Volume 14, Issue 3, Pages 382-391 (September 2006) Adenoviral Gene Vector Tethering to Nanoparticle Surfaces Results in Receptor- Independent Cell Entry and Increased Transgene Expression  Michael Chorny, Ilia Fishbein, Ivan S. Alferiev, Origene Nyanguile, Richard Gaster, Robert J. Levy  Molecular Therapy  Volume 14, Issue 3, Pages 382-391 (September 2006) DOI: 10.1016/j.ymthe.2006.03.023 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Synthesis of the polyallylamine–benzophenone–pyridyldithiocarboxylate (PBPC) and polyallylamine–benzophenone–maleimidocarboxylate (PBMC) photoreactive polymers (see Methods) based upon the multifunctional derivatization of polyallylamine under the reaction conditions shown. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Transmission electron micrograph of (left) a free NP compared to (right) a NP–Ad complex formed via tethering Ad to the surface of PLA nanoparticles using D1–Ad affinity binding. Electron microscopy (FEI Tecnai G2 electron microscope, Netherlands) was performed after negative staining with 2% (w/v) uranyl acetate. The original magnification was ×50,000. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 The uptake and retention of complex-forming and control NP (D1NP and nIgGNP, respectively). The NP were incubated with Ad at a dose of 1.3 × 108 viral particles/well and added to the cells for 2 h. The NP uptake by (A) A10 cells, (B) BAEC, and (C) H5V cells was determined 24 h posttreatment as a function of the NP dose (0–3.2 μg PLA/well) and was found to be significantly dose-dependent (P < 0.05). (D) The change in the intracellular levels with time (24–72 h) was determined for NP applied at a dose of 1.6 μg PLA/well and was not statistically significant (P > 0.2). The measurements were performed fluorimetrically in live cells using λem/λex 544 nm/580 nm. Error bars indicate standard deviation. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 The levels of GFP transgene expression following NP–Ad complex administration in cultures of (A, D, G) A10 cells, (B, E, H) BAEC, and (C, F, I) H5V cells. Increasing amounts of NP were combined with GFP-encoding Ad at a dose of 1.3 × 108 viral particles/well. The top (A–C) shows merged green-fluorescent and bright-field images of respective cells treated with D1NP–Ad complexes at a NP dose of 1.6 μg PLA/well. The original magnification is ×100. The middle (D–F) shows GFP amount in cells 72 h posttreatment as a function of NP dose used to form the complexes, with D1 tethering associated with significantly greater GFP expression for all NP doses (P < 0.001). The bottom (G–I) shows GFP amount in cells treated with D1NP–Ad complexes at a NP dose of 1.6 μg PLA/well as a function of time posttreatment, demonstrating significantly greater expression at all time points (P < 0.001) in comparison to Ad applied with or without control nIgGNP. Error bars indicate standard deviation. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 The effects of recombinant knob fiber protein on the transduction and uptake of complex-forming NP in A10 cells. The cells were pretreated with knob dissolved in the medium at 5 μg/ml for 1 h prior to addition of (A) free Ad (2 × 108/well) or Ad in the presence of (B) nIgGNP or (C) D1NP (4 μg PLA/well). Knob-containing cell medium was aspirated, and the cells were washed with PBS and incubated for 2 h with the formulations. Gene expression was assayed fluorimetrically (λem and λex 485 nm and 535 nm, respectively) in live cells 2 days posttreatment with the above formulations. (D) The comparative effect of knob pretreatment on the intracellular level of NP was measured using λem/λex 544 nm/580 nm. Knob pretreatment had no significant effect on NP cellular uptake (P = 0.18). Ad immobilization on D1NP resulted in the rescue of gene expression from knob inhibition, while nIgGNP had no significant effect (P < 0.03 and P > 0.1, respectively). Error bars indicate standard deviation. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 (A) The effects of NP surface modification with D1 using surface activation with PBPC vs PBMC (see Methods and Fig. 1). Thiolated D1 reacts with the NP surface activated with PDT groups and MI groups (using PBPC and PBMC, respectively) to form a biodegradable disulfide (S–S) or a nonbiodegradable thioether (C–S) bond, respectively. The NP were incubated with Ad at a dose of 1.3 × 108 viral particles/well and added to the cells for 2 h. The effects of the bond character on (B) NP uptake and (C) GFP expression of the complexes was measured fluorimetrically using λem/λex 544 nm/580 nm and 485 nm/535nm, respectively, 24 and 72 h posttreatment, using nIgGNP as a control. (D) The time course of the gene expression mediated by the complexes formed at a NP dose of 1.6 μg PLA/well was followed for 72 h, showing no significant differences between PBMC- and PBPC-modified NP (P > 0.4). Error bars indicate standard deviation. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 7 GFP expression in A10 cells treated with NP–Ad complexes employing (A) anti-knob Ad (AKNP–Ad) vs (B) D1 (D1NP–Ad) as vector tethering agent as a function of NP and Ad dose assayed fluorimetrically 48 h posttreatment and representative fluorescence microscopic images showing GFP expression and NP–Ad uptake in A10 cells employing binding with either (C, F, I) D1 or (D, G, J) anti-knob Ad in comparison with (E, H, K) free Ad included as a control. Ad at a dose of 3.36 × 108 viral particles/well was added to the cells for 2 h with or without preincubation with the respective NP at a dose of 4.0 μg PLA/well (C–K). The first row shows GFP expression (C–E) and the second row shows respective cellular localization of the complex forming red fluorescence-labeled NP (F–H) observed 48 h posttreatment. Cell nuclei were stained with DAPI following fixation with glutaraldehyde. Note the perinuclear localization pattern and the substantially higher amount of the red fluorescence associated with D1NP–Ad vs AKNP–Ad complexes (F vs G) that correspond to a higher level of GFP expression in D1NP–Ad-treated cells (C vs D). Note the absence of red fluorescence in the cells treated with free Ad (H). The A10 cells treated with NP–Ad and the free Ad exhibited their normal morphology (I–K). The original magnification was ×200. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 8 The A10 cell growth-inhibitory effects of iNOSAd in the presence of NP modified with (A) D1 (D1NP) and (B) nonimmune IgG (nIgGNP) in comparison to the reporter gene expression mediated by respective formulations prepared with GFPAd vector (E, F). A10 growth inhibition was also studied in comparison with control formulations including NP–Ad formulated with (C) GFPAd and (D) NULLAd. The reporter gene expression and cell growth inhibition were assayed 48 h posttreatment using direct cell fluorimetry (λem/λex 485 nm/535 nm) and the Alamar blue cell growth assay (λem/λex 544 nm/580 nm), respectively. Molecular Therapy 2006 14, 382-391DOI: (10.1016/j.ymthe.2006.03.023) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions