A. Molecular engineering approach for developing molecules targeting disease markers <box 560px center grey></box|Figure 1. Selection of antibodies (A) and nucleic acid aptamers (B) against human proteins> We have developed a streamlined approach to discovering physiological ligands, antibodies, and aptamers against disease markers with high specificity and affinity (Fig 1). In addition to animal immunization for developing antibodies, we use in vitro platform consisting of functional display of human proteins in yeast and screening of phage library of human antibodies as well as RNA library against antigens on yeast and in micro-fluidic system. As an example of using physiological ligands to target a disease marker, we have engineered a protein domain in integrin known as I domain to target intercellular adhesion molecule (ICAM)-1, which is overexpressed in many tumors and rapidly induced under inflammation

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Figure 2. Integrin LFA-1 (A) expressed in leukocytes interacts with ICAM-1 (B). ICAM-1 binding domain in LFA-1 is called the Inserted or I domain, which was engineered by mutations to adopt a high affinity conformation (C&D).

B. Smart nanoplatforms for imaging and drug delivery

Once the molecules specific to disease markers are isolated, they are combined with appropriate nanoparticles of 50-150 nm in diameter. The nanoparticles of this size have been found to be optimal in vivo in consideration of maximization of targeted delivery, minimization of off-target effect, and long circulation time. Three types of nanoplatforms are being investigated for targeting cancer and inflammation. SPIO-micelle is designed for MRI as well as for optical imaging (Fig 3). UAN is highly versatile polymeric nanoparticles for encapsulation of chemotherapy and anti-inflammatory drugs and attachment of target recognition molecules (Fig 4). Liposomes are being investigated as a carrier of water-soluble and water-insoluble drugs.

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Figure 3. SPIO-micelle formation, protein attachment, and visualization with transmission electron microscopy (TEM) (A). Black arrow in the left TEM image indicates the density of nickel ions around the SPIO micelle. White arrow in the right TEM image indicates the protein bound to SPIO-micelle after a negative staining with 1% uranyl acetate. Size distribution of the I domain-coupled SPIO-micelle was analyzed by dynamic light scattering, which estimated ~60 nm in size (B).

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Figure 4: Synthesis and characterization of UAN. (A) Synthesis of UAN nanoparticle via hydrophobic interactions. (B) TEM images of UAN nanoparticles stained with uranyl acetate (C) Ni-NTA-UAN functionality test to confirm presence and activity of Ni-NTA on surface of UAN nanoparticles. (D) Release kinetics of materials of varying hydrophobicity.

C.In vitro and in vivo validation of target-specific nanoplatforms.

Nanoparticles targeting tumor for optical and MR imaging (Fig 5) and the delivery of chemotherapeutic drugs are being validated in both in vitro and in vivo systems.

<box 380px left grey></box>	Figure 5. Specific delivery of ICAM-1 targeting SPIO-micelle into cervical tumor (HeLa) in vitro (A) and in vivo (B-D). Whole body near-infrared fluorescence imaging (B) together with ex vivo imaging of the major organs and tumor tissue (C) and histology of the tumor tissue (D) confirm specific targeting of nanoparticles to the tumor.
<box 380px left grey></box>	Figure 6. In vitro cell cytotoxicity assay. (A) I domain-UAN particles encapsulating celastrol (20 or 125 μM) or (B) camptothecin were delivered to HeLa cells. Affinity dependent cell cytotoxicity was observed for both CPT and celastrol.