Surfactant-stripped Micelles for NIR-II Photoacoustic Imaging through 12 cm of Breast Tissue and Whole Human Breasts

Abstract

Surfactant-stripped micelles are formed from a commercially available cyanine fluroalkylphosphate salt dye (CyFaP) and used for high contrast photoacoustic imaging in the second near infrared window (NIR-II). The co-loading of Coenzyme Q10 into surfactant-stripped CyFaP (ss-CyFaP) micelles improves yield, storage stability and results in a peak absorption wavelength in the NIR-II window close to the 1064 nm output of Nd-YAG lasers used for photoacoustic imaging. Aqueous ss-CyFaP dispersions exhibit intense NIR-II optical absorption, calculated to be greater than 500 at 1064 nm. ss-CyFaP is detected through 12 cm of chicken breast tissue with PAI. In preclinical animal models, ss-CyFaP is visualized in draining lymph nodes of rats through 3.1 cm of overlaid chicken breast tissue. Following intravenous administration, ss-CyFaP accumulates in neoplastic tissues of mice and rats bearing orthotopic mammary tumors without observation of acute toxic side effects. ss-CyFaP is imaged through whole compressed human breasts in three female volunteers at depths of 2.6 to 5.1 cm. Taken together, these data show that ss-CyFaP is an accessible contrast agent for deep tissue photoacoustic imaging in the NIR-II window.

Photoacoustic imaging (PAI) is a hybrid optical-ultrasound imaging modality that has garnered interest for imaging in deep tissues. Exogenous contrast agents have been explored extensively in PAI.^[1–4] These enable techniques for molecular imaging^{[5, 6]} and multiplexed imaging^[7], and improving imaging depth, relative to other optical imaging modalities, is a driving attribute. Generally, advanced nanomaterials have excellent potential for PAI owing to strong near infrared (NIR) absorption and can be used not only for photoacoustic contrast imaging but also photothermal therapy.^{[8, 9]} Silver nanocrystals^[10] and transition-metal chalcogenides^[11] have been demonstrated as PAI agents. Methylene blue (667 nm) has been used in clinical studies to detect sentinel lymph nodes (SLN) in breast cancer to approximately 1–2 cm in depth.^[12] ICG (800 nm) was demonstrated to detect clinical melanoma in SLNs.^[13]

The second near infrared window (NIR-II) is appealing for in vivo optical imaging, due to reduced light scattering at longer wavelengths.^[14] In recent years, interest in NIR-II contrast agents has increased.^[14–32] For PAI, the use of contrast agents with absorption at 1064 nm matches the output of Nd:YAG pulse lasers, has a higher American National Standards Institute (ANSI) safety limit for laser pulse power, and reduces scattering for deep tissue imaging. Many contrast agents have been explored for PAI at 1064 nm including copper sulfide^[33], semi-conducting polymers^[34–37], gold nanorods^{[38, 39]} and others.

Previously, we demonstrated that a Tween formulation of a NIR-II-absorbing phosphorous phthalocyanine dye could be imaged through 11.6 cm of chicken breast tissue with PAI.^[40] That approach used a non-commercial dye with low synthetic yield (in our hands), and was not amenable to surfactant stripping. In this work, we formulate a commercial dye (available at the gram scale) into surfactant-stripped (ss) micelles with extreme NIR absorption. Hydrophobic cargos, such as pheophytin and naphthalocyanines, can be subjected to surfactant-stripping using Pluronic (Poloxamer) F127 triblock copolymer surfactant (F127).^[41–44] As shown in Figure 1A, hydrophobic cargo remains stable in suspension while low-temperature surfactant-stripping removes all free and loose F127 with a membrane, resulting in concentrated cargo dispersions. This approach is enabled by the unique temperature-sensitive properties of F127, resulting in the conversion of micelles to unimers at low temperature.

Figure 1: Surfactant-stripped CyFaP (ss-CyFaP).

A) Illustration of the surfactant stripping process. Pluronic micelles and cargos are shown as indicated. Lowering the temperature of the solution changes the critical micelle concentration (CMC) so that bulk micelles dissociate into unimers that can be stripped away with a membrane. B) Structure of cyanine fluoroalkylphosphate (CyFaP), a commercial NIR-II dye; C) Normalized absorbance of CyFaP in organic solvent (DCM), and in Pluronic F127 before and after stripping process. D) Calculated absorption at 1064 nm of CyFaP in commonly used pharmaceutical surfactants following attempted concentration by centrifugal filtration. E) Absorbance of CyFaP in ss-micelles compared to CyFaP solubilized with DMPC liposomes.

Since we introduced the concept of surfactant-stripping, other groups have used the surfactant-stripping approach for generating concentrated hydrophobic cargo dispersions for biomedical^{[45, 46]} and other^[47] applications. Given that there is interest in NIR-II imaging and that surfactant-stripping can induce micelle dispersions with high absorption contrast in a simple and scalable fashion, we set out to generate surfactant-stripped (ss) NIR-II dye micelles for PAI. In this work, we find that addition of a stabilizing hydrophobic co-cargo was essential for ss-micelle production and we further demonstrate these micelles are effective for PAI at multi-centimeter depths in breast chicken phantoms, rodent mammary tumor model, lymphatic mapping and whole compressed breast PAI in human volunteers.

We assessed surfactant-stripping using a commercially available NIR-II dye, a cationic cyanine fluroalkylphosphate salt; CyFaP (Figure 1B). Stable ion paring renders the dye soluble in organic solvents such as dichloromethane (DCM). CyFaP micelles were formed by dropping a DCM solution of CyFaP into an aqueous stirring solution of 10% F127 (w/v), followed by DCM evaporation. The absorbance spectra of CyFaP in DCM and F127 (“Pre-strip”) is shown in Figure 1C. In DCM, the CyFaP absorption peak was close to 1013 nm, in the NIR-II window. CyFaP dispersed in Pluronic F127 micelles exhibited a slightly shifted peak around 1020 nm. F127-solubilized CyFaP was subjected to surfactant-stripping carried out by washing the micelles with excess water (pH 6.5) using centrifugal filtration units with 100 kDa MWCO at 4 °C. Following surfactant-stripping, the absorbance spectra of CyFaP following exhibited a shift with two peaks apparent around 1040 and 1120 nm.

Owing to the unique temperature-sensitive CMC properties of Pluronics, only this class of surfactants is capable of low-temperature surfactant stripping. Figure 1D shows that F127, but not other common pharmaceutical surfactants (i.e., Cremephor EL and Tween 80), could effectively concentrate CyFaP solutions at low temperatures using centrifugal filtration. The calculated absorption at 1064 nm was close to 500. Figure 1E shows the efficiency of ss-CyFaP for generating high NIR-II contrast compared to a typical liposomal preparation, which did not effectively solubilize CyFaP. Surfactant-stripping provided orders of magnitude higher NIR-II absorption, which is useful for PAI contrast imaging. To ascertain if closely packed CyFaP molecules introduced any concentration-dependent changes in the absorbance spectra, we measured the absorbance spectra of ss-CyFaP micelles in cuvette of different path lengths (10 μm and 1 cm). The 1 cm pathlength measurements were carried out with dilute samples, whereas the 10 μm pathlength measurements used the samples without dilution. As shown in Figure S1, the spectra did not exhibit significant concentration-dependent spectral changes.

Unfortunately, during refrigerated storage, ss-CyFaP aggregated, with visible precipitation becoming apparent after several days. We previously reported the use of hydrophobic “co-loaders” for enhancing the stability of ss-micelles.^[41] Other hydrophobic molecules that are loaded together with the active cargo can potentially inhibit micelle aggregation (schematic shown in Figure 1A). Various co-loaders including Vitamin D3 (D3), beta-carotene (β-car), alpha-tocopherol (α-toc) and Coenzyme Q10 (CoQ) were assessed for stabilizing ss-CyFaP. As shown in Figure 2A, CyFaP micelles co-loaded with CoQ or α-toc improved dye retention during the stripping process whereas vitamin D3 and β-car had minimal impact. A CoQ co-loading mass ratio of at least 0.6:1 appeared to enhance ss-CyFaP yield (Figure 2B) A 1:1 mass ratio of co-loader to CyFaP was used for subsequent studies. Since CoQ and α-toc are safe and biocompatible molecules widely used vitamin supplements, and also induced yield enhancements during surfactant-stripping, these co-loaders were assessed further.

Figure 2: Coenzyme Q co-loading enhances ss-CyFaP yield and stability.

A) CyFaP yield in Pluronic F127 micelles co-loaded with hydrophobic cargo. (B) Calculated absorbance of ss-CyFaP micelles at indicated CoQ mass ratios (C) Stability of ss-CyFaP with indicated co-loader, in storage at 4 °C (D) Absorbance spectra of ss-CyFaP micelles with no co-loader or with CoQ as a co-loader (E) Stripping of free and loose Pluronic F127 as a function of centrifugal washing cycles at 4 °C (F) Negative-stained electron micrograph of CoQ-coloaded ss-CyFaP micelles.

To assess whether co-loading hydrophobic vitamins or vitamin-like molecules improves stability, ss-micelles were prepared with either CyFaP alone or CyFaP co-loaded with α-toc or CoQ. Samples were stored at 4 °C and dispersed dye stability was monitored for 4 weeks (Figure 2C). Stability of the formulations was assessed by the NIR-II absorbance of the samples. ss-CyFaP micelles exhibited aggregation in the first week leaving only 70 % of the dye encapsulated in dispersed micelles. α-toc co-loaded ss-CyFaP micelles showed slightly higher stability after the first week, but approximately 40% of the dye aggregated at the end of second week. On the other hand, CoQ co-loaded ss-micelles showed less than 5 % aggregation after 4 weeks of storage. The mechanism of CoQ co-loading enhanced stability is not clear; however it is presumably based on interaction with CyFaP in the hydrophobic core of the micelle, resulting in reduced aggregation. As shown in Figure 2D, inclusion of CoQ with ss-CyFaP resulted in slight shift of the CyFaP absorption peak. For all subsequent studies, CoQ was included in ss-CyFaP unless otherwise noted.

To quantify F127 surfactant-stripping, the cobalt thiocyanate assay was used to measure F127 (Figure 2E). An F127 standard curve was made to estimate the amount of Pluronic in the filtrate (Figure S2). After five washing cycles of CyFaP-CoQ micelles at pH 6.5, the free and loose F127 was nearly completely eliminated. In addition, CyFaP and CoQ were retained with ss-micelles with minimal loss. Electron microscopy (EM) suggested that ss-CyFaP micelles were spherical with a varying diameter between 10–30 nm, as shown in Figure 2F. Size measurements from DLS corroborated EM measurements (Figure S3). The stability of ss-CyFaP micelles in a biological environment was assessed in vitro by incubating ss-CyFaP micelles with 50% fetal bovine serum at 37 °C. After 24 hours, there was no change in NIR-II absorbance, indicating serum stability in biological media in vitro (Figure S4).

To study the deep tissue imaging with ss-CyFaP, we performed PAI studies in vitro. A phantom containing ss-CyFaP micelles was placed between chicken breast tissues and imaged. A large plastic container was used to hold chicken breasts for PAI studies. Approximately 1.25 mL of ss-CyFaP micelles (15 mg ml⁻¹ dye) or water were placed in plastic tubes with a 5 mm inner diameter and positioned in the middle of the container stacked with 12 cm of chicken breast tissue on top as well as beneath the tubes as shown in Figure 3A. Chicken breast tissue surrounded the tube in all directions for 12 cm to rule out light leakage from any direction. Figure 3B shows a photograph of the setup for phantom PAI studies. Overlaid PA/US images of the phantom containing a tube filled with water (left) or ss-CyFaP (right) placed 12 cm beneath chicken breast tissue are shown in Figure 3C. The US signal (gray scale) originating from the phantom containing water (left) is clearly visible at 11.6 cm depth, but no PA signal is detected. However, for the tube filled with ss-CyFaP, both US (gray scale) and PA (color scale) are clearly visible in the phantom, showing the deep tissue PA constrast imaging capabilities. PA signals from depths more than 12 cm were found to be obscured by noise. To improve the signal to noise ratio (SNR), we acquired 100 frames at the same depth and averaged them. By removing the chicken breast layers, SNR was computed for different penetration depths. At 12 cm depth, the SNR was determined to be 24.3 dB (Figure 3D). To ensure there was no interference from CoQ at 1064 nm, CoQ alone micelles were prepared at 15 mg mL⁻¹ and imaged. A tube containing ss-CyFaP exhibited a strong PAI signal whereas CoQ itself was not detected (Figure S5).

Figure 3: Deep tissue photoacoustic imaging of ss-CyFaP micelles.

A) Schematic of the imaging setup. B) Photograph of the container filled with chicken breasts. A tube filled with 15 mg mL⁻¹ ss-CyFaP (or water) was surrounded by at least 12 cm of chicken breast tissues on all sides. C) Photoacoustic (PA) image (color scale) overlaid with ultrasound (US) image (gray scale) of tube filled with water (left) or ss-CyFaP micelles (right). 45 mJ cm⁻² laser energy was used. D) Signal to Noise ratio of PA signal as a function of tissue thickness on top of the tube.

Noninvasive imaging techniques like PAI have been employed for sentinel lymph node detection.^{[48, 49]} To demonstrate lymph node imaging capabilities of ss-CyFaP micelles, rats were injected with 5 mg kg⁻¹ ss-CyFaP micelles (dye basis) via footpad. Photoacoustic imaging was performed 30 minutes after injection by placing 1 – 5 cm of chicken breast tissue on top of lymph node region as shown in Figure 4A. PA signal from ss-CyFaP micelles was clearly visible in the lymph node through a 3.1 cm chicken breast stack placed on top of lymph node area (Figure 4B). Accumulation of ss-CyFaP in the lymph node was confirmed by ex vivo visualization (Figure S6), although the color was faint given the minimal absorption of CyFaP in the visible spectra. PA signal to noise ratio was found to decrease (slope of −9.8 dB cm⁻¹) with increasing penetration depth. The signal to noise ratio of the PA response in the lymph node through 3.1 cm external tissue was 26.5 dB (Figure 4C). PA amplitude generated from the lymph node region with dye injection was found to be nearly 6 folds higher compared to the control signal (Figure 4D).

Figure 4: In vivo imaging using CyFaP.

A) Setup for lymph node imaging of rats injected with 5 mg kg⁻¹ ss-CyFaP. Chicken breast tissue was stacked on top of the rat. B) Overlaid PA/US image of lymph node in rat stacked with 3.1 cm chicken breast tissue imaged 30 min post footpad injection. C) Signal to noise ratio of PA signal generated from lymph node as a function of depth. D) Comparison of PA amplitude in the lymph node before and after injection of ss-CyFaP. E) Pharmacokinetics of ss-CyFaP after intravenous injection of 60 mg kg⁻¹ dye in mice. F) Biodistribution in mice bearing orthotopic 4T1 tumors 6 hours after intravenous injection with 60 mg kg⁻¹ ss-CyFaP. G) Representative in vivo PA tumor images of mice bearing orthotopic 4T1 tumors, 6 h post injection of 60 mg kg⁻¹ ss-CyFaP (right) and control tumor-bearing mice without injection (left). H) In vivo PA tumor imaging of a rat bearing an orthotopic mammary tumor 6 h post injection of 55 mg kg⁻¹ ss-CyFaP (right) and control rats without injection (left).

To determine pharmacokinetic properties of ss-CyFaP, 60 mg kg⁻¹ of dye was intravenously injected in healthy ICR mice. Blood was sampled at various intervals and the amount of dye in blood was estimated based on the absorbance of serum at 1064 nm. Figure 4E shows the absorbance of ss-CyFaP micelles in blood at various time points. The half-life of ss-CyFaP micelles was found to be 5.6 hours based on non-compartmental analysis. Next, to determine where the dye localizes following administration, female BALB/c mice bearing orthotopic 4T1 tumors were injected by tail vein with 60 mg kg⁻¹ ss-CyFaP micelles. After 6 hours, mice were sacrificed and liver, spleen, kidneys, heart, and tumor was harvested. Tissue was homogenized, centrifuged at 1500 rcf, and absorbance of the homogenate supernatant was measured. Approximately 10% of the injected dose per gram accumulated in the tumor at the 6 h time point (Figure 4F). Most of the CyFaP was found to accumulate in liver and spleen.

To further demonstrate the versatility of ss-CyFaP as a PAI imaging contrast agent, we carried out tumor imaging studies in mice and rats. Tumor imaging studies in mice were carried out in subjects bearing orthotopic 4T1 tumors. Mice were injected with 60 mg kg⁻¹ of ss-CyFaP and imaged 6 hours after injection. The optical setup used for mice and rat tumor imaging is shown in Figure S7. Imaging at 1064 nm showed strong photoacoustic signal in the tumor 6 hours after injection (Figure 4G), consistent with the biodistribution data showing accumulation of ss-CyFaP. Tumors in control mice that did not receive contrast administration did not show PA signal. The NIR-I and NIR-II spectral features of CyFaP micelles were found to be similar to the ex-vivo photoacoustic spectra of excised tumors, suggesting that the CyFaP dye remains intact after accumulating in the tumor (Figure S8). Next, we performed tumor imaging studies in a rat model to assess PAI capabilities of ss-CyFaP micelles in larger animals with tumors. Rats bearing large orthotopic R3230 mammary tumor were injected with 55 mg kg⁻¹ ss-CyFaP micelles via tail vein and imaged after 6 hours. As shown in Figure 4H, PA signal was observed only in the tumor of rats administered ss-CyFaP. These results demonstrate the in vivo capability of ss-CyFaP micelles for PA tumor imaging. For in vivo rodent tumor imaging experiments, light intensity originating from PAI system was approximately 12 mJ cm⁻² which is several folds below the American National Standards Institute safety limit of 100 mJ cm⁻².^[50] To determine if the 1064 nm pulse laser used in PAI resulted in photothermal heating, the temperature of concentrated ss-CyFaP in a tube was monitored throughout 10 minutes of laser irradiation at 12 mJ cm⁻², the energy used for the in vivo animal experiments. Less than a 3 °C increase was observed compared to saline control (Figure S9). This suggests that photothermal heating is a possibility that should be considered with the concentrated contrast agent, in some circumstances.

To assess potential cytotoxicity of ss-micelles in vitro, ss-CyFaP micelles were incubated with U87 cells at various concentrations, along with methylene blue, a common dye used in PAI (Figure S10). Methylene blue showed toxicity at low absorption values while ss-CyFaP showed no signs of cytotoxicity even at calculate NIR-II absorption as high as 100. Next, ss-CyFaP micelles were administered to healthy BALB/c mice at a dose of 60 mg kg⁻¹. Following intravenous administration, the weight of mice was monitored for two weeks. After two weeks, mice were sacrificed and blood was collected for serum chemistry profile and complete blood count (CBC) analysis. Key organs were harvested for H&E staining to assess the toxicity induced by ss-CyFaP micelles in mice. Serum chemistry profile and CBC results of ss-CyFaP micelles injected mice were compared to healthy mice and the results are as displayed in Figure 5A and 5B. Differences in eosinophils (EOS) and red cell distribution width (RDW) % were found to be statistically significant by two-tailed t-test. However, both EOS and RDW were found to be within the expected range for healthy mice. As shown in Figure 5C, H&E staining of organs did not reveal any obvious toxicity to liver, spleen, kidneys, lungs or heart. This suggests the mouse dose of 60 mg kg⁻¹ (a dose sufficient to provide an 1064 nm absorption of 40, when the injection volume is diluted in 1 mL of water) of ss-CyFaP micelles is tolerated in mice with intravenous injection. Mice injected with ss-CyFaP micelles showed statistically significant weight loss on the first day post injection but recovered completely in the following days and remained healthy until they were sacrificed after two weeks (Figure 5D). Additional studies are warranted to assess toxicity, ss-CyFaP dose response, and maximum tolerated doses.

Figure 5: Assessing the toxicity of ss-CyFaP micelles.

A) Serum chemistry and B) complete blood count of control and treated mice (n=5 per group) 2 weeks post intravenous injection of 60 mg/kg ss-CyFap. C) H&E staining of organs harvested 2 weeks after injection. Asterisk indicates statistically significant groups when unpaired t-test was performed. D) Weight data for mice injected with 60 mg kg⁻¹ ss-CyFaP micelles or control mice. Asterisk indicates statistically significant difference in weight when compared to the initial weight based on paired student t-test. There were no statistically significant differences in weight change between the two groups at any time, based on unpaired t-test.

Given the high NIR-II absorption of ss-CyFaP micelles and promising results obtained with in vitro deep tissue imaging, we attempted to image the dye through whole human breasts. The setup for human breast PAI studies is shown in Figure 6A. A tube containing ss-CyFaP micelles was placed beneath the breast of healthy adult female volunteers. PA/US imaging was performed and the results were reconstructed using MATLAB. Figure 6B shows photoacoustic signals of the tube through compressed breast tissue of healthy human volunteers. PAI of ss-CyFaP enabled detection of the tube through all the volunteers with at the following breast depths: 2.6 cm (left, volunteer # 1 with C-cup breast size), 3.8 cm (middle, volunteer # 2 with D-cup breast size) and 5.1 cm (right, volunteer # 3 with C-cup breast size). At the greatest imaging depths, some non-specific background signal was visible above the tube, but the photoacoustic contrast in the tube was clearly detectable. The laser energy irradiated on the human breast surface was measured to be 21 mJ/cm², which is considerably lower than the 100 mJ/cm² ANSI safety limit at 1064 nm, so that increasing laser power may be able to further improve signal to noise and increase imaging depth. These results demonstrate the potential of ss-CyFaP micelles as a contrast agent for deep-tissue PAI in humans.

Figure 6: Photoacoustic imaging of ss-CyFaP through the breast of healthy human adult volunteers.

A) Schematic drawing of the PAI setup for in vivo imaging. B) US, PA overlaid images of tube containing ss-CyFaP placed beneath the breast of three different human adult female volunteers with indicated cup size.

In summary, ss-CyFaP micelles are formed using a commercially available dye and are stabilized with the co-loading of CoQ. This approach yields a colloidal photoacoustic contrast agent with extreme NIR-II contrast. ss-CyFaP was imaged through 12.0 cm of chicken breast tissue which is, to the best of our knowledge, amongst the deepest tissue optical imaging reported to date. Studies performed in mice and rats show the capability for lymphatic and tumor imaging, with minimal observed toxicity. The use of ss-CyFaP for the first demonstration of imaging across whole human breasts further demonstrates capability for deep-tissue imaging. Taken together, these data show that ss-CyFaP is a readily prepared NIR-II contrast agent for photoacoustic contrast imaging.

Experimental

Materials:

Pluronic F127 (Sigma # P2443), Cremophor EL (Sigma # C5135) were ordered from Sigma and Polysorbate 80 (Cat # EM-9490) from VWR. Coenzyme Q10 (Sigma # 45-C9538), Carotene (TCI America, #C0560), Vitamin D3 (Alfa Aesar, #B22524), α-toc (Sigma, #T3251), methylene chloride (fisher), polysorbate 80 (VWR # EM-9490) and Cremophor EL (Sigma # C5135) were purchased. CyFaP was obtained from Spectrum Info Ltd. (#S8734).

Micelle Preparation:

For small scale studies, 1 mg of dye was dissolved in 4 mL of methylene chloride (DCM) and sonicated for 5 seconds to ensure the dye is dissolved completely. Dye in DCM was added to 10% (w/v) Pluronic F127 (28 mL) dropwise under constant stirring. Pluronic solution was stirred until the DCM evaporated, while the solution turned from milky-white to clear. For Tween 80 and Cremophor EL micelles, dye in DCM was added to 10% w/v surfactant solutions and stirred for 4 hours. When the samples became clear, they were centrifuged for 15 min at 3000 rcf to remove free dye. The pellet, if any, from the previous step was discarded and the supernatant was subjected to centrifugation using Amicon centrifugal filtration units (MWCO: 100,000 Da) for 20 min at 4 °C. Post washing, cool water was added to retentate and washing was continued for 4 additional cycles. After the last washing cycle was complete the retentate was diluted (to 1 mL) to normalize the concentration. For large scale studies involving more than 10 mg dye, a diafiltration membrane (Sartorius Vivaflow 200, 100 kDa MWCO, #VF20H4) was used. To maintain the tubing, membrane and sample at 4 °C, the entire setup (except the peristalsis pump) was placed in ice. After 5–6 washing cycles, sample volume was reduced to 50 mL, and centrifugal filtration was used to further concentrate the samples to less than 2 mL. For all studies detailed below, ss-CyFaP represents ss-micelles co-loaded with CoQ (1:1) unless otherwise specified. No dye loss was observed during the large scale stripping process. ss-CyFaP exhibited a yield of 59% with filtration through a 0.22 μm sterile filter. An Agilent Cary 7000 Universal Measurement Spectrophotometer was used for absorbance measurements beyond 1100 nm. A Perkin Elmer Lambda 365 spectrophotometer was used for all other absorbance measurements.

Pluronic F127 was quantified as described previously.^[44] In brief, cobalt thiocynate was prepared by adding cobalt nitrate hexahydrate (0.3 g) and ammonium thiocynate (1.2 g) in 3 mL water. Further, cobalt thiocynate, Pluronic standard solution (known concentration), ethanol and ethyl acetate were mixed in 1:0.4:2:0.8 volume ratios and centrifuged at 14,000 g for 2 minutes. Pellet at the bottom was washed multiple times and then dissolved in acetone and absorbance was measured at 623 nm. For ss-CyFaP micelles, after every wash using membrane filters, the filtrate was allowed to react with cobalt thiocynate as described above and pellet was dissolved in acetone and percentage of Pluronic removed was measured. Dye encapsulated in micelles was estimated based on the absorbance of dye in the sample. Concentration of CoQ was estimated based on the absorbance peak at 275 nm. The final ratio of Pluronic F127 to CyFaP in ss-micelles was found to be 2.3:1.

Cargo co-loaded micelles:

Cargo co-loaded dye micelles were prepared as described in the methods above, except before the addition of dye dissolved in DCM to Pluronic, the dye in DCM was mixed with the indicated co-loaders. Samples were stirred until the solution turned clear from milky white. Dye containing Pluronic solution was centrifuged at 3000 rcf for 15 min to remove any unloaded dye. The supernatant was then subjected to centrifugal filtration with 15 mL centrifugal filtration units for 5 cycles of 25 min each at 4 °C. After the final wash samples were concentrated to 200 μL and absorbance of the sample was measured. Dye retention capacity conferred by the co-loaders was estimated by comparing the initial absorbance to final absorbance.

To determine the maximum absorbance, varying ratios of CyFaP to CoQ were prepared by co-loading CyFaP and CoQ in ss-micelles. The mass ratio of CyFaP to DCM to Pluronic was 1:4:28 and micelles were prepared as described in the previous section. Dye (3 mg) was dissolved in DCM (12 mL) and dropped in to 10% (w/v) Pluronic F127 (84 mL). All samples were centrifuged using centrifugal filtration units (MWCO = 100 kDa) until the sample volume was reduced to 200 μL. Absorbance of the samples at 1064 nm was measured by diluting the samples in water and the dilution factor was taken into account to arrive at the calculated absorbance. To measure the absorbance spectra of ss-CyFaP micelles at high optical density, concentrated samples were either diluted in water (for 1 cm cuvette) or directly measured using 10 μm path length cuvettes. ss-CyFaP micelles and ss-CyFaP- α-toc (1:1) micelles were prepared by co-loading method as detailed before. After the samples were washed and concentrated, samples were filtered through a 0.2 μm filter and stored at 4 °C for 4 weeks. Samples were centrifuged at 3500 rcf every week. Pellet (if any) was discarded and absorbance of the supernatant was measured to monitor the dye retention and micelle stability.

Liposomes were prepared by addition of 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) and CyFaP in 1:19 molar ratio. Chloroform was evaporated from the lipid-dye mixture to form a lipid film. Lipid-CyFaP mixture was hydrated with phosphate buffered saline (PBS) (500 μL) and subjected to sonication for 15 minutes. Liposome samples along with ss-CyFaP micelles were then subjected to lyophilization overnight to enable further concentration. The dried samples were dissolved in 50 μL water and absorbance of the sample was measured.

Hydrodynamic size of the micelles was measured using DLS (Nanobrook 90 plus PALS). Transmission electron microscopy of ss-CyFaP micelles was performed using JEM-2010 electron microscope with 1% uranyl acetate as negative staining agent.

In vitro studies:

Serum stability studies were performed by incubating ss-CyFaP micelles with 50% fetal bovine serum for 24 hours at 37 °C. Samples were pipetted out at indicated time points and absorbance of the sample was recorded using Perkin Elmer spectrophotometer. CyFaP retention was calculated with absorbance at t₀ as reference. For cell viability studies, human glioblastoma (U87) cells were procured from ATCC. Cells were grown in DMEM with 10% FBS and 1% antibiotics. Cells were seeded in to a 96-well plate at 10,000 cells per well concentration. Cells were allowed to adhere and 24 hours later they were incubated with ss-CyFaP micelles or methylene blue samples at same OD for 24 hours. Cells were washed with PBS twice post incubation and fresh media with FBS was added and incubated at 37 °C. After 24 hours, cells were subjected to XTT assay to assess the cell viability.

Cell viability assay was performed by removing the media from the 96-well plate and adding 100 μL of XTT stock solution into each well. XTT stock solution was prepared by adding 50 μg mL⁻¹ of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 30 μg mL⁻¹ of N-methyl dibenzopyrazine methyl sulfate to PBS. After adding XTT stock solution to 96-well plate, the plate was incubated at 37 °C for 2 hours. TECAN plate reader was used to read the 96-well plate at 450 nm with 630 nm subtracted as background. Cell viability of treated cells was calculated relative to untreated cells. In vitro cell viability experiment was performed for n=3. Error bars indicate mean +/− standard deviation for n=3.

Deep tissue photoacoustic imaging:

To estimate the depth of penetration that could be achieved using the ss-CyFaP micelles, we imaged a 5 mm diameter tube loaded with 600 OD (1.25 mL of 15 mg ml⁻¹ dye) ss-CyFaP micelles. The length of the tube filled with ss-CyFaP micelles was 4.5 cm. We filled a 31 × 27 × 27.5 cm container with chicken breasts up to a height of 12 cm from the bottom. The tube containing ss-CyFaP micelles was placed exactly at the center of the container. Chicken breasts of about 4.5 cm thicknesses were placed on top layer by layer, and tube was imaged after addition of chicken breast tissue each time until the total depth reached 12 cm. A custom-made 128-element 2.25 MHz linear array transducer (Imasonic, Inc.) with 2.25 MHz central frequency was used for ultrasound and photoacoustic detection. The tube was illuminated as shown in Figure 3A, and the surface light intensity was 45 mJ cm⁻², which is less than half of the ANSI safety limit at 1064 nm.^[50] 1064 nm laser irradiation area was 15 cm². Photoacoustic results were reconstructed using universal back-projection algorithm.^[51] Results were plotted in color and overlaid on top of grayscale ultrasound images.

A tube containing CyFaP (15 mg mL⁻¹) or Coenzyme Q alone (15 mg mL⁻¹) was prepared. For 1064 nm imaging tube containing samples were placed parallel to each other with a 2 cm distance between them. The excitation source was a 10 ns Nd:YAG laser, with 10 Hz pulse repetition rate at 1064 nm output wavelength. A custom-made 128-element linear array transducer (Imasonic Inc.) was placed perpendicular to a linear output fiber bundle; and a dichroic mirror (97 % transmission at 1064 nm, cold mirror, Edmund Optics Inc.) was placed at 45° to each component. This design allowed for co-planar light illumination and acoustic detection. The tubes were scanned along their length at a scan speed of 1 mm/s for 40 s. The PA data acquired was reconstructed using universal back-projection algorithm.^[51]

Transient temperature monitoring:

A thermocouple was used to determine the increase in temperature. 1064 nm laser with pulse duration of 10 ns was used for these studies. Excitation source for the system was an Nd:YAG laser, with 10 Hz pulse repetition rate and 10 ns pulse duration. A circular fiber bundle of input and output diameter 1.4 cm was used for light delivery. Temperature probe was dipped in the tube containing dye and temperature was recorded every minute for 10 minutes. Similar procedure was followed for PBS which served as control. Samples were irradiated at 12 mJ cm⁻² which corresponds to the energy delivered for in vivo animal studies. Transient temperature monitoring results indicate the mean values for triplicate measurements.

Animal studies:

All animal experiments were performed in compliance with the animal protocol approved by Institutional Animal Care and Use Committee (IACUC) at University at Buffalo. For pharmacokinetic studies, ICR mice were intravenously injected with 60 mg kg⁻¹ ss-CyFaP micelles and blood samples were collected by retroorbital location at 0.5, 1, 2, 4, 8, 24 h post injection. Blood samples were centrifuged for 15 min. at 1500xg. 10 μL of serum was extracted of which 2 μL was diluted in 1 mL water and absorbance was measured using spectrophotometer. For pharmacokinetic studies, error bars indicate mean +/− standard deviation for n=3.

For biodistribution studies, 60 mg kg⁻¹ (in an injection volume sufficient to produce an absorption at 1064 nm of 40, if diluted into 1 mL volume and measured in a 1 cm path length cuvette) ss-CyFaP micelles was injected in mice via tail-vein and mice were sacrificed after 6 hours. Tumor, liver, spleen, heart, and kidneys were harvested and dye accumulated in the tissue was analyzed. All the organs were weighted and approximately 100 mg of each tissue was homogenized in DCM using Bullet Blender. Homogenized samples were centrifuged at 3000g and absorbance of the supernatant was measured at 1018 nm. Samples were diluted in DCM and dilution factor was accounted for while calculating the final absorbance. Total CyFaP in each organ per gram of the tissue was calculated. For biodistribution studies, error bars indicate mean +/− standard deviation for n=6.

For toxicity studies, BALB/c mice were either injected with 60 mg kg⁻¹ (40 OD) of ss-CyFaP micelles or saline. Mice were observed on daily basis to monitor if they remained healthy for 14 days. Weight of the mice was monitored at regular intervals for 14 days and on day 15 they were sacrificed. Liver, spleen, kidneys, lungs, and heart were harvested and blood was collected. Tissues were immediately transferred to formalin bath and were further processed for H&E staining. Blood was collected via cardiac puncture and the serum was analyzed and complete blood count and serum chemistry studies were performed. Error bar indicates mean +/− standard deviation for n=5.

Tumor imaging:

The excitation source for PAI system was an Nd:YAG laser, whose output was 1064 nm with 10 Hz pulse repetition rate and 10 ns pulse duration. For this experiment, a water tank with a window at the bottom was used. The window was sealed with the thin plastic membrane. During experiments, rodents were anesthetized using isoflurane (1.5–2.5%) and placed underneath the window with ultrasound gel as coupling medium. To image the whole tumor, we scanned the L7–4 array (ATL/Philips 5 MHz central frequency, 128 elements) over 4 cm with a step size of 0.1 mm. The excitation light (1064 nm) was routed to the imaging region through a bifurcated optical fiber bundle with one circular input and two linear outputs. The light intensity on the skin surface of mice was around 12 mJ cm⁻², which is more than 8 folds lower compared to American National Standards Institute (ANSI) safety limit (100 mJ cm⁻² at 1064 nm).^[50] Laser irradiation area of the 1064 nm laser was calculated to be 8 cm². The received PA signals were amplified (by 54 dB) and digitized by a 128-channel ultrasound data acquisition (DAQ) system (Vantage, Verasonics) with 20 MHz sampling rate. After each laser pulse, the raw channel data was reconstructed using the universal back-projection algorithm, and was displayed in real-time during experiments. BALB/c mice bearing 4T1 orthotopic mammary tumors were used. 60 mg kg⁻¹ (40 OD) of ss-CyFaP micelles was injected via tail-vein. After 6 hours, mice were anesthetized and the region of tumor was imaged using the PACT system as described in the previous section. Rat mammary adenocarcinoma (R3230) was induced by injecting 1×10⁷ R3230 cells subcutaneously into rats. 55 mg kg⁻¹ of ss-CyFaP was intravenously injected into rats and the tumor was imaged using PACT system after 6 hours as described above.

Tumor ex vivo imaging:

For acquiring PA spectral data, we used the PACT system. The excitation source for the system was an Nd:YAG laser, with 10 Hz pulse repetition rate and 10 ns pulse duration. When the tumor size reached 8–10 mm, 60 mg kg⁻¹ ss-CyFaP was intravenously injected and 6 h later mice were sacrificed and tumor was excised. For this experiment, a water tank with a window at the bottom was used. The window was sealed with the thin plastic membrane. For acoustic coupling, ultrasound gel was applied on the excised tumor. A custom-made 128-element linear array transducer (Imasonic Inc.) with central frequency of 2.25 MHz was used for PA signal acquisition. A circular fiber bundle of input and output diameter 1.4 cm was used for light delivery. We acquired data at 750 nm, 850 nm, 950 nm and 1064 nm wavelengths. The laser output power at each wavelength was measured and the signal was normalized. To improve SNR, 50 frames of PA images were acquired and averaged.

Lymph node imaging:

Institutional animal care and use committee (IACUC) of Pohang University of Science and Technology (POSTECH) approved all the procedures of lymph node imaging. Nd:YAG laser (Surelite III-10, Continuum) generates pulsed laser beam with a wavelength of 1064 nm, a pulse width of 4 ns, and a repetition rate of 10 Hz was used. Healthy Sprague Dawley rats were prepared to acquire PA images of lymph nodes. A vaporized isoflurane system (VIP3000, Midmark) was used to anesthetize the animals. We used mixture of isoflurane (2%) in oxygen (100%) with a flow rate of 1 L min⁻¹. Once the animals were anesthetized, we used a heating pad to maintain the body temperature. Imaging area was shaved before the mice were injected. Rats were then placed on the imaging stage to acquire the image. For increased depth imaging, chicken tissue layers were stacked on the rats. The output laser beam was delivered to the top of the chicken tissue layers to generate PA signals. The pulse energy of laser was 62 mJ cm⁻², which is below the ANSI safety limit at 1064 nm (100 mJ cm⁻²).^[50] Laser irradiation area was estimated to be 4.9 cm². We used a clinical ultrasound (US) imaging system (EC-12R, Alpinison Medical Systems) to acquire PA and US images. The system is equipped with a128-element linear array transducer (L3–12, Alpinion Meidcal Systems) with a center frequency of 8.5 MHz and a fractional bandwidth (−20 dB) of 95%. By synchronizing the US machine and the laser, we simultaneously acquired both PA and US image and raw data acquired was subjected to post-processing. To generate both PA and US images, conventional delay-and-sum reconstruction algorithm was used. To acquire images of lymph nodes, we injected 5 mg kg⁻¹ (100 μL) ss-CyFaP micelles via footpad. By comparing the images before and after injection, we could identify the PA signals from the ss-CyFaP micelles. To assess the deep tissue imaging capability, we acquired PA and US data at various imaging depths (0.9, 2.3, 3.1 cm). To improve the SNR, we averaged 100 frames of PA images.

Imaging through whole human breasts:

All procedures involving human volunteers were in compliance with the University at Buffalo Institutional Review Board protocol. A 5 mm diameter tube was filled with ss-CyFaP micelles and placed underneath the compressed breast healthy female human volunteers. Mild compression was achieved by a small water container whose bottom was lined with FEP plastic film. To ensure acoustic coupling, we applied ultrasound gel between the top-breast surface and the plastic film, as well as between the bottom-breast surface and the tube. The photoacoustic excitation source was a 10 ns Nd:YAG laser with pulse repetition rate of 10 Hz and 1064 nm output. Light delivery and photoacoustic signal detection were achieved by a linear-output fiber bundle and a custom-made 128-element linear transducer array (Imasonic, Inc.) with 2.25 MHz central frequency. The two components were combined with acoustic reflectors to achieve coplanar light illumination and acoustic detection.^[52] We imaged two adult human healthy volunteers for this study. The surface light intensity during experiments was approximately 21 mJ cm⁻² and laser irradiation area was 450 cm². During the experiment, we first used ultrasound to identify the location of the tube and then turned on photoacoustic acquisition with 100 frames of averaging. Photoacoustic results were reconstructed using universal back-projection algorithm.^[51] Results were plotted in color and overlaid on top of grayscale ultrasound images.