Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 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

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

ACS Paragon Plus Environment

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

Page 2 of 29

2 1

ABSTRACT

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

imaging and encourage further optimization of FND for fluorescence brightness.

20 21

KEYWORDS: Nanodiamond, Vascular Endothelial Growth Factor, Targeted Fluorescence

22

Imaging, Oncology, Angiogenesis.

23


Page 3 of 29 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


3 24

INTRODUCTION

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

for targeted tumor imaging utilizing their intrinsic fluorescence.

33

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

34

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

35

VEGFRs causes dimerization of transmembrane receptor proteins and subsequent activation of

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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



Page 4 of 29

4 47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57 58

RESULTS

59

FND functionalization for click chemistry

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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


Page 5 of 29 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


5 70

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

71

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

72

obtained after removal of background Rayleigh particle scattering by polynomial subtraction

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

particles were used for further validation or in vivo work.

82 83

Scheme 1

84 85

86 87



Page 6 of 29

6 88 89 90 91 92 93 94 95

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).

96

Biocompatibility and functional activity of derivatized FND in tissue culture.

97

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

98

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

99

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

100

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

101

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

102

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

103

104 105 106 107 108 109 110 111

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.

112

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

113

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

114

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


Page 7 of 29 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


7 115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

TCO to pelleted FND-mTz or Ag-Tz.

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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



Page 8 of 29

8 138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

FND-conjugated scVEGF.

153 154

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


Page 9 of 29

9 A)

Cells exposed to SLT-VEGF with following competitors None

FND-mTz(high)

B) 100

Survival, % control

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


scVEGF FND-scVEGF

75

FND-mTz

50 25 0 0.1

scVEGF

FND-scVEGF(high)

293KDR cells (untreated controls)

1

scVEGF, nM

10

C)

157 158 159 160 161 162 163 164 165 166 167 168 169

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.

170

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

171

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

172

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

173

human dermal microvascular endothelial cells, and human dermal lymphatic microvascular

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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



Page 10 of 29

10 182

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

183

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

184

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

185 186 187 188 189 190 191

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).

192

Imaging of FND-scVEGF accumulation in tumors

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

untargeted FND-PG (p

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth

Recommend Documents