gd-DO3A)-Mce6
resulted in relatively short blood circulation and high liver uptake possibly due to the hydrophobic interaction of mesochlorin e6
with the reticuloendothelial system. The modification of PGA-(Gd-DO3A)-Mce6
with mPEG of 2 kDa significantly reduced the non-specific liver uptake of the photosensitizer conjugate at the same Gd(III) dose. The pegylated conjugate resulted in more prolonged blood enhancement than PGA-(Gd-DO3A)-Mce6
despite the relatively low relaxivity of the former. Generally, higher contrast enhancement in tissues corresponds to higher concentration of contrast agents as a result of increased accumulation. The prolonged circulation of pegylated conjugate in the blood can be attributed to reduced recognition by liver macrophages (Kupffer cells) and splenic macrophages (34
). This could also account for their higher accumulation in tumor tissues over 18 hours than non-pegylated and control conjugates.
High tumor accumulation with low non-specific tissue uptake of the pegylated conjugate, as observed in , resulted in more effective tumor enhancement and site-directed photodynamic therapy. Upon injection at the same mesochlorin e6 dose, the pegylated conjugate showed a higher contrast enhancement in tumors despite the lower Gd (III) dose, indicating a better tumor uptake profile (). Consistent with the higher tumor accumulation, the pegylated conjugate resulted in more significant tumor growth inhibition than the non-pegylated conjugate PGA-(Gd-DO3A)-Mce6 after a single dose treatment ().
It appears that the timing of photodynamic therapy is also critical for the bifunctional conjugates. Contrary to the result in our previous publication (17
showed a lower therapeutic efficacy in this study. This difference in therapeutic efficacy could be attributed to the difference in the timing of laser irradiation. In the previous study, the tumors were irradiated at 2 and 18 hours post-injection. The PDT at 2 hours post-injection might cause tumor vascular damage due to the presence of the conjugate at high concentration in the blood pool in addition to PDT-related cytotoxicity. In this study, however, the irradiation time points were 18 and 24 hours post-injection for all conjugates. It is expected that the blood concentration of the conjugate is significantly reduced at these time points. The damage to tumor tissues would most likely be caused by the PDT-related cytotoxicity inside the lesions and little or no efficacy would be associated with the damage to tumor microvasculature. It has also been reported that outcome of PDT is determined to a large extent by vascular response (35
). Therefore, therapeutic efficacy could be further enhanced by irradiating at early time points when the concentration of the photosensitizer conjugate would be higher within the tumor vasculature.
DCE-MRI with a biodegradable macromolecular MRI contrast agent was effective for non-invasive assessment of tumor response to photodynamic therapy based on the tumor vascular permeability. It has been demonstrated in various preclinical and clinical studies that DCE-MRI can provide timely and non-invasive assessment of the therapeutic efficacy in anti-angiogenesis therapy, which is comparable to morphological parameters (36
). However, DCE-MRI with macromolecular contrast agents is advantageous for the characterization of tumor vascularity because they can selectively permeate through the hyperpermeable microvasculature of tumor tissues without extravasating through normal endothelium (38
). (Gd-DTPA) cystine copolymers are a biodegradable macromolecular MRI contrast agent that behaves initially as a macromolecular agent and can then degrade and excrete from the body after the MRI studies (39
). The vascular permeability maps of tumor tissues calculated from the DCE-MRI data correlated well to the tumor growth and histological analysis in different treatment groups. The tumor tissues treated with the polymer conjugates with the photosensitizer had significantly lower contrast agent uptake as observed on DCE-MRI. This could be attributed to the lower vascular permeability, lower MVD on histology and slower growth rate than those treated with the control.
Pegylation of PGA-(Gd-DO3A)-Mce6 significantly modified its pharmacokinetics and biodistribution. The pegylated bifunctional conjugate containing both photosensitizer and MRI contrast agent had prolonged blood circulation, reduced liver uptake and improved tumor targeting as shown by non-invasive three-dimensional high-resolution MRI. Correspondingly, the pegylated conjugate showed more significant tumor enhancement and better therapeutic efficacy than the non-pegylated conjugate for MRI-guided photodynamic therapy. Contrast-enhanced MRI can effectively detect changes in tissue distribution of polymer conjugates and guide site-directed irradiation of target tissues. DCE-MRI with the biodegradable macromolecular MRI contrast agent was also effective in non-invasive assessment of tumor response to photodynamic therapy. Contrast-enhanced MRI-guided photodynamic therapy with the pegylated bifunctional polymer conjugate is promising for minimally invasive cancer treatment.