Croconaine rotaxane for acid activated photothermal heating and ratiometric photoacoustic imaging of acidic pH

Article abstract of DOI:10.1039/c5cc08317f

Absorption of 808 nm laser light by liposomes containing a pH sensitive, near-infrared croconaine rotaxane dye increases dramatically in weak acid. A stealth liposome composition permits acid activated, photothermal heating and also acts as an effective nanoparticle probe for ratiometric photoacoustic imaging of acidic pH in deep sample locations, including a living mouse.

Full text of DOI:10.1039/c5cc08317f

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Croconaine rotaxane for acid activated
photothermal heating and ratiometric
Cite this:DOI: 10.1039/c5cc08317f
photoacoustic imaging of acidic pH†
Received 6th October 2015,
Accepted 20th October 2015
Samit Guha,a Gillian Karen Shaw,a Trevor M. Mitcham,b Richard R. Bouchardb and
Bradley D. Smith*a
DOI: 10.1039/c5cc08317f
www.rsc.org/chemcomm
Absorption of 808 nm laser light by liposomes containing a pH and have short excited state lifetimes with little fluorescence
sensitive, near-infrared croconaine rotaxane dye increases dramatically emission, singlet oxygen generation, or dye photobleaching.
in weak acid. A stealth liposome composition permits acid activated, We have described a supramolecular encapsulation strategy
photothermal heating and also acts as an effective nanoparticle probe that modulates a croconaine’s NIR absorbance wavelength, but
for ratiometric photoacoustic imaging of acidic pH in deep sample this method is susceptible to the concentration dependence
locations, including a living mouse.
mentioned above.6a Here we report a conceptual advance that is
based on the pH dependent croconaine (Croc) dye shown in
Molecules and materials that can absorb near-infrared (NIR) Fig. 1a.7 The dye’s absorption profile can be switched between
laser light and create localized heating are potentially valuable an anionic basic form (lmax B 660 nm) and a zwitterionic acidic
for numerous therapeutic applications.1 These photothermal form (lmax B 794 nm). An important spectral feature is the
agents can also enhance photoacoustic imaging, an emerging relatively narrow bandwidths which permit large ampli-
modality in molecular imaging with many performance features tude switching of molar absorptivity at the two wavelengths.
that are superior to fluorescence imaging and more amenable to To utilize the lipophilic Croc dye for biological applications we
clinical translation.2 To date, most NIR photothermal agents incorporated it within liposome membranes and employed
have absorbance profiles that cannot be chemically modulated. supramolecular strategies to achieve two crucial photothermal
However, many photothermal and photoacoustic applications and photoacoustic performance features: stable ratiometric
would be improved if the light absorbing properties could be absorption response that is unaltered by laser irradiation,
switched on by specific local conditions. A good example is and fine-tuning of the dye pKa to match the local pH.
the tissue acidosis associated with pathological states such as
A preliminary study using a 808 nm diode laser showed that
cancer, infection, inflammation, and fibrosis.3 There are a few irradiation of an ethanol solution of Croc in its basic form
reports of NIR agents that can undergo changes in absorbance created very little sample heating, but conversion to the acidic
cross-section due to triggered self-aggregation but an inherent
drawback with this approach is a dependence on local concen-
tration which can be hard to control.4 New NIR absorbing
agents are needed with chromophores that can be altered
directly by the local chemical environment. A logical strategy
is to design appropriate dyes with switchable absorbance,
but there are very few NIR chromophores with the correct
combination of chemical and photophysical properties.5 Recently,
we discovered that croconaine dyes exhibit excellent laser heating
properties.6 They strongly absorb NIR light (e 4 105 MÀ1 cmÀ1
)
a Department of Chemistry and Biochemistry, 236 Nieuwland Science Hall,
University of Notre Dame, IN 46556, USA. E-mail: smith.115@nd.edu
b Department of Imaging Physics, University of Texas MD Anderson Cancer Center,
Houston, TX 77054, USA
Fig. 1 (a) pH sensitive croconaine (Croc). (b) UV/Vis absorption of Croc-SL
in buffer at various pH values, [Croc] = 3 mM. (c) UV/Vis absorption plots of
Croc-SL in buffer at pH 5.0, spectra were acquired initially (solid line) and
after standing for 1 hour (dashed line) at 4 1C.
† Electronic supplementary information (ESI) available: Chemical structures,
synthesis and characterization; liposome data; photoacoustic imaging data. See
DOI: 10.1039/c5cc08317f
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form greatly enhanced laser induced heating (Fig. S1, ESI†).
Formulation of the Croc dye for operation in aqueous solution,
was achieved by incorporating it into stealth liposomes as an
amphiphilic ion-pair with 1,2-dioleoyl-3-trimethylammonium-
propane (DOTAP) as the counter cation.‡ Stealth liposomes
have polyethylene glycol chains protruding from the membrane
surface which inhibit liposome aggregation and extend circu-
lation time in the blood stream.8 Initially, stealth liposomes
doped with Croc dye (Croc-SL) were prepared as unilamellar
vesicles (diameter = 194 nm) with a composition of POPC : Chol-
PEG600 : DOTAP : Croc 84 : 10 : 3 : 3 (Fig. S3, ESI†).‡ The sample
color was blue at pH 7.4 due to the Croc conjugate base
absorption at 660 nm. Titration with acid abolished the
blue color and an intense new peak appeared at 794 nm with
a pKa of 6.5 (Fig. 1b and Fig. S3, ESI†). Irradiation of cuvettes
containing Croc-SL with an 808 nm diode laser produced
acid activated photothermal heating (Fig. S3b, ESI†). Dynamic
light scattering measurements indicated that Croc-SL do not
change size in acidic pH, but the absorption spectrum
slowly broadened with time (Fig. 1c, Fig. S3 and S4, ESI†).
The absorption broadening is consistent with self-aggregation
of the zwitterionic Croc conjugate acid within the liposome
membrane and is a potential drawback for important applica-
tions, such as repetitive photothermal heating and ratiometric
Fig. 2 (a) pH sensitive croconaine rotaxane (CrocRot). (b) UV/Vis absorption
of CrocRot-SL at various pH values, [CrocRot] = 3 mM. (c) UV/Vis absorption
photoacoustic imaging of pH (see below). We hypothesized plots of CrocRot-SL in buffer at pH 5.0, acquired initially (solid line) and after
standing for 24 hour (dashed line) at 4 1C. (d) Temperature changes during
that the problem could be circumvented by trapping the Croc
dye within a tetralactam macrocycle and generating an inter-
locked croconaine rotaxane (CrocRot) (Fig. 2a). Even under
laser irradiation (808 nm, 3.5 W cmÀ2) of CrocRot-SL samples at different pH
values. (e) Multiple heating cycles of CrocRot-SL at pH 5.0 due to periodic
laser irradiation (808 nm).
self-aggregation conditions, we expected the surrounding
macrocycle in CrocRot to prevent electronic coupling of the
encapsulated dye.6b Initial attempts to prepare CrocRot by a small biomolecules such as glutathione, cysteine, and H2O2
direct slippage process in organic solvent or within liposome (Fig. S16b, ESI†). Furthermore, CrocRot-SL were not toxic to
membranes were unsuccessful, presumably due to the steric cultured mammalian cells (Fig. S16c, ESI†). These results
bulk of the indolenine dimethyl substituents. Thus, a Leigh- demonstrate that CrocRot-SL are robust biocompatible nano-
type templated clipping reaction was used to convert Croc dye particles that can be switched reversibly between acid and base
to CrocRot and the rotaxane structure was elucidated using forms and that they survive strong and repeated laser heating.
a battery of spectroscopic methods (Fig. S5–S11, ESI†).9 They likely can be used for photothermal therapy and controlled
As expected, rotaxane formation produced a 22 nm red-shift release in disease states that have tissue acidosis.10 In addition,
of the dye absorption maxima.6 The interlocked CrocRot mole- they can also be utilized for another important application,
cule does not unthread in competitive organic solvents or namely, ratiometric photoacoustic imaging of acidic pH.
when it is located within a liposome membrane (Fig. S9, ESI†).
Photothermal agents facilitate photoacoustic imaging by
We prepared CrocRot doped stealth liposomes (CrocRot-SL) absorbing short-duration, low power density laser pulses
composed of POPC : Chol-PEG600 : DOTAP : CrocRot 84 : 10 : 3 : 3 (B0.06 W cmÀ2) and emitting ultrasound waves which are
(Fig. S12, ESI†) and found that they exhibited very similar pH scattered less than light waves and thus penetrate further
dependent changes in absorption spectra as above, with a slightly through biological media to produce higher resolution
decreased dye pKa of B6.0 (Fig. 2b and Fig. S13, ESI†). We were images.2 There is a need for new types of exogenous probes
pleased to find that the spectral stability of CrocRot-SL was for enhanced photoacoustic imaging. The current literature
greatly improved; for example, there was very little spectral contains several reports of probes that can be activated by
change after sitting for 24 hours in acidic pH (Fig. 2c and cleavage enzymes to produce changes in signal intensity, but
Fig. S14, ESI†). As shown in Fig. 2d, pH dependent photothermal to the best of our knowledge, there is no NIR photoacoustic
heating was observed using an 808 diode laser (3.5 W cmÀ2), and probe with a rapid ratiometric pH response.11 A preliminary
the high spectral stability of the CrocRot-SL enabled multiple evaluation of phantoms (narrow tubes oriented parallel to the
laser heating cycles using the same sample with no measurable face of the transducer, see Fig. S17, ESI†) containing ethanol
change in the absorption profile and photothermal heating solutions of Croc or CrocRot showed that they generated about
response (Fig. 2e and Fig. S15, ESI†). CrocRot-SL are highly stable the same photoacoustic signal intensities as the commonly
in the presence of serum, biological salts (Fig. S16a, ESI†), and used NIR dye indocyanine green (ICG) (Fig. S18, ESI†).12 We
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Fig. 3 (a) Photoacoustic scans of CrocRot-SL at pH 7.4 (black) and pH 5.0
(red), mean (N = 3) and SD shown. (b) Ratiometric photoacuostic images of
two phantoms containing CrocRot-SL in buffer at pH 7.4 or 5.0, proximally
located at 11–13 mm depth in light scattering media. See Fig. S17 (ESI†) for
a diagram of the transverse orientation of the phantoms.
then examined two phantoms containing CrocRot-SL at pH 7.4
or 5.0 and observed that the photoacoustic and absorption
spectra at each pH value were closely matched (Fig. 3a and
Fig. S19, ESI†). Additional photoacoustic imaging studies exam-
ined the two phantoms at different depths in a light scattering
medium (0.7% milk fat solution) that is opaque to normal
fluorescence imaging methods. We found that the difference in
phantom pH could easily be distinguished by comparing the
Fig. 4 (a) UV/Vis absorption spectra of CrocRot-IVSL at various pH values,
[CrocRot] = 3 mM. (b) Crossectional B-mode ultrasound image (sagittal plane)
of living mouse abdomen (grayscale), co-registered with the photoacoustic
response at 740 nm; magenta and green arrows denote two ROIs of
CrocRot-IVSL accumulation within peritoneal cavity. (c) Photoacoustic
spectra of CroRot-IVSL in buffer pH 7.4 before injection (blue/black trace)
and in the two ROIs within the mouse peritoneal cavity (magenta and green).
ratio of photoacoustic signal intensities at 812 and 690 nm. The photoacoustic imaging. The image in Fig. 4b is comprised of a
image in Fig. 3b shows a large difference in ratiometric response B-mode ultrasound image (grayscale) clearly showing the perito-
(more than 5-fold) for the two phantoms, each located at a depth neal cavity and an overlay (red) depicting the corresponding
of 11–13 mm from the transducer face. Additional imaging photoacoustic response when the excitation wavelength was
experiments at phantom depths of 9 and 26 mm confirmed that 740 nm. There are three photoacoustic spectra in Fig. 4c. One
the photoacoustic signal-to-noise decreased with depth, but the spectrum corresponds to the sample of CrocRot-IVSL in buffer at
pH dependent 812/690 signal ratio hardly changed.
pH 7.4 before injection into the mouse, and the other two spectra
With these proof-of-concept imaging results in hand we correspond to the different regions-of-interest (ROI) in the mouse
moved to a more demanding biological system to assess the peritoneal indicated by the arrows in Fig. 4b. A comparison of the
potential of acid sensitive CrocRot-doped liposomes. In some two in vivo ratiometric photoacoustic scans with the UV/absorp-
diseases, the tissue pH can be as low as 5.5 (e.g., intracellular tion plots indicates a peritoneal pH of 6.0–6.5.¶ Thus, the in vivo
endosomes, inflamed tissue) but in others it is closer to 6.5 (e.g., imaging correctly identified the weakly acidic pH of the mouse
extracellular matrix of solid tumors).3 This means it will be peritoneal. With further development, this photoacoustic method
necessary to optimize the pKa of the CrocRot for each case, to may become a new in vivo technique for measuring the pH of
ensure a significant degree of absorbance switching. After examin- peritoneal fluid, which is known to decrease with pathological
ing the literature,13 we reasoned that the pKa can be fine-tuned in a conditions such as bacterial peritonitis, a frequent complication
rational way by simply adding controlled amounts of appropriately in patients on peritoneal dialysis.15 It should also be effective at
charged amphiphiles to the liposome formulation. To demonstrate identifying local regions of weakly acidic tissue associated with
the utility of this concept we purposely raised the CrocRot pKa by other types of disease.3 In addition, the liposome architecture can
substituting the neutral Ch-PEG600 in the CrocRot-SL membrane be further customized by incorporating drugs or additional
with anionic DSPE-PEG2000.‡ After a short sequence of trials, imaging reporter groups to make a wide array of novel laser
we found that a modified liposome formulation composed of responsive therapeutic and diagnostic agents.16
POPC:DSPE-PEG2000 : DOTAP : CrocRot 79 : 15 : 3 : 3 and given
the descriptor name, CrocRot-IVSL, exhibited a pKa of B6.5 Foundation Advancing Basic Cancer Research Grant (2013/14)
(Fig. 4a and Fig. S20, ESI†).§ administered by the Harper Cancer Research Institute (USA)
We are grateful for funding support from the Walther Cancer
To demonstrate the capability of CrocRot-IVSL for in vivo and the NIH (GM059078 to B. D. S., and P30 CA016672, S10
photoacoustic imaging we chose to image the pH of peritoneal OD010403 to R. R. B.).
fluid in a living mouse which is known to be in the range of 6.1–
6.3.14 Following a protocol that was approved by the appropriate
animal care and use committee, a single dose of CrocRot-
IVSL was injected into the peritoneal cavity of a living mouse
(N = 2), and the sagittal plane of the mouse abdomen was imaged
Notes and references
‡ Abbreviations: Cholesterol-PEG600 n = 10–11 (Ch-PEG600), croconaine
(Croc), croconaine rotaxane (CrocRot), 1,2-dioleoyl-3-trimethylammonium-
propane (chloride salt) (DOTAP), N-(carbonyl-methoxypolyethyleneglycol-
using co-registered B-mode ultrasound and multi-wavelength 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE-PEG2000),
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Indocyanine Green (ICG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC). See Fig. S2 (ESI†) for chemical structures.
§ This result highlights an important technical advantage of pH sensor
production by nanoparticle self-assembly versus covalent synthesis of a
discrete molecular sensor. Fine-tuning the pKa of a molecular sensor
requires a much longer and more labour intensive cycle of structure
redesign and chemical synthesis.
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photoacoustic spectra are slightly red-shifted when compared to the
spectrum in buffer before injection. This is attributed to in vivo
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