Beth Anne Allison

Liposomal delivery of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD), to tumor tissue in a mouse tumor model.

Abstract— Biodistribution studies were carried out on 14C‐labeled benzoporphyrin derivative monoacid ring A (BPD), which had been formulated as a unilamellar liposome or taken from a stock solution in dimethyl sulfoxide diluted into phosphate‐buffered saline immediately before intravenous injection into DBA/2 mice. By and large the general distribution of BPD to various organs and tissues was comparable for both formulations. It was noted, however, that liposomal material appeared to enter tissues more rapidly and to be cleared more rapidly, as demonstrated by shorter half‐lives for a number of tissues including skin, lung and fat, and generally lower levels in most tissues 24 h following administration. Accumulation in tumor tissue was slightly higher with liposomal BPD, and clearance rates for this tissue were equivalent (half‐lives 16.1 h for liposomal BPD and 16.9 h for aqueous BPD). When the two preparations were tested in a bioassay in tumor‐bearing mice, photodynamic therapy (PDT) with liposomal BPD proved to be superior to the aqueous preparation when PDT was administered 3 h following intravenous administration of BPD. Plasma distribution studies in vitro demonstrated that 91.1 ± 0.3% of the liposomal BPD distributed to the lipoprotein fraction within the first hour of mixing, whereas only 49.1 ± 2.6% of nonliposomal BPD was associated with lipoprotein under the same conditions. Furthermore, while lipoprotein‐associated liposomal BPD distributed evenly between all three types of lipoprotein (high, low and very low density), a majority of nonliposomal BPD associated with the high‐density lipoprotein fraction.

The effects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative.

The influence of lipoprotein association on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative (BPD) has been investigated in M‐1 tumor bearing mice. The association of benzoporphyrin mono acid ring A with either low or high density lipoprotein increased tumor cell killing in an in vivolin vitro cytotoxicity assay performed 3 h post intravenous drug administration. Eight hours following photosensitizer injection only low density lipoprotein (LDL) mixtures produced significant (P≤ 0.005) increases in tumor cell killing compared to BPD in unfractionated plasma. The efficacy of in vivo photosensitization in the presence of lipoproteins correlated with the in vivolin vitro cytotoxicity. Association of BPD with low or high density lipoproteins resulted in delayed tumor regrowth and higher cure rates when light exposure (125J/cm2) was performed 3 h post drug administration. When light exposure was performed 8 h post‐injection only LDL‐BPD mixtures led to enhanced tumor eradication compared to BPD administered in aqueous solution or unfractionated plasma.

THE PLASMA DISTRIBUTION OF BENZOPORPHYRIN DERIVATIVE and THE EFFECTS OF PLASMA LIPOPROTEINS ON ITS BIODISTRIBUTION

Abstract— The plasma distribution and biodistribution of benzoporphyrin derivative were examined. Two analogs of benzoporphyrin derivative were mixed with human plasma in vitro and recovered in the lipoprotein fractions upon separation by chromatography or ultracentrifugation. The majority of both analogs was recovered with high density lipoprotein. The effect of prebinding benzoporphyrin derivative to lipoproteins on the biodistribution of the drug in vivo was studied in tumor bearing DBA/2J mice. At 3, 8 and 24 h post‐injection, tumor and tissue samples were excised and analyzed for benzoporphyrin derivative content. Precomplexing benzoporphyrin derivative with low density lipoprotein or high density lipoprotein led to significantly (P < 0.05) greater tumor accumulation than in aqueous solution.

Effects of Native, Triglyceride-Enriched, and Oxidatively Modified LDL on Plasminogen Activator Inhibitor-1 Expression in Human Endothelial Cells

Whereas VLDL has consistently been shown to induce a concentration-dependent increase in the expression of plasminogen activator inhibitor-1 (PAI-1) in human umbilical vein endothelial cells (HUVECs) and liver cells, variable effects have been reported for native and oxidatively modified LDL. In the present study, activation of PAI-1 protein and mRNA expression by native LDL (nLDL), UV-oxidized LDL (uvLDL), and triglyceride (TG)-enriched LDL was studied in HUVECs by using different incubation times and a wide range of lipoprotein concentrations. No significant increase of PAI-1 protein expression was observed after 4 hours of incubation with nLDL or uvLDL. However, PAI-1 protein secretion from HUVECs was markedly enhanced after 18 hours of incubation with uvLDL (200% increase at 10 microg/mL). Stimulation of PAI-1 protein expression in HUVECs by nLDL was seen, however, after increasing the TG content of the LDL particle. LDL enriched in phospholipid had no effect on PAI-1 secretion. PAI-1 mRNA levels on northern blot increased in parallel with the activation of PAI-1 protein expression by native and modified forms of LDL. Low concentrations of TG-enriched LDL (10 microg/mL) and higher concentrations of nLDL and uvLDL (100 microg/mL) were found to increase the binding of a VLDL-inducible transcription factor to the PAI-1 promoter. These results indicate that the TG content of the LDL particle influences PAI-1 expression in endothelial cells. Low concentrations of uvLDL enhanced PAI-1 protein and mRNA expression in the HUVECs after an 18-hour incubation but did not influence the VLDL-inducible transcription factor. This suggests that low levels of oxidized LDL increase PAI-1 expression by a different mechanism than VLDL and TG-enriched LDL.

Enhanced photodynamic killing of target cells by either monoclonal antibody or low density lipoprotein mediated delivery systems

Abstract Photodynamic therapy (PDT) is now being tested and employed in a growing number of medical centers for the treatment of malignant tumors. This form of therapy selectively destroys cancer cells by activation of retained photosensitizer molecules by absorption of light at appropriate wavelengths. One major limitation of PDT is the potential damage of normal tissue due to nonspecific retention of photosensitizers. In an attempt to address this problem, two delivery systems, monoclonal antibody (MoAb) mediated and low density lipoprotein (LDL) mediated, have been tested. In this study, we first showed that the photosensitizer benzoporphyrin derivative monoacid ring A (BPD) could be covalently bound to a MoAb (5E8 ) via a modified polyvinyl alcohol (PVA ). We then demonstrated that both BPD-5E8 conjugate and BPD-LDL mixtures exhibited enhanced photodynamic killing on target cells. Finally, we showed that internalized BPD-5E8 and BPD-LDL displayed much higher cytotoxicity than that of surface associated BPD-5E8 and BPD-LDL, respectively. These results suggest that monoclonal antibody or LDL mediated delivery increases the selectivity and cytotoxicity of BPD. They also indicate that internalization of BPD via either delivery mode produces significantly enhanced cell killing.

Delivery of benzoporphyrin derivative,a photosensitizer,into atherosclerotic plaque of Watanabe Heritable Hyperlipidemic Rabbits and balloon-injured New Zealand rabbits

Abstract— In this study we compared the plasma distribution and arterial accumulation of a photosensitizer, benzoporphyrin derivative (BPD), in two models of atherosclerosis: the spontaneous lesions of the Watanabe heritable hyperlipidemic (WHHL) rabbit and induced lesions of the balloon‐injured, cholesterol‐fed New Zealand white (NZW) rabbit. Selective uptake and retention of a photosensitizer by the abnormal portion of a vessel is a necessity in order for photodynamic therapy to become a successful modality for inhibition of intimal hyperplasia, selective removal of atherosclerotic tissue or imaging of diseased arteries. Liposome‐based formulations were compared to freshly isolated native low density lipoprotein (LDL) and acetylated‐LDL (Ac‐LDL) as delivery vehicles for BPD. Plasma distribution of the photosensitizer was analyzed by KBr density gradient ultracentrifuga‐tion. Although the delivery vehicle influenced plasma distribution immediately postinjection, BPD subsequently partitioned according to the plasma concentration of the lipoproteins. Photosensitizer level in plaque and normal artery specimens was determined by ethyl acetate extraction and spectrofluorometric measurement. The measurement of BPD in normal and atherosclerotic arterial tissue demonstrated a selective accumulation in atherosclerotic tissue. Preassociation with LDL and Ac‐LDL enhanced accumulation of BPD in atherosclerotic tissue when compared with normal artery (mean ratios of 2.8 and 4.1 were achieved, respectively). These results indicate that the preferential uptake of BPD by atherosclerotic plaque can be enhanced by preassociation with plasma lipoproteins, suggesting that light activation could lead to a highly selective destruction of diseased vascular tissue.

Efficacy of photodynamic killing with membrane associated and internalized photosensitizer molecules

Many photosensitizers under investigation for possible use in PDT share the property of selective accumulation in malignant or abnormal tissues. However, the mechanisms by which this occurs is not understood. Although it has been established that singlet oxygen is responsible for cell killing once a photosensitizer has reached the target tissue, the cellular targets and molecular mechanisms also remain unknown. The mechanisms of selective accumulation and cytotoxicity may depend upon the photosensitizer used. In attempts to develop technology to improve selective uptake by target tissues, the authors address the question of cellular targets at a preliminary level. These studies were performed using one benzoporphyrin derivative analogue, benzoporphyrin mono-acid ring A (BPD-MA). Plasma distribution of BPD-MA shows that this lipophilic photosensitizer has a propensity to associate with plasma lipoproteins in blood. Biodistribution studies indicate that preincubation of BPD-MA with low density lipoprotein (LDL) leads to significantly greater tumor accumulation than BPD-MA in aqueous solution. Further, the technology for conjugating BPD-MA to monoclonal antibodies has been established in the laboratory. Selective photodynamic killing of a human squamous cell carcinoma cell line (A549) has been demonstrated using such a conjugate. The monoclonal antibody (MoAb) used, 5E8, has specificity for a glycoprotein detected on human squamous cell carcinoma of the lung. In this study these two delivery systems are used to compare the cytotoxicity of membrane bound and internalized photosensitizer at the cellular level. The results indicate that LDL or MoAb mediated delivery increases the selectivity and cytotoxicity of BPD-MA. Internalization of BPD-MA via either delivery mode produces significantly enhanced cell killing.