Well-Defined Nanoscopic Embedded Therapeutics Nystro€m and Wooley FIGURE 2. SCKs having fluorescent labels and Tat peptides were prepared by conjugation of the active units onto pre-established SCKs. Reprinted with permission from ref 11. Copyright 2008 American Chemical Society. of the PEGylation of these nanocarriers, prohibiting effective targeting due to interference with receptor binding.8 In most cases, the PEG shell is critical and cannot be excluded, as the NP construct would otherwise rapidly activate mononuclear phagocyte system (MPS) clearance mechanisms. However, other important factors have not been studied in great detail in vivo with respect to targeting, such as how particle shape9,10 and flexibility affect targeting with the same ligand.11,12 In such studies, nonbinding ligands of similar shape, size, and charge, for example, scrambled peptides for peptide-based targeting ligands, must be used as controls. In the case of SCKs of varying shapes, the inherent carboxylic acid functionality of the hydrophilic poly(acrylic acid) shell layer has been utilized via the formation of amide bonds for cross-linking and the attachment of imaging agents, PEGs for circulation enhancement, and targeting components. Given that the SCK nanostructure contains thousands of carboxylic acids or activated esters,13 there are many sites available, but the chemistry must be con- ducted with care to avoid loss of ligand binding function. For example, with peptides, this requires that the N-terminus is the only amino group reacted, in order to avoid intra- and intermolecular cross-linking. Conjugation of the protein transduction domain of human immunodeficiency virus type 1 Tat protein, a commonly employed cell penetrating peptide sequence having the sequence GYGRKKRRQRRR, onto spherical and cylindrical SCKs was accomplished by using protected Tat peptides that could then be deprotected 972 ’ ACCOUNTS OF CHEMICAL RESEARCH ’ 969–978 ’ 2011 ’ Vol. 44, No. 10 (Figure 2).11 It was found that smaller spherical Tat-SCKs (ca. 10 nm) were internalized by cells more rapidly than were longer cylindrical (ca. 10 30 nm diameter but lengths of g200 nm) nanostructures of the same composition. Differ- ing degrees of Tat functionalization were also studied, for which it was observed that the highest Tat peptide concen- tration resulted in a 5-fold increase in cellular uptake, as compared to the naked SCK. In contrast, the opposite behavior was observed for spherical versus cylindrical SCKs labeled with folate targeting ligands. The folate receptor (FR), or membrane-associated folate- binding protein, is one of the cellular transport systems that binds folates with high affinity in the nanomolar range and undergoes receptor-mediated endocytosis. Because the fo- late receptor is well characterized and is overexpressed in several cancer types with limited expression in normal tissue, the FR is an attractive target for nanoparticles to treat tumors.14 Three studies have been reported for folate- labeled SCKs: one that involved synthetic methodology development,15 another primarily in vivo oriented,16 and one in vitro focused.12 In the first studies, folate was placed on the terminus of a 1.6 kDa PEG spacer that was conjugated to the surface of pre-established SCKs, using their surface available COOH groups. These particles were further labeled with fluorophores and 64Cu radioisotopes for in vitro and in vivo studies.16 The in vitro experiments indicated rapid uptake of the folate-functionalized SCKs within 4 h at 37 C and only a surface bound fraction at 4 C, each of