which could be blocked with the addition of free folic acid. The BioD data in vivo indicated rapid and high MPS uptake (53% in liver and spleen in 10 min), suggesting that the folate-PEG did not provide a sufficient stealth layer and should be complemented with unfunctionalized PEG chains at a higher surface density. The BioD data showed no evidence of the benefit of folate conjugation on in vivo accumulation of nanoparticles in FR-overexpressing large tumors, for which the EPR effect appeared to dominate SCK uptake. However, there was some indication that smaller, less developed tumors may have been targeted by folate SCK-to-folate receptor interactions. In the most recent study, the focus was on investigation of how the in vitro cellular internalization was affected by the nanomorphology for spherical versus cylindrical SCKs.12 Contrary to the results observed for the Tat-modified cylinders, it was found that folate-functionalized cylinders were better candidates for cell internalization than were folate-functionalized spheres. This effect may be a result of receptor clustering, where the cylinders are more capable of multivalent interactions due to their (“longer”) size, and thereby triggering of multiple receptor-mediated endocytotic processes. In order to explore the use of diverse and biologically complex targeting ligands, new conjugation methodologies have been developed for SCKs. One such example is the utilization of protected thiols, placed on the termini of PEG grafts presented topologically on the outer surface of the SCK nanoparticle, that can be deprotected/activated for further conjugation with thiol- or maleimide-containing targeting groups to form reversible disulfide or thioether linkages, respectively.17 These SCKs were shown to have high thiol reactivity and could be used to attach the model protein albumin with good coupling efficacy. The thiol maleimide Michael addition or furan maleimide Diels Alder conjugation are highly attractive conjugation strategies for the attachment of antibodies, since site-specific Fc-introduction of maleimide groups on antibodies is well established and demonstrated on nanoparticles to maintain binding efficacy in vitro.18 Although not tested yet on the SCK scaffold, thiol groups can also be utilized in high yielding thiol ene couplings.19 In our experience with SCK nanoparticles, it has been clear that achieving efficient and quantifiable targeting (including results not published) is indeed difficult. Our hypothesis is that the ligand presentation is not optimum in many targeted NPs and that the chemical design of the carriers must be more sophisticated. If the ligand is placed on a spacer unit such as PEG, the remaining PEG (necessary for Well-Defined Nanoscopic Embedded Therapeutics Nystro€m and Wooley circulation enhancement) must be of sufficiently shorter length in order not to interfere with binding to the receptor. In addition, covalent attachment of the ligand to the spacer molecule must be such that the binding characteristics are not hampered. In the antibody, antibody fragment or pro- tein case, this requires site specific modification reactions, and the employment of rapid, efficient and orthogonal (REO) ligation methods for covalent attachment to NPs.20 With small molecule ligands, control is achieved more easily. Similarly, the nature of the spacer is worthwhile to consider, as one can imagine that a more rigid spacer with longer persistence length may well present the targeting ligand in a better conformation for receptor interaction. Jiang and Cao have demonstrated that zwitterionic polymers offer advan- tages over PEG for inhibition of protein adsorption,21 and these polymers are quite attractive for SCKs because they also offer functionalizability. Strategies that employ cleava- ble or other linkages to bury the ligands until the target site (e.g., tumor) is reached may offer advantages for cell uptake and so on, but they fail to address the challenges associated with getting to that site. Control over the route of administration of the nanocon- struct is a highly attractive means by which to exploit the advantage that the specific organ provides combined with selected targeting approaches. For example, localized ther- apy of urinary tract or lung infections may be achieved via catheter delivery to the bladder or inhalation delivery of aerosols to the lung, respectively. We are currently devoting significant efforts toward the treatment of infectious dis- eases by the direct administration of antimicrobial-loaded SCKs22 to the lung and bladder. Preliminary in vivo tests have shown outstanding performance. Nasal delivery has been suggested as another direct administration method for achieving delivery in the brain, utilizing the olfactory path- way where the nervous system is in contact with the surrounding environment.23 A directed approach to actively target tissues in vivo, without the use of targeting ligands, utilizes superparamag- netic nanoparticles together with an external magnetic field to localize the drug carrier to the tissue of choice.24 This method requires that the diseased tissue is known and can be visualized for guidance procedures. The concept is indeed interesting for some clinical applications, but several problems still exist, such as scaling up from the animal models, embolization of blood vessels in the area, controlling the magnetic field geometry, and dealing with biological clearance issues for nondegradable inorganic nanoparticles. Vol. 44, No. 10 ’ 2011 ’ 969–978 ’ ACCOUNTS OF CHEMICAL RESEARCH ’ 973