PHOTOFRIN II UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS. FLUORESCENCE AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS ON POST-MORTEM AORTA

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.     

UPTAKE and PHOTODYNAMIC EFFICIENCY OF HEM ATOPORPH YRIN, HYDROXYETHYLVINYLDEUTEROPORPHYRIN and HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II®): A STUDY WITH ISOLATED MITOCHONDRIA

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.     

PORPHYRIN ACCUMULATION BY ATHEROMATOUS PLAQUES OF THE AORTA

Abstract A complex mixture of porphyrins termed hematoporphyrin derivative (HPD) has been clinically useful for tumor localization. When sections of human aorta containing atheromatous plaques were incubated with HPD, accumulation of fluorescent porphyrin was observed within the plaques. Analytic studies showed that the plaques had accumulated hematoporphyrin (HP), which is substantially more hydrophilic than that HPD fraction generally associated with tumor localization. Fluorescence spectra suggest that the plaque binding sites of HP resemble the relatively aqueous micelles formed by the detergent sodium dodecylsulfate. This result has implications for tumor-localization procedures, since accumulation of hydrophilic porphyrins by tumors has been reported.     

Correlation between Site ll-Specific Human Serum Albumin (HSA) Binding Affinity and Murine in vivo Photosensitizing Efficacy of Some Photofrin Components

Human serum albumin (HSA) is one of the key components in human blood that may influence drug distribution. As such, it is important to know the affinity of any drug for albumin. Previously, Photofrina mixture of monomeric, dimeric and oligomeric porphyrins, has been subjected to HSA binding studies. However, due to its complex nature, binding studies on Photofrin or other hematoporphyrin derivatives with HSA are inconclusive. In this report, the binding properties of some components (dimers and trimers) of Photofrin® and the relationship between murine photosensitizing efficacy and those binding properties were investigated. The interaction of these porphyrins with HSA was investigated by direct ultrafiltration and fluorescent titration techniques with fluorescent probes such as dansyl-L-proline (DP), which is known to interact selectively with site II on HSA. Porphyrins also were tested for antitumor activity in a mouse model following intravenous administration and exposure to laser light. Together, the results suggest that the photosensitizers that were preferentially bound to site II of HSA were most effective at controlling murine tumor regrowth     

UPTAKE AND RETENTION OF PHOTOFRIN® BY CULTIVATED HUMAN LYMPHOBLASTIC CELLS (Reh6): PREFERENTIAL AFFINITY OF THE CELLS FOR A MINOR COMPONENT DEMONSTRATED BY NORMAL PHASE CHROMATOGRAPHY

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.     

Binding studies of porphyrins to human serum albumin using affinity capillary electrophoresis

The present work demonstrates that affinity capillary electrophoresis (ACE) can be employed as a valuable and powerful tool for studying the interactions between porphyrins and proteins in biological and biomedical research, such as the development of porphyrins and related compounds as efficient and selective photosensitizers in the photodynamic therapy of cancers. Binding constants of human serum albumin (HSA) to four biological porphyrins (uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, protoporphyrin IX), which possess a wide range of hydrophobicity, were estimated by ACE. Based on 1:1 molecular association between these individual porphyrins and HSA, the change of the electrophoretic mobility of HSA as a function of porphyrin concentration in the run buffer was measured and the binding constants were calculated from the slope of the Scatchard plots. The binding constant values were found to be 8.80 +/- 0.51 x 10(4) M(-1), 2.39 +/- 0.16 x 10(5) M(-1), 1.61 +/- 0.11 x 10(6) M(-1), and 9.34 +/- 0.30 x 10(6) M(-1) for uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, and protoporphyrin IX, respectively, and most of these results are in good agreement with those reported in the literature using conventional methods for binding measurements. Additionally, experimental binding constant data obtained using ACE was found to exhibit very good correlation with theoretical hydrophobicity values calculated using the Rekker's hydrophobic fragmental constant method, thus further supporting the hypothesis that the hydrophobicity of the porphyrin side chains play an important role in governing the hydrophobic interaction of porphyrins with serum proteins such as HSA.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: II. THE EFFECTS OF HUMAN versus MOUSE AND BOVINE SERUM

The effects of human serum (HS), mouse serum (MS) and fetal bovine serum (FBS) on cellular delivery and retention of Photofrin were examined using human lung tumor cells (A549) cultured in vitro. The results show that these three kinds of sera exhibit substantial differences in: (i) degree of inhibition of Photofrin cellular uptake, (ii) retention capacity of Photofrin delivered to the cells in their presence and (iii) efficacy of promoting the clearance of Photofrin from the cells. It is suggested that these differences originate from unequal interaction of each of the sera with Photofrin material, which in turn is the consequence of variability in composition and in the levels of serum proteins in HS, MS and FBS. The highest degree of Photofrin disaggregation and and competitive binding of its constituents was attributed to HS. The lowest degree of Photofrin disaggregation, and the competitive binding limited mostly to monomeric porphyrin forms was implicated for FBS. For MS, the spectroscopic and cellular data indicated a lesser degree of Photofrin disaggregation than with HS, with little if any consequence in Photofrin retention characteristics. The implication of this comparative analysis is that in vitro studies using FBS may underestimate the extent of interaction of Photofrin with serum proteins in humans, and overestimate the retention capacity of the photosensitizer in human tissues. Studies in vivo using a mouse model may also underestimate the degree of disaggregation of Photofrin in human circulation, and give different photosensitizer tissue retention levels than in humans.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: III. ROLE OF PLASMA PROTEINS IN PHOTOSENSITIZER CLEARANCE FROM CELLS

Abstract— Human plasma proteins, albumin, globulins and low density (LDL), high density (HDL) and very low density (VLDL) lipoproteins were tested for their effects on retention of Photofrin and three other photosensitizers in cultured cells. This was assessed by incubating the cells, subsequent to the exposure to Photofrin, in the photo-sensitizer-free medium containing various concentrations of different plasma proteins. Photofrin clearance levels differed with individual plasma proteins and also were dependent on concentration of these proteins in the incubation medium. All of the proteins except VLDL promoted clearance of Photofrin taken up by the cells in the presence of 5% human serum. Subsequent to some Photofrin exposure conditions (in the presence of 5% fetal bovine serum, or in protein-free medium), albumin, in contrast to LDL, HDL and globulins, exhibited decreased capacity for promoting the photosensitizer clearance from the cells. The VLDL showed very little or no effect in promoting cellular clearance of Photofrin, tetraphenyl porphine tetrasulfonate (TPPS₄), and di- and tetrasulfonated chloroaluminum phthalocy-anine (AlPcS₂ and AlPcS₄, respectively). The LDL seem to be particularly effective in promoting clearance of Photofrin and AlPcS₂ from the cells, whereas albumin and globulins were shown to be more effective than LDL and HDL in promoting the cellular clearance of TPPS₄.     

ACCUMULATION OF PORPHYRINS IN CELLS: INFLUENCE OF HYDROPHOBICITY AGGREGATION AND PROTEIN BINDING

Abstract— For a variety of chemically defined, synthetic and natural porphyrins, the tendency for self aggregation, binding to serum albumin, distribution coefficient betweenoctanol–1 and water and uptake in V79 Chinese hamster cells have been determined. A strong correlation was found between cell uptake and distribution into octanol. None of the other factors could be correlated with cell uptake. These observations might have an impact on the use of porphyrins in photodynamic and boron neutron capture therapy of tumors.     

Identification and partial characterization of an unusual distribution of the photosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin) in human plasma

Temoporfin (m-THPC) is an extremely powerful photosensitizing drug, more than 100-fold more photocytotoxic than Photofrin and many other drugs. The reasons for this are not yet known but are likely to be associated with the mechanism of uptake of the drug and its intratumoral and intracellular localization. Uptake itself is likely to be dependent upon the plasma binding of the drug following administration. In the current work, we have shown that the addition of m-THPC to human plasma in vitro at clinically relevant doses of sensitizer and administration solvent (diluant) gives rise to a protein-binding pattern quite different to that of Photofrin and other hydrophobic drugs as judged by density-gradient ultracentrifugation. Analysis of the binding immediately after addition to human plasma has shown that lipoprotein binding accounts for only a minor proportion of the sensitizer, which is mainly associated with a high-density protein fraction that is not coincident with serum albumin. The m-THPC protein complex does not fluoresce significantly even on dilution. This binding pattern is highly dependent on administration conditions and storage. Over a period of 6-8 h at 37 degrees C the m-THPC that is associated with this unidentified fraction redistributes to the plasma lipoproteins. Plasma collected from rats after intravenous administration of m-THPC also contains this low fluorescent complex, showing that this phenomenon is not limited to human plasma and also occurs in vivo. It is postulated that the m-THPC bound to the unknown protein fraction is highly aggregated and that it is likely to be taken up into tissues in this form. This unusual uptake may possibly be associated with the very high activity of m-THPC and also to the recent finding of a second peak in the plasma pharmacokinetics of the drug.     

DETERMINANTS OF PHOTOSENSITIZATION BY PURPURINS

The human colon adenocarcinoma cell line, WiDr, was exposed to Photofrin II, hematoporphyrin derivative (HPD), hematoporphyrin (HP) or tetrasodium-meso-tetra(4-sulfonatophyenyl)porphine (TPPS4) followed by irradiation with light. Clonogenicity was determined and the resultant survival curves compared and shown to be qualitatively similar in shape. However, for equal amounts of drug in the medium, there were large differences in photosensitizing efficiency with Photofrin II approximately 5, 25 and 50 fold more effective than HPD, HP and TTPS4, respectively. For the same power used, all drugs were less efficient photosensitizers under red light (600-1100 nm) than under white light (300-110 nm). For all drugs this could be explained in terms of changes in light absorption over the two wavelength ranges. Differences in clonogenic cell survival could not be explained in terms of differences in singlet oxygen production (from published values). A reduction in drug uptake into the cells was sufficient to explain the differences between Photofrin II, HPD and HP, while TPPS4 was 5-fold less effective compared to other drugs than would be expected from drug uptake measurements. Two methods for measuring drug uptake were compared and shown to give different results for Photofrin II. Measurements of drug fluorescence in 0.1 N NaOH yielded 5-fold lower values than when measurements were in 1 N HCl following heat treatment to monomerise aggregated drug. Clearly the reliability of the method used in determining drug uptake must be carefully ascertained.     

The interaction of cationic and anionic porphyrins with the bovine serum albumin in borate buffer

The interaction of water-soluble porphyrins with the bovine serum albumin in borate buffer at pH 8.6 has been studied. The localization of porphyrins in the protein globule has been determined. It was established that the native conformation of albumin upon binding with the porphyrins is preserved, however, the anionic porphyrins are exhibit wedging effect on the albumin domains. The binding constants were obtained from fluorescence spectroscopy data.     

On the correlation between hydrophobicity, liposome binding and cellular uptake of porphyrin sensitizers

A crucial factor in choosing a porphyrin or analogous photosensitizer for photodynamic therapy (PDT) is its ability to incorporate into the cells. For hydrophobic compounds that partition passively into the cytoplasmic membrane, a partition coefficient between an organic solvent and water, P, is one factor that could be used to predict the molecule's ability to diffuse into biomembranes. We synthesized several porphyrins, modified with two, three or four meso-substituents and studied their spectroscopic and photophysical properties. The octanol-water partitioning coefficients, log P, were calculated as a parameter for hydrophobicity. We found these porphyrins to be very hydrophobic, with log P values in the range of 8.9-11.8. These were correlated with the binding constants of these porphyrins into liposomes, K(b), as well as to their uptake by cells. The correlation between the estimated log P and K(b) is nearly linear but negative, indicating, apparently, that there is lesser binding to liposomes with increased hydrophobicity. On the other hand, all of the studied porphyrins are taken up by cells, but there is no clear correlation between cellular uptake and the log P or K(b). Lipinski's pharmacological "rule of 5" predicts poor permeation of drugs into cells when log P is greater than five. This may be relevant for diffusional binding to liposomes, where aqueous aggregation can interfere strongly with cellular uptake. In such extreme conditions, neither liposome binding nor other rules seem to predict porphyrin behavior in vitro.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

PICOSECOND FLUORESCENCE OF R3230AC MAMMARY CARCINOMA MITOCHONDRIA AFTER TREATMENT WITH HEMATOPORPHYRIN DERIVATIVE and PHOTOFRIN® II in vivo

Hematoporphyrin derivative (HPD) and other porphyrin samples were excited by 20-ps 532-nm laser pulses. Fluorescence was detected using a low-jitter streak camera. Data were fitted to a sum of exponential decay times on the order of picoseconds. Fluorescence of porphyrins in aqueous solution show various behaviors depending on the hydrophobicity of the porphyrins. The most hydrophilic porphyrins show long decays only (greater than 500 ps). Porphyrins intermediate in hydrophobicity have intensity-dependent fast decays. The most hydrophobic have fast decays (less than 20 ps). Picosecond fluorescences of mitochondria prepared from rat tumors treated in vivo with HPD or Photofrin II show an increase in the ratio of fast to slow decays when compared to the injected porphyrins. These results are consistent with the concentration of the more hydrophobic porphyrins in mitochondria in photosensitization treatment. Thus picosecond fluorescence studies of porphyrins may provide a means to obtain photoproperties which differentiate between effective and ineffective in vivo photosensitizers.     

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated “stealth” liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.     

QUANTITATIVE ANALYSIS OF INTRACELLULAR BEHAVIOUR OF PORPHYRINS

Abstract Fluorometric analysis performed on L 1210 cells after treatment with Photofrin indicated that the interactions with cellular structures induce a significant modification of the equilibria among the different porphyrin species. This modification turned out to be dependent on the uptake and release processes. Thus, a comparative analysis of the dynamic aspects of the drug accumulation process was performed on cells treated with hematoporphyrin, Photofrin and Photofrin II. The results obtained were interpreted taking into account the different chemical composition of the drugs employed. The porphyrin species mainly released seem to be the monomeric ones and 'unfolded oligomers'. The release process results in further modifications of the aggregation and/or configu-rational state of intracellular porphyrins due to altered internal equilibrium.     

INTERACTION OF HUMAN SERUM LOW DENSITY LIPOPROTEINS WITH PORPHYRINS: A SPECTROSCOPIC AND PHOTOCHEMICAL STUDY

The incorporation of proto-, uro- and hematoporphyrin in low density lipoproteins (LDL) of human blood has been studied by equilibrium dialysis, fluorescence and absorption spectroscopy. The lipoproteins may efficiently compete with albumin in the binding of protoporphyrin to human blood proteins in patients suffering from protoporphyria. It can be concluded that hydrophobic porphyrins bind to blood proteins.
The complexation of hydrophobic porphyrins in LDL is responsible not only for efficient photodynamic effect at the lipoprotein level, but also for photoinduced lipid peroxidation and for consumption of β-carotene incorporated into LDL which are one of their natural carriers. The water-soluble uroporphyrin, although an efficient photosensitizer for the LDL apoprotein photoinactivation, is much less efficient for lipid peroxidation and β-carotene bleaching. The 353 nm laser flash photolysis shows that porphyrin triplet states are not affected by the physiological β-carotene content of LDL but are fully accessible to oxygen.     

THERMODYNAMICS OF THE BINDING OF HEMATOPORPHYRIN ESTER, A HEMATOPORPHYRIN DERIVATIVE-LIKE PHOTOSENSITIZER, AND ITS COMPONENTS TO HUMAN SERUM ALBUMIN, HUMAN HIGH-DENSITY LIPOPROTEIN AND HUMAN LOW-DENSITY LIPOPROTEIN

The phenomena of the high affinity of porphyrins to the human serum proteins, albumin, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) is well established. Yet, evaluation of the activities of these proteins as endogenous porphyrin carriers, especially with respect to receptor-mediated porphyrin uptake into tumor cells, the merits of which are still in dispute, requires more quantitative protein-porphyrin binding data. As a continuation of previous studies on this issue, the binding of several porphyrin systems to each of the three proteins, employing previously developed spectral methodologies, was studied. The specific systems reported here are hematoporphyrin ester (HPE), which is a novel hematoporphyrin derivative (HPD)-like system, two porphyrin trimers (denoted O1 and O2) and a porphyrin dimer (denoted O3) isolated from HPE. Human serum albumin (HSA) was found to have a single high-affinity site for the monomeric components of HPE, with an equilibrium binding constant of 3.6 × 10⁶. The equilibrium parameters determined for the binding of the three HPE-isolated oligomers to each of the serum proteins are: (1) Binding constants (K_b') of 2.3 × 10⁶, 6.9 × 10⁴ and 1.5 × 10⁴ and number of sites per protein molecule (n) of 3, 1 and 5, for the binding of 01, 02 and 03, respectively, to HSA. (2) K_b’values of 15.5 × 10³, 15.3 × 10³ and 6.6 × 10³ and n values of 1, 2 and 2, for the binding of O1, O2 and O3, respectively, to HDL. (3) K_b’values of 3.3 × 10³, 2.28 × 10⁴ and 8.0 × 10³ and n values of 50, 20 and 16 for the binding of O1, O2 and O3, respectively, to LDL. These data are direct and clear support not only for the high affinity of porphyrins to serum proteins but specifically of stable oligomers that have been assigned critical roles in the photodynamic treatment of tumors. Of the three proteins, LDL is clearly the best camer, providing the highest drug payload with a moderate affinity (enough to bind and not too much to prevent release). These data are suggested to be promising for the postulated role of LDL in porphyrin uptake into tumor cells and to be useful in the future as benchmarks for novel porphyrin systems.