BODIPY derivatives as molecular photoacoustic contrast agents

Samir Laoui,1 Seema Bag,2 Olivier Dantiste,1 Mathieu Frenette,2 Maryam Hatamimoslehabadi,1

Stephanie Bellinger-Buckley,2 Jen-Chieh Tseng,3 Jonathan Rochford,2† Chandra Yelleswarapu1* 3Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston,

MA 02215. 2 Department of Chemistry, 1 Department of Physics, University of Massachusetts

Boston, 100 Morrissey Blvd, Boston, MA 02125.

ABSTRACT

Photoacoustic imaging (PAI) is emerging as a key in vivo imaging technique. Endogenous contrast agents alone are

insufficient to obtain high contrast images necessitating a need for synthetic exogenous contrast agents. In recent years

a great deal of research has been devoted to the development of nanoparticle based contrast agents with little effort on

molecular systems. Here we report on the design and evaluation of BODIPY inspired molecular photoacoustic contrast

agents (MPACs). Through chemical modification of the established BODIPY fluorophore, increasing its vibrational

freedom and appending with non-emissive functionalities, it is demonstrated that the S0S1 absorbed excitation energy is redirected towards a nonradiative excited-state decay pathway. Optical and photoacoustic characterization

of the modified BODIPY MPACs demonstrates a stronger photoacoustic signal compared to the corresponding

fluorescent BODIPY probes.

Keywords: Molecular contrast agents, Fluorescence quenching, Photoacoustic imaging, Bodipy, Nonlinear

photoacoustics

† Jonthan.rochford@umb.edu, phone 1 617-6133; fax 1 617-287-6030. * Chandra.yelleswarapu@umb.edu, phone 1 617-287-6063; fax 1 617-287-6053.

INTRODUCTION

Cancer is the most prevalent disease throughout the world. For successful diagnosis and treatment, a complete

understanding of cancer tumor growth is necessary. Photoacoustic imaging (PAI; aka photoacoustic tomography) is

emerging as a key in vivo imaging technique to aid in the understanding of cancer growth by providing tumor location

and metabolic activity [1, 2]. It represents a best of both worlds approach, combining high optical contrast with low

scattering of ultrasound in biological media. PAI is based on the photoacoustic effect, i.e. the conversion of optical

energy into acoustic energy. When laser pulses are incident on biological tissue, photons of appropriate energy may

be absorbed by endogenous chromophores, e.g. melanian and hemoglobin, and nonradiative decay takes place with

the release of localized heat. This produces a small temperature rise, characterized by the specific heat capacity of the

medium leading to a volumetric thermal expansion of the optical interaction region. Usually the optical absorption of

endogenous contrast agents show a substantial PA response to allow vascular tissue imaging. In many scenarios

however, such as the detection of early stage tumors, an endogenous contrast agent alone is insufficient to provide

enough information. In these cases, optimized exogenous contrast agents were employed to provide better

signal/contrast for photoacoustic imaging [3-10]. However, in order to capture high resolution images of a tumor in

deep tissue, the availability of an effective contrast agent is essential.

Nanomaterials have attracted much attention as contrast agents because they can be engineered in various sizes and

shapes to generate high aspect ratio and tunable optical, magnetic and biological properties [8, 11-22]. Gold

nanostructures (nanoparticles, nanoshells, nanocages, nanorods etc.) are being extensively investigated for biomedical

applications in view of their favorable photonic properties and scope of functionalization, e.g. bioconjugation [18, 23-

33]. Gold nanorods in particular have been targeted as potential contrast agents for PAI due to their strong tunable

NIR absorption and enhancement of their photothermal properties which arise from the plasmon resonance effect [34,

35]. For example, near-infrared (NIR) absorbing gold nanocages have been successfully studied as a new class of

tracers for photoacoustic sentinel lymph node mapping on a rat model [36]. Furthermore, single-walled carbon

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VI, edited by Samuel Achilefu, Ramesh Raghavachari, Proc. of SPIE Vol. 8956, 895609 · © 2014

SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2040057

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nanotubes have also shown promise as molecular contrast agents for the photoacoustic imaging of tumors when

functionalized with cyclic Arg-Gly-Asp (RGD) peptides and PEGylated dendrons[9, 37, 38]. In contrast to the diverse

nature of nanoparticulate based PA contrast agents studied to date, studies of molecular based systems are rare and

are typically based upon commercially available NIR fluorescent dyes [39-46]. For example, in vivo PAI studies on

indocyanine green and its derivatives are rare and have been mostly limited to non-specific investigations of vascular

lymph tissue or specific targeting of integrin in U87 xenografts [47]. The indocyanine green derivative IRDye800-

NHS has been mostly studied due to its commercial availability, strong NIR absorption (max = 800 nm, = 2.7 x 105

M-1 cm-1), poor fluorescence quantum yield (Fl = 0.10) and N-hydrosuccinimide functionality allowing for protein

conjugation via lysine residues [48]. PAI using the rhodamine based AlexaFluor750 dye has also been reported

recently with some success; however it remains that the only option currently available for PAI contrast agents is to

choose existing NIR absorbing fluorescent probes with the hope that they may generate a substantial PA response.

The caveat here is that fluorophores such as AlexaFluor750 have been designed with an optimized fluorescence

emission response and are not anticipated to generate a substantial photoacoustic response. The limited studies of

molecular based PA contrast agents is in stark contrast to the large catalogues of commercially available contrast

agents available today for fluorescence imaging applications. Thus there exists an urgent need to develop a broader

catalogue of PA contrast agents with tailored absorption profiles, improved conjugation strategies for targeted

imaging, and of course an optimized PA response.

In this study a series of molecular based photoacoustic contrast agents (MPACs) are presented whose design is inspired

by the well-established 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore. BODIPY is well

documented for its use as a fluorescence probe for in vivo imaging due to its strong, sharp absorption in the visible

region (max = 500 nm, = 1.20 x 105 M-1 cm-1), small Stokes shift (392 cm-1) and large fluorescence quantum yield

(fl ~ 0.9). However, by increasing the vibrational freedom of the BODIPY molecule and appending non-emissive

fluorescence quenching functionalities in conjugation with BODIPY it is here demonstrated that the S1 excited-state

energy is redirected towards a nonradiative decay pathway which significantly increases the photoacoustic response.

METHODOLOGY

To the best of our knowledge, there are no prior reports in the scientific literature which have investigated the PA

properties of any BODIPY system. This is unsurprising however given the reputation of BODIPY as an efficient

fluorophore whose unique photophysical properties make it unsurpassed as an in vivo fluorescence probe. As such we

take advantage of its highly attractive absorption properties but, by way of understanding its structure-function

properties, have successfully redirected its S1 excited-state energy towards a nonradiative relaxation pathway

contributing significantly towards a desired PA response.

The PA response of any system is determined by a photoinduced change in enthalpy (H) of that system from its

equilibrium thermodynamic state giving rise to heat dissipation and volume expansion (V). These enthalpic and

volumetric changes can be described at the molecular level by both internal (structural/electronic) and external

(environmental) factors, i.e. molecular vibration and solvent/ion reorganization energies, respectively. Having no

control over the environmental factors in vivo our basic design principle is to target structures capable of undergoing

significant structural and/or electronic distortion upon population of their S1 excited states with low energy visible

radiation. Relaxation of the metastable excited-state to the S0 singlet ground-state for the basic meso-aryl-1,3,5,7-

tetramethyl-BODIPY molecule 1 (Fig. 1) occurs predominantly via direct S0S1 radiative decay due to the highly

rigid conformation of its atomic scaffold. By taking advantage of Knoevanagel based -extension strategies at the 1

and 7 positions of 1 with mono- or bis-styryl functionalization, nonradiative decay pathways can be accessed from the

S1 excited-state by increasing the structural flexibility and degrees of freedom for the molecule; in effect taking a

detour en-route to the S0 ground state via the lesser travelled vibrational pathways.

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Figure 1. Structural representation of the reference BODIPY system 1, the extended bis-styryl p-methoxyphenyl (2) and bis-

styryl ferrocenyl BODIPY (3) constrast agents.

On comparing the organic MPACs 1 and 2 successive extension of the -system by introduction of the organic p-

methoxy styrylbenzene substituents gives rise to a 140 nm red-shift in the electronic absorption maximum from 500

(5.45 x105 M-1 cm-1) to 640 nm (4.34 x105 M-1 cm-1) with a slight increase in molar extinction coefficient for

the lowest energy * electronic transition. Accordingly, there is a comparable red-shift of 144 nm observed in the

fluorescence emission spectra (Figure 2, Table 1). The comparable oscillator strengths of this S0S1 electronic

excitation for 1 and 2, and minimal broadening of their full-width-half-maxima (fwhm = 40 cm-1), indicates little

divergence between their ground and excited state geometries even in the presence of both styryl -conjugated groups

for 2. In fact, the non-styryl BODIPY 1 dye displays a lower fluorescence quantum yield (Fl = 0.306) relative to

compound 2 (Fl = 0.719), most probably due to localization of the S0 and S1 electronic wavefunctions in close

proximity to the rotating meso-aryl substituent (Fig. 2b). On comparing the organic MPACs 2 and 3, substitution with

the organometallic, electron rich, ferrocenyl substituent a further red-shift is observed with a two-fold drop in molar

extinction coefficient (max = 687 nm; 2.51 x105 M-1 cm-1). This correlates with a dramatic five-fold increase of

the fwhm for the lowest energy (S0S1) d*, i.e. metal-to-ligand charge transfer (MLCT), absorption band relative

to the (S0S1) *, electronic absorption band for 2. This peak broadening is highly significant as it indicates an

increased Boltzmann population of vibrational levels in the S1 excited-state, no doubt as a consequence of increased

vibrational freedom upon introduction of the substituents. Such extensive broadening is characteristic of transition

metal d* charge transfer (MLCT) transitions. As anticipated, 3 displays zero fluorescence signal due to strong

vibronic-coupling of the BODIPY S1 excited-state with the ferrocenyl based MLCT excited state. Ferrocene

complexes are known to undergo rapid nonradiative decay via ligand field electronic excited states at the iron center.

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Figure 2. (a) UV/Vis electronic absorption spectra for MPACs 1, 2 and 3 and (b) fluorescence emission spectra for MPACs 1 and

2 recorded at room temperature in spectrophotometric grade acetonitrile.

Table 1. UV/Vis electronic absorption and fluorescence emission data for MPACs 1, 2 and 3 recorded at room temperature in

spectrophotometric grade acetonitrile (all fluorescence samples were thoroughly degassed with argon prior to measurement).

a estimated following deconvolution of MLCT absorption bands.

To gain further insight into the electronic structure of these systems preliminary computational investigations were

conducted to ascertain the extent of -conjugation in these complex systems. Computational analysis was conducted

using the density functional theory B3LYP functional with 6-31g* (H, B, C, N, O, F) and LANL2DZ (Fe) basis sets.

Due to space considerations only the highest occupied (HOMO) and lowest unoccupied (LUMO) frontier molecular

orbitals are shown in Figure 3. Trends in the UV/Vis absorption and fluorescence emission spectra can be better

understood by analysis of the frontier orbitals and their respective energies (viz. electrochemical analysis) with the

HOMOLUMO band-gaps decreasing upon increased conjugation via the p-methoxystyrylbenzene substituents in the

1 and 7 positions of the BODIPY ring. Delocalization of electron density from the ferrocene unit onto the styryl-

BODIPY system is clearly evident also confirming the MLCT nature of the low energy electronic transitions in 3.

UV/Vis (max, nm)

(x105 M-1 cm-1) fwhm (cm-1)

emission

(max, nm)

Stokes shift

(cm-1) fl

1 310 (0.48), 364 (0.34), 500 (5.45) 806 510 392 0.306

2 318 (0.93), 368 (2.70), 590 (1.54), 640 (4.34) 813 654 334 0.719

3 280 (1.12), 344 (2.60), 559 (1.22), 687 (2.51) 3929, 2223 a ~ ~ ~

450 500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0 1 BODIPY

2 (MeO)2BODIPY

Flu

ore

scen

ce Q

ua

ntu

m Y

ield

(

Fl)

Wavelength (nm)

300 400 500 600 700 800 900

0

1

2

3

4

5

1 BODIPY

2 (MeOPh)2BODIPY

3 Fc2BODIPY

(x

10

4 M

-1 c

m-1)

Wavelength (nm)

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LUMO

-1.49 V

2.23 eV

0.74V

LUMO

-1.30 V

fi1.67 eV

0.37 V

1 2

LUMO

-1.38 V

1.45 eV

0.07 V

2.01 eV

0.63 V

Figure 3. Computational analysis using the density functional theory B3LYP functional with 6-31g* (H, B, C, N, O, F) and

LANL2DZ (Fe) basis sets for MPACs 1, 2 and 3. Energy levels are estimated on the basis of electrochemical data.

An important observation here is that delocalization or increase vibrational freedom alone is not sufficient enough

to quench BODIPY fluorescence; while extended -conjugation is observed to increase the fluorescence quantum

yield upon comparison of 1 and 2, energy level alignment of the ferrocenyl redox centres in 3 is deemed responsible

for complete quenching of the BODIPY fluorescence by a photoinduced electron-transfer event.[49]

Photoacoustic characterization of contrast agents: For these studies we used our recently developed photoacoustic

z-scan technique [50]. A schematic of the experimental setup is shown in Scheme 1. A frequency doubled Nd:YAG

laser (exc = 532 nm) is focused onto the sample with a 20 cm focal length lens. The sample is placed in a 2 mm quartz

cuvette and is mounted in a custom made cell that contains water for ultrasound coupling. As the visible laser pulse is

incident on the sample, some of the energy delivered is absorbed by the HOMOLUMO electronic transition described

above, and subsequently dissipated as a mixture of fluorescence and/or heat, depending upon the sample in question.

Heat production (H) by the excited state molecule produces pressure transients and thus wideband ultrasonic

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Nd: YAG Laser1. =532 nm

Lensf =20cm

1

v

Sample>

Water

Photoacoustictransducer

r--,

optical

Detector

emission in its local environment. The ultrasonic waves (PA signal) are then detected using a 10 MHz focused water

immersion transducer. The sample cell is placed at 45° with respect to the incident laser beam. The whole system is

mounted on a XYZ translation stage and aligned carefully so that the PA signal collected by the transducer is optimum.

The sample is then translated along the direction of the laser beam in discrete steps such that the focal region (on either

side of the focal point) of the beam scans the sample along the Z-direction. Concurrently the laser beam transmitted

through the samples is measured using a photodiode. This arrangement enables simultaneous monitoring of both

optical transmission and photoacoustic response of the sample under investigation.

Scheme 1. An illustration of the experimental assembly for conducting Z-scan experiments to elucidate MPACs PA response.

The experimental setup was initially optimized with standard nonlinear absorbing materials such as

zinc(II)phthalocyanine and C60-fullerene. The optical and photoacoustic response of MPACs 1, 2 and 3 were then

investigated in acetonitrile with a uniform optical density of 0.5 at 532 nm. The reference BODIPY system 1 shows

very weak absorption at 532 nm and as such a high concentration of sample was required. In any case, due to the

favored fluorescence response of this system a negligible photoacoustic response was observed. Upon excitation with

the 532 nm laser pulse a strong fluorescence response was also evident for 2. As stated above any fluorescence

response was completely absent for the ferrocenyl derivative 3. A plot of PA response vs. laser intensity for selected

MPACs is presented in Figure 4. In the case of 2, an estimated 28.1 % of the excited state energy is available for

contribution to a PA response (Fl = 0.719), as least in a linear optical absorption regime. The bis-ferrocenyl derivative

3 shows zero fluorescence response which correlates to an almost 4-fold increase in the PA signal relative 2.

Significantly, for 3 the PA signal displays a linear increase with increasing laser flux. This is in stark contrast to

MPACs 2 (MeOPh)2BODIPY which shows evidence of a non-linear absorption which saturates beyond ~1x1012 J/m2.

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120 -

100 -

8 0 -

6 0 -

40 -

.

20-

0

5-Fc2BODIPY3{AI eOPh)=BO DIP Y

.. ...:..- *: 'A'#+t' ,

.....

,

.-

0.........................

0 1 2

Intenstiy (x 101" J/m2)

Figure 5. Plot of PA response vs. laser intensity for MPACs (MeOPh)2BODIPY and Fc2BODIPY recorded in methanol

by the Z-scan technique with optical densities of 0.5.

CONCLUSION

In conclusion we have successfully designed and characterized BODIPY inspired molecular photoacoustic contrast

agents (MPACs). By chemically modifying -extensions at the 1 and 7 positions of 1 with styryl methoxyphenyl or styryl ferrocene systems, the fluorescence quantum yield was tuned from 0.859 to nil. Concurrently the photoacoustic

response is amplified ultimately achieving a four-fold increase.

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