1 Introduction Apart from its previous history in pharmaceutics, nanotechnology has recently become a major paradigm for the delivery of anticancer drugs, imaging agents, and genetic material. Pharmaceutical nanosystems have shown beneficial therapeutic efficacy with reduced side effects in treating diseases when compared to traditional dosage forms. For example, delivery of high doses of therapeutic and/or diagnostic agents to target cancer sites has been achieved using nano-sized carrier systems. This effect is primarily attributed to passive accumulation in solid tumors and inflamed regions by the EPR effect and the size (20��� 200 nm) of the carriers, followed by passive diffusional release of the drug in the extracel- lular space and/or active internalization into the cells via various entry mechanisms. To further improve local high-dose therapy, nanosystems need to be inert, meaning minimal interactions with biological components and negligible drug release while circulating in the blood stream. However, upon reaching their target sites, the nano- systems should switch their nature to induce aggressive cellular interaction, rapid localization at their intracellular destination, and enhanced drug-release kinetics. The switching property could be endowed by employing stimuli-sensitive components when constructing nanocarriers. It is anticipated that the responsive systems would reach their target sites more effectively by overcoming biological barriers such as drug-resistant mechanisms and entrapment in harsh lysosomal compartments. A summary of recent stimuli and stimuli-sensitive nanosystems for effective drug/gene delivery are presented in this chapter, with particular emphasis on tumor treatment. While pharmaceutical stimuli-sensitive nanocarriers for drug/gene deliv- ery include various carriers constructed from polymers, phospholipids, lipids, and their hybrids, this chapter will focus on polymeric nanocarriers. 2 Stimuli and Stimuli-Sensitive Polymers Nanocarriers constructed by self-assembling processes can have stimuli-sensitive properties induced by a variety of environmental changes covering a broad range of stimuli. Within the body, stimuli are limited to physiological signals such as Stimuli-Sensitive Nanosystems: For Drug and Gene Delivery Han Chang Kang, Eun Seong Lee, Kun Na, and You Han Bae V. Torchilin (ed.), Multifunctional Pharmaceutical Nanocarriers, 161 �� Springer Science + Business Media, LLC 2008

162 H.C. Kang et al. temperature, pH, redox gradients, ionic species, and concentration, which are often linked to biological and pathological events. External physical signals such as light, temperature, and ultrasound can also be employed. As shown in Fig. 1, internal and external stimuli affect the physical/chemical properties of materials or nanosystems causing ���first responses.��� This includes swelling/deswelling (see Cammas et al., 1997 Chung et al., 2000), disruption/aggregation (see Lee et al., 2003a,b, 2005a,b Na et al., 2003, 2004), chain cleavage (see Kaneko et al., 1991 Sawant et al., 2006), and complexation/decomplexation (see Sethuraman and Bae, 2007 Sethuraman et al., 2006). The ���first responses��� successively activate ���second responses��� such as drug/gene release (see Takeda et al., 2004), cell interactions (see Sethuraman et al., 2006), cell membrane destabilization (see Kang and Bae, 2007), and ligand���receptor interactions (see Lee et al., 2005b) for effective drug/gene delivery. 2.1 Temperature and Thermo-Sensitive Polymers Although changes in local body temperature are occasionally induced by pathologi- cal conditions (i.e., fever and irregular metabolism), external control of local body temperature by physical means offers a more consistent signal and provides broader application of thermo-sensitive pharmaceutical nanosystems. Hyperthermic ther- apy is one physical process that is known to kill or weaken cancer cells and/or Fig. 1 Stimuli for drug/gene delivery-induced physical/chemical changes of pharmaceutical nanosystems as ���first responses,��� successively leading to ���second responses���

Stimuli-Sensitive Nanosystems: For Drug and Gene Delivery 163 facilitate radiation and anticancer drug treatment. To heat target sites, microwave (see Yokoyama, 2002), ultrasound (see Tacker and Anderson, 1982), and magnetic devices (see Alexiou et al., 2006) have been routinely applied, and most internal organs and tissues can be accessed from outside of the body. As shown in Fig. 2, locally heating solid tumors to 40���45��C increases blood flow and vascular perme- ability, causing increased accumulation of pharmaceutical nanosystems and improved antitumor therapeutic effects (see Ponce et al., 2006). In addition, normal cells are unaffected by local hyperthermic conditions, which greatly influence the biological functions of cancer cells. Cancer cells show decreased DNA synthesis, heat shock protein expression, microtubule disruption, alteration in receptor expression and changed cell morphology (see Gerlowski and Jain, 1985 Jain, 1987 Ponce et al., 2006 Song, 1978). In contrast to heating, results obtained by cooling specific sites are rather limited. Using catheters with a cooling mechanism could be one option because catheters can access most internal organs and tissues via blood vessels for short-term applications. Prolonged cooling may reduce vari- ous biological functions of tissues including protein synthesis and gene regulation (see Yokoyama, 2002). Thermo-sensitive nanosystems or polymers often utilize thermal phase transi- tions (i.e., coil-to-globule transition). Most thermo-responsive polymers are water- soluble below their lower critical solution temperature (LCST) and become water-insoluble (hydrophobically collapsed or aggregated) upon raising the tem- perature above the LCST. Poly(N-isopropylacrylamide) (poly(NIPAAm) ) is a rep- resentative thermo-sensitive polymer. This polymer forms hydrogen bonds between its amine groups and water molecules, hydrating of the N-isopropyl groups below the LCST, giving the polymer a hydrophilic character. However, above the LCST, Fig. 2 Tumor-specific accumulation of thermo-sensitive nanocarriers under local hyperthermia

164 H.C. Kang et al. poly(NIPAAm) is dehydrated because of decreased interaction with water mole- cules (see Yuk and Bae, 1999). Polymers with LCST characteristics include poly(NIPAAm) (see Chung et al., 2000), polyester block copolymers (see Jeong et al., 1999 Na et al., 2006), and elastin-like polypeptides (ELP) (see Dreher et al., 2003 Furgeson et al., 2006 Matsumura and Maeda, 1986 Rodriguez-Cabello et al., 2006 Urry, 1997). Their chemical structures are presented in Fig. 3. The poly- mers and their copolymers showed LCST in the range of 30���50��C (poly(NIPAAm) and its copolymers), 20���100��C (polyester block copolymers), and 27���40��C (ELP). For specific applications, phase transition temperatures can be tuned by controlling comonomer composition, hydrophilic/hydrophobic balance, stereochemistry (see Chung et al., 1998, 1999, 2000 Kikuchi and Okano, 2002) and additives (i.e., salts and surfactants) (see Makhaeva et al., 1998). Drugs or genes can be incorporated into the thermo-responsive nanosystems, which can be constructed from thermo-sensitive polymers only or polymers modi- fied to include drug/gene-interacting segments via chemical linkages, electrostatic attraction, and hydrophobic interactions. For example, block copolymers composed of a thermo-sensitive block and a hydrophobic block are used for water-insoluble drug loading, while positively charged blocks are used for loading negatively charged genetic material. For water-insoluble anticancer drug delivery, typical hydrophobic blocks include methacrylic acid stearoyl ester (see Cammas et al., 1997), stearoyl chloride (see Kikuchi and Okano, 2002), poly(styrene (St) ) (see Chung et al., 2000 Gaucher et al., 2005), poly(n-butyl methacrylate (BMA) ) (see Chung et al., 2000 Kikuchi H2 C H C C HN CH H3C CH3 O Poly(N-isopropylacrylamide (poly(NIPAAm)) H2 C C C HN CH3 H2C CH O CH3 C CH O H3C O C CH O CH3 OH Poly(N-(2-hydroxypropyl) methacrylamide lactate) O H2 C H2 C O C H C O C H2 C O H2 C CH3 H2 C O O PEG/PLGA/PEG triblock copolymer Val Pro Gly Val Gly an Elastin-like polypeptide (ELP) O Fig. 3 Typical thermo-sensitive polymers having LCST characteristics

Stimuli-Sensitive Nanosystems: For Drug and Gene Delivery 165 and Okano, 2002), and poly(d,l-lactide-co-glycolide) (PLGA) (see Liu et al., 2005). Below the LCST, amphiphilic block copolymers form nano-sized micelles or nanoparticles depending on the copolymer architecture (di-, tri-, and multi- blocks, random, and graft copolymers). Diblock copolymers (i.e., poly(NIPAAm)- b-stearoyl chloride and poly(NIPAAm)-b-polySt) formed a typical core-shell micellar structure, and their structural transition temperatures were close to the LCST of the thermo-sensitive blocks (i.e., poly(NIPAAm) ) (see Kikuchi and Okano, 2002). Micelle formation might be caused if there is no interfering interac- tions between the hydrophilic poly(NIPAAm) chains and the hydrophobic core (see Heskins and Guillet, 1968). However, unlike diblock types, poly(NIPAAm-co- methacrylic acid stearoyl ester) random copolymers showed aggregation at tem- peratures lower than the LCST of poly(NIPAAm) (see Cammas et al., 1997). This might be caused by incomplete phase separation between the hydrophobic and hydrophilic segments. For genetic material, thermo-sensitive polymers have been linked to cationic segments such as poly(l-lysine) (PLL) (see Oupicky et al., 2003), polyethylene- imine (PEI) (see Bisht et al., 2006 Lavigne et al., 2007 T��rk et al., 2004), and dimethylaminoethyl methacrylate (DMAEMA) (see Kurisawa et al., 2000 Takeda et al., 2004). Genes can also be chemically conjugated to thermo-responsive poly- mers (see Murata et al., 2003a,b). The block copolymers form nanocomplexes with genes via electrostatic interactions or self-assembly. Above the LCST of polymers, the loaded genes were protected from nucleases because of the hydrophobic thermo-sensitive blocks, whereas temperatures lower than the LCST made the thermo-sensitive blocks more hydrophilic and caused gene release from the swol- len nanocomplexes. Thus, combining the applications of thermo-responsive materials and tempera- ture modulation (i.e., heating and cooling) has the potential to effectively deliver chemical drugs and therapeutic genes. Specific examples will be introduced in Sects. 3.1. and 4.1. 2.2 pH and pH-Sensitive Polymers Table 1 compiles the specific pH of various organs, intracellular compartments, and body fluids in normal and pathological conditions (see Diessemond et al., 2003 Kang et al., 2005 Na and Bae, 2005 Okada and Hillery, 2001 Owen and Katz, 2005 Schmaljohann, 2006 Tannock and Rotin, 1989). Under nonpathological conditions, the gastrointestinal tract (i.e., stomach, duodenum, and colon) and intra- cellular compartments (i.e., early endosomes, late endosomes, lysosomes, cytosol, and Golgi) within a single cell have their own specific ranges of pH. pH can be a signal to trigger the release of a drug/gene from carriers at a particular location of the GI track or in select intracellular compartments. Specific pHs are also found in wounds (~pH 5.5���8.7) (see Diessemond et al., 2003), semen (~pH 7.5) (see Owen and Katz, 2005), and vaginal fluid (~pH 4���5) (see Okada and Hillery, 2001).