Effective Encapsulation and Biological Activity of Phosphorylated Chemotherapeutics in Calcium Phosphosilicate Nanoparticles for the Treatment of Pancreatic Cancer Welley S. Loc, Samuel S. Linton, Zachary R. Wilczynski, Gail L. Matters, Christopher O. McGovern, Thomas Abraham, Todd Fox, Christopher M. Gigliotti, Xiaomeng Tang, Amra Tabakovic, Jo Ann Martin, Gary A. Clawson, Jill P. Smith, Peter J. Butler, Mark Kester, James H. Adair PII: DOI: Reference:
S1549-9634(17)30125-9 doi: 10.1016/j.nano.2017.06.017 NANO 1616
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
23 January 2017 23 May 2017 20 June 2017
Please cite this article as: Loc Welley S., Linton Samuel S., Wilczynski Zachary R., Matters Gail L., McGovern Christopher O., Abraham Thomas, Fox Todd, Gigliotti Christopher M., Tang Xiaomeng, Tabakovic Amra, Martin Jo Ann, Clawson Gary A., Smith Jill P., Butler Peter J., Kester Mark, Adair James H., Effective Encapsulation and Biological Activity of Phosphorylated Chemotherapeutics in Calcium Phosphosilicate Nanoparticles for the Treatment of Pancreatic Cancer, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.06.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effective Encapsulation and Biological Activity of Phosphorylated Chemotherapeutics in Calcium Phosphosilicate Nanoparticles for the Treatment of Pancreatic Cancer
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Welley S. Loc, PhD a, b ,k , Samuel S. Linton c, k , Zachary R. Wilczynski d, Gail L. Matters, PhD e, Christopher O. McGovern e, Thomas Abraham, PhD f, Todd Fox, PhD g, Christopher M. Gigliotti d, Xiaomeng Tang, PhD a, b, Amra Tabakovic, PhD b, Jo Ann Martin h , Gary A. Clawson, MD/PhD i, Jill P. Smith, MD j, Peter J. Butler, PhD d, Mark Kester, PhD g, James H. Adair, PhD b, c, d, * Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
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Department of Materials Science and Engineering, Pennsylvania State University, University Park, PA 16802, USA c
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Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA d
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Department of Biomedical Engineering/Bioengineering, Pennsylvania State University, University Park, PA 16802, USA e
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Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
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Department of Neural and Behavioral Sciences and the Microscopy Imaging Facility, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA g
Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
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Biomedical Engineering, University of Florida, Gainesville, FL 32611, USA
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Department of Pathology and Gittlen Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA j
Department of Medicine, Georgetown University, Washington DC 20007, USA
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Authors contributed equally to this work.
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*Corresponding Author James H. Adair Professor of Materials Science & Engineering, Biomedical Engineering, and Pharmacology 205 Steidle Building Pennsylvania State University University Park, PA 16803 Email: [email protected] Office Phone: (814) 863-6047
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Statement of Funding and Conflicts of Interest: This study was funded by the National Institutes of Health (NIH) grants R01CA167535 and R21CA170121 from the National Cancer Institute (NCI). The project was also supported in part under a grant with the Pennsylvania Department of Health using Tobacco CURE funds (SAP#4100072562 to GLM and JHA). The department specifically disclaims responsibility for any analyses, interpretations or conclusions. WSL was supported, in part, by the grants UL1 TR000127 and TL1 TR000125 from the National Center for Advancing Translational Sciences (NCATS). Penn State Research Foundation has licensed CPSNP technology to Keystone Nano, Inc. (PA, USA). JHA and MK are co-founders of Keystone Nano and are CSO and CMO, respectively. All other authors declare that there are no conflicts of interest.
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Word count for abstract = 147/150 Word count for manuscript = 4983 Number of references = 56 Number of figures = 8 Number of tables = 1 Number of Supplementary online-only files: 1
ACCEPTED MANUSCRIPT ABSTRACT Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and
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nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive,
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phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated
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drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is
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essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained
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biological activity. Analysis of orthotopic tumors from mice treated systemically with tumortargeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells
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and release of active drug cargos intracellularly. These findings demonstrate a novel
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methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to
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effectively deliver them to pancreatic tumor cells.
ACCEPTED MANUSCRIPT KEYWORDS. FdUMP, 5-fluorouracil, gemcitabine, nanodelivery, dFdCMP
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ABBREVIATIONS
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CPSNP, calcium phosphosilicate nanoparticle; mPEG, methoxy-polyethylene glycol; cPEG, carboxy-polyethylene glycol; malPEG, maleimide-polyethylene glycol; Cit, citrate; NT, no
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treatment; TS, thymidylate synthase; THF, 5,10-methylene-tetrahydrofolate; FdUMP, 5-
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fluorodeoxyuridine monophosphate; 5-FU, 5-fluorouracil; FUdR, 5-fluorodeoxyuridine; dFdC, gemcitabine or 2’,2’-difluorodeoxycytidine; dFdCMP, gemcitabine monophosphate or 2’,2’-
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difluorodeoxycytidine monophosphate; ATP, adenosine triphosphate; 5-CU, 5-chlorouracil; SEM, standard error of the mean; TEM, transmission electron microscopy; CCKBR,
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cholecystokinin B receptor
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ACCEPTED MANUSCRIPT BACKGROUND Applications of nanoparticle (NP) delivery systems to medical oncology include the use of
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NPs for encapsulated therapeutic drug delivery and for non-invasive tumor imaging. Due to the
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small size and versatile surface chemistry, NPs are ideal platforms to penetrate poorly vascularized and fibrotic tumors (1). Reports on NP-based delivery systems encompass a
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diversity of materials that include liposomes (2-4), dendrimers (5, 6), gold NPs (7), and quantum
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dots (8, 9). The design and composition of NPs reflect their abilities to load and protect agents in circulation and to provide a clearance mechanism for excess NPs (10).
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Amorphous calcium phosphosilicate nanoparticles (CPSNPs) are composed of bioresorbable materials (10, 11) that have been used to deliver therapeutic (12-14) and imaging agents (13, 15-
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18) to tumor cells. The NP matrix provides protection of encapsulated bioactive agents (such as
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chemotherapeutic drugs) from metabolic breakdown and rapid clearance. Further, surface decoration of CPSNPs with polyethylene glycol (PEG) mitigates non-specific interactions that
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cause agglomeration and facilitates bioconjugation of tumor targeting agents such as peptides,
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antibodies or aptamers (14, 15, 17-20). After cellular uptake, the low pH of late-stage endosomes triggers CPSNP dissolution and the endosomes rupture from a change in osmotic pressure to release active agents into the cytosol (13, 18). Treating pancreatic cancer with standard chemotherapeutic agents is challenging. Most pancreatic adenocarcinoma (PDAC) patients respond poorly to chemotherapeutic drugs, and even new drug combinations have demonstrated only a modest improvement in patient survival (21). This lack of efficacy has been attributed in part to inadequate drug delivery and metabolic drug inactivation (22). Two chemotherapeutics commonly used to treat PDAC, 5-fluorouracil (5-FU) and gemcitabine (dFdC), act by blocking key enzymes in nucleotide synthesis. 5-FU is
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ACCEPTED MANUSCRIPT metabolized to 5-fluorodeoxyuridine monophosphate (FdUMP) which, in the presence of 5,10methylenetetrahydrofolate (CH2 THF), irreversibly inhibits thymidylate synthase (TS) (23). TS
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inhibition results in nucleotide pool imbalances, impaired DNA synthesis, and a reduction in
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DNA repair (24). Similarly, the prodrug dFdC is phosphorylated intracellularly by deoxycytidine kinase to form gemcitabine monophosphate (dFdCMP), diphosphate (dFdCDP) and triphosphate
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(dFdCTP) (25). Gemcitabine has multiple modes of action; dFdCDP inhibits ribonucleotide
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reductase, which is responsible for producing the deoxynucleotides required for DNA synthesis and repair. This favors incorporation of dFdCTP into DNA, resulting in stalled DNA replication
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forks and apoptosis (26). While both 5-FU and dFdC are activated within tumor cells by conversion to phosphorylated metabolites, direct delivery of phosphorylated metabolites is
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limited by cell impermeability, lipid insolubility and precipitation.
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Systemic administration of many chemotherapeutics is limited by the need for high dosing regimens due to metabolic inactivation and rapid clearance. For example, in vivo studies have
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shown that less than 20% of the 5-FU becomes activated to FdUMP while more than 80% of 5-
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FU is converted to the inactive 5-FU metabolite 5-fluorodihydrouracil by the liver enzyme dihydropyrimidine dehydrogenase (DPD) (27). Likewise, dFdC can be inactivated by cytidine deaminase and rapidly cleared from the body (28-30). Both dFdC and 5-FU are transported into tumor cells by nucleoside transporter systems, including human equilibrative nucleoside transporters (hENT), which have low affinity for FdUMP and dFdCMP (31). Tumor targeted, NP-based delivery of phosphorylated drug metabolites could avoid drug inactivation processes and enhance drug uptake by tumor cells. This study compares the encapsulation efficiency of both phosphorylated and non-phosphorylated forms of 5-FU and dFdC and outlines a novel approach to deliver the chemotherapeutics directly to pancreatic tumors using targeted CPSNPs.
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ACCEPTED MANUSCRIPT METHODS CPSNP Synthesis and Characterization. CPSNPs were prepared in reverse-micelles and
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isolated by HPLC as described by Barth et al. and Morgan et al. (13, 17, 18). The materials,
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synthetic procedure, and additional sample preparation methods are outlined in supplementary information. Citrate-functionalized (Cit) CPSNPs were preheated to 50°C and stirred at 550 rpm
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for PEGylation. To yield mPEG-CPSNPs, 1 ml of EDC (1 mg/ml) was first added drop-wise to
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10 ml of Cit-CPSNPs. After 5 min, 1 ml of each Sulfo-NHS (15 mg/ml) and 2 kDa methoxyPEG-amine (6 mg/ml) were transferred. The reaction proceeded for 15 hr. PEGylated CPSNPs
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were purified via filtration for 2-3 min at 5000 g (Amicon Ultra-15 30 kDa). Gastrin-16 peptide (G16, CSGGQQQQQQAYGWMDF, Genscript) was conjugated via 2 kDa maleimide-PEG-
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amine coupling (18). The peptide was added to the malPEG-CPSNPs after filtration and stored at
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4°C overnight. The aptamer (AP, TriLink Biotechnologies) was conjugated via 2 kDa carboxyPEG-amine coupling, first reported by Clawson et al. (19). A second EDC/NHS coupling
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CPSNPs).
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reaction followed after filtration with an aliquot of 0.2 mM aptamer solution (5 ul per ml cPEG-
Surface modification of CPSNPs was characterized by the Brookhaven Instruments zeta potential analyzer (ZetaPlus v. 3.23, Holtsville, NY). Particles were imaged on the FEI Tecnai G2 Spirit BioTWIN TEM operating at 120kV. The drug concentration in CPSNPs was quantified by LC-MS/MS (supplementary information). To calculate the payload, the total number of particles (Ntotal) was first determined on the Nanotrac Wave II DLS (Microtrac Flex 11.1.0.2, York, PA). Measurements were taken from an average of five runs, 10 s per run. Treating the NPs as spheres, the products of Ntotal and probability of particle sizes based on TEM distributions
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ACCEPTED MANUSCRIPT were used to determine the total NP volume in suspension. The number of drug molecules per
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nm3 of CPSNP were averaged with 95% confidence intervals.
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Cell Proliferation Assay. PANC-1 (in Dulbecco’s modified Eagle medium/10% fetal bovine serum) and BxPC-3 (in RPMI medium/10% fetal bovine serum) cells (ATCC) were seeded onto
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96-well plates at 5,000 cells/well. After 24 hr, treatment with vehicle (1x PBS), free drug,
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mPEG-CPSNPs or drug-loaded CPSNPs were initiated. Viable cell determinations were made after 72 hr using an alamarBlue® assay (Life Technologies). Relative proliferation for all
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treatment groups were normalized to vehicle controls.
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Thymidylate Synthase Immunoblotting. PANC-1 and BxPC-3 cells were seeded onto 6-well
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dishes and incubated for 24 hr in one of the following treatment groups: no treatment, vehicle, free FdUMP, mPEG-FdUMP-CPSNPs, and mPEG-CPSNPs. Lysates were collected by
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aspirating the media, washing with 1x PBS, and adding RIPA buffer containing Complete Mini
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protease cocktail (Roche). Protein concentration was determined by microBCA assay (Thermo Scientific) and 20 g of total protein was separated by gel electrophoresis. After transfer to HyBond ECL and blocking for 1 hr in 5% BSA, blots were probed overnight with anti-TS antibody (#9045 Cell Signaling Technology, 1:1000) or beta-actin antibody (#A2228; Sigma, 1:10,000). Membranes were washed, incubated with secondary antibody coupled to horseradish peroxidase (Amersham), and visualized using an enhanced chemiluminescent substrate (Pierce). Quantitation of scanned blots was done using Image-J software (NIH). After normalizing to βactin, % active TS is the amount of uncomplexed TS divided by the amount of total TS (active TS and TS-FdUMP ternary complex).
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Cell Cycle Analysis. PANC-1 cells were treated with vehicle, free FdUMP, mPEG-FdUMP-
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CPSNPs or an equal volume of mPEG-CPSNPs, or left untreated for 72 hr (32). Cells were
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fixed in 75% ethanol overnight and immediately prior to analysis treated with 1 g/mL RNase A
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and 50 g/mL propidium iodide. Cellular DNA content was determined using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed with Cellquest (Verity Software,
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Topsham, ME).
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In vivo assessment of aptamer-targeted FdUMP-CPSNP up take by PDAC tumor cells. Animal protocols were approved by the Penn State College of Medicine IACUC committee. All
Orthotopic PANC-1 tumors were established in male, athymic (nu/nu) mice (Charles
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animals.
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procedures were done in accordance with institutional guidelines for the humane care of the
River). Pancreata were injected with 5x10 6 cells and treatment with CSPNPs was initiated at one
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week post-surgery. Treatment groups (n= 4-5 mice per group, with two experimental replicates)
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included mPEG-CPSNPs, mPEG-FdUMP-CPSNPS, gastrin-targeted G16-malPEG-FdUMPCPSNPs, and aptamer-targeted AP-cPEG-FdUMP-CPSNPs. CPSNPs were administered at a FdUMP dose of 100 g/kg, or an equal volume of mPEG-CPSNPs, twice weekly via tail vein injection. After six weeks of treatment, animals were sacrificed. Blood was removed by cardiac puncture, and complete blood cell counts and serum chemistries were done by the Department of Comparative Medicine Diagnostic Laboratory (PSU-Hershey). A portion of each tumor was flash frozen in liquid nitrogen and was powdered by grinding in a pre-chilled mortar and pestle. RIPA buffer containing Complete Mini protease cocktail (Roche) was added to each sample following grinding; the resuspended tissue was sonicated for 15 s and centrifuged to pellet
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ACCEPTED MANUSCRIPT debris. Protein concentrations in supernatants were determined by micro BCA protein assay (Thermo Scientific), and 20 µg of protein from each sample was separated by gel
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electrophoresis. Thymidylate synthase immunoblots were performed as described above.
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Another portion of each tumor was fixed and embedded in OCT, cryo-sectioned and stained for Ki-67 (Abcam #92742) according to the manufacturer’s instructions; nuclei were counterstained
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with Hoechst 33432. Images were acquired with a Leica AOBS SP8 laser scanning confocal
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microscope.
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Statistical Analysis. Results were expressed as means ± standard error. Student t-tests were used to evaluate statistical significance with a p < 0.05 considered to be significant. To calculate
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EC50 ± 95% CI, nonlinear regression analysis was performed to generate the curve of best fit for
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the data according to a Sigmoidal regression using a 4-parameter logistic curve calibration [Y =
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Yo + (a/(1+((X/Xo )b))] in SigmaPlot 12 (Systat, Inc., San Jose, CA).
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ACCEPTED MANUSCRIPT RESULTS CPSNP Characterization. Preparation of CPSNPs (Figure 1) in reverse micelles yielded sub-
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100 nm particles (13-18, 33, 34); a size range that is optimal for NP penetration into fibrotic
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pancreatic tumors (18, 35). The lognormal mean of the size distribution for mPEG-FdUMPCPSNPs was 8.4 nm (𝜎𝑧 =0.2) and 10.5 nm (z=0.4) for mPEG-dFdCMP-CPSNPs (Figures 2A
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and 2B). Aptamer-targeted AP-cPEG-FdUMP-CPSNPs in Figure 2C were about 8.0 nm
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(𝜎𝑧 =0.3) and 17.2 nm (𝜎𝑧 =0.1) for gastrin-targeted G16-malPEG-FdUMP-CPSNPs in Figure 2D. Because CPSNPs depend on citrate (Cit) and/or PEG to maintain dispersion (33), the zeta
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potential is a common metric to verify changes in surface terminal groups and colloidal stability (Figure 3). The mean values for Cit-CPSNPs, Cit-FdUMP-CPSNPs and Cit-dFdCMP-CPSNPs
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were ‒37±6 mV, ‒44±5 mV and ‒28±7 mV, respectively. After PEGylation, the uncharged
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methoxy group termination at the NP surface exhibits near-zero net charge, so mPEG-CPSNPs, mPEG-FdUMP-CPSNPs and mPEG-dFdCMP-CPSNPs displayed mean values of ‒1±5 mV, ‒
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4±6 mV and 1±9 mV, respectively. Bioconjugation of the aptamer to cPEG-FdUMP-CPSNPs
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resulted in a reduction of the zeta potential magnitude from ‒35±5 mV to ‒22±5 mV. An increased magnitude from ‒8±5 mV to ‒29±9 mV was observed for the conjugation of the gastrin to malPEG-FdUMP-CPSNPs.
Encapsulation efficiency of chemotherapeutics by CPSNPs. We hypothesized that chemotherapeutic drugs such as 5-FU or dFdC would be less effectively encapsulated into CPSNPs than FdUMP and dFdCMP (Figure 4). To address this hypothesis, the encapsulation efficiencies (EE) for 5-FU, ATP:5-FU complex, FUdR (5-fluorodeoxyuridine), FdUMP, dFdC, and dFdCMP (Table 1) were experimentally determined. The EE is defined by the relationship,
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ACCEPTED MANUSCRIPT 𝑚
(1)
𝑚𝑜𝑙% 𝐸𝐸 = 𝑚𝑓 × 100 𝑖
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where m i is the initial moles of drug and mf is the moles present in the purified CPSNPs,
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quantified by LC-MS/MS. CPSNP encapsulation of 5-FU was only 4.1 (±1.8) ×10-7 M, or 0.11 (±0.04) mol% (Table 1), and extended reaction times failed to improve 5-FU encapsulation.
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FUdR, the deoxynucleoside analog of 5-FU, was incorporated in CPSNPs even less effectively than 5-FU, with an EE of
S1549-9634(17)30125-9 doi: 10.1016/j.nano.2017.06.017 NANO 1616
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
23 January 2017 23 May 2017 20 June 2017
Please cite this article as: Loc Welley S., Linton Samuel S., Wilczynski Zachary R., Matters Gail L., McGovern Christopher O., Abraham Thomas, Fox Todd, Gigliotti Christopher M., Tang Xiaomeng, Tabakovic Amra, Martin Jo Ann, Clawson Gary A., Smith Jill P., Butler Peter J., Kester Mark, Adair James H., Effective Encapsulation and Biological Activity of Phosphorylated Chemotherapeutics in Calcium Phosphosilicate Nanoparticles for the Treatment of Pancreatic Cancer, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.06.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Effective Encapsulation and Biological Activity of Phosphorylated Chemotherapeutics in Calcium Phosphosilicate Nanoparticles for the Treatment of Pancreatic Cancer
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Welley S. Loc, PhD a, b ,k , Samuel S. Linton c, k , Zachary R. Wilczynski d, Gail L. Matters, PhD e, Christopher O. McGovern e, Thomas Abraham, PhD f, Todd Fox, PhD g, Christopher M. Gigliotti d, Xiaomeng Tang, PhD a, b, Amra Tabakovic, PhD b, Jo Ann Martin h , Gary A. Clawson, MD/PhD i, Jill P. Smith, MD j, Peter J. Butler, PhD d, Mark Kester, PhD g, James H. Adair, PhD b, c, d, * Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
b
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Department of Materials Science and Engineering, Pennsylvania State University, University Park, PA 16802, USA c
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Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA d
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Department of Biomedical Engineering/Bioengineering, Pennsylvania State University, University Park, PA 16802, USA e
f
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Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
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Department of Neural and Behavioral Sciences and the Microscopy Imaging Facility, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA g
Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
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Biomedical Engineering, University of Florida, Gainesville, FL 32611, USA
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Department of Pathology and Gittlen Cancer Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA j
Department of Medicine, Georgetown University, Washington DC 20007, USA
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Authors contributed equally to this work.
ACCEPTED MANUSCRIPT
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*Corresponding Author James H. Adair Professor of Materials Science & Engineering, Biomedical Engineering, and Pharmacology 205 Steidle Building Pennsylvania State University University Park, PA 16803 Email: [email protected] Office Phone: (814) 863-6047
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Statement of Funding and Conflicts of Interest: This study was funded by the National Institutes of Health (NIH) grants R01CA167535 and R21CA170121 from the National Cancer Institute (NCI). The project was also supported in part under a grant with the Pennsylvania Department of Health using Tobacco CURE funds (SAP#4100072562 to GLM and JHA). The department specifically disclaims responsibility for any analyses, interpretations or conclusions. WSL was supported, in part, by the grants UL1 TR000127 and TL1 TR000125 from the National Center for Advancing Translational Sciences (NCATS). Penn State Research Foundation has licensed CPSNP technology to Keystone Nano, Inc. (PA, USA). JHA and MK are co-founders of Keystone Nano and are CSO and CMO, respectively. All other authors declare that there are no conflicts of interest.
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Word Count
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Word count for abstract = 147/150 Word count for manuscript = 4983 Number of references = 56 Number of figures = 8 Number of tables = 1 Number of Supplementary online-only files: 1
ACCEPTED MANUSCRIPT ABSTRACT Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and
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nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive,
SC
phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated
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drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is
MA
essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained
ED
biological activity. Analysis of orthotopic tumors from mice treated systemically with tumortargeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells
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and release of active drug cargos intracellularly. These findings demonstrate a novel
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methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to
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effectively deliver them to pancreatic tumor cells.
ACCEPTED MANUSCRIPT KEYWORDS. FdUMP, 5-fluorouracil, gemcitabine, nanodelivery, dFdCMP
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ABBREVIATIONS
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CPSNP, calcium phosphosilicate nanoparticle; mPEG, methoxy-polyethylene glycol; cPEG, carboxy-polyethylene glycol; malPEG, maleimide-polyethylene glycol; Cit, citrate; NT, no
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treatment; TS, thymidylate synthase; THF, 5,10-methylene-tetrahydrofolate; FdUMP, 5-
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fluorodeoxyuridine monophosphate; 5-FU, 5-fluorouracil; FUdR, 5-fluorodeoxyuridine; dFdC, gemcitabine or 2’,2’-difluorodeoxycytidine; dFdCMP, gemcitabine monophosphate or 2’,2’-
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difluorodeoxycytidine monophosphate; ATP, adenosine triphosphate; 5-CU, 5-chlorouracil; SEM, standard error of the mean; TEM, transmission electron microscopy; CCKBR,
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cholecystokinin B receptor
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ACCEPTED MANUSCRIPT BACKGROUND Applications of nanoparticle (NP) delivery systems to medical oncology include the use of
T
NPs for encapsulated therapeutic drug delivery and for non-invasive tumor imaging. Due to the
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small size and versatile surface chemistry, NPs are ideal platforms to penetrate poorly vascularized and fibrotic tumors (1). Reports on NP-based delivery systems encompass a
SC
diversity of materials that include liposomes (2-4), dendrimers (5, 6), gold NPs (7), and quantum
NU
dots (8, 9). The design and composition of NPs reflect their abilities to load and protect agents in circulation and to provide a clearance mechanism for excess NPs (10).
MA
Amorphous calcium phosphosilicate nanoparticles (CPSNPs) are composed of bioresorbable materials (10, 11) that have been used to deliver therapeutic (12-14) and imaging agents (13, 15-
ED
18) to tumor cells. The NP matrix provides protection of encapsulated bioactive agents (such as
PT
chemotherapeutic drugs) from metabolic breakdown and rapid clearance. Further, surface decoration of CPSNPs with polyethylene glycol (PEG) mitigates non-specific interactions that
CE
cause agglomeration and facilitates bioconjugation of tumor targeting agents such as peptides,
AC
antibodies or aptamers (14, 15, 17-20). After cellular uptake, the low pH of late-stage endosomes triggers CPSNP dissolution and the endosomes rupture from a change in osmotic pressure to release active agents into the cytosol (13, 18). Treating pancreatic cancer with standard chemotherapeutic agents is challenging. Most pancreatic adenocarcinoma (PDAC) patients respond poorly to chemotherapeutic drugs, and even new drug combinations have demonstrated only a modest improvement in patient survival (21). This lack of efficacy has been attributed in part to inadequate drug delivery and metabolic drug inactivation (22). Two chemotherapeutics commonly used to treat PDAC, 5-fluorouracil (5-FU) and gemcitabine (dFdC), act by blocking key enzymes in nucleotide synthesis. 5-FU is
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ACCEPTED MANUSCRIPT metabolized to 5-fluorodeoxyuridine monophosphate (FdUMP) which, in the presence of 5,10methylenetetrahydrofolate (CH2 THF), irreversibly inhibits thymidylate synthase (TS) (23). TS
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inhibition results in nucleotide pool imbalances, impaired DNA synthesis, and a reduction in
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DNA repair (24). Similarly, the prodrug dFdC is phosphorylated intracellularly by deoxycytidine kinase to form gemcitabine monophosphate (dFdCMP), diphosphate (dFdCDP) and triphosphate
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(dFdCTP) (25). Gemcitabine has multiple modes of action; dFdCDP inhibits ribonucleotide
NU
reductase, which is responsible for producing the deoxynucleotides required for DNA synthesis and repair. This favors incorporation of dFdCTP into DNA, resulting in stalled DNA replication
MA
forks and apoptosis (26). While both 5-FU and dFdC are activated within tumor cells by conversion to phosphorylated metabolites, direct delivery of phosphorylated metabolites is
ED
limited by cell impermeability, lipid insolubility and precipitation.
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Systemic administration of many chemotherapeutics is limited by the need for high dosing regimens due to metabolic inactivation and rapid clearance. For example, in vivo studies have
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shown that less than 20% of the 5-FU becomes activated to FdUMP while more than 80% of 5-
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FU is converted to the inactive 5-FU metabolite 5-fluorodihydrouracil by the liver enzyme dihydropyrimidine dehydrogenase (DPD) (27). Likewise, dFdC can be inactivated by cytidine deaminase and rapidly cleared from the body (28-30). Both dFdC and 5-FU are transported into tumor cells by nucleoside transporter systems, including human equilibrative nucleoside transporters (hENT), which have low affinity for FdUMP and dFdCMP (31). Tumor targeted, NP-based delivery of phosphorylated drug metabolites could avoid drug inactivation processes and enhance drug uptake by tumor cells. This study compares the encapsulation efficiency of both phosphorylated and non-phosphorylated forms of 5-FU and dFdC and outlines a novel approach to deliver the chemotherapeutics directly to pancreatic tumors using targeted CPSNPs.
6
ACCEPTED MANUSCRIPT METHODS CPSNP Synthesis and Characterization. CPSNPs were prepared in reverse-micelles and
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isolated by HPLC as described by Barth et al. and Morgan et al. (13, 17, 18). The materials,
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synthetic procedure, and additional sample preparation methods are outlined in supplementary information. Citrate-functionalized (Cit) CPSNPs were preheated to 50°C and stirred at 550 rpm
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for PEGylation. To yield mPEG-CPSNPs, 1 ml of EDC (1 mg/ml) was first added drop-wise to
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10 ml of Cit-CPSNPs. After 5 min, 1 ml of each Sulfo-NHS (15 mg/ml) and 2 kDa methoxyPEG-amine (6 mg/ml) were transferred. The reaction proceeded for 15 hr. PEGylated CPSNPs
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were purified via filtration for 2-3 min at 5000 g (Amicon Ultra-15 30 kDa). Gastrin-16 peptide (G16, CSGGQQQQQQAYGWMDF, Genscript) was conjugated via 2 kDa maleimide-PEG-
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amine coupling (18). The peptide was added to the malPEG-CPSNPs after filtration and stored at
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4°C overnight. The aptamer (AP, TriLink Biotechnologies) was conjugated via 2 kDa carboxyPEG-amine coupling, first reported by Clawson et al. (19). A second EDC/NHS coupling
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CPSNPs).
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reaction followed after filtration with an aliquot of 0.2 mM aptamer solution (5 ul per ml cPEG-
Surface modification of CPSNPs was characterized by the Brookhaven Instruments zeta potential analyzer (ZetaPlus v. 3.23, Holtsville, NY). Particles were imaged on the FEI Tecnai G2 Spirit BioTWIN TEM operating at 120kV. The drug concentration in CPSNPs was quantified by LC-MS/MS (supplementary information). To calculate the payload, the total number of particles (Ntotal) was first determined on the Nanotrac Wave II DLS (Microtrac Flex 11.1.0.2, York, PA). Measurements were taken from an average of five runs, 10 s per run. Treating the NPs as spheres, the products of Ntotal and probability of particle sizes based on TEM distributions
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ACCEPTED MANUSCRIPT were used to determine the total NP volume in suspension. The number of drug molecules per
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nm3 of CPSNP were averaged with 95% confidence intervals.
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Cell Proliferation Assay. PANC-1 (in Dulbecco’s modified Eagle medium/10% fetal bovine serum) and BxPC-3 (in RPMI medium/10% fetal bovine serum) cells (ATCC) were seeded onto
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96-well plates at 5,000 cells/well. After 24 hr, treatment with vehicle (1x PBS), free drug,
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mPEG-CPSNPs or drug-loaded CPSNPs were initiated. Viable cell determinations were made after 72 hr using an alamarBlue® assay (Life Technologies). Relative proliferation for all
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treatment groups were normalized to vehicle controls.
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Thymidylate Synthase Immunoblotting. PANC-1 and BxPC-3 cells were seeded onto 6-well
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dishes and incubated for 24 hr in one of the following treatment groups: no treatment, vehicle, free FdUMP, mPEG-FdUMP-CPSNPs, and mPEG-CPSNPs. Lysates were collected by
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aspirating the media, washing with 1x PBS, and adding RIPA buffer containing Complete Mini
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protease cocktail (Roche). Protein concentration was determined by microBCA assay (Thermo Scientific) and 20 g of total protein was separated by gel electrophoresis. After transfer to HyBond ECL and blocking for 1 hr in 5% BSA, blots were probed overnight with anti-TS antibody (#9045 Cell Signaling Technology, 1:1000) or beta-actin antibody (#A2228; Sigma, 1:10,000). Membranes were washed, incubated with secondary antibody coupled to horseradish peroxidase (Amersham), and visualized using an enhanced chemiluminescent substrate (Pierce). Quantitation of scanned blots was done using Image-J software (NIH). After normalizing to βactin, % active TS is the amount of uncomplexed TS divided by the amount of total TS (active TS and TS-FdUMP ternary complex).
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Cell Cycle Analysis. PANC-1 cells were treated with vehicle, free FdUMP, mPEG-FdUMP-
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CPSNPs or an equal volume of mPEG-CPSNPs, or left untreated for 72 hr (32). Cells were
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fixed in 75% ethanol overnight and immediately prior to analysis treated with 1 g/mL RNase A
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and 50 g/mL propidium iodide. Cellular DNA content was determined using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed with Cellquest (Verity Software,
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Topsham, ME).
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In vivo assessment of aptamer-targeted FdUMP-CPSNP up take by PDAC tumor cells. Animal protocols were approved by the Penn State College of Medicine IACUC committee. All
Orthotopic PANC-1 tumors were established in male, athymic (nu/nu) mice (Charles
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animals.
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procedures were done in accordance with institutional guidelines for the humane care of the
River). Pancreata were injected with 5x10 6 cells and treatment with CSPNPs was initiated at one
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week post-surgery. Treatment groups (n= 4-5 mice per group, with two experimental replicates)
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included mPEG-CPSNPs, mPEG-FdUMP-CPSNPS, gastrin-targeted G16-malPEG-FdUMPCPSNPs, and aptamer-targeted AP-cPEG-FdUMP-CPSNPs. CPSNPs were administered at a FdUMP dose of 100 g/kg, or an equal volume of mPEG-CPSNPs, twice weekly via tail vein injection. After six weeks of treatment, animals were sacrificed. Blood was removed by cardiac puncture, and complete blood cell counts and serum chemistries were done by the Department of Comparative Medicine Diagnostic Laboratory (PSU-Hershey). A portion of each tumor was flash frozen in liquid nitrogen and was powdered by grinding in a pre-chilled mortar and pestle. RIPA buffer containing Complete Mini protease cocktail (Roche) was added to each sample following grinding; the resuspended tissue was sonicated for 15 s and centrifuged to pellet
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ACCEPTED MANUSCRIPT debris. Protein concentrations in supernatants were determined by micro BCA protein assay (Thermo Scientific), and 20 µg of protein from each sample was separated by gel
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electrophoresis. Thymidylate synthase immunoblots were performed as described above.
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Another portion of each tumor was fixed and embedded in OCT, cryo-sectioned and stained for Ki-67 (Abcam #92742) according to the manufacturer’s instructions; nuclei were counterstained
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with Hoechst 33432. Images were acquired with a Leica AOBS SP8 laser scanning confocal
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microscope.
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Statistical Analysis. Results were expressed as means ± standard error. Student t-tests were used to evaluate statistical significance with a p < 0.05 considered to be significant. To calculate
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EC50 ± 95% CI, nonlinear regression analysis was performed to generate the curve of best fit for
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the data according to a Sigmoidal regression using a 4-parameter logistic curve calibration [Y =
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Yo + (a/(1+((X/Xo )b))] in SigmaPlot 12 (Systat, Inc., San Jose, CA).
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ACCEPTED MANUSCRIPT RESULTS CPSNP Characterization. Preparation of CPSNPs (Figure 1) in reverse micelles yielded sub-
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100 nm particles (13-18, 33, 34); a size range that is optimal for NP penetration into fibrotic
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pancreatic tumors (18, 35). The lognormal mean of the size distribution for mPEG-FdUMPCPSNPs was 8.4 nm (𝜎𝑧 =0.2) and 10.5 nm (z=0.4) for mPEG-dFdCMP-CPSNPs (Figures 2A
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and 2B). Aptamer-targeted AP-cPEG-FdUMP-CPSNPs in Figure 2C were about 8.0 nm
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(𝜎𝑧 =0.3) and 17.2 nm (𝜎𝑧 =0.1) for gastrin-targeted G16-malPEG-FdUMP-CPSNPs in Figure 2D. Because CPSNPs depend on citrate (Cit) and/or PEG to maintain dispersion (33), the zeta
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potential is a common metric to verify changes in surface terminal groups and colloidal stability (Figure 3). The mean values for Cit-CPSNPs, Cit-FdUMP-CPSNPs and Cit-dFdCMP-CPSNPs
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were ‒37±6 mV, ‒44±5 mV and ‒28±7 mV, respectively. After PEGylation, the uncharged
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methoxy group termination at the NP surface exhibits near-zero net charge, so mPEG-CPSNPs, mPEG-FdUMP-CPSNPs and mPEG-dFdCMP-CPSNPs displayed mean values of ‒1±5 mV, ‒
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4±6 mV and 1±9 mV, respectively. Bioconjugation of the aptamer to cPEG-FdUMP-CPSNPs
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resulted in a reduction of the zeta potential magnitude from ‒35±5 mV to ‒22±5 mV. An increased magnitude from ‒8±5 mV to ‒29±9 mV was observed for the conjugation of the gastrin to malPEG-FdUMP-CPSNPs.
Encapsulation efficiency of chemotherapeutics by CPSNPs. We hypothesized that chemotherapeutic drugs such as 5-FU or dFdC would be less effectively encapsulated into CPSNPs than FdUMP and dFdCMP (Figure 4). To address this hypothesis, the encapsulation efficiencies (EE) for 5-FU, ATP:5-FU complex, FUdR (5-fluorodeoxyuridine), FdUMP, dFdC, and dFdCMP (Table 1) were experimentally determined. The EE is defined by the relationship,
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ACCEPTED MANUSCRIPT 𝑚
(1)
𝑚𝑜𝑙% 𝐸𝐸 = 𝑚𝑓 × 100 𝑖
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where m i is the initial moles of drug and mf is the moles present in the purified CPSNPs,
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quantified by LC-MS/MS. CPSNP encapsulation of 5-FU was only 4.1 (±1.8) ×10-7 M, or 0.11 (±0.04) mol% (Table 1), and extended reaction times failed to improve 5-FU encapsulation.
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FUdR, the deoxynucleoside analog of 5-FU, was incorporated in CPSNPs even less effectively than 5-FU, with an EE of
