Magnetic nanoparticles as a new approach to improve the efficacy of gene therapy against differentiated human uterine fibroid cells and tumor-initiating stem cells  Shahinaz Mahmood Shalaby, M.D., M.Sc., Mostafa K. Khater, Pharm.D., M.Sc., Aymara Mas Perucho, Ph.D., Sara A. Mohamed, M.D., M.Sc., Inas Helwa, Ph.D., Archana Laknaur, M.Sc., Iryna Lebedyeva, Ph.D., Yutao Liu, Ph.D., Michael P. Diamond, M.D., Ayman A. Al- Hendy, M.D., Ph.D.  Fertility and Sterility  Volume 105, Issue 6, Pages 1638-1648.e8 (June 2016) DOI: 10.1016/j.fertnstert.2016.03.001 Copyright © 2016 Terms and Conditions

Figure 1 Transmission electron micrographs of adenovirus Ad-RGD TK, magnetic nanoparticles (MNP), and MNP Ad-RGD-TK. Size characterization of adenovirus and MNPs by transmission electron microscopy. All samples were negatively stained with phosphotungstic acid solution and observed under transmission electron microscope at different magnifications. (A) Adenovirus. (B) MNPs. (C, D) Adenovirus conjugated to MNPs. Bar size is 50 nm in A and D, and 200 nm in B and C. The upper panel represents the color-enhanced version of the actual micrographs in the lower panel. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Figure 2 Controlled in vitro Ad-GFP magnetofection (incubation of adenovirus with magnetic nanoparticles [MNPs] on magnet for 20 minutes). Human fibroid tumor cells (1 × 106 in each of six wells) incubated for 20 minutes with MNP-conjugated adenovirus or regular virus with exposure to external magnetic wells. (A) MNPs boosted Ad-GFP transfection in human fibroid tumor cells by fluorescent microscopy. (B) Quantitative analysis to compare the transfection efficiency of Ad-GFP with or without MNPs. We noted a statistically significant increase in the transfection rate after conjugating Ad-GFP with MNPs at both multiplicities of infection (MOI) of 5 (*P<0.05) and 10 (*P<.005). Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Figure 3 Transfection of fibroid stem cells by Ad-LacZ X Gal staining of human fibroid stem cells after transfection with adenovirus with Ad-LacZ reporter gene at multiplicity of infection (MOI) of 10 (A) and 25 (B). (C, D) X-gal is an analogue of lactose and thus hydrolyzed by the B-galactosidase enzyme giving intensely blue products (arrows). DAPI (4′,6-diamidino-2-phenylindole), a fluorescent stain that binds strongly to A-T rich regions in DNA, is used as a general nuclear stain to demonstrate total numbers. (E) Numerical representation of results observed in A and B and calculated as described in Materials and Methods. Results represent three independent experiments. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Figure 4 (A) In vitro Ad-GFP magnetofection: (incubation of Ad-GFP with MNPs for 20 minutes) of fibroid stem cells (1 × 103/cm2 in each of six wells), followed by 20 minutes of exposure to the magnetic field. (B) We observed that magnetic nanoparticles (MNPs) significantly enhanced the transfection efficiency at the three different multiplicities of infection (MOI) *P<0.005; **P<0.001. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 1 Ad-RGD luciferase transfection against fibroid tumor cells with or without magnetic nanoparticles (MNPs). This experiment shows that MNPs statistically significantly enhanced the transfection ability of the genetically modified Ad-RGD against fibroid tumor cells (P<.005), with the bioluminescence at both multiplicities of infection (MOIs) of 5 and 10, respectively (*P<.005). Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 2 Magnetic nanoparticles (MNPs) enhanced the ability of Ad-RGD-TK/ganciclovir (GCV) to suppress the proliferation of human fibroid tumor cells. Fibroid tumor cells were transfected with three different multiplicities of infection (MOI 25, 50, and 75) of Ad-RGD-TK followed by GCV therapy at 10 μg/mL for 5 days. An MTT assay was used to evaluate the percentage of cell proliferation. The MNPs statistically significantly boosted the suppressive effect of Ad-RGD-TK/GCB on cellular proliferation (*P<.0001) at three different multiplicities of infection (MOIs). Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 3 Expression analysis by Western blot in fibroid tumor cells. Magnetic nanoparticle (MNP)-conjugated Ad-RGD-TK (A) statistically significantly decreased the expression of proliferation-related proliferating cell nuclear antigen (PCNA) and (B) increased the expression of apoptosis-related BAX in human uterine fibroid tumor cells compared with Ad-RGD-TK at 25 plaque-forming units (PFU)/cell and ganciclovir (GCV) at 10 μg/mL for 48 hours. Whole-cell lysates were analyzed by Western blot as described in Materials and Methods. (C) The intensity of each protein signal was quantified and normalized with corresponding β-actin. Data are indicative of two independent experiments. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 4 Suicidal gene therapy strategy in fibroid stem cells: Fibroid stem cells were transfected with three different multiplicities of infection (MOIs) of 25, 50, 75 of Ad-RGD-TK followed by ganciclovir therapy at 10 μg/mL for 5 days. An MTT assay was used to evaluate the percentage of cell proliferation. A statistically significant decrease in the cell proliferation rate after exposure to both MOIs of 50 (*P<.005) and 75 (**P<.0001) as compared with untreated cells was detected. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 5 Ad-RGD-TK/ganciclovir (GCV) bystander killing effect on human fibroid stem cells. The cell mixture of wild-type (WT) human fibroid stem cells and Ad-RGD-TK transfected fibroid stem cells (TK) of different ratios of transfected cells followed by GCV therapy at 10 μg/mL for 5 days. An MTT assay was performed, and the mean optical density was recorded. Statistically significantly lower proliferation rates were observed with respect to untransfected WT cells when transfected cell ratios were between 20 and 70% (**P<.001; ***P<.0001) of total cocultured cells. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 6 Evaluation of human fibroid stem cell viability after exposure to regular unconjugated or magnetic nanoparticles (MNPs) conjugated Ad-RGD-TK/ganciclovir (GCV) by MTT assay. A single multiplicity of infection (MOI) of 50 was used. Observations included [1] a statistically significant decrease in cell proliferation by 55% ± 12.3% after exposure to unconjugated Ad-RGD-TK/GCV (*P<.005), [2] a statistically significant decrease in cell proliferation by about 78% ± 16.75% when MNPs were conjugated to Ad-RGD-TK (**P<.001) as compared with unexposed cells, and [3] a statistically significant difference between MNP Ad-RGD-TK and regular Ad-RGD-TK (ˆP<.01). Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 7 Magnetic nanoparticles (MNPs)-Adenomag conjugated Ad-RGD-TK (A) decreased the expression of proliferation-related proliferating cell nuclear antigen (PCNA) and (B) increased the expression of apoptosis-related BCL2-associated X protein (BAX) in human fibroid stem cells. Human fibroid stem cells were transduced with Ad-RGD-TK and Adenomag conjugated Ad-RGD-TK at 25 plaque-forming units (PFU)/cell and ganciclovir (GCV) at 10 μg/mL for 48 hours. Lysates were analyzed by Western blot. The intensity of each protein signal was quantified and normalized with corresponding β-actin (C). Data are indicative of two different experiments. Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions

Supplemental Figure 8 In vitro assessment of caspase-3 activity comparing magnetofection versus regular transduction of Ad-RGD-TK/ganciclovir (GCV). An increase was observed in caspase-3 enzyme activity by threefold and sixfold in regular transfection and magnetofection groups, respectively (*P<.05). Fertility and Sterility 2016 105, 1638-1648.e8DOI: (10.1016/j.fertnstert.2016.03.001) Copyright © 2016 Terms and Conditions