Smart polymeric micelles for gene and drug delivery

Vol. 2, No. 1 2005

Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY

TODAY

TECHNOLOGIES

Drug delivery/formulation and nanotechnology

Smart polymeric micelles for gene and drug delivery Nobuhiro Nishiyama1, Younsoo Bae2, Kanjiro Miyata2, Shigeto Fukushima2, Kazunori Kataoka1,2,3,* 1

Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Department of Materials Science and Engineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 3 CREST, Japan Science and Technology Agency, Japan 2

Polymeric micelles, supramolecular assemblies of block copolymers, are useful nanocarriers for the systemic delivery of drugs and genes. Recently, novel polymeric micelles with smart functions, such as targetability and stimuli-sensitivity, have emerged as promising carriers that enhance the efficacy of drugs and genes with minimal side effects. This review focuses on the construction and characteristic behaviors of intracellular environment-sensitive micelles that selectively exert drug activity and gene expression in live cells. Introduction A polymeric micelle is a supramolecular assembly of block copolymers, having a characteristic core-shell structure; the drug-loaded core is surrounded by biocompatible poly(ethylene glycol) (PEG) outer shells (Fig. 1) [1,2]. During the past decade, polymeric micelles have demonstrated their utility in delivering drugs and are currently recognized as promising formulations for enhancing the efficacy of drugs [3,4]. Indeed, several micellar formulations encapsulating antitumor drugs are now undergoing clinical trials [5]. Recently, there has been a strong incentive to develop polymeric micelles with smart functions such as targetability to specific tissues [6–10] and chemical [7,11–13] or physical [14] stimulisensitivity. Such smart polymeric micelles are assumed to enhance the efficacy of the loaded drugs as well as to mini*Corresponding author: K. Kataoka ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddtec.2005.05.007

Section Editor: Daan J.A. Crommelin – Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Gerrit Borchard – Enzon Pharmaceuticals, Piscataway, NJ, USA mize side effects beyond current drug delivery formulations. In this review, the focus is placed on the development of smart polymeric micelles for the delivery of drugs and genes, which accomplish the therapeutic function by responding to low pH conditions in the endosomal or lysosomal compartment of the cells.

Main text Smart polymeric micelles for site-specific drug delivery Table 1 summarizes the polymeric micelles responding to intracellular signals. Among these intracellular signals, low pH in endosomes and lysosomes is a useful chemical stimulus for designing environmentally sensitive drug carriers because macromolecular carriers are taken up by the cells via endocytosis and finally localized in the endosomes and/or lysosomes. More than a 100-fold higher proton concentration in acidic vesicular organelles (pH 5.0) than the extracellular medium (pH 7.4) could allow a highly selective intracellular drug delivery. Thus, a minimal leakage of the loaded drug in the bloodstream should ensure the safety in their clinical use. Furthermore, the selective drug release in endosomes and lysosomes might circumvent the drug efflux by P-glycoproteins in multidrug-resistant cells, overcoming a multiple drug resistance in cancer chemotherapy [17]. The pH-sensitive www.drugdiscoverytoday.com

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Figure 1. Formation of pH-sensitive polymeric micelles from amphiphilic PEG-b-P(Asp-Hyd-DXR) copolymers, in which the antitumor drug, doxorubicin (DXR), was conjugated through acid-labile hydrazone linkers [reprinted with permission from Ref. [11] (ß2003 Wiley-VCH) and Ref. [12] (ß2005 American Chemical Society)].

polymeric micelles can be constructed by the use of an acidcleavable bond between the drug and the carrier polymer. Kataoka et al. at the University of Tokyo (Tokyo, Japan) (http://bmw.t.u-tokyo.ac.jp/english/index.html) have recently developed micelle-forming PEG-block-P(Asp-Hyd-DXR) copolymers, which are prepared by chemically conjugating doxorubicin (DXR) to the side chain of PEG-b-poly(aspartic acid) copolymers via an acid-labile hydrazone bond (Fig. 1) [11,12]. Such pH-sensitive polymeric micelles significantly released active DXR under low pH conditions (5.0) corresponding to late endosomes and lysosomes, whereas they stably retained drugs under a physiological pH condition [11]. The biodistribution study revealed that the micelles showed stable blood circulation owing to a minimal drug leakage, and thereby, they preferentially accumulated in solid

tumors [12]. Consequently, the micelles effectively suppressed tumor growth in mice over a broad range of injection doses while the toxicity remained extremely low, which is in marked contrast to free DXR with a narrow therapeutic window [12]. Bae et al. at the University of Utah (Salt Lake City, UT, USA) (http://www.pharmacy.utah.edu/pharmaceutics/groups/bae) have reported PEG-b-poly(L-histidine) copolymers possessing pKa values around a physiological pH, which act as amphiphilic copolymers to form polymeric micelles under a physiological pH condition while showing a hydrophilicity, biodegradability and fusogenic activity at the lower pH conditions (<7.0) [7]. This micelle showed an accelerated release of DXR when the pH decreased. The active targeting in conjunction with intracellular drug release might allow precise control of the drug distribution,

Table 1. Polymeric micelles responding to intracellular signals Signals

Researchers

University

URL

Refs

Low pH

Kataoka et al. Bae et al. Fre´ chet et al.

University of Tokyo University of Utah UC Berkeley

http://bmw.t.u-tokyo.ac.jp/english/index.html http://www.pharmacy.utah.edu/pharmaceutics/groups/bae http://ist-socrates.berkeley.edu/jfrechet/

[11,12] [7] [15]

GSHa

Kataoka et al.

University of Tokyo

http://bmw.t.u-tokyo.ac.jp/english/index.html

[13]

Katayama et al.

Kyushu University

b

PKA a

GSH, glutathion. b PKA, protein kinase A.

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[16]

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leading to enhanced therapeutic efficacy and reduced side effects. However, the effectiveness of the active targeting seems to be controversial, which might be associated with limited penetration of particulate drug carriers into avascular tumor tissues [18]. Thus, the eradication of tumor cells at an avascular site is a challenging issue in the targeted tumor therapy using drug carriers [19]. In this connection, the multicellular tumor spheroid (MCTS) with a diameter of 200 mm has attracted our focus because MCTS is reported as one of the most suitable in vitro experimental models for avascular tumor tissues to study the tissue penetration of particulate drug carriers [20,21], whereas the furthest distance between adjacent capillaries in an avascular solid tumor is known to be 200 mm or less [22]. The tumor permeability and intracellular drug release of the pH-sensitive micelles consisting of PEG-b-P(Asp-Hyd-DXR), which was observed by confocal laser scanning microscopy, are shown in Fig. 2 [12]. In this observation, the fluorescence of DXR, which is quenched in the micelle core owing to the locally increased concentration, becomes detectable accompanied by drug release. The micelles showed a time-dependent increase in the fluorescent intensity throughout the MCTS (Fig. 2a), whereas free DXR was localized in the nuclei after a 1-h exposure (data not shown). Importantly, the DXR fluorescence of the micelles was confined to the cytoplasm after 3 h, and then detected in the cell nuclei (Fig. 2b), suggesting the intracellular drug release following the internalization of the micelles. These results indicate that the pH-sensitive micelles would access every cell inside the MCST and exhibit an intracellular pH-

Drug Discovery Today: Technologies | Drug delivery/formulation and nanotechnology

triggered drug release. By contrast, long-circulating liposomes incoporating DXR (Doxil) (http://www.doxil.com/) showed modest fluorescence only in the periphery of the MCTS even after 24 h, although each study was independently performed using different tumors [23]. This result strongly suggests that Doxil might have a limited accessibility inside the MCST and difficulty in drug release, which might be associated with the lower efficiency of the active targeting using liposomal formulations [18]. The larger particle size of Doxil (100–150 nm) compared with the micelles (65 nm) might account for the discrepancy in the penetration into the MCST. Thus, the intracellular pH-sensitive polymeric micelles are expected to be a potential tumor-infiltrating drug carrier for the treatment of the avascular region of solid tumors in vivo, providing a future promise in the active targeting.

Polymeric micelles for systemic gene delivery The efficient gene vectors should be a key technology in the forefront of modern medicine owing to their potential use for the treatment of intractable diseases and tissue engineering. Recently, synthetic vectors based on cationic polymers (polyplex) have been recognized as an attractive alternative to viral vectors owing to their safety for clinical use, ease in manufacturing and mass production, and a variety of chemical designs for constructing vector systems with smart functions [24–26]. Cationic polymers (polycations) have the ability to mask the negative charge of the plasmid DNA (pDNA) and package it into a small particle (<200 nm); they can be taken up by the cells, while protecting pDNA from hydrolytic and

Figure 2. Confocal laser microscopic observation of tumor permeability and intracellular drug release of the pH-sensitive polymeric micelles in a multicellular tumor spheroid (MCTS) model. (a) The fluorescent intensity of DXR increased with the incubation time owing to the release of DXR. The images suggest that the micelle can access the inside of the MCTS and release the loaded drugs (bar = 100 mm). (b) The subcellular localization and drug release of the micelles were observed using a high-magnification 63 objective. The image indicates the intracellular drug release following the internalization of the micelles, and the released drugs eventually accumulated in the cell nuclei (bar = 50 mm) [reprinted with permission from Ref. [12] (ß2005 American Chemical Society)].

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enzymatic degradations. It is well known that polycations with a comparatively low pKa value, such as polyethylenimine (PEI), show a high transfection activity. One of the possible reasons for this behavior is that polycations buffer the endosomal acidification and cause an increase in the ion osmotic pressure in endosomes accompanied by the protonation of amines, leading to the disruption of the endosomal membranes to release their content into the cytoplasm (socalled proton sponge effect) [27]. However, such polycations require a high N/P ratio to form the polyplex with a high stability and efficient transfection activity. It is noted that the N/P ratio is defined as the ratio of the cationic amino groups in polycations to phosphate anions in DNA. In this connection, Ogris et al. recently demonstrated that the PEI polyplexes prepared at different N/P feed ratios always gave the identical N/P ratios of 2.5 after purification by size exclusion chromatography, regardless of the initial N/P feed ratios, and that free PEI not only substantially contributes to the increased gene expression but also mediates the toxic side effects [28]. It appears that such a polyplex system containing free polycations is not useful for the in vivo, particularly the systemic route, gene delivery owing to instability and toxicity concerns. By contrast, polycations with a high pKa value (>9.0), such as poly(L-lysine) (PLL), might form stable polyplexes even at a relatively low N/P ratio [24]. Putnam et al. [29] at Cornell University (Ithaca, NY, USA) introduced buffering units, such as imidazole groups, into the PLL segment to improve the transfection activity based on the aforementioned proton sponge effect. However, the simple introduction of the buffering units into the polyplex is unlikely to solve the problem of instability under physiological conditions because these buffering units are weak bases and eventually have a low affinity to DNA. Also, it is known that the protonation of the buffering polycations is facilitated during the complexation with DNA [30], resulting in a loss of the buffering capacity. From a toxicological viewpoint, the polyplexes, prepared particularly at a high N/P ratio, cause a lethal toxicity due to the embolization of the lung capillary after intravenous administration [28], highlighting the importance for the design of biocompatible polyplexes. In summary, the systemic gene delivery systems are assumed to solve the stability and toxicity issues, and have a buffering capacity for enhanced transfection without an excess of free polymers. Recently, Kataoka et al. at the University of Tokyo have developed an A–B–C type triblock copolymer, tandemly aligning two types of polycations with different pKa values in a single polymer strand to construct the biocompatible polyplexes (polyplex micelles) satisfying the stability, biocompatibility and transfection ability [31]. The triblock copolymer is composed of PEG as the biocompatible A-segment, poly[(3-morpholinopropyl) aspartamide] (PMPA) as the low pKa B-segment with a buffering capacity, and PLL as the high 24

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pKa C-segment to condense the DNA (Fig. 3a). At the Lys/ nucleotide ratio of 2, the PEG-b-PMPA-b-PLL copolymer formed a polyplex micelle having the size and z-potential of 90 nm and +7 mV, respectively. Importantly, an 1H-NMR study has revealed that the PLL segment preferentially contributes to the DNA condensation, and the uncomplexed PMPA segment remains in the micelle [31]. Also, it should be noted that the free PEG-b-PMPA-b-PLL copolymer might be minimal in the system. These results are consistent with the formation of the polyplex micelle featured by the distinctive three-layered structure, in which an inner core of the pDNA/PLL polyplex is successively wrapped with an intermediate layer of the low pKa PMPA segment and an outer layer of the biocompatible PEG segment, as illustrated in Fig. 3a. The PEG-b-PMPA-b-PLL polyplex micelle exhibited a one order of magnitude higher transfection efficiency against HuH-7 cells than the polyplexes from PEG-b-PLL or the mixture of PEG-b-PLL and PEG-b-PMPA (Fig. 3b). This enhancement of the transfection seems to be attributed to the buffering capacity of the uncomplexed PMPA segment in the polyplex micelle [31]. Thus, the three-layered polyplex micelle achieved efficient transfection under the condition of minimal free polymers, providing a new design of synthetic vectors suitable for the systemic gene delivery. To function as efficient gene vectors, polycations are required to have several conflicting roles such as stabilizing ability and buffering capacity. In the design of the triblock copolymer, such essential roles are assigned to separate blocks in a single polymer strand. The tandem alignment of two types of polycations with different pKa might allow the preferential interaction of the high pKa block with pDNA, preventing the low pKa block from the facilitated protonation during the complexation with DNA. Thus, this design might ensure the buffering capacity of the low pKa block under the condition of minimal free polymers. By contrast, an outer shell of the PEG segment might provide a biocompatible surface of the polyplex, preventing erythrocyte aggregation [28] and complement activation [32] in the bloodstream, which might be associated with the lethal toxicity of synthetic vectors. Also, the polyplex micelle from PEG-b-PLL revealed a high serum tolerability [33] and prolonged blood circulation [34] in previous studies. These properties of the polyplex micelles facilitate their future utility for in vivo gene delivery.

Future perspectives of smart polymeric micelles for drug and gene delivery To enhance the efficacy and minimize the side effects during drug targeting, the spatial control of the drug action in the body should be desirable, and might be achieved by a combination of a specific drug delivery to the target tissue and specific activation of the delivered drug in the target cell. The versatile design and engineering of the micelle-forming block

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Drug Discovery Today: Technologies | Drug delivery/formulation and nanotechnology

Figure 3. (a) Chemical structure of PEG-b-PMPA-b-PLL triblock copolymers and schematic illustration of the three-layered polyplex micelles with spatially regulated structure [reprinted with permission from Ref. [31] (ß2005 American Chemical Society)]. (b) In vitro transfection of luciferase gene to HuH-7 cells by polyplex micelles from di- or triblock copolymers. HuH-7 cells were incubated with each micelle in a medium containing 10% serum for 24 h, followed by an additional 24 h incubation without the micelles.

copolymers enable the preparation of polymeric micelles with smart functions, such as the targetability to specific tissues and a chemical or physical stimuli-sensitivity. To establish active targeting, polymeric micelles, of which the surface is modified with saccharides [6], folic acid [7], peptides [8,9] and monoclonal antibodies [10], have been reported. Pasqualini et al. at the University of Texas (Houston, TX, USA) have developed an in vivo phage display technique, facilitating the discovery of specific peptides available for constructing the actively targeted polymeric micelles [35]. Although the micelles described above are exploiting intracellular pH, other chemical stimuli are also available. For instance, Kataoka et al. at the University of Tokyo have recently developed the polyplex micelle with a disulfidecrosslinked core, which is characterized by the efficient release of the loaded pDNA responding to the reductive condition mimicking the intracellular environment, where the glutathione concentration is 50–1000 times higher than that in the extracellular milieu. As a result, this type of micelle induced efficient transcription in the cell while achieving the improved stability against destabilization by anionically charged biocomponents, such as serum albumin and extracellular matrices [13]. Meanwhile, Katayama et al. at the Kyushu University (Fukuoka, Japan) have utilized the peptide

cleavable by a specific enzyme (e.g. caspases) for construction of the polyplex for the cell type-specific gene expression [36]. Also, several physical stimuli might be useful for achieving the site-directed transfection in vivo. Yokoyama et al. at KAST (Kawasaki, Japan) have reported polymeric carriers responding to changes in the temperature for thermal activation of drugs [14] and genes [37]. Recently, PIC Biotech AS (Oslo, Norway) (http://www.pcibiotech.com/) has developed the technology of photochemical transfection, in which the endosomal escape of the polyplex to the cytoplasm is assisted by co-incubated photosensitizers photodamaging the endosomal membranes, thus enhancing the transfection efficiency [38]. In particular, for the development of nonviral vectors, the use of the chemical or physical stimuli-sensitive vectors in conjunction with the genes expressed under the control of the specific promoters to organ or tissue type [39] and chemical or physical signals [40] will lead to a highly specific gene transfer to somatic cells at the diseased site. In any case, the development of systemic gene delivery systems should be directed not only to obtain an efficient transfection but also to achieve effective gene delivery to the desired site. In conclusion, the development of smart polymeric micelles is considered to be a subject that attracts much attention and will be intensively studied in the next decade, and eventually, www.drugdiscoverytoday.com

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such systems will provide a new type of methodology to enhance the efficacy of drugs and genes in a safe and secure manner.

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Acknowledgement This work was supported in part by the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST).

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