1.1.

Optical Approaches to Protease Imaging

The most widely pursued strategy for in vivo protease imaging has involved pure optical imaging of activatable probes containing near-infrared (NIR) fluorophores. This approach has been pioneered by the group of Ralph Weissleder at Massachusetts General Hospital and has been recently commercialized for small animal imaging by VisEn Medical. The molecular probes typically comprise pairs of mutually quenching fluorophores or a fluorophore and a dedicated quencher that are linked to each other or to a backbone molecule by peptide spacers that are cleavage targets for the protease of interest.^{6, 10, 11, 12, 13, 14, 15, 16} In the uncleaved state, the molecule is relatively quiescent due to the fluorescence quenching, but upon cleavage by the target protease, the fluorescence activity increases several fold. The simplest such construct involves simply attaching fluorophores to either end of an appropriate protease-substrate peptide chain.¹⁷

A longer-circulating probe was obtained by use of a larger molecule featuring a polyethylene glycol protected graft co-polymer (PGC), comprising a poly-L-lysine backbone to which are attached methoxy polyethylene glycol (MPEG) side chains.¹⁸ Other side chains held mutually quenching, near-infrared fluorophores or combinations of fluorophores and dedicated quenchers.^{14, 19} The fluorophores are generally chosen to absorb and emit in the near-infrared $\left(700\text{to}1100\mathrm{nm}\right)$ range to maximize tissue penetration.

Once cleaved, these activatable probes can be imaged by a number of different optical approaches, including simple fluorescent reflectance imaging or fluorescent endoscopic imaging,^{20, 21} which involve stimulating fluorescence by illuminating the sample at appropriate wavelengths and then detecting the emitted fluorescence photons on the same side of the subject using appropriate filters. These approaches are simple and cost effective but provide neither fully three-dimensional (3-D) nor quantitative information.

Quantitative three-dimensional information can be obtained by a more sophisticated fluorescence molecular tomography (FMT) approach.^{13, 22, 23} In FMT, near-infrared light is directed into the subject from a number of different directions, and the resulting fluorescent photons as well as the scattered incident photons are detected outside the subject. The problem of reconstructing the distribution of fluorophores from this set of measurements is similar in principle to that in diffuse optical tomography, and it allows for formation of quantitative three-dimensional images, albeit with limited depth penetration, resolution, and sensitivity, as discussed in the following.

Optical techniques such as FMT have been used primarily in small-animal imaging and have practical limitations that may restrict their widespread application in human imaging. Due to the strong optical scattering of the both excitation and fluorescent light in biological tissues, the resulting images have resolution limited to the millimeter range. The best reported resolution is approximately $1\mathrm{mm}$ in a fairly optimized planar geometry with the fluorophore at a depth of $0.75\mathrm{cm}$ , centered between two imaging plates separated by $1.5\mathrm{cm}$ (Ref. 24). The resolution would be expected to worsen for a more deeply seated structure. In addition, the strong scattering and absorption of the fluorescent photons limits the sensitivity of FMT, which in the same planar imaging device just discussed, was shown to have a detection threshold of 100 fmol of fluorophore (Cy 5.5) at a depth of $0.75\mathrm{cm}$ (Ref. 24). Again, this sensitivity would fall off at greater depths, although overall sensitivity could conceivably be improved by advances in photon-detection technology.²⁴

1.2.

Potential Advantages of Optoacoustic Protease Imaging

Optoacoustic imaging can potentially overcome many of the limitations of the pure optical imaging approach. While OAT can, of course, be used to measure innate optical absorption properties of the body, it can also be used to detect strongly absorbing molecular probes that are introduced into the body.^{25, 26, 27} Kruger have shown that the basic fluorescing imaging agents employed in FMT also produce a detectable optoacoustic signal.^{25, 26, 27} In fact, because the fluorescence yield of many agents is relatively low (for Cy5.5, it is 0.28), more of the absorbed optical energy goes into producing heat, and thus into producing an optoacoustic signal, than goes into fluorescence. Kruger demonstrated that their OAT system can detect as little as 5 fmol of fluorophore (Cy 5.5) at $1.25\mathrm{cm}$ depth in a tissue mimicking phantom, which is over an order of magnitude better sensitivity than has been reported for FMT.^{25, 26, 27}

More recently, Razansky have shown that optoacoustic techniques can be used to image green fluorescent proteins at greater depth and sensitivity than pure optical detection techniques.²⁸

Wang’s group has demonstrated the use of such agents in vivo, performing optoacoustic imaging of live mice after injection of indocyanine green (ICG), which absorbs strongly in the near-infrared. They were able to detect as little as 7 pmol of ICG per resolution element against the strong absorption background of blood.²⁹ They have recently performed in vivo imaging of gene expression using the b-Gal/X-Gal system, whereby b-Gal is used as a reporter gene behind a promoter of interest and X-Gal as a cleavable substrate. When cleaved by b-Gal, X-Gal, which is originally colorless, produces a blue product that absorbs strongly in the near-infrared and can be imaged by OAT.³⁰

Naturally, the quenched fluorophore strategy employed in FMT for protease imaging cannot be applied directly in OAT, which is sensitive to the absorption rather than the fluorescence properties of the contrast agents. This is the motivation for the absorption-shifting cleavable probe that we are developing.³⁵

2.1.

Overview of Probe Design

We have designed a molecular probe for in vivo optoacoustic imaging of local protease activity. The molecule is designed so that its optical absorption properties are altered when it is cleaved by a protease of interest. Specifically, the probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules tend to deaggregate, giving rise to a spectrum characteristic of monomers.

2.2.

Background on Chlorophyll and Bacteriochlorophyll

The chlorophylls are a family of porphyrin-derived chromophores naturally found in the photosynthetic system of plants and photosynthetic bacteria.³¹ The most common form found in plants is chlorophyll $a$ (Chl $a$ ), while bacteriochlorophyll $a$ (Bchl $a$ ) is the most common in bacteria. Both compounds are strongly absorbent pigments with molar extinction coefficients over $\mathrm{10,000}{\mathrm{M}}^{-1}{\mathrm{cm}}^{-1}$ . As with other porphyrins, there are four electronic transitions, creating four absorption bands, with the lowest in energy being at approximately $680\mathrm{nm}$ for Chl $a$ and approximately $780\mathrm{nm}$ for Bchl $a$ (Ref. 31).

In order to use Chl $a$ or Bchl $a$ in an imaging probe, a coupling point, which is not present in their native forms, must be created. The ester linking the phytyl chain to the main ring is easily hydrolyzed to form a coupling point in either compound. While it is possible to remove the phytyl chain without affecting any other portion of the structure, it is cleaner to hydrolyze the ester with trifluoroacetic acid (TFA) and remove the central magnesium in the same step.³¹ This converts Chl $a$ to pheophorbide $a$ (Phe $a$ ) and Bchl $a$ to bacteriopheophorbide $a$ (Bphe $a$ ). The consequence of using Phe $a$ or Bphe $a$ is a blue shift of $20\mathrm{nm}$ in the absorption spectra relative to that in natural compound.

An absorption-shifting phenomenon can occur in any of the four aforementioned compounds upon aggregation. In this phenomenon, a shift in the optical absorption spectrum is observed when two or more of the chromophores are in close proximity. The extent of the optical shift is determined by the distance of separation and the relative orientations of the macrocycles.³² The chlorophylls, including Bchl $a$ , are particularly known for aggregating into dimers or oligomers under the right conditions, resulting in optical absorption spectra exhibiting large red shifts of the lowest electronic transition.³³ Increasing numbers of molecules aggregating does somewhat increase the red shift relative to that of the dimer, but with diminishing returns: the increase in absorption shift decays exponentially as more molecules are added to the aggregate, so most of the shift is explained by the difference between the monomer and dimer forms. This means that when oligomers are formed, large differences in the number of components have very little effect on the absorption spectrum. Lewis acid-base and $\pi \text{-}\pi$ interactions play significant roles in determining the structure of the aggregate.³³ The Lewis acid-base interaction occurs when the ketone group donates electron density to an empty orbital of the center magnesium. The $\pi \text{-}\pi$ interaction occurs when the flat rings stack on top of each other, allowing overlap between the $\pi$ orbitals. Typical optical spectra of monomer and dimer forms of Bchl $a$ will be shown in the following, indicating a $40\text{-}\mathrm{nm}$ shift. Depending on the system, as much as a $70\text{-}\mathrm{nm}$ shift has been observed for the red-most band of the optical spectrum of Bchl $a$ dimers and oligomers.³⁴

Similarly, the reaction center of the photosynthetic complex contains two chlorophyll $a$ molecules in close contact to each other and exhibits an absorption peak at $700\mathrm{nm}$ . Upon isolation of chlorophyll from the plant material, the absorption peak shifts to $662\mathrm{nm}$ . This change is due to a phenomenon associated with the aggregation of chromophores. When two or more chromophores—in this case, chlorophyll $a$ —are in close proximity, the electric field around each chromophore will change the energy associated with the absorption spectrum.

It is these aggregation-based shifts that we exploit in the design of the protease-sensitive optoacoustic probe.

2.3.

Details of the Probe Design

As depicted in Fig. 1 , the probe comprises simply a peptide chain with a Phe or Bphe attached at the N-terminus. The working design targets MMP-2 with the peptide sequence HRALMGLPG, where each letter stands for a specific amino acid. The protease cleaves the peptide between the methonine (M) and the glycine (G) residues. Prior to cleavage, the probe aggregates in semisoluble micelles. After the addition of MMP-2, the micelles are broken apart, and the cleaved portions become monomers. This is illustrated schematically in Fig. 2 .

Fig. 1

Probe design using the sequence HO-HRALMGLPGG-Phe.

Fig. 2

Schematic of the probe behavior. Prior to cleavage, the probe molecules tend to aggregate, bringing the Chl or Bchl into dimers, trimers, and higher-order aggregates. After cleavage, the cleaved moieties tend to float free, producing monomer Chl or Bchl.

3.1.

Solvent-Induced Aggregation of Bacteriochlorophyll $a$

We conducted a preliminary study to explore the absorption spectra that we could expect from the monomer and dimer forms of Bchl $a$ , since the difference between these two spectra is what will allow us to detect cleaving of our probe molecule by proteases, which results in the conversion of Bchl and Chl dimers and trimers to monomers. By carefully controlling the solvent conditions, aggregation of pure Bchl $a$ can be created or destroyed.

3.2.

Synthesis of the Probe

The probe was synthesized with standard solid-phase peptide synthesis techniques.³⁶ A custom synthesized fluorine labile resin was used as the solid support.³⁷ The peptide was then built sequentially from the c-terminus and capped with Phe $a$ . The sequence was cleaved from the resin, washed, and then evaporated to dryness. The resulting peptide was dissolved in the enzyme reaction buffer described in Sec. 3.3.

3.3.

Enzymatic Cleavage of the Imaging Probe

The cleavage procedure³⁸ follows the protocol provided by the enzyme producer, NovaBioChem. A solution of $5\mathrm{mM}$ NaCl, $5\mathrm{mM}$ $\mathrm{Ca}{\mathrm{Cl}}_2$ , and $0.01\mathrm{M}$ ethylenediaminetetraacetic acid (EDTA) in water is prepared. Two milliliters of the solution is added to approximately $200\mathrm{mg}$ of the dry, solvent-free imaging probe and stirred at $30°\mathrm{C}$ until fully dissolved. The 5 units of the MMP-2 enzyme at room temperature are added to the probe solution. The solution was stirred and complete cleavage generally took less than $15\min$ .

3.4.

Optical Spectroscopy

We used optical light spectroscopy to measure the molar extinction coefficient as a function of wavelength for the uncleaved and cleaved forms of the sample. A Shimadzu UV-1602 UV-visible spectrophotometer was coupled to a desktop PC and used for the acquisition of all extinction coefficient spectra. The purity of Bchl $a$ /Chl $a$ and carotenoid mixtures can be determined with optical spectroscopy along with the state of aggregation. Furthermore, the high extinction coefficient of Bchl $a$ ( $\epsilon =42.0\times {10}^3$ in methanol³¹) and Chl $a$ ( $\epsilon =70.5\times {10}^3$ in methanol³¹) allows for good absorption with extremely low concentrations.

After the synthesis of the probe, the sample was dissolved in methylene chloride and divided into two equal (by volume) aliquots. The methylene chloride was completely removed by vacuum evaporation, and the solid probe was dissolved in $2\mathrm{ml}$ of enzyme buffer solution. The absorption spectra were taken consecutively using either a $1\text{-}\mathrm{cm}$ , $5\text{-}\mathrm{mm}$ , or $1\text{-}\mathrm{mm}$ path length cuvette, depending on the concentration of the sample. Generally, the uncleaved samples required the usage of the $1\text{-}\mathrm{mm}$ or $5\text{-}\mathrm{mm}$ cuvette, while the cleaved sample performed well in the $5\text{-}\mathrm{mm}$ and $1\text{-}\mathrm{cm}$ cuvettes. The absorbance $A$ of each sample was calculated as

3.5.

Optoacoustic Microscopy

Optoaocoustic measurements were performed using the microscopic system described in Xie ³⁹ and illustrated in Fig. 3 . In this system, the illumination is focused to a diffraction-limited spot to provide the lateral resolution, and an unfocused transducer provides the depth resolution by mapping the measured temporal signals into the image volume in a B-scan mode. The illumination point can be scanned rapidly using a standard galvonometer scanner.

Fig. 3

(a) Schematic of the optoacoustic microscopy system. (b) Schematic of the sample.

The samples were the same as those used to obtain the optical extinction coefficient spectra, except they were transferred to a different imaging cell. A $0.5\text{-}\mathrm{mm}$ inside diameter, $0.7\text{-}\mathrm{mm}$ outside diameter Teflon tube was placed at the surface of a plastic weighting boat full of hot gelatin gel (gelatin from bovine skin, Sigma-Aldrich, St. Louis, Missouri). The gel was allowed to cool, trapping the tube at the surface of the gel, as shown schematically in Fig 3. Ultrasound gel (Clear Image, Sonotech, Bellingham, Washington) was spread over the top of the agar gel to facilitate ultrasonic coupling, and all visible air bubbles were carefully removed.

The sample was illuminated by laser pulses (pulse repetition rate: $1\mathrm{kHz}$ ; pulse duration: $6\mathrm{ns}$ ) from a tunable dye laser (Cobra, Sirah Laser and Plasmatechnik GmbH, Germany) pumped by an Nd:YLF laser (IS811-e, EdgeWave GmbH, Germany). The beam output from the dye laser, which is not spatially homogeneous, was spatially filtered by an iris and expanded to an $8\text{-}\mathrm{mm}$ diameter. The beam then passed sequentially through a neutral density filter and a $60\text{-}\mathrm{mm}$ focal length objective lens. The beam then passed into an imaging basin filled with water (depth: $30\mathrm{mm}$ ). The pulse energy was around $0.1\mu \mathrm{J}$ , and the energy density at the optical focus was around $200\mathrm{mJ}∕{\mathrm{cm}}^2$ . This energy density was higher than the ANSI maximum permissible exposure (MPE) for wavelengths shorter than $700\mathrm{nm}$ $(20\mathrm{mJ}∕{\mathrm{cm}}^2)$ but did not exceed the MPE for wavelengths longer than $700\mathrm{nm}$ $(200\mathrm{mJ}∕{\mathrm{cm}}^2)$ .

A single-element, unfocused transducer (V313, Olympus NDT; $15\mathrm{MHz}$ ; bandwidth: 80%; active element diameter: $6\mathrm{mm}$ ) was submerged in the water at an angle of $15\mathrm{deg}$ to the incidental laser beam at the sample. The recorded optoacoustic signals were amplified by $58\mathrm{dB}$ and were digitized and stored by high-speed digitizer (CS12400, Gage Applied, Lockport, Illinois) for further processing. At each optical wavelength, the optoacoustic signal was recorded 512 times, allowing for the calculation of a mean and standard deviation.

4.1.

Optical Properties of Bacteriochlorophyll

The spectra resulting from the experiment described in Sec. 3.1 are shown in Fig. 4 , where it can be seen that the monomer form of Bchl $a$ has an absorption peak at $782\mathrm{nm}$ , while the dimer absorption peak is shifted to approximately $820\mathrm{nm}$ .

Fig. 4

Optical absorption spectra of bacteriochlorophyll $a$ monomer and dimer. A: Bchl $a$ in semidry carbon tetrachloride. Water coordinated to central Mg prevents aggregation, resulting in blue-shifted monomers. Dimers of Bchl $a$ form when water is not coordinated to Mg, resulting in the red-shifted shoulder at about $820\mathrm{nm}$ . B: Same as solution A but with addition of 100-fold excess (relative to Bchl $a$ ) of pyridine that strongly coordinates with Mg, preventing any aggregation, resulting in a single transition peak centered at $782\text{-}\mathrm{nm}$ . C: Difference spectrum between solutions A and B, clearly showing the optical shift accompanying Bchl $a$ dimerization.

4.2.

Ultraviolet–Visible Light Spectroscopy of Complete Probe

The extinction coefficient spectra for the tubes containing the probe with and without cleavage by MMP-2 are shown in Fig. 5 . In its whole state, the probe produces aggregates (dimers and trimers) that give rise to a broad absorption spectrum with a peak at $726\mathrm{nm}$ . After addition of MMP-2, the probe is cleaved, and the aggregates are not reformed. The sharp peak in the spectra at $668\mathrm{nm}$ is characteristic of a monomer, as the energy levels are distinct, whereas an aggregate will have a broad absorption peak due to the various possible dipole angles between the Chl or Bchl in the aggregates. The absorption of an individual aggregate creates a peak of the same bandwidth as the monomer, although shifted to be centered at a different wavelength. As the angle, $\theta$ , between the two dipoles changes, the magnitude of the red shift in absorption increases or decreases. The superposition of all these shifted absorption peaks creates the broad overall absorption peak seen. The concentration of the samples was $\sim 140\mu \mathrm{M}$ , so the monomer peak corresponds to an extinction coefficient of approximately $\mathrm{35,000}{\mathrm{M}}^{-1}{\mathrm{cm}}^{-1}$ .

Fig. 5

Extinction coefficient spectra of the probe without (uncleaved) and with (cleaved) the addition of MMP-2.

4.3.

Optoacoustic Results

The measured, calibrated peak-to-peak optoacoustic signals obtained with each laser are plotted. The results obtained with the Oxazine 720 laser, which should cover the peak of the cleaved probe, are shown in Fig. 6 . As expected from the extinction coefficient spectra, the cleaved probe has a very strong peak centered near $666\mathrm{nm}$ . The uncleaved probe absorbs much less strongly than the cleaved probe in this range, as expected. In this range, the uncleaved probe signal is consistently twice as strong as that of the water in the control tube, however.

Fig. 6

Measurements made in the range $660\text{to}680\mathrm{nm}$ on tubes containing the probe before and after cleavage with MMP-2, as well as a tube containing water alone.

The results obtained with the LDS 722 laser, which should cover the peak of the uncleaved probe, are shown in Fig. 7 . The uncalibrated results, in which the differences among the curves are more clearly visible, are shown in Fig. 8 . The uncleaved probe produces a clear peak near 730, as expected from the extinction coefficient spectrum, although it is somewhat weaker than expected. The cleaved probe also appears to be absorbing much more strongly relative to the uncleaved probe than would have been expected from the extinction coefficient spectrum.

Fig. 7

Measurements made in the range $695\text{to}755\mathrm{nm}$ on tubes containing the probe before and after cleavage with MMP-2, as well as a tube containing water alone.

Fig. 8

Raw, uncalibrated measurements made in the range $695\text{to}755\mathrm{nm}$ on tubes containing the probe before and after cleavage with MMP-2, as well as a tube containing water alone.

There are several potential explanations for the relatively weak signal from the uncleaved form of the probe, although these are all necessarily speculative and will require further experimentation to resolve. One explanation is that the uncleaved probe has a smaller Grüneisen parameter than expected. The Grüneisen parameter $\left(\Gamma \right)$ is the efficiency of the conversion of absorbed light energy into acoustic energy. The interaction between the aggregates could be interfering with the thermal expansion by reducing the expansion of the absorber when irradiated. A second potential explanation would be the presence of transient absorption changes due to alternative pathways for the deactivation of the excited aggregates, a phenomenon that has actually been observed in porphyrin aggregates, which are chemically similar to the molecules we are employing.⁴⁰