1. Application of Polyglycerol Coating to Plasmid DNA Lipoplex for the Evasion of the Accelerated Blood Clearance Phenomenon in Nucleic Acid Delivery 
Amr S. Abu Lila, Yumi Uehara, Tatsuhiro Ishida, Hiroshi Kiwada 
Journal of Pharmaceutical Sciences 
Volume 103, Issue 2, Pages (February 2014)
DOI: /jps.23823
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

2. Figure 1
Anti-polymer IgM production. Mice were intravenously administered either PEG-coated CL (PEG–CL), PEG-coated pDNA–lipoplex (PEG–DCL), PG-coated CL (PG–CL), or PG-coated pDNA–lipoplex (PG–DCL) at the indicated doses. Five days later, blood was withdrawn from each treated mouse and serum was collected. The serum collected from naive mice, receiving saline instead of lipoplex, was used as the control. Anti-polymer IgM produced in response to (a) CLs or (b) pDNA–lipoplexes was detected with ELISA as described in the Materials and Methods. Each value represents the mean ± SD (n = 4). *p < 0.05, **p < 0.01.
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

3. Figure 2
The effect of a prior dose on the tumor accumulation of a fluorescence-labeled test dose of either PEG-coated pDNA–lipoplex (PEGDCL) or PG-coated pDNA–lipoplex (PG–DCL). C26 tumor-bearing mice received a single administration (a) or two administrations within a 5-day interval (b) of either pDNA–lipoplex (DCL), PEG-coated pDNA–lipoplex (PEG–DCL), or PG-coated pDNA–lipoplex (PG–DCL) (0.4 :MPL/mouse and 10 :g pDNA/mouse). The first dose was given on day 7 after tumor inoculation. To visualize the tumor accumulation, the dose was labeled with fluorescence (DiD). At 8, 12, and 24 h after the last injection, in vivo optical images were recorded. All fluorescence images were acquired using a 1/8 exposure time. A typical image from three independent experiments is expressed. (c) Mean fluorescence intensity per tumor tissue collected at 24 h after administration. Data are reported as the mean ± SD (n = 3). ***p <
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

4. Figure 3
Detection of inflammatory cytokines. At 4 h after administration of either CL, PEG-coated CL (PEG–CL), PG-coated CL (PG– CL), pDNA–lipoplex (DCL), PEG-coated pDNA–lipoplex (PEG–DCL), or PG-coated pDNA-lipoplex (PG–DCL) (0.4 :M PL/mouse and/or 3 :g pDNA/mouse), blood was withdrawn and inflammatory cytokines (IL- 6, INF-(, and TNF-") in the serum was determined with ELISA. The serum collected from na¨ıve mice was used as the control. Each value represents the mean ± SD (n = 4).
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

5. Figure 4
Amount of incorporated BrdU into splenic B-cells. Suspensionof splenic B-cells was prepared as described in the Materials and Methods and incubated with either CL, PEG-coated CL (PEG–CL), PG-coated CL (PG–CL), pDNA–lipoplex (DCL), PEG-coated pDNA– lipoplex (PEG-DCL), or PG-coated pDNA-lipoplex (PG–DCL) (0.004 :M PL/well and/or 0.1 :g pDNA/well) for 48 h. After incubation, the cells were treated with BrdU solution and then analyzed by in vitro BrdU incorporation assay. Each value represents the mean ± SD (n = 4). ***p < 0.005
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

6. Figure 5
Cellular binding/uptake of PEG-coated pDNA–lipoplex (PEG–DCL), or PG-coated pDNA–lipoplex (PG–DCL) by splenic Bcells. Suspension of splenic B-cells was prepared as described in the Materials and Methods. The cells were incubated for 48 h with either Rho-labeled CLs, PEG-coated CL (PEG–CL), PG-coated CL (PG–CL), pDNA–lipoplex (DCL), PEG-coated pDNA–lipoplex (PEG–DCL), or PGcoated pDNA–lipoplex (PG–DCL). After the treatment, the cells were divided into two fractions, and then one fraction was treated with trypan blue solution to quench the Rho bound to the cellular surface. The cells were finally analyzed by flow cytometry. (a) The amount of cellular surface-bound Rho-labeled liposomes/lipoplexes as calculated by subtracting the amount of trypan blue-treated cells from non trypan blue-treated cells. (b) The amount of cellular internalized Rho-labeled liposomes/lipoplexes. Data represent the mean of three independent experiments ± SD. ***p <
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

7. Figure 6
Cartoon depicting the possible mechanism underlying the differences in anti-polymer IgM production in response to (a) PEG-coatedpDNA–lipoplex (PEG–DCL), or (b) PG-coated pDNA–lipoplex (PG–DCL).
Journal of Pharmaceutical Sciences  , DOI: ( /jps.23823)
Copyright © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions