General Criteria for the Selection of Nanoparticles for Certain Applications

They are, in essence, polymeric backbones equipped with multiple functional groups to which all sorts of chemical moieties can be covalently conjugated. This allows different drug compounds to be concurrently delivered to the tumor site (Pinhassi et al., 2010), hence opening up the myriad possibilities of synergistic anticancer effect among separate classes of antineoplastic agents. In addition, they are not vulnerable to drug resistance, an impediment usually faced by chemotherapeutics (Ryan, 2013), by circumventing multidrug resistance transporters expressed by tumor cells (Lee et al., 2006a; Ljubimova et al., 2008). To revert to the issue of passive targeting, polymeric nanoconjugates are generally smaller than veteran NPs such as liposomes or micelles. Therefore, they are less prone to clearance by the spleen, less immunogenic and as a consequence more long lived in plasma circulation (Dhanikula and Panchagnula, 2005). Active targeting is not discussed in depth in this chapter since it is already covered elsewhere in the book (e.g., Chapter 14). A rather detailed list of targeting ligands investigated thus far can be found in an article by Loomis et al. (2011). Even though the list is by no means comprehensive, it does a great job in projecting the abundance of targeting ligands out there. Below is a summary table that condenses information on both passive and active targeting. 7.3 Nanoparticles as Delivery VehiclesIn the last few decades, NPs have been extensively investigated as potential transporters for both therapeutic and diagnostic purposes. The type of cargos to be delivered is another criterion vital for NPs selection process (Table 7.2). 7.3.1 The Raid on Chemoresistance: Combination

Therapy and siRNAGenexol-PMTM is a paclitaxel-containing polymeric micelle synthesized from the biodegradable block copolymer, monomethoxy poly(ethylene glycol)-block-poly(d,l-lactide). This formulation has undergone clinical trials on a handful of cancer types such as metastatic breast cancer, solid tumors, and non-small cell lung

Table 7.2 Types of cargo that can be carried by NPs Header Key points Authors

Therapeutic cargo Paclitaxel -Genexol-PM is a formulation of paclitaxel encapsulated in a polymeric micelle-Tested in several clinical trials on metastatic breast cancer, solid tumors, and non-small cell lung cancer Lee et al., 2008bKim et al., 2007aLim et al., 2010aCisplatin CNT Pt(IV) prodrug delivery using folate as targeting ligand Dhar et al., 2008Doxorubicin Positive results on HeLa, ZR-75-1, MCF-7, and H661 cancer cells Chen et al., 2012eCombination therapy

Widely employed to combat chemoresistance Agarwal et al., 2003-CNT-encapsulated cisplatin prodrug serves as a template to bind doxorubicin-Shown to be highly effective against endometrial adenocarcinoma cells Chin et al., 2014Combination with siRNA suggested to improve the efficacy of chemotherapy Wang et al., 2009Imagingagents cargo Unimodality Endohedral metallofullerenes are able to solubilize metallic agents fi MRI contrast agent Kato et al., 2003-Trimetallic nitride endohedral metallofullerene NP containing gadodiamide produced as much contrast as the control clinical agent but at much lower concentrations (0.013 mmol compared to 0.050 mmol)-Metallofullerene inhibits the release of toxic metal ions Fatouros et al., 2006Bimodality Polyamidoamine dendrimer-based nanoprobe for dual modality conjugated with: + Gd(III), MRI contrast agent + Cy5.5, a fluorescent marker

Goldberg et al., 2007

Header Key points Authors

Imagingagents cargo Imaging ofangiogenesis Endothelial precursor cells of hematopoietic stem cell origin labeled with dextran-coated superparamagnetic iron oxide NP, monitored by MRI

fi noninvasively track tumor vasculature Arbab et al., 2005Uses in diagnostic techniques otherthan MRI GNP functionalized with gum arabic fi contrast agent for X-ray imaging Kattumuri et al., 2007-Iodine-containing micelle fi CT imaging-Methoxy-polyethylene glycol and tri-iodobenzoic polylysine micelles fi improved X-ray signal Trubetskoy et al., 1997

Coencap-sulation Hydrophil ic cargo favored by nanogel

-Crosslinking prevents dissociation of micelle structures of diblock copolymers Rapoport et al., 1999-Large amount of water at the inner core fi a challenge to encapsulate hydrophobic drugs-Overcome with the use of pyridyl disulfide groups Liu and An, 2014Another strategy to allows entrapment of water-insoluble compounds makes use of cholesteryl groups Morimoto et al., 2012Hydrophobiccargo favored by CNT -Hydrophilic chemicals are not readily incorporated into the inside of CNTfi hydrophobic precursor of a hydrophilic drug helps address the issue Li et al., 2012dLiposome tolerate both Well suited to codeliver therapeutic and diagnostic agents, which can differ in their physicochemical properties Torchilin, 2005

Table 7.2 (Continued)

cancer (Lee et al., 2008b, Kim et al., 2007a, Lim et al., 2010a). The FDA has hitherto approved some pharmaceutical products which utilize NPs, thus far mainly liposomes, as delivery vehicles (Kateb et al., 2011). The apparent fact that all of the formulations utilize a sole therapeutic agent does not reflect the current research direction. Many researchers have started to shift their interest to combination therapy as a means to subdue chemoresistance (Agarwal et al., 2003). Simultaneous delivery of multiple therapeutic agents helps trigger varied intracellular pathways at once. At any instant, the probability that a particular cell has the genetic makeup requisite for protection of all those drug targets is definitely smaller than when a single therapeutic agent is utilized. This exact line of reasoning suffices to explain why combination therapy has become the new trend. An attempt to model this novel approach can be seen with CNTs functionalized with integrin-targeting cyclic peptide for codelivery of cisplatin and doxorubicin (Chin et al., 2014). Within the CNTs, cisplatin prodrug serves as an anchor to which doxorubicin is tethered. Inside tumor cells, intracellular reduction triggers release of both compounds. The system has been shown to be highly cytotoxic against endometrial adenocarcinoma cells. Another strategy employed to deal with drug resistance is codelivery of therapeutic agents with short interfering RNA (siRNA). Investigations on mouse lymphoma and lung cancer models have illustrated utility of siRNA. Suppression of error-prone translesion synthesis (TLS) activity in mammalian cells by deactivating either Rev1 or Rev3L genes inhibits drug-induced mutation of the genetic makeup. In effect, relapsed tumors retain vulnerability to said anticancer agents in ensuing treatments (Doles et al., 2010; Xie et al., 2010b). Moreover, siRNA has been shown to synergistically ameliorate cytotoxicity of the companion cargo-therapeutic agent (Wang et al., 2009). Xu et al. developed PLGA-PEG-based NP formulation for codelivery of siRNA and the Pt(IV) prodrug. Even though the xenograft model did not allow for examination of acquired chemoresistance, the observation that this delivery system was capable of silencing target TLS genes for at least three days after the administration is a testimony to its potential as a breakthrough in the escape from the chemoresistance hassle (Xu et al., 2013b). Thus far, we have provided many cases of drug-encapsulated NPs, but it is not the only manner in which NPs work as a delivery

vehicle. Therapeutic agents can be conjugated to the external surface of the NPs as well. For instance, Tripisciano and Borowiak-Palen functionalized cisplatin to the side walls of single-walled carbon nanotubes (SWCNTs) and managed to observe enhanced therapeutic effect compared to cisplatin control (Tripisciano and Borowiak-Palen, 2008). These two delivery methods are not equivalent alternatives. It has been suggested that internalization inside CNTs ensures that the cargo stays impervious to biochemical reactions with endogenous compounds that might undermine chemical integrity of the compounds (Hampel et al., 2008). Nonetheless, encapsulation of therapeutic molecules inside NPs is anything but the ultimate solution. The cargo might not be completely released from its nanocarrier, thereby being a waste of bioactive agent and a potential source of toxicity. 7.3.2 Charismatic Mediators: What It TakesCPX-351 is a liposomal formulation for codelivery of cytarabine and daunorubicin, recently investigated in clinical trials as a therapy for acute myeloid leukemia (Dicko et al., 2010). Being an anthracycline, daunorubicin is inherently fluorescent. It was confirmed on rats that has the drug not been entrapped inside the liposome, its optical activity would have been lost as the compound gets metabolized by xanthine oxidase in the liver (Dodion et al., 1987). In this way, daunorubicin doubles as an imaging tool for in vivo tracking of the NPs. Unfortunately, there are not that many versatile anticancer agents. An imaging agent commonly needs to tag along the NPs for monitoring of the in vivo distribution of the drug delivery system, when accompanied by therapeutic agents, or purely for diagnostic purposes, when it is administered alone. The vast majority of anticancer agents are hydrophobic (except some such as first-line therapy cisplatin), whereas imaging agents are mainly hydrophilic. Such contrast in chemical nature is a formidable challenge to the design of NPs. Then again, even in the case of CPX-351, whereby an imaging agent is missing, the rise of combination therapy renders simultaneous entrapment of various compounds, which might very well display polar opposite affinity for water, inevitable. In some other cases, encapsulation is not an option (e.g., polymeric nanoconjugates, dendrimers). Topping the hierarchy of favorites is none other than liposomes. They are well suited for carrying both hydrophobic and hydrophilic

compounds (Torchilin, 2005), requiring no more cargo nor vehicle modifications than the ones normally seen in other NPs intended for monotherapy. In fact, these NPs can trap water-soluble molecules in their inner core, whereas hydrophobic ones remain within the lipid bilayer. Having said so, we should not give other NPs too little credit. All it takes for them to be up for the task is some elegant tweaks in the design. Nanogel is a type of NPs that especially favor hydrophilic cargos as a consequence of the large water content located in its interior. In the formation of nanogel, crosslinking stabilizes the micellar structure of diblock copolymers and minimizes their dissociation in aqueous environment. In a series of studies (Ryu et al., 2010, Gonzalez-Toro et al., 2012, Matsumoto et al., 2013), a self-crosslinked nanogel was made from copolymer of polyethylene glycol monomethyl ether methacrylate and pyridyl disulfide ethyl methacrylate. Aside from construction of disulfide crosslinks, pyridyl disulfide groups are essential to form hydrophobic regions within the hydrophilic interior of the nanogel (Liu and An, 2014). Cholesteryl groups have proven to be capable of a similar role (Morimoto et al., 2012). Furthermore, nanogels have their own merits to offer. The most outstanding is probably their unusually high drug loading capacity; a nanogel can carry a cargo molecule with a mass eight times its own (Chen et al., 2010b). Right at the other extremity are CNTs and their highly hydrophobic interior. To encapsulate the hydrophilic cisplatin, many have resorted to the prodrug solution, coupled with enzymatic release (Dhar et al., 2008, Li et al., 2012c). The lipophilic Pt(IV) prodrug can be incorporated into CNTs with ease by nanoextraction. Transported in this dormant form all the way until the vehicle has penetrated into the tumor cells, the compound will then be converted by the endogenous reductants overexpressed inside tumor cells (e.g., glutathione) into the cytotoxic Pt(II) species. In this manner, the activated form is conveniently more hydrophilic and hence promptly released from the CNTs. 7.3.3 Cargo Release on DemandTriggered release refers to the ability of NPs to release the cargo in response to particular environmental cues (Aaron et al., 2011). It is another hotspot extensively delved into in recent years

(Table 7.3). Having substantial contribution to tumor-specific release of anticancer drugs, triggered release is undeniably one of the key determinants in the selection of NPs. The stimuli fostering the release of cargo can either come internally from the intracellular environment of the tumor cells or come externally from an artificial source. Among these the following four have attracted much interest: heat, pH, enzyme, and ultrasound. 7.3.3.1 Thermal trigger Heat for this purpose can be generated by irradiating gold nanoparticles (AuNPs) (Paasonen et al., 2007a, 2007b, Wu et al., 2008) or subjecting iron oxide nanoparticles (FeONPs) to alternating magnetic frequency (Pradhan et al., 2010, Tai et al., 2009). Both of these auxiliary NPs could be coadministered alongside the drug delivery system of interest. In another study, nondestructive high-intensity focused ultrasound was used to induce localized raise in temperature, which accelerated the release of doxorubicin from liposomes compared to nonthermosensitive analogues (Dromi et al., 2007). Despite the additional utility of increasing permeability of vasculature at the area of interest, external stimulation of hyperthermia is discouraged when dealing with tumor situated deep within vulnerable tissues (e.g., brain tissues). In such cases, FeONPs and AuNPs are favored by virtue of their relatively lower collateral damage (Loomis et al., 2011). As a bonus, the heat generated causes cancer cells to ablate and to become more susceptible to damage exerted by chemotherapeutics. Candidates for this category of triggered release include largely the types of NPs constructed from polymers (e.g., nanogel, micelles). Some examples of thermoresponsive polymers include poly(methoxydiethylene glycol methacrylate) (poly-(MeODEGM)) (Heyden et al., 2009), poly(N-isopropylacrylamide) (poly(NIPAM)) (Paasonen et al., 2007a, 2007b, Prevot et al., 2006), and poly(t-butyl acrylate)-b-poly(N-isopropylacrylamide) (Li et al., 2009b). Bhuchar et al. reported the synthesis of a nanogel whose hydrophobic core was composed of MeODEGM and 2-aminoethyl methacrylamide hydrochloride (AEMA) crosslinked with an acid-degradable crosslinker and a hydrophilic shell of poly(2-methacryloyloxyethyl phosphoryl-choline) (Bhuchar et al., 2012). AEMA provides cationic character to the nanogel core, which aids the encapsulation of negatively

charged proteins (e.g., insulin, bovine serum albumin, and β-galactosidase). Above the lower critical solution temperature, the thermoresponsive MeODEGM core becomes hydrophilic, hence promoting the release of the cargo. Nanogel as a drug carrier has other fortes as well, which were already discussed in Section 7.3.2. 7.3.3.2 pH trigger It has long been noted that the microenvironment within cancer cells is slightly more acidic than that in normal healthy cells (Song et al., 1993; Webb et al., 1999; Wike-Hookley et al., 1984). This serves as the basis for usage of acid-labile bonds that are readily cleaved under acidic conditions. The PEG stealth coating on nano-carriers can be anchored by acid-labile linker. Once the NPs reach the tumor site, the PEG layer is released under the presence of pH stimulus. This exposes the cellular membrane to membrane-desta-bilizing complexes previously hiding under the stealth coating and thus does not represent a threat to the cellular plasma membrane of nontumor cells (Kirpotin et al., 1996, Kono et al., 1997). These bonds have also been tapped on in polymer-drug conjugates (e.g., doxorubicin, paclitaxel) (Devalapally et al., 2007; Etrych et al., 2001; Potineni et al., 2003; Prabaharan et al., 2009a). Notable examples of bonds labile to hydrolysis under acidic pH include diorto esters, vinyl esters, disulfide bonds, double esters, and hydrazones (Ishida et al., 2006; Kirpotin et al., 1996). Interestingly, some nanosized systems have been fashioned to be pH sensitive without a need for acid-labile bonds. One such special system is the lipid-coated calcium phosphate NPs (liposome/calcium/phosphate, or LCP), which consists of a CaP inner core and a lipid outer layer (Li et al., 2010a; Li et al., 2012b). At the endosomal acidic pH level, LCP would dissolve to disassemble the NP. The resultant increase in os-motic pressure eventually causes endosomal swelling. Even though the role the pH trigger plays in this situation is not relevant to the ultimate goal of tumor selectivity, given the fact that endosomes can be found in both tumor and healthy cells, it is still worthy of being mentioned here. pH-triggered release within the endosomes actually ensures the escape of the cargo, siRNA in this study, and preserves its chemical integrity, which could otherwise be impaired by lysosomal enzymes encountered along the endocytic pathway.