NPs-DIP were specifically delivered to the tumor site (Figure 6IV) and co-localized with -tubulin by exhibiting merged yellow signals in the pancreatic tumor cells (Figure 6I, indicated by white arrow), whereas NPs showed low fluorescence signals both in red (Figure 6IV) and in yellow (Figure 6I) in the tumor site

NPs-DIP were specifically delivered to the tumor site (Figure 6IV) and co-localized with -tubulin by exhibiting merged yellow signals in the pancreatic tumor cells (Figure 6I, indicated by white arrow), whereas NPs showed low fluorescence signals both in red (Figure 6IV) and in yellow (Figure 6I) in the tumor site. applications. strong class=”kwd-title” Keywords: imaging, pancreatic cancer, PEPT1 transporter, SerCGlu, target ligand Introduction Nanoparticles (NPs) are an emerging field that offers great prospect for cancer imaging and therapy.1C4 Owing to the enhanced permeability and retention effect, NPs show a higher accumulation in tumor sites than in normal tissues after intravenous injection.5 In recent years, active target moieties have been engineered to improve NPs specificity to tumor.6C8 Although many ligands demonstrate highly specific targeting ability in vitro, only a small number of them practically enhance the tumor accumulation of systemically administered NPs.9C15 This limitation has inspired attempts to develop ligands for tumor-targeted Rabbit Polyclonal to TUBGCP6 applications with high efficiency. Peptides are amino acid sequences with less than approximately 50 residues. Because of Cardiolipin its simpler structures and smaller molecular sizes, it has enhanced stability and easier conjugation as well as better resistance to environment.16 In peptides functionalized NP fields, RGD (arginineCglycineCaspartic acid) peptide family maybe the most widely applied peptide ligand, Cardiolipin which can specifically bind to cancer overexpressed v3 integrin receptors.17,18 Peptide transporter 1 (PEPT1) is a Cardiolipin member of peptide transporters.19 Under healthy conditions, PEPT1 restrictedly existed in the epithelial cells of small intestine, kidney and bile duct, and nuclei and lysosomes of pancreas.20,21 Interestingly, PEPT1 was reported to be expressed in some human cancer cell lines such as pancreatic cancer AsPC-1,22 hinting the possibility that PEPT1 is a positive tumor biomarker. In the previous study, PEPT1 was used to target and inhibit cancer.23,24 Recently, a dipeptide SerCGlu was identified to have high affinity and specificity with PEPT1.25 Further, SerCGlu with a smaller molecular size may result in little characteristic alteration of NPs after conjugation. Based on these clues, we propose that specific recognition and binding between SerCGlu and PEPT1 might provide a biological base for designing a new ligand for tumor-targeted applications. In this work, PEPT1 was tested as a remarkable biomarker in pancreatic cancer cells comparing with normal cells. The dipeptide SerCGlu (DIP), as a specific PEPT1 ligand, was conjugated with polymer-based fluorescence NPs to form DIP-functionalized nanoparticles (NPs-DIP). NPs-DIP were evaluated in pancreatic cancer target imaging both in vitro and in vivo. Materials and methods Materials Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV; MW: 168,000 Da, 536512), silicon 2,3-naph-thalocyaninebis (trihexylsilyloxide) (NIR775, 389935), dipeptide SerCGlu and TrpCGly were purchased from Sigma-Aldrich (St Louis, MO, USA). PS-PEG-COOH (“type”:”entrez-protein”,”attrs”:”text”:”P15019″,”term_id”:”1729825″,”term_text”:”P15019″P15019-SEOCOOHcomb) was purchased from Polymer Source (Quebec, Canada). All other chemicals were purchased from Sigma-Aldrich unless otherwise noted. Synthesis of NPs Fluorescence NPs were prepared using a nanoscale precipitation technique with some modifications.26 Briefly, a solution of tetrahydrofuran (THF) consisting of 60 g/mL of PS-PEG-COOH, 40 g/mL of MEH-PPV, and 0.6 g/mL of NIR775 dye was initially prepared. Under vigorous sonication, each 2.5 mL of the mixture was then quickly dispersed into 5 mL of millipore water. Extra THF was evaporated under vacuum. The THF-free NPs solution was filtered through a 0.22 m filter. Bioconjugation was processed with carbodiimide chemistry between the amino groups exposed on SerCGlu and the carboxyl groups on the NPs. In a typical conjugation reaction, 100 L of 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid buffer (1 M) was added to 4.5 mL of NPs liquid (55.6 g/mL), then N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (14 mg) and N-Hydroxysuccinimide (NHS) (17 mg) were added. The reaction was processed for 1 hour. Subsequently, 200 L of SerCGlu solution (12 mg/mL) was added to the aforementioned mixture and stirred for 1 hour at 28C. Uncoupled SerCGlu combined with excess EDC and NHS was removed by several washes using a 100 kDa Amicon Ultra filter-4 (Millipore Corporation) under centrifugation at 3,000 rpm for 15 minutes at 4C. The final complex was kept at 4C. Characterization of nanoparticles The morphology and size of the NPs Cardiolipin were measured by Cardiolipin transmission electron microscopy (JEM-1400, JEOL, Japan). The hydrodynamic size of the NPs was tested by dynamic light scattering (DLS) using a Zetasizer NanoZS Instrument (Malvern Instruments, UK). The absorption and fluorescence spectra were analyzed using a SpectraMax (M5, Molecular Devices, Sunnyvale, CA, USA). Cell culture Human pancreatic cancer cell line AsPC-1 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, Peoples Republic of China). Human embryonic kidney cell line HEK 293 was maintained in.