The accelerated blood clearance (ABC) phenomenon: Clinical challenge and approaches to manage

Journal of Controlled Release 172 (2013) 38–47

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

The accelerated blood clearance (ABC) phenomenon: Clinical challenge and approaches to manage Amr S. Abu Lila a,b, Hiroshi Kiwada a, Tatsuhiro Ishida a,⁎ a Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima; 1-78-1, Sho-machi, Tokushima 770-8505, Japan b Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

a r t i c l e

i n f o

Article history: Received 21 June 2013 Accepted 29 July 2013 Available online 7 August 2013 Keywords: Accelerated blood clearance (ABC) phenomenon Anti-PEG IgM Polyethylene glycol(PEG) Repeated administration Splenic B cells

a b s t r a c t Despite the clinical introduction of an increasing number of polyethylene glycol (PEG)-conjugated substances, PEG has been named as the cause of an unexpected immunogenic response known as the “accelerated blood clearance (ABC) phenomenon.” This phenomenon has been extensively observed during the repeated administration of PEG-conjugated substances and PEGylated nanocarriers including PEGylated liposomes, PEGylated nanoparticles, PEGylated micelles, etc., resulting in the increased clearance and reduced efficacy of PEGconjugated substances/PEGylated nanocarriers. In this review, therefore, we focused on the possible mechanisms underlying the induction of such a phenomenon and emphasized the factors affecting its magnitude. In addition, the clinical implications of the ABC phenomenon on the therapeutic efficacy of PEG-conjugated substances/ PEGylated nanocarriers, along with the new approaches that can be applied to manage and/or abrogate the induction of the ABC phenomenon, are also discussed. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of the ABC phenomenon . . . . . . . . . . . . . . . . . . . . . . . . . . . . Correlation between complement activation and the ABC phenomenon . . . . . . . . . . . . Factors affecting the magnitude of the ABC phenomenon . . . . . . . . . . . . . . . . . . 4.1. Effect of time interval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Effect of a third dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Effect of PEG-surface density and PEG-chain length . . . . . . . . . . . . . . . . . . 4.4. Effect of size and surface charge . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Effect of a lipid dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Effect of route and mode of administration . . . . . . . . . . . . . . . . . . . . . 4.7. Effect of animal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Effect of an encapsulated drug . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1. Effect of encapsulated cytotoxic agents on the induction of the ABC phenomenon 4.8.2. Effect of encapsulated nucleic acids on the induction of the ABC phenomenon . 4.9. Effect of structure and components of nanocarriers . . . . . . . . . . . . . . . . . . 5. Approaches to abrogate/attenuate the induction of the ABC phenomenon . . . . . . . . . . . 5.1. Manipulation of the physicochemical properties of the PEGylated nanocarriers . . . . . 5.2. Modification of the PEG moiety . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Use of alternative polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Changing the administration regimen . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Encapsulation of drugs with immunosuppressive activity . . . . . . . . . . . . . . . 6. Clinical implications of the ABC phenomenon . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel./fax: +81 88 633 7260. E-mail address: [email protected] (T. Ishida). 0168-3659/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2013.07.026

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

39 39 39 40 40 41 41 42 42 42 42 42 42 43 44 44 44 44 44 45 45 45 45 46

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction PEGylation of nanocarriers is a promising approach applied to improve the pharmacokinetic characteristics of encapsulated drugs, and to achieve better therapeutic outcomes with fewer side effects [1–4]. Polyethylene glycol (PEG) is believed to provide a steric barrier, a hydration zone, around the nanocarriers because of its hydrophilicity that reduces the adsorption of opsonins onto the surfaces of the nanocarriers and thus reduces the particle uptake by the cells of the mononuclear phagocyte system (MPS) in the liver and spleen, and thus, prolonging their blood circulation time [3,5]. However, in contrast to the general assumption that PEG-conjugated substances lack immunogenicity and antigenicity, the PEG-coating, which is intended to confer relative ‘invisibility’ to liposomes against recognition by the cells of the MPS, has been found to trigger the recognition of PEG-stabilized “Stealth” liposomes by the cells of the MPS, upon repeated administration. This was an unexpected pharmacokinetic alteration brought about with a second dose of PEGylated liposomes — the so-called accelerated blood clearance (ABC) phenomenon [6–9]. With this phenomenon, a second dose of PEGylated liposomes is rapidly cleared from circulation when administered within a certain time interval from injection of the first dose due to an enhanced accumulation in the liver. This has been observed for a number of animal species [6,10,11]. Furthermore, it is worth noting that this phenomenon has been induced by pre-treatment with not only PEGylated liposomes but also PEG-containing microemulsions [12], polymeric micelles [13], polymeric nanoparticles [14] and even PEGylated proteins [15]. Therefore, the ABC phenomenon is of clinical concern because it decreases the therapeutic efficacy of encapsulated drugs or PEG-modified proteins upon repeated administration and may cause adverse effects due to the altered biodistribution of the drug. 2. Mechanism of the ABC phenomenon The first report concerning the ABC phenomenon came from Dams et al. [6]. They showed that upon injection of PEGylated liposomes, in rats or in a rhesus monkey, a second dose of PEGylated liposomes was cleared very rapidly from the blood circulation when the interval between the first and second injection was between 5 and 21 days. They suggested that a soluble serum factor is responsible for the induction of the ABC phenomenon. In addition, they demonstrated that the serum factor is a heat-labile non-antibody molecule, which was coeluted on a size extrusion column with a 150 KDa protein. Later on, Laverman et al. [7] identified 2 phases of the ABC phenomenon: the induction phase, following the first administration in which the biological system is “primed”; and, the effectuation phase, following the second, or subsequent, administration in which the PEGylated liposomes are rapidly cleared from the blood circulation. In contrast to the results observed by Dams et al., we revealed that in rats and mice the ABC phenomenon is mediated by soluble serum factors — most likely, low-specificity, anti-PEG immunoglobulin (Ig) M antibody (anti-PEG IgM) [8,16,17]. We emphasized that the level of anti-PEG IgM induced by a single injection of PEGylated liposomes is strongly correlated with the extent of the ABC phenomenon induction [16–20]. Further studies indicated that the spleen plays a critical role in the induction phase [21], and the PEG-specific IgM produced in the spleen is responsible for this unexpected phenomenon [22]. In addition, we showed that the immune reaction in the spleen against PEGylated liposomes extends over a period of at least 2–3 days following the first injection. This pattern of production of IgM is similar to that of the

39

46 9

splenic marginal zone (MZ) B cells, responsible for the first line of defense, and are able to produce large amounts of neutralizing antibodies in a short period (3–4 days) [23,24]. Furthermore, we showed that the ABC phenomenon is observed in BALB/c nu/nu (T cell-deficient) mice, but not in BALB/c SCID (T and B cells-deficient) mice and the anti-PEG IgM was induced only in BALB/c nu/nu via stimulation by the PEGylated liposomes [25]. A similar observation was reported by Semple et al. [26] with nude and SCID-Rag2 mice. They showed that B cells and immunoglobulins, but not T cells, are critical in the development of immune responses against PEGylated liposomes containing oligonucleotides. These data prove that anti-PEG IgM, produced by the splenic B cells that are independent of T-cell involvement, could play a significant role in the induction of the ABC phenomenon. The so-called class-2 of thymus-independent antigens (TI-2) may provide an additional explanation for the mechanism of the ABC phenomenon induced by PEGylated liposomes. TI-2 antigens can induce an immunological response by extensively cross-linking the cellsurface immunoglobulins of specific B cells, resulting in secretion of massive amounts of neutralizing antibodies including IgM and IgG from the B cells [27,28]. Cheng and co-workers [29,30] have demonstrated that anti-PEG antibody (IgM) obtained following immunization with PEGylated β-glucuronide recognizes the repeating \(O\CH2\CH2)n\ subunit (16 units) of PEG. This raises the assumption that PEG polymer in PEGylated nanocarriers acts as a TI-2 antigen and the repeating subunit may be an immunogenic epitope of PEG and a binding site for the derived anti-PEG IgM. It is well established that IgM, one of the major opsonins, has the potential to activate the complement system and consequently enhances the clearance of foreign materials such as pathogens via complement receptor-mediated endocytosis or phagocytosis [31]. Based on all the above-mentioned reports, the following mechanism was set to explain the ABC phenomenon (Fig. 1): Once the PEGylated liposomes (first dose) reach the spleen, they bind and crosslink to surface immunoglobulins on reactive B cells in the splenic marginal zone and consequently trigger the production of an anti-PEG IgM that is independent of T-cell help. Upon administration of the second dose, if anti-PEG IgM, produced in response to the first dose, still exists in the blood circulation, it binds to the PEG on the liposomes, and subsequently activates the complement system, resulting in opsonization by C3 fragments and enhanced uptake by Kupffer cells via complement receptor-mediated endocytosis. Nevertheless, it was also clear that splenectomy, removal of the spleen, failed to completely reverse the rapid clearance and increased hepatic accumulation of PEGylated liposomes to control levels. This suggests that another serum factor(s) or tissue(s) was involved in this phenomenon [21,25]. A recent study by Zhao et al. [32] showed that the ABC phenomenon was observed also after the subcutaneous injection of PEGylated liposomes. In that study, speculation pointed to the contribution of regional lymph nodes and lymphocytes in the initiation of an immune response and/or the production of antibodies against the PEG of PEGylated liposomes. Taken together, the induction of the ABC phenomenon by PEGylated liposomes seems to be a complicated process, for which more studies are needed to elucidate all the contributing factors or tissues. 3. Correlation between complement activation and the ABC phenomenon Complement activation is the major mechanism by which invading pathogens, pathogenic materials and immune complexes are cleared

40

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

Stimulation

Spleen

SL (First injection) Production & release of anti-PEG IgM

Association of anti-PEG IgM SL (Second injection) Complement

IgM-mediated complement activation Liver Complement-receptor mediated endocytosis by liver macrophages Fig. 1. Cartoon depicting the sequence of events leading from anti-PEG IgM induction to accelerated blood clearance of PEGylated liposomes.

subsequent dose of PEGylated liposome. However, the contribution of other serum factor(s) rather than complement in the induction of the ABC phenomenon should not be excluded. Activation of complement systems was also reported to promote the leakage of encapsulated drug from PEGylated liposomes. Yang et al. [36] have demonstrated that epirubicin encapsulated within PEGylated liposomes was rapidly released when liposomes were incubated with sera containing anti-PEG IgM. They attributed such rapid release of epirubicin from PEGylated liposomes to the activation of the complement system via the formation of a membrane attack complex by the assembly of a C5b-9 complex. They assumed that such membrane attack complex, inserted into the liposomal bilayer, generates pores of 10 nm in diameter and consequently triggers the leakage of epirubicin from PEGylated liposomes. Besides the crucial role of complement systems in the opsonization of PEGylated liposomes and/or enhancing the leakage of encapsulated drug in the ABC phenomenon, complement activation by PEGylated liposomes is accounted for the induction of PEGylated liposomerelated hypersensitivity reaction [38–40]. Chanan-Khan et al. [40] demonstrated that Doxil® therapy, a PEGylated liposomal formulation of doxorubicin, activated complement in 21 out of 29 patients and induced moderate to severe hypersensitivity reactions in 13 out of 29 patients. Serum complement terminal complex (SC5b-9) levels were elevated relative to the baseline in 80% of the patients who developed hypersensitivity reactions and 50% in the group with no hypersensitivity at 10 min post-infusion, suggesting that complement activation plays a casual role in hypersensitivity reactions caused by Doxil®. In addition, several reports have emphasized the existence of naturally occurring anti-PEG antibodies in normal donors [41–43]. These naturally occurring anti-PEG antibodies could prime a host's immune response against treatment with PEGylated materials, resulting in the induction of immunogenic responses including hypersensitivity reaction and/or reduced therapeutic effect. 4. Factors affecting the magnitude of the ABC phenomenon

from blood circulation [33]. Many studies have focused on the role of the complement system in the accelerated clearance of PEGylated liposomes from circulation [17,34–36]. We, in a series of our studies [17,21,34,37], have reported that considerable IgM binding and complement consumption upon incubation with PEGylated liposomes were observed only in sera from rats displaying rapid clearance of the second dose. We speculated that anti-PEG IgM, produced in response to a first dose of PEGylated liposome, has a strong potential to activate the classical pathway of complement system, which in turn can serve as a prominent opsonin, and consequently enhances the uptake of a subsequently injected dose of PEGylated liposome via complement receptor-mediated endocytosis or phagocytosis. In the same regard, we also confirmed that anti-PEG IgM-mediated complement activation significantly enhanced the hepatic uptake of PEGylated liposomes [37]. In that study, by using a single-pass liver perfusion technique, we showed that a first dose of PEGylated liposome did not enhance the intrinsic phagocytic activity of the Kupffer cells. On the other hand, the serum obtained from rats pre-treated with PEGylated liposomes was found to enhance the hepatic uptake of test dose of PEGylated liposome. Furthermore, this serumdependent uptake of a test dose by the liver was completely abolished by pre-treatment of the serum at 56 °C for 30 min, which inhibits the complement activity. These results strongly clarify the major role of anti-PEG IgM-mediated complement activation in initiating the accelerated blood clearance of PEGylated liposomes upon multiple administrations. Yang et al. [36] also measured the residual complement activity in serum following incubation with PEGylated liposomes. They demonstrated that a massive amount of complement was consumed in the serum obtained at early stages of the ABC phenomenon in a phospholipids dose-dependent manner; the less the injected dose, the greater the complement activation. These results suggest that complement activation is a major cause to induce the accelerated clearance of

Thus far, it has been reported that both the occurrence and the magnitude of the ABC induction by liposomes are influenced by the dose and physicochemical properties of the PEGylated liposomes, the time interval between repeated injections, and by the species of the encapsulated drugs. 4.1. Effect of time interval Previous reports on PEGylated liposomes have confirmed that the ABC phenomenon occurs in a time-dependent manner and the time interval between the first and second doses is a key factor to elicit the ABC phenomenon and to affect the extent of this phenomenon [6,9,10]. Goins et al. [10] have previously reported that sequential injections of PEGylated liposomes to rabbits in 6-week time intervals did not affect the pharmacokinetics of each of the injected liposomes. Similarly, Oussoren and Storm [44] also reported similar pharmacokinetics for four doses of PEGylated liposomes in rats during either 24 or 48 h intervals. Contradictory to these results, Dams et al. [6] had first shown that prior administration of PEGylated liposome in rats and a rhesus monkey induced the rapid clearance and enhanced the hepatosplenic accumulation of a second dose of PEGylated liposomes when administered 7-days apart from the first dose. Later on, in a series of our studies [9,22,45], we observed a similar phenomenon in rats and mice. We demonstrated that the ABC phenomenon of a second dose is much more pronounced within a time interval of 4 to 7 days following the first injection of PEGylated liposomes (Fig. 2) [22]. The extent of the ABC phenomenon correlated very well with the enhanced production of anti-PEG IgM by splenic B cells following priming by a first dose of PEGylated liposomes [17]. Recently, Ishihara et al. [14] investigated the pharmacokinetic behavior of PEG-modified polylactide (PLA) nanoparticles administered

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

A

120

100

Control

1 day

2 day

3 day

4 day

5 day

% Dose

80

60

40

20

0 0

5

10

15

20

25

30

Time (hr)

B

80

***

***

% Dose

60

41

nanoparticles containing prostaglandin E1. They found an inverse correlation between the dose frequency and the detected IgM level as well as the magnitude of the ABC phenomenon. The ABC phenomenon was less pronounced after the third dose in rats compared to that after a second dose of PEGylated nanoparticles. They attributed such an inverse correlation to the apoptosis and anergy of immune cells in response to cumulative amounts of polymer or encapsulated drugs. Suzuki et al. [48] also reported that, unlike a second dose of DXR-containing PEGylated liposomes, a third dose was retained in the blood circulation for a prolonged period. They assumed that, upon the rapid clearance of the second dose, DXR in the PEGylated liposomes taken up by B cells and Kupffer cells impaired the production of IgM from B cells and also impaired the endocytotic ability of Kupffer cells, thus resulting in a gradual recovery of the pharmacokinetics of the third dose in Beagle dogs. Recently, Saadati et al. [46] observed that, although the anti-PEG IgM level was still high, the pharmacokinetic parameters and the plasma-concentration time curves of a third dose of etoposide-loaded PEGylated nanoparticles were similar to those of the first dose. They concluded in their study that the extended circulation time of the third dose was a result of the saturation of the cells of MPS due to the accumulation of PEGylated nanoparticles after repeated administration, which further hindered the uptake of the nanoparticles by MPS cells.

4.3. Effect of PEG-surface density and PEG-chain length

***

40

20

0 5

4

3

2

1

C

y

y da

da

y da

y da

y da

ol tr on

Fig. 2. Accelerated blood clearance and enhanced hepatic accumulation of a second dose of PEGylated liposomes. Rats were pretreated with PEGylated liposomes (0.001 μmol phospholipids/kg). (A) Blood clearance of a second dose of radio-labeled PEGylated liposomes (5 μmol phospholipids/kg). (B) Hepatic accumulation at 24 h following the injection. Each value represents the mean ± S.D. (n = 3). ⁎⁎⁎p b 0.005 versus control. Modified from Ref. [22].

twice in rats at time intervals of 3, 5, 7, 14, or 28 days. They revealed that in the 7-day interval, the second dose was cleared more rapidly from circulation compared to the first dose or the second doses administered at time intervals of either 3, 14 or 28 days. Saadati et al. [46] have also examined the pharmacokinetic alteration of a second dose of PEGylated polymeric nanoparticles in rats at various time intervals after a first injection (3, 5, 7, 14, or 28 days). They revealed that the ABC phenomenon induced by PEGylated polymeric nanoparticles was most apparent at a time interval of 7 days between the first and second doses, and when the time interval between injections was extended to 28 days, the plasma concentration-time profile of the second dose was comparable to that of the first dose. 4.2. Effect of a third dose Many studies have emphasized the rapid clearance of a second dose of PEGylated nanocarriers after pretreatment with a first dose of the same nanocarriers. However, study of the effect of the injection of sequential doses (more than two) on the ABC phenomenon has been scant. Recently, Ishihara et al. [47] investigated an incidence of the ABC phenomenon upon the sequential administration of PEGylated

As mentioned above, PEGylated liposomes might elicit an anti-PEG IgM response in a T-cell-independent manner and PEG might act as an antigenic epitope [17,49]. Therefore, it was proposed that both the PEG surface density and the PEG chain length would affect the induction of the ABC phenomenon. We have reported that PEGylated liposomes containing 5 mol% mPEG2000-DSPE remarkably elicit a maximal ABC effect at a lower lipid dose (0.001 μmol phospholipids/kg) in rats [45]. While, at either low-surface density of PEG (b 5 mol%) or an excessively high density of PEG (N5 mol%), the ABC phenomenon is reduced. We proposed that the low-surface density of PEG (b5 mol%) is insufficient for the activation of splenic B cells; on the other hand, an excessively high density of PEG (N 5 mol%) resulted in a decrease in the reactivity of the splenic B cells [45]. The extent of the ABC effect induced by PEGylated liposomes with different chain lengths (M.W. 2000 and 5000) was comparable [18]. Contrary to our observation, Ishihara et al. [14] demonstrated that there was no apparent relationship between PEG content and the induction of the ABC phenomenon by PEG-modified nanoparticles where nanoparticles modified with varying concentrations of PEG-derived lipid (0, 7, 11, 18 and 30% mol) caused the ABC phenomenon irrespective of their PEG content. Recently, Li et al. [50] also found that liposomes modified with 9% PEG-derived lipid induced a more severe ABC phenomenon than those modified with 3% PEG-derived lipid despite similar anti-PEG IgM levels following the first dose. Antibody neutralization experiments revealed that a 9% PEG formulation showed a much higher affinity to anti-PEG IgM and thus was easily neutralized by anti-PEG IgM, produced in response to the first dose, compared to the low PEG (3% PEG) formulation, which consequently led to an increased severity of the ABC phenomenon. Similarly, Zhao et al. [51] demonstrated that solid lipid nanoparticles (SLNs) containing 10 mol% PEG produced a higher elimination rate and more hepatic and splenic uptakes of the second dose than SLNs containing 5 mol% PEG. They speculated that varying circulation times of the initial dose, based on PEG surface densities, would lead to a different magnitude of the ABC phenomenon. In addition, they assumed that the only slightly prolonged circulation time imparted by SLNs containing 5 mol% PEG hindered the efficient contact of PEGylated SLNs with splenic B cells, resulting in the secretion of a small amount of anti-PEG IgM. On the other hand, 10 mol% PEG–SLNs exhibited a more prolonged circulation time that enabled an efficient interaction with splenic B cells and the

42

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

subsequent production of larger amounts of anti-PEG IgM, leading to their accelerated blood clearance, compared to 5 mol% PEG–SLNs. 4.4. Effect of size and surface charge We investigated the effect of size and surface charge of PEGylated liposomes in the first injection on the induction of the ABC phenomenon in mice. We prepared liposomes in three different mean particle sizes (100, 400 and 800 nm) and with three different surface charges (+13.15, −46.15 and −1.51 mV). Results showed that changing the liposomal size and/or surface charge of the first dose had no effect on the induction of the ABC phenomenon [11]. Koide et al. [13] also investigated the dependency of the induction of the ABC phenomenon on the particle size of PEGylated polymeric micelles. They reported that mice pretreated with 50.2 nm polymeric micelles experienced an enhanced rapid clearance and hepatic accumulation of a subsequently injected test dose of PEGylated liposomes (127 nm). On the other hand, the pre-administration of both 9.7 and 31.5 nm polymeric micelles did not change plasma concentration and hepatic uptake of a test-dose of PEGylated liposomes. They speculated that the ABC phenomenon is not caused by pre-treatment with smaller-sized polymeric micelles (31.5 nm or less), while it was caused by pre-treatment with largersized polymeric micelles (50.2 nm or more). They assumed that largersized polymeric micelles (50.2 nm or more) could be easily recognized by immune cells and thus activate immune systems. By contrast, smaller-sized polymeric micelles (31.5 nm or less) could avoid the recognition by immune cells. 4.5. Effect of a lipid dose Many reports have confirmed that the lipid dose of PEGylated nanocarrier is critically important for an optimal priming of the immune system in the induction phase of the ABC phenomenon. We have previously reported a strong inverse relationship between the dose of initially injected PEGylated liposomes and the extent of the ABC phenomenon [45]. The ABC phenomenon was potentially manifested when lower phospholipid doses (0.001–0.1 μmol phospholipids/kg) of PEGylated liposomes were intravenously injected as the first dose. On the other hand, higher phospholipid doses (≥5 μmol phospholipids/kg) substantially abrogated the induction of the ABC phenomenon. At a low dose of phospholipids, the extent of B-cell receptor cross-linking by lowdose PEGylated liposomes might be sufficient to activate the cells and promote the production of specific antibody against PEG, i.e. antiPEG IgM. On the other hand, higher doses of PEGylated liposomes (≥5 μmol/kg) might cause MZ-B cells to induce an immune tolerance or to be anergic; unable to mount a complete response against PEGylated nanocarriers. These observations could explain the disappearance of the ABC phenomenon upon repeated administration of the recommended dose intensity (15 μmol phospholipids/kg or higher) of Doxil®/Caelyx® for patients [52]. Nevertheless, the results of a recent study by Ishihara et al. [14] demonstrated that the induction of the ABC phenomenon by PEGylated nanoparticles was not clearly affected by the initial dose. A similar observation with PEGylated PLGA nanoparticles was reported by Saadati et al. [46] who observed that by increasing the dose from 0.1 to 20 mg/mouse, the magnitude of the ABC phenomenon was not changed. However, both studies dealt with polymeric nanoparticles instead of liposomes, and, therefore, the physicochemical properties of the PEGylated nanocarrier in addition to the dose might have contributed to the induction of the ABC phenomenon. 4.6. Effect of route and mode of administration The induction of the ABC phenomenon was mainly studied after a bolus intravenous injection of PEGylated liposomes or nanocarriers in animals. Hence, little is known about the effect of the route and/or the

mode of administration of the nanocarriers on the induction of this phenomenon. Li et al. [50] recently demonstrated that the magnitude of induction of the ABC phenomenon is significantly influenced by the administration mode of PEGylated liposomes. A slow infusion of PEGylated liposomes with a low lipid concentration was found to induce a more severe ABC phenomenon, compared to a bolus intravenous administration with the same dose of PEGylated liposomes. The authors assumed that a slow infusion of PEGylated liposomes might facilitate the binding of antibody and the subsequent clearance of liposomes from blood circulation. Once a small amount of liposomes entered the blood, the anti-PEG IgM produced in response to the first dose of PEGylated liposomes might be in excess relative to the injected second dose, and, thus, might lead to a significant clearance of the second dose. In contrast, if a large dose of liposomes was rapidly injected into an animal, the IgM levels might not be sufficient to neutralize such a high dose of liposomes or might subsequently lead to significant alterations in the pharmacokinetic profiles. Interestingly, Zhao et al. [32] recently declared that the induction of the ABC phenomenon is not restricted only to the intravenous injection of PEGylated nanocarriers (SLNs), but also to the subcutaneous injection of PEGylated SLNs. They demonstrated that the first subcutaneous injection of PEGylated SLNs in rats efficiently induced the rapid clearance of a second intravenously administered dose of PEGylated SLNs from circulation. In addition, the ABC index, the ratio of AUC(0→t) for the second injection to that of the control for a subcutaneous injection was equivalent to or even lower than that following the first intravenous injection. These observations potentially emphasize the implications of the route/mode of administration of PEGylated products on the induction of the ABC phenomenon. 4.7. Effect of animal species Researchers have used different animal models to study the ABC phenomenon, including rhesus monkeys, rats, mice and rabbits among others. Dams et al. found that that rhesus monkeys and rats could demonstrate the ABC phenomenon following repeated injection of PEGylated liposomes, whereas mice could not [6]. In 2003, we reported the same phenomenon in mice, although we found accelerated elimination with a time interval of 10 days between doses, which differed from the 7-day interval for rats [9]. By contrast, Goins et al. excluded the occurrence of the ABC phenomenon in rabbits following repeated administration of PEGylated liposomes at a time interval of 6 weeks [10]. Recently, Suzuki et al. [48] used Beagle dogs to study the ABC phenomenon. They revealed that the Beagle dogs were more sensitive to PEGylated liposomes than were rats in terms of the anti-PEG antibody response and the induction of the ABC phenomenon. They found that dogs that received a higher lipid dose of empty PEGylated liposomes (16.7 μmol phospholipids/kg) continued to produce a higher titer of anti-PEG IgM, and the ABC phenomenon was consequently induced. These results were contradictory to those of previous studies, which showed that when rats were pretreated with a high dose (more than 5 μmol phospholipids/kg) of empty PEGylated liposomes, the induction of the ABC phenomenon was weakened [22,25,45]. Consequently, difference between species as a cause of the ABC phenomenon is another important issue that should be addressed by pre-clinical study of the PEGylated formulation. 4.8. Effect of an encapsulated drug 4.8.1. Effect of encapsulated cytotoxic agents on the induction of the ABC phenomenon PEGylated liposomes are frequently utilized for cancer drug delivery because of their tumor selective accumulation via the EPR (enhanced permeability and retention) effect [53–56]. However, the induction of the ABC phenomenon upon repeated administration of PEGylated

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

liposomes may present a tremendous challenge to their clinical use, as multiple injections are generally needed in clinical settings. Nevertheless, it is worth noting that in clinical practice the repeated administration of Doxil®, a PEGylated liposomal formulation of the cytotoxic drug doxorubicin (DXR) within the dose range of 10–60 mg/m2 every 28 days, did not induce the ABC phenomenon [57]. Laverman et al. [7] demonstrated, in a murine model, that repeated injections of Caelyx® did not induce the ABC phenomenon, while empty PEGylated liposomes did. They assumed that the toxic effect of DXR on the hepatosplenic macrophages might have impaired the role of these cells in the induction of the phenomenon. In a subsequent study, we proposed that the abrogation of the ABC phenomenon, upon repeated administration of DXR-containing PEGylated liposomes, was attributed to the reduced production of anti-PEG IgM caused by the inhibition of splenic B cell proliferation and/or the killing of proliferating B cells by DXR released from the liposomes after the first injection [34]. This assumption was supported by the observation that free DXR did not alleviate the induction of the ABC phenomenon by “empty” PEGylated liposomes [48]. In addition, many studies have emphasized that the encapsulation of cytotoxic agents such as mitoxantrone [58] and oxaliplatin [20,59] significantly abrogates the induction of the ABC phenomenon. These results suggest that PEGylated liposomes containing cytotoxic drugs do not cause the ABC phenomenon under multiple-dosing regimens. Consequently, the ABC phenomenon has no clinical implications for cytotoxic drug-containing liposomes and is only restricted to “empty” PEGylated liposomes or PEGylated liposomes containing non-cytotoxic drugs [8,9,49,60]. However, Deng and his colleagues [50,61] have recently demonstrated that, unlike DXR and mitoxantrone, pre-dosing with topotecancontaining PEGylated liposomes still induces a strong ABC phenomenon for the second dose in Wistar rats, Beagle dogs and mice. They assumed that topotecan, a cell-cycle phase-specific drug, could only inhibit the population of B cells in the splenic marginal zone occupying the S phase of the cell cycle, and thus, its toxic effect on B cells was dramatically limited [61]. In addition, they offered another assumption that the lipophilicity of topotecan and unsatisfactory retention inside PEGylated liposomes may trigger the formation of “empty” liposomes in blood circulation and the inefficient delivery of topotecan to splenic B cells leading to an immunostimulatory, rather than an immunoinhibitory, effect to B cells by such “empty” PEGylated liposomes [50]. In the same context, Saadati et al. [46] have recently investigated the effect of the cytotoxic agent, etoposide, encapsulated in PEGylated poly (lacticco-glycolic acid) (PLGA) nanoparticles on the induction of the ABC phenomenon in rats. They demonstrated that the magnitude of the ABC phenomenon as well as the level of anti-PEG IgM in rats pre-treated with etoposide-loaded PEGylated PLGA nanoparticles were equal to those in rats pre-treated with “empty” nanoparticles. They concluded that, etoposide, like topotecan, a cell cycle-specific drug, only affected the B cells that are in the G2/M phase, and, hence, the drug-loaded PEGylated nanoparticles did not inhibit the entire B cell population, which produces the anti-PEG IgM, in the splenic marginal zone. Besides the mode of action of a cytotoxic agent, the administered dose of the cytotoxic agent encapsulated within PEGylated liposomes was found to affect the magnitude of the ABC phenomenon. In the case of Doxil®, Suzuki et al. [48] recently reported, in a dog study, that at lower doses (2 mg DXR/m2 or lower), Doxil® strongly induced the ABC phenomenon, but that a high Doxil® dose, which is similar to a conventional clinical dose, abrogated the induction of the ABC phenomenon. It seems that, unlike a higher dose, a lower dose of Doxil® instead activates the B cells and thereby enhances the secretion of anti-PEG IgM following the first injection. A similar observation was demonstrated by Nagao et al. [20] who reported that at a low dose of oxaliplatin (l-OHP)-containing PEGylated liposomes (0.023 μg l-OHP/kg), the l-OHP triggered a relatively higher anti-PEG IgM response. On the other hand, at higher doses of l-OHP-containing PEGylated liposomes (2.3–2300 μg l-OHP/kg), the l-OHP instead tended to inhibit the anti-PEG IgM response.

43

They proposed that, at a low dose of l-OHP-containing PEGylated liposomes, l-OHP stimulates a host's immune system, resulting in the induction of anti-PEG IgM production presumably via its immunomodulatory effect on splenic B-cells. On the other hand, at higher doses of l-OHP-containing PEGylated liposomes, the l-OHP released from liposomes accumulating in the spleen inhibited B cell proliferation and/or killed proliferating B cells, thus impairing the production of anti-PEG IgM. Recently, sequential low-dose chemotherapy has received great attention for its unique advantages in attenuating the multi-drug resistance of tumor cells. However, this raises the risk of producing new problems associated with the ABC phenomenon, particularly with multiple injections of PEGylated liposomes. Yang et al. [36] examined the effect of the sequential administration of PEGylated liposomes containing the cytotoxic agent, epirubicin, on the induction of the ABC phenomenon. They demonstrated that the first or sequential injections of PEGylated epirubicin liposomes in Wistar rats within a certain range (1–15 μmol phospholipids/kg, corresponding to 0.08– 1.2 mg epirubicin/kg) induced the rapid clearance of subsequently injected PEGylated epirubicin liposome resulting in the abolishment of therapeutic efficacy in S180 tumor-bearing mice. Taken together, the above-mentioned data emphasize the necessity of examining the possibility of the occurrence of the ABC phenomenon during doseescalating studies undertaking the preclinical evaluation of new therapeutic cytotoxic agents encapsulated in PEGylated nanocarriers. 4.8.2. Effect of encapsulated nucleic acids on the induction of the ABC phenomenon Cationic liposomes represent promising alternatives to viral vectors as gene delivery vehicles [62–64]. However, due to their low in vivo transfection efficiency, repeated administration is required in order to obtain the optimum therapeutic outcome. Hence, the ABC phenomenon encountered upon repeated administration of PEGylated liposomes may constitute potential risks of non-immunogenic vectors (PEGylated cationic liposome) acting as a strong immune adjuvant, particularly when they carry immune-stimulating nucleic acids such as plasmid DNA (pDNA) or short interfering RNA (siRNA). Semple et al. [26] reported that repeated administration of PEGylated liposomes carrying oligonucleotides (ODN), pDNA or RNA ribozyme, induced a strong immune response that resulted in an enhanced blood clearance and an increase in the mortality of experimental mice. Judge et al. [65] also demonstrated that PEG-coated lipid nanoparticles encapsulating pDNA significantly enhanced anti-PEG IgM production when compared with empty PEGcoated nanoparticles and consequently diminished the gene expression relating to pDNA in tumor tissue following its second injection. Based on the above-mentioned results, we extensively studied the immunogenic response of PEGylated liposomes encapsulating nucleic acids such as pDNA [66] and siRNA [19,60]. We showed that PEGcoated pDNA-lipoplexes potentiate the production of anti-PEG IgM compared with PEG-coated cationic liposomes without pDNA [66]. In addition, we showed that the removal of the CpG motif from pDNA, which is a ligand of toll-like receptor 9 (TLR9), significantly attenuated the anti-PEG IgM production and allowed the accumulation of a second dose in tumor tissue. These results suggest that the adjuvant effect of nucleic acids on anti-PEG IgM production is mediated via TLR9activation by the CpG motif of pDNA. Similar results were revealed by Hemmi et al. who confirmed that CpG motifs in DNA have a strong adjuvant effect that contributes to an increase in immunogenicity via the stimulation of TLR9 [67]. Therefore, the use of non-CpG DNA may allow repeated administration of PEGylated formulations containing pDNA without the induction of strong immune reactions such as the ABC phenomenon, thereby promoting a better transfection and an enhanced therapeutic effect. In a subsequent study, we investigated the effect of siRNA-encapsulation within a PEGylated wrapsome (PEG-WS; a recently introduced PEGylated lipid nanocarrier [68]) as well as the effect of the siRNA-sequence on anti-PEG IgM production and the

44

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

occurrence of the ABC phenomenon [19]. We showed that siRNA encapsulated within PEG-WS barely induced the production of anti-PEG IgM compared to siRNA in conventional PEGylated lipoplexes. In addition, the incorporation of 2′-O-methyl (2′-OMe) uridine into the sequence of siRNA attenuated the production of anti-PEG IgM via inhibiting the production of cytokines such as IL-6 and TNF-α. Collectively, these results suggest that a proper design of the siRNA delivery system, by adequate chemical modification and masking the siRNA by lipid encapsulation into the core of PEGylated liposomes, could alleviate or lessen the adjuvant effect of siRNA via attenuating the activation of innate immunity by siRNA, which induces anti-PEG IgM production.

5. Approaches to abrogate/attenuate the induction of the ABC phenomenon

4.9. Effect of structure and components of nanocarriers

Many studies have emphasized the effect of the physicochemical properties of PEGylated nanocarriers, such as particle size, on the occurrence and magnitude of the ABC phenomenon. Koide et al. [13] reported that polymeric micelles with diameters of approximately 50 nm elicited the ABC phenomenon but that smaller ones with diameters of less than 30 nm did not. In addition, the ABC phenomenon was not induced in humans upon repeated injection of PEGylated interferon with a molecular weight of 40 KDa [72]. Thus, the size of PEGylated formulations may be a determinant for induction of the ABC phenomenon. However, obtaining liposomal formulations with diameters lower than 30 nm seems to be technically difficult on account of the lower physical stability of the liposomes and the difficulty of encapsulating drugs in such small nanocarriers with high efficiency. Therefore, it is necessary to find another way to suppress the ABC phenomenon rather than affecting the size of the colloidal nanocarriers.

The ABC phenomenon represents a barrier against the development of efficient drug delivery systems and against the clinical use of not only PEGylated liposomes but also other PEGylated nanocarriers, such as nanoparticles [14,46], microemulsions [12], polymeric micelles [13] or even PEGylated proteins [15]. Lu et al. [15] have found that cationic bovine serum albumin-conjugated polyethylene glycol–polylactide (PEG–PLA) nanoparticles could induce the ABC phenomenon with repeated injection of the PEGylated nanoparticles. Similarly, Ishihara et al. [14] also found that the ABC phenomenon occurred when PEG– PLA-modified nanoparticles were administered. Nevertheless, many recent studies have confirmed that, in addition to PEG, the colloidal structure of nanocarriers strongly contributes to the induction of the ABC phenomenon. Koide et al. [13] reported the accelerated clearance of PEGylated liposomes in mice pre-administered empty polyethylene glycol-b-poly(β-benzyl L-aspartate) (PEG–PBLA) polymeric micelles. On the other hand, Ma et al. [69] revealed that repeated injections of the gadolinium (Gd)-containing PEG-poly(L-lysine)-based polymeric micelles (Gd–micelles) with a 7-day time interval did not cause accelerated clearance for the second dose. The ABC phenomenon of the second dose of the Gd-containing PEGylated liposome (Gd-liposome) was induced by the first dose of both the Gd-liposome and the empty PEGylated liposome, but not by the first injection of the Gdmicelle. Such controversial results might be attributed to the differences in the structure and components of nanocarriers. From a structural perspective, the Gd-micelle has no hydrophobic portion. In contrast, the PEG–PBLA micelle is composed of both a hydrophilic part, PEG, and a hydrophobic part, PBLA. In a similar manner, PEGylated liposomes possess a hydrophilic PEG chain and a hydrophobic bilayer membrane. Therefore, those researchers suggested that the hydrophobic core of the micelle or lipid bilayer of PEGylated liposomes has a major effect on the induction of the ABC phenomenon. Recently, Yokoyama and his group [70] reported the results of a study on the ABC phenomenon induced by PEGylated polymeric micelles. For that study, two types of PEG-containing polymeric micelles were prepared: a micelle with a hydrophobic inner core (PEG–PBLA); and, a micelle with a hydrophilic inner core (PEGP(Lys-DOTA-Gd)). The results indicated that PEG-P(Lys-DOTA-Gd) induced neither an anti-PEG IgM response nor the ABC phenomenon, on the other hand, a PEG–PBLA micelle did. These results confirmed the assumption that, although both types of polymer micelles have a PEG outer shell, the absence of the hydrophobic part is a key for abrogation of the ABC phenomenon. Another study declaring the impact of the colloidal structure of a nanocarrier on the induction of the ABC phenomenon has been presented by Hara et al. [71]. They showed that lactosome, which is a polymer micelle composed of poly(lactic acid)-b-poly(sarcosine), was cleared from the bloodstream after a second dosage by trapping in the liver, although lactosome contains no PEGylated component. They concluded that the occurrence of the ABC phenomenon was mediated via the production of anti-lactosome IgM and IgG3 through the immune response related to B-lymphocyte cells. They emphasized that, the antigenic epitope capable of generating anti-lactosome IgM is the repeating poly(sarcosine) subunit, which covers the surface of the lactosome.

Many researchers are now paying an increasing amount of attention to the ABC phenomenon. Several strategies have been proposed to alleviate the induction of the phenomenon, but no feasible solution has been devised to avoid or abrogate this problem. In this section, we will focus on some of these approaches. 5.1. Manipulation of the physicochemical properties of the PEGylated nanocarriers

5.2. Modification of the PEG moiety Many approaches have been applied to minimize the immunogenicity of the PEG moiety upon repeated administration. Ambegia et al. [73] and Webb et al. [74] considered that the activity of liposomes in vivo could be adjusted by using a dissociable PEG-lipid with a different alkyl chain length. They showed that PEG-lipids with a shorter alkyl chain could dissociate more rapidly from the lipid bilayer than conventional PEG-lipids, such as mPEG-DSPE and mPEG-CHOL, and, thus, could attenuate the occurrence of the ABC phenomenon. Judge [65] and Semple et al. [26] used PEGylated lipids with smaller C14 lipid anchors in the preparation of PEGylated liposomes and found that the ABC phenomenon was avoided by the dissociation of PEG from the particles. However, this lipid exchange would result in defects in the membranes of the liposomes and thus may induce a pre-mature release of the encapsulated payload. Another attempt has focused on altering the linkage between the lipids and PEG. Xu et al. [75] synthesized two PEG-lipid derivatives (PEG-CHMC and PEG-CHEMS) that were linked via a single ester bond so that PEG could be cleaved by the esterase in circulating blood. They showed that PEGylated liposome prepared using such cleavable PEGlipid derivatives could attenuate the occurrence of the ABC phenomenon [76]. Later on, Chen et al. [77] introduced a novel cleavable PEGlipid derivative (mPEG-Hz-CHEMS) in which the PEG moiety was linked to cholesterol by two ester bonds and one pH-sensitive hydrazone (Fig. 3). They emphasized that liposomes modified with this novel PEG-lipid derivative showed prolonged blood circulation characteristics, compared with liposomes modified with an mPEG-CHEMS lipid derivative. In addition, upon repeated administration of mPEG-HzCHEMS-modified liposomes, no ABC phenomenon was observed. 5.3. Use of alternative polymers Although PEG remains the gold standard for the steric stabilization of liposomes, many attempts have focused on the use of other polymers to prepare long-circulating liposomes and to avoid the occurrence of the ABC phenomenon. Romberg et al. [78] reported that liposomes coated with poly(hydroxyethyl L-glutamine) or poly(hydroxyethyl L-asparagine) showed similar stealth properties and reduced the occurrence of the ABC

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

Attenuation of the ABC phenomenon

45

Blood vessel

were observed with PEGylated nanocarriers carrying cytotoxic agents such as l-OHP [20,59] and mitoxantrone [58]. Encapsulation of immunosuppressive agents within a PEGylated nanocarrier, therefore, could be a useful approach to abrogate the induction of the ABC phenomenon upon repeated administration. However, this strategy significantly limits the utility of the PEGylated nanocarriers because it is expected that, in addition to agents having immunosuppressive activity, these carriers will also be used for different agents with no immunosuppressive activity, and for chemically unstable drugs such as siRNA. 6. Clinical implications of the ABC phenomenon

Cleavage

Esterace

pH

Ester Chol

hz Ester PEG

mPEG-Hz-CHEMS Fig. 3. Schematic diagram of the strategy for double cleavable smart mPEG-Hz-CHEMSmodified liposomes. Modified from Ref. [77].

phenomenon compared with PEG-coated liposomes. However, when those liposomes were injected at low doses, elicitation of the ABC phenomenon persisted. Ishihara et al. [47] have reported that a coating of nanoparticles with poly(N-vinyl-2-pyrrolidone) (PVP) led to an extended residence of the first dose of nanoparticles in the bloodstream of rats, although their circulating times were shorter than that of PEG-coated nanoparticles. The ABC phenomenon was not induced upon repeated injection of PVP-coated nanoparticles at various time intervals due to the lack of anti-PVP IgM production. In the same manner, we recently showed that modification of the liposome surface with a polyglycerol (PG)-derived lipid, instead of PEG, elicits neither an anti-polymer immune response nor the ABC phenomenon upon repeated administration, resulting in an enhanced therapeutic efficacy of encapsulated DXR in a tumor-bearing mouse model. We assumed that the hydroxymethyl side group in the repeating \(O\CH2\CH(CH2OH))n\ subunit of PG sterically hinders the interaction and/or effective binding to surface immunoglobulins of reactive splenic B-cells, resulting in the absence of the specific stimulation of splenic B cells. This could afford the attenuation of anti-PG IgM production, which may be responsible for eliciting the ABC phenomenon. In a subsequent study, we confirmed that the surface modification of pDNA-lipoplex with PG, instead of PEG, was also efficient in attenuating the immunogenic response encountered with PEGylated lipoplex. 5.4. Changing the administration regimen Many studies have emphasized that the administration of high doses of PEGylated nanocarriers and the prolongation of a time interval between injections can considerably reduce the magnitude of the ABC phenomenon, as mentioned earlier [35,79,80]. However, these approaches may cause severe side effects at high doses and/or affect the therapeutic efficacy of the encapsulated drug. 5.5. Encapsulation of drugs with immunosuppressive activity Repeated injections of Doxil®/Caelyx®, a PEGylated liposomal formulation of DXR, did not induce the ABC phenomenon because of the immunosuppressive activity of the encapsulated DXR [7]. Similar results

It is generally believed that PEGylated nanocarriers have no, or a lower, degree of immunogenicity. However, results from the aforementioned research have indicated that an unexpected immune response, the “ABC phenomenon,” occurs with such PEGylated nanocarriers. Such immunogenicity of PEG-coated nanocarriers presents a serious concern in their development and use in the clinic since the establishment of an antibody response can severely compromise both the safety and the efficacy of an associated drug payload such as DNA, RNA or proteins. Despite the fact that the ABC phenomenon does not constitute a problem in cancer chemotherapy [7,52,53,81] from the use of a PEGylated liposome, because of either the higher lipid dose used or the cytotoxic effect of an encapsulated agent on antibody-secreting cells such as splenic B cells [34], the ABC phenomenon could be substantially emphasized upon repeated administration of non- or moderately cytotoxic agents or immuno-stimulatory agents such as pDNA and RNA [60,61,66]. Furthermore, currently, a relatively new and promising application of nanocarrier systems is their use in molecular and diagnostic imaging. “Theranostic” is a common term for the realization of efficient therapies using imaging techniques [82–84]. Two types of theranostic agents currently exist. In the first type, diagnostic and therapeutic agents are individually loaded into separate nanocarriers. In the second type, however, both therapeutic and diagnostic agents are loaded onto the same nanocarrier. PEGylation is commonly applied to both types of systems to prolong their residence time in blood circulation. Therefore, if the system triggers the activation of an immune response to PEG in the first dose, the targeting effect of the second dose will be critically lost. Consequently, the induction of the ABC phenomenon indicates that we will have only one chance to obtain either a diagnostic or therapeutic effect upon the application of a theranostic strategy. 7. Conclusion The PEGylation technique has been applied for more than 30 years in the pharmaceutical field [85,86]. PEGylation has been acknowledged for increasing the blood circulation time and decreasing the immunogenicity of modified protein drugs and drug-delivery vehicles [1,3,4,87,88]. Despite these achievements, a number of limitations and challenges still hamper complete deployment of PEG [89]. The ABC phenomenon, observed upon repeated administration of PEGylated nanocarriers, is one such limitation, and it imposes great impediments to the clinical application of PEGylated nanocarriers that require repeated administration [90]. Therefore, the development of a strategy to attenuate and/or abrogate the immunogenicity of PEG-coated nanocarriers without significantly compromising their in vivo performance would be highly desirable for the further development of this promising drug delivery system. Abbreviations ABC accelerated blood clearance AUC area under the plasma versus time curve CHOL cholesterol DXR doxorubicin EPR enhanced permeability and retention Gd gadolinium IL-6 interleukin 6

46

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47

mPEG2000-DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n[methoxy(polyethylene glycol)-2000] MPS mononuclear phagocyte system MZ marginal zone ODN oligonucleotides pDNA plasmid DNA PEG polyethylene glycol PEG-PBLA polyethylene glycol-b-poly(β-benzyl L-aspartate) PG polyglycerine PLA polylactide PLGA poly(lactic-co-glycolic acid) PVP poly(N-vinyl-2-pyrrolidone) siRNA short interfering RNA SLNs solid lipid nanoparticles TI thymus-independent TNF-α tumor necrosis factor alpha Acknowledgment We thank Mr. J.L. McDonald for his helpful advice in writing the manuscript. This study was supported, in part, by a Grant-in-Aid for Scientific Research (B) (23390012), the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References [1] A.L. Klibanov, K. Maruyama, V.P. Torchilin, L. Huang, Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes, FEBS Lett. 268 (1990) 235–237. [2] J. Senior, C. Delgado, D. Fisher, C. Tilcock, G. Gregoriadis, Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles, Biochim. Biophys. Acta 1062 (1991) 77–82. [3] V.P. Torchilin, Polymer-coated long-circulating microparticulate pharmaceuticals, J. Microencapsul. 15 (1998) 1–19. [4] C. Allen, N. Dos Santos, R. Gallagher, G.N. Chiu, Y. Shu, W.M. Li, S.A. Johnstone, A.S. Janoff, L.D. Mayer, M.S. Webb, M.B. Bally, Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol), Biosci Rep. 22 (2002) 225–250. [5] D.D. Lasic, F.J. Martin, A. Gabizon, S.K. Huang, D. Papahadjopoulos, Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times, Biochim. Biophys. Acta 1070 (1991) 187–192. [6] E.T. Dams, P. Laverman, W.J. Oyen, G. Storm, G.L. Scherphof, J.W. van Der Meer, F.H. Corstens, O.C. Boerman, Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes, J. Pharmacol. Exp. Ther. 292 (2000) 1071–1079. [7] P. Laverman, M.G. Carstens, O.C. Boerman, E.T. Dams, W.J. Oyen, N. van Rooijen, F.H. Corstens, G. Storm, Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection, J. Pharmacol. Exp. Ther. 298 (2001) 607–612. [8] T. Ishida, R. Maeda, M. Ichihara, Y. Mukai, Y. Motoki, Y. Manabe, K. Irimura, H. Kiwada, The accelerated clearance on repeated injection of pegylated liposomes in rats: laboratory and histopathological study, Cell. Mol. Biol. Lett. 7 (2002) 286. [9] T. Ishida, R. Maeda, M. Ichihara, K. Irimura, H. Kiwada, Accelerated clearance of PEGylated liposomes in rats after repeated injections, J. Control. Release 88 (2003) 35–42. [10] B. Goins, W.T. Phillips, R. Klipper, Repeat injection studies of technetium-99mlabeled peg-liposomes in the same animal, J. Liposome Res. 8 (1998) 265–281. [11] T. Ishida, T. Ichikawa, M. Ichihara, Y. Sadzuka, H. Kiwada, Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice, J. Control. Release 95 (2004) 403–412. [12] M. Joshi, S. Pathak, S. Sharma, V. Patravale, Solid microemulsion preconcentrate (NanOsorb) of artemether for effective treatment of malaria, Int. J. Pharm. 362 (2008) 172–178. [13] H. Koide, T. Asai, K. Hatanaka, T. Urakami, T. Ishii, E. Kenjo, M. Nishihara, M. Yokoyama, T. Ishida, H. Kiwada, N. Oku, Particle size-dependent triggering of accelerated blood clearance phenomenon, Int. J. Pharm. 362 (2008) 197–200. [14] T. Ishihara, M. Takeda, H. Sakamoto, A. Kimoto, C. Kobayashi, N. Takasaki, K. Yuki, K. Tanaka, M. Takenaga, R. Igarashi, T. Maeda, N. Yamakawa, Y. Okamoto, M. Otsuka, T. Ishida, H. Kiwada, Y. Mizushima, T. Mizushima, Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles, Pharm. Res. 26 (2009) 2270–2279. [15] W. Lu, J. Wan, Z. She, X. Jiang, Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle, J. Control. Release 118 (2007) 38–53. [16] T. Ishida, X. Wang, T. Shimizu, K. Nawata, H. Kiwada, PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner, J. Control. Release 122 (2007) 349–355.

[17] X. Wang, T. Ishida, H. Kiwada, Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes, J. Control. Release 119 (2007) 236–244. [18] T. Ishida, H. Kiwada, Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes, Int. J. Pharm. 354 (2008) 56–62. [19] T. Tagami, Y. Uehara, N. Moriyoshi, T. Ishida, H. Kiwada, Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA, J. Control. Release 151 (2011) 149–154. [20] A. Nagao, A.S. Abu Lila, T. Ishida, H. Kiwada, Abrogation of the accelerated blood clearance phenomenon by SOXL regimen: promise for clinical application, Int. J. Pharm. 441 (2013) 395–401. [21] T. Ishida, M. Ichihara, X. Wang, H. Kiwada, Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes, J. Control. Release 115 (2006) 243–250. [22] T. Ishida, M. Ichihara, X. Wang, K. Yamamoto, J. Kimura, E. Majima, H. Kiwada, Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes, J. Control. Release 112 (2006) 15–25. [23] F. Martin, A.M. Oliver, J.F. Kearney, Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens, Immunity 14 (2001) 617–629. [24] A. Zandvoort, W. Timens, The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general first-line defense against blood-borne antigens, Clin. Exp. Immunol. 130 (2002) 4–11. [25] M. Ichihara, T. Shimizu, A. Imoto, Y. Hashiguchi, Y. Uehara, T. Ishida, H. Kiwada, Anti-PEG IgM response against PEGylated liposomes in mice and rats, Pharmaceutics 3 (2011) 1–11. [26] S.C. Semple, T.O. Harasym, K.A. Clow, S.M. Ansell, S.K. Klimuk, M.J. Hope, Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic acid, J. Pharmacol. Exp. Ther. 312 (2005) 1020–1026. [27] Q. Vos, A. Lees, Z.Q. Wu, C.M. Snapper, J.J. Mond, B-cell activation by T-cellindependent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms, Immunol. Rev. 176 (2000) 154–170. [28] Z. Sha, R.W. Compans, Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus, J. Virol. 74 (2000) 4999–5005. [29] T.L. Cheng, B.M. Chen, J.W. Chern, M.F. Wu, S.R. Roffler, Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy, Bioconjugate Chem. 11 (2000) 258–266. [30] T.L. Cheng, P.Y. Wu, M.F. Wu, J.W. Chern, S.R. Roffler, Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM, Bioconjugate Chem. 10 (1999) 520–528. [31] K.B. Reid, R.R. Porter, The proteolytic activation systems of complement, Annu. Rev. Biochem. 50 (1981) 433–464. [32] Y. Zhao, C. Wang, L. Wang, Q. Yang, W. Tang, Z. She, Y. Deng, A frustrating problem: accelerated blood clearance of PEGylated solid lipid nanoparticles following subcutaneous injection in rats, Eur. J. Pharm. Biopharm. 81 (2012) 506–513. [33] A.J. Bradley, D.V. Devine, The complement system in liposome clearance: can complement deposition be inhibited? Adv. Drug Deliv. Rev. 32 (1998) 19–29. [34] T. Ishida, K. Atobe, X. Wang, H. Kiwada, Accelerated blood clearance of PEGylated liposomes upon repeated injections: effect of doxorubicin-encapsulation and high-dose first injection, J. Control. Release 115 (2006) 251–258. [35] K. Taguchi, S. Ogaki, H. Watanabe, D. Kadowaki, H. Sakai, K. Kobayashi, H. Horinouchi, T. Maruyama, M. Otagiri, Fluid resuscitation with hemoglobin vesicles prevents Escherichia coli growth via complement activation in a hemorrhagic shock rat model, J. Pharmacol. Exp. Ther. 337 (2011) 201–208. [36] Q. Yang, Y. Ma, Y. Zhao, Z. She, L. Wang, J. Li, C. Wang, Y. Deng, Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy, Int. J. Nanomedicine 8 (2013) 1257–1268. [37] T. Ishida, S. Kashima, H. Kiwada, The contribution of phagocytic activity of liver macrophages to the accelerated blood clearance (ABC) phenomenon of PEGylated liposomes in rats, J. Control. Release 126 (2008) 162–165. [38] A.H. Brouwers, D.J. De Jong, E.T. Dams, W.J. Oyen, O.C. Boerman, P. Laverman, T.H. Naber, G. Storm, F.H. Corstens, Tc-99m-PEG-liposomes for the evaluation of colitis in Crohn's disease, J. Drug Target. 8 (2000) 225–233. [39] J. Szebeni, L. Baranyi, S. Savay, M. Bodo, D.S. Morse, M. Basta, G.L. Stahl, R. Bunger, C.R. Alving, Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction, Am. J. Physiol. Heart Circ. Physiol. 279 (2000) H1319–H1328. [40] A. Chanan-Khan, J. Szebeni, S. Savay, L. Liebes, N.M. Rafique, C.R. Alving, F.M. Muggia, Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions, Ann. Oncol. 14 (2003) 1430–1437. [41] Y. Liu, H. Reidler, J. Pan, D. Milunic, D. Qin, D. Chen, Y.R. Vallejo, R. Yin, A double antigen bridging immunogenicity ELISA for the detection of antibodies to polyethylene glycol polymers, J. Pharmacol. Toxicol. Methods 64 (2011) 238–245. [42] J.K. Armstrong, G. Hempel, S. Koling, L.S. Chan, T. Fisher, H.J. Meiselman, G. Garratty, Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients, Cancer 110 (2007) 103–111. [43] A.W. Richter, E. Akerblom, Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins, Int. Arch. Allergy Appl. Immunol. 70 (1983) 124–131. [44] C. Oussoren, G. Storm, Effect of repeated intravenous administration on circulation kinetics of poly(ethyleneglycol) liposomes in rats, J. Liposome Res. 9 (1999) 349–355.

A.S. Abu Lila et al. / Journal of Controlled Release 172 (2013) 38–47 [45] T. Ishida, M. Harada, X.Y. Wang, M. Ichihara, K. Irimura, H. Kiwada, Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes, J. Control. Release 105 (2005) 305–317. [46] R. Saadati, S. Dadashzadeh, Z. Abbasian, H. Soleimanjahi, Accelerated blood clearance of PEGylated PLGA nanoparticles following repeated injections: effects of polymer dose, PEG coating, and encapsulated anticancer drug, Pharm. Res. 30 (2013) 985–995. [47] T. Ishihara, T. Maeda, H. Sakamoto, N. Takasaki, M. Shigyo, T. Ishida, H. Kiwada, Y. Mizushima, T. Mizushima, Evasion of the accelerated blood clearance phenomenon by coating of nanoparticles with various hydrophilic polymers, Biomacromolecules 11 (2010) 2700–2706. [48] T. Suzuki, M. Ichihara, K. Hyodo, E. Yamamoto, T. Ishida, H. Kiwada, H. Ishihara, H. Kikuchi, Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs, Int. J. Pharm. 436 (2012) 636–643. [49] H. Koide, T. Asai, K. Hatanaka, S. Akai, T. Ishii, E. Kenjo, T. Ishida, H. Kiwada, H. Tsukada, N. Oku, T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes, Int. J. Pharm. 392 (2010) 218–223. [50] C. Li, J. Cao, Y. Wang, X. Zhao, C. Deng, N. Wei, J. Yang, J. Cui, Accelerated blood clearance of pegylated liposomal topotecan: influence of polyethylene glycol grafting density and animal species, J. Pharm. Sci. 101 (2012) 3864–3876. [51] Y. Zhao, L. Wang, M. Yan, Y. Ma, G. Zang, Z. She, Y. Deng, Repeated injection of PEGylated solid lipid nanoparticles induces accelerated blood clearance in mice and beagles, Int. J. Nanomedicine 7 (2012) 2891–2900. [52] O. Lyass, B. Uziely, R. Ben-Yosef, D. Tzemach, N.I. Heshing, M. Lotem, G. Brufman, A. Gabizon, Correlation of toxicity with pharmacokinetics of pegylated liposomal doxorubicin (Doxil) in metastatic breast carcinoma, Cancer 89 (2000) 1037–1047. [53] A. Gabizon, A. Dagan, D. Goren, Y. Barenholz, Z. Fuks, Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice, Cancer Res. 42 (1982) 4734–4739. [54] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [55] A. Chonn, P.R. Cullis, Recent advances in liposomal drug-delivery systems, Curr. Opin. Biotechnol. 6 (1995) 698–708. [56] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004) 1818–1822. [57] N.M. La-Beck, B.A. Zamboni, A. Gabizon, H. Schmeeda, M. Amantea, P.A. Gehrig, W.C. Zamboni, Factors affecting the pharmacokinetics of pegylated liposomal doxorubicin in patients, Cancer Chemother. Pharmacol. 69 (2012) 43–50. [58] J. Cui, C. Li, C. Wang, Y. Li, L. Zhang, H. Yang, Repeated injection of pegylated liposomal antitumour drugs induces the disappearance of the rapid distribution phase, J. Pharm. Pharmacol. 60 (2008) 1651–1657. [59] A.S. Abu Lila, N.E. Eldin, M. Ichihara, T. Ishida, H. Kiwada, Multiple administration of PEG-coated liposomal oxaliplatin enhances its therapeutic efficacy: a possible mechanism and the potential for clinical application, Int. J. Pharm. 438 (2012) 176–183. [60] T. Tagami, K. Nakamura, T. Shimizu, T. Ishida, H. Kiwada, Effect of siRNA in PEG-coated siRNA-lipoplex on anti-PEG IgM production, J. Control. Release 137 (2009) 234–240. [61] Y. Ma, Q. Yang, L. Wang, X. Zhou, Y. Zhao, Y. Deng, Repeated injections of PEGylated liposomal topotecan induces accelerated blood clearance phenomenon in rats, Eur. J. Pharm. Sci. 45 (2012) 539–545. [62] K. Nakamura, A.S. Abu Lila, M. Matsunaga, Y. Doi, T. Ishida, H. Kiwada, A double-modulation strategy in cancer treatment with a chemotherapeutic agent and siRNA, Mol. Ther. 19 (2011) 2040–2047. [63] M. Sainlos, M. Hauchecorne, N. Oudrhiri, S. Zertal-Zidani, A. Aissaoui, J.P. Vigneron, J.M. Lehn, P. Lehn, Kanamycin A-derived cationic lipids as vectors for gene transfection, ChemBioChem 6 (2005) 1023–1033. [64] G.D. Schmidt-Wolf, I.G. Schmidt-Wolf, Non-viral and hybrid vectors in human gene therapy: an update, Trends Mol. Med. 9 (2003) 67–72. [65] A. Judge, K. McClintock, J.R. Phelps, I. Maclachlan, Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes, Mol. Ther. 13 (2006) 328–337. [66] T. Tagami, K. Nakamura, T. Shimizu, N. Yamazaki, T. Ishida, H. Kiwada, CpG motifs in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNA-lipoplexes, J. Control. Release 142 (2010) 160–166. [67] H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira, A toll-like receptor recognizes bacterial DNA, Nature 408 (2000) 740–745. [68] N. Yagi, I. Manabe, T. Tottori, A. Ishihara, F. Ogata, J.H. Kim, S. Nishimura, K. Fujiu, Y. Oishi, K. Itaka, Y. Kato, M. Yamauchi, R. Nagai, A nanoparticle system specifically designed to deliver short interfering RNA inhibits tumor growth in vivo, Cancer Res. 69 (2009) 6531–6538.

47

[69] H. Ma, K. Shiraishi, T. Minowa, K. Kawano, M. Yokoyama, Y. Hattori, Y. Maitani, Accelerated blood clearance was not induced for a gadolinium-containing PEGpoly(L-lysine)-based polymeric micelle in mice, Pharm. Res. 27 (2010) 296–302. [70] K. Shiraishi, M. Hamano, H. Ma, K. Kawano, Y. Maitani, T. Aoshi, K.J. Ishii, M. Yokoyama, Hydrophobic blocks of PEG-conjugates play a significant role in the accelerated blood clearance (ABC) phenomenon, J. Control. Release 165 (2013) 183–190. [71] E. Hara, A. Makino, K. Kurihara, F. Yamamoto, E. Ozeki, S. Kimura, Pharmacokinetic change of nanoparticulate formulation “Lactosome” on multiple administrations, Int. Immunopharmacol. 14 (2012) 261–266. [72] E. Formann, W. Jessner, L. Bennett, P. Ferenci, Twice-weekly administration of peginterferon-alpha-2b improves viral kinetics in patients with chronic hepatitis C genotype 1, J. Viral Hepat. 10 (2003) 271–276. [73] E. Ambegia, S. Ansell, P. Cullis, J. Heyes, L. Palmer, I. MacLachlan, Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression, Biochim. Biophys. Acta 1669 (2005) 155–163. [74] M.S. Webb, D. Saxon, F.M. Wong, H.J. Lim, Z. Wang, M.B. Bally, L.S. Choi, P.R. Cullis, L.D. Mayer, Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine, Biochim. Biophys. Acta 1372 (1998) 272–282. [75] H. Xu, Y. Deng, D. Chen, W. Hong, Y. Lu, X. Dong, Esterase-catalyzed dePEGylation of pH-sensitive vesicles modified with cleavable PEG-lipid derivatives, J. Control. Release 130 (2008) 238–245. [76] H. Xu, K.Q. Wang, Y.H. Deng, W. Chen da, Effects of cleavable PEG-cholesterol derivatives on the accelerated blood clearance of PEGylated liposomes, Biomaterials 31 (2010) 4757–4763. [77] D. Chen, W. Liu, Y. Shen, H. Mu, Y. Zhang, R. Liang, A. Wang, K. Sun, F. Fu, Effects of a novel pH-sensitive liposome with cleavable esterase-catalyzed and pH-responsive double smart mPEG lipid derivative on ABC phenomenon, Int. J. Nanomedicine 6 (2011) 2053–2061. [78] B. Romberg, C. Oussoren, C.J. Snel, M.G. Carstens, W.E. Hennink, G. Storm, Pharmacokinetics of poly(hydroxyethyl-L-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration, Biochim. Biophys. Acta 1768 (2007) 737–743. [79] K. Taguchi, Y. Urata, M. Anraku, H. Watanabe, D. Kadowaki, H. Sakai, H. Horinouchi, K. Kobayashi, E. Tsuchida, T. Maruyama, M. Otagiri, Hemoglobin vesicles, polyethylene glycol (PEG)ylated liposomes developed as a red blood cell substitute, do not induce the accelerated blood clearance phenomenon in mice, Drug Metab. Dispos. 37 (2009) 2197–2203. [80] C. Li, X. Zhao, Y. Wang, H. Yang, H. Li, W. Tian, J. Yang, J. Cui, Prolongation of time interval between doses could eliminate accelerated blood clearance phenomenon induced by pegylated liposomal topotecan, Int. J. Pharm. 443 (2013) 17–25. [81] P.G. Tardi, E.N. Swartz, T.O. Harasym, P.R. Cullis, M.B. Bally, An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin, J. Immunol. Methods 210 (1997) 137–148. [82] H. Koo, M.S. Huh, I.C. Sun, S.H. Yuk, K. Choi, K. Kim, I.C. Kwon, In vivo targeted delivery of nanoparticles for theranosis, Acc. Chem. Res. 44 (2011) 1018–1028. [83] M. Kumagai, M.R. Kano, Y. Morishita, M. Ota, Y. Imai, N. Nishiyama, M. Sekino, S. Ueno, K. Miyazono, K. Kataoka, Enhanced magnetic resonance imaging of experimental pancreatic tumor in vivo by block copolymer-coated magnetite nanoparticles with TGF-beta inhibitor, J. Control. Release 140 (2009) 306–311. [84] H. Cabral, N. Nishiyama, K. Kataoka, Supramolecular nanodevices: from design validation to theranostic nanomedicine, Acc. Chem. Res. 44 (2011) 999–1008. [85] A. Abuchowski, J.R. McCoy, N.C. Palczuk, T. van Es, F.F. Davis, Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase, J. Biol. Chem. 252 (1977) 3582–3586. [86] A. Abuchowski, T. van Es, N.C. Palczuk, F.F. Davis, Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol, J. Biol. Chem. 252 (1977) 3578–3581. [87] W. Sun, W. Zou, G. Huang, A. Li, N. Zhang, Pharmacokinetics and targeting property of TFu-loaded liposomes with different sizes after intravenous and oral administration, J. Drug Target. 16 (2008) 357–365. [88] S. Hussain, A. Pluckthun, T.M. Allen, U. Zangemeister-Wittke, Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system, Mol. Cancer Ther. 6 (2007) 3019–3027. [89] G. Knop, R. Margaria, Cardiac pheochromocytoma: a new case reported, J. Thorac. Cardiovasc. Surg. 132 (2006) 1230–1231. [90] N.J. Ganson, S.J. Kelly, E. Scarlett, J.S. Sundy, M.S. Hershfield, Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase, Arthritis Res. Ther. 8 (2006) R12.