The authors declare that there is no conflict of interest.. in cell migration. In vivo U-101017 tumor growth assays for subcutaneous xenografts in nude mice also revealed a significantly enhanced suppression of tumor growth in the treated group suggesting that these novel CAPE-MotAb nanoparticles may serve as a potent anticancer nanomedicine. 0.05, ** 0.01, and *** 0.001. 3. Results 3.1. Generation and Characterization of CAPE-MotAb Nanoparticles Polymeric micelles have attracted considerable attention as an effective delivery system for anticancer drugs that face poor water solubility issues [55,56]. Polyethylene glycol (PEG) is the most commonly used hydrophilic segment of polymeric micelles due to its biocompatibility and biodegradability [57]. Herein, we employed phospholipid PEG conjugates that can react with main amine groups (DSPE-PEG-NHS) and anti-mortalin antibody U-101017 (MotAb) to encapsulate CAPE U-101017 in PEG-stabilized polymeric micelles and explored their characteristics (Physique 1A). The schematic illustration of CAPE-MotAb structure is shown in Physique 1B. The polymeric micelles made up of CAPE were very easily synthesized through a unique self-assembly behavior of amphiphilic block copolymers that have polar or hydrophilic groups as well as nonpolar or hydrophobic portions when dissolved in the solvent. In a hydrophilic solvent, the hydrophobic portions are clustered in a core, away from the solvent and the hydrophilic portions are aligned towards solvent [58]. Hydrophobic CAPE was encapsulated in the nanoparticles composed of an inner hydrophobic domain name (DSPE) and an outer hydrophilic part (PEG-modified with NHS). CAPE-MotAb was expected to have a prolonged circulation time, actively enter and accumulate at the tumor site, and have high loading capacity. Once in the tumor, these CAPE-MotAb nanoparticles were anticipated to rapidly release CAPE in acidic endo/lysosomes and subsequently deliver the drug to the cytoplasm and nucleus (illustrated in Physique 1C). We subjected the nanoparticles to non-reducing SDS-PAGE analysis (Physique 1D). As shown, the antibody was visible at the ~250-kDa molecular excess weight. Of notice, the SMARCA4 CAPE-MotAb nanoparticles showed higher molecular excess weight suggesting successful conjugation of MotAb to DSPE-PEG-NHS. The UV-Vis-NIR spectrum of CAPE-MotAb showed characteristic peaks of MotAb at 280 nM and CAPE at 335 nM confirmed the successful encapsulation of CAPE in MotAb-conjugated polymeric micelles (Physique 1E). The encapsulation efficiency of CAPE improved with an increasing amount of DSPE-PEG-NHS and reached the highest value of 84.88% 8.66% at 1:20 ratio of CAPE to DSPE-PEG-NHS (Table 1). The loading efficiency of CAPE reached the highest value of 19.65% 0.96% when CAPE and DSPE-PEG-NHS were used in a 1:1 ratio and found to decrease with an increase in polymer amounts (Table 2). The encapsulation and loading efficiency were both acceptable with a ratio of 1 1:5 for CAPE and DSPE-PEG-NHS; hence it was selected as the optimum ratio for further experiments. These results strongly suggested that this DSPE-PEG-NHS could efficiently solubilize CAPE in water. As size and morphology have a wide influence around the biological applications of nanoparticles, we examined these aspects by transmission electron microscopy (TEM). The TEM observations revealed that CAPE-MotAb are monodisperse with spherical morphology (Physique 1F). We also calculated the size distribution of these nanoparticles from your TEM images and found that after conjugation with DSPE-PEG-NHS and MotAb, the nanoparticles are in the size ranging from 9 to 19 nm (Physique 1G). Furthermore, we examined the stability of CAPE-MotAb nanoparticles by UV-Vis-NIR spectrum of CAPE and Mot Ab at 335 nm and 280 nm, respectively. As shown in Physique S1, CAPE-MotAb nanoparticles were found to be U-101017 stable even after eight days of incubation at 4 C. Having confirmed the easy preparation, high stability, and reproducibility of CAPE-MotAb by multiple experiments, we then evaluated the in vitro and in vivo targeting efficiency, cytotoxicity, and anticancer properties of CAPE-MotAb nanoparticles. Open in a separate window Open in a separate window Physique 1 Schematic.