@article{vrabel_schulman_gillam_mantooth_nguyen_zaharoff_2023, title={Focal Cryo-Immunotherapy with Intratumoral IL-12 Prevents Recurrence of Large Murine Tumors}, volume={15}, ISSN={["2072-6694"]}, DOI={10.3390/cancers15082210}, abstractNote={Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but they often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies, capable of safely eliminating residual tumor cells, are therefore of great clinical interest. Interleukin-12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers, including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with a CS/IL-12 formulation could prevent tumor recurrence after cryoablation (CA). Tumor recurrence and overall survival rates were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node (dLN) samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced complete durable regression of large tumors in 80–100% of treated animals. Additionally, CS/IL-12 prevented lung metastases when delivered as a neoadjuvant to CA. However, CA plus CS/IL-12 had minimal antitumor activity against established, untreated abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal tumors. Transcriptome analyses revealed early immunological changes in the dLN, followed by a significant increase in gene expression associated with immune suppression and regulation. Cryo-immunotherapy with localized CS/IL-12 reduces recurrences and enhances the elimination of large primary tumors. This focal combination therapy also induces significant but limited systemic antitumor immunity.}, number={8}, journal={CANCERS}, author={Vrabel, Maura R. and Schulman, Jacob A. and Gillam, Francis B. and Mantooth, Siena M. and Nguyen, Khue G. and Zaharoff, David A.}, year={2023}, month={Apr} }
 @article{zaharoff_nguyen_2022, title={IDENTIFICATION OF VASOACTIVE INTESTINAL PEPTIDE (VIP)-SPECIFIC SINGLE-CHAIN ANTIBODY FRAGMENTS (SCFVS) VIA YEAST SURFACE DISPLAY}, volume={10}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2022-SITC2022.1371}, abstractNote={<h3>Background</h3> Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide expressed in various tissues including the pancreas, intestines and central nervous system.<sup>1, 2</sup> The overexpression of VIP and its receptors is associated with increased growth and metastasis of breast, prostate, and lung malignancies.<sup>3</sup> In addition, the interaction of VIP with its receptors on activated T cells results in immune suppression which further supports tumor growth.<sup>4, 5</sup> Furthermore, tumor-supporting regulatory T cells have been found to be promoted by VIP-dependent mechanisms.<sup>6</sup> Altogether, prior literature implies that blockade of VIP signaling may inhibit tumor-mediated immune suppression and augment antitumor immune responses. Recent preclinical studies in acute myeloid leukemia and T lymphoblastic leukemia demonstrated that VIP receptor antagonists increase T cell-dependent anti-tumor responses.<sup>2</sup>. Unfortunately, the short-half lives of peptide antagonists limit their clinical utility. A more translatable approach is the development of long circulating antibodies that bind VIP and inhibit its immunosuppressive activities. <h3>Methods</h3> In this study, we utilized a yeast display of a non-immune human single-chain variable fragment (scFv) library to identify VIP-binding scFvs.<sup>7-9</sup> VIP binders were screened by several rounds of selection using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The enriched binder population was cloned into single colonies of yeast cells by limited dilution. The binding affinities of VIP-binding clones were evaluated via flow cytometry by titrating fluorescence-labeled VIP. Clones with high binding affinity (Kd < 500 nM) were selected for sequencing (figures 1-5). <h3>Results</h3> Sequences of the isolated scFv revealed that a unique section of complementarity-determining region 3 (CDR3) of the heavy chain played an important role in VIP binding. Multiple clones with similar but distinct CDR3 sequences produced a useful range of binding affinities for further development (figure 6). <h3>Conclusions</h3> Yeast display is an effective technology for identifying human scFvs that bind to the immunosuppressive neuropeptide, VIP. CDR3 of scFv heavy chains were influential in VIP recognition. Ongoing studies are focused on the production, purification, and validation of novel anti-VIP human antibodies. <h3>Acknowledgements</h3> We thank Dr. K. Dane Wittrup (Massachusetts Institute of Technology) for kindly providing yeast libraries, Drs. Edmund K. Waller and Jens Wrammert (Emory University) for helpful discussions and comments, and Dr. Ryan W. Paerl (North Carolina State University) for technical advice on cell sorting. <h3>References</h3> Petersen CT, Li JM, Waller EK. Administration of a vasoactive intestinal peptide antagonist enhances the autologous anti-leukemia T cell response in murine models of acute leukemia. <i>Oncoimmunology</i>. 2017;<b>6</b>(5):e1304336. Epub 2017/06/24. Li JM, Darlak KA, Southerland L, Hossain MS, Jaye DL, Josephson CD, <i>et al</i>. VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice. <i>PLoS One</i>. 2013;<b>8</b>(5):e63381. Epub 2013/06/01. Moody TW, Nuche-Berenguer B, Jensen RT. Vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide, and their receptors and cancer. <i>Curr Opin Endocrinol Diabetes Obes</i>. 2016;<b>23</b>(1):38–47. Epub 2015/12/26. Forghani P, Petersen CT, Waller EK. Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity. <i>Oncotarget</i>. 2017;<b>8</b>(47):81873–9. Epub 2017/11/16. Liu L, Yen JH, Ganea D. A novel VIP signaling pathway in T cells cAMP-->protein tyrosine phosphatase (SHP-2?)-->JAK2/STAT4-->Th1 differentiation. <i>Peptides</i> 2007;<b>28</b>(9):1814–24. Epub 2007/04/28. Delgado M, Chorny A, Gonzalez-Rey E, Ganea D. Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo. <i>J Leukoc Biol</i> 2005;<b>78</b>(6):1327-38. Epub 2005/10/06. Angelini A, Chen TF, de Picciotto S, Yang NJ, Tzeng A, Santos MS, <i>et al</i>. Protein engineering and selection using yeast surface display. <i>Methods Mol Biol</i> 2015;<b>1319</b>:3–36. Epub 2015/06/11. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, <i>et al</i>. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. <i>Nat Biotechnol</i>. 2003;<b>21</b>(2):163–70. Epub 2003/01/22. Kelly RL, Le D, Zhao J, Wittrup KD. Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries. <i>J Mol Biol</i>. 2018;<b>430</b>(1):119–30. Epub 2017/12/01.}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Zaharoff, David and Nguyen, Khue}, year={2022}, month={Nov}, pages={A1424–A1425} }
 @article{nguyen_mantooth_vrabel_zaharoff_2022, title={Intranasal Delivery of Thermostable Subunit Vaccine for Cross-Reactive Mucosal and Systemic Antibody Responses Against SARS-CoV-2}, volume={13}, ISSN={["1664-3224"]}, DOI={10.3389/fimmu.2022.858904}, abstractNote={Despite the remarkable efficacy of currently approved COVID-19 vaccines, there are several opportunities for continued vaccine development against SARS-CoV-2 and future lethal respiratory viruses. In particular, restricted vaccine access and hesitancy have limited immunization rates. In addition, current vaccines are unable to prevent breakthrough infections, leading to prolonged virus circulation. To improve access, a subunit vaccine with enhanced thermostability was designed to eliminate the need for an ultra-cold chain. The exclusion of infectious and genetic materials from this vaccine may also help reduce vaccine hesitancy. In an effort to prevent breakthrough infections, intranasal immunization to induce mucosal immunity was explored. A prototype vaccine comprised of receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution induced high levels of RBD-specific antibodies in laboratory mice after 1 or 2 immunizations. Antibody responses were durable with high titers persisting for at least five months following subcutaneous vaccination. Serum anti-RBD antibodies contained both IgG1 and IgG2a isotypes suggesting that the vaccine induced a mixed Th1/Th2 response. RBD vaccination without CS formulation resulted in minimal anti-RBD responses. The addition of CpG oligonucleotides to the CS plus RBD vaccine formulation increased antibody titers more effectively than interleukin-12 (IL-12). Importantly, generated antibodies were cross-reactive against RBD mutants associated with SARS-CoV-2 variants of concern, including alpha, beta and delta variants, and inhibited binding of RBD to its cognate receptor angiotensin converting enzyme 2 (ACE2). With respect to stability, vaccines did not lose activity when stored at either room temperature (21-22°C) or 4°C for at least one month. When delivered intranasally, vaccines induced RBD-specific mucosal IgA antibodies, which may protect against breakthrough infections in the upper respiratory tract. Altogether, data indicate that the designed vaccine platform is versatile, adaptable and capable of overcoming key constraints of current COVID-19 vaccines.}, journal={FRONTIERS IN IMMUNOLOGY}, author={Nguyen, Khue G. and Mantooth, Siena M. and Vrabel, Maura R. and Zaharoff, David A.}, year={2022}, month={May} }
 @article{paidi_troncoso_harper_liu_nguyen_ravindranathan_rebello_lee_ivers_zaharoff_et al._2022, title={Raman spectroscopy reveals phenotype switches in breast cancer metastasis}, volume={12}, ISSN={["1838-7640"]}, DOI={10.7150/thno.74002}, abstractNote={The accurate analytical characterization of metastatic phenotype at primary tumor diagnosis and its evolution with time are critical for controlling metastatic progression of cancer. Here, we report a label-free optical strategy using Raman spectroscopy and machine learning to identify distinct metastatic phenotypes observed in tumors formed by isogenic murine breast cancer cell lines of progressively increasing metastatic propensities. Methods: We employed the 4T1 isogenic panel of murine breast cancer cells to grow tumors of varying metastatic potential and acquired label-free spectra using a fiber probe-based portable Raman spectroscopy system. We used MCR-ALS and random forests classifiers to identify putative spectral markers and predict metastatic phenotype of tumors based on their optical spectra. We also used tumors derived from 4T1 cells silenced for the expression of TWIST, FOXC2 and CXCR3 genes to assess their metastatic phenotype based on their Raman spectra. Results: The MCR-ALS spectral decomposition showed consistent differences in the contribution of components that resembled collagen and lipids between the non-metastatic 67NR tumors and the metastatic tumors formed by FARN, 4T07, and 4T1 cells. Our Raman spectra-based random forest analysis provided evidence that machine learning models built on spectral data can allow the accurate identification of metastatic phenotype of independent test tumors. By silencing genes critical for metastasis in highly metastatic cell lines, we showed that the random forest classifiers provided predictions consistent with the observed phenotypic switch of the resultant tumors towards lower metastatic potential. Furthermore, the spectral assessment of lipid and collagen content of these tumors was consistent with the observed phenotypic switch. Conclusion: Overall, our findings indicate that Raman spectroscopy may offer a novel strategy to evaluate metastatic risk during primary tumor biopsies in clinical patients.}, number={12}, journal={THERANOSTICS}, author={Paidi, Santosh Kumar and Troncoso, Joel Rodriguez and Harper, Mason G. and Liu, Zhenhui and Nguyen, Khue G. and Ravindranathan, Sruthi and Rebello, Lisa and Lee, David E. and Ivers, Jesse D. and Zaharoff, David A. and et al.}, year={2022}, pages={5351–5363} }
 @article{horton_su_yi_wang_xu_pagadala_zhang_zaharoff_pearce_linhardt_et al._2021, title={Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins}, volume={31}, ISSN={["1460-2423"]}, DOI={10.1093/glycob/cwaa068}, abstractNote={Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.}, number={3}, journal={GLYCOBIOLOGY}, author={Horton, Maurice and Su, Guowei and Yi, Lin and Wang, Zhangjie and Xu, Yongmei and Pagadala, Vijayakanth and Zhang, Fuming and Zaharoff, David A. and Pearce, Ken and Linhardt, Robert J. and et al.}, year={2021}, month={Mar}, pages={188–199} }
 @article{mantooth_zaharoff_mantooth_2021, title={INJECTABLE CHITOSAN HYDROGEL FOR LOCALIZED DELIVERY OF IMMUNE CHECKPOINT INHIBITORS}, volume={9}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2021-SITC2021.259}, abstractNote={Background Systemic delivery of checkpoint inhibitors risks the development of immune-related adverse events (irAEs) in up to 85% of patients. 1 Localized delivery methods with slow-release kinetics have the potential to avoid systemic exposure and reduce irAEs. Direct tumor injection is extremely difficult, as saline-based solutions are rapidly excluded from the high-pressure tumor environment. Utilizing hydrogels as a delivery medium and local depot can address this shortcoming. To this end, we developed an injectable chitosan-based hydrogel for intratumoral delivery of checkpoint antibodies. Methods Hydrogel Low-viscosity, 80% deacetylated chitosan (Heppe Medical Chitosan; Halle, Germany) was reacted with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence of β-glycerophosphate at room temperature for 48 hours. The mixture was then washed with ethanol and dried at 60°C. The resulting solid was dissolved in phosphate buffered saline (PBS) at concentrations from 35–70 mg/mL.In vitro release. 300 μg/mL bovine serum albumin (BSA) labeled with fluorescein-5-isothiocyanate (FITC) as a model protein drug was loaded into the hydrogel. The hydrogel was injected through a 28g needle and incubated with PBS. Samples were taken over a week period. Release kinetics were analyzed by fitting fluorescence data to zero-order, first-order, and Korsmeyer-Peppas models. To visualize retention after injection, dye-loaded hydrogels or dye in PBS alone were injected into a 0.6 wt% agar tissue phantom.In vivo imaging and tumor treatment. Flank MC38 tumors will be established in C57BL/6 mice. At tumor volumes of 50–100 mm3, 200 ug of fluorescently labeled aCTLA-4 and aPD-L1 included in the chitosan hydrogel will be delivered intratumorally. Images will be captured using an In Vivo Imaging System (IVIS). Antitumor activity will be assessed in a separate cohort using unlabeled antibodies. Results The chitosan hydrogel was found to be injectable in needles as thin as 28g. After exiting the needle, the hydrogel reformed (figure 1A). Upon injection into the tissue phantom, dyed PBS immediately leaked out, primarily through the needle track, while the dyed hydrogel was retained (figure 1B). In vitro release studies demonstrated long-term, nearly zero-order, week-long sustained release (figure 1C). In vivo retention and tumor treatment studies are ongoing. Abstract 259 Figure 1 Injectable chitosan hydrogel. (A) Re-formed BSA-FITC hydrogel in 1x PBS; (B) (i) Retained hydrogel in agar tissue phantom, (ii) Excluded 1x PBS in agar tissue phantom; (C) Release kinetics in 1x PBS. Conclusions A novel injectable chitosan hydrogel was found to provide sustained release of a large model protein over a 1–2 week period with favorable in vitro kinetics. Importantly, this hydrogel can be engineered to provide faster or slower release as needed. Ongoing studies in vivo will quantify intratumoral retention, systemic dissemination, and antitumor activity. Acknowledgements This work is supported by the National Science Foundation Graduate Research Fellowship. Reference Hommes J, Verheijden R, Suijkerbuijk K, Hamann D. Biomarkers of checkpoint inhibitor induced immune-related adverse events—a comprehensive review. Front Oncol 2021; 10 :1–16. Ethics Approval Animal use was in compliance with the Public Health Service Policy on Human Care and Use of Laboratory Animals. All experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee at North Carolina State University (Protocol #19–795).}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Mantooth, Siena and Zaharoff, David and Mantooth, Siena}, year={2021}, month={Nov}, pages={A281–A281} }
 @article{vrabel_zaharoff_mantooth_2021, title={ONCO-IMMUNOLOGICAL MECHANISMS OF FOCAL ABLATION AND LOCALIZED IL-12 IMMUNOTHERAPY}, volume={9}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2021-SITC2021.620}, abstractNote={Background Unresectable solid malignancies are responsible for a major proportion of total cancer-related mortalities, making focal ablation an attractive alternative. Nevertheless, there are high rates of recurrence after ablation [1,2]. The addition of an immune agonist to ablation has the potential to prevent this recurrence and improve treatment outcomes. The goal of this study is to determine if localized interleukin-12 (IL-12) can prevent primary tumor recurrence after cryoablation in both minimal ablation and metastasis models. Methods LLC (LL/2) (ATCC) and MC38 (NCI) were implanted in 6–9 week old C57BL/6 mice. All tumors were treated at volumes of 200–500 mm3. LLC tumors with treated with three cycles of freeze/thaw and then monitored for tumor recurrence and lung metastasis 22–25 days after implantation. MC38 tumors were treated with a minimal cryoablation protocol where the tumor undergoes one cycle of freezing at 100% intensity up to the tumor margin by visual inspection, followed by one cycle of active thaw until the cryo probe can be removed. Ablation was performed using the Argon-Helium Visual-ICETM Cryoablation System (Boston Scientific). Interleukin-12 (IL-12) in 1.5% (w/v) chitosan acetate (CS) dissolved in dPBS was injected intratumorally within an hour after cryoablation, or as indicated. The dose of IL-12 was 1 ug unless otherwise indicated. Results We established a model of 100% recurrence using the minimal cryoablation protocol. Using this protocol, we demonstrated that a single intratumoral injection of CS/IL-12 within an hour after cryoablation prevents recurrence in 7/8 mice while only 3/8 mice remained tumor-free without CS/IL-12 (Figure 1). Studies evaluating the impact of CS/IL-12 on tumor-specific T cell responses following cryoablation are ongoing. In the spontaneously metastatic LLC model, delivering CS/IL-12 two days before treatment, either resection or cryoablation, reduced the number of metastatic lung nodules and furthermore prevented the recurrence of the primary tumor after resection (Figure 2). Abstract 620 Figure 1 CS/IL-12 prevents tumor recurrence after minimal cryo Abstract 620 Figure 2 CS/IL-12 before cryo or surgery prevents metastasis Conclusions Due to the high percentage of solid malignancies that are unresectable at diagnosis, focal ablation is an attractive alternative, yet has a high rate of recurrence. We demonstrated that intratumoral neoadjuvant CS/IL-12 protects not only against primary recurrence after cryoablation, but also protects against lung metastasis and recurrence after resection. Further studies are necessary to explore the immune populations responsible for this therapeutic effect. Acknowledgements This work is supported by Boston Scientific, the NC State University Provost’s Fellowship, the NSF Graduate Research Fellowship and startup funds provided by the College of Engineering at NC State University. References Weld KJ, Landman J. Comparison of cryoablation, radiofrequency ablation and high-intensity focused ultrasound for treating small renal tumours. BJU Int. 2005;96(9):1224–1229. doi:10.1111/j.1464–410X.2005.05848.x. Guenther E, Klein N, Zapf S, et al. Prostate cancer treatment with Irreversible Electroporation (IRE): Safety, efficacy and clinical experience in 471 treatments. PloS One. 2019;14(4):e0215093. doi:10.1371/journal.pone.0215093 Ethics Approval The Institutional Animal Care and Use Committee at North Carolina State University approved of all animal protocols (#19–795) in compliance with The Guide for Care and Use of Laboratory Animals (National Research Council).}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Vrabel, Maura and Zaharoff, David and Mantooth, Siena}, year={2021}, month={Nov}, pages={A650–A650} }
 @article{nguyen_wagner_vrabel_mantooth_meritet_zaharoff_2021, title={Safety and Pharmacokinetics of Intravesical Chitosan/Interleukin-12 Immunotherapy in Murine Bladders}, volume={7}, ISSN={["2352-3735"]}, DOI={10.3233/BLC-211542}, abstractNote={BACKGROUND: Intravesical administration of interleukin 12 (IL-12) co-formulated with the biopolymer, chitosan (CS/IL-12), has demonstrated remarkable antitumor activity against preclinical models of bladder cancer. However, given historical concerns regarding severe toxicities associated with systemic IL-12 administration in clinical trials, it is important to evaluate the safety of intravesical CS/IL-12 prior to clinical translation. OBJECTIVE: To evaluate the pharmacokinetics as well as the local and systemic toxicities of intravesical CS/IL-12 immunotherapy in laboratory mice. METHODS: Local inflammatory responses in mouse bladders treated with intravesical IL-12 or CS/IL-12 were assessed via histopathology. Serum cytokine levels following intravesical and subcutaneous (s.c.) administrations of IL-12 or CS/IL-12 in laboratory mice were compared. Systemic toxicities were evaluated via body weight and liver enzyme levels. RESULTS: Intravesical IL-12 and CS/IL-12 treatments did not induce significant local or systemic toxicity. IL-12 dissemination and exposure from intravesical administration was significantly lower compared to s.c. injections. Weekly intravesical CS/IL-12 treatments were well-tolerated and did not result in blunted immune responses. CONCLUSIONS: Intravesical CS/IL-12 is safe and well-tolerated in mice. In particular, the lack of cystitis and acute inflammation justifies continued investigation of intravesical CS/IL-12 immunotherapy in larger animals and patients with bladder cancer.}, number={4}, journal={BLADDER CANCER}, author={Nguyen, Khue G. and Wagner, Ethan S. and Vrabel, Maura R. and Mantooth, Siena M. and Meritet, Danielle M. and Zaharoff, David A.}, year={2021}, pages={427–437} }
 @article{vrabel_schulman_zaharoff_2020, title={INTRATUMORAL INTERLEUKIN-12 ADMINISTERED AFTER CRYOABLATION DOES NOT IMPROVE SURVIVAL IN MULTIPLE BILATERAL MURINE MODELS}, volume={8}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2020-SITC2020.0457}, abstractNote={Background Clinically, cryoablation is used to treat certain early stage prostate, liver, and kidney tumors in addition to bone and soft tissue sarcomas. However, for late-stage cancers, ablation is only an auxiliary step before complete resection. This leaves a gap of patients with advanced and inoperable tumors, where up to 90% of all pancreatic, and 80–85% of all prostate cancers are unresectable at diagnosis. 1 2 Because cryoablation can release large amounts of antigen, it is uniquely capable of not only treating advanced, unresectable tumors, but also may induce an in situ vaccination response when combined with the appropriate immunotherapy. Previously, our results in single primary tumor models indicated that the addition of interleukin-12 (IL-12) to cryoablation (cryo) improved tumor burden and survival We hypothesized that intratumoral injection of IL-12 after cryo would activate a strong T cell response and induce systemic immunity in bilateral tumor models. Methods Panc02 cells were purchased from ATCC; MC38 and MB49 cells were acquired from the NIH. Female C57BL/6 mice were purchased from Jackson Laboratory. For primary tumor implantation, 1 × 10 6 Panc02 cells and 3 × 10 5 MC38 cells were injected subcutaneously (s.c.) in the right flank. For rechallenge, the same dose of cells was implanted on the left flank of cured mice. For bilateral models, in both the MB49 and MC38 models, 3 × 10 5 and 1.5 × 10 5 cells were injected s.c. in the right and left flanks respectively on the same day. For the Panc02 model, 1 × 10 6 cells were implanted s.c. on both the right and left flanks on the same day. Tumor volume was calculated as 0.5*a*b 2 given the perpendicular long (a) and short (b) dimensions. Tumors measuring between 150–300 mm 3 were cryoablated with three cycles of freeze/thaw using the Visual-ICETM Cryoablation System (Boston Scientific). The dose of IL-12 was 1 µg/mouse in 1.5% (w/v) chitosan acetate (CS) dissolved in DPBS, and then injected intratumorally within an hour after cryoablation. For the anti-PD-1 and isotype antibodies (BioXCell, clone: RMP1.14). 300µg was injected intraperitoneally every 3 days starting on the day of cryoablation for a total of 4 doses. Results In the bilateral MB49 mouse bladder cancer model, the median survival for the cryo alone group was 20 days post treatment (p.t.) compared to 23 days p.t. for cryo + CS/IL-12, which was not significant, and 12 days for the untreated control group. In the bilateral Panc02 model, the median survival for both the cryo alone and cryo + CS/IL-12 groups was the same at 20.5 days p.t., compared to 10 days p.t. for the untreated control. In the bilateral MC38 model, the addition of anti-PD-1 to cryo + CS/IL-12 did not significantly improve survival compared to isotype + cryo + CS/IL-12, with a median survival of 24 days p.t. and 16 days p.t. respectively (p=0.53, Log-rank test) (figure 1). However, addition of anti-PD-1 did significantly delay abscopal tumor growth up to 500 mm 3 when compared to the isotype + cryo + CS/IL-12 (p=0.0398, Unpaired t test) (figure 2). Finally, the addition of IL-12 worsens memory in the MC38 model, where 100% of rechallenged cryo alone mice were protected (5/5) compared to only 43% protected of the cryo + CS/IL-12 group (3/7). Abstract 457 Figure 1 Survival of bilateral MC38MC38 cells were implanted s.c. in the right and left flanks of C57/BL6 mice at 3 × 105 and 1.5 × 105 cells per 100 uL respectively. The tumor was allowed to grow to between 150 to 300 mm3 prior to cryoablation. CS/IL-12 was injected intratumorally within an hour after cryoablation. Criteria for euthanasia was a tumor burden greater than or equal to 2000 mm3 or if mouse became moribund. The median survival for each group was 8 days p.t. (Isotype), 11 days p.t. (a-PD-1), 15 days (Iso + Cryo), 14 days p.t. (a-PD-1 + Cryo), 16 days p.t. (Iso + Cryo + CS/IL-12), 24 days p.t. (a-PD-1 + Cryo + CS/Il-12), p < 0.0001 (Log-rank test). The number of subjects for each group is given in parenthesis in the legend. Abbreviations: CS, chitosan acetate; p.t., post treatment Abstract 457 Figure 2 Days p.t. for untreated MC38 to reach 500 mm3Days post treatment to reach 500 mm3 was determined as the day on which the untreated tumor volume measurement exceeded 500 mm3 for the first time and growth continued to increase afterward for at least two days. Statistics: One-way ANOVA * p<0.05, ** p<0.01 Conclusions Conclusions: While cryoablation in combination with immunotherapy has the potential to treat advanced, unresectable primary tumors and distant untreated tumors, the addition of a single injection of IL-12 is not enough to induce a strong abscopal effect. Furthermore, it may actually worsen the establishment of effector memory cells. The addition of anti-PD-1 only slows abscopal tumor growth. Future work is needed to understand the mechanism of T cell priming in the context of the post-ablative tumor. Acknowledgements This work is supported by Boston Scientific, the NC State University Provost’s Fellowship, the NSF Graduate Research Fellowship and startup funds provided by the College of Engineering at NC State University. References van der Geest LGM. et al. Trends in treatment and survival of patients with nonresected, nonmetastatic pancreatic cancer: a population-based study. Cancer Med 2018; 7, 4943–4951. Takaki H. et al. Thermal ablation and immunomodulation: from preclinical experiments to clinical trials. Diagn. Interv. Imaging 2017; 98:651–659.}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Vrabel, Maura and Schulman, Jake and Zaharoff, David}, year={2020}, month={Nov}, pages={A277–A278} }
 @article{smith_griffith_zaharoff_2019, title={Analyzing the effects of instillation volume on intravesical delivery using biphasic solute transport in a deformable geometry}, volume={36}, ISSN={["1477-8602"]}, DOI={10.1093/imammb/dqy004}, abstractNote={Ailments of the bladder are often treated via intravesical delivery-direct application of therapeutic into the bladder through a catheter. This technique is employed hundreds of thousands of times every year, but protocol development has largely been limited to empirical determination. Furthermore, the numerical analyses of intravesical delivery performed to date have been restricted to static geometries and have not accounted for bladder deformation. This study uses a finite element analysis approach with biphasic solute transport to investigate several parameters pertinent to intravesical delivery including solute concentration, solute transport properties and instillation volume. The volume of instillation was found to have a substantial impact on the exposure of solute to the deeper muscle layers of the bladder, which are typically more difficult to reach. Indeed, increasing the instillation volume from 50-100 ml raised the muscle solute exposure as a percentage of overall bladder exposure from 60-70% with higher levels achieved for larger instillation volumes. Similar increases were not seen for changes in solute concentration or solute transport properties. These results indicate the role that instillation volume may play in targeting particular layers of the bladder during an intravesical delivery.}, number={2}, journal={MATHEMATICAL MEDICINE AND BIOLOGY-A JOURNAL OF THE IMA}, author={Smith, Sean G. and Griffith, Boyce E. and Zaharoff, David A.}, year={2019}, month={Jun}, pages={139–156} }
 @article{kerr_agrawal_maity_koppolu_jayanthi_kumar_gundampati_mcnabb_zaharoff_kumar_2019, title={Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity}, volume={518}, ISSN={["1090-2104"]}, DOI={10.1016/j.bbrc.2019.08.029}, abstractNote={Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.}, number={2}, journal={BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, author={Kerr, Rebecca and Agrawal, Shilpi and Maity, Sanhita and Koppolu, Bhanuprasanth and Jayanthi, Srinivas and Kumar, Gayatri Suresh and Gundampati, Ravi Kumar and McNabb, David S. and Zaharoff, David A. and Kumar, Thallapuranam Krishnaswamy Suresh}, year={2019}, month={Oct}, pages={191–196} }
 @article{yao_rabbani_sattler_nguyen_zaharoff_walker_gamcsik_2019, title={Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia}, volume={91}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.9b03709}, abstractNote={Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Yao, Ming and Rabbani, Zahid N. and Sattler, Tyler and Nguyen, Khue G. and Zaharoff, David A. and Walker, Glenn and Gamcsik, Michael P.}, year={2019}, month={Dec}, pages={15032–15039} }
 @article{nguyen_gillam_hopkins_jayanthi_gundampati_su_bear_pilkington_jalah_felber_et al._2019, title={Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity}, volume={294}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.RA118.006193}, DOI={10.1074/JBC.RA118.006193}, abstractNote={Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.}, number={12}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Nguyen, Khue G. and Gillam, Francis B. and Hopkins, Jared J. and Jayanthi, Srinivas and Gundampati, Ravi Kumar and Su, Guowei and Bear, Jenifer and Pilkington, Guy R. and Jalah, Rashmi and Felber, Barbara K. and et al.}, year={2019}, month={Jan}, pages={4412–4424} }
 @article{fix_koppolu_novell_hopkins_kierski_zaharoff_dayton_papadopoulou_2019, title={ULTRASOUND-STIMULATED PHASE-CHANGE CONTRAST AGENTS FOR TRANSEPITHELIAL DELIVERY OF MACROMOLECULES, TOWARD GASTROINTESTINAL DRUG DELIVERY}, volume={45}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2019.02.004}, abstractNote={The gastrointestinal (GI) tract presents a notoriously difficult barrier for macromolecular drug delivery, especially for biologics. Herein, we demonstrate that ultrasound-stimulated phase change contrast agents (PCCAs) can transiently disrupt confluent colorectal adenocarcinoma monolayers and improve the transepithelial transport of a macromolecular model drug. With ultrasound treatment in the presence of PCCAs, we achieved a maximum of 44 ± 15% transepithelial delivery of 70-kDa fluorescein isothiocyanate-dextran, compared with negligible delivery through sham control monolayers. Among all tested rarefactional pressures (300-600 kPa), dextran delivery efficiency was consistently greatest at 300 kPa. To explore this unexpected finding, we quantified stable and inertial cavitation energy generated by various ultrasound exposure conditions. In general, lower pressures resulted in more persistent cavitation activity during the 30-s ultrasound exposures, which may explain the enhanced dextran delivery efficiency. Thus, a unique advantage of using low boiling point PCCAs for this application is that the same low-pressure pulses can be used to induce vaporization and provide maximal delivery.}, number={7}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Fix, Samantha M. and Koppolu, Bhanu P. and Novell, Anthony and Hopkins, Jared and Kierski, Thomas M. and Zaharoff, David A. and Dayton, Paul A. and Papadopoulou, Virginie}, year={2019}, month={Jul}, pages={1762–1776} }
 @article{davis_gundampati_jayanthi_anderson_pickhardt_koppolu_zaharoff_kumar_2018, title={Effect of extension of the heparin binding pocket on the structure, stability, and cell proliferation activity of the human acidic fibroblast growth factor}, volume={13}, ISSN={2405-5808}, url={http://dx.doi.org/10.1016/J.BBREP.2017.12.001}, DOI={10.1016/J.BBREP.2017.12.001}, abstractNote={Acidic human fibroblast growth factor (hFGF1) plays a key role in cell growth and proliferation. Activation of the cell surface FGF receptor is believed to involve the glycosaminoglycan, heparin. However, the exact role of heparin is a subject of considerable debate. In this context, in this study, the correlation between heparin binding affinity and cell proliferation activity of hFGF1 is examined by extending the heparin binding pocket through selective engineering via charge reversal mutations (D82R, D84R and D82R/D84R). Results of biophysical experiments such as intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy suggest that the gross native structure of hFGF1 is not significantly perturbed by the engineered mutations. However, results of limited trypsin digestion and ANS binding experiments show that the backbone structure of the D82R variant is more flexible than that of the wild type hFGF1. Results of the temperature and urea-induced equilibrium unfolding experiments suggest that the stability of the charge-reversal mutations increases in the presence of heparin. Isothermal titration calorimetry (ITC) data reveal that the heparin binding affinity is significantly increased when the charge on D82 is reversed but not when the negative charge is reversed at both positions D82 and D84 (D82R/D84R). However, despite the increased affinity of D82R for heparin, the cell proliferation activity of the D82R variant is observed to be reduced compared to the wild type hFGF1. The results of this study clearly demonstrate that heparin binding affinity of hFGF1 is not strongly correlated to its cell proliferation activity.}, journal={Biochemistry and Biophysics Reports}, publisher={Elsevier BV}, author={Davis, Julie Eberle and Gundampati, Ravi Kumar and Jayanthi, Srinivas and Anderson, Joshua and Pickhardt, Abigail and Koppolu, Bhanu prasanth and Zaharoff, David A. and Kumar, Thallapuranam Krishnaswamy Suresh}, year={2018}, month={Mar}, pages={45–57} }
 @article{davis_alghanmi_gundampati_jayanthi_fields_armstrong_weidling_shah_agrawal_koppolu_et al._2018, title={Probing the role of proline-135 on the structure, stability, and cell proliferation activity of human acidic fibroblast growth factor}, volume={654}, ISSN={["1096-0384"]}, DOI={10.1016/j.abb.2018.07.017}, abstractNote={Human acidic fibroblast growth factor 1 (hFGF1) is a protein intricately involved in cell growth and tissue repair. In this study, we investigate the effect(s) of understanding the role of a conserved proline (P135), located in the heparin binding pocket, on the structure, stability, heparin binding affinity, and cell proliferation activity of hFGF1. Substitution of proline-135 with a positively charged lysine (P135K) resulted in partial destabilization of the protein; however, the overall structural integrity of the protein was maintained upon substitution of proline-135 with either a negative charge (P135E) or a polar amino acid (P135Q). Interestingly, upon heparin binding, an increase in thermal stability equivalent to that of wt-hFGF1 was observed when P135 was replaced with a positive (P135K) or a negative charge (P135E), or with a polar amino acid (P135Q). Surprisingly, introduction of negative charge in the heparin-binding pocket at position 135 (P135E) increased hFGF1's affinity for heparin by 3-fold, while the P135K mutation, did not alter the heparin-binding affinity. However, the enhanced heparin-binding affinity of mutant P135E did not translate to an increase in cell proliferation activity. Interestingly, the P135K and P135E double mutations, P135K/R136E and P135/R136E, reduced the heparin binding affinity by ∼3-fold. Furthermore, the cell proliferation activity was increased when the charge reversal mutation R136E was paired with both P135E (P135E/R136E) and P135K (P135K/R136E). Overall, the results of this study suggest that while heparin is useful for stabilizing hFGF1 on the cell surface, this interaction is not mandatory for activation of the FGF receptor.}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, author={Davis, Julie Eberle and Alghanmi, Arwa and Gundampati, Ravi Kumar and Jayanthi, Srinivas and Fields, Ellen and Armstrong, Monica and Weidling, Vanessa and Shah, Varun and Agrawal, Shilpi and Koppolu, Bhanu Prasanth and et al.}, year={2018}, month={Sep}, pages={115–125} }
 @article{ravindranathan_nguyen_kurtz_frazier_smith_koppolu_rajaram_zaharoff_2018, title={Tumor-derived granulocyte colony-stimulating factor diminishes efficacy of breast tumor cell vaccines}, volume={20}, ISSN={["1465-542X"]}, DOI={10.1186/s13058-018-1054-3}, abstractNote={Although metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness.The relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed.Mice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity.Breast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential.}, journal={BREAST CANCER RESEARCH}, author={Ravindranathan, Sruthi and Nguyen, Khue G. and Kurtz, Samantha L. and Frazier, Haven N. and Smith, Sean G. and Koppolu, Bhanu Prasanth and Rajaram, Narasimhan and Zaharoff, David A.}, year={2018}, month={Oct} }
 @article{smith_baltz_koppolu_ravindranathan_nguyen_zaharoff_2017, title={Immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer}, volume={6}, ISSN={["2162-402X"]}, DOI={10.1080/2162402x.2016.1259050}, abstractNote={ABSTRACTThere is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each t...}, number={1}, journal={ONCOIMMUNOLOGY}, author={Smith, Sean G. and Baltz, John L. and Koppolu, Bhanu Prasanth and Ravindranathan, Sruthi and Nguyen, Khue and Zaharoff, David A.}, year={2017} }
 @article{jayanthi_koppolu_nguyen_smith_felber_kumar_zaharoff_2017, title={Modulation of Interleukin-12 activity in the presence of heparin}, volume={7}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-017-05382-1}, abstractNote={Abstract Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold. Conversely, other GAGs did not demonstrate significant binding, nor did their addition affect hIL-12 bioactivity. Biophysical studies demonstrated that heparin induced only minor conformational changes while size-exclusion chromatography and small angle X-ray scattering studies indicated that heparin induced dimerization of hIL-12. Heparin modestly protected hIL-12 from proteolytic degradation, however, this was not a likely mechanism for increased cytokine activity in vitro . Flow cytometry studies revealed that heparin increased the amount of hIL-12 bound to cell surfaces. Heparin also facilitated hIL-12 binding and signaling in cells in which both hIL-12 receptor subunits were functionally deleted. Results of this study demonstrate a new role for heparin in modulating the biological activity of IL-12.}, journal={SCIENTIFIC REPORTS}, author={Jayanthi, Srinivas and Koppolu, Bhanu Prasanth and Nguyen, Khue G. and Smith, Sean G. and Felber, Barbara K. and Kumar, Thallapuranam Krishnaswamy Suresh and Zaharoff, David A.}, year={2017}, month={Jul} }
 @article{zaharoff_jewell_2017, title={Strategic Directions in Immunoresponsive Biomaterials in Tissue Engineering INTRODUCTION}, volume={23}, ISSN={["1937-335X"]}, DOI={10.1089/ten.tea.2017.0395}, number={19-20}, journal={TISSUE ENGINEERING PART A}, author={Zaharoff, David A. and Jewell, Christopher M.}, year={2017}, month={Oct}, pages={1042–1043} }
 @article{koppolu_smith_ravindranathan_jayanthi_suresh kumar_zaharoff_2015, title={Corrigendum to ‘Controlling chitosan-based encapsulation for protein and vaccine delivery’ [Biomaterials 35 (2014) 4382–4389]}, volume={73}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/J.BIOMATERIALS.2015.09.029}, DOI={10.1016/J.BIOMATERIALS.2015.09.029}, journal={Biomaterials}, publisher={Elsevier BV}, author={Koppolu, Bhanu prasanth and Smith, Sean G. and Ravindranathan, Sruthi and Jayanthi, Srinivas and Suresh Kumar, Thallapuranam K. and Zaharoff, David A.}, year={2015}, month={Dec}, pages={308} }
 @article{smith_koppolu_ravindranathan_kurtz_yang_katz_zaharoff_2015, title={Intravesical chitosan/interleukin-12 immunotherapy induces tumor-specific systemic immunity against murine bladder cancer}, volume={64}, ISSN={0340-7004 1432-0851}, url={http://dx.doi.org/10.1007/S00262-015-1672-X}, DOI={10.1007/S00262-015-1672-X}, abstractNote={Bladder cancer is a highly recurrent disease in need of novel, durable treatment strategies. This study assessed the ability of an intravesical immunotherapy composed of a coformulation of the biopolymer chitosan with interleukin-12 (CS/IL-12) to induce systemic adaptive tumor-specific immunity. Intravesical CS/IL-12 immunotherapy was used to treat established orthotopic MB49 and MBT-2 bladder tumors. All mice receiving intravesical CS/IL-12 immunotherapy experienced high cure rates of orthotopic disease. To investigate the durability and extent of the resultant adaptive immune response, cured mice were rechallenged both locally (intravesically) and distally. Cured mice rejected 100 % of intravesical tumor rechallenges and 50–100 % of distant subcutaneous rechallenges in a tumor-specific manner. The ability of splenocytes from cured mice to lyse targets in a tumor-specific manner was assessed in vitro, revealing that lytic activity of splenocytes from cured mice was robust and tumor specific. Protective immunity was durable, lasting for at least 18 months after immunotherapy. In an advanced bladder cancer model, intravesical CS/IL-12 immunotherapy controlled simultaneous orthotopic and subcutaneous tumors in 70 % of treated mice. Intravesical CS/IL-12 immunotherapy creates a robust and durable tumor-specific adaptive immune response against bladder cancer. The specificity, durability, and potential of this therapy to treat both superficial and advanced disease are deserving of consideration for clinical translation.}, number={6}, journal={Cancer Immunology, Immunotherapy}, publisher={Springer Science and Business Media LLC}, author={Smith, Sean G. and Koppolu, Bhanu prasanth and Ravindranathan, Sruthi and Kurtz, Samantha L. and Yang, Lirong and Katz, Matthew D. and Zaharoff, David A.}, year={2015}, month={Mar}, pages={689–696} }
 @article{koppolu_smith_ravindranathan_jayanthi_suresh kumar_zaharoff_2014, title={Controlling chitosan-based encapsulation for protein and vaccine delivery}, volume={35}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/J.BIOMATERIALS.2014.01.078}, DOI={10.1016/J.BIOMATERIALS.2014.01.078}, abstractNote={Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation-coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, were inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters.}, number={14}, journal={Biomaterials}, publisher={Elsevier BV}, author={Koppolu, Bhanu prasanth and Smith, Sean G. and Ravindranathan, Sruthi and Jayanthi, Srinivas and Suresh Kumar, Thallapuranam K. and Zaharoff, David A.}, year={2014}, month={May}, pages={4382–4389} }
 @article{jayanthi_koppolu_smith_jalah_bear_rosati_pavlakis_felber_zaharoff_kumar_2014, title={Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag}, volume={102}, ISSN={1046-5928}, url={http://dx.doi.org/10.1016/J.PEP.2014.07.002}, DOI={10.1016/J.PEP.2014.07.002}, abstractNote={Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ∼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.}, journal={Protein Expression and Purification}, publisher={Elsevier BV}, author={Jayanthi, Srinivas and Koppolu, Bhanu prasanth and Smith, Sean G. and Jalah, Rashmi and Bear, Jenifer and Rosati, Margherita and Pavlakis, George N. and Felber, Barbara K. and Zaharoff, David A. and Kumar, Thallapuranam Krishnaswamy Suresh}, year={2014}, month={Oct}, pages={76–84} }
 @article{smith_yang_vo_zaharoff_2013, title={583 INTRAVESICAL IMMUNOTHERAPY WITH CHITOSAN AND INTERLEUKIN-12 INDUCES SYSTEMIC TUMOR-SPECIFIC IMMUNITY}, volume={189}, ISSN={0022-5347 1527-3792}, url={http://dx.doi.org/10.1016/J.JURO.2013.02.1979}, DOI={10.1016/J.JURO.2013.02.1979}, number={4S}, journal={Journal of Urology}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Smith, Sean and Yang, Lirong and Vo, Jimmy and Zaharoff, David}, year={2013}, month={Apr} }
 @article{kim_galanzha_zaharoff_griffin_zharov_2013, title={Nanotheranostics of Circulating Tumor Cells, Infections and Other Pathological Features in Vivo}, volume={10}, ISSN={1543-8384 1543-8392}, url={http://dx.doi.org/10.1021/mp300577s}, DOI={10.1021/mp300577s}, abstractNote={Many life-threatening diseases are disseminated through biological fluids, such as blood, lymph, and cerebrospinal fluid. The migration of tumor cells through the vascular circulation is a mandatory step in metastasis, which is responsible for ∼90% of cancer-associated mortality. Circulating pathogenic bacteria, viruses, or blood clots lead to other serious conditions including bacteremia, sepsis, viremia, infarction, and stroke. Therefore, technologies capable of detecting circulating tumor cells (CTCs), circulating bacterial cells (CBCs), circulating endothelial cells (CECs), circulating blood clots, cancer biomarkers such as microparticles and exosomes, which contain important microRNA signatures, and other abnormal features such as malaria parasites in biological fluids may facilitate early diagnosis and treatment of metastatic cancers, infections, and adverse cardiovascular events. Unfortunately, even in a disease setting, circulating abnormal cells are rare events that are easily obscured by the overwhelming background material in whole blood. Existing detection methods mostly rely on ex vivo analyses of limited volumes (a few milliliters) of blood samples. These small volumes limit the probability of detecting CTCs, CECs, CBCs and other rare phenomena. In vivo detection platforms capable of continuously monitoring the entire blood volume may substantially increase the probability of detecting circulating abnormal cells and, in particular, increase the opportunity to identify exceedingly rare and potentially dangerous subsets of these cells, such as circulating cancer stem cells (CCSCs). In addition, in vivo detection technologies capable of destroying and/or capturing circulating abnormal cells may inhibit disease progression. This review focuses on novel therapeutic and diagnostic (theranostic) platforms integrating in vivo real-time early diagnosis and nano-bubble based targeted therapy of CTCs, CECs, CBCs and other abnormal objects in circulation. This critical review particularly focuses on nanotechnology-based theranostic (nanotheranostic) approaches, especially in vivo photoacoustic (PA) and photothermal (PT) nanotheranostic platforms. We emphasize an urgent need for in vivo platforms composed of multifunctional contrast nanoagents, which utilize diverse modalities to realize a breakthrough for early detection and treatment of harmful diseases disseminated through the circulation.}, number={3}, journal={Molecular Pharmaceutics}, publisher={American Chemical Society (ACS)}, author={Kim, Jin-Woo and Galanzha, Ekaterina I. and Zaharoff, David A. and Griffin, Robert J. and Zharov, Vladimir P.}, year={2013}, month={Feb}, pages={813–830} }
 @article{yang_zaharoff_2013, title={Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity}, volume={34}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/J.BIOMATERIALS.2013.02.031}, DOI={10.1016/J.BIOMATERIALS.2013.02.031}, abstractNote={Local delivery systems that provide sustained, high concentrations of antitumor cytokines in the tumor microenvironment while minimizing systemic dissemination are needed to realize the potential of cytokine-based immunotherapies. Recently, co-formulations of cytokines with chitosan solutions have been shown to increase local cytokine retention and bioactivity. In particular, intratumoral (i.t.) injections of chitosan/IL-12 can eliminate established tumors and generate tumor-specific immune responses. In the present study, we explored the mechanisms by which chitosan potentiated IL-12's antitumor activity. The location of chitosan/IL-12 injection was found to be critical for optimal cytokine delivery. I.t. injections eliminated 9 of 10 MC38 adenocarcinomas while contralateral and peritumoral injections delayed tumor growth but could not eliminate tumors. Microdosing studies demonstrated that IL-12 depots, simulated through daily i.t. injections with IL-12 alone, were not as effective as weekly i.t. chitosan/IL-12. 50–75% of mice receiving daily IL-12 microdoses and 87.5% of mice receiving weekly chitosan/IL-12 were cured of MC38 tumors. Chitosan was found to increase IL-12-mediated leukocytic expansion in tumors and tumor-draining lymph nodes (TDLNs) by 40 and 100%, respectively. Immunophenotyping studies demonstrated that chitosan co-formulation amplified IL-12-induced increases in important effector populations, such as CD8+IFN-γ+ and NKT cells, in tumors and dendritic cell populations in TDLNs. Remarkable increases in Gr-1+CD11b+ tumor infiltrates were also observed in mice receiving chitosan or chitosan/IL-12. This population does not appear be suppressive and may facilitate the local antitumor response. Presented data suggest that chitosan-mediated depot formation and enhanced local cytokine retention is significantly, but not entirely, responsible for increased cytokine bioactivity.}, number={15}, journal={Biomaterials}, publisher={Elsevier BV}, author={Yang, Lirong and Zaharoff, David A.}, year={2013}, month={May}, pages={3828–3836} }
 @article{koppolu_zaharoff_2013, title={The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells}, volume={34}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/j.biomaterials.2012.11.066}, DOI={10.1016/j.biomaterials.2012.11.066}, abstractNote={Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. In this study, the influence of chitosan encapsulation on antigen uptake, activation and presentation by antigen presenting cells (APCs) is explored. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and ovalbumin (OVA) were used as model protein antigens and encapsulated in chitosan particles via precipitation-coacervation at loading efficiencies >89%. Formulation conditions were manipulated to create antigen-encapsulated chitosan particles (AgCPs) with discrete nominal sizes (300 nm, 1 μm, and 3 μm). Uptake of AgCPs by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs.}, number={9}, journal={Biomaterials}, publisher={Elsevier BV}, author={Koppolu, Bhanuprasanth and Zaharoff, David A.}, year={2013}, month={Mar}, pages={2359–2369} }
 @article{heffernan_zaharoff_fallon_schlom_greiner_2011, title={In vivo efficacy of a chitosan/IL-12 adjuvant system for protein-based vaccines}, volume={32}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/j.biomaterials.2010.09.058}, DOI={10.1016/j.biomaterials.2010.09.058}, abstractNote={Vaccines based on recombinant proteins require adjuvant systems in order to generate Th1-type immune responses. We have developed a vaccine adjuvant system using a viscous chitosan solution and interleukin (IL)-12, a Th1-inducing cytokine. The chitosan solution is designed to create a depot of antigen and IL-12 at a subcutaneous injection site. We measured the in vivo immune response of a vaccine containing 0.25, 1, or 4 μg murine IL-12 and 75 μg ovalbumin (OVA), formulated in a 1.5% chitosan glutamate solution. The chitosan/IL-12/OVA vaccine, in comparison to chitosan/OVA, IL-12/OVA, or OVA alone, elicited greater antigen-specific CD4+ and CD8+ T-cell responses, as determined by CD4+ splenocyte proliferation, Th1 cytokine release, CD8+ T-cell interferon-γ release, and MHC class I peptide pentamer staining. The combination of chitosan and IL-12 also enhanced IgG2a and IgG2b antibody responses to OVA. Co-formulation of chitosan and IL-12 thus promoted the generation of a Th1 immune response to a model protein vaccine.}, number={3}, journal={Biomaterials}, publisher={Elsevier BV}, author={Heffernan, Michael J. and Zaharoff, David A. and Fallon, Jonathan K. and Schlom, Jeffrey and Greiner, John W.}, year={2011}, month={Jan}, pages={926–932} }
 @article{rogers_zaharoff_hance_perkins_hursting_schlom_greiner_2008, title={Exercise enhances vaccine-induced antigen-specific T cell responses}, volume={26}, ISSN={0264-410X}, url={http://dx.doi.org/10.1016/j.vaccine.2008.07.081}, DOI={10.1016/j.vaccine.2008.07.081}, abstractNote={Regular moderate exercise has been proposed to enhance immune function, but its effects on immunity and their consequences have not been well studied. Mice without (AL) or with access (AL + EX) to voluntary running wheels were vaccinated with a model antigen (ovalbumin (OVA)) via intranasal or subcutaneous routes to target the mucosal and systemic immune compartments, respectively. EX enhanced OVA-specific CD4+ T cell cytokine production and proliferation in all lymphoid organs examined without changes in cell distribution in any organ. These results suggest that coupling moderate exercise with vaccination may enhance vaccine efficacy for the prevention and/or therapy of numerous diseases.}, number={42}, journal={Vaccine}, publisher={Elsevier BV}, author={Rogers, Connie J. and Zaharoff, David A. and Hance, Kenneth W. and Perkins, Susan N. and Hursting, Stephen D. and Schlom, Jeffrey and Greiner, John W.}, year={2008}, month={Oct}, pages={5407–5415} }
 @article{zaharoff_rogers_hance_schlom_greiner_2007, title={Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination}, volume={25}, ISSN={0264-410X}, url={http://dx.doi.org/10.1016/j.vaccine.2006.11.034}, DOI={10.1016/j.vaccine.2006.11.034}, abstractNote={The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosan's biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freund's adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 9% of a protein antigen, when delivered in saline, remained at the injection site after 8 h. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery.}, number={11}, journal={Vaccine}, publisher={Elsevier BV}, author={Zaharoff, David A. and Rogers, Connie J. and Hance, Kenneth W. and Schlom, Jeffrey and Greiner, John W.}, year={2007}, month={Mar}, pages={2085–2094} }
 @article{zaharoff_rogers_hance_schlom_greiner_2007, title={Chitosan solution enhances the immunoadjuvant properties of GM-CSF}, volume={25}, ISSN={0264-410X}, url={http://dx.doi.org/10.1016/j.vaccine.2007.10.037}, DOI={10.1016/j.vaccine.2007.10.037}, abstractNote={Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12–24 h. Furthermore, a single s.c. injection of 20 μg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 μg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 μg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 μg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4+ proliferation, peptide-specific CD8+ pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF.}, number={52}, journal={Vaccine}, publisher={Elsevier BV}, author={Zaharoff, David A. and Rogers, Connie J. and Hance, Kenneth W. and Schlom, Jeffrey and Greiner, John W.}, year={2007}, month={Dec}, pages={8673–8686} }
 @article{henshaw_zaharoff_mossop_yuan_2007, title={Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo}, volume={71}, ISSN={1567-5394}, url={http://dx.doi.org/10.1016/j.bioelechem.2007.07.005}, DOI={10.1016/j.bioelechem.2007.07.005}, abstractNote={Local pulsed electric field application is a method for improving non-viral gene delivery. Mechanisms of the improvement include electroporation and electrophoresis. To understand how electrophoresis affects pDNA delivery in vivo, we quantified the magnitude of electric field-induced interstitial transport of pDNA in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chambers. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 microm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of ten electric pulses was ineffective for macroscopic delivery of genes in tumors. However, the induced transport was more efficient than passive diffusion.}, number={2}, journal={Bioelectrochemistry}, publisher={Elsevier BV}, author={Henshaw, Joshua W. and Zaharoff, David A. and Mossop, Brian J. and Yuan, Fan}, year={2007}, month={Nov}, pages={233–242} }
 @article{henshaw_zaharoff_mossop_yuan_2006, title={A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport of genes}, volume={69}, ISSN={1567-5394}, url={http://dx.doi.org/10.1016/j.bioelechem.2006.03.006}, DOI={10.1016/j.bioelechem.2006.03.006}, abstractNote={The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.}, number={2}, journal={Bioelectrochemistry}, publisher={Elsevier BV}, author={Henshaw, Joshua W. and Zaharoff, David A. and Mossop, Brian J. and Yuan, Fan}, year={2006}, month={Oct}, pages={248–253} }
 @article{mossop_barr_henshaw_zaharoff_yuan_2006, title={Electric Fields in Tumors Exposed to External Voltage Sources: Implication for Electric Field-Mediated Drug and Gene Delivery}, volume={34}, ISSN={0090-6964 1573-9686}, url={http://dx.doi.org/10.1007/S10439-006-9151-3}, DOI={10.1007/S10439-006-9151-3}, number={10}, journal={Annals of Biomedical Engineering}, publisher={Springer Science and Business Media LLC}, author={Mossop, Brian J. and Barr, Roger C. and Henshaw, Joshua W. and Zaharoff, David A. and Yuan, Fan}, year={2006}, month={Aug}, pages={1564–1572} }
 @article{henshaw_zaharoff_mossop_yuan_2006, title={Electric field-mediated enhancement of in vivo transport of plasmid DNA in tumor interstitium}, volume={39}, ISSN={0021-9290}, url={http://dx.doi.org/10.1016/S0021-9290(06)84518-5}, DOI={10.1016/S0021-9290(06)84518-5}, journal={Journal of Biomechanics}, publisher={Elsevier BV}, author={Henshaw, J.W. and Zaharoff, D.A. and Mossop, B.J. and Yuan, F.}, year={2006}, month={Jan}, pages={S377} }
 @article{mcguire_zaharoff_yuan_2006, title={Nonlinear Dependence of Hydraulic Conductivity on Tissue Deformation During Intratumoral Infusion}, volume={34}, ISSN={0090-6964 1573-9686}, url={http://dx.doi.org/10.1007/S10439-006-9136-2}, DOI={10.1007/S10439-006-9136-2}, number={7}, journal={Annals of Biomedical Engineering}, publisher={Springer Science and Business Media LLC}, author={McGuire, Sarah and Zaharoff, David and Yuan, Fan}, year={2006}, month={Jun}, pages={1173–1181} }
 @article{zaharoff_yuan_2004, title={Effects of pulse strength and pulse duration on in vitro DNA electromobility}, volume={62}, ISSN={1567-5394}, url={http://dx.doi.org/10.1016/j.bioelechem.2003.10.011}, DOI={10.1016/j.bioelechem.2003.10.011}, abstractNote={Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.}, number={1}, journal={Bioelectrochemistry}, publisher={Elsevier BV}, author={Zaharoff, David A and Yuan, Fan}, year={2004}, month={Apr}, pages={37–45} }