This study developed a targeted drug delivery system using Multistage Nanovectors (MSV) conjugated with VEGFR2 antibodies to specifically target blood vessels that support tumor growth. MSV were functionalized with VEGFR2 antibodies and shown to bind endothelial cells expressing VEGFR2 in vitro, including under flow conditions mimicking blood flow. The MSV were also internalized by the endothelial cells. This targeted delivery system aims to improve cancer therapy by efficiently transporting anti-angiogenic antibodies to their VEGFR2 targets on endothelial cells, preventing blood vessel formation and limiting tumor growth. Future work will investigate this system's potential for dual-function delivery of anti-angiogenic antibodies and anti-cancer drugs to tumors.

1. VEGFR2 Specific Delivery of Multistage Nanovectors:
A nanomedicine-based approach to treat cancer by targeting blood vessel formation
This work was conducted at the Houston Methodist Hospital Research Institute,
Department of Nanomedicine, Houston, Texas

2. Introduction
Cancer Therapy
 Cancer causes the of death of over 500,000 Americans every
year.
VEGF
VEGFR2 Receptor
 A promising approach for the treatment of cancer is preventing
angiogenesis (the formation of new blood vessels) that supports
tumor growth.1
 When VEGF (Vascular Endothelial Growth Factor) binds to its
receptor (VEGFR2) on endothelial cells, a complex signaling
cascade starts, resulting in angiogenesis.
 Therapeutic antibodies against VEGF or VEGFR2 prevent
angiogenesis and are currently used for cancer treatment.2
 These treatments have limited success however, since only a
small percentage of the antibodies reach their target because of
degradation and other biological barriers.3
Angiogenesis
1. Kim, K.J., et al., 1993; 2. Ferrara, N. et al., 2004; 3. Crawford, Y. et al. 2009.

3. Nanomedicine
 Nanoparticles represent a new way of delivering
therapeutic agents to tumors and to blood vessels
around tumors.
 A next-generation nanoparticle that was produced
at our Institute is called Multistage Nanovector (MSV).1
 These MSV, with their special physical and chemical
properties, are designed to avoid biological barriers and
therefore, successfully deliver therapeutic agents to cells.2
Tasciotti , E. et al. 2008
The diagram shows intravenously injected MSV
(grey disks) that efficiently move to the
endothelial cells of the target tissue by avoiding
biological barriers and entrapment.
1. Tasciotti , E. et al. 2008; 2. Martinez, J. et al., 2013

4. Purpose
To use the unique properties of MSV, together with the
powerful targeting potential of VEGFR2, in order to
develop a specific and efficient transport system for
VEGFR2 antibodies to blood vessels.
Importance
If successful, this research will improve the delivery of
therapeutic antibodies that prevent angiogenesis and hence
tumor growth.
Diagram of VEGFR2 mediated targeting of MSV
to endothelial cells of a blood vessel at a tumor site
Approach
Preparation of MSV conjugated with anti-VEGFR2 antibodies (VEGFR2-MSV) and then the use of
these particles to target the VEGFR2 receptor of an endothelial cell line (PAEC) in vitro.

5. Methods: Overview
1.
Preparation and characterization of VEGFR2 antibody
conjugated MSV (VEGFR2-MSV)
2.
Preparation and characterization of VEGFR2
expressing endothelial cells
3.
In vitro targeting of VEGFR2 on endothelial cells by
VEGFR2-MSV
a.) Static conditions (MSV added to cells and incubated)
b.) Dynamic flow conditions (MSV continuously flowed over
the cells at 100 µL/min to mimic the force of blood flow
4.
Internalization of VEGFR2-MSV by endothelial cells

6. Methods:
1.
Detailed
Preparation and characterization of VEGFR2-MSV
Porous silicon micro-particles (3.2 µm in size, with 15 nm pores) were
modified with APTES, functionalized with SMCC, conjugated with
anti-VEGFR2 antibody and labeled with fluorescent dye (Figure 1).
Stabilities of the colloidal solutions of MSV were determined by
measuring Zeta potentials at each step of the conjugation process with
a Malvern Zetasizer instrument (Table 1).
The MSV particles were labeled with a Cy5 fluorescence dye (purple).
VEGFR2 antibodies were labeled with TRITC fluorescence dye (red).
Imaging was done with a Nikon fluorescence microscope (Figure 2).

7. Methods:
2.
Detailed (continued)
Preparation and characterization of VEGFR2 expressing endothelial cells
Porcine aortic endothelial cells (PAEC) were transfected with genes for:
human VEGFR2, neomycin resistance, and yellow fluorescent protein
(YFP). PAEC clones expressing VEGFR2 were selected with G418 to
choose the cells with high VEGFR2 expression.
Western blot analysis was used to determine VEGFR2 protein expression
in PAEC after transfection with the VEGFR2 gene (Figure 3). PAEC
were imaged with a Nikon fluorescence microscope (Figure 4).

8. Methods:
3.
Detailed (continued)
In vitro targeting of VEGFR2 on endothelial cells by VEGFR2-MSV
PAEC (wild-type or expressing VEGFR2) grown on culture slides were
incubated with MSV (with or without VEGFR2 antibody) and analyzed at 15
and 60 minutes. Cell nuclei were labeled with DAPI (blue); VEGFR2 on PAEC
with YFP (green); MSV with AlexaFluor 647(red). The fluorescent intensities
of imaged cells were analyzed with Nikon Elements. (Figure 5).
For dynamic flow experiments, cells were grown on slides in an induction
chamber set at 37 C and 5% CO2 and were flowed with 3x107 MSV at 100
uL/min for 30 min (with or without VEGFR2 antibody). MSV were labeled
with AlexaFluor 647 (red). Cells were continuously imaged using time-lapse
microscopy, merging transmitted light and fluorescence (Figure 6).

9. Methods:
4.
Detailed (continued)
Internalization of VEGFR2-MSV by endothelial cells
PAEC (wild-type or expressing VEGFR2) were grown on culture slides and
incubated with VEGFR2-MSV. Cytoskeleton (α -tubulin) was labeled with
FITC and VEGFR2-MSV were labeled with AlexaFluor 647. Cells were
imaged with a Nikon fluorescence microscope (Figure 7).

10. Results
1. Preparation and characterization of VEGFR2-MSV
Figure 1: Conjugation of VEGFR2 antibody to MSV
Tasciotti et al., 2008

11. Table 1: Zeta potential of MSV
Nanovectors
Zeta potential
measurements
1
Oxidized MSV
APTES-modified MSV
SMCC-modified MSV
VEGFR2-MSV
2
3
Zeta
potential
(average)
-23.10 -23.50 -23.20 -23.27 + 0.21*
5.59
6.26
5.50 + 0.81
-9.95 -7.27
-7.94
-8.39 + 1.39
4.65
-28.80 -30.00 -30.20 -29.67 + 0.76
*Triplicate measurements with calculated averages
Zeta potential, a measure of the surface charge of particles, was used to determine the
stability of solutions of nanoparticles.
Results show:
The VEGFR2-MSV had the most negative Zeta potentials, indicating greatest stability
because repulsive forces between negative charges keep particles dispersed in solution.
These results were proof of successful conjugation and good stability of colloidal solutions
of VEGFR2-MSV.

12. Figure 2: Fluorescent microscopy of MSV with and without the VEGFR2 antibody
MSV without VEGFR2 antibody
Bright field
MSV
MSV with VEGFR2 antibody
ANTIBODY
Bright field
MSV
ANTIBODY
MSV were labeled with Cy5 (purple) and VEGFR2 antibodies were labeled with TRITC (red). Imaging was
done using a Nikon fluorescence microscope.
Results show:
Proof of conjugation of the VEGFR2 antibody to the MSV is seen by the red fluorescence signal of the
conjugated particle.

13. 2. Preparation and Characterization of VEGFR2 Expressing Endothelial Cells
Figure 3: Western blot of VEGFR2 protein expression in PAEC clones
 Porcine aortic endothelial cells (PAEC) were transfected with: humanVEGFR-2, neomycin
resistance and yellow fluorescent protein (YFP) genes. PAEC clones were selected with G418.
 Figure shows western blot analysis of VEGFR2 protein. Positive control: HUVEC cells; negative
control: 4T1GFP and wild-type (WT) PAEC; protein loading control: β-actin.
Results show:
 PAEC clones expressed the human VEGFR2 protein. Clones 20 & 23 were used in subsequent
experiments.

14. Figure 4: Fluorescence microscopy of VEGFR2
transfected PAEC
1
1.
2.
3.
4.
Cell nuclei labeled with DAPI nucleic acid stain (blue).
VEGFR2 on PAEC labeled with YFP (green)
VEGFR2 antibody labeled with TRITC (red)
Merged images
Results show:
2
 PAEC transfected with the VEGFR2 gene expressed the
receptor on the cell surface (2).
 VEGFR2 was recognized by the VEGFR2 antibody (3).
3
 The receptor and antibody co-localized on the cell surface
(4) indicating that the VEGFR2 was functional and the
VEGFR2 antibody worked.
4

15. 3 In vitro targeting of VEGFR2 on endothelial cells by VEGFR2-MSV
Figure 5: In vitro targeting of VEGFR2 on PAEC by VEGFR2-MSV: Static conditions
A
VEGFR2 expressing PAEC clones
B
MSV without antibody
Intensity/# of cells
MSV without antibody
Wild-type PAEC
15
10
WT
5
Clone
0
15
60
C
D
Intensity/# of cells
MSV with antibody
Time (min)
18
16
14
12
10
8
6
4
2
0
MSV with antibody
WT
Clone
15
60
Time (min)

Results show:
PAEC incubated with MSV without VEGFR2 antibody, showed no difference in the MSV bound to wild-type (A) and VEGFR2
expressing PAEC (B), indicating minimal non-specific adherence to control cells. Graphs show intensity of fluorescence at two time
points (15 and 60 mins).

PAEC incubated with MSV, with VEGFR2 antibody, showed significantly more MSV bound to VEGFR2 expressing PAEC (D)
compared to wild-type (C), as measured by the intensity of the red dye, indicating specific targeting of VEGFR2 at 15 and 60 mins.

The data show in vitro targeting of VEGFR2 on PAEC by VEGFR2-MSV.

16. Figure 6: In vitro targeting of VEGFR2 on PAEC by VEGFR2-MSV: dynamic flow conditions
B
MSV without antibody
Intensity/mm2
A
VEGFR2 expressing PAEC clones
C
1.6
0.6
-0.4
WT
0
10
20
30
Clone
Time (min)
D
MSV with antibody
Intensity/mm2
MSV with antibody
MSV without antibody
Wild-type PAEC
1.6
0.6
-0.4
WT
0
10
20
30
Clone
Time (min)
Results show:
 PAEC incubated with MSV, without VEGFR2 antibody, showed no difference in the MSV bound to wild-type (A) and VEGFR2
expressing PAEC (B) indicating minimal non-specific adherence to control cells. Graphs show time course (0 to 30 min) of intensity
of fluorescence.

PAEC incubated with MSV, with VEGFR2 antibody, showed significantly more MSV bound to VEGFR2 expressing PAEC (D)
compared to wild-type (C), after 20 min, as measured by the intensity of the red dye, indicating specific targeting of VEGFR2.

The data show in vitro targeting of VEGFR2 by VEGFR2-MSV, under dynamic flow conditions that mimic blood flow.

17. 4.
Cellular Internalization of VEGFR2-MSV by PAEC
Wild-type PAEC
A
VEGFR2 expressing PAEC clones
B
Results show:

VEGFR2-MSV (AlexaFluor 647, red) co-localized with the cytoskeleton (TRITC, blue) of PAEC (B).

The data show that VEGFR2-MSV were not only targeted to cell surface receptors, but were also
internalized by the cells, suggesting that the VEGFR2 antibody will have a potential therapeutic effect
in preventing angiogenesis.

18. Summary
The results shows that MSV conjugated with VEGFR2 antibodies bind to the
VEGFR2 of endothelial cells in vitro and deliver the therapeutic antibodies to
their target in a specific manner.
VEGFR2-MSV mediated targeting was also demonstrated under dynamic flow
conditions that mimicked the flow of blood in blood vessels.
VEGFR2-MSV mediated targeting resulted in internalization of the particles.

19. Conclusions
Multistage nanovector mediated targeting of the VEGF receptor on endothelial
cells is a promising technology for specific and efficient delivery of antibodies
that can prevent angiogenesis around tumors and therefore, may have important
clinical applications for cancer therapy.
As a first step toward this goal, in this study, we established an in vitro
system to test the targeting of VEGFR2-MSV to endothelial cells.

20. Future Directions
Experiments underway:
Quantitative analyses of VEGFR-2 expression in PAEC
Quantitative analyses of VEGFR2-MSV targeting to PAEC
Cell viability and toxicity studies designed to test the safety of
VEGFR2-MSV targeting.
In vivo studies to validate our in vitro experiments. Mice, with
tumors, will be injected with VEGFRR2-MSV and live imaging of
the animals will be performed to examine in vivo targeting.

21. Future Directions
Long-term goals:
MSV will be loaded with anti-cancer drugs that will be
released from the pores of the MSV at the target tissue.
This system will have the dual function of preventing
angiogenesis with the VEGFR2 antibody on its surface
and directly destroying cancer cells with the drugs
inside it.
Tumor
Multistage nanovector
Stage 1 particle
Stage 2 particle
VEGFR2
Antibody to VEGFR2
VEGF
Endothelial cell lined blood vessel
No Angiogenesis
+
Drug Delivery

22. References
•
Blanco, E., Hsiao, A., Ruiz-Esparza, G.U., Landry, M.G., Meric-Bernstam, F. and Ferrari, M. Moleculartargeted nanotherapies in cancer: Enabling treatment specificity. Molec. Oncol., 5:6 (2011), pp. 492–503.
•
Carmeliet. Angiogenesis in health and disease Nat. Med., 9 (2003), pp. 653–66.
•
Crawford, Y. and Ferrara, N. Tumor and stromal pathways mediating refractoriness/resistance to antiangiogenic therapies. Trends Pharmacol. Sci., 30 (2009), pp. 624–630.
•
Ferrara, N., Hillan, K.J., Gerber, H.P., Novotny, W. Discovery and development of bevacizumab, an antiVEGF antibody for treating cancer. Nat. Rev. Drug Discov.(2004) 391-400.
•
Ferrari, M. Cancer nanotechnology: opportunities and challenges. Nat. Rev. Cancer, 5 (2005), pp. 161–
171.
•
Kim, K.J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H.S. and Ferrara, N. Inhibition of vascular
endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, (1993), pp.
841 – 844; doi:10.1038/362841a0.
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Martinez, J.O., Chiappini, C., Ziemys A., Faust, A.M., Kojic, M., Liu, X., Ferrari, M. and Tasciotti E.
Engineering multi-stage nanovectors for controlled degradation and tunable release kinetics.
Biomaterials, 34(33) (2013) pp. 8469-77. doi: 10.1016/j.biomaterials.2013.07.049. Epub 2013 Jul 30.
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Tasciotti, E., Liu, X., Bhavane, R., Plant, K., Leonard, A.D., Price, B.K., Cheng, M.M., Decuzzi, P., T
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23. Acknowledgements
I would like to thank:
Dr. Ennio Tasciotti, my research mentor, for giving me the opportunity to work in
his laboratory.
Jonathan O. Martinez (graduate student) for guiding my work every step of the way.
Vivek Karun (undergraduate student) for helping me with many experiments.
Joshua Wang (high school student) for collaborating on many experiments.
This study was supported by Dr. Ennio Tasciotti‘s research grants.