Theoretical Considerations of RES-Avoiding Liposomes

Advanced Drug Delivery Reviews 32 (1998) 119138
Theoretical considerations of RES-avoiding liposomes: Molecular mechanics and chemistry of liposome interactions
Mikhail I. Papisov*
Department of Radiology, Division of Nuclear Medicine, Massachusetts General Hospital and Harvard Medical School, Radiology EDR-517, MGH, Boston, MA 02114, USA
Abstract The development of long-circulating, RES-avoiding liposomes has become a remarkable milestone in the progress of contemporary pharmacology. Drugs incorporated in such liposomes are protected from fast metabolization and clearance, and can be further targeted to a desired tissue site. Ideally, future developments should result in drug carriers which can identify and act upon their targets with even higher efciency and selectivity, preferably close to or exceeding that of the natural immune cells. Further increasing carrier inertness with regard to the normal biological milieu is the major requirement for future success. The ability of natural blood components to circulate with blood for several days and weeks presents both the motivation and the challenge for further research. Today, even the best available preparations are inferior to natural proteins and cells with regard to their ability to remain in circulation by approximately two orders of magnitude. In view of the above, it seems vitally important to determine the mechanisms responsible for glycolipid- or polymermodied liposome protection against RES, and whether any potentially useful mechanisms have been underutilized. Furthermore, identication of quantitative dependencies between liposome structure and pharmacokinetics (and mechanisms underlying such dependencies) would benet future research and reduce the cost of development. This paper discusses the relationships between liposome structure and circulation with respect to the theoretical mechanistic models of mass transfer, liposome interactions with cells and blood proteins, and boundary effects resulting from surface modication. Special attention is paid to the practical application and limitations of the models. 1998 Elsevier Science B.V. Keywords: Drug carrier; Drug delivery; Drug targeting; Liposome; Macromolecule; Modeling; Pharmacokinetics; Polymer; Steric protection
Contents 1. Introduction ............................................................................................................................................................................ 2. Mechanistic aspects of liposome circulation .............................................................................................................................. 2.1. Circulation time as a measure of liposome interactions in vivo ............................................................................................ 2.2. Pseudohomogenous model of long-circulating liposome biokinetics..................................................................................... 3. Liposome protection by molecular brushes ................................................................................................................................ 3.1. Liposome interaction in vivo: The most general aspects ...................................................................................................... 3.2. Recognition proteins of plasma ......................................................................................................................................... 3.2.1. Major opsonins ...................................................................................................................................................... 120 120 120 121 122 123 124 125
*Tel.: 1 1 617 724 9655; fax: 1 1 617 724 8315; e-mail: misha@nmr.mgh.harvard.edu 0169-409X / 98 / $32.00 1998 Elsevier Science B.V. All rights reserved. PII S0169-409X( 97 )00135-X
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3.2.2. Minor opsonins ...................................................................................................................................................... 3.2.3. Animal vs. human and normal vs. pathology models................................................................................................. 3.3. The surface of long-circulating liposomes .......................................................................................................................... 3.4. Mathematical modeling of molecular brushes ..................................................................................................................... 3.5. Mechanics of surface protection by molecular brushes ........................................................................................................ 3.5.1. The type 1 hindrance (steric shielding) .................................................................................................................. 3.5.2. The type 2 hindrance (steric obstruction) ............................................................................................................... 3.5.3. Molecular brush: Structure and protective properties................................................................................................. 4. Interactions of molecular brushes in vivo .................................................................................................................................. 4.1. Interactions of protective polymers with components of biological systems .......................................................................... 4.2. Approaches to the development of better liposome surface modiers ................................................................................... 5. Conclusions ............................................................................................................................................................................ Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................
1. Introduction Over the last few years, methods of regulating liposome pharmacokinetics via surface modication have gone through signicant development and reassessment. The dependence of biokinetics on surface characteristics was discovered early in liposome development. The pioneering studies related mostly to the biological signicance of liposome size, dose and general surface characteristics, such as charge, hydrophilicity, uidity and lipid composition. The development of surface-modied long-circulating liposomes was the most signicant achievement of the last decade (reviewed in other papers in this issue and elsewhere [13]). The availability of this novel group of drug carriers with fundamentally new pharmacokinetic features seems to open an opportunity to raise drug development to a highly rational level, where the relationships between carrier structure and biokinetics are known, and the pharmacological objectives of drug development can be reached, at least in part, on the basis of quantitative mathematical forecasting. Developing such quantitative methods for predicting biological properties of liposomes on the basis of their structure is the major objective of theoretical analysis. This paper analyses the available data on longcirculating liposome transfer and interactions in vivo and the concepts of the underlying mechanisms of these interactions, and discusses the approaches to the mechanistic description and modeling of various stages of liposome pharmacokinetics.
2. Mechanistic aspects of liposome circulation In general, liposome biokinetics can be formalized on the basis of existing empirical and semi-empirical methods (reviewed in Ref. [4]). These methods provide excellent quantitative tools for data description where no or little information regarding the elementary mechanisms of biokinetics is available. Physiological mechanisms of liposome transport in vivo further link the pharmacokinetics with fundamental liposome properties, such as composition and size [5]. Several aspects of liposome interactions in vivo, which contribute to the processes of liposome transfer and uptake, remain obscure. However, as discussed below, liposome blood kinetics readily provide a gross measure for liposome interactions in vivo. Furthermore, biokinetics of long-circulating (unlike the other types) liposomes can be expressed in terms of pseudo-homogenous kinetics, which signicantly simplies the connections between liposome structure and pharmacokinetics. The quantitative relationships between liposome pharmacokinetics and interactions in vivo described in this section can provide simple approaches to data processing and calculation of absolute kinetic parameters of the processes occurring in vivo.
2.1. Circulation time as a measure of liposome interactions in vivo

In our previous publications, we have shown that biokinetics of intravenously injected particles and
M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138
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polymers can be described by relatively simple mechanistic models of mass transfer [6]. Furthermore, models with localized parameters can be readily transformed into two-compartmental models with strictly monoexponential kinetics (in the absence of saturation). This results in simple equations for large (non-extravasating) polymers and particles. For example, polymer concentration in plasma (C ) can be expressed as: D0 2 C 5 ]e a t V and polymer accumulation in target i (Di ) is: k i Fi 2 Di 5 D0 ](1 2 e a t ) Va (2) (1)
where Fi and k i are blood ow through tissue i and the capture coefcient in this target, respectively. A capture coefcient reects interactions of a particular preparation with a particular tissue (regardless of their mechanism) and is expressed via the probability of binding to the tissue during a single passage of preparation-carrying blood. V is plasma volume, D0 is injected dose, and 1 a 5] V
Ok F
i i
(3)
Experimental evaluation using radiolabeled iron oxide nanoparticles has shown that kinetic data ts the theoretical equations with R . 0.95, often R . 0.99 [7,8]. It is notable that, within this model, the experimentally measured blood half-life t1 / 2 5 ln(2) /a. This immediately makes blood half-life a convenient and mathematically exact measure of polymer (particle) interactions in vivo, because a is a function of all interactions of a particular preparation in all tissues (Eq. (3)). Local capture coefcients can be easily calculated as soon as the blood ow through the tissue of interest is known. We have also shown how the experimentally measured parameters of carrier circulation can be used to model the pharmacokinetics of the carrier-transported drug [9].
2.2. Pseudohomogenous model of long-circulating liposome biokinetics

Interestingly, the above model can be further simplied for preparations with long blood half-life.
During the initial period of circulation, intravenously injected liposomes are distributed almost exclusively to the blood compartment [5]. Redistribution to other tissue compartments is usually very slow (e.g., t1 / 2 . 20 h in rats [10]). Any signicant lymphatic return of intact liposomes has not been reported. Due to these facts, in particular because blood recirculation is much faster than transfer to other tissues, the blood compartment can be considered to be ideally mixed during the entire length of the circulation. Accordingly, the bolus effect should be considered negligible. For the same reason, if liposome interaction with tissues is not blood ow-limited in any signicant target (otherwise the blood clearance would be fast), all parameters related to the circulation in blood can be excluded from the transfer model. As a result, long-circulating liposome interaction with cells and tissues exposed to the blood can be handled as a system of conventional chemical reactions in ideally mixed media. Liposome transfer to other tissue compartments, for example by extravasation via transcytosis or endothelial leakage, can be accounted for in exactly the same way. However, this would require experimental data on liposome extravasation, which is not yet available, although some pilot experiments have already been performed [11]. This most direct connection of biokinetics with chemical reactions and other interactions at the liposome surface suggests that long-circulating liposome pharmacokinetics would be readily and mathematically precisely predictable if we only knew the liposome counteragents in vivo or, at least, the most important of them. Unfortunately, this information is not yet available. To determine what missing information would be most important, let us analyze a more detailed scheme of liposome interactions in vivo as depicted in Fig. 1. It is notable that most of the processes in Fig. 1 are conventional chemical reactions that can be described in terms of conventional kinetics. For example, recognition protein binding (see Section 3.2) can be described as typical chemical reactions, either reversible (e.g., immunoglobulin) or irreversible (e.g., complement-3). Binding of both unopsonized and opsonized liposomes to cells should be considered reversible, while endocytosis is an irreversible process (unless retroendocytosis has been experimentally established). True liquid-phase endo-
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liposomes and liposome complexes can be further resolved (either analytically or numerically) if the individual elementary processes are known and their parameters can be experimentally determined. Intimate details of the mechanisms of liposome interaction with recognition proteins (for which little or no data is available) can be even more important for optimizing the liposome surface, as discussed in the next section.
3. Liposome protection by molecular brushes The mechanism for the long circulation of glycolipid- and polymer-modied liposomes was rst related to the effect of surface hydrophilization, similar to that described for polystyrene particles. Later, more specic hypotheses were presented, of which the hypothesis of steric protection against interactions with the biological milieu has become most widely accepted. Most often, the term steric protection is used with regard to liposome interactions with opsonins, i.e. proteins enhancing liposome phagocytosis upon binding. Before discussing the theoretical aspects of the mechanisms of liposome protection against RES, we should note that the term steric protection is not self-explanatory. The word steric is as vague as having to do with spatial arrangement of the atoms in a molecule [12]. Therefore, a sentence like surface-bound PEG sterically protects liposome surface means nothing more specic than surfacebound PEG protects the liposome surface because it is surface-bound. One of the rst physical explanations of steric protection with regard to drug carriers was presented on the basis of the steric repulsion mechanism, where polymer chains repel protein molecules due to polymer chain constriction [13]. This provides a much more specic mechanistic explanation. However, it would be desirable to translate the protective effects of surface modiers (both polymers and glycolipids) into kinetic parameters of liposome reactions with biological milieu. This would directly link the protective layer structure with liposome biokinetics and, therefore, immensely streamline the process of liposome optimization.
Fig. 1. General scheme of liposome clearance. Stages: (I) Intact liposome. (II) Liposome complexes with recognition proteins. (III) Liposome associated with cell surface (directly and via recognition proteins. (IV) Phagocytosed liposome. RP, recognition protein. k i , kinetic constants of liposome interaction with RP1, subsequent binding and phagocytosis. Note that recognition protein binding results in a variety of complexes containing the same or different proteins.
cytosis can be described as a simple irreversible rst-order process. The stage of phagocytosis is signicantly more complex regarding the kinetic description. The act of phagocytosis consists of at least two stages (triggering, which includes receptor clustering, and endocytosis, which includes cytoskeleton rearrangement). A detailed mechanistic kinetic description of phagocytosis is hardly possible, at least at the current level of knowledge. However, the overall kinetics of phagocytosis can be described by kinetic equations similar to those used for the description of enzymatic reactions. Their particular form will depend on the values of the kinetic constants and the number of reactive centers at each stage. For example, theoretically the blood kinetics can be purely monoexponential, Michaelis-like, linear (zero-order) or even sigmoidal (depending also on the blood-borne liposome forms studied; see also Ref. [5]), which can provide valuable information regarding the uptake mechanism. The complex kinetics of the in vivo interactions of
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3.1. Liposome interaction in vivo: The most general aspects

Another important question, as already mentioned, relates to the nature of the liposome counteragents which mediate liposome withdrawal from the circulation. While phagocytic cell populations are denitely the nal destination of liposomes and there is a strong correlation between liposome uptake by phagocytes in vitro and in vivo [14], the intermediate liposome complexes with blood proteins are poorly studied. Besides, phagocyte properties with regard to circulating particulates may vary signicantly in different phagocyte populations [15,16]. Such differences in the response of different phagocyte populations to liposome surface modication in particular, have also been suggested [14]. It has also been reported that liposomes of different composition bind different pools of plasma proteins [17]. The nonspecic liposome binding to a variety of tissues also should be taken into account, as non-specic interactions were suggested to be a major factor of nanoparticulate biokinetics [18]. The nature and physical character of liposome interactions with components of biological milieu can be critically important for developing methods of liposome optimization. Two remarks should be made here regarding the commonly used terminology. One relates to the term non-specic binding. The term specic interactions implies the presence of a
high-afnity center. The term non-specic most often refers to interactions where such a center can not be identied. Instead, non-specic interactions involve either the entire structure or a certain domain of a molecule or particle. With regard to macromolecular and supramolecular structures, which liposomes are, this can be more specically dened in terms of physical chemistry as a cooperative system of weak associations, or cooperative binding. The action of high-afnity centers also involves cooperative processes, but in the case of non-specic interactions the interacting moieties are distributed over the molecule (particle) rather than organized in any active center (Fig. 2). To further illustrate the difference with a macroscopic example, we could compare the non-specic binding with the action of Velcro, while specic binding could be compared with a button or a snap. The second remark relates to the term opsonization. As many other researchers, we refer this term to liposome interactions with specialized recognition proteins (rather than any plasma proteins). Recognition proteins have clearly dened binding sites, which immediately relates their interactions to the class of specic binding. They have important functions in either immune or other systems and can trigger a variety of events via interactions with cell receptors. Other proteins, that may bind liposomes via direct or indirect non-specic interactions, clearly belong to a different class, both mechanistically and functionally.
Fig. 2. Scheme illustrating the difference between specic and non-specic interactions of a macromolecule with liposome surface or another macromolecule.
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Recognition proteins and cell receptors comprise well organized active centers which recognize and bind (with the exception of some lectins) relatively small molecular fragments. Their interactions, therefore, are focused within a relatively small ( , 1 nm 2 ) area. Immunoglobulins, mannose-binding protein, pentraxins, conglutinins and many other recognition proteins of blood have several binding centers. However, they bind their counteragents via a few focused areas of their multiple active centers. Recognition protein binding does not require any continuous close contacts (possibly with the exception of bronectins and pentraxins; see Section 3.2). The same is true for most of the individual cell receptors. In contrast, non-specic cooperative interactions do require continuous contacts between the counterparts. The overall stability of cooperative associations can be expressed in common terms of physical chemistry, for example via the association constant K 5 [liposomesubstrate] / [free liposome][free substrate], where concentrations are expressed in moles. The stability constant depends on association energy as K 5 e 2D G / RT (where G is Gibbs free energy, T the absolute temperature, and R the universal constant). In a cooperative bond system the energy is additive, so Kcoop 5 e 2 ( o D G ) / RT , and for a system of n equal bonds Kn 5 e 2 n (D G / RT ) . Thus, the cooperative binding constant grows exponentially with the number of associations (which can be proportional, for example, to the liposomesubstrate contact area). A simple calculation shows that the association constant for a system of n bonds can reach remarkable values even at low single-bond energies. Fig. 3 illustrates that collective low-energy interactions can result in association constant values as high as 10 20 M 2 1 , i.e. non-specic interactions can be even more efcient than typical high-afnity interactions in vivo (where K usually does not exceed 10 15 M 2 1 ). In summary, liposomes in vivo can participate in two distinctively different types of binding. The rst type involves one or a few single-point contact(s) with soluble or membrane-bound recognition proteins; the second type requires continuous surface-tosurface contact. This difference will be further discussed within the context of the mechanistic analysis of liposome protection by hydrophilic molecular brushes.
Fig. 3. Cooperative association constants as a function of the number of associations. Calculated for single association energies from 0.15 to 12 kJ mol 2 1 .
3.2. Recognition proteins of plasma

Boundary interactions in vivo are often mediated by components of biological uids (blood, interstitial liquid), cells and the extracellular matrix. For example, particle clearance from blood is most often attributed to uptake by phagocytes, e.g. in liver and spleen. Phagocytosis may be mediated by specialized recognition proteins, such as immunoglobulins [19], bronectins [2022], complement system [2326], soluble lectins [2730], vitronectin [31], etc. Several recognition proteins have been shown to be able to bind liposomes, cell membranes or phospholipids. Opsonin-mediated phagocytosis may have an element of cooperativity, because the phagocytosis rate depends on the number of opsonin molecules in the immunocomplex [32]. In some cases, opsonins trigger phagocytosis only in cooperation with other factors present in the immunocomplex [33]. The distinctive feature of recognition proteins relates to their capability to enhance polymer binding and uptake by phagocytes [34]. Specialized opsonins contain at least one receptor-recognizable site, and often more than one binding site.
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Specialized opsonins bound to a liposome will expose their receptor-recognizable sites and, thus, mediate further interaction of the liposomeprotein complex with cells. The latter process depends on numerous factors. In particular, the fate of a complex depends on the number and type(s) of incorporated opsonin molecules. Some immunocomplexes (e.g., containing a small number of opsonin molecules) are known to circulate for hours and even days [35], while others are immediately arrested by phagocytes (e.g., in liver, spleen, bone marrow or lungs [36]).
3.2.1. Major opsonins Major specialized opsonins of normal blood, such as immunoglobulins, proteins of complement system and bronectin, are often mentioned in publications in the eld of macromolecular and supramolecular pharmacology. Immunoglobulins are the most specialized opsonins that can recognize and selectively bind specic fragments of macromolecular species. Immunoglobulins have high afnity for antigens known to the immune system of a certain individual. The major concern regarding liposome pharmacokinetics (and integrity) relates to opsonins specic to phospholipidprotein complexes [37,38]. Fibronectins are large proteins abundant in plasma and extracellular matrix. There are indications that the bronectin molecule in solution is a disk, which has a diameter and thickness of 30 and 2 nm, respectively [39]. However, the bronectin molecule consists of several domains connected by relatively exible joints and is believed to be capable of unfolding [40], in particular as a result of substrate binding [41]. Fibronectins are believed to participate in hepatic uptake of particulates [42] and may play a role in the pharmacokinetics of liposomes [43]. Complement comprises a set of proteins that work to eliminate microorganisms and other antigens from blood [44]. The task is achieved by complement cooperation with cells that express complement receptors and, often, with antibodies and other opsonins. Proteins of complement have historically assigned numbers which do not completely correspond to the sequence of their actions. The third complement protein, C3, has a central role in complement function. One of the most important func-
tions of complement is to mark antigens with fragments of C3, thus making them recognizable by phagocytes bearing C3 receptors. There are indications, however, that phagocytosis may not be triggered by C3 alone and may require an additional signal [33]. To be bound to an antigen, C3 should rst be activated by C3 convertases (which are antigenbound) that produce C3b, a C3 fragment containing an active cyclothioester group; the latter is chemically active and may covalently bind nucleophilic functional groups, preferentially OH groups [45]. C4, which is less abundant, has a similar thioester group but preferentially binds amino groups [46]. C3 convertases are protein complexes formed either with participation of activated C1 complex, C2 and C4 (C4b2a, classical activation pathway) or by C3 itself and factors B and D (C3bBb, alternative pathway). Subsequent C3 activation produces more antigenbound C3b and more C3 convertase complexes. Particle ability to bind complement presumably depends on the presence of nucleophilic functional groups that may bind C3 or C4, and / or domains that may bind C1. The C1 complex consists of three proteins, C1q, C1r and C1s, where C1q is the component responsible for substrate recognition. C1q binds to a wide variety of substances, including IgG and IgM-containing immune complexes, lipopolysaccharide and porins from Gram-negative bacteria, ligand-bound C-reactive protein, nucleic acids and other highly charged polymers [47]. It is notable that even if C1 is not activated, C1q binding may alter polymer / particle pharmacokinetics due to C1q interactions with bronectin, C1q receptors, etc. Some information on complement interactions with liposomes is available [48,49].
3.2.2. Minor opsonins Several proteins present in blood in small concentrations are also recognition proteins bearing binding sites, receptor-recognizable domains and, sometimes, sites for binding another recognition molecule. Some minor opsonins are acute-phase proteins (i.e., their concentration in plasma increases as a result of inammation or trauma). Many minor opsonins trigger complex responses that often include histamine release. Most minor opsonins are
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lectins. For example, conglutinin is a 330 kDa lectin with four binding sites and high specicity to terminal (a1 2) oligomannose [50]. Mannose-binding protein (MBP), a protein that was recently found to be identical to a component of Ra-reactive factor (RaRF), binds terminal non-reducing mannose-, N acetylglucosamine and glucose residues. MBP was reported to enhance phagocytosis mediated by C1q receptor; in addition, MBP was found to trigger complement activation via activating the C1r 2 C1s 2 complex [51]. MBA is suggested to be an acutephase protein, showing concentrations in normal sera from 0 to 870 mg l 2 1 . Lipopolysaccharide binding protein (LBP) was recently shown to directly recognize lipopolysaccharides and to trigger remarkable biological responses [52,53]. Vitronectin, a component of the extracellular matrix, participates in complement reactions (often called protein S) and probably may also act as an opsonin [54]. Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure, identical with the non-brillar amyloid P component found in amyloid deposits. SAP is composed of 10 (according to some data, ve) non-covalently associated globular subunits, each of 25 kDa, arranged as two face-to-face cyclic pentameric discs [55]. SAP binds to a number of apparently unrelated substances, including phospholipids [56]. Another pentraxin, Creactive protein (CRP), is an acute-phase protein. Human CRP interacts with bronectin, brinogen and phosphorylcholine; rat CRP also binds to phosphorylethanolamine [57]. In view of the recent publication of Cullis [58], b2-glycoprotein I ( b2GPI) has become one of the most interesting minor proteins participating in liposome removal from blood. Although the exact biological function of this protein has not been established, the presence of a specic anionic phospholipid-binding site on one of the ve domains [59] suggests b2GPI participation in recognition of apoptotic cells [60]. An anticoagulatory function has also been suggested [61]. b2GPI is a reverse acutephase protein (concentration in blood during acute phase decreases [61]). Binding to the lipid membrane is followed by a signicant conformation change [59]. It has been shown that the liposome clearance rate is in excellent correlation with b2GPI amount on the surface, and that anti-b2GPI antibodies suppress the opsonic activity of the protein [58].
3.2.3. Animal vs. human and normal vs. pathology models Data available from the most recent publications suggest that the specicity of some recognition proteins (especially minor) may signicantly vary from species to species. For example, human and rodent pentraxins show different phospholipid specicities [62]. The same may be true for some receptor systems. Unfortunately, there is very little information regarding the extent of these differences, and almost no quantitative data on the receptor ligand association constants in different animals. As a result, it is difcult to predict precisely how different the behavior of liposomes can be in man as compared to animal models. This problem can be further complicated in pathology models by the appearance of acute-phase proteins and variations in concentration of other signicant proteins, such as b2GPI. Acute-phase proteins comprise a variety of binding sites and, therefore, can potentially alter liposome pharmacodynamics. Although liposome surface modication is intended to diminish liposome interactions, the potential effects of opsonins should not be disregarded, because, most likely, none of the surface modication techniques will provide absolute surface protection. The results of these interactions in man are difcult to predict, because (1) the specicity of some acutephase proteins is species-dependent, (2) concentrations of these proteins in animal pathology models are not particularly well studied and (3) their interactions with liposomes are also insufciently studied. With respect to future liposome development, it would be particularly interesting to determine how these proteins interact with liposomes, how molecular brushes alter the overall biological effects of these interactions, and what corrections should be used in extrapolating animal data to man. 3.3. The surface of long-circulating liposomes
The success in developing long-circulating liposomal preparations is due to liposome surface modication with glycolipids and polymers [3]. Despite the differences in chemical structures between, for example, G M1 and PEG derivatives, liposomes modied with these substances have one common feature: a brush of elongated hydrophilic molecules on the surface. Similar brushes are present
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on all eukaryotic cell surfaces, and on surfaces (interfaces) formed by the extracellular matrix [63]. The natural molecular brushes, which consist of a variety of glycoproteins and glycolipids, have an informational function [63] and, at least some of them, a protective function. The effects of brushes planted on the liposome surface can be described in the same terms. Thus, the protective function would relate to the steric effects hindering liposome interactions with biological milieu, while the informational function would relate to the interactions of the brush itself (e.g., glycolipid recognition and binding by natural lectins) that can alter the overall biological reaction to the liposome. Obviously, the desired minimization of the overall biological reactivity of liposome requires minimization of brush reactivity and maximization of the protective properties. The major problem, however, relates to the facts that (a) the intimate mechanisms of steric hindrance remain largely obscure, and (b) there are no rational ways of minimizing brush interactions in vivo beyond the limit set by the current state of knowledge on macromolecule interactions in vivo. It has been shown that macromolecule- and surface-grafted hydrophilic polymer molecules create relatively diffuse boundary layers. While their roots are attached to the foundation, the rest of the chain retains freedom of movement and, therefore, remains dissolved and randomly coils and moves around the attachment point [64]. The resultant constantly changing conformation has been referred to as a mushroom or a molecular cloud. Multiple molecules form a brush. Some authors make a distinction between the mushroom and the brush, although there is no fundamental contradistinction between the former and the latter. The term mushroom may relate to the semi-spherical volume occupied by a single exible elongated molecule. Brush simply means that there is more than one molecule on the surface, whether they form lone standing mushrooms or form a dense network of entangling chains. Preferably, classication of brushes should be made with regard to some specic parameter important for protective properties (for which not much information is available as yet). In fact, the theory of molecular brushes itself is far from being complete (reviewed in Ref. [64]).
Hypothetically, molecular brushes sterically protect their foundation from interactions with a biological substrate, thus diminishing the overall reaction with the biological system. To date, this hypothesis has no reasonable alternatives. However, knowledge of the molecular brush action in vivo has a fragmentary character. The most commonly cited hypothesis suggests that polymer brushes sterically stabilize liposomes or other particles [65] or diminish opsonization [14,66]. The suggested mechanisms include (i) repulsion resulting from polymer chain constriction [13]; (ii) enhanced binding of disopsonins (plasma components that hypothetically prevent the recognition and uptake by macrophages) [67]; (iii) difculties in modeling antibodies around a exible polymer molecule by the immune system [2]; (iv) formation of a molecular cloud which shields the surface from opsonins [68]; (v) formation of a hydrated shell that prevents opsonin entrance [69], etc. Despite the variety of approaches to the explanation of the protective properties and the selected terms, all these hypotheses do not contradict each other and relate to different aspects of the same mechanism. All these approaches appear to be well justied (possibly with a very small exception which relates to a few recent publications where the limits of applicability of the concepts are overextended). Identifying brush parameters that show even rough correlations with protective properties in vitro and in vivo would be highly valuable for liposome surface engineering. One of the most basic obstacles in the understanding of molecular brush behavior in vivo is related to the lack of satisfactory models of molecular brushes that could be used for studying quantitative correlations between brush structure and protective properties.
3.4. Mathematical modeling of molecular brushes

Molecular brushes used in liposome surface protection are short (polymerization degree , 500) and xed at the surface. Physico-chemical methods developed for describing conformations of long polymers in solution [70] are not applicable for their analysis. Models describing dense brushes [64] also have limited use, because poly(ethyleneglycol) and other polymers provide signicant blood clearance prolongation in the form of a relatively loose brush
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[71]. Dense and loose molecular brushes have to be described by distinctively different methods [64,72]. To avoid problems associated with polymer statistics and thermodynamics, we utilized computational modeling of brush behavior, initially using a very simple three-dimensional segmental model [68]. Subsequently, we developed a less simplistic model based on an atom-by-atom calculation on the basis of realistic bond lengths, angles and atom diameters [73]. In this model, torsional angles are randomly generated, and locations of new atoms are checked for coincidence with the previously generated atoms (within Van der Waals diameters) and for location below the hx, y j plane. The impossible positions are re-generated. Thus, the model generates molecule conformations in the indifferent solvent, where moleculesolvent and moleculeself interactions are mutually balanced (Fig. 4). Water does not differ much from the indifferent solvent for several hydrophilic uncharged polymers (the experimentally determined segment length differs no more than 20% from the theoretical) [74]. In our ongoing project, the inuence of good solvent will be accounted for (which is expected to result in relatively insignicant changes). The statistics of conformational data (obtained via generation of thousands of random conformations) allows us to calculate several parameters of the brush, such as average surface and volume densities, average effective distance between brush elements, etc. For example, the statistics of conformations has shown that exible polymer molecules (e.g., PEG) occupy signicantly less space, locating mostly within 10% of their length from the grafting point. Rigid molecules, such as oligo- and polysaccharides,
are signicantly less coiled and reside within 50% of their full length. As a result, the rigid polymer density near the grafting point appeared to be signicantly lower [75]. Modeling has further shown that brush density per unit of liposome surface area signicantly depends on the brush length. The efcacy of steric protection apparently should depend on brush density [74]. Unexpectedly, the effective brush concentration per unit of the liquid phase near grafting points was found to be practically independent of brush length and very insignicant beyond ca. 1 nm from the grafting point [73]. The latter result demonstrates a high brush inhomogeneity (much higher than either mushroom or molecular cloud models can tolerate), which is an important factor in assessing protective properties. On the other hand, the availability of realistic conformations of brush molecules provides the unique opportunity to visualize a polymer-modied liposome. The average area occupied by a lipid molecule on the liposome surface is approximately 1 nm 2 [76,77]. The number of brush elements per liposome of any size can be readily calculated for any given graft density. For example, a 100-nm liposome would carry approximately 400 polymer molecules per each molar percent of PEG, assuming that all PEG molecules reside on the external side of the membrane. The positions of grafting points can be randomly generated (Fig. 5), and the brush molecules planted to these points. One such image is presented in Fig. 6. As can be readily seen, a PEG brush created by 4.4 kDa PEG at 0.2% PEG / lipid ratio does not appear to be dense at all. For example, a practically naked site at the right ( . 500 nm 2 ) is potentially as vulnerable to opsonin attack as an unmodied liposome. However, even liposomes modied with brushes of such densities were reported to have signicantly increased circulation times [81], and liposomes with more than 5 mol% PEG are generally suboptimal [78]. It seems to be important to determine what mechanisms can be responsible for this effect.
3.5. Mechanics of surface protection by molecular brushes

Fig. 4. Example of a simulated random conformation of a poly(ethylene glycol) molecule (MW 4400 Da) grafted to a surface (at the left), and the molecule projection to the surface (shadow).
Liposome surface protection by molecular brushes has been related to repulsion by constricted brush elements [13], which satisfactorily explains the phys-
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Fig. 5. Example of random distributions of protective lipid conjugates over a 100-nm liposome surface. Calculated for 0.2, 1, 2 and 7 mol% modication (top left to bottom right). Note the presence of large ( . 10 nm 2 ) areas of unmodied surface at all but the largest modication degree.
ical nature of the forces responsible for protection (although the fundamental differences in the mechanisms of constriction of macroscopic physical bodies and exible polymer chains must be taken into account). Unfortunately, it is almost impossible to translate such forces into kinetic or thermodynamic constants of interaction in any but the most elementary systems, which molecular brushes are not. To open a way to quantitative approaches to studying brush effects, the protective action of molecular brushes should be described in terms of
either chemical kinetics or thermodynamics. This can be done within the context of the classical kinetic denition of a steric factor, which refers to the fraction of collisions that result in a productive interaction. Alternatively, it can be done via calculation of interaction energies that can be easily translated into equilibrium constants. Recently, we introduced a novel mechanistic concept of steric protection by molecular brushes. Elements of the brush (elongated exible or rigid molecules) have been proposed to act as purely mechanical objects. Their protective action has been
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Fig. 7. Steric shielding. Collision of a recognition protein (RP) with the protected liposome surface can involve either the surface itself (1) or a randomly moving brush element (2). In the latter case, the collision is non-productive. RP, recognition protein molecules.
Fig. 6. Example of a simulated liposome image. Liposome diameter: 100 nm. PEG (4400 Da) content: 0.2 mol%. PEG conformations are simulated as described in the text (see also Fig. 4). Some conformations are used more than once. Liposome surface: dot size approximately corresponds to the area occupied by one phospholipid molecule.
attributed to the character of their positioning, movement and collisions with biological objects (cells, extracellular matrix and proteins). It was found convenient to analyze the steric hindrance or repulsion of two different types.
and P0 are the probabilities of collision with the polymer and of any collision, respectively. It is easy to see that F is also the ratio between the kinetic constants of surfacebiomolecule collision rates: F 5 k protected / k unprotected , both of which can be experimentally determined. It is notable that steric factor F is independent of concentration, which makes it a convenient parameter for in vitro surface testing and subsequent modeling. Within this mechanism, the number of accessible reactive centers on the protected surface remains the same, and the [active center][biomolecule] association energy does not change.
3.5.1. The type 1 hindrance ( steric shielding) This type of hindrance occurs as a result of the collision of an incoming molecule (e.g., opsonin) with a segment of the molecular brush, which redirects the molecule from the liposome surface. In other words, the molecule bounces back after the collision with the brush, not reaching the liposome surface. The issue of polymer attachment to the liposome surface does not play any role here, except that polymer molecules are retained near the surface. This mechanism, in general, is similar to the mechanisms decreasing diffusion rates in polymer solutions. Brush elements randomly move around their grafting points and can intervene in the acts of collision between the surface and a substrate (Fig. 7); consequently, the biomoleculesurface collision rate is decreased by a factor F 5 (P0 2 Pp ) / P0 , where Pp
3.5.2. The type 2 hindrance ( steric obstruction [79] ) Brush molecules can be positioned on the surface in such a way that direct contacts between the surface and the substrate become mechanically impossible or difcult, especially at high brush densities. Steric obstruction critically depends on the ratio between the reagent size (cross-section) and the size of free gaps between the brush elements. The reagent shape can play a very important if not the critical role. For example, at graft densities where free gaps between polymer roots just allow liposome surface contacts with relatively small molecules, e.g. IgG, larger protein molecules may not be able to contact the surface at all (possibly except those comprising active centers on the ends of long narrow protrusions; see Section 3.2). Steric obstruction is particularly effective against non-specic multi-point interactions, where the formation of a cooperative bond system requires con-
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Fig. 8. Steric obstruction of non-specic binding. (1) An unprotected macromolecule readily associates with the surface via a system of weak bonds (see also Figs. 2 and 3). (2) Molecular brush restricts the full-length contact of an analogous protected molecule. M, macromolecule
tinuous surfacesubstrate contacts (Fig. 8). The stability of cooperative complexes crucially depends on the number of associations. The association constant Kn for a cooperative system of n bonds depends on the system energy as Kn 5 e 2D G / RT 5 e O D Gi / RT (4)
where DG is the Gibbs free energy of the system, DGi is the free energy of the i th bond, and o is the energy summation over all n bonds. For a cooperative system of n equal bonds, the total association constant depends on the number of bonds exponentially: Kn 5 e 2 (n D G ) / RT 5 e n (2D G / RT ) (5)
where DG is the free energy of a single bond. If formation of continuous contacts between the surface and the substrate is impossible, the cooperative association constant is sharply decreased for two reasons: (a) the maximal allowed number of associations n max decreases; and (b) several intermediate states (with n , n max ) become impossible (the experimentally determined association constant incorporates all states of adsorption with 1 , n , n max ). It is expected that, due to the steric obstruction, the association rate can be decreased, the dissociation rate can be increased, and some (or all) reactive centers can become completely inaccessible for a counteragent.
3.5.3. Molecular brush: Structure and protective properties According to the proposed mechanisms, steric shielding and steric obstruction depend on fundamentally different structural characteristics of the molecular brush. The efcacy of steric shielding depends on the probability of polymer chain interference with the surfacesubstrate collision. Therefore, steric shielding is expected to increase with concentration of brush material near the liposome surface, so both graft density and brush length can increase the efcacy of steric shielding. Some data on the connections between polymer length and the efcacy of steric protection is available [80]. On the other hand, steric shielding depends on the substrate molecule size. Small molecules readily penetrate the brush, while larger molecules meet signicant resistance [81]. Steric shielding should also correlate with the uniformity of the dynamic molecular cloud formed by the brush over the surface. Conformational mobility of the brush results in higher cloud density and uniformity, which can enhance the shielding effect [68]. In model experiments, poly(ethylene glycol) grafted to liposomes decelerated protein interaction with the liposome surface, whereas liposome-grafted dextran (a more rigid polymer) did not affect protein-to-liposome interaction [82]. A similar experiment conducted using graft copolymers of dextran showed, however, that dextran also may provide effective steric protection [75]. Steric obstruction is expected to increase with brush density, rigidity and length. The substrate size, as compared to the average distance between brush molecule roots (grafting points), should be a critically important factor. Cooperative non-specic liposomesubstrate interactions, e.g. as in liposome cell surface or liposomecellular matrix interactions, should be extremely sensitive to steric obstruction [73,79]. The dependence of steric obstruction on brush density has not been studied. In general, dense brushes can be expected to have greater protective properties with the possible presence of a critical density. On the other hand, dense brushes can hypothetically have a greater intrinsic capability of non-specic binding (due to the presence of a larger number of polymer domains per surface square); therefore, the existence of a certain optimal brush
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Table 1 Steric hindrance as a function of the properties of a molecular brush and a substrate. Steric hindrance increases ( 1 ) or decreases (2) with increase in the factor in the left column Factor Brush density Brush rigidity Brush molecule length Substrate size Cooperative character of interactions Steric shielding 1 2 1 1 2 Steric obstruction 111 11 11 11 1111
density interval can be expected. Besides, polymer modied liposomes become unstable if a certain brush density limit has been exceeded [83]. The hypothetical relationships between brush characteristics and overall protection efcacy are given in Table 1. According to the modeling results, the overall biological effect of molecular brushes depends on the interplay of interactions and steric effects of all types. In turn, the latter depend on both brush structure and carrier type.
4. Interactions of molecular brushes in vivo
4.1. Interactions of protective polymers with components of biological systems

Besides the protective properties, discussed in the previous section, materials for liposome modication should meet another obvious requirement: their own interactions with biological milieu should be minimal. In other words, they should belong to the group of long-circulating polymers [6]. Poly(ethylene glycol) (PEG) demonstrates, apparently, the best currently known combination of properties that provide long liposome circulation. The advantages of PEG and other low-reactive long-circulating polymers are utilized via assembling brush-like interfaces or various coatings around liposomes and other drug carriers [8487]. The currently available materials and methods for liposome surface modications are reviewed elsewhere [2,88]. The main feature of long-circulating polymers can be dened as minimal presence of biologically reactive functional groups on their interface with the liquid phase. The term biologically reactive refers here to polymer ability to form chemical bonds and to participate in either high- or low-energy interactions of any type with any components of biological
systems. As in the case of the liposome surface, it is convenient to discuss the high-afnity (specic) and non-specic interactions separately. Polymer fragments and functional groups capable of interactions with recognition proteins may demonstrate potent effects on circulation via opsonization and increase in vascular permeability. For this reason, the biokinetics of liposomes modied by such polymers would be more vulnerable to changes caused by pathological processes (e.g., increased vascular permeability and appearance of acute-phase proteins in blood). Several macromolecules of biological origin and their components are known to interact with recognition proteins. Antigens interact with complementary immunoglobulins; several carbohydrates interact with a variety of recognition proteins (lectins); hydroxyl and amino groups may bind C3 and C4, respectively [6]. Carbohydrates are recognized by several receptors, including receptors present in large numbers in liver (to name a few, receptors binding galactose, mannose and fucose derivatives [88,89]). Scavenger receptors of phagocytes may bind a variety of compounds [90]. Various receptors bind peptides and several other compounds. Although receptor concentration in tissues (except RES) appears to be too low to affect polymer circulation at all but the smallest doses, polymer cross-reactivity with such receptors may cause various side effects and should, in general, be avoided. Dextran B512-F (practically linear a-D(1 6)glucose) is considered to be one of the most biologically inert biopolymers [91,92]. Most of the other polysaccharides contain receptor-recognizable sites [9398], which hardly make them useful for carrier modication. Most synthetic polymers lack any receptor- and opsonin-binding groups. However, all functional groups without any exceptions are capable of cooperative interactions in vivo, for example based on
133
Van der Waals, hydrophobic, dipoledipole, electrostatic interactions and formation of hydrogen bonds. Polymers containing several charged groups or hydrophobic sites are most reactive in vivo; the longest circulation times were reported for neutral or slightly negatively charged polymers. Uncharged hydrophilic functional groups with low nucleophilicity appear to be most suitable for liposome protection [2,6,86]. Because the components of any molecular brush are not completely inert, they can interact with the biological substrate via systems of collective weak associations. Obviously, long polymer chains and dense brushes are more vulnerable to cooperative interactions because of the presence of a greater number of groups forming weak bonds (see Fig. 3). There are indications that excessively long brushes (PEG, . 5 kDa) are in fact inferior in their ability to prolong circulation time [78]. We have shown also that a polymer that has a long circulation time and resistance to RES recognition can become well recognizable as a component of a brush. Graft copolymers of dextran and dextran-modied nanoparticles, unlike dextran, are readily recognized by some phagocyte populations and accumulate in lymph nodes after intravenous administration [99 101]. Based on the above, the brush should be as short and loose as possible, regardless of the polymer used. This obviously confronts the requirements directed to optimizing the protective properties of the same brush (see Section 3). Therefore, an optimal brush density providing minimal overall liposome interactions should exist and can be determined experimentally for each polymer type and liposome composition.
4.2. Approaches to the development of better liposome surface modiers

Considering the intrinsic vulnerability of polymers to cooperative interactions, the question remains: how the natural supramolecular and macromolecular structures (blood cells, plasma proteins) can remain in the circulation for weeks and months, if their components cannot avoid cooperative interactions? The answer would be undoubtedly important for liposome development: possibly, the mechanisms involved in this long-term retention could be utilized
for developing ultra-long circulating liposomal drug carriers with exceptional target specicity. Although there is no direct experimental evidence, it seems possible that long circulation of natural cells and proteins, which are well known to be heavily modied by oligosaccharides, is due to saturation of the potential sites of non-specic binding. The total effective amount of circulating oligosaccharides consisting of structurally similar fragments is so high that all potential sites of non-specic binding must be occupied (while high-afnity centers specically recognizing certain structural domains of the oligosaccharides remain vacant). If the above hypothesis is correct, polymers assembled of fragments structurally similar to those of carbohydrates (but lacking any receptor- and opsonin-recognizable domains) should be most resistant to non-specic binding, because all possible binding sites are already occupied by natural glycoconjugates. Obviously, it is important to avoid even the slightest cross-reactivity with recognition proteins and receptors, because cooperative weak interactions with several binding centers may still result in polymer recognition. Structures common for oligosaccharides include, in general, aliphatic secondary and primary OH groups, short carbon chains, and aliphatic acetal groups (in general, aliphatic acetal carbon and ether type oxygen). The structure of the most long-circulating sialated oligosaccharides also contains N acetyl groups. The sites of branching often participate in recognition. It is further notable that all oligosaccharides share approximately the same structures in the back of the carbohydrate units (as they are conventionally drawn), for example carbon 1 (the acetal carbon) and carbons 5 and 6 in pyranoses (Fig. 9). The front part of the ring (carbons 24) forms various structures that are recognized, in accordance with their conformation and substitution, by a variety of receptors. Therefore, it was interesting to test if an aliphatic polymer assembled of carbon 1 carbon 6 carbon 5 like structures 1 shows desirable effects as a component of protective brushes. Polyacetals of this type can be synthesized either by polymerization of
1
We are not using the standard nomenclature to avoid confusion due to the similarity of abbreviations used for complement proteins and carbohydrate carbons.
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Fig. 9. Hexose ring as an object of recognition. a-D-Glucopiranose is shown as a part of a larger molecule (1,6 modied). Several oligosaccharides share the structure formed by the back part of the ring (oxygencarbon 6 carbon 5 oxygencarbon 1 ). The front side of the ring (carbons 24 and their substituents) is variable and often involved in recognition by lectins.
gous polyethyleneglycol derivatives. The effect of Fleximer on liposome circulation was similar (to be published elsewhere). Another important factor of liposome viability relates to the stability of the protective brush (as well as the liposome itself). Due to a variety of interactions, the protective polymer can be chemically altered or removed from the liposome surface. The composition of the underlying lipid bilayer can also be altered as a result of lipid exchange, which may cause, in turn, signicant changes in the protective brush. In vivo, such alterations are well known [104,105] and can be repaired by cells, for example via production of new membrane components and either endocytosis or exocytosis (shedding) the weared ones [106108]. These phenomena are being studied and will probably provide valuable new approaches for drug carrier development.
substituted 1,3-dioxolanes or by elimination of carbon 2 carbon 4 domains via periodate oxidation with subsequent reduction. A polymer of this type (Fleximer, or poly(hydroxymethylethylene hydroxymethylformal) has been synthesized in our laboratory [102] and tested as a protective brush component in a graft copolymer and nanoparticle models [103]. The rst results have shown that Fleximer prolongs circulation of the model carriers much better than dextran (one of the most biologically inert polysaccharides). The observed circulation times (half-life . 25 h) and biodistribution (see Table 2) were similar (or better) to those of analoTable 2 Biodistribution of dextran-protected and Fleximer-protected polylysine in rat (% dose / g tissue), 24 h after intravenous administration of 1 mg / kg body weight Tissue Protective polymer Dextran Blood Lymph nodes Paraaortic Mesenteric Spleen Liver Kidney Muscle Heart Lung Pyers patch 0.3 58.9 81.8 19.9 9.0 2.7 0.1 0.3 0.2 0.2 Fleximer 3.7 0.9 0.8 1.3 2.1 3.7 0.4 0.9 1.2 0.7
5. Conclusions The objective of theoretical analysis is to identify the most efcient approaches to the rational development of long-circulating RES-avoiding liposomes. The relationships between liposome structure and pharmacokinetics, which constitute the underlying basis for rational development, can be reliably evaluated only on the basis of detailed mechanistic analysis of each elementary processes of liposome transfer and interactions with biological milieu. Mechanistic models of mass transfer, liposome interaction with cells and blood proteins, and boundary effects resulting from surface modication provide quantitative approaches to relating the observed biological effects to structural factors, such as liposome composition and surface structure. The availability of such approaches can streamline the development of liposome technology on the basis of clinical objectives. Rational technology development requires further expansion of the factual basis. The latter includes, in the rst place, identication of recognition proteins capable of liposome binding; quantitative (physicochemical) data on liposome interactions with these recognition proteins; quantitative data on liposome protein complex and liposome interactions with cells; data on recognition protein content in normal and pathological conditions; and data on variations in
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recognition protein specicity among species. Optimization of the protective surface modiers requires further mechanistic studies on the molecular brush behavior, and on the interplay of steric and cooperative effects in the interactions of surfacemodied liposomes. The development of novel exible and rigid hydrophilic polymers with minimized ability of binding to any components of the biological milieu would also benet further developments.
[12]
[13]
[14]
[15]
Acknowledgements This work was supported by The Whitaker Foundation.
[16]
[17]
[18]
References
[1] G. Gregoriadis (Ed.), Liposome Technology, 2nd ed., CRC Press, Boca Raton, FL, 1993. [2] M.C. Woodle, D.D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta 1113 (1992) 171199. [3] M.C. Woodle, Sterically stabilized liposome therapeutics, Adv. Drug Deliv. Rev. 16 (1995) 249266. [4] P.R. Chaturvedi, Pharmacokinetics of microparticular systems, in: S. Cohen, H. Bernstein (Eds.), Microparticulate Systems for the Delivery of Proteins and Vaccines, Marcel Dekker, New York, 1996. [5] T.M. Allen, C.B. Hansen, D.E.L. de Menzes, Pharmacokinetics of long-circulating liposomes, Adv. Drug Deliv. Rev. 16 (1995) 267. [6] M.I. Papisov, Modeling in vivo transfer of long-circulating polymers (two classes of long circulating polymers and factors affecting their transfer in vivo), Adv. Drug Deliv. Rev. 16 (1995) 127137. [7] M.I. Papisov, V.Y. Savelyev, V.B. Sergienko, V.P. Torchilin, Magnetic drug targeting (I) In vivo kinetics of radiolabeled magnetic drug carriers, Int. J. Pharm. 40 (1987) 201206. [8] B.S. Schaffer, C. Linker, M.I. Papisov, E. Tsai, N. Nossiff, T. Shibata, A.A. Bogdanov Jr., T.J. Brady, R. Weissleder, MION-ASF: Biokinetics of an MR receptor agent, J. Magn. Res. Imag. 11 (1993) 411417. [9] M.I. Papisov, V.P. Torchilin, Magnetic drug targeting (II) Targeted drug transport by magnetic microparticles: Factors inuencing therapeutic effect, Int. J. Pharm. 40 (1987) 207 214. [10] M.C. Woodle, K.K. Matthay, M.S. Newman, J.E. Hidayat, L.R. Collins, C. Redemann, F.J. Martin, D. Papahadjopoulos, Versatility in lipid composition showing prolonged circulation with sterically stabilized liposomes, Biochim. Biophys. Acta 1105 (1992) 193200. [11] F. Yuan, M. Dellian, D. Fukumura, M. Leunig, D.A. Berk,
[19]
[20]
[21]
[22]
[23] [24]
[25]
[26] [27]
[28]
V.P. Torchilin, R.K. Jain, Vascular permeability in a human tumor xenograft: Molecular size dependence and cutoff size, Cancer Res. 55(17) (1995) 37523756. V. Neufeltd (Ed.), Webster New World Dictionary of American English, third college edition, Webster New World, New York, 1988, p. 1314. S.L. Jeon, J.H. Lee, J.D. Andrade, P.G. DeGennes, Protein surface interactions in the presence of polyethylene oxide, J. Colloid Interface Sci. 142 (1991) 149166. T.M. Allen, G.A. Austin, A. Chonn, L. Lin, K.C. Lee, Uptake of liposomes by cultured mouse bone marrow macrophages: Inuence of liposome composition and size, Biochim. Biophys. Acta 1061 (1991) 5664. J.W.B. Bradeld, A new look at reticuloendothelial blockade, Br. J. Exp. Pathol. 61 (1980) 617623. S.M. Moghimi, H.M. Patel, Differencial properties of organ specic serum opsonins for liver and spleen macrophages, Biochim. Biophys. Acta 984 (1989) 379384. D.O. Oja, S.C. Semple, A. Chonn, P.R. Cullis, Inuence of dose on liposome clearance: Critical role of blood proteins, Biochim. Biophys. Acta 1281 (1996) 3137. S.M. Moghimi, C.J.H. Porter, I.S. Muir, L. Illum, S.S. Davis, Non-phagocytic uptake of intravenously injected microspheres in rat spleen: Inuence of particle size and hydrophylic coating, Biochem. Biophys. Res. Commun. 177 (1991) 861866. J.P. Atkinson, M.M. Frank, Interaction of IgM antibody and complement in the immune clearance and destruction of erythrocytes in man, J. Clin. Invest. 54 (1974) 339348. K.W. Walton, T.J. Almond, M. Robinson, D.L. Scott, An experimental model for the study of the opsonic activity of bronectin in the clearance of intravascular complexes, Br. J. Exp. Pathol. 65 (1984) 191200. M.J. Benecky, C.G. Kolvenbach, R.W. Wine, J.P. DiOrio, M.W. Mosesson, Human plasma bronectin structure probed by steady-state uorescence polarization: Evidence for a rigid oblate structure, Biochemistry 29 (1990) 30823091. M.Y. Khan, M.S. Medow, S.A. Newman, Unfolding transitions of bronectin and its domains, Biochem. J. 270 (1990) 3338. T. Kinoshita, Biology of complement: The overture, Immunol. Today 12 (1991) 291301. S.K. Law, N.A. Lichtenberg, R.P. Levine, Covalent binding and hemolytic activity of complement proteins, Proc. Natl. Acad. Sci. USA 77 (1980) 71947198. S.K. Law, T.M. Minich, R.P. Levine, Covalent binding efcacy of the third and fourth complement proteins in relation to pH, nucleophilicity, and availability of hydroxyl groups, Biochemistry 23 (1984) 32673272. R.B. Sim, K.B.M. Reid, C1: Molecular interactions with activating systems, Immunol. Today 12 (1991) 307311. N.M. Young, M.A. Leon, The carbohydrate specicity of conglutinin and its homology to proteins in the hepatic lectin family, Biochem. Biophys. Res. Commun. 143 (1987) 645 651. R.A.B. Ezekowitz, L.E. Day, G.A. Herman, A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins, J. Exp. Med. 167 (1988) 10341047.
136
M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138 [44] T. Kinoshita, Biology of complement: The overture, Immunol. Today 12 (1991) 291301. [45] S.K. Law, N.A. Lichtenberg, R.P. Levine, Covalent binding and hemolytic activity of complement proteins, Proc. Natl. Acad. Sci. USA 77 (1980) 71947198. [46] S.K. Law, T.M. Minich, R.P. Levine, Covalent binding efcacy of the third and fourth complement proteins in relation to pH, nucleophilicity, and availability of hydroxyl groups, Biochemistry 23 (1984) 32673272. [47] R.B. Sim, K.B.M. Reid, C1: Molecular interactions with activating systems, Immunol. Today 12 (1991) 307311. [48] S.C. Semple, A. Chonn, P.R. Cullis, Inuence of cholesterol on the association of plasma proteins with liposomes, Biochemistry 35 (1996) 25212525. [49] C. Mold, E.I. Walter, M.E. Medof, The inuence of membrane components on regulation of alternative pathway activation by decay-accelerating factor, J. Immunol. 145 (1990) 38363841. [50] N.M. Young, M.A. Leon, The carbohydrate specicity of conglutinin and its homology to proteins in the hepatic lectin family, Biochem. Biophys. Res. Commun. 143 (1987) 645 651. [51] R.A.B. Ezekowitz, L.E. Day, G.A. Herman, A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins, J. Exp. Med. 167 (1988) 10341047. [52] S.D. Wright, P.S. Tobias, R.J. Ulevitch, R.A. Ramos, Lipopolysaccharide (LPS) binding protein opsonizes LPSbearing particles for recognition by a novel receptor on macrophages, J. Exp. Med. 170 (1989) 12311241. [53] P.S. Tobias, K. Soldau, L. Kline, J.D. Lee, K. Kato, T.P. Martin, R.J. Ulevitch, Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein, J. Immunol. 150 (1993) 30113021. [54] J. Savill, I. Dranseld, N. Hogg, C. Haslett, Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis, Nature 343 (1990) 170173. [55] L. Pinteric, S.M. Assimeh, D.I.C. Kells, R.H. Painter, The ultrastructure of C1t, a subcomponent of the rst component of complement: An EM and ultracentrifuge study, J. Immunol. 117 (1976) 7983. [56] U. Saxena, A. Nagpurkar, P.J. Dolphin, S. Mookerjea, A study on the selective binding of apoprotein B- and Econtaining human plasma lipoproteins to immobilized rat serum phosphorilcholine-binding protein, J. Biol. Chem. 262 (1987) 30113016. [57] E. Kottgen, B. Hell, A. Kage, R. Tauber, Lectin specicity and binding characteristics of human C-reactive protein, J. Immunol. 149 (1992) 445453. [58] A. Chonn, S.C. Semple, P.R. Cullis, b2-Glycoprotein I is a major protein associated with very rapidly cleared liposomes in vivo, suggesting a signicant role in the immune clearance of non-self particles, J. Biol. Chem. 270 (1995) 25845 25849. [59] M. Igarashi, E. Matsuura, Y. Igarashi, H. Nagae, K. Ichikawa, D.A. Triplett, T. Koike, Human beta2-glycoprotein I as an anticardiolipin cofactor determined using mutants
[29] S.D. Wright, P.S. Tobias, R.J. Ulevitch, R.A. Ramos, Lipopolysaccharide (LPS) binding protein opsonizes LPSbearing particles for recognition by a novel receptor on macrophages, J. Exp. Med. 170 (1989) 12311241. [30] P.S. Tobias, K. Soldau, L. Kline, J.D. Lee, K. Kato, T.P. Martin, R.J. Ulevitch, Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein, J. Immunol. 150 (1993) 30113021. [31] J. Savill, I. Dranseld, N. Hogg, C. Haslett, Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis, Nature 343 (1990) 170173. [32] A.G. Ehlenberger, V. Nussenzweig, The role of membrane receptors for C3b and C3d in phagocytosis, J. Exp. Med. 145 (1977) 357371. [33] G.D. Ross, J.A. Cain, P.J. Lachmann, Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin and functions as a receptor for zymozan and rabbit erythrocytes as well as a receptor for iC3b, J. Immunol. 134 (1985) 33073315. [34] D.L. Brown, P.J. Lachmann, J.V. Dacie, The in vivo behavior of complement-coated red cells: Studies in C6-decient, C3-depleted and normal rabbits, Clin. Exp. Immunol. 7 (1970) 401422. [35] J.P. Atkinson, M.M. Frank, Interaction of IgM antibody and complement in the immune clearance and destruction of erythrocytes in man, J. Clin. Invest. 54 (1974) 339348. [36] D.L. Brown, P.J. Lachmann, J.V. Dacie, The in vivo behaviour of complement-coated red cells: Studies in C6decient, C3-depleted and normal rabbits, Clin. Exp. Immunol. 7 (1970) 401422. [37] J. Senior, J.A. Waters, G. Gregoriadis, Antibody-coated liposomes. The role of non-specic antibody adsorption, FEBS Lett. 196 (1986) 5458. [38] J. Vazquez-Mellado, L. Llorente, Y. Richaud-Patin, D. Alarcon-Segovia, Exposure of anionic phospholipids upon platelet activation permits binding of beta 2 glycoprotein I and through it that of IgG antiphospholipid antibodies. Studies in platelets from patients with antiphospholipid syndrome and normal subjects, J. Autoimmun. 7 (1994) 335348. [39] M.J. Benecky, C.G. Kolvenbach, R.W. Wine, J.P. DiOrio, M.W. Mosesson, Human plsama bronectin structure probed by steady-state uorescence polarization: Evidence for a rigid oblate structure, Biochemistry 29 (1990) 30823091. [40] M.Y. Khan, M.S. Medow, S.A. Newman, Unfolding transitions of bronectin and its domains, Biochem. J. 270 (1990) 3338. [41] C. Wolff, C.S. Lai, Fluorescence energy transfer detects changes in bronectin structure upon surface binding, Arch. Biochem. Biophys. 268 (1989) 536545. [42] K.W. Walton, T.J. Almond, M. Robinson, D.L. Scott, An experimental model for the study of the opsonic activity of bronectin in the clearance of intravascular complexes, Br. J. Exp. Pathol. 65 (1984) 191200. [43] M.J. Hsu, R.L. Juliano, Interactions of liposomes with the reticuloendothelial system. II: Nonspecic and receptor-mediated uptake of liposomes by mouse peritoneal macrophages, Biochim. Biophys. Acta 720 (1982) 411419.
M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138 expressed by a baculovirus system, Blood 87 (1996) 3262 3270. B.E. Price, J. Rauch, M.A. Shia, M.T. Walsh, W. Lieberthal, H.M. Gilligan, T. OLaughlin, J.S. Koh, J.S. Levine, Antiphospholipid autoantibodies bind to apoptotic, but not viable, thymocytes in a beta 2-glycoprotein I-dependent manner, J. Immunol. 157 (1996) 22012208. T.A. Brighton, P.J. Hogg, Y.P. Dai, B.H. Murray, B.H. Chong, C.N. Chesterman, Beta 2-glycoprotein I in thrombosis: Evidence for a role as a natural anticoagulant, Br. J. Haematol. 93 (1996) 185194. R.A. Schwalbe, B. Dahlback, J.E. Coe, G.L. Nelsestuen, Pentraxin family of proteins interact specically with phosphorylcholine and / or phosphorylethanolamine, Biochemistry 31 (1992) 49074915. D.D. Roberts, R.P. Mecham (Eds.), Cell Surface and Extracellular Glycoconjugates. Structure and Function, Academic Press, San Diego, 1993. S.T. Milner, Polymer brushes, Science 251 (1991) 905914. A. Gabizon, D. Papahadjopoulos, Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors, Proc. Natl. Acad. Sci. USA 85 (1988) 69496953. D.D. Lasic, F.J. Martin, A. Gabizon, S.K. Huang, D. Papahajopoulos, Sterically stabilized liposomes: A hypothesis on the molecular origin of the extended circulation times, Biochim. Biophys. Acta 1070 (1991) 187192. S. Moghimi, L. Illum, S. Davis, V. Kolb-Bachofen, Coating particles with a block copolymer (poloxamine 908) suppresses opsonization but permits the activity of dysopsonins in the serum, Biochim. Biophys. Acta 1179 (1993) 157165. V.P. Torchilin, M.I. Papisov, Why do PEG-coated liposomes circulate long?, J. Liposome Res. 4 (1994) 725739. A.A. Bogdanov Jr., R. Weissleder, H.W. Frank, A.V. Bogdanova, N. Nossiff, B.K. Schaeffer, E. Tsai, M.I. Papisov, T.J. Brady, A new macromolecule as a contrast agent for MR angiography: Preparation, properties, and animal studies, Radiology 187 (1993) 701706. P.J. Flory, W.R. Krigbaum, Thermodynamics of high polymer solutions, Annu. Rev. Phys. Chem. 2 (1951) 383416. T.M. Allen, C.B. Hansen, D.E.L. de Menzes, Pharmacokinetics of long-circulating liposomes, Adv. Drug Deliv. Rev. 16 (1995) 267284. P.G. De Gennes, Polymer solutions near an interface. 1. Adsorption and depletion layers, Macromolecules 14 (1981) 16371644. M.I. Papisov, L. LaPointe, S. Myagchilov, K. Poss, R. Weissleder, T.J. Brady, Underlying mechanisms of surface protection by molecular brushes: Models and reality, in: Twenty-third International Symposium on Controlled Release of Bioactive Materials, 1996, pp. 681682. M. Kurata, Y. Tsunashima, Viscositymolecular weight relationships and unperturbed dimensions of linear chain molecules, in: J. Brandup, H.E. Himmelgut (Eds.), Polymer Handbook, Wiley, New York, chapt. VII. M.I. Papisov, K. Poss, R. Weissleder, T.J. Brady, Intravenous lymphotropic polymers: The SPAR effect, in: Proceedings of
137
[60]
[76]
[77]
[61]
[78]
[62]
[79]
[63]
[80]
[64] [65]
[81]
[66]
[82]
[67]
[68] [69]
[83]
[84]
[70] [71]
[85]
[86]
[72]
[87]
[73]
[88]
[89]
[74]
[75]
[90]
the Second Meeting of the Society of Magnetic Resonance, Berkley, CA, 1994, p. 388. C. Huang, J.T. Mason, Geometric packaging constraints in egg phosphatidylcholine vesicles, Proc. Natl. Acad. Sci. USA 75 (1978) 308310. H.G. Enoch, P. Strittmatter, Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles, Proc. Natl. Acad. Sci. USA 76 (1979) 145149. F.K. Bedu-Addo, L. Huang, Interaction of PEGphospholipid conjugates with phospholipid: Implications in liposomal drug delivery, Adv. Drug Deliv. Rev. 16 (1995) 235248. M.I. Papisov, C. Martin, K. Poss, R. Weissleder, T.J. Brady, Amplication of steric effects in cooperative systems, in: Twenty-second International Symposium on Controlled Release of Bioactive Materials, 1995, pp. 442443. A. Mori, A.L. Klibanov, V.P. Torchilin, L. Huang, Inuence of the steric barrier of amphypatic poly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo, FEBS Lett. 284 (1991) 263266. M.I. Papisov, K. Poss, R. Weissleder, T.J. Brady, Effect of steric protection in lymphotropic graft copolymers, in: Seventh International Symposium on Recent Advances in Drug Delivery Systems (abstracts), Salt Lake City, UT, 1995, University of Utah 1995, pp. 171172. V.P. Torchilin, V. Omelyanenko, M.I. Papisov, A.A. Bogdanov Jr., V.S. Trubetskoy, D.I.N. Herron, C.A. Gentry, Poly(ethyleneglycol) on the liposome surface: On the mechanism of polymer-coated liposome longevity, Biochem. Biophys. Acta 1195 (1994) 1120. D.D. Lasic, M.C. Woodle, F.J. Martin, T. Valentincic, Phase behavior of Stealthlipidlecithin mixtures, Period. Biol. 93 (1991) 287290. R. Gref, A. Domb, P. Quelec, P. Blunk, R.H. Muller, J.M. Verbavatz, R. Langer, The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres, Adv. Drug Deliv. Rev. 16 (1995) 215234. S. Zalipsky, Chemistry of polyethyleneglycol conjugates with biologically active molecules, Adv. Drug Deliv. Rev. 16 (1995) 157182. V.P. Torchilin, V.S. Trubetskoy, Which polymers make nanoparticular drug carriers long-circulating?, Adv. Drug Deliv. Rev. 16 (1995) 141156. C. Deluged, G.E. Francis, D. Fisher, The uses and properties of PEG-linked proteins, Crit. Rev. Ther. Drug Deliv. Syst. 9 (1992) 249304. K.G. Rice, Y.C. Lee, Modication of triantennary glycopeptide into probes for the asialoglycoprotein of hepatocytes, J. Biol. Chem. 265 (1990) 1842318428. R.S. Haltiwanger, M.A. Lehrman, A.E. Eckardt, R.L. Hill, The distribution and localization of the fucose-binding lectin in rat tissues and the identication of a high afnity form of mannose / N -acetylglucosamine-binding lectin in rat liver, J. Biol. Chem. 261 (1986) 74337439. L. Rohrer, M. Freeman, T. Kodama, M. Penman, M. Krieger, Coiled-coil brous domains mediate ligand binding by
138
M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138 macrophage scavenger receptor type II, Nature 343 (1990) 570572. C. Larsen, Dextran prodrugs structure and stability in relation to therapeutic activity, Adv. Drug Deliv. Rev. 3 (1989) 103154. A. Jeanes, Immunochemical and related interactions with dextrans reviewed in terms of improved structural information, Mol. Immunol. 23 (1986) 9991028. R.B. Sim, K.B.M. Reid, C1: Molecular interactions with activating systems, Immunol. Today 12 (1991) 307311. R.S. Haltiwanger, M.A. Lehrman, A.E. Eckardt, R.L. Hill, The distribution and localization of the fucose-binding lectin in rat tissues and the identication of a high afnity form of mannose / N -acetylglucosamine-binding lectin in rat liver, J. Biol. Chem. 261 (1986) 74337439. N.M. Young, M.A. Leon, The carbohydrate specicity of conglutinin and its homology to proteins in the hepatic lectin family, Biochem. Biophys. Res. Commun. 143 (1987) 645 651. S.D. Wright, P.S. Tobias, R.J. Ulevitch, R.A. Ramos, Lipopolysaccharide (LPS) binding protein opsonizes LPSbearing particles for recognition by a novel receptor on macrophages, J. Exp. Med. 170 (1989) 12311241. P.S. Tobias, K. Soldau, L. Kline, J.D. Lee, K. Kato, T.P. Martin, R.J. Ulevitch, Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein, J. Immunol. 150 (1993) 30113021. U. Saxena, A. Nagpurkar, P.J. Dolphin, S. Mookerjea, A study on the selective binding of apoprotein B- and Econtaining human plasma lipoproteins to immobilized rat serum phosphorilcholine-binding protein, J. Biol. Chem. 262 (1987) 30113016. [99] M. Papisov, R. Weissleder, Drug delivery to lymphatic tissue, Crit. Rev. Ther. Drug Carrier Syst. 13 (1996) 5784. [100] M.I. Papisov, R. Weissleder, T.J. Brady, Diagnostic targeting of lymph nodes with polymeric imaging agents, in: V.P. Torchilin (Ed.), Targeted Delivery of Imaging Agents, CRC Press, Boca Raton, FL, 1995, pp. 385402. [101] R. Weissleder, M.I. Papisov, Pharmaceutical iron oxides for MR imaging, Rev. Magn. Reson. 4 (1992) 120. [102] M.I. Papisov, L. Garrido, K. Poss, C. Wright, R. Weissleder, T.J. Brady, A long-circulating polymer with hydrolyzable main chain, in: Twenty-third International Symposium on Controlled Release of Bioactive Materials, 1996, pp. 107 108. [103] M. Papisov, unpublished data, 1995. [104] L. Gattegno, F. Fabia, D. Bladier, P. Cornillot, Physiological ageing of red blood cells and changes in membrane carbohydrates, Biomedicine 30 (1979) 194199. [105] E. Zocchi, L. Guida, U. Benatti, M. Canepa, L. Borgiani, T. Zanin, A. De Flora, Hepatic or splenic targeting of carrier erythrocytes: A murine model, Biotechnol. Appl. Biochem. 9 (1987) 423434. [106] P. Comfurius, J.M. Senden, R.H. Tilly, A.J. Schroit, E.M. Bevers, R.F. Zwaal, Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase, Biochim. Biophys. Acta 1026 (1990) 153160. [107] U.J. Dumaswala, T.J. Greenwalt, Human erythrocytes shed exocytic vesicles in vivo, Transfusion 24 (1984) 490492. [108] C.P. Muller, M. Shinitzky, Passive shedding of erythrocyte antigens induced by membrane rigidication, Exp. Cell Res. 136 (1981) 5362.
[91]
[92]
[93] [94]
[95]
[96]
[97]
[98]

Theoretical Considerations of RES-Avoiding Liposomes

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Advanced Drug Delivery Reviews 32 (1998) 119138

2.1. Circulation time as a measure of liposome interactions in vivo

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

2.2. Pseudohomogenous model of long-circulating liposome biokinetics

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

3.1. Liposome interaction in vivo: The most general aspects

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

3.2. Recognition proteins of plasma

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

3.4. Mathematical modeling of molecular brushes

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

3.5. Mechanics of surface protection by molecular brushes

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

4. Interactions of molecular brushes in vivo

4.1. Interactions of protective polymers with components of biological systems

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

4.2. Approaches to the development of better liposome surface modiers

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

M.I. Papisov / Advanced Drug Delivery Reviews 32 (1998) 119 138

Acknowledgements This work was supported by The Whitaker Foundation.

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