There are theoretically 60 free amines on BSA through which Gd-DOTA could be conjugated; however some fraction of these must be occupied by maleyl groups for recognition by the scavenger receptor. The number of maleyl groups incorporated, however, must be balanced with the number of Gd-DOTA that must be attached to achieve significant contrast enhancement. To evaluate the minimum number of maleyl groups to satisfy receptor recognition we synthesized a series of mal-BSA molecules with varying degrees of maleylation. Significant conformation changes with maleylation are believed to facilitate access to lysines that are buried in unmodified BSA.(32
) These molecules were also tagged with FITC and applied to macrophages in culture to assess uptake. As shown in optimal uptake is achieved with greater than 80% of the free amines modified by maleyl groups. present confocal images for cells incubated for one hour with mal-BSA from 0 to 89% maleylation carrying ~1 FITC per molecule. Confocal images were obtained on a BioRad system using 488nm excitation. Significant increases in fluorescence intensity are observed as the degree of maleylation is increased, indicating increased uptake. Although optimal at 80% maleylation, cellular internalization is still significant for 40-80% maleylated molecules. These results are consistent with previous studies performed using radiolabeled ligands.(33
) In a study with rat endothelial sinusoidal liver cells, SRA uptake activity correlated with different degrees of BSA modification labeled with 125
). Scavenger receptor mediated endocytosis (uptake) of maleylated BSA indicated that 60% maleylation, or >37 lysyl amino residues, sharply increased ligand activity. Radioligand data supported that all lysyl amino groups are accessible for maleylation or DOTA conjugation. Based on the results of , subsequent labeling with Gd-DOTA was performed on 60% maleylated BSA in order to maximize the number of free-amines for Gd-DOTA conjugation. At 60% maleylation, there should be up to 24 available free amines for conjugation of Gd-DOTA groups.
Figure 1 Macrophage uptake is dependent on degree of maleylation. Cells were incubated with FITC labeled derivatives of mal-BSA bearing different numbers of maleyl groups. Signal intensity increases with increasing maleylation, with significant uptake at greater (more ...)
Additions of increasing numbers of DOTA groups to mal-BSA (Gd-DOTA)n
increased the number of paramagnetic centers, improving the relaxivity of the contrast agent as shown in . Observed relaxivities for the mal-BSA contrast agents are summarized in . Incorporation of DOTA groups was verified by mass spectrometry and Gd content was analyzed by ICP-MS. Close agreement between the DOTA:BSA ratio, determined by mass spec, and Gd:BSA ratio, determined by ICP-MS, suggests formation of the Gd:DOTA chelate with no non-specific binding to the protein despite reported affinity sites for gadolinium on the BSA molecule.(34
) Relaxivity of Mal-BSA(GdDOTA) increases with increasing Gd-DOTA per molecule then plateaus at >15 Gd/molecule, attaining a maximum of 33mM-1
. The relaxivity values are comparable to those for literature reported and commercial agents such as isothiocyanato-benzyl-diethylenetriamine-pentaacetic acid (ITCB-DTPA-Gd) albumin (30.3mM-1
at 29Mhz, 24°C)(35
), and MS-325, a Gd chelate that reversibly and non-covalently binds native albumin with relaxivity at maximum loading of albumin equal to 33mM-1
and relaxivity under in vivo
conditions of 17.3mM-1
) Relaxivities for Mal-BSA(GdDOTA)>15
are significantly higher than those reported for a related albumin-(Gd-DTPA)19
at 10.7Mhz, 37°C)(37
) or for Magnevist, a small molecule gadolinium chelate (3.4mM-1
at 42MHz, 37°C). The reported values for the albumin compounds were obtained a slightly higher temperature and with different field strength instruments, so the values are not directly translatable. However, these results show that the relaxivities for the prepared agents are on the order or better than expected values. R1
values are also improved over free Gd-DOTA preparations such as Magnevist. Conjugation of Gd-DOTA to larger molecules is known to increase relaxivity by reducing rotational correlation time.(38
) This has been observed for lipophilic Gd-DOTP and Gd-DOTA complexes that bind to albumin(39
), as well as for other Gd-DOTA conjugates(41
Relaxivities of prepared agents compared to other reported agents. Relaxivities increase with increasing Gd-DOTA incorporation, reaching an apparent plateau above 15 Gd/molecule.
MR images of solutions of the contrast agent at 7T demonstrated that these levels of labeling with Gd-DOTA were sufficient to affect contrast at physiologically reasonable concentrations. As shown in contrast was increased in a concentration dependent manner by the Gd-DOTA conjugates, with greater enhancement by more heavily labeled molecules. presents mal-BSA that has been conjugated to 18 or 10 Gd-DOTA, greater contrast is seen with 18 attached Gd-DOTA for top row of solutions, as expected. For the 18 Gd-DOTA molecule, significant contrast enhancement is seen for concentrations greater than 50μM.
Figure 2 Contrast enhancement correlates with number of Gd-DOTAs bound. Solutions of mal-BSA (Gd-DOTA)n with n = 10 (bottom row) or 18 (top row) in distilled, deionized water, pH 7.0 were imaged at 7T at ambient temperature (25°C). Signal intensity increases (more ...)
Uptake by cells was assessed on P388D1 macrophages in culture. Cells were incubated with 15 and 10 Gd-DOTA labeled mal-BSA for one hour and then lysed for MR imaging. As shown in macrophages show increasing contrast in an MRI image with increasing concentration of the Gd-DOTA labeled compounds. Incubation with greater than 50μM of either labeled agent was sufficient to produce observable contrast in the MR image. Results from ICP-MS indicate that femtomoles of gadolinium could be found inside a cell after one hour incubation.
Figure 3 Mal-BSA (Gd-DOTA)n is taken up by macrophages. P388D1 cells were incubated with n = 15 (top row) or n = 10 (bottom row) mal-BSA (Gd-DOTA)n for one hour then imaged at 7T. Contrast increases in a concentration dependent manner (right to left) indicating (more ...)
That uptake by cells is specific was confirmed by incubating cells with mal-BSA carrying 22 Gd-DOTA, or with a Gd-DOTA matched BSA control. These control molecules were prepared from the same batch of Gd-DOTA labeled BSA but were not maleylated for scavenger receptor recognition. Images at 7T, , from incubated cells show that there is no uptake of the BSA compound (bottom row) while the maleylated partner is taken up in a concentration dependent manner (top row). Specificity of uptake was further confirmed by competition studies in which cells were incubated with a fixed concentration of mal-BSA (Gd-DOTA)15 and increasing excess of unlabeled mal-BSA. If uptake were receptor mediated the excess unlabeled ligand should compete away uptake. Nonspecific uptake is strictly concentration dependent and would not be affected by additional ligands in the solution. Receptor mediated uptake is confirmed by the results seen in , increasing amounts from 0-20 fold excess competitor reduce uptake as expected for a receptor-mediated process.
Figure 4 Uptake of mal-BSA (Gd-DOTA)n is specific. P388D1 cells were incubated with either mal-BSA (Gd-DOTA)n or a matched BSA control, n = 22 for both. Cells were imaged at 7T. Macrophages demonstrate uptake of the maleylated agent (top row) but do not recognize (more ...)
Figure 5 Uptake of mal-BSA (Gd-DOTA)n is receptor-mediated. P388D1 cells were incubated with a fixed concentration of mal-BSA (Gd-DOTA)15 and increasing molar excesses of unlabeled mal-BSA. Representative samples are shown with molar excesses of competitor as (more ...)
One of the primary concerns regarding the use of biomolecules labeled with gadolinium chelates as contrast agents is whether sufficient amounts of gadolinium can be accumulated in sites of interest to be visible on an MR image.(43
) The final amount of contrast agent required to produce sufficient contrast in an MRI scan depends on a host of factors including the precise pulse sequence and parameters used for acquisition.(2
) Nevertheless, this issue has been studied by a number of investigators and estimations can be made. One of the earliest theoretical models for the minimum amount of Gd(III) complex, of a given relaxivity, to produce detectable contrast enhancement was tested against microinjections of Gd(III) in to Xenopus eggs(44
). These trials established that measurable contrast was visualized when the concentration of agent in the egg is on the order of 10-100μM (at 500MHz). The Xenopus embryo system is unique, and while similar to some types of human tissue, the eggs are quite fatty and are different from many tissues of interest in mammals. More recent empirical results at 7T have shown that for contrast agents with relaxivities in the range of 5-7mM-1
the minimum amount of Gd(III) to produce contrast is on the order of 2 × 109
molecules per cell; while for an agent with relaxivity on the order of 80mM-1
(20MHz) contrast is observed for concentrations on the order of 4 × 107
Gd(III) per cell(45
). These observations were made using apoferritin as a carrier for the Gd(III), which is taken up by receptors found on the cell surface at a density of 4 × 104
per cell, Kd = 1nM.
Relaxivities are also field dependent. NMRD curves for Gd-DOTA show that relaxivity does not change much up to about 2MHz and then begins to fall off rapidly, so the relaxivity at typical clinical field strengths (1.5-3T) should actually be a bit higher than that observed on typical research instruments (4.7-11.7T)(40
). Thus, the amount of Gd-DOTA required would be less at lower field strengths. We estimated that the relaxivity for our multi-labeled mal-BSA molecules would be similar to that observed for Gd-labeled albumin (26:1 Gd:albumin) = 192mM-1
(per mM agent) at 4.7T(46
). At 1.5T this value would be higher. With this estimate of relaxivity, based on the criteria just described, the amount of Gd(III) required to produce contrast at 1.5T should be less than 4 × 107
molecules per cell.
The scavenger receptor is a high affinity (pM-nM, depending upon ligand) receptor present in high numbers on macrophages, on the order of 5 × 105
receptors per cell I P388D1.(18
) Thus, ~20-80 Gd(III) must be taken up per receptor during the course of incubation (depending on cell type used) to reach the concentrations required per cell. Scavenger receptors uptake and process modified lipoproteins through a classic coated pit pathway, similar to the LDL pathway, wherein the receptor takes up ligand, brings ligand into the cell, dissociates from the ligand, and recycles back to the surface in a highly efficient manner. The scavenger receptor has an estimated t1/2
for internalization of the ligand-receptor complex of 2.8-4min(22
). Recycling of receptors in the scavenger receptor pathway has not been fully characterized; but in the LDL pathway receptors recycle on the order of 10 minutes(49
). If this is similar for the scavenger receptor pathway then 500,000 receptors can uptake ~3 × 106
ligands per hour. Based on the values given above, this would be sufficient for contrast with 13:1 Gd:ligand complex and one hour incubation. As our results show, modification with ten and greater Gd-DOTA per mal-BSA was sufficient to produce contrast with micromolar applications to cells. Moreover, the cell densities used for these studies is on the order of that found for macrophages in isolated human plaques, indicating that these imaging results will be applicable to the in vivo
There are number of examples in the literature of efficient cellular uptake of Gd(III) complexes using Gd-DOTA derived molecules(57
). More recent efforts have sought to maximize the load of Gd delivered per receptor by tethering carrier molecules to dendridic polymers containing high numbers of Gd(III)(61
), uptake through the folate receptor has been demonstrated by this means as has avidin-biotin antibody-based targeting(59
). Again, definitive measurement of the amount of Gd required to produce contrast depends upon the imaging parameters employed and these must also be evaluated when testing the new contrast agents. But on the basis of the approximate calculations shown here, and these similar studies in the literature, we predicted that MRI contrast could be achieved for the scavenger receptor system using feasible ratios of Gd:ligand and this was confirmed by the results reported here.
Recent literature has investigated MRI contrast agents targeted to other molecular markers in plaque, but these tend to be geared for imaging of more advanced stages of disease. For example, 5-HT-DOTA(Gd) targets myeloperoxidase (MPO), a heme-containing enzyme secreted by a number of neutrophils, for selective imaging of MPO activity in a model system(64
). Myeloperoxidase secretion has been correlated with plaque instability. The described agent is a serotonin derivative of Gd-DOTA that polymerizes after oxidation by myeloperoxidase-catalyzed reduction of hydrogen peroxide. Polymerization resulted in increased relaxivity attributed to decrease in rotational correlation time and demonstrated contrast enhancement in presence of at least 650U of enzyme. This agent is geared toward more advanced plaques that would have significant secretion of MPO and may be vulnerable to rupture. Other examples in the literature describe the use of Gd-DTPA labeled fibrin binding peptides to label thrombi in animal models(65
). Each agent carries four gadolinium chelates and binds to mural thrombi, rather than atherosclerotic plaque. Thrombosis occurs after plaque rupture and these agents are designed to aid in guiding thrombolytic therapy.
Fewer reports describe MRI agents able to detect earlier stages of disease. One example is low density lipoprotein (LDL) complexed with manganese-mesoporphyrin (MnMeso)(67
). These agents are also targeted to macrophages in that the modified LDL was demonstrated to be taken up by foam cell cultures. However, the MnMeso is not covalently bound to the lipoprotein, and therefore may transfer off LDL to other plasma proteins and lipoproteins. In addition, there is concern about possible cardiotoxicity and accumulation of Mn2+
agents in the brain after intravenous injection(68
). Dextran coated iron oxide nanoparticles have also been observed to be taken up by macrophages in plaques, due to the phagocytic nature of the cells to engulf particles(70
). However these rely on nonspecific uptake of the nanoparticles, which an inefficient process, outside of the liver, and the agent must be delivered in several fold excess of standard clinical doses in order to accumulate enough contrast agent in the plaque after clearance through by renal system.
A possible limitation to administering mal-BSA (Gd-DOTA)n in a clinical setting is possible humoral immune response to this macrocyclic chelated agent.(71
) By targeting macrophages, we are in effect aiming for increased recognition by these immune cells. However, our aim is for internalization of mal-BSA and removal from blood without negative systemic effects. Studies on methods to increase immunogenicity of antigens for vaccine development have examined maleylation(21
). In those studies, immunogenicity, i.e.
uptake by macrophages, of maleylated antigens was increased compared to unmodified antigens upon repeated injections in mice without adjuvants present(21
). Upon repeated exposure, animals exposed to mal-BSA shows higher serum anti-immunogen antibody concentration than those exposed to BSA. In addition, maleylation disrupts native B cell epitopes on the antigens, for example, maleylated diphtheria toxoid (DT) is not recognized by antibodies to native DT. New epitopes seem to created that can cross-react between various maleylated proteins regardless of amino acid sequence. It is likely then that the epitopes are purely maleylation dependent. While mice produced antibodies against the antigen, no systemic toxicity was reported. Possible toxic or other negative immune responses of mal-BSA require further study. The scavenger receptor recognizes a number of different ligands(9
), these can also be investigated as carriers should mal-BSA prove undesirable in vivo
From the MTT cell toxicity studies, dose response curves suggest that mal-BSA (Gd-DOTA)15
has a low toxicity when compared to chlorpromazine, as shown in . Chlorpromazine is a phenothiazine drug, primarily used as a psychotropic agent since the 1950's, with a low toxicity (73
). Its toxicity has been well documented and therefore, it has been employed as a standard in toxicity assays(74
). The EC50
can be interpolated from these plots and used to directly compare the toxicity of mal-BSA (Gd-DOTA)15
to chlorpromazine. From , EC50
is not reached at even the highest concentration tested for 4 hour incubations with mal-BSA (Gd-DOTA)15
. For 24 hour incubations, EC50
was found to be 40μM. Both values are less toxic than those found for chlorpromazine, , which showed EC50
= 84μM for 4 hour, and EC50
= 24μM for 24 hour incubations, respectively.
Figure 6 Toxicity of mal-BSA. Cytotoxicity was assessed for mal-BSA (Gd-DOTA)15 and chlorpromazine as a positive control. The assays were carried out in parallel and in triplicate. Cells were counted and plated evenly in a 96-well plate. After a 24-hour adhesion (more ...)
Bio-distribution studies show a very fast clearance from the blood stream, more than 97% of the injected dose of radio labeled contrast agent is cleared out within two minutes as seen in . From the PET studies of biodistribution it was found that the main sites for accumulation are the liver, kidneys and the urinary bladder. The highest accumulation is in the liver. After one hour about 50% of the injected dose remains in the liver. During the next 3h the activity drops to 30% of the injected dose. This is equivalent to 30μM agent in the liver after one hour and 18μM after four hours. The activity then remains stable at 18μM. Comparing these accumulated concentrations to the concentrations from the cell toxicity studies, EC50
= 40μM for 24 hour incubation, suggests that in these experiments in vivo
accumulation is at less than toxic levels. Accumulation observed in the other organs is even less, and thus, also below toxic EC50
. For the kidneys, about 1% of the injected dose is found in each kidney and the levels are stable for the duration of the whole experiment. In the urinary bladder 2.5% of the injected dose remains after one hour; this clears to approximately 0.2-0.5% of injected dose during the first 24h and falls to undetectable levels after 48h. By comparison, the blood pool agent albumin(Gd-DTPA)30
is largely retained in the intravascular space with a half-life of 88min after intravenous injection and can remain in the bloodtstream and extravascular compartment two weeks after injection.(77
) These undesirably long retention times have precluded its use in the clinic.
Figure 7 Blood clearance. Blood samples were collected by tail nicking at 2, 15, 33, 60 and 200 minutes post injection of contrast agent and quantitated by gamma counting. Clearance from the blood is rapid with ~ 97% of the injected agent cleared out from (more ...)
In conclusion, we describe the preparation, characterization and successful in vitro
targeting of a paramagnetic contrast agent, mal-BSA (Gd-DOTA)n
, for use in MRI. This novel contrast agent builds upon previously reported studies on anionic modifications of BSA and knowledge of the SR-A receptor. Mal-BSA (Gd-DOTA)n
has physical properties that permit localization to the target and demonstrated high specific uptake of agent by macrophages, also low toxicity and fast clearance dynamics are shown. The synthesis process outlined here allows controllable degrees of maleylation and paramagnetic labeling of known lysines on the BSA molecule. Mal-BSA (Gd-DOTA)n
demonstrated relaxation effects comparable to or better than similar commercial and experimental plaque-targeted or albumin-based agents. An optimal payload of 22 Gd-DOTA with mal-BSA (Gd-DOTA)22
showed contrast effects comparable to albumin (Gd-DTPA)30
, which is used as a standard to evaluate performances of novel intravascular agents. (79
Though further studies are required to evaluate immunogenicity of the agent, the high specificity of mal-BSA (Gd-DOTA)n shows promise for reducing dosage of administration and toxicity compared to high dose nonspecific contrast agents currently employed in the clinic. Future work includes isolating the regional domains of maleylated BSA that afford targeting in order to circumvent immune responses that may be provoked by full length protein. Through the modification with other diagnostic agents, such as the 64Cu positron emitter or fluorophores, this carrier system shows potential to act as a multimodality imaging agent for diagnosis and study of atherosclerotic and restenotic lesions.