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

Cancer causes the of death of over 500,000 Americans every year.

A promising approach for the treatment of cancer is preventing angiogenesis (the formation of new blood vessels) that supports tumor growth.

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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

Cancer Therapy

Introduction

1. Kim, K.J., et al., 1993; 2. Ferrara, N. et al., 2004; 3. Crawford, Y. et al. 2009.

VEGF

VEGFR2 Receptor

Angiogenesis

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

Nanomedicine

1. Tasciotti , E. et al. 2008; 2. Martinez, J. et al., 2013

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.

Diagram of VEGFR2 mediated targeting of MSVto endothelial cells of a blood vessel at a tumor site

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.

Purpose

Importance

If successful, this research will improve the delivery of therapeutic antibodies that prevent angiogenesis and hence tumor growth.

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.

Approach

Preparation and characterization of VEGFR2 antibodyconjugated MSV (VEGFR2-MSV)

Preparation and characterization of VEGFR2 expressing endothelial cells

In vitro targeting of VEGFR2 on endothelial cells byVEGFR2-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

Internalization of VEGFR2-MSV by endothelial cells

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Methods: Overview

Preparation and characterization of VEGFR2-MSV

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Methods: Detailed

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).

Preparation and characterization of VEGFR2 expressing endothelial cells

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Methods: Detailed (continued)

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).

In vitro targeting of VEGFR2 on endothelial cells by VEGFR2-MSV

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Methods: Detailed (continued)

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).

Internalization of VEGFR2-MSV by endothelial cells

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Methods: Detailed (continued)

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).

Figure 1: Conjugation of VEGFR2 antibody to MSV

Preparation and characterization of VEGFR2-MSV

Results

1.

Tasciotti et al., 2008

Table 1: Zeta potential of MSV

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 stabilitybecause 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.

Nanovectors Zeta potential measurements

Zeta potential(average)

1 2 3

Oxidized MSV -23.10 -23.50 -23.20 -23.27 + 0.21*

APTES-modified MSV 4.65 5.59 6.26 5.50 + 0.81

SMCC-modified MSV -9.95 -7.27 -7.94 -8.39 + 1.39

VEGFR2-MSV -28.80 -30.00 -30.20 -29.67 + 0.76*Triplicate measurements with calculated averages

MSV with VEGFR2 antibodyMSV without VEGFR2 antibody

Bright field ANTIBODYMSV Bright field ANTIBODYMSV

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.

Figure 2: Fluorescent microscopy of MSV with and without the VEGFR2 antibody

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.

Preparation and Characterization of VEGFR2 Expressing Endothelial Cells

Figure 3: Western blot of VEGFR2 protein expression in PAEC clones

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1. Cell nuclei labeled with DAPI nucleic acid stain (blue).2. VEGFR2 on PAEC labeled with YFP (green)3. VEGFR2 antibody labeled with TRITC (red)4. Merged images

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Results show:

PAEC transfected with the VEGFR2 gene expressed the receptor on the cell surface (2).

VEGFR2 was recognized by the VEGFR2 antibody (3).

The receptor and antibody co-localized on the cell surface (4) indicating that the VEGFR2 was functional and the VEGFR2 antibody worked.

Figure 4: Fluorescence microscopy of VEGFR2 transfected PAEC

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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.

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

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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.

Figure 6: In vitro targeting of VEGFR2 on PAEC by VEGFR2-MSV: dynamic flow conditions

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.

Wild-type PAEC VEGFR2 expressing PAEC clones

A B

Cellular Internalization of VEGFR2-MSV by PAEC 4.

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.

Summary

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.

Conclusions

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.

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.

VEGFR2

Antibody to VEGFR2

Multistage nanovector

VEGF

Tumor

Endothelial cell lined blood vessel

No Angiogenesis + Drug Delivery

Stage 1 particle Stage 2 particle

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.

Future Directions

Long-term goals:

References

• Blanco, E., Hsiao, A., Ruiz-Esparza, G.U., Landry, M.G., Meric-Bernstam, F. and Ferrari, M. Molecular-targeted 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 anti-angiogenic 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 anti-VEGF 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.

• 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.

• Tasciotti, E., Liu, X., Bhavane, R., Plant, K., Leonard, A.D., Price, B.K., Cheng, M.M., Decuzzi, P., Tour, J.M., Robertson, F. and Ferrari, M. Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications. Nat. Nanotechnol., 3 (2008), pp. 151–157.

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.

Acknowledgements