International Journal of Pharmaceutics 307 (2006) 93–102
`
`Review
`Opsonization, biodistribution, and pharmacokinetics
`of polymeric nanoparticles
`Donald E. Owens III a, Nicholas A. Peppas a,b,c,∗
`
`a Department of Chemical Engineering, University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA
`b Department of Biomedical Engineering, University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA
`c Department of Pharmaceutics, University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA
`Received 8 July 2005; received in revised form 11 October 2005; accepted 12 October 2005
`Available online 21 November 2005
`
`Abstract
`
`The process of opsonization is one of the most important biological barriers to controlled drug delivery. Injectable polymeric nanoparticle carriers
`have the ability to revolutionize disease treatment via spatially and temporally controlled drug delivery. However, opsonin proteins present in the
`blood serum quickly bind to conventional non-stealth nanoparticles, allowing macrophages of the mononuclear phagocytic system (MPS) to easily
`recognize and remove these drug delivery devices before they can perform their designed therapeutic function. To address these limitations, several
`methods have been developed to mask or camouflage nanoparticles from the MPS. Of these methods, the most preferred is the adsorption or grafting
`of poly(ethylene glycol) (PEG) to the surface of nanoparticles. Addition of PEG and PEG-containing copolymers to the surface of nanoparticles
`results in an increase in the blood circulation half-life of the particles by several orders of magnitude. This method creates a hydrophilic protective
`layer around the nanoparticles that is able to repel the absorption of opsonin proteins via steric repulsion forces, thereby blocking and delaying the
`first step in the opsonization process.
`© 2005 Elsevier B.V. All rights reserved.
`
`Keywords: Opsonization; Poloxamer; Poloxamine; Poly(ethylene glycol); PEGylation; Stealth nanoparticles
`
`Contents
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`1.
`2. Opsonization and phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.
`PEGylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4. Biodistribution and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`93
`94
`95
`98
`99
`100
`100
`
`1. Introduction
`
`Through spatial and temporal controlled drug delivery,
`injectable nanoparticle carriers have the ability to revolutionize
`disease treatment. Spatially localizing the release of toxic and
`other potent drugs only at specific therapeutic sites can lower
`the overall systemic dose and damage that these drugs would
`
`∗
`
`Corresponding author. Tel.: +1 512 471 6644; fax: +1 512 471 8227.
`E-mail address: peppas@che.utexas.edu (N.A. Peppas).
`
`0378-5173/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
`doi:10.1016/j.ijpharm.2005.10.010
`
`otherwise produce. Temporally controlling the release of a drug
`can also help decrease unwanted side effects that might other-
`wise occur due to the natural circadian fluctuations of chemical
`levels throughout the body (Hermida et al., 2001). The overall
`benefit of these improvements in disease treatment would be
`an increase in patient compliance and quality of life. In order
`for a drug delivery device to achieve these desired benefits it
`must be present in the bloodstream long enough to reach or
`recognize its therapeutic site of action. However, the opsoniza-
`tion or removal of nanoparticulate drug carriers from the body
`
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`D.E. Owens III, N.A. Peppas / International Journal of Pharmaceutics 307 (2006) 93–102
`
`by the mononuclear phagocytic system (MPS), also known as
`the reticuloendothelial system (RES), is a major obstacle to the
`realization of these goals.
`The macrophages of the MPS have the ability to remove
`unprotected nanoparticles from the bloodstream within sec-
`onds of intravenous administration, rendering them ineffective
`as site-specific drug delivery devices (Gref et al., 1994). These
`macrophages, which are typically Kupffer cells, or macrophages
`of the liver, cannot directly identify the nanoparticles them-
`selves, but rather recognize specific opsonin proteins bound to
`the surface of the particles (Frank and Fries, 1991). Broadly
`speaking, opsonins are any blood serum component that aids
`in the process of phagocytic recognition, but complement pro-
`teins such as C3, C4, and C5 and immunoglobulins are typically
`the most common. Several methods of camouflaging or mask-
`ing nanoparticles have been developed, which allow them to
`temporarily bypass recognition by the MPS and increase their
`blood circulation half-life (Illum and Davis, 1984; Gref et al.,
`1994; Kaul and Amiji, 2002). Many of these systems make use
`of surface treatments that interfere with the binding of opsonin
`proteins to the particle surface as a means of imparting stealth,
`or MPS-avoidance characteristics to nanoparticles. This review
`focuses on those systems that utilize poly(ethylene glycol) and
`PEG-containing surface treatments because these systems seem
`to hold the most promise and show the lowest occurrence of
`harmful effects in vivo.
`
`2. Opsonization and phagocytosis
`
`Opsonization is the process by which a foreign organism or
`particle becomes covered with opsonin proteins, thereby making
`it more visible to phagocytic cells. After opsonization, phagocy-
`tosis can occur, which is the engulfing and eventual destruction
`or removal of foreign materials from the bloodstream. Together
`these two processes form the main clearance mechanism for the
`removal of undesirable components larger than the renal thresh-
`old limit from the blood. In the case of polymeric nanoparticles,
`which cannot normally be destroyed by the phagocytes, seques-
`tration in the MPS organs typically occurs. If the polymeric
`nanoparticle is non-biodegradable, then accumulation of parti-
`cles in these organs, most commonly the liver and spleen, can
`occur leading to toxicity and other negative side effects (Illum
`et al., 1986; Peracchia et al., 1999a; Plard and Bazile, 1999).
`Opsonization typically takes place in the blood circulation
`and can take anywhere from a matter of seconds to many days
`to complete. The exact mechanism through which this process
`is activated is very complicated and not yet full understood,
`but the important components involved are, for the most part,
`well known. Immunoglobulins and components of the comple-
`ment system such as C3, C4, and C5 are known to be common
`opsonins as well as other blood serum proteins such as laminin,
`fibronectin, C-reactive protein, type I collagen and many others
`(Frank and Fries, 1991; Johnson, 2004). The importance of these
`proteins in the clearance process has been indirectly demon-
`strated in many in vivo animal studies of inherited and induced
`C3 deficient animal models. For instance, research has shown
`that these animal models are often times more susceptible to cer-
`
`tain diseases which are easily controlled by phagocytosis in non-
`C3 deficient animal models (Singer et al., 1994). The opsonins,
`which are present throughout the blood, are thought to come into
`contact with injected polymeric nanoparticles typically by ran-
`dom Brownian motion. However, once sufficiently close to the
`surface of a particle, any of several attractive forces including
`van der Walls, electrostatic, ionic, hydrophobic/hydrophilic, and
`others can be involved in the binding of opsonins to the surface
`of the nanoparticle.
`After opsonization has occurred, the next step in the clearance
`process is the attachment of the phagocyte to the nanoparticle
`via surface bound opsonins. Without the presence of surface
`bound or adsorbed opsonin proteins, the phagocytes will typ-
`ically not be able to bind or recognize the foreign particles.
`One method of attachment occurs when the bound opsonin pro-
`teins undergo conformational changes from an inactive protein
`present in the blood serum to an activated protein structure
`that can be recognized by phagocytes. Phagocytic cell surfaces
`contain specialized receptors that interact with the modified con-
`formation of these various opsonins thus alerting them to the
`presence of a foreign material.
`A second method of phagocyte attachment is the non-specific
`adherence of phagocytes to surface adsorbed blood serum pro-
`teins which can result in the stimulation of phagocytosis as well
`(Frank and Fries, 1991). This process is typically due to the asso-
`ciation of opsonin proteins with a more hydrophobic particle
`surface. The third significant method of phagocyte attachment
`is complement activation. The complement system can be acti-
`vated by one of several mechanisms including the classical,
`alternative, and lectin pathway. The exact details of these mech-
`anisms are beyond the scope of this review, but several excellent
`sources are available on this subject (Frank and Fries, 1991;
`Singer et al., 1994; Morgan, 1995; Johnson, 2004). Regardless
`of the pathway of complement activation, the final result is the
`binding and phagocytosis of the foreign particle by the mononu-
`clear phagocytes.
`The third and final step in the clearance process is the inges-
`tion of foreign materials by phagocytes. This step in the process
`typically involves the endocytosis of the particle or foreign
`material by a phagocyte. Following endocytosis of the par-
`ticle, the phagocytes will begin to secret enzymes and other
`oxidative-reactive chemical factors, such as superoxides, oxy-
`halide molecules, nitric oxide, and hydrogen peroxide, to break
`down the phagocytosed material (Mitchell, 2004). Unfortu-
`nately, most non-biodegradable polymeric nanoparticles cannot
`be degraded significantly by this process and, depending on their
`relative size and molecular weight, will either be removed by the
`renal system or sequestered and stored in one of the MPS organs.
`As a first approximation, removal by the renal system occurs
`only for molecules with a molecular weight of around 5000 or
`less, but can be as high as 100,000 for more dense polymers
`such as dendrimers. Therefore, non-biodegradable particles and
`degradation molecules with a molecular weight higher than
`the renal threshold, typically become sequestered in the MPS
`organs. The final biodistribution of this sequestration depends
`on several factors and is discussed in more detail in the biodis-
`tribution and pharmacokinetics section of this paper.
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`95
`
`Since the initial opsonization of particles is so critical to the
`process of phagocytic recognition and clearance from the blood-
`stream, most research in the area of stealth drug delivery has
`focused on trying to stop or block this step of the process. There
`are no absolute rules or methods available to completely and
`effectively block the opsonization of particles, but research over
`the last 30 years has found some trends and methods that can
`be effective at slowing this process, thus increasing the blood
`circulation half-life and effectiveness of stealth devices. As a
`general rule, the opsonization of hydrophobic particles, as com-
`pared to hydrophilic particles, has been shown to occur more
`quickly due the enhanced adsorbability of blood serum proteins
`on these surfaces (Carstensen et al., 1992; Muller et al., 1992;
`Norman et al., 1992).
`A correlation between surface charge and opsonization has
`also been demonstrated in vitro, with research showing that neu-
`trally charged particles have a much lower opsonization rate than
`charged particles (Roser et al., 1998). Therefore, one widely used
`method to slow opsonization is the use of surface adsorbed or
`grafted shielding groups which can block the electrostatic and
`hydrophobic interactions that help opsonins bind to particle sur-
`faces. These groups tend to be long hydrophilic polymer chains
`and non-ionic surfactants. Some examples of polymer systems
`that have been tried in the literature as shielding groups include
`polysaccharides, polyacrylamide, poly(vinyl alcohol), poly(N-
`vinyl-2-pyrrolidone), PEG, and PEG-containing copolymers
`
`PEG chains are always available even after the degradation of
`surface layers. The purpose of these PEG chains is to create a bar-
`rier layer to block the adhesion of opsonins present in the blood
`serum, so that the particles can remain camouflaged or invisible
`to phagocytic cells. Experimental research using freeze-fracture
`transmission electron microscopy (TEM) has even been able to
`demonstrate visually the protein rejecting capabilities of PEGy-
`lated surfaces (Peracchia et al., 1999b).
`Many different types of PEG-containing polymers have been
`tested for their ability to impart stealth characteristic to poly-
`meric nanoparticles. The basic repeating units of poly(ethylene
`glycol) and poly(propylene glycol) are shown below. Because of
`the chemical structure of the repeating units, these polymers are
`also known as poly(ethylene oxide) (PEO) and poly(propylene
`oxide) (PPO).
`
`Tables 1 and 2 contain a representative listing of PEG-
`containing polymers for adsorbed and covalently attached sur-
`face coatings, (adapted from Storm et al., (1995)). From Table 1,
`it is evident that the vast majority of research in PEG surface
`coatings has involved surface adsorbed poloxamers and polax-
`amines.
`
`such as poloxamers, poloxamines, polysorbates, and PEG copo-
`lymers. Of all the polymers tested to date, the most effective and
`most commonly used are the PEG and PEG-containing copoly-
`mers. These polymers are typically very flexible and highly
`hydrophilic, which can help shield even hydrophobic or charged
`particles from blood proteins. They are also typically charge
`neutral, which lessens the effect of electrostatic interactions.
`
`3. PEGylation
`
`As previously mentioned, the preferred method of impart-
`ing stealth, or sterically stabilized properties to nanoparticles is
`through the PEGylation of these particles. PEGylation simply
`refers to the decoration of a particle surface by the covalently
`grafting, entrapping, or adsorbing of PEG chains. Also, in the
`case of biodegradable nanoparticles, PEG chains can be incorpo-
`rated as copolymers throughout the particle so that some surface
`
`These polymers are amphiphilic block copolymers consist-
`ing of blocks of ethylene oxide (EO) and propylene oxide (PO)
`monomer units, which are typically formed by anionic polymer-
`ization.
`The important difference between these structures is the
`additional methyl group of the PO unit, which makes it more
`hydrophobic, while the EO unit is more hydrophilic. Therefore,
`the hydrophobic sections of the polymer which contain PO units
`can be used to adsorb and anchor the surfactant molecule to
`the nanoparticle surface, while the hydrophilic EO containing
`polymers or PEG sections can extend into solution and shield
`the surface of the particle. This method has the advantage of
`being fairly simple to achieve and can impart increased MPS-
`avoidance characteristics to the particles. Conversely, it has the
`draw back that surface adsorbed PEG polymers can also desorb,
`leaving holes in surface coverage where opsonins can bind (Neal
`et al., 1998). The situation is even worse when PEG polymers
`are surface adsorbed on biodegradable polymer nanoparticles.
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`
`Table 1
`Studies of the opsonization of polymeric nanoparticles with surface adsorbed PEG and PEG containing polymer layers
`
`Nanoparticle
`
`Poly(butyl 2-cyanoacrylate) (PBCA)
`
`Poly(␧-caprolactone) (PCL)
`
`Poly(␤-hydroxybutyrate) (PHB)
`
`Poly(lactic acid) (PLA)
`
`Surface coating
`
`Poloxamer-338
`Poloxamine-908
`
`PEG (6000, 20,000)
`Poloxamer-407
`
`Poloxamer (338, 407)
`Poloxamine-908
`
`PEG (6, 20 kDa)
`Poloxamer-188
`Poloxamer-338
`Poloxamer-407
`Poloxamine-908
`
`Poly(lactic-co-glycolic acid) (PLGA)
`
`PEG (2000 or 5000)-b-PLA
`Poloxamer (184, 188, 388)
`Poloxamer-407
`
`Poly(lactic acid):
`poly(ethylene-co-vinyl acetate)
`(PLA:EVA) 50:50
`Poly(methyl methacrylate) (PMMA)
`
`Polystyrene (PS)
`
`Poloxamine-904
`Poloxamine-908
`
`Poloxamer-407
`
`Poloxamer-184
`Poloxamer-188
`Poloxamer-338
`Poloxamer-407
`Poloxamine-904
`Poloxamine-908
`
`Poloxamine-1508
`Polysorbate (20, 60, 80)
`Polyxyethylene (23) lauryl ether (Brij 35)
`
`PEG (2000)
`PEG (22,000)
`PEG (550)-b-BSA (Bovine Serum Albumin)
`PEG (5000)-b-BSA (Bovine Serum Albumin)
`PEG (5000)-b-IgG (Rat)
`PEG (2000 or 5000)-b-PLA
`Poloxamer-184
`Poloxamer-188
`
`Poloxamer-235
`Poloxamer-237
`
`Poloxamer-238
`Poloxamer-338
`
`Poloxamer (401, 402)
`Poloxamer-407
`
`Poloxamine-904
`Poloxamine-908
`
`Poloxamine-1508
`
`Reference
`
`Douglas et al. (1986)
`Douglas et al. (1986)
`
`Leroux et al. (1995)
`Jackson et al. (2000)
`
`Muller and Wallis (1993)
`Muller and Wallis (1993)
`
`De Jaeghere et al. (2000)
`Vittaz et al. (1996)
`Muller and Wallis (1993)
`Muller and Wallis (1993); Jackson et al. (2000)
`Muller and Wallis (1993)
`
`Stolnik et al. (1994)
`Muller and Wallis (1993)
`Muller and Wallis (1993); Dunn et al. (1997); Neal et al. (1998);
`Park et al. (2003)
`Muller and Wallis (1993); Dunn et al. (1997); Neal et al. (1998)
`Stolnik et al. (1994); Dunn et al. (1997)
`
`Jackson et al. (2000)
`
`Troster et al. (1990)
`Leu et al. (1984); Troster et al. (1990)
`Troster et al. (1990); Troster and Kreuter (1992)
`Troster et al. (1990); Jackson et al. (2000)
`Troster and Kreuter (1992)
`Troster et al. (1990); Troster and Kreuter (1992); Troster et al.
`(1992)
`Troster and Kreuter (1992); Troster et al. (1992)
`Troster et al. (1990)
`Troster et al. (1990); Troster and Kreuter (1992)
`
`Harper et al. (1991)
`Tan et al. (1993)
`Moghimi (2002)
`Gbadamosi et al. (2002); Moghimi (2002)
`Moghimi (2002)
`Stolnik et al. (1994)
`Illum et al. (1987b); Blunk et al. (1993); Muller and Wallis (1993)
`Illum et al. (1986, 1987b); Blunk et al. (1993); Muller and Wallis
`(1993)
`Norman et al. (1992)
`Illum et al. (1987b); O’Mullane et al. (1990); Norman et al.
`(1992)
`Illum et al. (1987b); Harper et al. (1991); Norman et al. (1992)
`Illum and Davis (1983, 1984); Illum et al. (1986, 1987b);
`O’Mullane et al. (1990); Watrous-Peltier et al. (1992); Muller
`and Wallis (1993); Tan et al. (1993)
`Moghimi (2003)
`Davis and Illum (1988); Moghimi et al. (1991); Norman et al.
`(1992); Porter et al. (1992a,b); Blunk et al. (1993); Muller and
`Wallis (1993); Moghimi and Gray (1997); Neal et al. (1998);
`Stolnik et al. (2001); Moghimi (2003)
`Muir et al. (1991)
`Illum et al. (1987a,b); Davis and Illum (1988); Moghimi et al.
`(1991); Muir et al. (1991); Norman et al. (1992); Watrous-Peltier
`et al. (1992); Moghimi et al. (1993a,c); Muller and Wallis (1993);
`Tan et al. (1993); Dunn et al. (1994); Stolnik et al. (1994);
`Moghimi and Gray (1997); Neal et al. (1998); Moghimi et al.
`(2003)
`Muir et al. (1991); Tan et al. (1993)
`
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`97
`
`Table 2
`Studies of the opsonization of polymeric nanoparticles with covalently bonded or entangled surface PEG and PEG containing polymer layers
`
`Nanoparticle
`
`Albumin (BSA)
`Gelatin (Type-B)
`Polyalkylcyanoacrylate (PACA)
`
`Poly(␧-caprolactone) (PCL)
`
`Poly(isobutyl 2-cyanoacrylate)
`(PIBCA)
`Poly(lactic acid) (PLA)
`
`Poly(lactic-co-glycolic acid) (PLGA)
`
`Polystyrene (PS)
`
`Surface coating
`
`PEG (1750)
`PEG (5000)
`PEG (2000)-b-polyhexa decylcyanoacrylate
`
`PEG (5000)-b-PCL
`
`PEG (12,000, 20,000)-b-PCL
`Poloxamer-188
`
`Poloxamer-338
`Poloxamer (188, 237, 238, 407)-b-PCL
`
`PEG (4500)-PIBCA
`
`PEG (2000)-b-PLA
`
`PEG (5000)-b-PLA
`
`PEG (10,000 or 15,000)-b-PLA
`PEG (20,000)-b-PLA
`
`PLA-b-PEG (6000 or 20,000)-b-PLA
`Poloxamer-188
`
`PEG (2000 or 5000)-b-PLA
`PEG (5000)-b-PLGA
`
`PEG (12,000 or 20,000)-b-PLGA
`Poloxamer-407
`Poloxamine-904
`Poloxamine-908
`
`PEG (1500)-PS
`PEG (3400 or 5000)-PS
`PEG (2000)-PS
`PS NH CH2
`(CHOH)2 PEG (linear 250,
`500, 1000, 1500, 4000, 19,000)
`PS NH CH2
`(CHOH)2 PEG (branched
`1000, 1700, 6000)
`
`Reference
`
`Ayhan et al. (2003)
`Kaul and Amiji, 2002 (2004)
`Peracchia et al. (1999a,b)
`
`Gref et al. (1994, 2000); Mosqueira et
`al. (2001); Ameller et al. (2003a)
`Gref et al. (1994)
`Chawla and Amiji (2002); Shenoy and
`Amiji (2005)
`Shenoy and Amiji (2005)
`Ha et al. (1999)
`
`Peracchia et al. (1997)
`
`Bazile et al. (1995); Vittaz et al.
`(1996); De Jaeghere et al. (2000); Gref
`et al. (2000)
`Bazile et al. (1995); De Jaeghere et al.
`(2000); Gref et al. (2000); Mosqueira
`et al. (2001); Ameller et al. (2003a,b)
`Gref et al. (2000)
`Gref et al. (2000); Zambaux et al.
`(2000); Mosqueira et al.
`(2001);
`Ameller et al. (2003a,b)
`De Jaeghere et al. (2000)
`Bazile et al. (1995)
`
`Stolnik et al. (1994)
`Gref et al. (1994, 2000); Mosqueira
`et al. (2001); Panagi et al. (2001);
`Ameller et al. (2003a); Avgoustakis et
`al. (2003)
`Gref et al. (1994)
`Dunn et al., (1997)
`Dunn et al. (1997)
`Stolnik et al. (1994); Dunn et al.
`(1997)
`
`Meng et al. (2004b)
`Meng et al. (2004a,b)
`Harper et al. (1991); Dunn et al. (1994)
`Bergstrom et al. (1994)
`
`Bergstrom et al. (1994)
`
`In this case, not only can desorption occur, but biodegradation
`of the particle can also increase the loss of surface bound PEG
`moieties. Because of these issues, several different methods have
`been developed in the literature, see Table 2, to covalently attach
`PEG chains to the surface of nanoparticles. Some research has
`directly shown that particles with covalently bound PEG chains
`achieve longer blood circulation half-lives than similar particles
`with only surface adsorbed PEG (Harper et al., 1991; Bazile et
`al., 1995). Nevertheless, there are some disadvantages to this
`method as well. It is sometimes hard to ensure that covalently
`binding of the PEG chains occurs at the surface and not in the
`bulk of the material, if surface coverage is the goal. Also, as
`a result of this, it can be much more difficult to control and
`optimize the surface coverage density and conformation. On the
`other hand, the covalent bonding of PEG chains throughout the
`
`particle maybe preferred for biodegradable particles, due to the
`availability of surface exposed PEG chains during the entire
`degradation and erosion process.
`To create these types of nanoparticle systems, most
`researchers use a copolymer of PEG with another biodegradable
`polymer, such as poly(lactic acid), poly(lactic acid-co-glycolic
`acid), or poly(alkylcyanoacrylates). In this case, a surface PEG
`layer is typically created by addition of PEG containing copoly-
`mers to the reaction mixture prior to polymerization. Since these
`reactions typically employ an emulsion, precipitation or disper-
`sion polymerization in aqueous media, the PEG portion of the
`copolymer is able to orient itself within the non-reacting water
`phase, while the biodegradable portion of the copolymer is cova-
`lently bonded or physically entangled inside the polymerizing
`nanoparticle matrix. Alternatively, PEG moieties might also be
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`covalently bonded to fully formed nanoparticles after polymer-
`ization by various “living” polymerization techniques, such as
`ATRP and iniferter, or through traditional surface functional
`group chemistry. However, their has only been a small number
`of stealth nanoparticle systems studied that utilize these more
`difficult methods of PEGylation (Bergstrom et al., 1994; Dunn
`et al., 1994).
`Several theories have been proposed to explain the appar-
`ent protein resistance and stealth characteristics imparted to
`materials by the incorporation of surface bound PEG. Alterna-
`tively, some theories have implied that PEGylated nanoparticles,
`added in excess, simply overload the opsonization and clear-
`ance systems of the body, thereby giving the particles the false
`appearance of stealth properties (Moghimi and Szebeni, 2003).
`However, the most widely accepted of these theories is one based
`on the interactions between proteins and PEGylated surfaces,
`which supports the hypothesis that PEGylation can add pro-
`tein resistant (i.e. opsonization resistant) properties to materials
`(Jeon et al., 1991).
`This theory makes the argument that the hydrophilic and flex-
`ible nature of the surface PEG chains allows them to take on a
`more extended conformation when free in solution. Therefore,
`when opsonins and other proteins are attracted to the surface of
`the particle, by van der Waals and other forces, they encounter the
`extended surface PEG chains and begin to compress them. This
`compression then forces the PEG chains into a more condensed
`and higher energy conformation. This change in conformation
`creates an opposing repulsive force that, when great enough,
`can completely balance and/or over power the attractive force
`between the opsonin and the particle surface. It is important
`to note that for effective blocking or repulsion of opsonins to
`occur, the surface coating layer needs to exceed a minimum
`layer thickness. The exact thickness of the layer required can
`vary depending on the situation and is sometimes hard to con-
`trol. Therefore, layer thickness is usually correlated to other
`factors such as PEG molecular weight, surface chain density,
`and conformation.
`Most research indicates that a surface PEG chain molecular
`weight of 2000 or greater is required to achieve increased MPS-
`avoidance characteristics. This minimum MW is most likely
`due to the loss in flexibility of shorter PEG chains. Also, it has
`been shown that as molecular weight is increased above 2000,
`the blood circulation half-life of the PEGylated particles is also
`increased, which may be due in part to the increased chain flex-
`ibility of higher MW PEG polymers (Gref et al., 1994; Leroux
`et al., 1995; Peracchia et al., 1997; Peracchia, 2003). In addition
`to chain molecular weight, surface chain density and confor-
`mation are also critical factors to achieving improved stealth
`characteristics, although these two aspects are much more inter-
`related. For instance, at low surface coverage, the PEG chains
`have a larger range of motion and will typically take on what is
`termed a “mushroom” configuration, where on average they will
`be located closer to the surface of the particle. Very low surface
`coverage can also lead to gaps in the PEG protective layer where
`opsonin proteins can freely bind to the nanoparticle surface. On
`the other hand, at high surface coverage the PEG chains range
`of motion will be greatly restricted and they will most often
`
`Fig. 1. Schematic diagrams of PEG configurations on the upper hemisphere of
`a polymeric nanoparticle. In (a), the low surface coverage of PEG chains leads
`to the “mushroom” configuration where most of the chains are located closer to
`the particles surface. In (b), the high surface coverage and lack of mobility of
`the PEG chains leads to the “brush” configuration where most of the chains are
`extended away from the surface.
`
`exhibit a semi-linear or “brush” configuration. Although a high
`surface coverage ensures that the entire surface of nanoparticle
`is covered, this method also decreases the mobility of the PEG
`chains and thus decreases the steric hindrance properties of the
`PEG layer (Storm et al., 1995). A 3D schematic diagram of the
`PEG “brush” and “mushroom” configurations is illustrated in
`Fig. 1.
`Therefore, the optimal surface coverage is located some-
`where in between the “mushroom” and “brush” configurations,
`where most chains are in a slightly constricted configuration,
`but are present at a high enough density to ensure that no gaps
`or spaces on the particle surface are left uncovered. As a gen-
`eral guideline, researchers have pointed to a minimum effective
`hydrodynamic layer thickness of roughly 5% of the particle’s
`diameter, or one that is greater than twice the hydrodynamic
`radius of the polymer coil in its dilution solution conformation
`(Stolnik et al., 1995; Storm et al., 1995). It should also be noted
`that this analysis of surface coverage was developed primarily
`for solid surfaces, which is not always the case in drug delivery
`systems. For instance, when the surface PEG chains of swollen
`hydrogel materials are compressed, there is a finite probability
`that these chains will penetrate back into the hydrogel matrix
`itself, instead of being compressed into a higher energy confor-
`mation, thereby making the surface coating layer less effective
`(Huang et al., 2001). Currently, this effect has not been fully
`studied in stealth nanoparticles and should therefore be taken
`into consideration when designing stealth hydrogel systems.
`
`4. Biodistribution and pharmacokinetics
`
`Typically once a polymeric nanoparticle is opsonized and
`removed from the bloodstream, it is sequestered in one of the
`MPS organs. In the case of “naked” nanoparticles, or nanopar-
`ticles that have not been PEGylated and lack stealth properties,
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`sequestration in the MPS organs is very rapid, typically a mat-
`ter of minutes, and usually concentrates in the liver and spleen
`(Illum et al., 1987a; Gref et al., 1995; Panagi et al., 2001). How-
`ever, for PEGylated stealth nanoparticles the speed of clearance
`and final biodistribution is dependant on many factors.
`Research has shown that particle size plays a key role in
`the final biodistribution and blood clearance of stealth particles.
`As discussed earlier, molecules that have a molecular weight
`less than 5000, or even higher for dense polymers such as den-
`drimers, can be removed from the body via the renal system.
`For large molecules and particles that can not be removed by the
`renal system, research has shown that particles with hydrody-
`namic radii of over 200 nm typically exhibit a more rapid rate of
`clearance than particles with radii under 200 nm, regardless of
`whether they are PEGylated or not (Moghimi et al., 1993b). In
`other words, a 250 nm PEGylated nanoparticle would be cleared
`from the blood stream much more rapidly than a 70 nm PEGy-
`lated particle. Likewise a 250 nm “naked” nanoparticle would be
`removed more quickly than a 70 nm “naked” nanoparticle, but
`both “naked” nanoparticles and the 250 nm PEGylated particle
`would be removed orders of magnitude more quickly than the
`70 nm PEGylated nanoparticle. Besides blood clearance rate, the
`final biodistribution is also affected by particle size. In the case
`of PEGylated nanoparticles, a hydrodynamic radius of less than
`150 nm was shown to produce an increased uptake of particles
`in the bone marrow of rabbits, where as particles of 250 nm in
`diameter where mostly sequestered in the spleen and liver, with
`only a small fraction of uptake by the bone marrow (Porter et
`al., 1992b).
`Researchers have hypothesized that differences in the uptake
`and biodistribution of stealth particles indicates the presence
`of opsonins that are specific to only a certain type of phago-
`cyte. For instance, Moghimi and Patel (1988) hypothesized that
`an increased accumulation of cholesterol-rich liposomes in the
`spleen was due to the presence of opsonins specific to splenic
`phagocytes, which exhibited stronger binding on chole