Assembly of Macrocycle Dye Derivatives into Particles for

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Assembly of Macrocycle Dye Derivatives into Particles for Fluorescence and Photoacoustic Applications Hoang Dung Lu, Tristan L. Lim, Shoshana Javitt, Andrew Heinmiller, and Robert K Prud'homme ACS Comb. Sci., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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ACS Combinatorial Science

Assembly of Macrocycle Dye Derivatives into Particles for Fluorescence and Photoacoustic Applications Hoang D. Lu,† Tristan L. Lim,† Shoshana Javitt,† Andrew Heinmiller,‡ and Robert K. Prud’homme*,† †

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

KEYWORDS Nanoparticle, dye, photoacoustic, fluorescence, imaging

ABSTRACT Optical imaging is a rapidly progressing medical technique that can benefit from the development of new and improved optical imaging agents suitable for use in vivo. However, the molecular rules detailing what optical agents can be processed and encapsulated into in vivo presentable forms are not known. We here present the screening of series of highly hydrophobic

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porphyrin, phthalocyanine, and naphthalocyanine dye macrocycles through a self-assembling Flash NanoPrecipitation process to form a series of water dispersible dye nanoparticles (NPs). Ten out of nineteen tested dyes could be formed into dense poly(ethylene glycol) coated nanoparticles 60-150 nm in size, and these results shed insight on dye structural criteria that are required to permit dye assembly into NPs. Dye NPs display a diverse range of absorbance profiles with absorbance maxima within the NIR region, and have absorbance that can be tuned by varying dye choice or by doping bulking materials in the NP core. Particle properties such as dye core load and the compositions of co-core dopants were varied, and subsequent effects on photoacoustic and fluorescence signal intensities were measured. These results provide divergent guidelines for designing NPs optimized for photoacoustic imaging and NPs optimized for fluorescence imaging. This work provides important details for dye NP engineering, and expands the optical imaging tools available for use.

Optical and hybrid optical imaging techniques have become increasingly important for the diagnosis and management of diseases.1-2 Optical based methods are attractive for routine and point-of-care imaging, since these methods have rapid acquisition times, are non-invasive, and are non-ionizing, unlike other imaging methods such as X-ray, CT, PET, or SPECT.3-4 Recently, increasing attention has also been given to fluorescent and photoacoustic (PA) imaging in particular for applications in advanced medical techniques, such as tumor phenotyping or image-guided surgery.5-8 For these applications, optical dyes are injected and localize to tissues of interest. Selective localization is achieved by conjugating dyes to ligands that bind to cell-


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surface receptors of tumors.9 Targeted dyes provide important information for predicting optimal methods of treatment, or for helping physicians to spatially identify tumors for removal. Several design criteria of optical agents used for PA and fluorescent imaging vary due to the intrinsic differences between the imaging techniques. Photoacoustic imaging (PAI) relies upon absorption of pulsed light and the conversion of adsorbed energy into heat.10-13 Local heating causes thermal expansion and contraction, and resultant pressure waves are then detected by conventional ultrasound transducers to construct 3D images. Whereas conventional ultrasound imaging provides physiological information, photoacoustic imaging also provides chemically specific information. Fluorescence imaging relies upon optical emission from irradiated and excited-state dyes.14 For both techniques, five factors should be optimized. (1) Optical agents should have favorable pharmacokinetic profiles such as long circulations times for prolonged imaging and diagnosis. (2) Agents should have narrow optical absorption/excitation bands with minimal spectral overlaps to facilitate the simultaneous use of multiple contrast agents, or to facilitate dye multiplexing.15 (3) Dyes should absorb strongly within the near infrared (NIR) region to minimize tissue attenuation and to enable deep imaging depths. However, optical agents should have different mechanisms of energy loss upon irradiation in photoacoustic and fluorescence applications. (4) For photoacoustic imaging, agents must absorb light and undergo non-radiative energy loss for maximum heat generation and minimum fluorescence. (5) For fluorescence imaging, agents must absorb light and re-emit energy through energy radiative loss for high fluorescence generation and minimum heat generation. Currently, there are few optical dyes or constructs that satisfy these outlined criteria for photoacoustic or fluorescence imaging, which severely limits the capabilities of optical imaging.16


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Gold nano rods have been used as contrast agents, but are usually formed with broad absorbance profiles over the NIR region due to variation in aspect ratios during synthesis.17-20 Indocyanine green (ICG) is an FDA approved and commonly used dye for optical imaging in the NIR region, but rapidly binds to serum proteins and is cleared within minutes upon injection, as are most small molecule dyes.21 The development of new and improved dye constructs suitable for use in biomedical applications can expand the potential applications of optical imaging. The two main methods of developing novel and improved optical dyes for imaging and satisfying the first three requirements involve either modifying the chemical structure of the dye, or encapsulating dyes into nanoparticles. In the first method, dye backbones are modified with pendant functional groups that alter the optical properties of the compound. The backbone of the compound can also be tuned to afford improved fluorescence/PA properties. Porphyrins, phthalocyanines, and naphthalocyanines are poorly soluble macrocycle dyes that have been heavily derivatized for use in optical applications, due to their strong absorbance in the NIR region.22 However, phthalocyanines/naphthalocyanines and their derivatives still suffer from poor in vivo pharmacokinetic properties typical of unencapsulated small molecules, such as rapid renal clearance for soluble dyes, or rapid absorption to serum proteins and subsequent hepatic clearance for poorly soluble dyes.23-24 Encapsulating dyes into nanoparticles can be used to improve the pharmacokinetic properties of dyes or particle brightness by clustering many dye molecules together. ICG has been surface conjugated or encapsulated into several nanocarriers (NCs) such as micelles, liposomes, and nanocapsules for such purposes.

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However, the

optical properties of these ICG or dye loaded NCs are predominately restricted to the absorbance and fluorescence properties of the parent dye itself. The development of a platform technology that can encapsulate a diverse set of dye derivatives, such as phthalocyanines and


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naphthalocyanines, with varying optical properties into NCs that are suitable for in vivo use is highly attractive. Flash NanoPrecipitation (FNP) is a scalable and block-copolymer directed self-assembly process that has recently been shown to be capable of encapsulating hydrophobic dyes for imaging applications.31-35 In this process, hydrophobic dyes are dissolved with amphiphilic block copolymers such as polystyrene-block-polyethylene glycol (PS-b-PEG) and rapidly micromixed with water, resulting in dye precipitation. The hydrophobic section of the block copolymer absorbs onto precipitating dyes, and the hydrophilic section of the block co-polymer sterically crowds the surface of the precipitating dyes. This arrests precipitation and growth, and stabilizes the dyes into water dispersible nanoparticles (NPs).35 The use of PEG or ligand-modified PEG block copolymers additionally affords NPs with long circulation times, mucus-penetrating capabilities, or targeting properties.36-38 An advantage of FNP includes dye sequestration in the NP core, which allows for dye mass loadings greater than 75% weight. This is in contrast to other liposomal systems, whose dye loading is limited to within the liposomal hydrophobic bilayer, or on the bilayer exterior for surface conjugated systems.39-41 Core sequestration limits negative effects that surface presentation of dyes can have on NP pharmacokinetics, targeting, or toxicity. Additional co-core materials can be encapsulated with precipitated dyes in the FNP system, such as active pharmaceutical ingredients to create theranostic NPs, or inactive bulking agents to tune NP fluorescence/PA properties. We here report on forming NPs with greatly expanded absorbance, fluorescence, and PA properties, by screening and forming NPs with a diverse set of 19 phthalocyanines and naphthalocyanines dyes. We additionally assess the effect of NP co-core compositions to finely tune NP optical properties, and lay guidelines for forming NPs that are ultimately NIR absorbing, optimized for fluorescence brightness, optimized for PA


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activity, water dispersible, and suitable for in vivo use. This work creates new tools for use in optical imaging. EXPERMENTAL Materials Phthalocyanines and naphthalocyanines dyes (Sigma Aldrich, Figure 1, Table S1), 1.6 kDa polystyrene-block-5 kDa polyethylene glycol (PS-b-PEG, Polymer Source Inc.), 1.8 kDa polystyrene homopolymer (PS, Polymer Source Inc.), alpha tocopherol (VitE, Sigma-Aldrich), and regenerated 6-8 kDa MWCO regenerated cellulose membrane (Spectra/Por, Spectrum Labs) were purchased and used as received.


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Figure 1. Structures of dyes tested. Names of dyes are detailed in Table S1. Dyes that could be formulated in stable nanoparticles are underlined.


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Nanoparticle (NP) formation NPs with defined levels of encapsulated dye and co-core were formed through Flash Nanoprecipitation (FNP) processing as previously described.31 In brief, dye, PS-b-PEG, PS, and VitE were dissolved at defined compositions in organic solvent, and rapidly micromixed against an equivalent volume of deionized water in a confined impingement jet mixer. Impinged particle streams were collected and diluted tenfold in a water collection bath. Particle formation was screened using THF, DMF, and DMSO as organic solvents. NPs of 668 and 727 were formed using DMSO, and THF was used to process the other dye NPs. Compositions of all NP formulations used in this study are described in Tables S2 and S3. Particles with varying levels of dye-core loadings were formed by varying feed concentrations of PS and VitE. Dye core load is defined as the mass fraction of hydrophobic dye over total hydrophobic materials in the NP core, where the hydrophobic block of the copolymer is included as a hydrophobic material:

Core load % =

100 ∗ (mass dye) 1.6 (mass dye) + (mass co core) + 6.6 (mass stabilizer)

The 1.6 kDa hydrophobic block of the 1.6 kDa polystyrene-block-5 kDa polyethylene glycol accounts for the 1.6/6.6 core fraction of the stabilizer used. NPs of 762 dye were made with dye core-loads between 2-86 wt%, when there were no additional co-core dopant excipients. 762 NPs doped with PS homopolymer co-core were formed with dye core-loadings between 0.5-60 wt%. To form NPs with defined dye core loading, PS-b-PEG and PS concentrations were held constant while varying 762 concentrations during NP formation.


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Nanoparticle characterization Particle sizes were determined by dynamic light scattering (Malvern Zetasizer Nano, Malvern Instruments). Size distributions were determined with backscattering measurements and displayed as intensity-weighted distributions. Particle absorbance properties were determined with UV-Vis spectroscopy (Evolution 3000, Thermo Electron Corporation) after particles were diluted into deionized water at linear-absorbance range concentrations. Particle fluorescence properties were determined by fluorometry (SpectraMax i3x, Molecular Devices). Particle fluorescence of 762 NPs in the NIR region was assessed by excitation at 740 nm, and emission measurements at 790 nm. 762 NP fluorescence from UV illumination was assessed by excitation at 330 nm, and emission measurements at 495 nm and 790 nm. Particle PA activities were determined by PA imaging (Vevo LAZR, FUJIFILM VisualSonics) using a 21 MHz transducer. Particles were placed in polyethylene tubes (PE20, Becton Dickinson) submerged in water for imaging.To avoid multiple scattering and “shadowing” NP concentrations were kept low for the absorbance and photoacoustic measurements. Dilutions were made so that the optical absorbance measurements were linear in concentration of the NP sample. For the PA measurements, the NPs were diluted ten-fold from the original concentrations used in the FNP process, that is, those in Tables S2-S3.

RESULTS AND DISCUSSION Dye screening for nanoparticle formation: To form NPs with a wide range of optical properties, nineteen porphyrin, phthalocyanine, and naphthalocyanine dyes with absorbance


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maxima between 650 nm and 900 nm were screened with FNP. The dyes tested include unmodified phthalocyanine (698) and naphthalocyanine (712) compounds, as well as their metal ligand chelated and pendant side chain modified derivatives (Figure 1). Only modified porphyrins were assessed since unmodified porphyrin has absorbance maxima in the UV/Visible outside the NIR-range. Out of all the dyes tested, ten could be formed into NPs with sizes between ~40-160 nm in diameter using FNP (Figure 2, 3a-b), which are sizes that are well suited for systemic injection and circulation. The other nine dyes tested formed visible aggregates upon FNP, rendering them unsuitable for in vivo use. All dyes formed visible aggregates when FNP was performed without the use of the block co-polymer PS-b-PEG, highlighting the necessity of an amphiphilic stabilizer to form NPs.

Figure 2. Nanoparticle sizes. Representative intensity-weighted dynamic light scattering size distributions of nanoparticles, formed with cores comprising of 762 dye, 762 dye plus polystyrene co-core, and 762 dye plus Vitamin-E co-core. The exact compositions of these particles are described in Table S2.


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Figure 3. Nanoparticle assembly and formation of dyes. (A) Z-average and intensity weighted nanoparticle diameters, and (B) cumulants fit-based polydispersity indexes of NPs determined by


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dynamic light scattering analysis. (C) Nanoparticle absorbance extinction coefficients. (D) Maximum absorbance wavelength peak shift upon nanoparticle encapsulation. Extinction coefficients are determined based on the mass dye in the NP, and the NPs absorbance maxima. Absorbance peak shifts are absorbance maxima wavelengths of free dye dissolved in THF subtracted from that of nanoparticles suspended in water. The compositions of these particles are described in Table S2.

Notably, unmodified phthalocyanine and naphthalocyanine (698, 712) could not be formed into NPs, while tetra-tert-butyl or butoxy modified variants (695, 784; 762, 867 respectively) derivatives could be. This is likely due to the strong tendency of the planar unmodified dyes to undergo pi-pi stacking to form large aggregates, which could be disrupted through the addition of flanking tert-butyl/butoxy side chains to permit NP assembly.42 However, the functional group and location of flanking chains are important, as a similar octyloxy modification (701) is an insufficient modification to permit NP assembly. While magnesium and manganese chelated phthalocyanines (668, 727) could also be formed into NPs, iron, copper, and vanadyl chelated phthalocyanines (654, 678, 817) could not. The addition of central chelation groups is known to disrupt stacking tendencies, and these results highlight that the chelate choice is important for enabling dye assembly into functional NPs. The exact mechanism of how functional groups and chelates affect dye aggregation propensities is interesting and can be elucidated in future molecular dynamics studies. Dye NP absorbance profiles: The absorbance extinction coefficients (ε) of dye NPs are important measures of optical activity. When compared to unencapsulated dyes dissolved in


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organic solvent, NP encapsulated dyes suspended in water exhibit lower ε, decreasing by ~33% in the case of 867 NPs, or as much as ~600% in the case of 808 NPs (Table S2, Figure 3c). The lower NP ε values are not surprising, as dyes are suspensions of densely packed cores when in NP form. Attenuation of light inside the densely crowded dye-cores decreases the efficiency of light absorption, , when compared to homogeneously dissolved dye solutions in organic solvents. The optical density inside a single particle is significantly higher than the optical density of the dispersion itself. NP absorbance spectral shifts can shed important information on the state of the encapsulated dye. For example, butoxy modified phthalocyanines and naphthalocyanines (762, 867) NPs have absorbance maxima shifts less than 10 nm, and have overall absorbance profiles that closely match that of the free dye. (Figure 3d, Figure 4). Tert-butyl modified phthalocyanines and naphthalocyanines (695, 784) in contrast have significantly blue-shifted absorbance maxima and spectra upon encapsulation, shifting as much as ~90 nm and ~80 nm respectively. Blue-shifted absorbance profiles are typically caused by face-face molecular stacking that results in the formation of higher energy absorption bands, in a process known as H-type aggregation.43 Red-shifted absorbance profiles in contrast are typically caused by headtail molecular stacking that results in lower energy absorption bands, in a process known as Jtype aggregation. These results suggest that tert-butyl modification sufficiently disrupts planar pi-pi stacking of phthalocyanine/naphthalocyanine dyes to permit NP assembly, but is still conductive for head-head dye molecular interactions when packed into a dense solid core. Butoxy modification, in contrast, disrupts molecular interactions between dyes so that absorbance maxima are shifted less than 10 nm. In general, metal-chelated dyes exhibited


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spectral broadening in both the red and blue regions, suggesting that these dyes aggregated into a variety of states, or aggregated into slipped cofacial structures upon encapsulation.44

Figure 4. Absorbance of NPs with dyes and co-core materials. (A-I) Normalized absorbance spectra of dyes dissolved in THF, and of NPs that could be successfully formed through FNP process and which have absorbance maxima within the NIR window. Particles were formed without the addition of a co-core, with a PS co-core, or with VitE-co core. The exact compositions and core-loads of these particles are described in Table S2.


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Interestingly, 668 dye exhibits a secondary maximum at 820 nm when encapsulated with a VitE co-core. This maximum is not present when 668 is dissolved in THF, or when encapsulated with or without a PS co-core. This suggests that 668 is capable of forming head-tail stacks in a liquid-like VitE environment, and cannot when packed in a solid and crystalline PS environment. The exact molecular mechanisms for the change in optical properties upon dye encapsulation might require X-ray scattering experiments to clarify the nature of the stacking. Altogether, these results highlight how dye and co-core interactions can have strongly influence the dye optical properties upon encapsulation. Varying optical properties from tuning core-load: A useful handle to tune the optical properties of a dye NP is by varying core-loading. To assess how varying dye-load affects the PA or fluorescence activity of NPs, 762 NPs were formed with core loadings between 2-86 wt% when no co-core was added, and with core loadings between 0.4-60 wt% when PS was included as a co-core. These NPs were formed by holding PS-b-PEG/PS-b-PEG plus PS concentrations constant while varying 762 concentrations in the organic stream during FNP processing. 762 was included at concentrations as low as 0.025 mg mL-1 to as high as 7.9 mg mL-1 to achieve these compositions (Table S3). The relative concentrations of stabilizing polymers, and 762 dye plus co-core materials dictate NP size and optical properties. The absorbance of NPs formed with this method increases as 762 feed stream concentrations and, subsequently, core load increase (Figure 5a). The addition of the neutral PS co-core material does not significantly alter absorbance; it is determined by the dye loading (Figure S2). The extinction coefficient, ε, depends on dye concentration and not significantly on the PS co-core loading (Figure 5b).


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Figure 5. Absorbance of 762 NPs as a function of dye composition. (A) Peak absorbance values and (B) extinction coefficients of 762 NPs made with varied 762 feed concentrations during FNP, with and without the addition of PS co-core bulking agent materials. The compositions and core-loads of these particles are described in Table S3. Extinction coefficients of NPs are based on the mass of 762 in NP formulations.

The 762 phthalocyanine dye is also fluorescent, which makes it an interesting candidate for combined optical imaging and photoacoustic imaging. It has a long wavelength emission at 790 nm, and when excited by shorter wavelength light, an emission at 495 nm. Excited dyes should predominately transfer energy through radiative mechanisms that result in photon emission in fluorescence applications. To investigate how dye core load or co-core materials affect fluorescence, the emission profiles of various 762 NPs were measured upon irradiation at 740 nm and 280 nm. In contrast to the absorbance and PA scenarios, where signal increases with concentration, the fluorescence of 762 NPs increases as 762 loadings increases and then decreases at higher concentrations due to quenching mechanisms. (Figure 6.


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The longer NIR emission at 790 nm would be of interest for in vivo studies, where penetration and avoidance of background fluorescence are important. The optimal 762 loading for maximum NIR fluorescence occurs at ~0.005 mg mL-1 dye feed, which corresponds to ~1% and ~4% dye core-load for NPs with and without PS co-core respectively (Table S3, Formulations 48 and 57). Thus, while high dye loadings are optimal for PA imaging, intermediate dye loads are optimal for fluorescence imaging. These results are consistent with previous reports of optimal dye encapsulations loads between 0.5-4 wt% cores when using other dyes.45 Thus, for combined PA and optical imaging a mixed particle population comprising both high and low concentrations of dye might be indicated. Both particles can be engineered to have identical sizes and PEG surface densities.

Figure 6. NIR and VIS Fluorescence of 762 NPs as a function of dye concentration in feed. Fluorescence intensity of 762 NPs made with varied 762 feed concentrations, with and without the addition of PS co-core materials, (A) at NIR excitation at 740 nm and NIR emission at 790 nm, (B) and at UV excitation at 280 nm and visible-range emission at 495 nm. The compositions and core-loads of these particles are described in Table S3.


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The shorter wavelength fluorescence occurs at 495 nm when illuminated at 280 nm (Figure S3). As the core load of 762 increases, the 495 nm fluorescence of NPs increases up to 0.58 mg mL-1 and 2.2 mg mL-1 762 dye feed compositions for PS co-core and no co-core NPs, respectively (Figure 6B), whereupon fluorescence rapidly drops with further 762 addition. The formulations with the 495 nm fluorescence maxima correspond to ~9 core wt% dye for PS cocore NPs, and ~64 core wt% for NPs without additional core material. Since the higher energy 495 nm fluorescence is likely due to dimer/multimer formation, higher core loading promotes the formation of dimers that fluoresce at 495 nm, up until the point where these dimers are quenched at even higher concentrations. The increased prominence of a blue-shifted absorbance shoulder (Figure S2) is consistent with increasing dimer/multimer formation with increased dye loading. The fluorescence of NPs with PS co-core is also higher than that of NPs without co-core; the dilution by the co-core material decreases quenching. This may also, at least in part, account for the higher PA activities of NPs without co-core. These results highlight how nanoparticle compositions can be tailored to optimize fluorescence in the visible and NIR regimes. The optimal core properties of NPs for use in PA applications would contrast those for use in fluorescence applications due to the different mechanisms of signal generation. The PA intensities of 762 NPs increase with higher dye core-loading (Figure 7a). However, unlike in the case of absorbance, 762 NPs without the addition of PS co-core material exhibits higher PA activity when compared to 762 NPs with co-core. For example, at 2.2 mg mL-1 762 dye feed concentrations, 762 NPs without co-core had a PA activity of 1.8 intensity units, while 762 NPs with PS co-core had a PA activity of 0.55 intensity units. NPs with only the 762 dye had ~3.3 fold increase in PA activity over NPs that had the same concentration of dye, but also added core


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material. At 2.2 mg mL-1 762 dye feed concentration,

NPs with no co-core material have 64

core wt% dye composition, while those NPs with added PS co-core, at the same dye concentration of 2.2 mg mL-1 , have 26 core wt% dye.. Since PA activity is produced from heat generation upon dye irradiation due to non-radiative energy transfer, the dye core environment influences to what extent dyes generate heat through this mechanism. To assess if the differences in PA activity are a result of varied NP absorbance properties, the PA intensities of NPs with varying 762 loads were plotted against their corresponding absorbance values (Figure 7b). The linear slope of PA activity against absorbance is significantly higher for 762 NPs without PS cocore at 1.3 PA units absorbance-1 (R2=0.97) opposed to 0.36 PA units absorbance-1 (R2=0.92) for that of 762 NPs containing PS co-core. In addition to showing that greater levels of PA activity are generated per dye molecule when dyes are not diluted with co-core, this demonstrates that the increase of PA activity is not solely due to an enhanced absorbance affect. These results are consistent with previous reports that molecular crowding or encapsulation of dyes such as ICG result in enhanced imaging activities.39, 46


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Figure 7. Photoacoustic activity of 762 NPs. (A) Photoacoustic intensity of 762 NPs made with varied 762 feed concentrations during FNP, with and without the addition of PS co-core material. The compositions and core-loads of these particles are described in Table S3. (B) Photoacoustic intensity plotted against optical absorbance of 762 NPs made with varied 762 feed concentrations. The linear slope of photoacoustic intensity vs absorbance for 762 NPs is 1.3 PA units per absorbance (R2=0.97), and that of 762 + PS NPs is 0.36 PA units per absorbance (R2=0.92).

CONCLUSION The applications of biomedical imaging in medicine can be greatly expanded by the development of new and improved optical imaging agents. While fluorescence is the most widely used bio-imaging modality, newer photoacoustic (PA) imaging techniques hold significant promise for advancing bio-imaging. The use of small molecules for imaging is generally limited by poor pharmacokinetic profiles and rapid in vivo clearance, which can be mitigated by encapsulation into nanoparticles. Nanoparticle imaging agents have been limited by the range of optical dyes available, especially in the NIR region. The molecular sizes required for resonance at longer wavelengths have generally meant that these molecules are either not water soluble, or if water soluble, are complex enough to interact with proteins and cell surface receptors in vivo. Rather than placing the dyes on the surfaces of nanoparticles, our approach is to encapsulate the imaging agent in the interior of the nanoparticle. The large, hydrophobic porphyrin, phthalocyanine, and naphthalocyanine dye molecules are ideally suited for encapsulation into the cores of nanoparticles.


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We have applied the block copolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to screen a wide range of highly hydrophobic dyes to form a series of 10 new particles that display absorption maxima in the wavelength range of 600-860 nm. At high loadings, the dyes provide strong PA responses. The relatively sharp absorption bands in the NIR region enable multiplexed PA imaging; whereby several NP types can be measured simultaneously.47 At lower loading, where the dyes do not self-quench, the 762 phthalocyanine dye fluoresces, and the combined PA response, long wavelength NIR fluorescence (790 nm), and short wavelength fluorescence (495 nm) make this an attractive dye for multimodal imaging. The particles are densely coated with poly(ethylene glycol), which makes them suitable for in vivo use. We have demonstrated the ability to incorporate drugs48-52, MRI imaging agents50 ,and PET/SPEC imaging agents53 into the cores of the FNP NPs. In addition, the PEG chains on the NP surfaces can be functionalized to enable NP targeting.54-55

Therefore, any of these

combination approaches can be used with the new PA agents presented here. The FNP particle formation process is scalable, modular, and tunable. This work provides engineering guidelines for tuning NP photochemical properties to develop new imaging agents for PA and optical imaging applications.

ASSOCIATED CONTENT Supporting Information.


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Details on dye identities, formulations, and physical characterizations of all nanoparticles discussed in this paper.

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). This work was supported by the Princeton University SEAS grant from the Old School Fund (RKP). REFERENCES 1. Kaplan, E.; Prashanth, A. K.; Brennan, C.; Sirovich, L. Optical Imaging: A Review. Opt. Photonic News 2000, 11, 26-30. 2. Costas, B. Review of biomedical optical imaging—a powerful, non-invasive, nonionizing technology for improving in vivo diagnosis. Meas. Sci. Technol. 2009, 20, 104020. 3. Fass, L. Imaging and cancer: A review. Mol. Oncol. 2008, 2, 115-152. 4. Frangioni, J. V., New Technologies for Human Cancer Imaging. J. Clin. Oncol. 2008, 26, 4012-4021. 5. Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van de Velde, C. J. H.; Frangioni, J. V. Image-guided cancer surgery using near-infrared fluorescence. Nat. Rev. Clin. Oncol. 2013, 10, 507-518.


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6. Mondal, S. B.; Gao, S.; Zhu, N.; Liang, R.; Gruev, V.; Achilefu, S. Real-time Fluorescence Image-Guided Oncologic Surgery. Adv. Cancer Res. 2014, 124, 171-211. 7. Xi, L.; Zhou, G.; Gao, N.; Yang, L.; Gonzalo, D. A.; Hughes, S. J.; Jiang, H. Photoacoustic and Fluorescence Image-Guided Surgery Using a Multifunctional Targeted Nanoprobe. Ann. Surg. Oncol. 2014, 21, 1602-1609. 8. Cleary, K.; Peters, T. M. Image-Guided Interventions: Technology Review and Clinical Applications. Annu. Rev. Biomed. Eng. 2010, 12, 119-142. 9. Xi, L.; Jiang, H. Image-guided surgery using multimodality strategy and molecular probes. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2016, 8, 46-60. 10. Xu, M.; Wang, L. V. Photoacoustic imaging in biomedicine. Rev. Sci. Instrum. 2006, 77, 041101. 11. Beard, P. Biomedical photoacoustic imaging. Interface Focus 2011, 1, 602-631. 12. Li, C.; Wang, L. V. Photoacoustic tomography and sensing in biomedicine. Phys. Med. Biol. 2009, 54, R59-R97. 13. Wang, L. V.; Hu, S. Photoacoustic tomography: In vivo imaging from organelles to organs. Science 2012, 335, 1458-1462. 14. Ntziachristos, V. Fluorescence Molecular Imaging. Annu. Rev. Biomed. Eng. 2006, 8, 133. 15. Lu, H. D.; Wilson, B. K.; Heinmiller, A.; Faenza, B.; Hejazi, S.; Prud’homme, R. K. Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging. ACS Appl. Mater. Interfaces 2016, 8, 14379-14388. 16. Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K. Review of LongWavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores, and Multifunctional Nano Carriers. Chem. Mater. 2012, 24, 812-827. 17. Kim, C.; Cho, E. C.; Chen, J.; Song, K. H.; Au, L.; Favazza, C.; Zhang, Q.; Cobley, C. M.; Gao, F.; Xia, Y.; Wang, L. V. In vivo molecular photoacoustic tomography of melanomas targeted by bioconjugated gold nanocages. ACS Nano 2010, 4, 4559-4564. 18. Chen, Y. S.; Frey, W.; Kim, S.; Homan, K.; Kruizinga, P.; Sokolov, K.; Emelianov, S. Enhanced thermal stability of silica-coated gold nanorods for photoacoustic imaging and imageguided therapy. Opt. Express 2010, 18, 8867-8877. 19. Lu, W.; Huang, Q.; Ku, G.; Wen, X.; Zhou, M.; Guzatov, D.; Brecht, P.; Su, R.; Oraevsky, A.; Wang, L. V.; Li, C. Photoacoustic imaging of living mouse brain vasculature using hollow gold nanospheres. Biomaterials 2010, 31, 2617-2626. 20. Pan, D.; Pramanik, M.; Senpan, A.; Allen, J. S.; Zhang, H.; Wickline, S. A.; Wang, L. V.; Lanza, G. M., Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons. FASEB J. 2011, 25, 875-882. 21. Kosaka, N.; Mitsunaga, M.; Longmire, M. R.; Choyke, P. L.; Kobayashi, H. Near infrared fluorescence-guided real-time endoscopic detection of peritoneal ovarian cancer nodules using intravenously injected indocyanine green. Int. J. Cancer 2011, 129, 1671-1677. 22. Ghani, F.; Kristen, J.; Riegler, H. Solubility Properties of Unsubstituted Metal Phthalocyanines in Different Types of Solvents. J. Chem. Eng. Data 2012, 57, 439-449. 23. Feng, B.; LaPerle, J. L.; Chang, G.; Varma, M. V. S., Renal clearance in drug discovery and development: molecular descriptors, drug transporters and disease state. Expert Opin. Drug Meta.b Toxicol. 2010, 6, 939-952. 24. Lee, W.; Kim, R. B. Transporters and renal drug elimination. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 137-166.


23


Page 24 of 26

25. Devoisselle, J. M.; Soulie-Begu, S.; Mordon, S. R.; Desmettre, T.; Maillols, H. Fluorescence properties of indocyanin green: I. In-vitro study with micelles and liposomes. Proceedings of the SPIE, 1997, 2980, 453-460. 26. Kim, T. H.; Chen, Y.; Mount, C. W.; Gombotz, W. R.; Li, X.; Pun, S. H. Evaluation of Temperature-Sensitive, Indocyanine Green-Encapsulating Micelles for Noninvasive NearInfrared Tumor Imaging. Pharm. Res. 2010, 27, 1900-1913. 27. Kirchherr, A. K.; Briel, A.; Mäder, K. Stabilization of Indocyanine Green by Encapsulation within Micellar Systems. Mol. Pharm. 2009, 6, 480-491. 28. Rodriguez, V. B.; Henry, S. M.; Hoffman, A. S.; Stayton, P. S.; Li, X.; Pun, S. H. Encapsulation and stabilization of indocyanine green within poly(styrene-alt-maleic anhydride) block-poly(styrene) micelles for near-infrared imaging. J. Biomed. Opt. 2008, 13, 014025. 29. Sun, G.; Berezin, M. Y.; Fan, J.; Lee, H.; Ma, J.; Zhang, K.; Wooley, K. L.; Achilefu, S. Bright fluorescent nanoparticles for developing potential optical imaging contrast agents. Nanoscale 2010, 2, 548-558. 30. Zheng, X.; Xing, D.; Zhou, F.; Wu, B.; Chen, W. R. Indocyanine Green-Containing Nanostructure as Near Infrared Dual-Functional Targeting Probes for Optical Imaging and Photothermal Therapy. Mol. Pharm. 2011, 8, 447-456. 31. Johnson, B. K.; Prud'homme, R. K. Flash NanoPrecipitation of Organic Actives and Block Copolymers using a Confined Impinging Jets Mixer. Aust. J. Chem. 2003, 56, 1021-1024. 32. Pustulka, K. M.; Wohl, A. R.; Lee, H. S.; Michel, A. R.; Han, J.; Hoye, T. R.; McCormick, A. V.; Panyam, J.; Macosko, C. W. Flash Nanoprecipitation: Particle Structure and Stability. Mol. Pharm. 2013, 10, 4367-4377. 33. Zhu, Z. Flash Nanoprecipitation: Prediction and Enhancement of Particle Stability via Drug Structure. Mol. Pharm. 2014, 11, 776-786. 34. Shen, H.; Hong, S.; Prud’homme, R. K.; Liu, Y. Self-assembling process of flash nanoprecipitation in a multi-inlet vortex mixer to produce drug-loaded polymeric nanoparticles. J. Nanopart. Res. 2011, 13, 4109-4120. 35. Johnson, B. K.; Prud'homme, R. K. Mechanism for rapid self-assembly of block copolymer nanoparticles. Phys. Rev. Lett. 2003, 91, 118302. 36. Lu, H. D.; Spiegel, A. C.; Hurley, A.; Perez, L. J.; Maisel, K.; Ensign, L. M.; Hanes, J.; Bassler, B. L.; Semmelhack, M. F.; Prud’homme, R. K. Modulating Vibrio cholerae QuorumSensing-Controlled Communication Using Autoinducer-Loaded Nanoparticles. Nano Lett. 2015, 15, 2235-2241. 37. D'Addio, S. M.; Saad, W.; Ansell, S. M.; Squiers, J. J.; Adamson, D. H.; Herrera-Alonso, M.; Wohl, A. R.; Hoye, T. R.; Macosko, C. W.; Mayer, L. D.; Vauthier, C.; Prud'homme, R. K. Effects of block copolymer properties on nanocarrier protection from in vivo clearance. J. Control. Release 2012, 162, 208-217. 38. D'Addio, S. M.; Baldassano, S.; Shi, L.; Cheung, L.; Adamson, D. H.; Bruzek, M.; Anthony, J. E.; Laskin, D. L.; Sinko, P. J.; Prud'homme, R. K. Optimization of cell receptorspecific targeting through multivalent surface decoration of polymeric nanocarriers. J. Control. Release 2013, 168, 41-49. 39. Beziere, N.; Lozano, N.; Nunes, A.; Salichs, J.; Queiros, D.; Kostarelos, K.; Ntziachristos, V. Dynamic imaging of PEGylated indocyanine green (ICG) liposomes within the tumor microenvironment using multi-spectral optoacoustic tomography (MSOT). Biomaterials 2015, 37, 415-424.


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Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40. Jeong, H.-S.; Lee, C.-M.; Cheong, S.-J.; Kim, E.-M.; Hwang, H.; Na, K. S.; Lim, S. T.; Sohn, M.-H.; Jeong, H.-J. The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node. J. Liposome Res. 2013, 23, 291-297. 41. Cook, J. R.; Frey, W.; Emelianov, S. Quantitative Photoacoustic Imaging of Nanoparticles in Cells and Tissues. ACS Nano 2013, 7, 1272-1280. 42. Claessens, C. G.; Hahn, U.; Torres, T. Phthalocyanines: From outstanding electronic properties to emerging applications. Chem. Rec. 2008, 8, 75-97. 43. Herz, A. H., Aggregation of sensitizing dyes in solution and their adsorption onto silver halides. Adv. Colloid Interface Sci. 1977, 8, 237-298. 44. Morisue M.; Ueda S.; Kurasawa M.; Naito M.; Kuroda Y. Highly Fluorescent SlippedCofacial Phthalocyanine Dimer as a Shallow Inclusion Complex with α-Cyclodextrin. J. Phys. Chem. A. 2012, 116, 5139–5144. 45. Pansare, V. J.; Bruzek, M. J.; Adamson, D. H.; Anthony, J.; Prud'homme, R. K. Composite Fluorescent Nanoparticles for Biomedical Imaging. Mol. Imaging Biol. 2014, 16, 180-188. 46. Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. In vivo Molecular Imaging of Cancer with a Quenching Near Infrared Fluorescent Probe Using Conjugates of Monoclonal Antibodies and Indocyanine Green. Cancer Res. 2009, 69, 1268-1272. 47. Lu, H. D.; Wilson, B. K.; Lim, T. L.; Heinmiller, A.; Prud’homme, R. K., Real-Time and Multiplexed Photoacoustic Imaging of Internally Normalized Mixed-Targeted Nanoparticles. ACS Biomaterials Science & Engineering 2017, 3 (3), 443-451.47. 48. Ansell, S. M.; Johnstone, S. A.; Tardi, P. G.; Lo, L.; Xie, S.; Shu, Y.; Harasym, T. O.; Harasym, N. L.; Williams, L.; Bermudes, D.; Liboiron, B. D.; Saad, W.; Prud’homme, R. K.; Mayer, L. D. Modulating the Therapeutic Activity of Nanoparticle Delivered Paclitaxel by Manipulating the Hydrophobicity of Prodrug Conjugates. J. Med. Chem. 2008, 51, 3288-3296. 49. D'Addio, S. M.; Reddy, V. M.; Liu, Y.; Sinko, P. J.; Einck, L.; Prud'homme, R. K. Antitubercular Nanocarrier Combination Therapy: Formulation Strategies and In Vitro Efficacy for Rifampicin and SQ641. Mol. Pharm. 2015, 12, 1554-1563. 50. Pinkerton, N. M.; Gindy, M. E.; Calero‐del, V. L.; Wolfson, T.; Pagels, R. F.; Adler, D.; Gao, D.; Li, S.; Wang, R.; Zevon, M. Single‐Step Assembly of Multimodal Imaging Nanocarriers: MRI and Long‐Wavelength Fluorescence Imaging. Adv. Healthcare Mater. 2015, 4, 1376-1385. 51. Pinkerton, N. M.; Grandeury, A.; Fisch, A.; Brozio, J.; Riebesehl, B. U.; Prud'homme, R. K. Formation of Stable Nanocarriers by in Situ Ion Pairing during Block-Copolymer-Directed Rapid Precipitation. Mol. Pharm. 2013, 10, 319-328. 52. Weissmueller, N. T.; Lu, H. D.; Hurley, A.; Prud'homme, R. K., Nanocarriers from GRAS Zein Proteins to Encapsulate Hydrophobic Actives. Biomacromolecules 2016, 17, 3828– 3837. 53. Tang, C.; Edelstein, J.; Mikitsh, J. L.; Xiao, E.; Hemphill, A. H.; Pagels, R.; Chacko, A.M.; Prud’homme, R. Biodistribution and fate of core-labeled 125 I polymeric nanocarriers prepared by Flash NanoPrecipitation (FNP). J. Mater. Chem. B 2016, 4, 2428-2434. 54. D'Addio, S. M.; Baldassano, S.; Shi, L.; Cheung, L. L.; Adamson, D. H.; Bruzek, M.; Anthony, J. E.; Laskin, D. L.; Sinko, P. J.; Prud'homme, R. K. Optimization of cell receptorspecific targeting through multivalent surface decoration of polymeric nanocarriers. J. of Control. Release 2013, 168, 41-49.


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55. Lu, H. D.; Yang, S. S.; Wilson, B. K.; McManus, S. A.; Chen, C. V.-H.; Prud’homme, R. K. Nanoparticle targeting of Gram-positive and Gram-negative bacteria for magnetic-based separations of bacterial pathogens. Appl. Nanosci. 2017, 7, 1-11.

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