MECHANISM OF LCM UPTAKE BY TUMOR CELLS (AND/OR NEURO-INJURY SITES): INDIRECT EVIDENCE FOR TARGETED DRUG-DELIVERY VIA CERTAIN "LIPOPROTEIN RECEPTOR"-MEDIATED ENDOCYTIC PATHWAYS
Abstract
INTRODUCTION
RELATED EXPERIMENTAL DATA
CONCLUSION
REFERENCES
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Numerous published studies have demonstrated, in rats, the selective affinity of intravenously administered, lipid-coated microbubbles (LCM) for tumor cells. Based upon the structure of LCM and their known physicochemical properties, it appears likely that injected LCM can bind apolipoprotein B-100 (apo B-100), apolipoprotein E (apo E), and/or lipoprotein lipase (LPL) in the bloodstream. Apo B-100 is known to mediate low-density lipoprotein (LDL) binding to cellular LDL receptors, and it is widely reported that many tumor cells show increased LDL receptor expression and activity. Consequently, the proposed binding of plasma apo B-100 by LCM could influence the biodistribution of those LCM, because of the increased LDL receptor-mediated endocytosis occurring within the tumor tissue. Enhanced receptor-mediated uptake of LCM by tumor tissue may, therefore, explain the marked rapidity and high selectivity of LCM accumulation in tumors, as compared to findings with liposomes.
Moreover, one other of the known LDL receptor gene-family members may well participate in this receptor-mediated uptake of LCM by tumor tissue. For example, apo E and LPL binding to various lipid particles (as similarly proposed above for LCM) is widely reported to facilitate the uptake of the lipid particles [e.g., apo E-enriched B-VLDL or LPL-enriched VLDL, B-VLDL, and chylomicrons] by a large "multi-ligand" endocytic receptor known as "LDL receptor-related protein" or LRP.
In addition, LCM could likely bind (as do LDL with high affinity) to another multi-ligand receptor type, the "macrophage scavenger receptors". This group of receptors could well account for the observed preferential uptake of LCM by neuro-injury sites as well (-- since macrophage infiltration/proliferation is significantly increased in response to neuro-injury in the CNS). Similarly, these "macrophage scavenger receptors" participate in the pathogenesis of neuronal degeneration observed in aging and Alzheimer's disease, and accordingly such scavenger receptors have been identified in the literature as potential therapeutic targets in Alzheimer's disease (as well as in atherosclerotic lesions).
Several published studies in the early 1990s have reported lipid-coated microbubbles (LCM) to be an effective neurosonographic contrast agent for early detection, and localization, of experimental tumors in rats (1-3). Most of the tumors examined, in the initial diagnostic studies with LCM, were located in the brain (1,2), but additional sonographic studies examined tumors located in the liver (3) and subcutaneous tissues of the rat (4). In all these experimental tumors, the observed sonographic enhancement of the tumor image suggested LCM accumulated rapidly and selectively within the tumor mass. This belief was subsequently confirmed by counter-staining the histological samples, from each of the above tumor locations, with either of two lipid-specific stains (2-4); the results provided micrographic documentation of lipid-stained disc-like structures (having the same particle-diameter distribution reported for LCM in vitro (5,6)) accumulated in the tumor mass but not in the tissues in which the tumors are embedded.
More recent tumor studies with LCM have sought to utilize the above findings for a second medical application of LCM, i.e., targeted chemotherapy of tumors. Initial evaluation of this LCM drug-delivery system has been carried out on glioblastoma (C6) and gliosarcoma (9L), both in tissue culture and in vivo using rats (7,8). After labeling LCM with the fluorescent lipophilic dye “diO”, Barbarese et al. (7) report that “analysis of LCM interactions [by confocal laser scanning microscopy] with C6 and 9L cells in culture showed that LCM first adsorb at the surface of the cells, and with time became localized inside the cells. Binding and internalization proceeded faster at 37° C than at room temperature”. Similar analysis of such tumors in vivo, from rats injected with diO-labeled LCM, again revealed that labeled LCM were actually inside the tumor cells (7). Barbarese et al. further found that “staining of live cells [in tissue culture with] DAMP, a dye that recognizes acidic compartments, showed that the majority of internalized LCM was associated with compartments containing DAMP. If the same uptake mechanism were operative in vivo, it would indicate that a portion of LCM bypasses the reticuloendothelial system and become endocytosed directly by tumor cells” (7).
The first antineoplastic agent to actually be employed, in these animal studies using the LCM drug-delivery vehicle, was the lipid-soluble drug Taxol® (Bristol-Myers Squibb Co., NYC, NY) for brain tumor therapy in rats. Ho et al. (8) reported in 1997 that “When compared with either a saline control group or a group receiving Taxol in an oil [cremophor] vehicle, Taxol-LCM reduced tumor progression in Fischer 344 rats inoculated with 9L glioma. The most profound effect was observed with rats treated with three treatment cycles (five daily injections/cycle) separated by two rest periods (2 d/period). Both in vitro and in vivo data indicate that Taxol can be incorporated into LCM, can be delivered to the tumor site, and can exert a measurable antitumor biological effect” (8).
The subcellular mechanism of affinity of the LCM for tumors can probably be best deduced from LCM’s biochemical similarity with naturally occurring (surfactant-stabilized) microbubbles (9-18), and by reviewing the protein-binding characteristics previously documented for the latter (see below).
LCM compared with naturally occurring , stabilized microbubbles
LCM have similarities with, and have been modeled after, the naturally occurring and very stable microbubble populations found in oceans and other natural waters. Various physicochemical and biochemical studies on these long-lived natural microbubbles were conducted, and published, in the 1970s and early 1980s (9-17).
These early studies revealed that while different classes of organic matter (glycopeptide-acyl lipid complexes) coated and thereby stabilized natural microbubbles, it was actually an underlying monolayer of lipids (surrounding the gas bubble) which provides the long-term stability to such coated microbubbles (18). The key structural categories of these stabilizing natural lipids were eventually identified (18), and then “off-the-shelf” substitute lipids were used instead to produce ultrastable artificial microbubbles (18,19). These LCMs range in diameter from about 0.5 to 4 um, with the vast majority having a diameter of approximately 0.5 um (5,6,20) or less (57).
LCM and natural microbubbles compared with plasma lipoprotein particles
LCM and natural microbubbles resemble in several ways (including their having a low dielectric material in their core) the “chylomicrons” in mammalian blood plasma. The mainly 0.1-1 um diameter sizes of chylomicrons (21) closely resemble the primary size distribution of both LCM (approx. 0.5 um) and natural surfactant-stabilized microbubbles (usually <3 um) (18). Moreover, the protein content of chylomicrons even though low (about 2% by weight) is a very consistent feature (21,22), as is also true for natural microbubble surfactant where the protein content is less than 5% by weight (18). Further analogies in the protein content of natural microbubble surfactant versus chylomicrons, and other plasma lipoproteins, become apparent upon amino-acid analysis (see below).
Considering first the plasma lipoproteins as a group, “Apolipoprotein B (apo B) is the quantitatively dominating, nonexchangeable apolipoprotein of the cholesterol- and triacylglycerol-rich lipoproteins in plasma (i.e., chylomicrons, very low (VLDL), intermediate (IDL), and low density lipoproteins (LDL))” (23). Human and rat plasma apo B exists in two primary forms designated apo B-100 and apo B-48 (24). Apo B-100 (which is produced in the liver in humans) is required for the synthesis and secretion of very low density lipoproteins (VLDL) (25). “After release of nascent VLDL particles into the bloodstream, the initial phase of their metabolism resembles that of chylomicrons. The triglyceride-rich core of VLDL particles is hydrolyzed by lipoprotein lipase along the capillary endothelium. … By the time the particles are metabolized to the size and density of the LDL range, they have become enriched in cholesteryl esters, and virtually the only remaining protein component is apo B-100. The average residence time of LDL is 2-3 days; about 80% of LDL is removed from the circulation by the interaction of apo B-100 with the LDL receptor and the remainder by nonreceptor pathways. Approximately one half of the LDL is removed from human plasma by the liver; the other half is removed by extrahepatic tissues. Once LDL is bound and internalized by cells, it is directed to the lysosome, where both its protein and lipid components are digested. … More than 90% of the apo B-100 within the plasma of normolipidemic individuals is contained within the LDL fraction” (25).
Hence, it can be seen from the above discussion that “When nearly all of the triglycerides have been removed and the cholesteryl esters have increased sufficiently, the VLDL becomes an LDL particle” (26) -- which has a diameter of approximately 0.02 um (27). As a result, “Each LDL particle contains a nonpolar core composed of approximately 1,500 cholesterol molecules that are esterified to long-chain fatty acids. This cholesteryl ester core is surrounded by a polar coat of phospholipid, unesterified cholesterol, and protein” (specifically apolipoprotein B-100) (27).
Since the lipid components of LCM consist of glycerides, cholesterol and cholesterol esters (18,19), it is evident that the LCM composition is similar to the lipid composition of the LDL particle. Based on this similarity, it appears reasonable to expect that injected LCM would readily bind apolipoprotein B-100 in the bloodstream. (Since more than 90% of the apo B-100 within the blood plasma is associated with LDL particles (25), any binding of apo B-100 by injected LCM would essentially be tantamount to LDL-particle binding to these LCM. The subsequent biodistribution of LCM could be influenced, after such LDL-particle binding, via the LDL receptor-mediated endocytic pathway (26,27) (see sections below).)
Further support for the notion of LCM probably binding apo B-100 can be found in comparing the amino acid composition of apo B-100 (24) with that determined for the above-mentioned protein content of natural microbubbles (15). Table I summarizes the results of this comparison. Data is given for 17 different types of amino acids accounting for over 90% of the weight of apoprotein B-100 and approximately 80% of the protein fraction of natural microbubble surfactant (isolated from either forest soil or seaweed (agarose) gels (15)). It can be seen from Table I that the relative amounts of these 17 different amino acids follow almost the same pattern for both apo B-100 and natural microbubbles. Since LCM were closely modeled (see above) after natural microbubbles, the correlation displayed in Table I implies that LCM are readily capable of binding apolipoprotein B-100 -- either alone or in association with the LDL-particle itself (see preceding paragraph).
TABLE 1 - Relative amounts of different amino acids in apolipoprotein B-100 versus natural coated microbubbles
Natural microbubbles (from soil or seaweed):
Asp + Asn, Glu + Gln, Ser >
Leu, Ala, Ile, Lys, Thr, Val > Phe, Pro > Arg, His, Tyr > Met
Apo B-100 (& apo B-48):
Asp + Asn, Glu + Gln, Leu > Ser, Ala, Ile,
Lys, Thr, Val > Phe, Pro > Arg, His, Tyr > Met
Pharmacokinetics/biodistribution of LCM versus liposomes
As alluded to above, there is good reason to believe that LCM binding of apo B-100 (either alone or more likely already attached to LDL-particles) will influence the subsequent biodistribution of those LCM. This belief derives from the fact that “apo B is the LDL component which mediates LDL binding to cellular receptors” (23), i.e., the LDL-receptors which in turn are involved in receptor-mediated endocytosis (25,27). “The reason why LDL [or, in the present case, the LDL-binding ‘LCM’ structure] is of special interest as a carrier for cytotoxic drugs is that leukemic cells isolated from patients with acute myelogenous leukemia have much higher LDL receptor activities than normal white blood cells and nucleated bone marrow cells (28). Gynecologic cancer cells also possess high LDL receptor activity both when assayed in monolayer culture and in membrane preparations from tumor-bearing nude mice (29). Recently, an enhanced receptor-mediated uptake of LDL by tumor tissue in vivo was demonstrated in an animal model (30)” and, subsequently, by solid tumors in vivo in humans (31). Accordingly, LDL particles have actually already been used “as a carrier for toxic compounds in order to kill tumor cells with high LDL receptor activity (32,33)” (34).
Such enhanced LDL receptor-mediated uptake of LDL particles, and also of LCM due to their bound apo B-100, by tumor tissue may therefore explain the extreme rapidity and high selectivity of the LCM accumulation observed within tumors (7), as compared to findings with liposomes. “Most 9L and C6 tumor cells appear to have LCM in their cytoplasm 2 min after LCM administration, while only a small proportion of sterically stabilized liposomes appears to be present intracellularly even after 24 hr of administration (35,36)” (7). Similarly, “When sterically stabilized liposomes were used to deliver encapsulated doxorubicin to tumors, it was shown that the maximum tissue accumulation of the drug occurs at 24 hours (36). In contrast, our data suggest LCM to be rapidly removed from the brain circulation by tumor cells. The maximum accumulation of LCM in the tumor area occurs within the first 30 min after administration, and LCM can no longer be detected in the same region or in any brain region after 6 hours” (7).
These rapid kinetics for LCM uptake are quite consistent with the well-documented kinetics long-known for the LDL receptor-mediated endocytic pathway (27). Goldstein, Anderson & Brown reported as follows: “LDL-ferritin bound to coated pits at 4°C is rapidly internalised when the fibroblasts are warmed to 37°C (37). In this process the coated pits invaginate to form coated endocytic vesicles. After 5 to 10 min at 37°C, LDL-ferritin is seen in lysosomes as the result of their fusion with the incoming coated vesicles (37). The rapid sequence of events visualised in [electron micrographs] precisely parallels biochemically derived data on the rapid uptake and degradation of [radiolabeled] LDL” (38,39).
Several published sonographic and histological studies have demonstrated, in rats, the selective affinity of intravenously administered LCM for tumors. Analysis of the tumor by confocal laser scanning microscopy revealed that labeled LCM were inside the tumor cells. The time course of internalization, the temperature dependency of the process, and other data all suggested an endocytic pathway. The identity of this pathway as well as the molecular basis of LCM affinity for tumor cells may now be deducible by comparing the structure of natural (surfactant-stabilized) microbubbles, after which LCM were closely modeled, with the known composition of plasma LDL. LCM, natural microbubbles, and LDL particles all resemble each other in lipid content; furthermore, there is considerable similarity in amino acid content between the bound protein material on natural microbubbles and the bound protein (essentially all apo B-100) on LDL particles. Based on these similarities, it appears likely that injected LCM can bind apo B-100 in the bloodstream. (Related data, not included in the present review, further suggest that injected LCM also bind apolipoprotein E (apo E) and/or lipoprotein lipase (LPL) in the blood (57).) Apo B-100 is known to mediate LDL binding to cellular LDL receptors, and it is widely reported that many tumor cells show increased LDL receptor expression and activity. Consequently, the proposed binding of plasma apo B-100 by LCM could influence the biodistribution of those LCM, because of the increased LDL receptor-mediated endocytosis occurring within the tumor tissue. Enhanced receptor-mediated uptake of LCM by tumor tissue may, therefore, explain the marked rapidity and high selectivity of LCM accumulation in tumors (40), as compared to findings with liposomes.
In addition to all the above considerations relating to the LDL receptor, it should also be noted that one other of the known LDL receptor gene-family members may well participate in this receptor-mediated uptake of LCM by tumor tissue (cf. 57). For example, apo E and LPL binding to various lipid particles (as similarly proposed above for LCM) is widely reported to facilitate the uptake of the lipid particles [e.g., apo E-enriched B-VLDL (41) or LPL-enriched VLDL, B-VLDL, and chylomicrons (41,42)] by a large "multi-ligand" endocytic receptor known as "LDL receptor-related protein" or LRP (43).
Further, LCM could likely bind (as do LDL with high affinity) to another multi-ligand receptor type, the "macrophage scavenger receptors" (43). This group of receptors could well account for the observed preferential uptake of LCM by neuro-injury sites (44-46) as well [-- since macrophage infiltration/proliferation is significantly increased in response to neuro-injury in the CNS (44-46)]. Similarly, these "macrophage scavenger receptors" participate in the pathogenesis of neuronal degeneration observed in aging and Alzheimer's disease (47-51), and accordingly such scavenger receptors have been identified in the literature as potential therapeutic targets in Alzheimer's disease (48,49)(as well as in atherosclerotic lesions (52-56)). [For further discussion and additional data regarding the above conclusions, see Chapter 14 of ref. 57.]