I. Introduction

Top Previous Next References

Macrophages are widely distributed and strategically placed in many tissues of the body to recognize and clear altered and senescent cells, invading particulates, as well as macromolecular ligands via a multitude of specialized plasma membrane receptors (Gordon, 1995). This propensity of macrophages for endocytosis/phagocytosis of foreign particles in the past has provided an opportunity for the efficient delivery of therapeutic agents to these cells with the aid of colloidal drug delivery systems (usually in the form of liposomes, polymeric nanospheres, micelles, and oil-in-water emulsions), following parenteral administration (Poznansky and Juliano, 1984). For example, macrophages that are in contact with blood or lymph serve as sites of proliferation for certain microbes during some or all of the infection process. Numerous logical strategies for using colloidal drug carriers in the treatment of certain infectious diseases involving macrophages have been developed, based on analysis of the pathogenesis of microbial diseases (Alving, 1988; Agrawal and Gupta, 2000). For instance, the proposed lysosomotropic-parasitotropic process for delivery of liposome-encapsulated drugs to Leishmania within macrophages involves uptake of the phagocytosed liposome and delivery to a lysosome, fusion of lysosome with the parasitophorous vacuole, and delivery of the liposome to a lysosome within the parasite (Alving, 1988). Ambisome (Gilead, Boulder, CO), a liposomal formulation of amphotericin B, is an example of a world-wide marketed drug delivery product available for treatment of visceral leishmaniasis or confirmed infections caused by specific fungal species.

Some pathogens have also evolved ways of evading intracellular killing to survive within the cell; one example is by escaping the phagosome, which is followed by direct entry into cytoplasm (Provoda and Lee, 2000). To allow further cytoplasmic delivery, new strategies have been developed. A classical example is the use of a pH sensitive liposome (Horwitz et al., 1980). Such vesicles maintain stable phospholipid bilayers at neutral pH or above but destabilize and become fusion-competent at the acidic pH of endosomes and subsequently release their entrapped contents first into endosomes and then into cytoplasm. However, the efficiency of cytoplasmic delivery has further been enhanced by employing natural mechanisms that breach the endosomal membrane once the liposomal contents are delivered into the endosomal lumen. For example, Lee et al. (1996) have adopted a strategy used by a facultative intracellular pathogen (Listeria monocytogenes) that mediates bacterial passage from the phagosome into cytosol. After being internalized into phagosomes, L. monocytogenes permeabilizes phagosomal membranes with the aid of listeriolysin O to enter the cytosolic space of host cells. Indeed, coencapsulation of listeriolysin O along with protein-based antigens in liposomes resulted in efficient delivery of protein antigens to the cytosolic pathway of antigen processing and presentation in macrophages. This approach seems highly desirable in vaccine production to polysaccharide antigens that are poorly immunogenic. Similarly, breaching of the endosomal membrane is also critical for survival of DNA after delivery to macrophages.

Metabolic processes within the mature macrophage have also served as targets for drug carriers. These included degradation of accumulated macromolecules within lysosomes (storage diseases) and iron overloading (Poznansky and Juliano, 1984). Apart from therapeutic goals, colloidal carriers have proved to be useful for diagnostic purposes, for example, to assess macrophage phagocytic and clearance functions. Similarly, particulate colloids tagged with a suitable radiopharmaceutical or contrast agent were shown to be helpful for imaging certain pathologies (e.g., deep-seated tumors) not via targeting but through macrophage loading (Seltzer, 1989; Tilcock, 1995; Kostarelos and Emfietzoglou, 1999). By this approach, it is the surrounding parenchyma, and not the pathology, that will change in intensity.

The rapid sequestration of intravenously injected colloidal particles from the blood by hepatic midzonal and periportal Kupffer cells is problematic for efficient targeting of drug carriers or diagnostic agents to a desired macrophage population (e.g., splenic red pulp macrophages) as well as to nonmacrophage sites. As a result, there has been a growing interest in the engineering of colloidal carrier systems that upon intravenous injection avoid rapid recognition by Kupffer cells and adequately remain in the blood. It is the aim of this article to critically discuss and evaluate the developments in the field of long-circulating and target-specific colloidal carrier systems for intravenous administration (and in selected cases with regard to subcutaneous injection). We have viewed the application of long-circulating particles in sustained-release of drugs within vasculature, transfusion medicine (artificial erythrocytes), and targeting. The latter is defined either as passive or active. With regard to passive targeting, this article identifies and discusses the opportunities afforded by certain physiological and pathological conditions that are amenable for treatment or diagnosis by using long-circulating colloidal carriers. In relation to active targeting, attention is focused toward identification of potential and accessible tissue-specific antigens and their ligands. For particle engineering, we have taken a lead from nature. That is, exploring the surface mechanisms that afford red blood cells long-circulatory lives and the ability of certain pathogenic microorganisms to evade macrophage recognition. Our analysis is centered where such strategies have been translated and fabricated to design a wide range of particulate carriers (e.g., nanospheres, liposomes, micelles, oil-in-water emulsions) with prolonged circulation and target specificity. Throughout our approach, we critically evaluate these engineered systems on the basis of biodistribution, target specificity, therapeutic end-points, biological activities, and toxicity.

	II. Theoretical Applications of Long-Circulating Particulate Carriers in Experimental and Clinical Medicine

Top Previous Next References

Similarly, one can also foresee long-circulating nanoparticles as carriers of radiopharmaceuticals or contrast agents for use in the imaging of vasculature. These may include blood-pool imaging, detection of vascular malformations, and gastrointestinal bleeding. Technetium-99m-labeled red blood cells are used routinely in nuclear medicine for blood-pool imaging, but technetium-99m is not strongly bound to the red cell and dissociates, resulting in the clearance of free technetium through the kidney and bladder (Phillips, 1998). This dissociation greatly interferes, for example, with the detection of sites of bleeding in the lower abdomen. This, together with the potential hazards associated with blood handling, justifies efforts for the development of synthetic long-circulatory particles in nuclear medicine.

The unique structural changes associated with a given vascular pathophysiology could also provide opportunities and insights for the use as well as engineering of long-circulating particulate carrier systems. For example, particle escape from the circulation is normally restricted to sites where the capillaries have open fenestrations as in the sinus endothelium of the liver (Roerdink et al., 1984) or when the integrity of the endothelial barrier is perturbed by inflammatory processes (e.g., rheumatoid arthritis, infarction, infections) (Turner and Wright, 1992) or by tumors, although such defects are not a consistent feature of all tumors (Jain, 1989; Hobbs et al., 1998). In the liver, the fenestrae-associated cytoskeleton controls the hepatic function of endothelial filtration, where the size of fenestrae can be as large as 150 nm (Braet et al., 1995). The size of fenestrae in certain inflammatory vessels as well as tumor capillaries can be up to 700 nm. Currently, there is evidence in support of liposome extravasation to hepatic parenchyma (Roerdink et al., 1984) as well as increased capillary permeability to liposomes and polymeric nanospheres (in the size range of 50-200 nm) during inflammation (Boerman et al., 1997a,b; Dams et al., 1998, 1999, 2000b) and in specific cancers (Poznansky and Juliano, 1984; Yuan et al., 1994; Hobbs et al., 1998; Drummond et al., 1999) based on observations in experimental animals and in humans. However, the efficiency and kinetics of particle delivery varies from one model to another. Particles of less than 10 nm can also leave the systemic circulation through the permeable vascular endothelium in lymph nodes (Wiessleder et al., 1994; Moghimi and Bonnemain, 1999). The sinus endothelium of bone marrow is also capable of removing small-sized particles from the systemic circulation. Here, the sinus endothelium can remove particles from the circulation by both transcellular and intracellular routes (Moghimi, 1995a). The transcellular route is through the diaphragmed fenestrae of endothelial walls, whereas the intercellular route is associated with the formation of bristle-coated pits containing matter on the luminal surface of the endothelium. However, regional dilation increased branching of sinuses, and even complete loss of bone marrow sinus endothelium can occur in certain pathological conditions thus resulting in particulate accumulation in affected areas. For instance, marked fragmentation of endothelial cytoplasm, as well as complete loss of the endothelium of bone marrow sinusoids, have been reported for dogs infected with a canine hepatitis virus (Linblad and Bjorkman, 1964).

Certainly one would expect this because of the prolonged residence in blood, long-circulating carriers with the appropriate sizes have a better chance of reaching the above-mentioned targets, resulting in improved treatment or diagnosis. Another topic that deserves investigation is the use of a tissue-specific pharmacological mediator that, by opening intercellular connections in selected vessels, may facilitate long-circulating colloidal entities to escape from the circulation and extravasate into the tissues of organs whose postcapillary endothelia contain receptors for the mediator (Rosenecker et al., 1996). In spite of the preceding evidence, unless the means are found to actively control local permeability and access, the issue of tissue selectivity is not properly addressed by the use of engineered nanoparticulate complexes, and significant therapeutic gain is unlikely.

Given the potency (and toxicity) of modern pharmacological agents, tissue selectivity is a major issue. In the delivery of chemotherapeutic agents to solid cancers, this is particularly critical, since the therapeutic window for these agents is often small and the dose-response curve is steep. Therefore, the idea of exploiting the well documented vascular abnormalities of tumors, restricting penetration into normal tissue interstitium while allowing freer access to that of the tumor, becomes particularly attractive. This has been the subject of considerable research; however, the data suggest that the solution to this problem will depend upon more than simply reducing mechanical barriers to trans-endothelial passage. Let us examine the idea of passive targeting more closely with regard to the process of extravasation of long-circulating particles from the blood to a solid tumor, a target that has received the most attention in the drug delivery field. A solid tumor comprises two major cellular components. These are the tumor parenchyma and the stroma, which incorporates the vasculature and other supporting cells. To meet the metabolic requirements of the expanding population of tumor cells, the pre-existing blood vessels become subject to intense angiogenic pressure (Folkman, 1995). Several factors produced by tumor cells are believed to signal the development of new capillaries from these vessels (angiogenesis). Scanning electron microscopy of microvascular corrosion casts has allowed visualization of the geometry of tumor architecture. From these studies, it has become apparent that tumor vessels are highly irregular and show gross architectural changes (e.g., the presence of interrupted endothelium and an incomplete basement membrane) with increased blood vessel tortuosity as well as abnormal and heterogeneous vessel density (Jain, 1988). As a consequence of these abnormalities, there may be profound physiological changes in blood flow within tumors and in transport properties of the tumor vessel. Due to the vascular heterogeneity of solid tumors, the blood flow, and consequently tissue oxygenation, tends to be nonuniform. Therefore, solid tumors usually contain well perfused, rapidly growing regions, and poorly perfused, often necrotic areas (Jain, 1988). As in normal tissues, diffusive and convective forces govern the movement of molecules into the interstitium of tumors. However, diffusion is believed to play a minor role in the movement of solutes across the endothelial barrier compared with bulk fluid flow. Examination of pressure gradients in experimental tumors has suggested that the movement of drugs and particulates out of tumor blood vessels into the extra-vascular compartment is remarkably limited (Jain, 1989). This has been attributed to a higher-than-expected interstitial pressure, in part due to a lack of functional lymphatic drainage, coupled with lower intravascular pressure. In addition, interstitial pressure tends to be higher at the center of solid tumors, diminishing toward the periphery, creating a mass flow movement of fluid away from the central region of the tumor.

These pathophysiological characteristics have serious implications for the systemic delivery of not only low-molecular weight drugs and macromolecules (e.g., antibodies, polymer conjugate therapeutics), but also particulate delivery vehicles (e.g., liposomes), and simply enhancing the plasma half-life of these agents will not necessarily lead to an increase in therapeutic effect. However, it is likely that long-circulating particles may have a better safety profile in terms of normal tissue toxicity than free anticancer agents, as well as anticancer drugs encapsulated in phagocyte-prone carriers. The utility of conventional colloidal carriers as vehicles for drug delivery in cancer treatment is inappropriate since the class of drug being used is able to induce apoptosis in macrophages of the RES (Daemen et al., 1995). In the liver, restoration of Kupffer cells may take up to periods of 2 weeks (Daemen et al., 1995). A potentially harmful effect is that bacteriemia may occur during the period of Kupffer cell deficiency (Daemen et al., 1995).

The ability to target drugs and gene therapeutics to nonmacrophage cells within the vasculature has been one of the most sought after goals in clinical therapeutics. Therefore, attachment of specific (but not macrophage-recognizable) ligands onto the surface of macrophage-evading carriers will open the possibility of targeting specific cell types or subsets of cells within the vasculature and even elements of vascular emboli and thrombi. One example is the abnormal lymphocyte differentiation antigens on leukemia cells (Raso et al., 1982), which could serve as target molecules for ligand-directed targeting of macrophage-evading carriers containing anticancer agents. Another important target is vascular endothelial cells. The vascular endothelium is remarkably heterogeneous; endothelial cells from different tissues of the body differ in expression of surface antigens and receptors (Kumar et al., 1987; Belloni and Nicolson, 1988; Rajotte et al., 1998; Thorin and Shreeve, 1998). Endothelial cells also display phenotypic changes in response to various cytokines and growth factors (Pober and Cotran, 1990; Bevilacqua, 1993; Kraling et al., 1996; Murray, 1997). For example, ELAM-1 (E-selectin) is not detectable on the surface of quiescent endothelial cells of normal vessels, but is strongly up-regulated on the luminal surface by pro-inflammatory cytokines such as IL-1 and TNF- (Lin et al., 1992). E-selectin induction on endothelial cells is clearly associated with inflammatory and immune processes (Pober and Cotran, 1990) as well as some noninflammatory angiogenic states (Kraling et al., 1996). E-selectin is known to be involved in the arrest of monocytes, neutrophils, and subsets of T-lymphocytes within the circulation. Therefore, it is not surprising that this molecule has become a potential target for several strategies designed to enhance delivery of therapeutic agents to, or to improve imaging of, particular vascular beds. For example, Wickham et al. (1997) have demonstrated that the enhanced expression of E-selectin may be used as a means to target entry of adenoviral vectors into endothelial cells. A bi-specific antibody, which reacts with both E-selectin and with a "FLAG" epitope genetically incorporated into one of the adenoviral coat proteins, was able to direct binding and entry of the adenovirus into endothelial cells pretreated with the TNF- but not into quiescent nonstimulated cells. Another antibody against E-selectin, 1.2B6, has been used for radionuclide imaging of rheumatoid arthritis in humans (Jamar et al., 1997). In this study, the sensitivity of anti-E-selectin coupled to indium-111 was compared with a nonspecific antibody conjugated with technetium-99m, in terms of their ability to detect synovitis. This study showed that targeting with the E-selectin antibody was more intense and specific than with a nonspecific antibody (Jamar et al., 1997). Furthermore, the anti-E-selectin antibody was able to detect joint abnormalities in areas that were clinically silent.

Biochemical differences between the vasculature of the tumor and the normal tissues are also noteworthy. Indeed, various approaches have been taken to identify differences in luminal protein expression between the endothelial cells of tumor vessels and those of normal tissues. These include two-dimensional gel electrophoresis of cell surface proteins isolated from cultured endothelial cells (P. Hewett and J. C. Murray, manuscript submitted), screening of monoclonal antibodies on tissue sections (Hagemeier et al., 1986; Rettig et al., 1992), in vivo lectin labeling (Belloni and Nicolson, 1988), and the application of bacteriophage display technology in vivo (Rajotte et al., 1998) to study endothelial heterogeneity. These markers, which are usually up-regulated on the surface of tumor-associated endothelial cells, include proteins involved in cell-cell or cell-matrix interactions (e.g., E-selectin, vascular cell adhesion molecule-1, and the _v3 integrin complex) and growth factors receptors (Cheresh, 1987; Matthews et al., 1991; Kim et al., 1993; Brooks et al., 1994; Dumont et al., 1994; Sato et al., 1995; Max et al., 1997; Pasqualini et al., 1997; Charpin et al., 1998; Lauren et al., 1998; Lin et al., 1998; Maurer et al., 1998; Ellerby et al., 1999). The growth factor receptors of potential interest are the vascular endothelial growth factors-2 (KDR-2), which are up-regulated in a wide range of solid tumors (Matthews et al., 1991), and the Tie family of endothelial receptor tyrosine kinases (Tie-1 and Tie-2) (Lauren et al., 1998). Targeted disruption of gene function in murine embryos indicates that Tie-2 plays a pivotal role in both angiogenesis and vascular remodeling/stability, whereas Tie-1 is required for the maintenance of vascular integrity (Dumont et al., 1994; Lin et al., 1998). The angiopoietin growth factors have been identified as the ligands for Tie-2, whereas Tie-1 remains an orphan receptor. Since the development of new blood vessels is essential for tumor growth (Griffioen and Molema, 2000), an attractive antitumor strategy is to exploit these biochemical differences and aim at the tumor vasculature for therapeutic intervention with ligand-bearing long-circulating particles. This strategy might overcome the physiological differences within tumors that have been described as heterogeneous and unpredictable. One must also be cautious with regard to heterogeneity in angiogenic stages in human tumor vasculature. This is because in the animal model, the majority of tumor vasculature is usually in a proangiogenic state, whereas in human tumors, the percentage of proangiogenic vessels is variable and usually low (Griffioen and Molema, 2000). Hence, such antiangiogenic approaches may only affect a minority of the tumor vasculature.

Even if engineered colloidal carriers can be targeted successfully to specific subsets of circulating blood cells or endothelia, subsets may differ significantly in their capacity to internalize particles bound to the cell surface. Therefore, in selected cases, it may be necessary to use particle-immobilized ligands that upon binding and signal transduction initiate local effects (e.g., apoptosis or cytokine production).

	III. Rational Approaches in the Design of Long-Circulating Particles

Top Previous Next References

If monocytes and macrophages in contact with blood are not the desired target, then how can we avoid their phagocytic "barrier"? To date, a classical approach has been to administer large doses of placebo carriers in an effort to impair the phagocytic capacity of macrophages in contact with the blood, thereby allowing subsequently administered particles containing active material to remain in systemic circulation for prolonged periods or to reach designated targets (Abra et al., 1980; Souhami et al., 1981; Moghimi and Davis, 1994). Another strategy is transient apoptotic destruction of liver and spleen macrophages by prior administration of gadolinium chloride (Hardonk et al., 1992) or liposomes with entrapped clodronate (Naito et al., 1996; Schmidt-Weber et al., 1996; van Rooijen et al., 1997). The latter approach has received some attention for the in vivo gene transfer protocols involving adenoviruses (Wolff et al., 1997). These strategies, although successful in experimental models, have little justification in clinical practice as they suppress the essential defense system of the body.

"If one way be better than another, you may be sure it is nature's way" [Aristotle]. It seems appropriate to design a long-circulating carrier based on nature's principles. For example, healthy erythrocytes evade the macrophages of the immune system and fulfill their function of transporting oxygen with a life span of 110 to 120 days. A multitude of physicochemical and physiological factors are believed to control the life span of red blood cells. These include surface characteristics (e.g., surface charge, membrane phospholipid composition, surface antigens) as well as bulk properties (e.g., shape and their extent of deformability) (Weiss and Tavassoli, 1970; Schnitzer et al., 1972; Chen and Weiss, 1973; Moghimi, 1995b; Oldenborg et al., 2000). For instance, red blood cells may avoid macrophage surveillance with the protection of a barrier of oligosaccharide groups. Furthermore, their deformable nature allows them to bypass the human splenic filtration process at the IES in the walls of venous sinuses. In the same context, two decades ago Densen and Mandell (1980) pointed out, "it is naive to assume that microbes passively accept their fate at the hands of phagocytes". In fact, for the successful growth, certain pathogens have deployed a clever array of surface strategies to avoid phagocytosis by macrophages (Ram et al., 1998; Alcami and Koszinowski, 2000). Perhaps we should also learn to translate feasible microbial surface strategies in the engineering of long-circulating or macrophage-evading nanoparticles.

A. The First Few Steps

1. Physicochemical Characteristics of Nanoparticles and Their Effect on Protein Adsorption and Opsonization. It has been repeatedly emphasized that the clearance behavior and tissue distribution of intravenously injected particulate drug carriers are greatly influenced by their size and surface characteristics (Poznansky and Juliano, 1984; Patel, 1992; Moghimi and Davis, 1994). These physicochemical parameters can control the degree of particle self-association (Ahl et al., 1997) in the blood as well as particle opsonization in biological fluids. The size of a particle may change substantially upon introduction into a protein-containing medium (e.g., plasma). Therefore, in the blood, particles and their aggregates should be small enough so that they are not removed from the circulation by simple filtration in the first capillary bed encountered (e.g., rat or mouse lung following tail vein injection). The opsonization process is the adsorption of protein entities capable of interacting with specific plasma membrane receptors on monocytes and various subsets of tissue macrophages, thus promoting particle recognition by these cells (Chonn et al., 1992; Moghimi and Davis, 1994; Gref et al., 1995; Moghimi and Patel, 1998; Moghimi and Hunter, 2000a). Classical examples of opsonic molecules include various subclasses of immunoglobulins, complement proteins like C1q and generated C3 fragments (C3b, iC3b), apolipoproteins, von Willebrand factor, thrombospondin, fibronectin, and mannose-binding protein (Absolom, 1986; Patel, 1992; Serra et al., 1992; Chonn et al., 1995; Moghimi and Patel, 1996; Szebeni, 1998). On exposure to blood, particles of differing surface characteristics, size, and morphology attract different arrays of opsonins as well as other plasma proteins, the content and conformation of which may account for the different pattern in the rate and site of particle clearance from the vasculature (Moghimi and Patel, 1998). Since opsonization plays a major role in particle clearance from the blood, then interindividual variations in blood opsonic activity and concentration must also be considered. Undoubtedly, a clear understanding of such events is the first rational step for the design of colloidal carriers that target not only a relevant macrophage population (see Section III.A.2.) but also for the engineering of long-circulating or macrophage-evading particles. It should also be emphasized that the interaction of particles with blood protein may have effects beyond opsonization. These may include interference with the blood-clotting cascade, a process that may lead to fibrin formation, and anaphylaxis because of complement activation.

2. Macrophage Heterogeneity, Physiological Status, and Species Differences. It is important to realize that not all macrophages and monocytes are identical; considerable heterogeneity with respect to phenotype and physiological properties (e.g., phagocytosis) exist between different types of phagocytes and even among phagocytic cells of the same tissue (Gordon et al., 1992; Naito, 1993; Rutherford et al., 1993). For example, rat Kupffer cells are heterogeneous with regard to phagocytosis and accessory functions such as cytokine production, tumoricidal capability, and antigen presentation to T cells (Sleyster and Knook, 1982; Hoedemakers et al., 1994). Kupffer cells in the periportal area are larger and have a higher lysosomal enzyme activity (on a per cell basis as compared with cells in other regions of the liver), which reflects their higher level of phagocytic and scavenging activities than those of central macrophages. On the other hand, macrophages of the pericentral region are more active in cytokine production and have a higher tumoricidal capability than the larger periportal Kupffer cells (Hoedemakers et al., 1994). With regard to phagocytic receptors, human Kupffer cells and splenic macrophages serve as good examples, Table 1 (Buckley et al., 1987; Tomita et al., 1994). For instance, in human liver the expression of the IgG-coated particle receptor (FcRIIIA) is restricted only to Kupffer cells located in the central area of the liver lobule (Tomita et al., 1994). In murine, macrophages found within the marginal zone, which surrounds the lymphoid regions of the spleen, differ in morphology, endocytosis, and receptor expression from those in the red pulp. For example, unlike other splenic macrophages, the marginal zone macrophages express a specific scavenger receptor (known as MARCO) that is distinct from type 1 and type 2 scavenger receptors (Ito et al., 1999). It is therefore conceivable that the particular populations of macrophages may respond differently to particulate delivery systems and, hence, employ one particular recognition mechanism. Therefore, understanding of macrophage properties, heterogeneity, and recognition mechanisms could provide new insights and opportunities for the design of long-circulating particles as well as carriers that can selectively deliver drugs and therapeutic materials to particular macrophage subpopulations (e.g., for optimizing delivery to recruited monocytes versus local resident macrophages, and selective and apoptotic ablation of macrophages serving as reservoirs of infectious agents, such as HIV and tuberculosis). Here, consideration must also be given toward the physiological status of macrophages of the RES. A responsive phagocyte can become primed and later selectively activated for one or more functions (Adams and Hamilton, 1992). Examples of such functions include enhanced phagocytosis, chemotaxis, antigen processing and presentation, and destruction of tumor cells. The capacity to accomplish a complex function is dependent, in turn, upon acquisition of the requisite capacities such as enhanced mobility of a given population of plasma membrane receptors for a given chemotactic agent or an immobilized opsonin, increase in the number or selective expression of a particular group of scavenging receptors (e.g., the expression of plasma membrane FcRIA which is restricted to human Kupffer cells at the sites of inflammation and is apparently unrelated to the type of liver disease), and the amount of mediators secreted etc. There are strong indications that activated macrophages can recognize engineered long-circulating particles from the blood (Moghimi et al., 1993a; Moghimi and Murray, 1996; Schmidt-Weber et al., 1996; Moghimi and Gray, 1997) (discussed under Section VII.). Hence, such long-circulating particles may act as vehicles for delivery of therapeutic or diagnostic agents to such macrophage populations.

3. Splenic Filtration. The size and the deformability of particles plays a critical role in their clearance by the sinusoidal spleens of humans and rats. Particles must be either small or deformable enough to avoid the splenic filtration process at the IES in the walls of venous sinuses (Chen and Weiss, 1973; Moghimi et al., 1991). The IES in sinusoidal spleens provides resistance to flow through the reticular meshwork. The endothelial cells of the sinus wall have two sets of cytoplasmic filaments: a set of loosely associated tonofilaments and a set of filaments tightly organized into dense bands in the basal cytoplasm containing actin and myosin, which can probably vary the tension in the endothelial cells and, hence, the size of IES (Drenckhahn and Wagner, 1986). However, the slit size rarely exceeds 200 to 500 nm in width, even with an erythrocyte in transit (Chen and Weiss, 1973). Hence, retention of blood cells and blood-borne particles at the IES depends on their bulk properties, such as size, sphericity, and deformability. These cell slits are the sites where erythrocytes containing rigid inclusions (e.g., Heinz bodies, malarial plasmodia) are believed to be "pitted" of their inclusions, which are eventually cleared by the red pulp macrophages (Weiss and Tavassoli, 1970; Schnitzer et al., 1972; Groom, 1987). Idealy, the size of an engineered long-circulatory particle should not exceed 200 nm. If larger, then the particle must be deformable enough to bypass IES filtration. Alternatively, long-circulating rigid particles of greater than 200 nm may act as splenotropic agents (Moghimi et al., 1991).

4. Confinement to Vasculature. Earlier we discussed the possibility of particle extravasation from the blood to selected sites in the body (Section II.D.). However, if we intend to keep long-circulating particles within the vasculature, as in certain cases (e.g., a circulating drug reservoir), then a lower size window in the design of long-circulating particles must also be considered. There is a clear-cut relationship between particle size (e.g., as in the case of liposomes) and the extent to which they reach the hepatic parenchyma. For instance, it has been demonstrated that the smaller the liposomes (usually those of below 100 nm in diameter), the larger the contribution of hepatocytes in total hepatic uptake (Roerdink et al., 1984). This is probably a reflection of the size of the fenestrations in the hepatic sinusoidal endothelium, which are ~100 to 150 nm in diameter (Braet et al., 1995). Even highly deformable vesicles of up to 400 nm in diameter can also reach hepatocytes after intravenous injection by a process of "extrusion" through endothelial fenestrations (Romero et al., 1999). Therefore, as a rough approximation, the size of long-circulating particles, providing that they are rigid structures, should be in the range of 120 to 200 nm in diameter to substantially avoid particle trapping in space of Disse and hepatic parenchyma.

	IV. Translation of Microbial and Related Mammalian Technologies to Nanoparticle Engineering

Top Previous Next References

Considerable information has become available from pathogenic microorganisms that employ various surface strategies to avoid recognition by macrophages. For example, mucoid strains of Pseudomonas aeruginosa from patients with cystic fibrosis display a polyuronic acid polysaccharide that interferes with phagocytosis by virtue of its hydrophilicity (Cabral et al., 1987). The external envelope glycoproteins of HIV type-1 and its simian counterpart are heavily glycosylated. Recent studies have demonstrated that these carbohydrates form a barrier to help shield the virus from immune recognition and limit effective antibody responses to the virus (Reitter et al., 1998). A major constituent of various microbial envelope glycoproteins is sialic acid (Ram et al., 1998). Such sialyated proteins favourably bind factor H (Meri and Pangburn, 1994). The binding of factor H contributes to pathogenicity by inhibiting complement-mediated destruction; factor H acts as a cofactor for factor I-mediated cleavage of C3b and also inactivates alternative pathway convertase by dissociating Bb from the C3b, Bb complex (Fearon and Austen, 1977; Sim et al., 1981). Examples of factor H binding proteins include the streptococcal M6 protein (Fischetti et al., 1995), the Yad A protein of Yersinia enterocolitica (China et al., 1993), and also the envelope glycoproteins 120 and 41 of HIV-1 (Stoiber et al., 1996; Reitter et al., 1998). Microorganisms such as certain species of Staphylococcus aureus fix complement in a cryptic subcapsular location inaccessible for ligation by complement receptors. Eukaryotic infectious agents, such as Candida albicans and related species, subvert C3-mediated opsonization processes by expressing a surface protein that is antigenically, structurally, and functionally related to the mammalian integrin CR3 and p150,95 (Heidenreich and Dierich, 1985). Several viruses express complement regulatory proteins to reduce complement attack (Alcami and Koszinowski, 2000).

Can coating of synthetic nanoparticles with a hydrophilic microbial polysaccharide, factor H, or a factor H ligand fend off the body's defense system? Can such constructs release their entrapped materials in a controlled manner within the systemic circulation or at the desired pathological sites? And, finally, what are the toxicological variables inherent with these types of systems? One of the earliest attempts undertaken was the coating of liposomes with linear dextrans, which are polymers of -D-glucose units (Pain et al., 1984). Linear dextrans are produced by the fermentation of sucrose with a strain of the bacterium Leuconostoc mesenteroides followed by hydrolysis and fractionation to give dextrans with different average molecular weights. Linear dextrans have frequently been used as plasma expanders in medicine; they remain in the systemic circulation for extended periods of time which are proportional to their molecular weights. The circulatory persistence of drugs (e.g., doxorubicin) and proteins such as carboxypeptidase G, superoxide dismutase, and arginase have all been increased following conjugation to dextran (Melton et al., 1987a,b; Wileman, 1991). Liposomes coated with dextran (molecular weight of 70,000) were also more efficient in retaining the entrapped radioactive markers within the circulation than uncoated liposomes (Pain et al., 1984). The rate of clearance of dextran-coated liposomes was dependent on the density of dextran molecules on the liposome surface. The steric brushes of the dextran macromolecules are believed to reduce protein adsorption, resulting in enhanced stability of liposomes in the blood (Pain et al., 1984). Perhaps, one of the most successful applications of dextran in nanoparticulate engineering is the development of ultrasmall superparamagnetic iron oxide particles, which are used in magnetic resonance imaging (Bengele et al., 1994; Guimaraes et al., 1994; Wiessleder et al., 1994; Moghimi and Bonnemain, 1999). These particles consist of hexagonally shaped iron cores of 4 to 5 nm in diameter surrounded by 20 to 30 hydrated "brush-like" structures composed of dextran molecules. By laser light scattering, such crystals exhibit a unimodal hydrodynamic radius of 20 to 25 nm. Because of their physical properties (small size and hydrophilic nature), iron oxide particles are poorly recognized by Kuppfer cells and splenic macrophages and, concomitantly, exhibit prolonged circulation in the blood with a half-life of 3 to 4 h (Moghimi and Bonnemain, 1999). The eventual macrophage recognition of such particles may be attributed to gradual opsonization with antidextran antibodies and complement activation. These partricles have found clinical applications in lymph node imaging (see Section VIII.C.).

Another example is the bacterial exopolysaccharide pullulan (an -D-glucan). Cholesterol-pullulan conjugates with various degrees of substitution as well as monoalkyl(hexadecyl)-pullulan have all been shown to confer some degree of protection to the liposome surface and moderately reduce vesicle uptake by both the liver and spleen macrophages in vivo (Kang et al., 1997). The capsular polysaccharide of N-acetyl neuraminic acid (polysialic acids) from Escherichia coli K1 and K92, Neisseria meningitidis B and C as well as their shorter chain derivatives should also be considered for nanoparticle surface engineering. Indeed, preliminary experiments have confirmed that long-chain polysialic acids exhibit prolonged circulation half-lives (Gregoriadis et al., 1993; Fernandes and Gregoriadis, 1997); due to their high hydrophilicity, little or no binding occurs between factor H and polysialic acids (Pangburn et al., 1991; Meri and Pangburn, 1994). Recently, successful attempts were made to increase the half-life of asparaginase following coupling to such polysialic acids (Fernandes and Gregoriadis, 1997). Theoretically, conjugation of liposomes or nanospheres to polysialic acids could be carried out by a variety of methods, depending on the reactive groups available on the interacting entities. However, caution is required for utilizing polysialic acids for human use. This is because coupling reactions could potentially alter or damage the tertiary structure of the long-chain polysialic acids and, hence, affect the clearance patterns of the engineered colloids. The bacterial polysialic acids C and K92 are immunogenic in humans whereas polysaccharide B and K1 are T-independent antigens and do not induce immunological memory (Moreno et al., 1985). However, it is possible that polysialic acids B and K1 could become T cell dependent antigens and induce memory when grafted to nanocarriers. Another concern is the possible antigenicity of polysialic acids; low levels of antibodies against polysialic acids exist in the circulation (Mandrell and Zollinger, 1982). Macrophages as well as other myeloid cells express several sialic acid binding receptors such as sialoadhesin, CD33, and siglec-5 (Crocker et al., 1991; Munday et al., 1999). The sialic acid-binding proteins are not phagocytic receptors (Munday et al., 1999). However, they could cooperate with phagocytic receptors to increase the efficiency of recognition and uptake, but they function primarily as ligands in cellular recognition events and signaling. Therefore, it will be of interest to determine whether interactions between sialic acid binding proteins and polysialic acids influence host defense functions.

Other successful approaches might be to tag drug carriers with complement regulatory proteins or specific complement inhibitors. Several novel complement inhibitors and chimeric or modified human complement regulatory proteins are currently available for pursuing such studies. Perhaps one of the easiest approaches is to enrich the surface of particulates with factor H ligands. For example, factor H has the ability to bind to polyanions such as heparin, and most sulfated glycosaminoglycans, to include dextran sulfate and chondroitin sulfate A (but not chondroitin sulfate C, keratan sulfate, hyaluronic acid, or polyaspartic acid) (Pangburn et al., 1991; Meri and Pangburn, 1994). It should be emphasized that the interaction between factor H and polyanions is specific and may depend upon the number, orientation, and polymeric arrangement of the anionic groups on nanoparticle surface. Recently, Passirani et al. (1998) covalently attached heparin to the surface of monodispersed poly(methyl methacrylate)-based nanoparticles with a mean diameter of 160 nm. Following intravenous injection into mice, heparin-coated nanoparticles exhibited an initial phase of elimination from the blood with a half-life of 5 h, the remaining heparin nanoparticles circulated for ~48 h. The prolonged circulation time of heparin nanoparticles was suggested to arise from the inhibition of complement activation (Passirani et al., 1998). Although, complement inhibition was only demonstrated in human serum, it is unlikely that the inhibition of complement activation in mice can explain the prolonged circulation time of nanoparticles and their poor hepatic localization. In the event of complement activation, resident murine Kupffer cells lack complement receptors with scavenging functions (Lepay et al., 1985; Lee et al., 1986; Gordon et al., 1992). The prolonged circulation time of nanoparticles in mice is most likely due to their high degree of surface hydrophilicity. It is also likely that the vascular endothelial cells may eventually play a critical role in the clearance of heparin-coated particles because these cells possess heparin receptors, although species variation must be considered (Patton et al., 1995).

Heparin-like polysaccharides can also be obtained from microorganisms. For example, E. coli serotype K5 synthesizes a form of a relatively low molecular mass (~50 kDa) desulfatoheparin (Vann et al., 1981). This molecule has repeating units of 4--D-glucuronosyl-1 and 4--N-acetyl-D-glucosamine, which is similar to N-acetylheparosan, a biosynthetic precursor of heparin. The bacterial product resembles type II glycosaminoglycuronan chains, which are synthesized in the Golgi complex of eukaryotes and then joined to core proteins. Unlike the eukaryotic products, the bacterial polymer lacks L-iduronosyl and sulfate residues. Because of its structural similarity to heparin and the abundance of the negatively charged glucronic acid, these heparin-like molecules may be worthy of particle-surface modification. A related and successful strategy has been the grafting of polyglucuronide to a liposome (100 nm) surface that conferred some degree of vesicle invisibility to macrophages of the RES of both mice and rats (Namba et al., 1992; Oku et al., 1992; Oku and Namba, 1994). For example, at 22 h after intravenous administration into rats, the ratios of the total amount of liposomal associated radioactive marker in the liver and the spleen to that in the blood were 2.4 and 6.3 for liposomes containing 20 mol% palmitylglucuronide and control formulation, respectively.

Sialic acid is an essential component of eukaryotic cell surfaces that plays an important role in preventing destruction of host tissue by constant low-grade activation of the alternative pathway. This is evident from desialyation of erthyrocyte membranes, which results in their conversion from nonactivators to activators of the alternative complement pathway due to reduction in factor H binding (Pangburn and Muller-Eberhard, 1978; Kazatchkine et al., 1979). Surolia and Bachhawat (1977) conducted an elegant approach to rendering liposomes invisible to macrophages by mimicking the mammalian cell surface. Here, the liposome surface was enriched with sialic acid following the incorporation of cell derived glycolipids into the liposomal bilayer. This work initiated a major breakthrough in the liposome field; since then, various modifications of vesicles with gangliosides (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Allen et al., 1989; Chonn et al., 1992; Liu et al., 1995), ganglioside derivatives (Park and Huang, 1993; Yamauchi et al., 1993, 1995) and glycophorin (Yamauchi et al., 1993) have been made and investigated. Indeed, sialic acid containing gangliosides (e.g., ganglioside GM1), Fig. 1, when incorporated into liposomes, inhibit the alternative complement pathway as it promotes the binding of factor H to liposome-bound C3b (Michalek et al., 1988). In mice, DSPC or DPPC vesicles of 70 to 200 nm in size, containing high concentrations of cholesterol (>30 mol%) and GM1 at 5 to 7 mol%, behave as long-circulatory (Allen and Chonn, 1987; Gabizon and Papahadjopoulos, 1988; Allen, 1994a). Interestingly, in rats, GM1-containing vesicles are cleared rapidly from the circulation by hepatic Kupffer cells (Liu et al., 1995). The presence of naturally occurring anti-GM1 IgM in rat (but not in mouse) blood, through the activation of the classical pathway of the complement system, is thought to be responsible for the rapid detection of GM1-incorporated vesicles by Kupffer cells (Wassef et al., 1991; Alving and Wassef, 1992; Liu et al., 1995). What mechanisms are therefore responsible for prolonged circulation time of GM1 liposomes in mice? GM1 at 5 to 7 mol% is miscible with both DPPC and DSPC bilayers and forms the most stable vesicles in the presence of cholesterol (Bedu-Addo and Huang, 1996). The stability of the bilayer reduces vesicle susceptibility to lysis by mouse plasma components (e.g., lipoproteins) and to perturbation or penetration by plasma or cell surface proteins. Furthermore, due to the inhibition of complement activation, the assembly of the membrane attack complex at the vesicle bilayer is prevented. Gabizon and Papahadjopoulos (1988) postulated that the negative charge in GM1 is "shielded" by a bulky, neutral, hydrophilic sugar moiety that contributes to macrophage avoidance by decreasing or preventing protein adsorption or opsonization processes. This hypothesis is further supported by the fact that liposomal incorporation of other negatively charged glycolipids, where the negative charge is shielded by neutral sugars (e.g., phosphatidylinositol and sulfatides), also prolongs the blood retention time of the vesicle. Replacing phosphatidylinositol with phosphatidylinositol phosphate, in which an "exposed" charged phosphate is added to the inositol molecule, or replacing GM1 with G_T1, in which two silaic acid molecules with exposed carboxyl groups are present, enhances the hepatic clearance of liposomes (Gabizon and Papahadjopoulos, 1988, 1992). In spite of these experiments, the validity of this hypothesis is still questionable. Several studies have demonstrated that negatively charged glycolipids or phospholipids with exposed and unshielded carboxylic groups such as ganglioside GM3 (Fig. 1) (Yamauchi et al., 1993), [2-(2-palmitoylamido-1-ethyl)-5-acetoamide-3,5-dideoxy-D-glycero--D-galacto-2- nonulipyranoside]onate (a synthetic derivative of sialic acid) (Yamauchi et al., 1995), N-glutaryldioleoylphosphatidylethanolamine, and N-adipyl dioleoylphosphatidyl-ethanolamine (Park et al., 1992)] show a considerable ability to prolong the circulation time of sub 200 nm liposomes in mice. Furthermore, derivatives of GM1 have also been synthesized such that their carboxyl group is either methylated or reduced (Park and Huang, 1993). These derivatives showed considerable ability in preventing hepatic sequestration of liposomes in mice. Reductive amination of a GM1 derivative containing a C7 analog of sialic acid in the presence of -alanine adds to GM1 oligosaccharide an additional carboxyl group, which is not shielded by the neutral sugar residues, and yet the -alaninyl GM1 had an activity very similar to native GM1 (Park and Huang, 1993). In contrast to GM1, ganglioside GM3 containing liposomes are long-circulatory in rats (Yamauchi et al., 1993). Therefore, it appears that particular stereo organization of the ganglioside sugar residues can control the extent of opsonization and the macrophage recognition of glycolipid-incorporated liposomes.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structure of gangliosides GM1 and GM3.

Studies by Moghimi and Patel (1989a,b, 1998) demonstrated that rat serum displays a dual role in Kupffer cell recognition of liposomes. In addition to stimulating liposome uptake by Kupffer cells via a noncomplement-mediated process, serum was shown to contain at least two heat-stable proteins that suppressed liposome recognition by Kupffer cells. Subsequently, it was suggested that a balance between the opsonic molecule and these suppressive proteins (dysopsonins) could regulate the quantity and the rate of clearance of liposomes from the blood by the hepatic macrophages (Moghimi and Patel, 1998). Dysopsonins could modulate the rate of liposome uptake by reducing the amount of liposome-bound opsonin and, hence, protect the phagocytic cells from being destroyed by excessive binding and ingestion of liposomes, particularly those vesicles which are more resistant toward lysosomal esterases. On the basis of this hypothesis, Park and Huang (1993) suggested that the prolonged circulation time of GM1-incorporated vesicles in mice may also be due to the binding of a putative dysopsonin onto the liposome surface leading to a reduced level of both opsonization and/or inactivation of bound opsonins. The dysopsonin hypothesis therefore suggests another possibility for enhancing the circulation time of liposomes (and perhaps other particulates). If dysopsonins play a significant role in vivo, then vesicles enriched with these proteins should be expected to display long circulation times. This approach warrants the identification of these blood dysopsonic molecules and their mode of action with regard to different types of colloidal carriers.

Attempts have also been made to introduce sialic acid groups onto the surface of polymeric nanospheres. One example was the noncovalent adsorption of glycoproteins rich in sialic acid such as orsomucoid onto the surface of poly(isobutylcyanoacrylate) nanoparticles (Olivier et al., 1996). This attempt was disappointing, since the half-life of these particulates was similar to those of uncoated systems. This was presumably due to insufficient density or altered conformation of the clustered glycans on the nanoparticle surface. Alternatively this could have been due to the desorption and exchange of sialic acid rich glycoproteins as soon as the particles were introduced into the systemic circulation.

Another potentially interesting mammalian system for disguising foreign particles as "self" is the integrin-associated protein CD47. This molecule has been suggested to function as a marker of self on murine red blood cells and lymphocytes since in the absence of CD47 the cells are cleared from the bloodstream by the splenic red pulp macrophages (Oldenborg et al., 2000). This marker protects the cells against elimination by binding to the SIRP. CD47 analogues are also encoded by smallpox and vaccina viruses (Lindberg et al., 1994). It seems that these pathogens are taking advantage of SIRP signaling to disable normal defenses. Therefore, CD47-SIRP may represent a viable approach for the design of macrophage-evading colloidal carriers.

	V. Synthetic Polymers in Colloid Engineering

Top Previous Next References

From the preceding discussion it is now apparent that a relatively successful approach for prolonging the circulation times of colloidal particles in the blood is to create a steric surface barrier of sufficient density. Because of the possible immunological consequences associated with some bacterial polysaccharides and the high cost of recombinant complement regulators, tremendous efforts have been directed to design synthetic polymers that can fulfill these criteria. The design of long-circulating particles can be traced back to the late sixties. It has long been known that stabilization of emulsion systems may be achieved at the interface by the addition of an emulsifying agent or a surfactant. In a pioneering experiment Geyer (1967) demonstrated that intravenously injected lipid emulsions prepared with high molecular weight members of POE/POP copolymer nonionic surfactants (poloxamers and poloxamines) as emulsifiers remained in the blood for relatively long periods. This behavior was initially thought to arise from the interference of surfactants with the lipoprotein-lipase activity (Hart and Payne, 1971). Later, it was suggested that high molecular weight POP/POE surfactants in some way prevent lipid particles from sticking to the blood vessel endothelium as well as inhibiting recognition by macrophages (Jeppsson and Rossner, 1975).

Poloxamers consist of a central POP block that is flanked on both sides by two hydrophilic chains of POE (Fig. 2). A slightly different structure is exhibited by the poloxamines that are tetrafunctional block copolymers with four POE/POP blocks joined together by a central ethylene diamine bridge (Fig. 2). Numerous investigators have now demonstrated that such copolymers adsorb onto the surface of oil-in-water emulsions or any hydrophobic nanoparticulate systems [e.g., polystyrene, gold, PLGA, poly(phosphazene), poly(methyl methacrylate) and poly(butyl 2-cyanoacrylate)] via their hydrophobic POP center-block (Troster et al., 1990; Moghimi et al., 1993c; Stolnik et al., 1994; Storm et al., 1995; Vandorpe et al., 1997; Monfardini and Veronese, 1998). This mode of adsorption leaves the hydrophilic POE side-arms in a mobile state as they extend outward from the particle surface and provide stability to the particle suspension by a repulsion effect through a steric mechanism of stabilization involving both enthalpic and entropic contributions (Moghimi et al., 1993c). The strength of polymer adsorption and the resultant polymer conformation is dependent on the proportion and the size of both POP and POE segments as well as the physicochemical properties of the nanoparticle surface (Moghimi and Hunter, 2000b). With the help of field-flow fractionation, electron spin resonance and conventional labeling techniques, detailed analytical characterization of the adsorption complexes formed between poloxamers and polystyrene beads of different sizes have been accomplished. These studies have also pointed toward the importance of nanoparticle surface curvature on polymer chain mobility and conformation (Li et al., 1994). For a given triblock polymer, it was found that both surface concentrations and adlayer thicknesses are strongly related to the particle size, such that smaller particles (sizes below 100 nm) take up fewer polymer molecules per unit area than the larger ones. The reduced crowding around each POE chain results in thinner adlayers and higher chain mobilities. Therefore, the surface density decreases with decreasing particle size. For a particle of a given size, it is the size of the surfactant's hydrophobic center block (POP), rather than its flanking tails, that determines the surface concentration or density. Thus, triblocks of similar POP size showed comparable surface concentration, while the longer POE chains were associated with thicker adlayers as well as greater chain dynamics (Li et al., 1994). Therefore, by keeping the particle size constant, one can gain insight into the effect of POE chain lengths on plasma protein adsorption and phagocytosis. Indeed, among the various copolymer members, poloxamine-908, poloxamine-1508, poloxamer-238, and poloxamer-407 (Fig. 2) have proved to be among the most effective copolymer nonionic surfactants for prolonging the circulation time of hydrophobic nanoparticles of 15 to 150 nm in mice and rats. For example, reported half-lives of poloxamine-908-coated nanospheres in mice and rats vary from a few hours to 1 to 2 days, depending on both the particle size and its initial surface hydrophobicity (Storm et al., 1995; Monfardini and Veronese, 1998; Moghimi and Hunter, 2000b).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   The structure of selected poloxamines (a) and poloxamers (b).

It has also been shown that surface modifications with poloxamers and poloxamines before intravenous injection is not really necessary for making nanoparticles long-circulatory. Intravenously injected uncoated 60 nm polystyrene nanoparticles (which are susceptible to phagocytosis by Kupffer cells) were converted to long-circulating entities in those rats that received a bolus intravenous dose of either poloxamer-407 or poloxamine-908, 1 to 3 h earlier (Moghimi, 1997, 1999). It can be argued that the altered biodistribution profile of nanoparticles is the result of cell-surface modification by the administered copolymers. For instance, block copolymers could adhere to cell membrane hydrophobic domains via their hydrophobic center block or act as an effective membrane-spanning entity (Watrous-Peltier et al., 1992). The extracellular steric constraints resulting from hydrophilic POE tails of copolymers will then prevent the interaction between an approaching particle and the cell. Interestingly, this was not the primary mechanism. Instead, nanoparticles were shown to acquire a coating of copolymer and/or copolymer-protein complexes in the blood (Moghimi, 1997); this event explains their phagocytic resistance.

Following intravenous injection to mice and rats, poloxamer- or poloxamine-coated sub-200 nm nanoparticles of albumin (Lin et al., 1994), poly(phosphazene) (Vandorpe et al., 1997), and PLGA (Stolnik et al., 1994) as well as liposomes (Woodle et al., 1992; Kostarelos et al., 1999) do not exhibit prolonged circulation times (reported half-lives are usually less than 2-3 h). Therefore, alternative coating of grafting materials have been tailored. The majority of these materials are based on PEG and its derivatives (Dunn et al., 1994; Lin et al., 1994, 1997, 1999; Gref et al., 1994; Stolnik et al., 1994; Vandorpe et al., 1996, 1997; Lacasse et al., 1998; Peracchia et al., 1997a,c, 1998; Monfardini and Veronese 1998; De Jaeghere et al., 2000). PEG is a linear polyether diol that exhibits a low degree of immunogenicity and antigenicity (Abuchowski et al., 1977). The polymer backbone is essentially chemically inert, and the terminal primary hydroxyl groups are available for derivatization. Usually, the hydroxyl groups are first activated and then reacted with the chosen molecule; PEG activation and functionalization methods have been exhaustively reviewed elsewhere (Zalipsky, 1995; Monfardini and Veronese, 1998). Surface modification of nanoparticles with PEG and its derivatives can be performed by adsorption, incorporation during the production of nanoparticles, or by covalent attachment to the surface of particles. Examples of currently available PEG conjugates for nanoparticle surface engineering includes PEG-R type copolymers, where R is PLA (Stolnik et al., 1994; Bazile et al., 1995), PLGA (Gref et al., 1994), and poly--caprolactone (Shin et al., 1998; Kim et al., 1998) with appropriate molecular weights. The molecular weight of the PEG segment varies between 2000 and 5000, which is necessary to suppress protein adsorption. Another related example is poly(phosphazene)-poly(ethyleneoxide)-5000 (Vandorpe et al., 1997). Despite these efforts, coating materials such as the two synthesized PLA-PEGs (PLA:PEG, 3:4, composed of methoxy-PEG-2000 and the weight average molecular weight of 5400 for the copolymer; and PLA:PEG, 2:5, composed of methoxy-PEG-5000 and the weight average molecular weight of 9500 for the copolymer) were found to be inferior to poloxamine-908 in keeping PLGA nanospheres in the rat blood (half-lives of less than 2 h in both cases of PLA-PEG) (Stolnik et al., 1994). On the other hand, biodistribution studies in rats have demonstrated longer retention time for poly(phosphazene) particles coated with poly(phosphazene)-poly(ethyleneoxide)-5000 (half-life of ~3 h) compared with poloxamine-908-coated counterparts (half-life of less than 30 min) (Vandorpe et al., 1997). A European Community Program in nanoparticle engineering (Brite/Euram, BE-3348-89) have further synthesized and used dextranox-methoxy-PEG (dextran-70,000 and methoxy-PEG-5000, total weight average molecular weight of 89,000) for cross-linking to preformed albumin nanospheres (Lin et al., 1994) as well as human serum albumin-poly(thioetheramido acid)-methoxy-PEG-2000 [albumin 49% w/w, PEG 38% w/w, and poly(thioetheramido acid) 13% w/w], albumin-poly(amidiamine)-PEG-2000 [albumin 50% w/w, PEG 40% w/w, and poly(thioetheramido acid) 10% w/w] and albumin-methoxy-PEG for incorporation during albumin nanosphere production (Lin et al., 1997, 1999). Physicochemical charaterization studies such as the measurement of in vitro protein adsorption, dynamic light scattering, turbidity ratio test, electrolyte and pH induced flocculation, and particulate electrophoretic mobility measurements have confirmed the presence of the steric barrier of PEG and dextran-PEG brush-like structures on the surface of albumin nanoparticles. Based on such observations it was proposed that these and structurally similar particles are expected to behave as long-circulatory entities (Lin et al., 1994, 1997, 1999). This, however, contradicts the earlier biodistribution data from the same group which demonstrated rapid localization of these particles in the rat liver (Dunn, 1995; Lin, 1996). Therefore, physicochemical characterization based on the described techniques can not predict the in vivo fate of nanoparticles. These assemblies presumably have an inadequate surface density of PEG which must be addressed.

Submicron sized PEG-ylated poly(isobutyl 2-cyanoacrylate) nanoparticles have also been produced by an emulsion/polymerization method (Peracchia et al., 1997c). Here, PEG was anchored to the nanoparticles through either only one end-group or through both end groups. The latter outcome surely limits the mobility of PEG chains and interaction with blood opsonins and, hence, particle retention time in the blood (Peracchia et al., 1997b). Efforts have also been made to form long-circulating nanospheres from diblock copolymers such as PLA-PEG (expressing a molecular weight of 20,000) in a one-step procedure, but again with rather disappointing results (Gref et al., 1994). Based on indium-111 labeling studies, the circulation half-life of PLA-PEG particles (90-150 nm) was less than 1 h in rats. Furthermore, the presented data indicated that at 90 min after injection, the circulatory pool of nanoparticles was less than 20% of the injected dose whereas the liver-associated activity was in the order of 30% of the initial dose (Gref et al., 1994). No explanation was provided for the missing 50% of the injected activity.

An interesting approach, however, has been the covalent attachment of semitelechelic poly[N-(2-hydroxylpropyl)methacrylamide]s of different molecular weights to nanospheres based on methyl methacrylate, maleic anhydride, and methacrylic acid (Kamei and Kopecek, 1995). One polymer preparation, with a weight average molecular weight of 18,800, was able to dramatically extend the circulation time of nanospheres (half-life of 12-15 h) in rats, whereas control preparations were cleared rapidly from the blood by the liver.

Multiblock copolymers such as POE-poly(L-lysine), POE-poly(-benzyl-L-aspartate, POE-poly(-caprolactone) and poly(acrylic acid)-poly(methyl methacrylate) as well as those that have been used in particle coatings (e.g., poloxamers, poloxamines, PEG-PLA, PEG-PLGA) also self-disperse in water to form spherical polymeric micelles with diameters in the size range of 15 to 80 nm (Yokoyama, 1992; Jones and Leroux, 1999). Some of these micellar structures have been suggested as promising long-circulating carriers of poorly water soluble and amphiphilic drugs, because of their small size and hydrophilic shell (Yokoyama et al., 1990, 1991; Yokoyama, 1992; Kwon et al., 1993, 1994; Hagan et al., 1996; Zhang et al., 1997a,b). Nonetheless, the effectiveness of such systems will depend on their critical micelle concentration. Upon intravenous injection, micelles are often diluted to less than their minimum micelle concentration and polymer molecules are known to behave in a dramatically different way. Furthermore, little information is known with regard to the stability of micellar systems in vasculature and their extent of interaction with blood and cellular components. Micellar stability may be enhanced by cross-linking procedures to produce a solid outer core (e.g., shell-cross-linked kendel-like particles, Thurmond et al., 1996; Bütün et al., 1999). However, such strategies may be rather premature for the design of a stable long-circulating micellar construct, as the reduced surface energy will probably result in rapid accumulation in liver macrophages.

For incorporation into the liposomal bilayer, numerous lipid derivatives of PEG have been made using lipids that, for example contain a primary amino group (particularly PE), an epoxy group or a diacylglycerol moiety (Blume and Cevc, 1990; Klibanov et al., 1990; Allen and Hansen, 1991; Allen et al., 1991a,b; Lasic et al., 1991; Mori et al., 1991; Maruyama et al., 1992; Parr et al., 1994; Kirpotin et al., 1996; Woodle, 1998). Alternatively, activated PEG can be anchored to reactive phospholipid groups of preformed liposomes (Senior et al., 1991). Another strategy has utilized the transfer of PEG-phospholipid conjugates from the micellar phase into the lipid bilayer of preformed vesicles (Uster et al., 1996). It was found by trial and error that PEG-grafted liposomes with extended circulation half-lives are in the size range of 70 to 200 nm and contain 3 to 7 mol% of methoxy-PEG-2000 grafted to DSPE or DPPE in addition to various amounts of phospholipids and cholesterol (Klibanov et al., 1990; Allen et al., 1991b; Woodle and Lasic, 1992; Woodle, 1998). The circulation half-lives of such vesicles is 15 to 24 h in rodents, and as high as 45 h in humans. To date, these are the best engineered long-circulating particles. Only recently, the biophysical basis of these observations was realized. It was shown that mixtures of PEG-phospholipid conjugates and phospholipids exist in primarily three physically distinct states (Bedu-Addo et al., 1996a). These included a lamellar phase with components exhibiting some miscibility, a lamellar phase with components phase separated, and mixed micelles. The relative proportion of the three states in a given mixture was dependent on PEG chain length, acyl chain length and the degree of unsaturation of the PEG-phospholipid conjugate, and the acyl chain composition of the phospholipid. For example, beyond 7 mol% short-chain PEG-DPPE conjugates (PEG molecular weight in the region of 1000-3000) show a strong tendency to form mixed micelles with DPPC. Long-chain PEG-DPPC conjugates (PEG molecular weight of either 5000 or 12,000) above 8 mol% formed phase separated lamellae with the phospholipid due to PEG chain-chain interactions; the PEG chain-chain interactions can be reduced by using PEG-dioleoylphosphatidylcholine due to the presence of the kink in the acyl chain. Changes in phospholipid composition also alters the miscibility of PEG-PE with phospholipids. Increasing phosphatidylcholine acyl chain length increases fatty acid chain-chain interaction and so the van der Waals cohesive force. This results in more tightly packed phosphatidylcholine and PEG-PE molecules and a greater tendency toward PEG chain-chain entanglement and micelle formation. The extent of demixing of PEG-PE in bilayers, therefore, decreases in the order of C18:0 > C16:0 > C14:0. These observations explain why long-circulatory PEG-ylated liposomes usually contain 3 to 7 mol% short-chain PEG-PE (PEG molecular weight in the range of 1000-3000). Large chain PEG-PE (PEG with a molecular weight of either 5000 or 12,000) seem unsuitable for preparing certain formulations of long-circulating liposomes; phase separation generates domains less enriched with PEG-PE and could lead to poor steric protection of the liposome surface and subsequent destabilization by lipoproteins and opsonic attack (Bedu-Addo et al., 1996a).

Inclusion of a high concentration of cholesterol (above 30 mol%) within the liposomal bilayer can further improve surface protection by PEG (molecular weight of 1000-3000)-PE (5-7 mol%). This is due to an increase in bilayer cohesive strength and, hence, a reduction in the formation of phase separated lamellae (Bedu-Addo et al., 1996b). Because of its relatively inflexible structure cholesterol thus acts as a spacer keeping lipid chains apart and reducing PEG chain-chain interaction. At higher concentrations of PEG-PE, solubilization of the bilayer occurs with preferential solubilization of cholesterol over phospholipid. For these reasons the most suitable formulations for a prolonged circulation time contains >30 mol% cholesterol and low concentrations (equal or less than 7 mol%) of short PEG-PE. Even in the presence of cholesterol the steric protection of long-chain PEG-PE is relatively poor. This is presumably due to the fact that reduction in the intramolecular expansion factor with increase in molecular weight could lead to coil shrinkage and, hence, reduced chain flexibility.

Apart from PEG lipid derivatives, other effective alternative materials for prolonging the circulation time of liposomes include phosphatidylpolyglycerols and phospholipid derivatives of amphiphilic poly(vinyl pyrrolidone), poly(vinyl alcohol), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(acryl amide), poly(acryloy morphine), and N-(2-hydroxylpropyl)methacrylamide (Torchilin et al., 1994, 1995; Maruyama et al., 1994; Woodle et al., 1994; Takeuchi et al., 1996; Zalipsky et al., 1996; Shtilman et al., 1999; Whiteman et al., 1999). In contrast to these materials, incorporation of 5 to 7 mol% PEG stearates (Klibanov et al., 1990; Moghimi, 1996) and Tergitols (S. M. Moghimi, unpublished observations) have proved to be ineffective in prolonging liposome circulation times. The biophysical basis for these observations remains to be elucidated.

Coating of liposomes with chitin derivatives such as carboxymethylchitin and carboxymethyl/glycochitin has also been achieved (Mobed and Chang, 1998). N-Acetylglucosamine is the repeating unit of these chitin derivatives and is rather similar to N-acetyl and N-glyconeuraminic acid of the red blood cell outer membrane (Mobed and Chang, 1991). The adsorption of carboxymethychitin (molecular weight 101,000) onto the surface of neutral liposomes not only resulted in electrostatic stabilization of the vesicles but also provided an effective barrier to macromolecular plasma protein adsorption (Mobed and Chang, 1998). However, the retention time of such constructs in the systemic circulation remains to be evaluated.

Apart from poloxamers and poloxamines, PEG-PE and Tweens have all been used as emulsifiers for the production of long-circulatory oil-in-water emulsions. Again, the blood retention time of such emulsions was shown to be related to PEG chain length and its surface density (Liu and Liu, 1995; Lundberg, 1997).

	VI. Essential Thoughts on Using Polymers in Nanoparticle Engineering

Top Previous Next References

An important consideration in clinical therapeutics is the ultimate fate of polymeric constituents used in nanoparticle engineering. To date this subject has received little attention. The fate of any polymer used will be determined by its method of synthesis, subsequent degree of purity and its behavior on administration into the individual biological system. Synthetic polymers exist as populations with a statistical distribution, i.e., they are polydisperse systems which vary in molecular weight (Fig. 3). The degree of polydispersity will tend to be greatest if the product has been synthesized by free radical polymerization, which is nonselective with chain termination occurring, by combination or diproportionation. On the other hand, starburst polyamidoamine dendrimers are a relatively new class of spherical macromolecules, which have been reported as monodisperse (Kukowska-Latallo et al., 1996). Ironically, the future usefulness of these monodisperse systems in medicine remains to be revealed, since in vivo studies so far have indicated their propensity to localize in the liver and spleen (Malik et al., 2000).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A comparison of two batches of poloxamine-908 obtained from the same manufacturer and analyzed by gel permeation chromatography. Clear differences are seen in both log molecular weight range and weight fraction of the polymers. Wn[log M], weight fraction of polymer; Mn, number average molecular weight; Mw, weight average number weight; Mz, molecular weight average.

There have been some preliminary studies on the effects of specific molecular weight fractions present within a polydisperse system to discern individual biological activities in vivo on both short-term and long-term administration (Emanuele et al., 1997; Toth et al., 1997). Variation in polymer molecular weight fractions has been linked to tissue toxicity (Toth et al., 1997). For example, Flocor (CytRx, GA, USA), a well defined fraction of poloxamer-188 (molecular weight 8964, polydispersity 1.0280) only reduced nephrotoxicity by 68% in a recent clinical trial (Emanuele et al., 1997) when compared with the native poloxamer-188 (RheothRx) following intravenous administration. This suggests that the nephrotoxic constituent(s), which are below the renal threshold, had not been removed or that the polymer is inherently toxic in its own right or a joint effect of the two (see also Section VI.C.). Limited studies with polydispersed radiolabeled POP/POE copolymers have indicated that the primary route of polymer excretion is renal and the minor route is biliary (Wang and Stern, 1975; Willcox et al., 1978; Rodgers et al., 1984). In addition, radiolabeled copolymers have been found in all organs, particularly in the liver, lung and skeletal muscles, 24 h after intravenous administration into the dog (Willcox et al., 1978). Long-term effects of accumulated polymers in these organs still remain to be evaluated. Furthermore, no studies so far have determined to what extent these copolymers are excreted intact or in a modified form from the starting material. Poloxamers and poloxamines can also adsorb on to the surface of blood cells and endothelia but the long-term significance has not been thoroughly investigated (Smith et al., 1987). For example, adsorption on to endothelial cells may lead to activation of NF-B or endothelial cells may respond with the expression of proteins stored in the cytoplasm.

Another classical example is the early work done with pyran copolymers. Here, the relation in variation between the polymer molecular weight and its LD₅₀ was illustrated by Breslow (1976) which may be of relevance to polydisperesed systems used in nanoparticle engineering. For example, the LD₅₀ for molecular weight fractions of 6900, 15,700, and 19,600 of pyran copolymer injected intravenously into mice were 127, 131, and 100 mg/kg, respectively. Molecular weight was found to affect the degree and type of physiological response. Below a molecular weight of ~15,000 stimulation of phagocytic activity of the RES was achieved, whereas with higher molecular weight polymers induced depression in phagocytic activity of the RES. Serum glutamate pyruvate transaminase was used as a marker of liver damage, higher levels were attained with increasing polymer molecular weight and greater polydispersity as was the inhibition of metabolism of the compounds aminopyrine and antipyrin. It is not clear from these studies to what extent the observed toxicities are related to the presence of impurities and/or oxidized contaminants in each polymer sample.

Most polymers are synthesized under relatively hostile conditions (elevated temperature and pressure) and are thus prone to processes of degradation during their formation. To counter the in-process environment processing modifiers (e.g., heat stabilisers and emulsifiers) are frequently used but readily separated from the polymer (Stevens, 1999). To ensure postprocessing polymer stability chemical property modifiers such as antioxidants and ultraviolet stabilisers are added. It must therefore be considered if these or other additives and/or low molecular weight degradative products or monomers are capable of inducing immunological (e.g., IgE-independent hypersensitivity reactions) and/or pharmacological events following parenteral administration (see also part C below). There is relatively little information available regarding the biological fate of antioxidants or their break-down products. Unfortunately the concentration and types of additives and low molecular weight contaminants (e.g., monomers, oligomers, homopolymers) present in commercial products is proprietary information, which is not readily available. An investigation into the effect of removal of the antioxidant butylated hydroxy toluene, a known skin sensitizing agent, from poloxamer-188 has been attempted (Bentley et al., 1989). Unfortunately a complete lack of data supporting the presence of the poloxamer following column chromatography rendered the results of this study meaningless. Other techniques such as supercritical fluid fractionation (Edwards et al., 1999) have been used to purify polymers; however, in this case there was evidence for macromolecular cleavage.

Incomplete polymerization of diblock copolymers may result in homopolymer contamination in the product, which are exceptionally difficult to remove and may explain the presence of homopolymers in some commercial samples (Moghimi and Hunter, 2000b). It has not yet been determined whether these contaminants are essential for the useful activity of the polymer or they have a synergistic role in combination with the copolymer. A possible future method to circumvent the deleterious consequences of hostile synthetic processing conditions and negate the use of specific additives may be achieved through milder microbiological methods. To date commercially bacterially derived polymers have included polyhydroxyacids and polyesters (Kunioka and Doi, 1990; Gogolewski et al., 1993b).

It should also be emphasized that different species may respond differently to the administered polymeric materials. For example an early study with polyelectrolyte poly(ethylene sulfonate) clearly demonstrated a broad spectrum of antitumor activity in mice following subcutaneous administration (Regelson and Holland, 1958). However, the antitumor activity of this polymer was found to be reduced in humans and it was also too toxic for clinical use (Regelson and Holland, 1958; Breslow, 1976). Variation in interspecies activity has also been observed with pyran copolymer. This material was able to induce interferon production in mice. A synergistic effect between pyran copolymer and the foot-and mouth-disease vaccine in mice (Campbell and Richmond, 1973) was observed. A 1.2 mg dose of pyran copolymer plus the vaccine protected 80% of the mice, with no antiviral activity observed for the copolymer alone. This activity was not carried over to cattle and pigs where dosages were too high and toxic side-effects were observed (Sellers et al., 1972; McVicar et al., 1973). These observations seem relevant to nanoparticle production and engineering with polymers and suggest caution in extrapolating animal model responses to humans. We may even experience variations among different individuals. For example, recently, we have demonstrated variation in complement activation in the sera of healthy individuals by various poloxamer and poloxamines (Szebeni et al., 2001). The surfactants failed to activate complement in the sera of some individuals, whereas in others complement activation did occur. Variation in the pathway of complement activation was also noticed among the positive population. We were not able to find any correlation between the molecular weight of the copolymers and the extent of complement activation among the positive population. Impurities in copolymers or derivatives of the copolymers generated in the manufacturing process have been proposed to be responsible for activation of the complement system (Ingram et al., 1993). Interestingly, removal of some impurities by dialysis or organic extraction resulted in further activation of complement system among some individuals. These treatments also activated complement in the sera of even some negative individuals (in those that no complement activation by the pretreated copolymers were observed). Complement activation may induce mild to severe hypersensitivity reactions. Therefore, future studies should explore the relationship between in vitro and/or in vivo complement activation by such copolymers and clinical signs and symptoms of hypersensitivity to poloxamer/poloxamine based nanoparticles. Similar to the above observations, variation in complement activation in serum of healthy individuals by PEG-bearing liposomes has also been observed (Szebeni et al., 2001).

The situation regarding the fate and activity of polymers is further complicated by the current focus on the development of biodegradable/bioerodable polymeric drug delivery vehicles for parenteral use. At present there is no study available, which demonstrates these polymers (e.g., PLA, PLGA) will degrade in a uniform manner in vivo. Clearly the in vivo environment will vary depending on the site of parenteral administration, composition of tissue fluids and disease state which further complicates the uniformity of the degradation process. PLGA polymers have been used widely as biomaterials for medical applications over the last 30 years and are regarded as "biocompatible" and "nontoxic". This has been due to the wide variety of materials achievable by varying the molar ratios of the lactic acid and glycolic acid moieties. For example, high molecular weight crystalline PLGA has been used effectively as surgical sutures and bone fixation nails and screws (Daniels et al., 1990; Pulapura and Kohn, 1992). Conversely low molecular weight amorphous PLGA has been researched widely for controlled drug delivery applications (Asano et al., 1990; Wang and Wu, 1997). An example of a successful pharmaceutical product for the controlled delivery of lutenizing hormone in the form of injectable depot is Zoladex (Zeneca, UK). However, there is little information available regarding the rate of degradation as well as toxicological problems associated with PLGA and related biodegradable particulate drug delivery systems following parenteral administration. A 2-fold increase in the rate of degradation of PLA has been observed in plasma compared with buffer or water at 37°C (Mason et al., 1981). The specific plasma constituents responsible were not identified. The large surface area of the colloid, which is gradually presented to opsonic and other immunoregulatory proteins, may potentiate these effects. This surface area will be markedly increased once the degradation process starts. It will be essential to control the rate of degradation in the in vivo environment to ensure even dosage delivery assuming that the polymer and its by products are completely inert in vivo. Faster in vivo rates of degradation have been attributed, in part, to the foreign body response (Tokiwa and Suzuki, 1977; Williams and Mort, 1977; Ali et al., 1994). Investigation into the phagocytosis of both whole ( 38 µm) and predegraded PLA particles following intraperitoneal administration into mice has been undertaken (Lam et al., 1993). Cells that had phagocytosed PLA particles showed increased signs of cell damage compared with the control which was nondegradable polytetrafluoroethylene particles of 4 to 9 µm. Predegradation of PLA particles induced cell damage (e.g., swollen mitochondria, widened endoplasmic reticulum, disappearance of membrane ruffling) on day 2 of the experiment with evidence of necrotic cells and cell debris on days 3 and 4. This clearly indicates that at least in the mouse model degradation products of the so called "bioinert" PLA can be cytotoxic to immune cells. Therefore, there is a clear need for a wide range of in vivo studies on biodegradable polymers to determine the nature and fate of the degradation product(s). The physicochemical nature of the particle (size, shape and porosity will also affect the degradation rate (Matlaga et al., 1976; Grizzi et al., 1995). The autocatalytic nature of some polymers will further accelerate degradation rates and this may be potentiated when the polymers are isolated or have restricted transport away from a region. For example, hydrolysis of the esteratic link of PLA will result in the formation of more acid groups and, hence, increased availability of protons to feed the esteratic hydrolysis. PLG polymers with the acid group capped (as an ester) have been demonstrated to have reduced rates of degradation compared with the free acid and that the length and type of capped moiety can be used to control the rate of degradation (Tracy et al., 1999). Perhaps the ideal drug delivery application for biodegradable polymer microparticulate systems is for the delivery of biologically active polymers. If controlled rates and site of degradation can be achieved it is an ideal opportunity for a sustained release "biologically active polymer-polymer therapeutic system".

The precise role of polymer stereochemistry on enzyme degradation and immunological response following parenteral administration has not yet been determined. Stereochemical preference for copolymer bonds has been demonstrated (Li et al., 2000), degradation of stereocopolymers (L-, DL- and meso-lactides) by proteinase K that showed preferential degradation of bonds with the stereochemical configuration of L-L, L-D and D-L bonds as opposed to D-D bonds. However enzymatic attack in the case of the meso form may have been facilitated by increase rates of water uptake. A comparison of the effect of poly-L- and D-lactides on inflammatory response was found to be reduced in the case of polymers containing the D-isomer (Gogolewski et al., 1993a). Whether different stereochemistry can modulate biological responses is still subject to speculation.

	VII. Why Are Polymer-Coated Long-Circulating Particles Eventually Cleared by Macrophages?

Top Previous Next References

All polymer-coated or -grafted long-circulating particles are at some point cleared from the blood of tested species by macrophages of the RES (Moghimi and Murray, 1996; Moghimi and Gray, 1997; Moghimi, 1998; Dams et al., 2000a). As discussed in the previous section, polymers are statistical macromolecules; any given sample usually contains several closely related species. In a particular polymer sample, the pattern as well as the strength of noncovalent binding of individual polymer macromolecules to a nanoparticle surface may vary from one to another. Therefore, it is possible that in the blood a fraction of the surface adsorbed polymers, depending on their physicochemical characteristics, is displaced by some plasma proteins (polymer dissociation by displacement). This partial displacement of the surface associated polymer decreases the steric shielding at particular surface sites and makes the nanoparticles prone to phagocytoses. In the case of long-circulating nanospheres with a polymeric texture, the processes of swelling, degradation and erosion of the polymer matrix may induce morphological and surface changes favorable for opsonization and/or phagocytosis. Similarly, with grafted systems (as in PEG liposomes), an alternative explanation is the chemical breakdown of the polymer-conjugate linkages (e.g., succinate, carbamate, amide) in the blood (Parr et al., 1994), rapid partitioning (e.g., PEG ceramides from the liposomes) (Webb et al., 1998) and complete transfer of the whole conjugate to vascular elements. These possibilities, at least, seem to be in agreement with two-phase partitioning of PEG-attached liposomes in the presence of plasma which resulted in gradual plasma protein build-up on the vesicle surface (Senior et al., 1991). The receivers of the coating materials may be the circulating lipoprotein particles or even cells such as erythrocytes and vascular endothelial cells (Smith et al., 1987; Howerton et al., 1990).

The majority of surface coating polymers used to date are rich in hydroxyl groups, which may eventually trigger the activation of both the alternative and classical pathways of the complement system. This results in particle fixation by opsonizing complement fragments (e.g., C3 degradative products) and stimulation of particle recognition by activated complement receptors of phagocytic cells. For example, Cremophor EL, as well as poloxamer and poloxamine copolymers can activate the human complement system, causing conversion of C3 through both the alternative and classical pathways (Hunter and Bennett, 1984; Szebeni et al., 1998, 2000, 2001). As for the explanation of classical pathway activation of complement by such polymeric materials, we have speculated a role for C-reactive protein and antibodies such as anti-cholesterol and anti-phospholipid antibodies (Szebeni et al., 2001); these antibodies are abundant in most humans (Alving and Swartz, 1991). For instance, the binding of naturally occurring anticholesterol antibodies to the hydroxyl-rich surface of poloxamer- or poloxamine-coated nanoparticles is possible, since the epitope they recognize contains a hydroxyl group.

Complement activation has also been suggested to be responsible for the rapid clearance of radiolabeled methoxy-PEG-coated liposomes by macrophages of the RES in two patients (Dams et al., 2000b). An intriguing observation, however, is the poor clearance of such complement (C3b and iC3b)-opsonized PEG-bearing liposomes in certain human individuals (circulation half-lives of more than 45 h) (Szebeni et al., 2000). Several possibilities may account for these observations. First, the binding of vesicle-bound monomeric and/or polymeric iC3b to macrophage receptor CD11b/CD18 (CR3) may be sterically hindered by PEG. Second, competition between vesicle-bound and free iC3b for CR3 can not be ruled out. Thirdly, such vesicles may bind to erythrocytes as they express receptors for C3b (CR1). These cells outnumber circulating leukocytes and the vast majority of CR1 receptors present in circulation are located on the erythrocytes (Cornacoff et al., 1983). It is plausible that erythrocytes will eventually transfer bound liposomes to liver and spleen macrophages during their passage through these organs. Finally, generation of liposome-bound C3bn complexes requires detailed analysis as PEG may protect C3bn-containing complexes from inactivation. Indeed, these complexes are among the most potent nucleation sites for the alternative C3 convertases.

Another interesting possibility arises from the ability of stimulated or activated macrophages to rapidly recognize and ingest long-circulating particles. The majority of tissue macrophages are in the quiescent state but some macrophage populations in the same tissue may exist in primed or activated states. One study (Moghimi et al., 1993a) has shown the ability of zymosan-stimulated macrophages to recognize and ingest long-circulating particles by an opsonic-independent process. This effect of zymosan was attributed to changes in macrophage cell surface hydrophobicity as well as changes in mobility of certain plasmalemma receptors, leading to recognition of some structural determinants of long-circulating particles in vivo. Therefore, it seems that activated or stimulated macrophages can overcome the steric barrier of long-circulating particles and proceed with the phagocytic ingestion. This may be of particular relevance with regard to the intravenous administration of long-circulating carriers in clinical situations with enhanced regional or global macrophage activity. To date, the majority of studies in determining the pharmacokinetics and tissue distribution of long-circulating particles have relied on healthy animals or have used disease models with suppressed macrophage activity.

It is also likely that in certain cases the coating polymer triggers the clearance of long-circulating particles from the blood by modulating some functions of the host immune system. Two lines of investigations are in agreement with this suggestion. Moghimi and Gray (1997) were the first to report that a single intravenous injection of poloxamine-908 or poloxamer-407-coated polystyrene particles (60 nm) into rats could dramatically affect the half-life and body distribution of subsequent doses. The degree of this alteration was dependent on the interval between the doses. An interval of 2 days between the two injections had no effect on the pharmacokinetics of the second dose. Interestingly, when the interval between the two injections was 3 to 10 days, the second dose of particles were rapidly cleared from the blood by Kupffer cells and splenic red pulp macrophages. Again, intervals of longer time periods had no effect on half-life or tissue distribution of the second dose of polymer-coated particles as they behaved as long-circulatory; these observations were not associated with macrophage depletions or defects in phagocytosis. Subsequently, the coating polymer was found to trigger particle clearance from the blood by resident ED2 positive Kupffer cells and certain subpopulations of splenic red pulp macrophages. These coplymers can also activate the phagocytic activity of neutrophils (Ingram et al., 1992). It is also possible that activated neutrophils phagocytose polymer-coated particles and subsequently home in to other organs such as the spleen and lungs. The mechanism of mononuclear phagocyte stimulation by these copolymers is not clear but may be directed primarily through cytokine production by these cells. For example, a poloxamer member (CRL-1072) was shown to stimulate the production of IL-8, TNF- and granulocyte-macrophage colony stimulating factor by macrophages in a dose-dependent manner (Jagannath et al., 1999).

In a similar line of investigation, Dams et al. (2000a) further demonstrated that a single intravenous injection of PEG-grafted long-circulating liposomes into rats or monkeys significantly alters the pharmacokinetic profile of subsequently injected PEG liposomes. The outcome was rather similar to the earlier reported studies on repeated administration of POP/POE copolymer-coated particles (Moghimi and Gray, 1997; Moghimi, 1998) with the exception that a minimum time interval of 5 days between the two liposome injections was required to evoke macrophage clearance of the second dose by an opsonic dependent process. The presented data suggested a role for complement activation by an unknown heat labile serum factor. The above evidence probably explains why the circulatory life time of all engineered long-circulating particles never exceeds 3 to 5 days.

	VIII. Experimental and Clinical Trials with Parenterally Administered Long-Circulating Particles: Achievements and Pitfalls

Top Previous Next References

To date, long-circulating polymeric nanospheres have received little attention for their ability to act as a sustained-release system for continuous and controlled release of their entrapped drugs over a period of days, because the current technology is not sophisticated or smart enough. First, with the exception of one or two examples, all long-circulating nanoparticles are cleared from the systemic circulation of experimental animals within the first 8 to 10 h after intravenous administration. Repeated administration of a system based on the current technology could also lead to rapid accumulation in macrophages of the RES and induce toxicity. Second, the complex processes of gradual nanosphere degradation and erosion (e.g., random-chain and end scission) within the vasculature and the kinetic of drug release into the blood have been poorly addressed. Earlier work with classical "macrophage-prone" nanoparticles has shown that the molecular and bulk properties of the matrix polymer such as melting and glass transition temperatures, molecular weight distribution, crystallinity, degree of crosslinking, chemical structure etc., all control the efficiency of drug encapsulation as well as the extent of nanosphere degradation and drug release (Si-Nang et al., 1973; Ziff and McGrady, 1985; Kehlen et al., 1988; Shah et al., 1992; Grandfils et al., 1996; Batycky et al., 1997). Therefore, the release rate of drug from nanoparticles is expected to vary, among other factors, with the thickness and porosity as well as the size of the core drug particles, the dispersion state of the drug, drug solubility and molecular weight. The release profile of any nanoparticulate population is also a function of the drug payload and rate constants of the individual nanoparticles (Benita, 1984; Donbrow et al., 1986; Hoffman et al., 1986; Benita et al., 1988). The above issues must be considered for the development and vascular performance of long-circulating polymeric nanoparticles if they are going to act as a circulating reservoir for a particular drug. Hence, not only the clearance kinetics of the nanoparticle from the blood should be considered, but also the in vivo process of gradual nanosphere matrix degradation and the extent of drug release must be worked out in relation to a pharmacologically desired free drug profile. Batch-to-batch reproducibility, both in terms of nanoparticle production and drug release, is also of major concern since polymers are heterogeneous with regard to both molecular weight and macromolecular architecture (e.g., degree of branching and crosslinking).

In contrast to nanoparticles, liposomes may serve as a better example of circulating drug reservoir in humans, simply because of their rather longer blood residence and the presumed intrinsic safety of their matrix-forming components over polymeric nanospheres. In an experimental study, intravenously injected PEG liposomes were tested for their efficacy as a slow release system within the vasculature for the antineoplastic drug ara-C in the treatment of murine L1210/C2 leukemia (Allen et al., 1992). This drug is rapidly inactivated in vivo by cytidine deaminases with an initial half-life of 16 to 20 min in mice (Borsa et al., 1969). This is very close to the value found in humans (Baguley and Falkenhaug, 1971). The PEG-liposomal formulation of the drug was found to be superior to the 24 h free drug infusions given at the same total drug dose as well ara-C encapsulated in non-PEG containing liposomes, and prolonged mean survival times of the mice. Thus such constructs appear at least to protect rapidly degraded drugs from breakdown in vivo, with release of the drugs in a therapeutically active form over relatively long time periods. Although not investigated, the ability of PEG liposomes to localize in bone marrow, liver, and spleen may also be contributing to their therapeutic effect: indeed, high numbers of tumor cells can be found in the bone marrow and spleen within a few hours following intravenous injection of leukemia cells. Similarly, the increased reported therapeutic activity of doxorubicin-entrapped PEG liposomes in various models of solid tumors, when compared with free doxorubicin or doxorubicin-entrapped in macrophage-prone vesicles (Cabanes et al., 1999; Drummond et al., 1999), might be attributed to the increased circulation lifetimes of such vesicles; these systems could act as slow release or systemic infusion delivery vehicles in the blood compartment. Furthermore, PEG incorporation into the liposome bilayer significantly attenuates the toxic effects of doxorubicin to hepatic macrophages (Daemen et al., 1997).

Long-circulating PEG liposomes and micelles with entrapped radiopharmaceuticals or contrast agents have been successful in blood-pool imaging. For example, an iodine-containing amphiphilic block copolymer consisting of methoxy-PEG and poly[,N-(triiodobenzoyl)]-L-lysine was shown to form stable long-circulating micelles of 80 nm in diameter with an iodine content of 35 to 40% wt (Torchilin et al., 1999). These micelles provided clear and informative images of the blood pool, using computer tomography. Similarly, PEG liposomes labeled with technetium-99m chelator hexamethylpropyleneamine, which are highly stable in vivo, have produced quality images of the heart and sites of gastrointestinal bleeding in experimental animals (Goins et al., 1996).

C. Passive Targeting

1. Pathologies with Leaky Vasculature: Solid Tumors. Numerous studies have confirmed passive accumulation of intravenously injected long-circulating particles in experimental and clinical cases of solid tumors (Papahadjopoulos et al., 1991; Huang et al., 1992; Yuan et al., 1994; Forssen et al., 1996; Northfelt et al., 1996; Parr et al., 1997; Drummond et al., 1999). The extent of particle extravasation, however, depends on the porosity of the angiogenic tumor vessels as well as tumor blood flow. Among the tested particles, PEG liposomes have received considerable attention and this subject has been examined by Drummond et al. (1999) in a recent issue of Pharmacological Reviews. From several experimental models as well as the Kaposi's sarcoma lesions in humans, it appears that the therapeutic activity of anticancer agents is substantially enhanced by entrapment in PEG liposomes (70-200 nm) when compared with the free drug and in some cases to drug-entrapped in unprotected liposomes (Gill et al., 1995; Harrison et al., 1995; Drummond et al., 1999). Undoubtedly, the encapsulation procedure reduces the free drug peak plasma concentration and drug exposure to susceptible tissue such as heart and macrophages of the liver. The question arises as to what extent passive accumulation of PEG liposomes in tumors accounts for the reported improvements in antitumor activity, particularly with respect to our earlier discussion (see Section II.). From the experimental tumor models it appears that the majority of localized liposomes do not interact with target cancer cells (Huang et al., 1992; Yuan et al., 1994). For example, in a human tumor xenograft implanted in severe combined immunodeficiency mice, the extravasated liposomes were found to be distributed heterogeneously where it formed perivascular clusters that did not move significantly and could be observed for up to 1 week (Yuan et al., 1994). Surely, for improved anti-tumor activity, the gradual process of vesicle extravasation must be followed by the efflux of drug from the liposomes, resulting in tumor cell exposure to drug molecules. The mechanisms responsible for drug leakage from extravasated liposomes in tumors has been poorly studied, primarily due to technical difficulties associated with monitoring drug release. However, several speculative mechanisms can induce drug release from extravasated vesicles. One possibility is the drug leakage induced by the interstitial fluid surrounding tumors. Such fluids usually contain oxidizing agents as well as lipases and other hydrolytic enzymes derived from dying tumor cells or the resident inflammatory cells (see next section). For example, in an in vitro study the leakage of doxorubicin from long-circulating liposomes was confirmed in the presence of fluid obtained from pleural malignant effusions (Gabizon, 1995).

2. Pathologies with Leaky Vasculature: Inflammatory and Infectious Sites. Surprisingly, little attention has been paid with regard to passive accumulation of long-circulating particles at inflammatory sites and infectious foci. However, a preliminary experiment has confirmed passive accumulation of intravenously injected PEG-1900 grafted liposomes at arthritic sites in rats (Corvo et al., 1999); the processes of drug release and therapeutic monitoring were not addressed in this study. Long-circulating radiolabeled liposomes (e.g., indium-111 or technetium-99m-labeled PEG-1900-coated liposomes of 100 nm in size) have also proved to be valuable tools in detection of experimental and clinical infectious and inflammatory foci by gamma scintigraphy (Goins et al., 1993; Boerman et al., 1995, 1997a,b; Dams et al., 1998, 1999, 2000b). In terms of diagnostic accuracy, the scintigraphic results seem to be similar between radiolabeled liposomes and radiolabeled leukocytes (the established method for diagnostic imaging). Therefore, liposome-based radiopharmaceuticals may be an attractive alternative for labeled leukocytes, because they can be prepared easily and handling of human blood is not required. Such radiopharmaceutical vesicles also show advantages over radiolabeled polyclonal antibodies in the detection of infectious and inflammatory foci. For example, in rats with focal S. aureus infection the abscess-to-muscle ratios with indium-111-labeled PEG liposomes were approximately four times as those obtained with indium-111-labeled polylclonal IgG (Boerman et al., 1995). Current limitations associated with radiolabeled IgGs include the inability to image vascular lesions due to slow clearance of immunoglobulins from the blood, moderate sensitivity in particular patient groups, a relatively long time-span between injection and final diagnosis, a high radiation burden, and poor stability of the radiolabel (as in technetium-99m-labeled IgG) and accumulation in nontarget tissue (e.g., the kidneys) (Oyen et al., 1991).

3. Spleen. There are several clinical advantages for efficiently redirecting intravenously injected colloids to the spleen (Moghimi, 1995b). For example, the design of spleen-specific radiopharmaceuticals and contrast agents may enhance visualization of abnormalities of the spleen number or position, and detection of residual tissue after splenectomy, which is otherwise hampered by the left hepatic lobe. Similarly, encapsulation of drugs such as antimicrobial, hemoxygenase inhibitors, vinca alkaloids, and cyclosporin in splenotropic carriers may offer an effective approach for the treatment of several splenic and hematological diseases and disorders (e.g., malaria, hairy-cell leukemia, idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia) as well as for prolonging survival of allografts.

View larger version (171K): [in this window] [in a new window]   Fig. 4.   Electron micrograph of a rat splenic red pulp macrophage showing massive accumulation of poloxamine-908-coated polystyrene nanospheres (220 nm) in lysosomal structures. Bar = 2 µm. Reproduced from Moghimi et al. (1993b) with permission of Elsevier Science (Oxford, UK).

View larger version (157K): [in this window] [in a new window]   Fig. 5.   Electron micrographs of poloxamine-908-coated polystyrene nanospheres (220 nm) incubated in the presence of rat serum (a) and a magnified view of a rat splenic red pulp macrophage lysosome with accumulated poloxamine-908-coated polystyrene particles (220 nm). Bar = 200 nm.

4. Lymph Nodes. The mammalian lymph nodes are aggregates of finely structured lympho-reticular tissues that arrest and eliminate antigenic materials, cells and microorganisms that have gained entry to the tissue fluids and have been conveyed to the nodes by their afferent lymph. In addition, direct passage of antigens or small foreign particles from the blood vessels to the lymphoid parenchyma occurs mainly through permeable vascular endothelium in lymph node vessels, although some species discrepancies do exist (Moghimi and Rajabi-Siahboomi, 1996). By taking advantage of these physiological processes colloidal carriers can be delivered to lymph nodes following intravenous, interstitial, and intramuscular injection. Experimental and clinical goals of delivering drugs and imaging agents to lymph node macrophages and other lymphatic elements are numerous and have been discussed in detail elsewhere (Moghimi and Rajabi-Siahboomi, 1996; Moghimi and Bonnemain, 1999). Here, we limit our discussion to the engineering aspects of long-circulating particles with regard to lymphatic targeting in two different scenarios of medical imaging to illustrate the role of the above physiological principles with regard to passive targeting and requirements in clinical medicine.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Footpad and lymph node distribution of uncoated and POP/POE copolymer-coated polystyrene nanospheres (60 nm) following subcutaneous injection into rat footpads. Copolymers were poloxamer-401, -402, -403, and 407. These poloxamers all share a central POP segment consisting of 67 propylene oxide units. Closed columns represent regional lymph nodes (popliteal and iliac nodes), and open columns represent footpads. For experimental details see Moghimi et al. (1994).

Active targeting increases the probability of redirecting long-circulating particles to a designated but accessible targets. Unlike passive targeting, in this case, ligands or homing devices that specifically bind to surface epitopes or receptors on the target sites, are coupled to the surface of the long-circulating carriers. Considerable attention has been focused on long-circulating liposomes for active or ligand-mediated targeting. For example, various endogenously occurring ligands and substances have been attached to PEG-grafted vesicles, which include oligosaccharides that mimic ligands for selectins (e.g., sialyl Lewis X) (Murohara et al., 1995; DeFrees et al., 1996), folic acid (Lee and Low, 1994; Lee and Huang, 1996; Goren et al., 2000), oligopeptides (Zalipsky et al., 1995, 1997) plasminogen (Blume et al., 1993) and interestingly antibodies (Mori et al., 1991; Torchilin et al., 1992; Ahmad et al., 1993; Allen et al., 1995b; Bloemen et al., 1995; Spragg et al., 1997) and their fragments (Park et al., 1995; Kirpotin et al., 1997; Drummond et al., 1999). The biological performance of such constructs has only been tested in selected cases.

1. Ligand Coupling. The most important criteria in the coupling procedure is to control the surface density of the ligand. Among the polymers discussed so far, poloxamers and PEG have received most attention. Derivatization of the end group of poloxamer POE chains with pyridyl disulfide has be achieved (Li et al., 1996). The disulfide bridge is resistant to hydrolysis. Release of the pyridyl thioketone can only take place following disulfide exchange with a thiol-containing moiety on the peptide or protein to be attached to the surface. By mixing derivatized with native poloxamer before nanoparticle coating, it may be possible to control the density of the surface functional groups, since there is no preferential adsorption of either species.

3. Antibody-Mediated in Vivo Targeting (the Cart in Front of the Horse?). Long-circulating immunoliposomes are interesting but puzzling constructs. Surely, the presence of a surface attached monoclonal antibody can stimulate vesicle clearance by the nonspecific macrophage Fc receptor. However, the circulation time of PEG-grafted immunoliposomes is influenced by the density of the attached antibody (Maruyama et al., 1990; Allen, 1994b; Allen et al., 1995a). At densities below 50 µg antibody/µmol phospholipid, immunoliposomes are cleared at faster rates than those seen for antibody free long-circulating liposomes and demonstrate some target binding activity (Allen et al., 1995a). At densities above 100 µg antibody/µmole phospholipid, clearance rates are extremely rapid with massive immunoliposome localization within macrophages of the RES (Allen et al., 1995a). Indeed, the concept of in vivo target binding of long-circulating immunoliposomes have been demonstrated successfully by Maruyama et al. (1990) and Mori et al. (1993). These investigators have targeted the murine pulmonary endothelium using monoclonal antibodies against the lung endothelial protein thrombomodulin. Similarly, Ahmad et al. (1993) and Allen et al. (1995a) have shown antibody-targeted delivery of doxorubicin entrapped in PEG-grafted liposomes for the eradication of a lung metastatic cancer in a murine model, where the targeting was associated with a significant decrease in the tumor burden. However, it should be emphasized that lung targeting is easy since following tail vein injection the long-circulating immunoliposomes encounter lung capillary beds first, where a significant fraction of the dose will be retained.

4. Folate-Mediated Targeting. To overcome some of the hurdles obstructing the traditional antibody based approaches in cancer targeting, several investigators have turned to folic acid (Goren et al., 2000). In principle, folate offers many advantages as a targeting ligand over antibodies. It is presumed nonimmunogenic due to its small size, has good stability, and is highly specific for tumors (Sudimack and Lee, 2000). The elevated expression of the folate receptor has frequently been observed in various types of human cancers including ovarian, endometrial, colorectal, breast, lung, and renal cell carcinoma as well as brain metastases derived from epithelial cancers (Sudimack and Lee, 2000). The receptor is generally absent in most normal human tissues with the exception of placenta, choroid plexus, lung, thyroid, and kidney. Folic acid has been attached to liposomes via a long PEG spacer (molecular mass of 3350 Da) to overcome the steric hindrance encountered at the cell surface (Lee and Low, 1994). Such conjugates are rapidly internalized into a low pH endosomal compartment by folate receptor-bearing cancer cells via receptor-mediated endocytosis and proved successful in delivering of protein toxins, anticancer agents, genetic materials as well as diagnostic agents. Again, the incorporation of folate is unlikely to affect the extent of liposome extravasation from the blood into the tumor interstitum. Whether this strategy has any therapeutic advantage over nontargeted long-circulating particulate systems remains to be shown.

An interesting case of "accidental" targeting was reported with respect to long-circulating polystyrene nanospheres coated with poloxamer-407 (Porter et al., 1992a,b). Among three different commercially available samples of poloxamer-407, only one poloxamer sample was found to be capable of directing polystyrene particles (60-150 nm) to sinus endothelial cells of the rabbit bone marrow 12 to 24 h after intravenous injection (Fig. 7), although they all suppressed particle recognition by macrophages of the RES (Porter et al., 1992a). A similar process was also reported with a batch of poloxamer-338 (Illum and Davis, 1984). This homing process is apparently exclusive to the rabbit, as bone marrow targeting can not be demonstrated in other species such as mice and rats (Porter et al., 1992a). These observations are interesting as these particular polymers render nanospheres both anti-adhesive (by virtue of the extend POE configuration on the particle surface which acts as a steric barrier) and pro-adhesive (by virtue of recognition by bone marrow sinus endothelium). Therefore, the process is analogous to the anti-adhesive and pro-adhesive behavior of mucins. The mechanisms of recognition still remain to be elucidated.

View larger version (182K): [in this window] [in a new window]   Fig. 7.   Electron micrographs showing the localization of poloxamer-407-coated latex nanospheres (150 nm) in sections through sinusoidal endothelial cells of male New Zealand White rabbits. The poloxamer was supplied by Ugine Kuhlman Company (Manchester, UK) and used as received. Particles (4.0 mg) were injected intravenously via the marginal ear vein. Bone marrow was extracted from a femoral bone and fixed by an immersion technique (Porter et al., 1992b). Poloxamer-coated nanoparticles are present in large vesicles designated as dense bodies (arrowheads). The endothelium of other bone marrow vessels remained indifferent to the presence of poloxamer-407-coated beads. In animals that received uncoated polystyrene particles, the sinus endothelial cells in bone marrow rarely participated in particle uptake from the blood. Micrographs courtesy of Dr. S. M. Moghimi. Bar = 1 µm.

An important question arises here, which must not be ignored, is the extent of reproducibility in biological targeting using polymeric materials. The main problem seems to be the exact composition of each polymer batch: the presence of a range of different molecular weight copolymers as well as homopolymers (Moghimi and Hunter, 2000b), which needs to be defined precisely. Nevertheless, hidden within this variability is the inference that exquisite levels of recognition and specificity are achievable with such polymers.

	IX. Conclusions

Top Previous Next References

In this article we have critically examined the wide range of approaches in the design and implementation of colloid based long-circulating drug carrier systems. The current focus of research in this area has generated a broad spectrum of carriers with the majority showing rather poor biological performance in terms of both circulation time and target specificity. Again, the concepts of drug incorporation and release from such particles has been poorly investigated. Also the ultimate fate of the constituent materials of the vehicles remain undefined. Our inadequate understanding of physicochemical interactions between a carrier and host immune system seems responsible for technical difficulties in constructing a multicomponent long-circulating system with optimal biological performance. However, the limited evidence suggests that the majority of polymers used in particle engineering can affect some macrophage accessory functions (e.g., cytokine release, major histocompatibility complex class II antigen expression). This may have important implications in vaccine design, particularly for subcutaneous and intramuscular protocols, using colloidal carriers. In designing long-circulating particles, attention must also be paid toward their application to a relevant pathology. Here, it is still unclear which approach to targeting is likely to yield the best results. Clearly much more needs to be learned about the physicochemical nature of interactions between polymers and the cell surface, in particular which components or combination of components on a given cell surface is responsible for these interactions. In terms of the endothelial cell surface, we have only begun to appreciate differences in the complement of molecules exposed on the surface.

We strongly believe that, understanding the nature of the scavenging properties of macrophages and more importantly the precise role of the different macrophage populations in these processes are fundamental to the rational design of long-circulating as well as target-specific colloidal entities. Although, the current limited evidence suggests that stimulated macrophages can recognize long-circulating carriers in vivo, the nature of the macrophage plasma membrane receptors involved in this process and their mode of action is still undetermined. It is interesting to note that in vivo a stimulated Kupffer cell will not phagocytose healthy erythrocytes. Is it therefore possible that an understanding of this mutual relationship between the stimulated phagocyte and the erythrocyte holds the key for the design of a truly synthetic long-circulating colloid. Perhaps, knowledge gained from research in this area could end the long search for an artificial red blood cell. This will require a greater understanding of erythrocyte membrane biochemistry and biophysics. For example, the unique biconcave disc shape in conjunction with the deformable nature of the erythrocyte may have an influential effect on the mode of interaction of red blood cells with other biological constituents.

At present the research focus tends to be on a single strategy rather than using a range of potentially complimentary tactics for engineering of long-circulatory particles. For example, inclusion of GM3 in liposomes has resulted in increased circulation time of the vesicles, whereas a combination of GM3 with complement regulatory proteins may further enhance vesicle longevity. Similarly, approaches with polymeric systems have rarely used a combination of coating polymers which may afford a higher energy steric barrier. The translation of microbial strategies to overcome macrophage recognition is another resource of elegant and evolutionary developed host uptake avoidance mechanism(s). However, caution must be exercised with respect to microbial surface expression which will vary depending on the exposed microenvironment (Cheung and Fischetti, 1988; Bayer et al., 1989).

To date synthetic polymers have been the materials of choice in the construction of long-circulating colloids. A deficiency in this field which have to be rectified is the precise characterization of these macromolecules in terms of molecular weight and purity. The problem with molecular weight determination is due to technical difficulties associated with the techniques available at present. For instance, aqueous gel permeation chromatography, used for the separation and molecular weight determination of water-soluble polymers, is a relatively new technique, and its use is hindered by the fact that there are very few molecular weight standards that can be used to calibrate the system. Also, little work has been done in determining the nature of polymeric degradation products, their biological activity and ultimate fate following parenteral administration.

Finally, a major factor that has been ignored is genetics. Future considerations must be given toward the immunogenetic and pharmacogenetic differences and related polymorphisms.