Optical Characteristics and Tumor Imaging Capabilities of Near

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Optical Characteristics and Tumor Imaging Capabilities of Near Infrared Dyes in Free and Nano-Encapsulated Formulations Comprised of Viral Capsids Yadir Guerrero, Sheela Singh, Turong Mai, Ravoori Murali, Leela Tanikella, Atta Zahedi, Vikas Kundra, and Bahman Anvari ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 22, 2017

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ACS Applied Materials & Interfaces

Optical Characteristics and Tumor Imaging Capabilities of Near Infrared Dyes in Free and Nano-Encapsulated Formulations Comprised of Viral Capsids †

†

†

Yadir Guerrero, Sheela P. Singh,§ Turong Mai, Ravoori K. Murali,§ Leela Tanikella, Atta †

Zahedi, Vikas Kundra,§ # and Bahman Anvari †

§

†*

Department of Bioengineering, University of California, Riverside, CA 92521, United States

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center,

Houston, TX 77030, United States #

Department of Radiology, The University of Texas MD Anderson Cancer Center, Houston, TX

77030, United States

KEYWORDS: Biomimetics, Fluorescence, Indocyanine Green, Peritoneal Metastasis, Plant Virus

ACS Paragon Plus Environment

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ABSTRACT: Near infrared (NIR) fluorescent molecules and nano-sized structures can serve as potential optical probes for image-guided removal of small tumor nodules (≈ < 1 mm diameter). While indocyanine green (ICG) remains as the only FDA-approved NIR dye, other organic dyes are under extensive development for enhanced imaging capabilities. One such dye is BrCy106NHS where bromine is substituted for aromatic structures in cyanine dyes. Herein, we investigate the absorption and fluorescence characteristics of ICG and BrCy106-NHS, and quantitatively assess their tumor imaging capabilities in free (non-encapsulated) and a nanoencapsulated form that utilizes the capsid protein (CP) from genome-depleted plant-infecting brome mosaic virus as the encapsulating shell. We refer to these nano-constructs as optical viral ghosts (OVGs). For example, when fabricated at CP to dye concentration ratio of 200, value of the spectrally integrated fluorescence emission for BrCy106-NHS-doped OVGs is ≈60 times higher than that of ICG-doped OVGs. Our analysis of homogenized mice intraperitoneal tumors indicate that the averaged total fluorescence emission associated with the use of BrCy106-NHSdoped can be at least about 44 times greater than that of ICG-doped OVGs. Our results suggest that OVGs containing BrCy106-NHS may potentially serve as effective optical probes for tumor imaging.


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INTRODUCTION The potential of fluorescent molecules and nano-sized structures as enabling technologies for use in image-guided surgery and other clinical application continues to be under extensive investigation.1-7 In particular, exogenous fluorescent materials that are activated by near infrared (NIR) wavelengths (in the range of ≈ 700-1450 nm) offer two key advantages towards these endeavors: (1) increased depth of optical penetration, on the order of ≈ 2-3 cm, resulting from reduced light absorption and scattering by water, proteins, and other macromolecules; and (2) enhanced imaging contrast resulting from reduced auto-fluorescence over NIR spectral band.8

Scheme 1. Molecular structures of (a) ICG, and (b) BrCy106-NHS. NHS-activated carboxyl groups in BrCy106 are shown in bold. One particular NIR imaging agent is indocyanine green (ICG) (Scheme 1a). ICG (molecular weight (MW) ≈ 775 Da) is composed of two nitrogen-containing polycyclic aromatic (benzoindotricarbocyanine) moieties, connected with a polyene bridge. The π-conjugation along the bridge between the two aromatic moieties gives rise to NIR absorption and fluorescence of


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the dye. ICG remains the only NIR fluorophore approved by United States Food and Drug Administration (FDA) for specific applications including cardiocirculatory measurements, liver function tests, and ophthalmological imaging.9-10 ICG has also been investigated for sentinel lymph node (SLN) mapping and staging in cancer patients,11-15 and lymphedema assessment.16-18 Despite its usage in clinical applications, ICG’s major drawbacks are its short half-life within plasma (≈ 2-4 minutes), and relatively weak fluorescence emission as quantified by a low fluorescence quantum yield, on the order of ≈ 0.03 when dissolved in water at low concentration (≈ 0.1-1 µM).19 Nano-encapsulation has been investigated as an approach to extend the circulation time of ICG. To-date, ICG has been encapsulated into various nano-sized constructs including those composed of micelles, liposomes, synthetic polymers,20-26 calcium phosphate,27 and silica and silicate matrices.28-30 We previously reported on encapsulation of ICG into polymeric nanoparticles, coated with poly ethylene glycol (PEG), as a technique to increase the blood circulation time of ICG and delay its hepatic accumulation up to an hour.31 Viruses have been investigated as platforms to incorporate imaging agents including quantum dots (QDs),32 gold,33 and magnetic nanoparticles.34 Virus-based protein cages, assembled from the cowpea chlorotic mottle virus,35 and the capsid of the MS2 bacteriophage coated with gadolinium have been investigated as MRI contrast agents.35-36 Constructs derived from replication deficient plant viruses are especially attractive since plant viruses do not infect humans,37 and may be biocompatible in mammalian systems.38-40 We previously demonstrated the first successful engineering of an optical nano-structured system consisting of capsid protein (CP) subunits derived from genome-depleted plant infecting


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brome mosaic virus (BMV) to encapsulate ICG,41 and reported their capability in NIR imaging,41-43 including fluorescence imaging of ovarian cancer cells in vitro.44 We refer to these constructs as optical viral ghosts (OVGs) since the genomic contents of the wild-type BMV are deleted, and only the CP subunits of the virus are utilized to encapsulate the fluorophore. In this study, we extend our previous works that focused on OVG-mediated in vitro imaging41,

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investigate the utility of OVGs in imaging implanted tumors models in mice. To address the second issue related to the low fluorescence quantum yield of ICG, development of alternative dyes is pursued by various investigators.45-46 Here we examine a new NIR cyanine dye, Brominated Cyanine 106 N-Hydroxysuccinimide (NHS) (MW ≈ 935 Da) (developed and manufactured by NanoQuantum Sciences, Bellevue, WA) (Scheme 1b). Briefly, the strategy in development of BrCy106-NHS is based replacement of the aromatic structures in cyanine dyes with heavier halogen elements such as F, Cl, Br, or I. In particular, BrCy106-NHS makes use of Br. Further background about such halogenated dyes is available.47 We utilized the NHS-activated form of this dye (referred to as BrCy106-NHS) throughout the studies presented in this manuscript (except for the results shown in Figure 2 where the dye with non-activated carboxyl group (BrCy106) was used). We pursue three main objectives in this manuscript. First, we compare the absorption and fluorescence characteristics of free (non-encapsulated) ICG and BrCy106-NHS. Results of such characterizations provide the baseline information that can be used to examine the effects of nano-encapsulation, and obtain quantitative information related to encapsulation efficiencies of the dyes. Next, we extend these characterizations to nano-encapsulated versions of the dyes formed as OVGs. In particular, we investigate the effects of varying the relative amounts of CP and the dye on the resulting CP utilization efficiency in forming OVGs, dye encapsulation


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efficiency, as well as the absorption and fluorescence characteristics and the relative number of ICG- and BrCy106-NHS-doped OVGs. Finally, we compare the effectiveness of ICG and BrCy106-NHS, in both free and nano-encapsulated OVG forms, in NIR imaging of intraperitoneal tumors implanted in mice, and discuss the potential utility and relevance of our findings for intraoperative NIR imaging of tumors. To the best of our knowledge, we report for the first time, the optical characteristics of BrCy106-NHS in free and nano-encapsulated forms, and present results related to its tumor imaging capability. Our findings from this study provide useful information towards the engineering of optimum formulations of OVGs with maximal fluorescence emission for use in potential future applications such as image-guided removal of tumors. RESULTS AND DISCUSSION Absorption and Fluorescence Characteristics of Free (Non-Encapsulated) Dyes. We present concentration-dependent absorption characteristics of free ICG and BrCy106-NHS when dissolved in OVG suspension buffer (50 mM sodium acetate, 8 mM magnesium acetate, pH=4.0) (Figure 1). Absorption spectra of 1 – 10 µg/ml ICG showed spectral peaks at 780 nm (Figure 1a), consistent with the 780 nm spectral peak of 0.6–13 µM ICG dissolved in deionized (DI) water,48 and attributed to the monomer form of ICG as previously indicated in literature.19, 49 With increased ICG concentrations to 5 and 10 µg/ml, where there is increased likelihood of ICG molecules to aggregate, a secondary peak at 715 nm emerged. Aggregation-induced split in the excited electronic state that results from a stacked (sandwich-like) arrangement of the individual chromophores, and their transition dipole moments constitutes H-aggregation.50-52 In H-aggregates, transitions to the upper level of the split-excited state are allowed, resulting in a


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blue spectral shift. The observed hypsochromic shift to 715 nm is consistent with spectral shifts induced by H-aggregates; hence, we attribute this shift to H-like aggregate forms of ICG. This H-like aggregate peak is also consistent with the 714 nm peak of ICG dissolved in DI water in similar concentration range.48

Figure 1. Absorption spectra of (a) ICG, and (b) BrCy106-NHS in OVG suspension buffer. In panel (c), we show χ, the ratio of the peak absorbance value of BrCy106-NHS to that of ICG with DI water and OVG suspension buffer as solvents.


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Absorption spectra of 1-10 µg/ml BrCy106-NHS dissolved in OVG suspension buffer showed a narrower absorption band than ICG with distinct spectral peak at 750 nm (Figure 1b), which may be associated with monomeric form of BrCy106-NHS. We present the quantity χ defined as the ratio of the peak absorbance value of BrCy106-NHS to that of ICG at concentrations of 1, 5, and 10 µg/ml (Figure 1c) (a listing of all symbols and their definitions is provided in Table S1 in Supporting Information). The respective ranges of χ were ≈ 1.7 – 2.6 when the dyes were dissolved in DI water, and 1.3 – 1.9 when dissolved in OVG suspension buffer.

Figure 2. Excitation-emission maps for (a) ICG, and (b) BrCy106 (the form of the dye with nonactivated carboxyl group) dissolved in DI water at concentrations of 7 µg/ml. We obtained the excitation and fluorescence emission maps of free ICG and BrCy106 dissolved in water (pH = 7.0) at concentration of 7 µg/ml at 4 °C (Figure 2). We attribute the “hot spot” peaked at 690 nm emission wavelength (indicated by the dark maroon color with intensity of 7.4E+04) and produced in response to photo-excitation at 675 nm to H-like aggregate forms of ICG (Figure 2a).19,

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Sweeping the excitation wavelength above 720 nm


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shifted the emission away from that produced by the H-like aggregate form of ICG, and resulted in the formation of the second hot spot, attributed to the monomeric forms of ICG. Peak emission intensity in the monomer hot spot was at 805 nm, and produced in response to 750 nm photoexcitation wavelength (Figure 2a). Photo-excitation of 7 µg/ml BrCy106 dissolved in DI water in the range of 600-800 nm produced a distinct emission peak at 775 nm (Figure 2b). The absence of a distinct secondary emission for BrCy108 dissolved in DI water suggests that in comparison to ICG, this dye is less prone to aggregation at the same concentration investigated. In response to photo-excitation of ICG at 680 nm and BrCy106-NHS at 730 nm, peak emission intensities at the respective spectral peaks (775 nm for ICG, and 780 nm for BrCy106), were produced at concentration of 8 µg/ml (Figure 3). The peak emission intensity value of BrCy106-NHS was ≈ 27 times higher than that of ICG at the same concentration (8 µg/ml) (Figure 3a). We attribute the increased fluorescence emission of BrCy106-NHS to the presence of Br in place of the aromatic structures to reduce or eliminate the non-radiative decay pathways involving C-H vibrational modes. Although the presence of Br can increase the rates of intersystem crossing to quench the emission, shifting the vibrational modes away from C-H resonance still leads to an increase in fluorescence emission. Value of the spectrally-integrated fluorescence emission (ϕ) (see Equation 1 in Experimental Section) over 735 – 900 nm band for free ICG dissolved in OVG suspension buffer was maximal at 8 µg/ml in response to photo-excitation at 720 nm, and declined at higher concentrations (Figure 3b). However, in response to photo-excitation at 720 nm, ϕ continued to rise with increasing concentration of free BrCy106-NHS dissolved in OVG suspension buffer,


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Figure 3. (a) Peak fluorescence emission intensity of free ICG and BrCy106-NHS in OVG suspension buffer at respective wavelengths 775 and 780 nm, and in response to corresponding excitation wavelengths of 680 and 730 nm. Arrows point to the corresponding axis for ICG (left axis) and BrCy106-NHS (right axis). (b) Spectrally-integrated values of fluorescence emission (ϕ) in the range of 735 – 900 nm with both dyes excited at 720 nm. (c) Values of ϕ for free BrCy106-NHS dissolved in DI water immediately after preparation, and after storage for the indicated times and temperatures. Measurements for the frozen samples were obtained after thawing them, and preparing the appropriate dilutions.


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and appeared to approach a plateau at concentration of 20 µg/ml. At this concentration, ϕ for BrCy106-NHS was ≈ 57 times higher than that of ICG. There was less than 10% variation in ϕ values when storing 3, 10, and 20 µg/ml of BrCy106-NHS dissolved in DI water for 60 hours at 4 °C, or for one year at -20 °C (Figure 3c). These results indicate that the fluorescence emission characteristics of BrCy106-NHS remain relatively stable for an extended time when stored at these temperatures. This stability is in contrast to that of ICG whose fluorescence emission tends to decrease by ≈ 96% after four days of storage at 8 °C in the dark.53 Absorption and Fluorescence Characteristics of ICG and BrCy106-NHS in NanoEncapsulated Forms Comprised of Viral Capsids. We used the CP of BMV to encapsulate ICG or BrCy106-NHS, and form OVGs. We define φ as the ratio of BMV CP concentration to that of the fluorophore in the encapsulation buffer solution during the OVGs fabrication process. We have previously presented transmission electron microscope images of OVGs,41, 43 and they are not presented again in this manuscript. OVGs resemble the morphology of wild type BMV, and are highly mono-dispersed. The mean diameter of the population ICG-doped OVGs formed at φ=4 or 200 is ≈24 nm.41, 44 We provide the measurements of the hydrodynamic diameters for BrCy106-NHS-doped OVGs in Figure S2c (Supporting Information). Absorption spectra for OVGs fabricated using 2 mg/ml of CP and 10 µg/ml of either ICG or BrCy106-NHS (i.e., φ=200) are shown in Figure 4a. As compared to free ICG (Figure 1a), the primary and secondary NIR peaks were red-shifted from 780 nm to 800 nm, and from 715 to 735 nm, respectively (Figure 4a). We attribute these bathochromic shifts to induced alterations in the transition dipole moments of ICG resulting from its conformational changes when encapsulated.


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BrCy106-NHS-doped OVGs also showed a red-shifted spectral peak from 750 nm to 760 nm without a distinct secondary peak (Figure 4a), suggesting that BrCy106-NHS may have been mostly present in monomeric form within the OVGs, but likely in an altered conformation.

Figure 4. Spectroscopic results for ICG- and BrCy106-doped OVGs fabricated at φ = 200 based on using 2 mg/ml and 10 µg/ml of CP and dye, respectively. (a) Absorption spectra for ICG-


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doped and BrCy106-doped OVGs, and their corresponding supernatants. (b) Fluorescence emission spectra for ICG- and BrCy106-NHS-doped OVGs, their corresponding supernatants, and filtrate associated with BrCy106-NHS-doped OVGs fabrication. Excitation wavelength was 680 nm. Arrows point to the corresponding axis for ICG-doped OVGs and its supernatant (left axis), and BrCy106-NHS-doped OVGs and its supernatant (right axis). (c) Spectrally-integrated values of fluorescence emission (ϕ) in the range of 700 – 900 nm for ICG- and BrCy106-NHSdoped OVGs, and their corresponding supernatants. The absorption peak at 280 nm (Figure 4a) for both ICG- and BrCy106-NHS-doped OVGs originates from the tyrosine, tryptophan, and cysteine residues in the CP,54

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consistent with our previous studies with ICG-doped OVGs.41, 44 Since the same concentration of CP was used to form both the ICG- and BrCy106-doped OVGs, the higher absorbance value at 280 nm for ICG-doped OVGs suggests that higher number of ICG-doped OVGs were formed as compared to BrCy106-NHS-doped OVGs. Specifically, the 280 nm absorbance value of BrCy106-NHS-doped OVGs was ≈ 82% that of ICG-doped OVGs (Figure 4a), suggesting that ≈ 18% fewer BrCy106-NHS-doped OVGs as compared to ICG-doped OVGs were formed. The absorbance values of BrCy106-NHS-doped OVGs in the spectral range of ≈ 700-820 nm were lower than those of ICG-doped OVGs. Notably, while χ at 10 µg/ml for the free dyes was ≈ 1.8 (Figure 1c), it was reduced to ≈0.62 in the encapsulated form (Figure 4a). The fact that the value of χ≈0.62 was smaller than the BrCy106-NHS-doped OVGs to ICG-doped OVGs 280 nm absorbance ratio of ≈ 0.82 suggests that in addition to fewer BrCy106-NHS-doped OVGs formed, the encapsulation efficiency of BrCy106-NHS was about 62% of ICG for this set of OVGs.


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The supernatant associated with fabrication of ICG-doped OVGs showed near zero absorption throughout the recorded spectral range, indicating that nearly all the CP and ICG were utilized to form this set of OVGs formed at φ=200. However, the supernatant associated with the fabrication of BrCy106-NHS-doped OVGs showed absorption peaks at 280 and 750 nm, indicating there were residual CP subunits and BrCy106-NHS in the supernatant that had not reassembled into OVGs. Specifically, based on the respective 280 nm absorbance values of ≈ 2, 1.4, and 0.47 corresponding to the purified CP at 2 mg/ml, BrCy106-NHS-doped OVGs, and the supernatant, we estimate that ≈ 23.5% (0.47/2) of the CP subunits remained within the supernatant, and the utilized fraction of purified CP to form these OVGs was ≈ 70% (1.4/2) of the total amount. In this case, the unaccounted fraction of CP (≈ 7%) could be associated with protein degradation, or protein settlement in the stock solution following purification, and ultimately not used during OVG fabrication process. Similarly, based on the 750 nm absorbance values of ≈ 1.28, 0.5, and 0.12 corresponding to free BrCy106-NHS at 10 µg/ml, BrCy106-NHS-doped OVGs, and the supernatant, respectively, we estimate that ≈ 9% (0.12/1.28) of BrCy106-NHS remained within the supernatant, and ≈ 39% (0.5/1.28) of the dye was incorporated into these OVGs. We attribute the un-accounted fraction of BrCy106-NHS dye (≈ 52%) to the non-incorporated amount of the dye during the encapsulation process, which permeated through the dialysis membrane. Fluorescence spectra of the filtrate showed NIR emission, consistent with that of BrCy106-NHS-doped OVGs (Figure 4b). In contrast, the near complete incorporation of ICG into this set of OVGs is suggestive of the more favorable interactions between ICG and CP subunits that result in its encapsulation and/or binding to CP.


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The fluorescence emission spectrum of BrCy106-NHS-doped OVGs showed a single peak at 765 nm in response to photo-excitation at 680 nm (Figure 4b). In contrast, ICG-doped OVGs produced emission peaks at 690 and 795 nm when photo-excited at 680 nm, respectively corresponding to the H-like aggregate and monomer forms of ICG in OVGs. Consistent with the absorption spectra (Figure 4a), the supernatant associated with fabrication of ICG-doped OVGs showed negligible fluorescence emission (Figure 4b), indicating that nearly all ICG molecules were loaded into the OVGs during encapsulation process. However, the supernatant associated with the fabrication of BrCy106-NHS-doped OVGs showed an emission peak at 765 nm (Figure 4b), another indication that not all BrCy106 molecules were loaded into the OVGs. Despite (1) the reduced encapsulation efficiency of BrCy106-NHS; (2) higher absorption of ICG-doped OVGs in the 700-820 nm range than BrCy106-NHS-doped OVGs; and (3) the likelihood that a greater number of ICG-doped OVGs were formed using the same concentrations of CP and fluorophore, the BrCy106-NHS-doped OVGs produced a more intense fluorescence emission intensity in response to 680 nm photo-excitation (Figure 4b). Specifically, the peak emission intensity for BrCy106-NHS-doped OVGs was nearly 22 times higher than that associated with the monomeric emission intensity peak of ICG-doped OVGs at 795 nm. The fluorescence emission of BrCy106-NHS-doped OVGs spectrally integrated over the 700-900 nm band was nearly 60 times higher than that of ICG-doped OVGs (Figure 4c). We point out that in the OVG form, the dye is encapsulated, and as such, the NHS groups on BrCy106 are unlikely to react with amino groups in proteins in vivo. More importantly, hydrolysis of NHS is the most important reaction that competes with reaction of esters with nucleophilic groups in proteins.56 For example, the reported hydrolysis rates of sulfosuccinimidyl propionate, as an illustrative NHS ester, in sodium phosphate ranges between ≈ 3.3-1.7 s-1 in the


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corresponding pH range of 7-8 at 25 °C.56 In another study, the reported range for hydrolysis of two different NHS esters adsorbed on gold surfaces is ≈ 0.045-0.61 M-1s-1.57 Given that the encapsulation process of BrCy106-NHS into OVGs in the aqueous media are on the order of hours, we expect that most of NHS undergoes hydrolysis. Therefore, it is unlikely that there would be any remaining NIH groups to react with amino groups. We investigated the effects of varying the φ value on the absorption and fluorescence characteristics of ICG- and BrCy106-NHS-doped OVGs, CP utilization efficiency in forming OVGs, dye encapsulation efficiency, and the relative number of particles fabricated. We varied φ in two manners. First, we kept the dye concentration fixed at 5 µg/ml while changing the CP concentration from 20 µg/ml to 5 mg/ml to fabricate OVGs at φ values in the range of 4 to 1000. In the second manner to vary φ, we changed the ICG concentration from 250 µg/ml to 1 µg/ml, and the BrCy106-NHS concentration from 50 µg/ml to 1 µg/ml, while fixing the CP concentration at 1 mg/ml in both cases. In doing so, we fabricated ICG-doped OVGs at φ values in the range of 4-1000, and BrCy106-NHS-doped OVGs at φ values in the range of 20-1000. Detailed results of these experiments are provided in Figures S1-S3 (Supporting Information). We summarize the results in Tables 1 and 2. NIR Imaging of Implanted Intraperitoneal Tumors in Mice. We investigated the effectiveness of free and nano-encapsulated ICG and BrCy106-NHS as fluorescent probes in imaging intraperitoneal tumors derived from ovarian cancer cells implanted in mice. We administered 150 µl of each of the agents ICG, BrCy106-NHS, ICG-doped OVGs, BrCy106NHS-doped OVGs, or RNA Assembly Buffer (control) via the tail vein. We set the administered free dye concentration to 75 µg/ml, and fabricated the dye-doped OVGs using φ=200 with 5


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µg/ml concentration for each of the two dyes. To achieve near equivalency in terms of administering the same effective concentration of dyes in all cases, we concentrated the ICG- or BrCy106-NHS-doped OVGs by combining independently obtained pellets. The effective dye concentrations were ≈ 62.5 µg/ml and 50 µg/ml for ICG- and BrCy106-NHS-doped OVGs, respectively. Table 1. Effects of varying φ by fixing the dye concentration at 5 µg/ml, and changing the CP concentration in the range of 20 µg/ml to 5 mg/ml. ___________________________________________________________________________ CP Utilization Efficiency to form OVGs

ICG Encapsulation Efficiency

BrCy106-NHS Encapsulation Efficiency

Ratio of the Number of BrCy106-NHS- to ICG-doped OVGs

___________________________________________________________________________ ≈ 100% for 4≤φ≤20 for both ICGand BrCy106-NHS-doped OVGs

≈ 45% - 90% for 4≤φ≤200

≈ 3% - 54% for 4≤φ≤1000

Formation of aggregates likely for φ≥300

≈ 15 % - 36% for 4≤φ≤ 20 ≈ 80 % - 95% for 50≤φ≤ 150

≈ 100 % - 127% for 200≤φ≤1000 ≈ 88% - 71% for 50≤φ≤1000 for ICG-doped OVGs ≈ 82% - 35% for 50≤φ≤1000 for BrCy106-NHS-doped OVGs except for φ=400 and 500 where CP utilization efficiency was ≈ 100% _____________________________________________________________________________________________________

Table 2. Effects of varying φ by fixing the CP concentration at 1 mg/ml, and changing the ICG and BrCy106-NHS concentrations in the ranges of 250 - 1 µg/ml and 50 - 1 µg/ml, respectively. ___________________________________________________________________________ CP Utilization Efficiency to form OVGs

ICG Encapsulation Efficiency

BrCy106-NHS Encapsulation Efficiency

Ratio of the Number of BrCy106-NHS- to ICG-doped OVGs

___________________________________________________________________________ ≈ 100% for 4≤φ≤ 1000 for ICG-doped OVGs. However, formation of aggregates is likely at certain φ values (φ=4, 50)

≈ 100% for 4≤φ≤ 1000

≈ 3% - 54% for 20≤φ≤ 1000

≈ 53 % - 95% for 20≤φ≤1000

Formation of aggregates likely for φ≤50

≈ 63% - 100% For 20≤φ≤1000 for BrCy106-NHS-doped OVGs. However, formation of aggregates is likely at certain φ values (φ=20, 50, 75) _____________________________________________________________________________________________________


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As an illustrative example of NIR in vivo imaging, we present whole body fluorescent images of intraperitoneal tumor bearing mice at two hours post-injection with each of the agents (Figure 5). When injected with any one of the optical agents, most of the fluorescence signals emanated from the abdominal regions, particularly the liver and bowel. In the case of BrCy106NHS and BrCy106-NHS doped OVGs, fluorescence emission from the gallbladder may be attributed to the greater emission intensity of BrCy106-NHS as compared to ICG. Based on these whole body fluorescent images, we did not observe fluorescent signals that could be specifically associated with tumors as emissions from the tumors were most likely attenuated by skin and overlying tissues.

Figure 5. Representative coronal in vivo NIR fluorescence imaging of intraperitoneal SKOV3 tumors two hours post injection with control (RNA assembly buffer) and various optical agents in immunodeficient mice. Short arrows point to gallbladder; long arrows to the bowl and liver. Excitation wavelength 710 = nm, and emission > 760 nm recorded.


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To examine the effectiveness of the optical agents in tumor imaging, we fluorescently imaged tumors extracted two hours post-injection (Figure 6a). The most intense fluorescent image corresponded to the emission from BrCy106-NHS-doped OVGs. Using the images, we determined I*, control-subtracted averaged total fluorescence emission from the tumors (see Equation 2 in Experimental Section), and found a statistically significant higher value associated with mice injected with BrCy106-NHS-doped OVGs (p

Optical Characteristics and Tumor Imaging Capabilities of Near

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