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Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging Hoang Dung Lu, Brian K. Wilson, Andrew Heinmiller, Bill Faenza, Shahram Hejazi, and Robert K Prud'homme ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03059 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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ACS Applied Materials & Interfaces

Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging Hoang D. Lu,† Brian K. Wilson,† Andrew Heinmiller,‡ Bill Faenza,γ Shahram Hejazi,§ and Robert K. Prud’homme*,† †

Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, United States; ‡FUJIFILM VisualSonics, Toronto, Ontario, Canada; γPersis

Science, Andreas, Pennsylvania 18211, United States; §Optimeos Life Sciences LLC, Princeton, New Jersey 08544, United States

KEYWORDS Nanoparticle, photoacoustic, optoacoustic, imaging, tumor, folate

ABSTRACT

Photoacoustic (PA) imaging is an emerging hybrid optical-ultrasound based imaging technique that can be used to visualize optical absorbers in deep tissue. Free organic dyes can be used as

ACS Paragon Plus Environment

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PA contrast agents to concurrently provide additional physiological and molecular information during imaging, but their use in vivo is generally limited by rapid renal clearance for soluble dyes and by the difficulty of delivery for hydrophobic dyes. We here report the use of the blockcopolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to form series of highly-hydrophobic optical dyes into stable, biocompatible, and water-dispersible nanoparticles (NPs) with sizes from 38-88 nm and with polyethylene glycol (PEG) surface coatings suitable for in vivo use. The incorporation of dyes with absorption profiles within the infrared range, that is optimal for PA imaging, produces the PA activity of the particles. The hydrophobicity of the dyes allows their sequestration in the NP cores, so that they do not interfere with targeting, and high loadings of >75 wt% dye are achieved. The optical extinction coefficients (ε(mLmg-1cm-1) were essentially invariant to the loading of the dye in NP core. Co-encapsulation of dye with vitamin E or polystyrene demonstrates the ability to simultaneously image and deliver a second agent. The PEG chains on the NP surface were functionalized with folate to demonstrate folatedependent targeting. The spectral separation of different dyes among different sets of particles enables multiplexed imaging, such as the simultaneous imaging of two sets of particles within the same animal. We provide the first demonstration of this capability with PA imaging, by simultaneously imaging non-targeted and folate-targeted nanoparticles within the same animal. These results highlight Flash NanoPrecipitation as a platform to develop photoacoustic tools with new diagnostic capabilities.

INTRODUCTION Photoacoustic (PA) imaging is a hybrid imaging modality based on the detection of ultrasound waves generated by the thermoelastic expansion of materials irradiated with short


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laser pulses.1-4 PA images can be considered as ultrasound images whose contrast is dependent on the optical absorption profiles of the materials assessed.5 These characteristics have made PA imaging a promising tool for diagnostic imaging, as PA imaging benefits from both the high spectral selectivity of laser excitation, and the high spatial resolution of ultrasound imaging. PA imaging also utilizes both diffuse and ballistic light for imaging generation, providing tissue penetration depths that are much greater than classical optical imaging. PA imaging has been used in a variety of biomedical applications, including breast, skin, cardiovasculature, brain, and tumor diagnosis/monitoring.6-9 Anatomical differences between healthy and unhealthy tissue can lead to differences in tissue optical absorbance properties, which can be detected with PA imaging.10-12 However, reliance on only the intrinsic differences in tissue absorbance properties limits the amount of information that may be gained with PA imaging. Exogenously added contrast agents increase sensitivity and provide additional physiological information, such as the presence of cell surface markers and tumor phenotypes (Figure 1).13, 14 Gold-based particles have been the most widely used nanoparticles (NPs) in PA imaging.8, 9, 12, 15 To create gold particles with optical absorption profiles shifted into the near infrared (NIR) range necessary for imaging, high aspect ratio gold structures must be synthesized, and the variation in aspect ratio causes broadening of the absorption peak. Organic optical dyes are promising PA contrast agents relative to gold nanorods or carbon nanotubes, as they can be readily synthesized with relatively sharp absorption peaks in the NIR window (600900 nm) optimal for PA imaging.16-18 The sharp absorption peaks are especially advantageous in multiplexed PA imaging, as will be demonstrated here, where several optical dyes can be imaged simultaneously.


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However, organic optical dyes that have absorption maxima in the NIR range are usually large, poly conjugated, or polyaromatic, and generally have delivery limitations. Dyes that are water soluble, such as indocyanine green (ICG), rapidly bind to serum proteins and are rapidly cleared.19

Hydrophobic NIR dyes typically contain large aromatic structures and are only

sparingly soluble in water, rending these dyes difficult to apply in vivo.20 The process we describe, Flash NanoPrecipitation, is a general methodology capable of processing poorly soluble dyes into long circulating PA contrast agents. The process enables the encapsulation of a variety of contrast agents, and the incorporation of targeting ligands on the NP surface. Flash NanoPrecipitation (FNP) is a continuous and scalable process, involving rapid micromixing and block-copolymer directed assembly of NPs.21-25 During the FNP process, hydrophobic actives are precipitated in an anti-solvent in the presence of amphiphilic block copolymers. The adsorption of the block copolymer sterically stabilizes the NP interface, arrests further aggregation, and controls NP size (Figure 2).25 Absorption of copolymers on precipitating actives is driven by hydrophobic interactions between the hydrophobic block of the polymer and hydrophobic aggregates. Particle stabilization is provided by steric crowding of the amphiphilic block of the polymer on the aggregate surface. Importantly, dyes encapsulated in this manner are located in the interior of the NP. This permits high NP dye loadings (>50-75% mass) with high PA contrast, when compared to liposomal NP systems whose loads with hydrophobic dyes are limited to surface conjugation or intercalation within membrane bilayers.26-29 Interior dye encapsulation also permits the tuning of surface properties such as targeting and clearance, simply by choice of stabilizer properties. Dense polyethylene glycol (PEG) steric layers minimize protein adsorption and slow reticuloendothelial clearance.30, 31 Also, the incorporation of targeting ligands on the end of a fraction of the PEG chains enables assembly of NPs with


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precisely known targeting chain densities. Our earlier studies on NP drug delivery and optical imaging involved adding fluorescent dyes to the core of the NPs; however these particles were formed and characterized for fluorescent, and for not for PA applications.32-36 Here we intentionally go to very high loading of the PA dye to maximize optical absorption, and consequently, PA signal. While optical dyes have been previously loaded into scaffolds, the broad absorption spectra of previous dyes have not enabled simultaneous multiplexed NP imaging; i.e. the ability to simultaneously inject two particle types and to optically de-mix the signals to track each population independently.26,

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The structure of the paper is to first

present the work on NP formation, and then the in vivo imaging results. We demonstrate that capability by simultaneously imaging one set of untargeted and one set of folate targeted NPs, with each set of NPs tagged with a unique optical dye. Also, the simultaneous in vivo imaging of three signals from oxygenated hemoglobin, deoxyhemoglobin and NP is demonstrated. The development of such constructs opens new opportunities for multiplexed NP and multiplexed PA-based diagnosis. EXPERMENTAL Nanoparticle (NP) formation and characterization PA NPs were formed using Flash NanoPrecipitation by mixing NP components dissolved in tetrahydrofuran (THF) against ultrapure water in a confined impingement jet, as previously described.21 The block co-polymer 1.6 kDa polystyrene-block-5 kDa polyethylene glycol (PS-bPEG, Polymer Source Inc., Dorval, Canada) was used as the interfacial stabilizer. The optical dyes used as the photoacoustic agents were obtained from Persis Sciences, Inc., Princeton, NJ: Par788, Par830, and Par900, are perylene, phthalocyanine, and naphthalocyanine-based compounds, respectively. In situations where co-core materials were included into NP cores, α-


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tocopherol (VitE, Sigma-Aldrich) or 1.8 kDa polystyrene homopolymer (PS, Polymer Source Inc.) was added to THF feed streams. THF solvent was removed by thorough dialysis against PBS with a 6-8 kDa MWCO regenerated cellulose membrane (Specra/Por, Spectrum Labs). For the formation of folic acid targeted NPs, 1.6 kDa polystyrene-block-5 kDa polyethylene glycolfolic acid (PS-b-PEG-fA) was synthesized. The synthesis scheme of PS-b-PEG-fA is described in the supplementary information. A blend of 25% PS-b-PEG-fA and 75% PS-b-PEG as the stabilizer was used to form 25% folic acid modified NPs. NP size was characterized with dynamic light scattering analysis (Malvern Zetasizer Nano, Malvern Instruments). Sizes were measured using backscattering measurements with 632 nm illumination. The sizes were analyzed by the normal mode analysis program and plotted as intensity-weighted distributions. NP absorbance profiles were determined with UV-Vis spectroscopy (Evolution 3000, Thermo Electron Corporation). To measure particle stability in buffer, NPs were diluted tenfold into PBS and incubated at 37°C whereupon size and absorbance measurements were periodically taken. Particles were diluted tenfold into 20% fetal bovine serum in PBS and incubated at 37°C to assess stability in the presence of serum components. Phantom Photoacoustic Imaging To characterize the PA spectral profiles of the NP formulations, solutions were put into polyethylene tubes (PE20, Intramedic, Becton Dickinson, Sparks, MD), submerged in water and imaged with the Vevo LAZR Photoacoustic Imaging System (FUJIFILM VisualSonics, Inc., Toronto, Canada) using a 21MHz transducer. Raw PA spectra were obtained and reported in the wavelength range of 680nm to 970nm in 5nm increments. In vivo Photoacoustic Imaging


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For in vivo PA imaging, athymic nude mice with KB tumors implanted subcutaneously on the hindlimb were anaesthetized with isofluorane, and placed on the imaging platform where their temperatures, heart rates and respiration rates were monitored for the duration of each imaging session. A tail-vein catheter was installed for intravenous injection of each NP formulation. Mice were injected with a 50 µL bolus of Par788 NPs at a concentration of 1.9 mg mL-1. Animals were imaged with the Vevo LAZR Photoacoustic Imaging System (FUJIFILM VisualSonics, Inc., Toronto, Canada) using a 21 MHz transducer. 2D and 3D micro-ultrasound and multispectral PA images of the tumors were obtained by imaging at 680, 710, 730, 750, 830, 870 and 950 nm wavelengths pre-IV NP injection, after injection and at 24 hrs and 96 hrs postinjection. Spectral unmixing was performed using the Vevo LAZR software based on photoacoustic spectra for each particle (as obtained from imaging the particles in a phantom as described above), oxygenated hemoglobin and deoxygenated hemoglobin, and resulting images were multiplexed and overlaid on the B-Mode ultrasound image. The relative photoacoustic signal intensity for each unmixed component was quantified using the software by drawing a region of interest around the tumor on image slices. The reported PA intensity is the average PA intensity signal between all image slices throughout the whole tumor volume. RESULTS AND DISCUSSION Nanoparticle formation and photoacoustic activity: NPs were made over a range of solids concentrations from 1.5 mg mL-1 to 9 mg mL-1 of the polymer, core material, and dye in the inlet streams, and at a series of ratios of components. Table S1 provides all experimental conditions, particle sizes, and optical properties for the NPs prepared. Spherical particles were formed with sizes were varied from 39 nm to 88 nm, and particle size distributions were narrow for all three Par788, Par830, and Par900 dyes (Figure 3A-C, Figure SI1, Table S1). The spectra


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of the dyes varied with their local microenvironment. Dyes in NP form have slightly broadened absorbance profiles, relative to the spectra for dye dissolved in THF (Figure 3D-F). The absorbance maximum for Par788 in NP form shifted downfield by 50 nm, whereas the maximum for Par830 is shifted up-field by 10 nm, and that of Par900 shifted upfield by 20 nm. This shifting of absorbance wavelengths is helpful because the absorption maxima become further separated, which facilitates spectral unmixing of optical signals. Dyes commonly exhibit shifted absorbance profiles when dissolved in varying solvents or when packed into a solid form due to differences in environmental polarity and dye structural conformation. The blueshift of Par788 absorbance when Par788 is densely packed into the NP core is comparable to that of ICG when the local concentration of ICG is increased. At high concentrations, ICG forms dimers and multimers that absorb light more strongly at higher wavelengths.39 Blueshifted dimers/aggregates are typically formed by face-face molecular stacking that result in the formation of higher energy absorption bands and are known as H-type aggregates.40 In contrast, head-tail molecular stacking typically results in the formation of lower energy absorption bands, and in subsequently redshifted structures known as J-type aggregates. The formation of stacked dyes and exclusion of solvents within the solid nanoparticle core likely leads to the shifted dye optical signatures. When delivering therapeutic actives via NPs it is sometimes desirable to include a cocore material to enhance the stability of the drug in the NP. For example, VitE enabled the encapsulation of 25 wt% progesterone, which otherwise would crystallize outside the NP.41, 42 In other instances the liquid VitE core resulted in quenching of fluorescence signals due to fluorophore stacking in the NP core. Quenching was avoided by using a solid co-core material such as polystyrene.36, 43 Both of VitE and PS were used as co-core materials to investigate how the addition of particle dopants would affect NP optical properties. The addition of either


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component in the NP core results in small shifts in absorption or absorption efficiency; the extinction coefficients for Par788 without dopants, with VitE co-core, and with PS co-core were 37.9, 38.6, and 43.2 mL mg-1 cm-1, respectively. See Table S1 and Figure S2 for complete data. The photoacoustic activity of Par788, Par830, and Par900 NPs were assessed by imaging the imaging constructs when suspended in polyethyelene tubing and submerged in water. The photoacoustic activity of particles corresponds with the absorbance profiles of its parent particle and optical dye (Figure 4). The PA activities of particles at different concentrations were also assessed to characterize NP-PA intensity relationships (Figure 5). Both PA intensity and optical absorbance signals are, essentially, linear over the range of concentrations studied. VitE or PS particles not loaded with dye exhibit little to no PA activity. The PA intensity for all formulations and all three dyes tested are summarized in Table S1. Modulating nanoparticle size and photoacoustic activity: NP size is an important design parameter that can affect NP clearance rates, tumor accumulation profiles, drug payload capacity, and biological activity. Particles with varying sizes were formed, using rules that have been presented previously.21 Briefly, the size is controlled by the competition between the growth rate of the particle by diffusion-limited aggregation, and the rate of particle stabilization by adsorbed block copolymer. Increasing the total solids concentration in the precipitation increases the rate of growth (Figure 6A), and particle size increases from 47 nm at 3 mg mL-1 Par788 feed concentration to 66 nm at 9 mg mL-1 Par788 feed concentration, while at a constant 1:1 Par788 dye to block copolymer mass ratio. Particle sizes decrease as the amount of block copolymer stabilizer increases (Figure 6B), since stabilizer adsorption quenches growth. Particle size decreases from 88 nm to 42 nm as the stabilizer-to-dye ratio increases from 0.33 to 2. Sizes and optical absorbance are stable over weeks whether in 20% FBS or PBS (Figure 3A-C, Figure


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S3-S4). This stability is much greater than that that of isocyanine dyes which are prone to photobleaching and degradation, such as ICG which can degrade ~18% within a day even when left in the dark.39 NP size may have an influence on PAI intensity. We have previously shown that the fluorescence in dye-loaded NPs scales with the volume of the particle, i.e. radius cubed.36 This indicates uniform distribution of the dye in the volume, without significant surface effects, as those depend on the surface-to-volume ratio. The photoacoustic response and specific photoacoustic activity (α, PA intensity divided by absorbance) is somewhat different (Figure 6C, Table S1), as seen among comparable Par788 NPs. The NPs ~90 nm in diameter had a 1.7x specific activity over NPs ~40 nm in diameter. Even though larger NPs had higher specific PA activity, the absorbance extinction coefficients (ε (mL mg-1 cm-1) based on dye loading) remained comparable between NPs of all sizes (Table S1, Figure S5), which demonstrates that the higher PA activity of larger NPs is not due to enhanced optical absorbance. The observation of increased PA activity with size is consistent with previous findings, that larger clustered PA agents exhibit higher PA signal, due to increased localized heat flux during laser irradiation.44 Indeed, the specific PA activity per particle scales slightly more strongly than the diameter cubed (D3) and rather at ~ D3.65 and with a power law regression R2 of 0.995 (Figure 6D). Therefore, the absorbance is not an absolute measure of PA response. While consistent with previous reports of increased PA activity based and particle size, the basis for this phenomenon would be an interesting question for further exploration. In vivo nanoparticle imaging: The ability to utilize the PA NPs in vivo was studied by imaging these constructs in a murine tumor model. When systematically delivered through a tail vein injection into mice harboring KB tumors, Par788 PA signals generated by NP contrast were


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easily seen on the tumors with high resolution (Figure 7). NP signals could be determined via multispectral scanning and wavelength deconvolution because the strong absorbance maxima at 715 nm and near baseline absorbance at 900 nm, which facilitates deconvolution from other tissue signals (Figure 7C). When overlaid with ultrasound images of the tumor (Figure 7D), the NP signal can be seen to be focused predominately around the periphery of the tumor. The clearance of the non-targeted NPs to the skin, through vasculature that might be compromised at the boundary of the tumor, has been observed in other NP systems.45, 46 Images of oxygenated hemoglobin (OxH) and deoxygenated hemoglobin (deOxH) can also be simultaneously captured and overlaid with ultrasound and NP images, since OxH and deOxH have unique absorbance profiles that can be deconvoluted from that of the Par788 signature.47,

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OxH is observed

predominately on the surface of the tumor, and deOxH is predominately on the tumor interior, as would be expected of tumors with hypoxic cores (Figure 7E). Thus, FNP based NPs are suitable for use in vivo PA imaging and to study tumor physiology. Knowledge of properties such as NP tumor accumulation and clearance rates is vital when designing NPs for the delivery of chemotherapeutics. The accumulation levels of Par788 NPs were tracked over time with PA imaging to study the pharmacokinetic properties of the NPs (Figures 8-9). The levels of NPs present in the tumor increased greatly over one day, with accumulation rates expected of stealthy PEG-coated NPs. The particles were also present in the tumor four days after injection, which highlights the ability to image tumors for remarkably long periods of time after NP injection. This is in contrast to most radionuclides agents that have more limited halftimes. These PA-based pharmacokinetic results are consistent with previous experiments that demonstrate the ability of FNP NPs to circulate for days.30 The use of NPs in PA imaging allows for real-time, rapid and non-invasive studies of NP pharmacokinetics without


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the need for blood sampling or biopsy, while providing additional physiological information such as blood oxygenation levels. This additional physiological information is not provided in alternative, non-invasive, NP monitoring techniques, such as X-ray, SPEC, PET, or MRI measurements. Mice exhibited no obvious adverse or toxic effects over the four-day experiments when injected with NPs. Together, these results show that the constructs formed are well suited for in vivo PA studies to characterize NP pharmacokinetics. The surface chemistries of NPs made with FNP can be easily tuned, such that NPs may be modified for the targeted delivery of PA contrast agents. To highlight the targeting capabilities of PA NPs, we demonstrate the proof-of-concept of multiplexed imaging using two types of particles in vivo. This is accomplished by simultaneous imaging a set of non-targeted and a set of folic acid targeted NPs, each of which are tagged with a different PA dye. Folic acid is a binding ligand for the folate receptor alpha, which is overexpressed on the surface of a variety of tumors, including KB cancer cell lines.49, 50 To first verify that folic acid modified NPs formed via FNP target folate receptor alpha, model polystyrene particles loaded with fluorescent dyes and with 25% of the end PEG chains functionalized with folic acid were formed (Table S1, Figure S6). NPs bound strongly to folate receptor alpha overexpressing KB cells, and minimally bound to A549 cells that do not express folate receptor alpha (Figures 10-11). Unmodified NPs with only PEG steric layers did not bind to either KB or A549 cells. The in vivo experiments were conducted with unmodified Par788 NPs that passively accumulated in tumors, alongside Par900 NPs modified with 25% folic acid (fol-Par900 NP). When both NPs were simultaneously injected into a mouse bearing KB tumors, Par788 NPs and fol-Par900 NPs showed differences in accumulation and spatial distribution (Figure 12). The Par788 PA signal, associated with nonspecific uptake by passive retention mechanisms, was focused predominately on the surface of


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the tumor as seen and described previously, while the folate-targeted fol-Par900 PA signal was more generally dispersed throughout the tumor. However, the hypoxic core had little NP signal, which would be consistent with tissue with poor interior circulation or high internal pressures. Irrespective of the biological mechanism, this result demonstrates that we are capable of simultaneously imaging two different sets of NPs with PA spectral demixing. The ability to simultaneously image two sets of NPs, each of which are modified with different surface markers, provides a capability for screening multiple tumor properties at once.

CONCLUSION Photoacoustic imaging is a young and emerging technique with promising capabilities for use in diagnostic imaging. The development of novel PA contrast agents with tunable optical and pharmacokinetic properties will greatly aid in the development and maturation of PA techniques. We have demonstrated the utility of using the block copolymer-directed, rapid precipitation process, Flash NanoPrecipitation (FNP), to prepare diagnostic NPs from highly hydrophobic dyes. The advantage of this approach is that the PA agent is encapsulated inside the NP core, and the high hydrophobicity guarantees the dye remains with the NP, and not in contact with the biological environment. The universality of encapsulation means that a wide variety of different PA imaging agents can be encapsulated in NPs of the same size and surface functionality as demonstrated by the encapsulation of the three dyes, Par788, Par830, and Par900. This enables multiplexing, i.e. the simultaneous injection of multiple NPs with different “colors”, which can be spectrally de-mixed to determine NP concentrations in vivo. In this study we produced NPs from 39 to 88 nm, which is considered the optimal size range for delivery to tumors.51-53


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The FNP process enables the simple assembly of targeted NPs with controllable surface functionality, as well as the co-encapsulation of multiple components in the core of the NPs. In this study we demonstrated the co-encapsulation of PA agents with VitE and polystyrene homopolymer. This highlights the ability to produce “theranostic” NPs to deliver therapeutics while remotely monitoring their fate and delivery. Also, new approaches to use light to both interrogate the NP location and to induce photodynamic reactive oxygen generation open up new avenues of investigation.54 In our future work we are investigating additional PA compounds with even narrower absorption bands. The goal is to produce PA imaging agents that can be multiplexed with 4 distinct signatures over the wavelength range 680-980 nm, and that are also biologically active. .


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FIGURES

Figure 1. Contrast enhanced photoacoustic imaging. Optical absorbers that serve as PA contrast agents are delivered and accumulate at target sites. Areas of interest are pulsed with laser irradiation at wavelengths specific to the PA contrast agent absorption profile. Contrast agents absorb incoming light to produce heat and undergo thermal expansion. Contrast agents and surrounding tissue subsequently cool and undergo thermal contraction to generate ultrasound waves. The emitted ultrasound waves are detected and deconvoluted to produce images of PA contrast agent distribution.


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Figure 2. Flash NanoPrecpitation assembly of photoacoustic NPs. Hydrophobic dyes are dissolved in the presence of an amphiphilic block-copolymer and rapidly mixed with water. Dyes precipitate and are stabilized by absorbed block-copolymers to form water-dispersible dye NPs suitable for use in in vivo applications. The procedure is generic and can encapsulate any sufficiently hydrophobic dye to form NPs with photo-active cores.


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Figure 3. Formation of photoacoustic NPs. Dynamic light scattering size distributions of NPs formed with (A) Par788, (B) Par830, and (C) Par900 dyes. Normalized absorbance spectra of (D) Par788, (E) Par830, and (F) Par830 dyes dissolved in THF or in NP encapsulated form. Particles are formed through the mixing of PS-b-PEG and dyes at 5 mg mL-1 concentration.


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Figure 4. Optical and photoacoustic profiles of NPs. (A) Normalized absorbance-wavelength profiles and (B) normalized photoacoustic-wavelength profiles of NPs assembled from Par788, Par830, and Par900 optical dyes.

Figure 5. Photoacoustic activities of NPs. (A) Photoacoustic activity-wavelength profiles and (B) peak photoacoustic activities (C) and absorbance profiles of Par788 NPs at varying NP concentrations. Measurements of zero Par788 dye NP concentrations are of dye-free polystyrenecore NPs.


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Figure 6. Modulating NP size and size-dependent photoacoustic activities. (A) Intensityweighted sizes of NPs assembled with different concentrations PS-b-PEG stabilizer and Par788 dye during precipitation. The NPs are formed using a constant 1:1 stabilizer to dye mass ratio, using without the addition of other co-core materials. (B) Intensity-weighted sizes of NPs assembled by tuning the ratio of PS-b-PEG stabilizer and Par788 during precipitation. Formulations are detailed in Table S1. (C) Specific photoacoustic activity of NPs with varying sizes identified in panels A-B, fit to a linear regression. (D) Specific photoacoustic activity per particle of NPs with varying size particles identified in panels A-B, fit to a power law regression. The specific PA activity per particle scales with size to the 3.65, with an R2 value of 0.995, as fitted to a power law trend.


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Figure 7. In vivo photoacoustic imaging of NPs and tumor physiology. (A) Optical image of an excised and (B) an in vivo ultrasound image of a murine implanted KB tumor. (C) Heat map of PA signals from Par788 NPs systematically injected in mice implanted with a KB tumor and (D) when overlaid with the ultrasound image. (E) Heat map of oxygenated hemoglobin and deoxygenated hemoglobin of the same KB tumor and when (F) overlaid with the ultrasound image. (G) Overlaid Par788 NP, oxygenated hemoglobin, and deoxygenated hemoglobin PA signals, and (H) when overlaid with the ultrasound image. Multiplexed imaging of particle accumulation and tumor physiology are of the same single mouse.


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Figure 8. Monitoring NP accumulation with photoacoustic imaging. Heat map of PA and PA-US overlay images respectively of Par788 NPs injected into mice bearing KB tumors after (A-B) 0 hrs, (C-D) 24 hrs, (E-F) and 96 hrs. Images of nanoparticle accumulation and tumor physiology are of the same single mouse.


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Figure 9. NP accumulation in tumors. Average PA intensity of Par788 NPs accumulated within KB tumors over time. Measurements are averages and error bars are standard deviations of >20 slices of images of tumors among two different mice.


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Figure 10. Nanoparticle targeting to folate receptor alpha negative A549 cells. Images of A549 cells incubated with non-targeted modified, and 25% folic acid modified NPs respectively at (A,B) brightfield, (C,D) eosin fluorescence channel in green, (E,F) NP fluorescence channel in red, and (G,H) overload eosin and NP fluorescence channel conditions. Scale bar = 50 µm.


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Figure 11. Nanoparticle targeting to folate receptor alpha overexpressing KB cells. Images of KB cells incubated with non-targeted modified, and 25% folic acid modified NPs respectively at (A,B) brightfield, (C,D) eosin fluorescence channel in green, (E,F) NP fluorescence channel in red, and (G,H) overload eosin and NP fluorescence channel conditions. Scale bar = 50 µm.


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Figure 12. Multiplexed dual-NP photoacoustic imaging. Heat map of PA and PA-US overlay images respectively of (A,B) non-targeted Par788 NPs, (C,D) 25% folate targeted Par900 NPs, (E,F) and both targeted + non-targeted NPs after injection into mice bearing KB tumors. Multiplexed imaging of nanoparticle accumulation and tumor physiology are of the same single mouse.


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ASSOCIATED CONTENT Supporting Information. Additional information on particle characterization is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes A.H. is employed by FUJIFILM VisualSonics, Inc. ACKNOWLEDGMENT We are grateful for support from the Princeton University Center for Health and Wellbeing (HDL), Woodrow Wilson School of Public and International Affairs Program in Science, Technology, and Environmental Policy (HDL), and National Science Foundation Graduate Research Program (BKW). This work was supported by the Princeton University SEAS grant from the Old School Fund (RKP). REFERENCES 1. Xu, M.; Wang, L. V. Photoacoustic imaging in biomedicine. Rev. Sci. Instrum. 2006, 77. 2. Beard, P. Biomedical photoacoustic imaging. Interface Focus 2011. 3. Li, C.; Wang, L. V. Photoacoustic tomography and sensing in biomedicine. Physics in Medicine and Biology 2009, 54, R59-R97. 4. Wang, L. V.; Hu, S. Photoacoustic tomography: In vivo imaging from organelles to organs. Science 2012, 335, 1458-1462. 5. Luke, G. P.; Yeager, D.; Emelianov, S. Y. Biomedical applications of photoacoustic imaging with exogenous contrast agents. Ann Biomed Eng 2012, 40, 422-437. 6. Yang, X.; Skrabalak, S. E.; Li, Z. Y.; Xia, Y.; Wang, L. V. Photoacoustic tomography of a rat cerebral cortex in vivo with Au nanocages as an optical contrast agent. Nano Letters 2007, 7, 3798-3802.


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