Polymeric Micelles Possessing Polyethyleneglycol as Outer Shell and Their Unique Behaviors in Accelerated Blood Clearance Phenomenon

Abstract

Polymeric micelles are assemblies of synthetic polymers and have been studied and developed as drug carriers for targeting. Polymeric micelles are composed of the inner core and the outer shell, and typically form from AB-type block copolymers in which two polymer blocks are connected in a tandem form. Polyethyleneglycol (PEG) has been most commonly used as one polymer block composing the outer shell. This review describes the reasons that PEG is used for the outer shell of the polymeric micelle carrier systems. On the other hand, accelerated blood clearance (ABC) phenomenon is a well-known immunological response of PEG-coated liposomes. Since the ABC phenomenon greatly influences targeting functions of carrier systems, elaborative studies on polymeric micelles’ ABC phenomenon have been done, and revealed different behaviors of the polymeric micelle systems from those of PEG-coated liposomes. These studies indicate that polymeric micelle systems are highly feasible tools for contrast agent targeting as well as theranostics.

1. INTRODUCTION

Polymeric micelles are nano-sized assemblies of synthetic polymers and have been actively studied as carriers of drugs and contrast agents since the 1980s. In this review, we describe correlations between polyethyleneglycol (PEG) and polymeric micelle carrier systems by focusing on two aspects: (1) the reasons that PEG has been most commonly used as the outer shell of the polymeric micelle systems; and (2) accelerated blood clearance (ABC) phenomenon of the polymeric micelles. The ABC phenomenon is an immunological response observed in multiple doses of PEG-possessing carrier systems.

Before going into the two aspect contents, we briefly explain polymeric micelles for drug and contrast agent carrier systems with descriptions of polymeric micelle’s chemistry as well as histories of research and clinical developments.

2. POLYMERIC MICELLE CARRIER SYSTEMS

A polymeric micelle is an assembly of synthetic polymers comprising an inner core and an outer shell. The polymeric micelles can have a spherical or cylindrical shape depending on chemical structures and chain lengths of the polymers. For drug carriers, most polymeric micelles studies have dealt with the spherical shape; only a very limited number of filamentous shape systems have been studied.^1,2) Figure 1 illustrates the formation of a spherical polymeric micelle from AB-type block copolymers in which two polymer blocks are connected in tandem form. Drug molecules are incorporated into the inner core of the micelle through chemical conjugation and physical entrapment. Such a micellar structure forms if one segment of the block copolymer can provide enough interchain cohesive interactions in an aqueous medium. For cohesive interactions, hydrophobic interactions are the most commonly used because many drug molecules possess a hydrophobic character. More detailed explanation of the polymeric micelle carriers can be obtained in other reviews.^3–10)

Fig. 1. Polymeric Micelle Carrier System

Research into polymeric micelles as drug carriers originated in a study of Ringsdorf and colleagues.^11,12) They reported the first application of polymeric micelles to drug carrier systems in 1984. A PEG-containing block copolymer conjugating drug and hydrophobic molecules was synthesized. Although micelle formation of this polymer–drug conjugate was suggested from data of dye-solubilization experiments, formation of micellar structure was not confirmed by more direct methods such as laser light scattering or gel-permeation chromatography. These researchers reported sustained release of conjugated drug from the block copolymer, and their study was limited to the in vitro stage.

Yokoyama et al.^13–19) designed polymeric micelle systems using PEG–poly(amino acid) block copolymers in the late 1980s with a clear focus on selective delivery to the target by this carrier system, and succeeded in obtaining drug targeting to solid tumors through enhanced permeability and retention (EPR) effect of these malignant tissues.

At that time, whereas studies of polymeric micelle drug carrier systems were very limited, by the 1990s a substantial increase in research activity was seen with a large number of publications by several research groups. In some papers,²⁰⁾ polymeric micelles were referred to as “nanospheres,” “nanoparticles,” or “nanoparticulates.” However, as far as the two-phase structure of the inner core and outer shell are formed from block or graft copolymers, this structure is correctly classified as polymeric micelles.

Owing to the substantial development of polymeric micelle studies, several clinical trials of targeting of anti-cancer drugs are curretnly ongoing. On the other hand, polymeric micelles were recently used as magnetic resonance imaging (MRI) contrast agent carriers. As described in Section 4, the ABC phenomenon is more important for polymeric micelle contrast agents.

3. WHY IS PEG MOST COMMONLY USED AS THE OUTER SHELL OF POLYMERIC MICELLE CARRIER SYSTEMS?

In most cases with a very limited number of exceptions,^21–23) PEG is used in the outer shell-forming polymer block for polymeric micelle carrier systems. In this section (Table 1), we explain the reasons for this very common use of PEG.

Table 1. The Reasons for PEG Use as the Outer Shell of Polymeric Micelle Carrier Systems

1 Biological reasons	Inertness
2 Chemical reasons
(2-1) Water-solubility
(2-2) Well-defined structure

First, PEG’s inertness in biological environments is well known, and this property has been utilized in many biomedical applications. This biological inertness includes low protein adsorption, low activation of contacted cells, low degree of cell adhesion, low cell uptake, low degree of inflammatory activation, and low toxicity. For drug carrier purposes, the low cell uptake, in particular by monocytes such as macrophages and liver Kupffer cells, is most important because the carrier’s targeting functions are lost if the monocytes’ uptake is enhanced.²⁴⁾ Enhanced monocyte uptake results in substantial accumulation of drug carriers in reticuloendothelial system organs such as liver and spleen, and consequently a very low concentration of the carriers in the bloodstream. As delivery to each organ and tissue is made through the bloodstream, the very low carrier’s concentration in blood greatly inhibits delivery to target organs.

On the other hand, water solubility is an essential prerequisite for polymeric micelles forming in an aqueous medium including blood. PEG is water-soluble, and therefore, the outer shell can be formed from PEG in physiological conditions. Another usuful chemical property of PEG is its well-defined structure including three desirable properties such as no branch structure, narrow molecular weight distribution, and high terminal functionality. PEG is synthesized through anionic polymerization of ethylene oxide. This polymerization is completely free from side reactions that produce branches in polymer structures of PEG. This is a significantly important advantage because most common radical polymerizations produce branch structures very frequently. Complex polymer structures such as branches are unfavorable for exact control of polymeric micelle physical properties such as size and critical micelle concentration. Polymeric micelles forming from AB-type block copolymers shown in Fig. 1 are highly suitable for exact control. Even if a water-soluble monomer containing a double bond (that can be polymerized in a radical polymerization) can produce a polymer suitable for the outer shell of polymeric micelle carriers, branch formation in its radical polymerization is a big disadvantage in comparison with PEG.

In general, synthetic polymers possess molecular weight distributions, while natural polypeptides possess only one molecular weight without its distribution. Narrower molecular weight distributions are preferable for exact control of polymeric micelles’ physical properties. The anionic polymerization that produces PEG is one of the optimal polymerization methods in terms of polymer production with narrow molecular weight distributions.

Another important property is high terminal functionality of PEG. Block copolymers containing PEG are prepared with polymerization of another monomer from a terminal of PEG. Two most commonly examined block copolymers, PEG-b-poly(amino acid)s¹⁹⁾ and PEG-b-polyesters,²⁵⁾ are synthesized from a primary amino terminal and from a hydroxyl terminal of PEG, respectively. A high terminal functionality (100% is ideal) makes it possible to obtain a high purity of block copolymer. If the terminal functionality is low, a considerable amount of PEG homopolymers may be contaminated in block copolymer products. The high terminal functionality of PEG is obtained owing to the following two observations: (1) in the polymerization for PEG, there is no side reaction that produces a wrong terminal; and (2) chemistry is well developed for terminal conversion²⁶⁾ (e.g., from a hydroxyl group to a primary amino group).

4. ABC PHENOMENON OF POLYMERIC MICELLES

One relevant and important aspect for polymeric micelle carriers is analysis of accelerated blood clearance (ABC) phenomenon. ABC is a phenomenon where clearance rates of carrier systems from the bloodstream are substantially raised after repeated injections.^27,28) This ABC phenomenon has been well studied with PEG-coated liposomes^29,30) that exhibit long-circulating characters at the first injection. As shown in Fig. 2, a PEG-coated liposome can circulate stably in blood for a long time-period at its first dose, whereas the same PEG-coated liposome injected at the second time rapidly decreases its concentration in blood²⁷⁾ if these doses are made in appropriate dose amounts and dose intervals. This phenomenon occurs owing to immunological activity induced at the first dose, and the change in clearance may be considerable. Since polymeric micelles with PEG outer shells have the same profile as that of the PEG-coated liposomes in terms of the PEG outer layer, it is of great interest to understand whether these polymeric micelles induce the ABC phenomenon.

Fig. 2. ABC Phenomenon

The ABC phenomenon was not observed³¹⁾ in cancer chemotherapy due to toxic side effects of cytotoxic anti-cancer drug to the immune system that provides the ABC phenomenon. Therefore the significance of the ABC phenomenon seems low for polymeric micelles carrying cytotoxic anti-cancer drugs. However, the significance becomes much higher once polymeric micelle systems carrying contrast agents are considered. If the ABC phenomenon happens for the polymeric micelle contrast agent systems, these contrast agents cannot be repeatedly used. Contrast agents permitting only one-time use are useless in clinics. Furthermore, a serious risk may arise in combination use of polymeric micelle contrast agent and polymeric micelle anti-cancer drug system in the wake of the ABC phenomenon. If the first dose of the contrast agent induces the ABC phenomenon, the second dose of the anti-cancer drug-carrying system can greatly accumulate in liver, and accordingly, severe hepatotoxic side effects could occur. This is a very bad scenario in the practice of theranostics, in which diagnosis and therapy are attempted with the same carrier. Furthermore, in the case of non-cytotoxic drug delivery, the presence of the ABC phenomenon substantially influences the efficacy of pharmacotherapy given in multiple doses. Even in cancer therapies, this aspect is important for non-cytotoxic anti-cancer drugs such as retinoids.^32–36) Therefore examination of the ABC phenomenon is important not only due to scientific interest but also from clinical viewpoints.

We reported the first observation of polymeric micelles’ display of the ABC phenomenon.³⁷⁾ We injected three kinds of polymeric micelles at the first dose, followed by injection of a PEG-coated liposome at the second injection. The ABC phenomenon was observed only for one polymeric micelle, whereas it was not observed for the other two polymeric micelles. This indicates that not all polymeric micelles induce the ABC phenomenon. This is an interesting observation because some polymeric micelles do not induce the ABC phenomenon even though these micelles possess PEG in the outer layer. More interestingly, blood concentrations of the ABC induction-positive polymeric micelle were not changed at the second dose.³⁸⁾ Once the ABC phenomenon is induced, the induced immune system enhances uptake of injected carrier systems by the liver, providing a significant decrease in the carrier system’s concentration in blood. However, this polymeric micelle did not exhibit the decrease, suggesting that although this micelle can induce the ABC phenomenon, it is not uptaken by the ABC-activated liver. This obseravation provides strong scientific interest concerning elucidation of the ABC phenomenon’s mechanism, and indicates great usefulness of polymeric micelle carrier systems in terms of favorable suppression of the ABC phenomenon in clinical settings.

We previously reported that a polymeric micelle MRI contrast agent did not exhibit the ABC phenomenon.^38,39) Not only did this polymeric micelle not induce the ABC phenomenon, nor was it uptaken by the ABC-activated liver. A distinctive difference is present in the inner core structure between this MRI contrast agent polymeric micelle and the drug carrier polymeric micelles stated above: the hydrophilic inner core for the former MRI contrast agent polymeric micelle and the hydrophobic inner core for the latter drug carrier polymeric micelles. (Ionic interactions are driving forces for cohesive interactions in the inner cores for the polymeric micelle MRI contrast agent.) Table 2 summarizes the above-mentioned results of the ABC phenomenon. We have not completely elucidated the immunological mechanism to explain polymeric micelles’ behaviors of the ABC phenomenon. Important keys are present at the interface between PEG and the inner core-forming polymer block, we believe, because chemical structures of this interface vary considerably in polymeric micelle cases in sharp contrast to the PEG-coated liposome cases that possess only one interface structure of PEG and lipid acyl chain. It is worth analyzing polymeric micelles’ ABC phenomenon for more clinical usefulness as well as elucidating underlying immune mechanisms that explain differences between polymeric micelles and liposomes.

Table 2. Polymeric Micelles’ Behaviors of ABC Phenomenon

Type	Inner core	Behaviors
1 Drug carrier	Hydrophobic	• Some micelles are ABC-positive, while some micelles are ABC-negative
		• Some micelles induce ABC, but are not uptaken by ABC(+) liver
2 MRI contrast agent carrier	Hydrophilic	ABC-negative

5. CONCLUSION

For polymeric micelles as drug targeting carriers, PEG is most commonly used as the outer shell-forming polymer block. The reason for this common use is its inertness in biological responses as well as its two chemical properties, water-solubility and well-defined structure. On the other hand, the ABC phenomenon is well known for PEG-coated liposomes. Since the ABC phenomenon greatly inhibits targeting efficiency in repeated doses, it is very important to investigate the ABC phenomenon of polymeric micelles. Interestingly, polymeric micelles exhibit very different behaviors for the ABC phenomenon from those of PEG-coated liposomes. Indeed, some polymeric micelles did not show any ABC phenomenon. This is a big advantage of polymeric micelle carriers.

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