1. Volume 25, Issue 7, Pages 1606-1615 (July 2017)
Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment 
Xiaohui Zhang, Austin Bohner, Sai Bhuvanagiri, Hironori Uehara, Arun Kumar Upadhyay, Lyska L. Emerson, Sailaja Bondalapati, Santosh Kumar Muddana, Daniel Fang, Miaoling Li, Zoya Sandhu, Alya Hussain, Lara S. Carroll, Michelle Tiem, Bonnie Archer, Uday Kompella, Rajkumar Patil, Balamurali K. Ambati 
Molecular Therapy 
Volume 25, Issue 7, Pages (July 2017)
DOI: /j.ymthe
Copyright © Terms and Conditions

2. Figure 1
Flt23k Overexpression Reduced Endogenous VEGF-A Expression in HeLa Cells
(A) As transfection control, pMaxGFP (0, 0.1, 0.5, 1.0, 2.5, and 5.0 μg) was transfected to HeLa cells with nucleofection. GFP expression increased with increasing plasmid concentration. (B) Western blot analysis demonstrates that Flt23k decreased endogenous VEGF expression, showing a dose-dependent trend.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

3. Figure 2 Flt23k.NP Inhibited HAEC Proliferation
Nanoparticle (NP) was dissolved in DPBS at 20 mg/mL. Then, 2 μL (40 μg NP) or 10 μL (200 μg NP) of NP solution were transfected to 2.0 × 106 human aortic endothelial cells (HAECs) with nucleofection. The next day, 5,000 cells were replated to a 24-well plate in 500 μL EGM2 medium. After day 3 and day 5, the cells were fixed with 4% PFA and stained with crystal violet. Crystal violet was eluted with 200 μL 10% acetic acid and measured at OD600. *p < 0.05, ***p < 0.001, ****p < 
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

4. Figure 3
RGD Functionalized Nanoparticles Reached the Highest Effective Dose above 30 μg RGD.Flt23k.NPs
(A) A pFlt23k-loaded nanoparticle dose from 0 to 60 μg was administrated in laser-induced CNV mice. CNV lesions were measured using FA and OCT. The reductions in CNV lesion sites were measured at 1, 2, and 3 weeks post-treatment with Flt23k plasmid. The left side of each panel shows the FA image, whereas the right side shows the IR image. (B) OCT images were used to calculate the CNV volume at 2 weeks post-treatment. ANOVA analysis was used to measure the significant difference in CNV volume regression at different doses of Flt23k plasmid. *p < 0.05, **p < 0.01, ****p < (C) Representative OCT images for laser CNV treated with different doses of RGD.Flt23k.NP.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

5. Figure 4
No Systemic Toxicity Was Detected after Treatment with RGD Functionalized Nanoparticles
(A) Mouse body weight with a single dose of 30 μg pFlt23k-loaded NP was recorded before treatment and at 1 month post-treatment and compared to that of control mice. (B) Mouse body weight for repeat doses of 30 μg pFlt23k and control mice was recorded before treatment and at 1, 2, and 3 months post-treatment. n = 10.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

6. Figure 5
No Systemic Toxicity Was Detected with RGD Functionalized Nanoparticles
(A) For single-dose treatment, organs (brain, heart, lung, liver, kidney, and spleen) were harvested and weights were measured. (B) For repeat-dose treatments, organs (brain, heart, lung, liver, kidney, and spleen) were harvested and measured at the endpoint of 3 months.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

7. Figure 6
No Hematological Toxicity or Systemic Inflammation Were Detected after Treatment with RGD Functionalized Nanoparticles
Mouse blood was collected and hemoglobin levels were measured using the Hemoglobin Colometeris Assay Kit (Cayman Chemical). (A) Hemoglobin (Hg) levels (in grams per deciliter) of mice treated with a single dose of 30 μg pFlt23k (p = ). (B) Hg levels (in grams per deciliter) of mice treated with repeat doses of 30 μg pFlt23k (p = ). Mouse blood was collected at the endpoint, and complement component 3 (C3) levels were assayed with the Complement C3 Mouse ELISA Kit (Abcam). (C) Single dose of 30 μg pFlt23k (p = ). (D) Repeat doses of 30 μg pFlt23k (p = ).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

8. Figure 7
Systemic Toxicity Was Not Detected after Treatment with RGD Functionalized Nanoparticles
Histology for all organs was performed with paraffin block and H&E staining. The slides were evaluated by a masked, experienced pathologist. n = 10. Scale bar, 200 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions

9. Figure 8
No Ocular Toxicity Was Detected after Treatment with RGD Functionalized Nanoparticles
(A and B) ERG and retinal thickness of eyes in mice treated with a maximum dose of 60 μg RGD.Flt23k.NPs (n = 4) in comparison to eyes of control mice (n = 9) (ERG a wave, p = ; b wave, p = ). (C) H&E staining (40×) of eyes in mice treated with a maximum dose of 60 μg RGD.Flt23k.NPs versus PBS control mice. (D) Retinal thickness as determined by OCT. Scale bar, 100 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © Terms and Conditions