# Steering the Clinical Translation of Delivery Systems for Drugs and Health Products
## 3 Poster Presentations
### Development of Novel Super Stealth Immunoliposomes for Anticancer Drug Delivery  
[PERSON] 1,*, [PERSON] 2, [PERSON] 3, [PERSON] 1, [PERSON] 1 and [PERSON] 1  
1 Department of Pharmaceutical and Pharmacological Sciences, University of Padua, 35122 Padua, Italy  
2 Institute of Oncology Research IOR, 6500 Bellinzona, Switzerland  
3 Department of Medicine, Pathology and Cytopathology Unit, University of Padua, 35122 Padua, Italy  
* Correspondence: email: [EMAIL_ADDRESS]  
Advancement in the field of liposomes has resulted in the development of a great variety of nanocarriers, including long-circulating PEG-coated liposomes and targeted-liposomes. This work seeks to formulate new Super Stealth Immunoliposomes (SSIL), which should be both stable in the bloodstream and capable of reaching selectively the tumor site. PEG dendron molecules were conjugated to 2 or 4 molecules of distearoylphosphatidylethanolamine (DSPE) [24] to increase the hydrophobic interactions with the phospholipid bilayer. The conjugation of these PEG dendron-lipids derivatives to the Fab' fragment of Trastuzumab, allowed to obtain liposomes selectively targeting the human epidermal growth factor receptor 2 (HER2), overexpressed on the surface of certain tumor cells. PEG dendron-lipids derivatives were synthetized starting either from mPEG5 kDa-NHS or Boc-NH-PEG5 kDa-NHS by derivatization with \(\beta\)-glutamic acid and coupling to 2 or 4 molecules of DSPE. Boc-PEG5 kDa-(DSPE)n derivatives, were further derivatized with N-(\(\beta\)-maleimidopropyl) succinimide ester (BMPS) to introduce a maleimide group. Trastuzumab was enzymatically digested with pepsin and reduced with cysteamine to yield the Fab' fragment, which was immediately coupled to the maleimide moiety of PEG dendron-lipids derivatives. mPEG-lipid(s) derivatives and ligand-coupled PEG-lipid(s) derivatives were included into doxorubicin-loaded pre-formed liposomes by post-insertion. Liposomes were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Drug leakage was quantified by measuring the fluorescence dequenching of doxorubicin (DXR) over the time. Preliminary cytotoxicity studies, pharmacokinetics and organ toxicity evaluation were performed to compare the in vitro and in vivo behavior of all the formulations. Results showed that after 2 months of incubation at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C all tested formulations were still stable and homogeneous. DXR was efficiently encapsulated (absence of leakage within 16 h). SSIL2 showed significantly prolonged elimination half-life (\(38.50\pm 8.53\) vs. \(12.49\pm 3.05\) h of SL, \(p<0.01\)) and reduced clearance rate (\(15.81\pm 3.90\) vs. \(48.04\pm 20.42\) mL/h-kg of SL, \(p<0.05\)). In HER-2 overexpressingcell lines (SK-BR3 and BT-474) SIL displayed a higher cytotoxic activity with respect to SL, thereby confirming the targeting effect of Trastuzumab. On the contrary, the IC50 of SSIL2 was higher than those of SL and SIL, probably because of PEG dendron molecules increasing the rigidity of the liposome bilayer thus affecting the uptake by the cells. Nevertheless in vivo SIL-treated rats showed numerous granulomatous lesions, sometimes associated with apoptotic bodies, whereas in SSIL2-treated animals only a few isolated granulomas could be observed. Neutrophil infiltration is indirectly confirmed by the increase of ROS concentration in the liver of rats treated with SIL (\(p<0.001\)). Accordingly, CCL2 and IL-10, markers of macrophage infiltration, do not change in SIL or SSIL2 with respect to control rats, whereas CXCL2, a chemokine involved in the recruitment of neutrophils, was significantly higher in SIL-treated animals (\(p<0.0001\)). Furthermore, the hepatic expression of IL-18 increased after treatment with immunoliposomes, although to a significantly lower extent in SSIL2-treated animals with respect to rats treated with SIL (\(p<0.05\)).