Targeting fluorescent nanodiamonds to vascular endothelial growth

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Targeting fluorescent nanodiamonds to vascular endothelial growth factor receptors in tumor Marco D Torelli, Ashlyn G. Rickard, Marina Backer, Daria S. Filonov, Nicholas Nunn, Alexander V. Kinev, Joseph M Backer, Gregory M. Palmer, and Olga A. Shenderova Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00803 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

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Bioconjugate Chemistry

Targeting fluorescent nanodiamonds to vascular endothelial growth factor receptors in tumor Marco D. Torelli, † Ashlyn G. Rickard, †† Marina V. Backer, ‡ Daria S. Filonov,§ Nicholas A. Nunn, † Alexander V. Kinev, § Joseph M. Backer,‡ Gregory M. Palmer, †† and Olga A. Shenderova. † Adámas Nanotechnologies, Inc., Raleigh, NC 27617, USA Duke University, Dept. of Radiation Oncology, Durham, NC 27710 USA ‡SibTech, Inc., Brookfield, CT 06804, USA §Creative Scientist, Inc., Research Triangle Park, NC 27509, USA †

††

*Corresponding author: Email: [email protected] Telephone: 919-881-0500 x239 Fax: 919-881-0440

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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The increased expression of vascular endothelial growth factor (VEGF) and its receptors

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is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective

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imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo

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imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their

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use in several applications involving long-term, continuous imaging. To address these problems,

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fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent

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biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in

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growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-

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chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-

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specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting

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targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF

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moieties through biochemical and tissue culture experiments and for selective tumor uptake in

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Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high

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affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of

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FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous

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determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy

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induced fluorescence. These results validate and invite the use of targeted FND for diagnostic

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imaging and encourage further optimization of FND for fluorescence brightness.

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KEYWORDS: Nanodiamond, Vascular Endothelial Growth Factor, Targeted Fluorescence

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Imaging, Oncology, Angiogenesis.

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INTRODUCTION

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Though fluorescence imaging plays a powerful role in research development and clinical

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diagnostics,1-5 the limited photostability and biocompatibility of current fluorophores constrain

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potential applications (e.g. longitudinal studies of cellular dynamics or continuous, long-term

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surgical imaging). In contrast, fluorescent nanodiamonds (FNDs) display true infinite

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photostability,6-9 and their biocompatibility is well-established.10-12 The advantages of FND have

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been explored in numerous applications, from fluorescence imaging to advanced sensing13-15 and

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including testing in several animal models.8, 16-18 However, to date, FNDs have not been explored

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for targeted tumor imaging utilizing their intrinsic fluorescence.

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Vascular endothelial growth factor A (VEGF) and its two main receptors VEGFR-1 and

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VEGFR-2 play important roles in normal and pathologic angiogenesis.19 Binding of VEGF to

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VEGFRs causes dimerization of transmembrane receptor proteins and subsequent activation of

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their tyrosine kinase activity within the cell, which initiates a number of signaling pathways. This

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signaling is critical for endothelial cell proliferation, viability, and function, regulating growth of

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new blood vessels (i.e., vasculogenesis and angiogenesis) and vascular permeability, as well as

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cell migration, inhibition of apoptosis, and recruitment of progenitor and hematopoietic cells from

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bone marrow to tumor.20, 21 Overexpression of VEGF and its receptors is associated with a number

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of pathologies, including the growth of primary tumors and metastatic lesions.22-25 Importantly,

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VEGFR-1 and VEGFR-2 may have different roles in cancer progression,26 whereby VEGFR-2 is

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a particularly well-characterized marker of tumor angiogenesis,27-31 while VEGFR-1 may be

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involved in setting protumorigenic microenvironments and contributing to metastatic growth.32-34

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VEGFRs are well-recognized as important therapeutic targets. To this end many drugs have

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been and are being developed to inhibit either VEGF binding to the receptors or the tyrosine kinase



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activity of these receptors.32 In turn, the therapeutic relevance of VEGF receptors motivates

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development of various tracers for targeted imaging of these receptors.35 Considering the

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importance of VEGF receptors and potential translational opportunities for effective VEGFR-

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targeting tracers, we selected these receptors for assessing FND’s potential for targeted imaging.

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We hypothesized that using re-engineered VEGF as a targeting moiety, we could employ FND for

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selective imaging of VEGF receptors. Herein, a methodology for facile click-chemistry

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conjugation of VEGFR-targeting ligands, specifically an engineered single chain (sc) VEGF, to

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FND was developed. The functional activity of the particle-conjugated scVEGF moiety was

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validated in vitro, and enhanced accumulation of FND-scVEGF versus untargeted FND was

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observed in a murine tumor model, highlighting the potential for FND for targeted imaging in vivo.

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RESULTS

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FND functionalization for click chemistry

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The emission spectrum for the starting FND is shown in Supplementary Figure S1. FND

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with a poly(glycerol) shell (FND-PG) was prepared as described previously.36-38 This shell

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increases the particle diameter by ~20 nm (Supplementary Figure S2) and increases the colloidal

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stability of FND in buffers. To activate FND-PG for the click-chemistry reaction between tetrazine

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and trans-cyclooctane (Scheme 1), FND-PG were functionalized with methyltetrazine (mTz)

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amine using carbonyldiimidazole (CDI)-mediated activation of poly(glycerol) hydroxyl groups in

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FND-PG to form a carbamate linkage.39 The increase in the concentration of CDI in reaction

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mixture led to the higher surface densities of conjugated mTz, which was readily visualized as an

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enhanced pink color in pelleted FND-mTz (Figure 1a). The chemical reactivity of FND-conjugated

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mTz moieties was confirmed by click-reaction (scheme 1) with Cy5-TCO, which is blue in color.


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The enhanced density of conjugated mTz led to an enhanced blue color in pelleted FND-Cy5

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(Figure 1b). UV-Vis absorption spectra of resuspended FND-Cy5 and control FND-PG were

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obtained after removal of background Rayleigh particle scattering by polynomial subtraction

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(Figure 1c). FND-Cy5 spectra, but not control FND-PG spectra, show an absorption maximum at

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650 nm with CDI-dependent intensity and a shoulder at 605 nm that were due to Cy5. Smaller

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peaks at 560 nm and 585 nm were observed in samples with higher mTz densities due to

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absorbance from these ligands. We then used the molar absorptivity of Cy5 at 650 nm to calculate

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the average density of reactive mTz per particle as a result of the different CDI reaction

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concentrations used (40, 30, 20, and 10 mg/mL CDI). The most concentrated sample activated

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with 40 mg/mL CDI exhibited up to 500 mTz per particle, however at this mTz density colloidal

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stability was affected. Thus, particles with lower densities (~200 mTz, 100 mTz, and 30 mTz) per

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particles were used for further validation or in vivo work.

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Scheme 1

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86 87



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Figure 1. Validation of mTz functionalized FND. a) color gradient (left to right) of pink pelleted FNDmTz particles with increasing mTz surface density correlates with the increase in CDI concentration in the conjugation reaction mixture (10-40 mg/mL) , b) color gradient (left to right) of blue pelleted FNDCy5 particles prepared via click-conjugation of Cy5-TCO to FND-mTz with different mTz surface density from Figure 1a, c) UV-Vis spectra of resuspended FND-Cy5 prepared from FND-mTz with different mTz surface density determine by concentration of CDI during preparation of FND-mTz (Contribution from Rayleigh scattering particles subtracted by polynomial subtraction).

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Biocompatibility and functional activity of derivatized FND in tissue culture.

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Three preparations of FND-mTz with approximate mTz surface densities of 30, 100, and

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200 mTz/FND, named FND-mTz Low, Middle, and High, were prepared as described above and

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used in tissue culture experiments. Although all FND-mTz formed dense precipitates on the cell

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surface, 293/KDR cell growth was not affected in the concentration range of 1-350 pM FND-mTz

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(Figure 2A). This is in agreement with the results previously reported for poly(glycerol) covered

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FND that were tested with colony forming endothelial cells (ECFCs).38

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Figure 2. a) FND-mTz with various mTz surface density do not affect 293/KDR cell viability (low, mid high respectively 30, 100, and 200 mTz/FND); b) SDS-PAGE analysis of scVEGF-TCO conjugation to FND-mTz. Lane 1: total amount of scVEGF-TCO. Lane 2-4: unreacted scVEGF-TCO left after Medium, Low, and High density FND-mTz. Lane 5: Control tetrazine agarose. Lane 6-8: amount of scVEGF-TCO coming off FND-scVEGF particles with high salt wash. Lane 9: control tetrazine agarose after high salt wash.

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For targeting VEGF receptors, preparations of FND-mTz with different surface density of

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mTz were functionalized with scVEGF-TCO, an engineered single-chain version VEGF121

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expressed with N-terminal cysteine-containing tag (Cys-tag), which was site-specifically


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derivatized with TCO. Click reactions were performed at concentrations of scVEGF-TCO twice

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higher than calculated mTz concentrations. As a positive control for such reaction we used

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agarose-Tz beads (Ag-Tz). After pelleting all FND from click-reaction mixtures, SDS-PAGE of

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supernatants indicated a significant decrease in the amounts of free scVEGF-TCO relative to the

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input scVEGF-TCO (compare intensity in lane 1 and lanes 2-5 in Figure 2b). Virtually no scVEGF-

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TCO was detected in high-salt washes of the corresponding pellets (lanes 6-9 in Figure 2b),

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indicating that association of scVEGF with FND was not due to a non-specific binding of scVEGF-

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TCO to pelleted FND-mTz or Ag-Tz.

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The amount of scVEGF-TCO click-conjugated to FND-mTz with different mTz surface

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density was estimated in two different assays. First, the upper limits of conjugated scVEGF-TCO

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was determined from the intensities of the residual scVEGF-TCO bands vs. the intensity of the

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input scVEGF-TCO band and was found to be in the range of 200-300 scVEGF/FND depending

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on initial mTz surface density in FND-mTz (Table 1). However, considering that not all surface-

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bound scVEGF-TCO can be simultaneously spatially accessible for interactions with the cellular

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VEGF receptors, we used a sandwich enzyme-linked immunosorbent assay (ELISA) to assess the

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fraction of scVEGF in FND-scVEGF that was capable of interacting with other proteins. Using

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free VEGF to calibrate ELISA, we found that the number of scVEGFs per FND that was

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responsible for binding of anti-VEGF antibodies to plate-bound FND-scVEGF was at least an

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order of magnitude lower than the total estimated amount of scVEGF per FND (Table 1).

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Next, we tested FND-scVEGF preparation with the highest scVEGF per FND, as determined

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by ELISA, in the cell protection assay that was previously developed for characterization of

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scVEGF and its conjugates. In this 72-hour assay, scVEGF-conjugates are tested for their ability

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to protect VEGFR-2 overexpressing 293/KDR cells from VEGFR-2 mediated cytotoxicity of



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Shiga-like-toxin (SLT)-VEGF fusion toxin containing Shiga-like toxin enzymatic subunit A

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genetically fused to VEGF121 isoform.40 The morphology of 293/KDR cells exposed to SLT-

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VEGF changes dramatically from a normal growing monolayer of cells to few clusters of dying

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cells (Figure 3a, upper left panel). As expected, untargeted FND-mTz did not rescue cells (Figure

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3a, upper right panel). In contrast, we found that FND-scVEGF, but not FND-mTz, protected cells

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from cytotoxic SLT-VEGF in a dose-dependent manner (Figure 3a lower panels and 3b). For dose-

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dependence analysis we were used the ELISA-based determination of ~40 accessible scVEGF

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moieties per FND. This surface density of accessible scVEGF led to a calculated EC50 for FND

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bound scVEGF of 3.5 nM, while EC50 for free scVEGF was 2 nM.

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We then tested the ability of FND-scVEGF prepared with FND-mTz high and middle to

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activate VEGF-mediated tyrosine autophosphorylation of the VEGFR-2 receptor in 293/KDR cells

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engineered to overexpress VEGFR-2.40 We found that both types of FND-scVEGF were active in

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this assay (Figure 3c). Although FND-scVEGF were somewhat less active than free scVEGF-

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TCO, tyrosine phosphorylation reached saturation at nanomolar concentrations of “accessible”

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FND-conjugated scVEGF.

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Table 1. scVEGF concentrations as determined by ELISA and compared to SDS-PAGE

Sample Non-Derivatized FND-scVEGF Low mTz FND-scVEGF Mid mTz FND-scVEGF High mTz

scVEGF per FND by SDS-PAGE None 230

scVEGF per FND by ELISA None 6

278

21

307

40

155 156


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Cells exposed to SLT-VEGF with following competitors None

FND-mTz(high)

B) 100

Survival, % control

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scVEGF FND-scVEGF

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FND-mTz

50 25 0 0.1

scVEGF

FND-scVEGF(high)

293KDR cells (untreated controls)

1

scVEGF, nM

10

C)

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Figure 3. a) Microscopy of cell morphology after SLT-VEGF incubation with either no competitor, 18 nM of scVEGF-TCO, FND-mTz , or 24 nM of FND-scVEGF with ELISA determined ~40 “accessible” scVEGF per FND, as compared to untreated controls. Note that 293/KDR cells treated with high concentrations of scVEGF are somewhat contracted and better separated (lower left panel) than untreated cells (lower right panel), as described previously for 293/KDR cells treated with recombinant VEGF41 b) dose-dependent increase in survival of SLT-VEGF treated 293/KDR cells in the presence of scVEGF, FND-scVEGF, but not FND-mTz. c) Western blot of VEGFR-2 tyrosine autophosphorylation induced by FND-scVEGF with different surface density of scVEGF and parental scVEGF. Concentration of scVEGF was based on ELISA measurements for FND-scVEGF preparations used in these experiments. Similar amount of 293/KDR were exposed to indicated concentrations of either free or FND bound scVEGF and processed as described in Material and Methods section.

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Finally, we tested cellular uptake of FND-scVEGF and control FND-PG in two primary

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human cell types: human endothelial colony forming cells (ECFCs, #CB002) and human foreskin

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fibroblasts (CCD1137). Human endothelial cells (e.g., human umbilical vein endothelial cells,

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human dermal microvascular endothelial cells, and human dermal lymphatic microvascular

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endothelial cells) express high level of VEGFR-1 and VEGFR-2 while primary fibroblasts express

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only low levels of VEGFR-1 and no VEGFR-2.42-44 Microscopy revealed readily detectable

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colocalization of FND-scVEGF, but not untargeted FND-PG with ECFCs, though were not present

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in nuclei (Figure 4a, Supplementary Figure S3). Although the intensity of FND-scVEGF per cell

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was variable, virtually all cells showed dose-dependent FND-scVEGF association, with saturation

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at ~ 2 pM (Supplementary Figure S4). Interestingly, the association of targeted FND-scVEGF was

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also readily detectable in experiments with CCD1137 fibroblasts, which express only low level of

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VEGFR-1 receptors. However, quantitative analysis of fluorescence intensity indicated a higher



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binding of FND-scVEGF in ECFCs (CB002) as compared to fibroblasts (CCD1137) (Figure 4B).

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Of note, the differential association of FND-scVEGF by ECFCs and CCD1137 was more

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prominent at higher FND-scVEGF concentrations (Supplementary Figure S5).

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Figure 4. A) representative images of ECFCs treated with FND-scVEGF or control (FND-PG) at 4 pM. The cells were treated for 3 hours, washed, and imaged with INCell 2200 high content imager. λex FND 475nm, λem FND 679nm. 5-Carboxyfluorescein diacetate was used as viability stain. B) Concentration dependent uptake of VEGFR-targeted FND or control FND in EFCF CB002 and CCD-1137 fibroblasts, which display differing amounts of VEGFR (n=3, 6 images analyzed/well).

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Imaging of FND-scVEGF accumulation in tumors

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Tumor-bearing mice were injected with targeted FND-scVEGF and untargeted FND-PG.

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Though whole-body imaging of mice was attempted, significant visualization of FND uptake over

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time with the particle size studied (140 nm) was not possible. To characterize tracer uptake, tumors

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were harvested, cryosectioned, and investigated by epifluorescence microscopy, with

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representative images shown in Figure 5A-C. Tumors harvested from FND-scVEGF injected mice

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showed increased fluorescence as compared to untargeted FND-PG control (Supplemental Figure

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S6). Quantitative analysis of cryosections revealed a significantly higher density of FND in tumors

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from animals injected with targeted FND-scVEGF relative to those from animals injected with

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untargeted FND-PG (p

Targeting fluorescent nanodiamonds to vascular endothelial growth

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