# Steering the Clinical Translation of Delivery Systems for Drugs and Health Products
## 3 Poster Presentations
### Development of a New Theranostic Agent for Inflammatory Diseases  
[PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] *  
DiFar, University of Genova, Viale Cembrano 4, 16148 Genova, Italy  
* Correspondence: [EMAIL_ADDRESS]  
Inflammation is a physiological response of our body, but it is also a pathological condition leading to the onset of different diseases like, among others, atherosclerosis, neurodegenerative disorders and cancer [92; 93; 94]. The onset of the inflammatory process involves several adhesion molecules: for example, the Vascular Cell Adhesion Molecule VCAM-1 is up-regulated during the initial step of the formation of the atherosclerotic plaque, allowing the accumulation of leukocytes in the expanding lesions. Recent studies have shown that VCAM-1 is also aberrantly expressed in different types of tumors, such as gastric, melanoma, ovarian, breast, and colorectal cancer [95]. Therefore, this adhesion molecule may be exploited as target for several inflammation-based diseases. For these pathologies, a non-invasive technique suitable for both VCAM-1 overexpression imaging and targeted delivery of actives might be advantageous for theragnostic purposes. To this aim, we have developed a new molecule (NAMP, Figure 24), based on a biotin derivative linked to a VCAM-1 binding peptide. By exploiting the well-known biotin/avidin affinity, this conjugate might be employed in a sequential treatment including avidin and a biotinylated tracer radiolabeled with \({}^{68}\)Ga, with the aim of obtaining a PET imaging of the site overexpressing VCAM-1. The conjugate has also been envisaged as pretargeting agent for biotinylated nanocarriers, such as liposomes, to deliver drugs to the inflammation sites (Figure 25). The VCAM-1 binding peptide that is included in NAMP structure is a 13 amino acid peptide in which seven amino acids have sequence homology with the alpha-chain of integrin VLA-4, the natural ligand of VCAM-1, while the other six are introduced as a linker sequence. By Orbitrap-MS we confirmed NAMP molecular structure, and in particular we verified that the peptide was linked to the biotinylated moiety through the carboxy-terminal cysteine. For the imaging application, we selected a double-chelating PET radiotracer which can be labeled with up to two radionuclides, thus increasing the signal deriving from the target and, consequently, the potentiality of the diagnostic approach. The three-step pre-targeting system was tested in vitro on HUVEC cells activated with TNF-alpha and subsequently incubated first with NAMP, then with avidin and finally with \({}^{68}\)Ga-radiotracer. The difference in radiosignal intensity shown by the activated cells compared to inactivated (control) cells was impressive. For the therapeutic application we selected, as nanocarrier, liposomes, because of their biocompatibility and their similarity with cell membranes. Biotinylated stealth liposomes were prepared with the well-known hydration method of a lipid film, followed by extrusion and purification by gel filtration. Liposome size (\(161\pm 12\) nm, polydispersity index between 0.04 and 0.1) was determined through photon correlation spectroscopy, while the zeta potential (\(-19.1\pm 0.6\) mV) was measured by micro-capillary electrophoresis. The concentration of the liposomal suspension, determined through a  
Figure 24: NAMP structure. Figure 25: Three-step Diagnostic system. Zetasizer Ultra, was \(114\times 10^{5}\) particles/mL. After two-week storage at 4 \({}^{\circ}\)C, the liposomes did not show any significant difference in the dimensional or surface electrical characteristics. A liposomal formulation including CM-Dil fluorescent dye was tested in vitro on human cancer cell cultures, applying the NAMP-avidin-liposomes protocol, and by live confocal fluorescence microscopy a preferential interaction of the biotinylated liposomes with the tumoral cells expressing VCAM-1 was observed. In conclusion, NAMP could be exploited as a theragnostic tool for targeting VCAM-1. The authors declare no conflict of interest.