Method for Real-Time Tissue Quantification of Indocyanine Green

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Method for Real-time Tissue Quantification of Indocyanine Green Revealing Optimal Conditions for Near Infrared Fluorescence Guided Surgery Ziyang Wang, Kena Ni, Xudong Zhang, Shichao Ai, Wenxian Guan, Huiming Cai, Yiqing Wang, Qian Lu, and Lucas A. Lane Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00480 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Analytical Chemistry

Method for Real-time Tissue Quantification of Indocyanine Green Revealing Optimal Conditions for Near Infrared Fluorescence Guided Surgery Ziyang Wang1,§, Kena Ni1,§, Xudong Zhang1, Shichao Ai2, Wenxian Guan2, Huiming Cai1, Yiqing Wang1,*, Qian Lu1,* and Lucas A. Lane1,* 1

Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu 210093, China. 2 Department of General Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu 210008, China. ABSTRACT: Near infrared fluorescence guided surgery (NIRFGS) offers better distinction between cancerous and normal tissues compared to surgeries relying on a surgeon’s senses of sight and touch. Due to the greater accuracy in determining tumor tissue margins, NIRFGS within clinics continues to grow. However, NIRFGS lacks standardization of the Indocyanine Green (ICG) dose and the preoperative period allowed after ICG administration. In an aim to find optimal doses and preoperative periods for NIRFGS standardization, we developed a method that quantitatively determines ICG levels within tissues in real-time. We find that not only do the dose and the preoperative periods influence tumor-to-background ratios (TBRs), but both also heavily influence subject-tosubject variances of these ratios. Optimal detection conditions are observed when larger than typical ICG doses are administered and longer than typical preoperative periods are allowed. Larger doses lead to increased TBRs, but longer preoperative periods are necessary to reduce TBR variances to those observed when using smaller doses. Our results suggest that a clinical investigation into maximum tolerable ICG doses and prolonging preoperative periods in NIRFGS is warranted.

For most patients afflicted with cancer comprising of solid tumors, surgery is the first line of treatment, seconded by chemotherapy and radiation. A complete resection, where the tumor and all of its constituent cancer cells are removed, is considered the most important factor in increasing patient survival.1,2 In common practice of surgical resections, surgeons rely on their senses of sight and touch to delineate the margin between the tumor and surrounding normal tissues. The human senses are highly subjective and successful surgical outcomes (i.e. no tumor recurrence) vary widely among surgeons and institutions.3 Around 40% of surgeries have evidence of residual tumor cells left within the surgical cavity after resection.4,5 These cells left unidentified can develop into another solid tumor leading to an increased risk of mortality. Thus, more sophisticated techniques are desired for attaining more precise determinations of tumor margins to diminish current recurrence rates after surgery. Near infrared fluorescence guided surgery (NIRFGS) is a rapidly growing technique that aids surgeons in determining tumor margins of several cancer types with greater precision and at detection sensitivities nearing a single cell.6-17 NIR wavelengths have less attenuation and background fluorescence from tissues,16,21-23 allowing greater tumor-tobackground ratios (TBRs) compared to other optical wavelengths. Though there are a variety of fluorescent probes which have been deemed useful for NIRFGS, to date only indocyanine green (ICG) is currently approved for clinical use.15,17-21 In a typical NIRFGS procedure, ICG is administered intravenously and allowed a preoperative period before the

surgical procedure. The length of the preoperative period is chosen to maximize signal to background ratios (TBRs), by allowing enough time for dye accumulation within the tumor (increasing signal) and allowing normal tissue clearance (decreasing background). After this period, the fluorescence is detected intraoperatively by an imaging device. Preferential accumulation of ICG within solid tumors over the surrounding normal tissues has been reported in several tumor types, thereby permitting precise margin determination.7,11,22-27 Despite the increase of NIRFGS devices and procedures under investigation or approved in the clinic,15,22-26 there remains a lack of standardization as to what doses and preoperative periods are optimal in terms of both TBRs and patient-topatient variances within these ratios. Though optimization is commonly perceived as maximizing the contrast between diseased and healthy tissues,28 large variabilities in TBRs complicate developing standardized device operation parameters during NIRFGS. Moreover, studies on optimizing ICG doses and preoperative times after injection tend to be end-point analyses which often miss important kinetic features of the dye within tissues over time. In this report, we present a method using mice tumor models that can quantitatively determine ICG tissue concentrations in real-time. This method revealed that while greater ICG administrations leads to an averaged increase in TBRs, there are much larger variations in tissue intensities within typical preoperative periods (12-24 hours after injection)29-31 than those observed using smaller doses. Smaller doses by having lower TBRs may necessitate devices with greater sensitivity. However, we find longer preoperative

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periods (> 48 hours) decrease the variabilities in the larger applied dose groups with only a slight sacrifice in the TBR. These results suggest that a clinical investigation into tolerable doses and longer preoperative periods after injection of ICG to minimize patient-to-patient variances while maintaining high TBRs is warranted.

Figure 1. A depiction of how our spectroscopic device records the fluorescence of intravenously administered ICG from the tumor, muscle, and liver tissues of a mouse over time.

MATERIALS AND METHODS Reagents. Pharmaceutical grade indocyanine green (ICG) for injection was purchased from Tianyi Biological Pharmaceutical Co., Ltd. (Liaoning China). Dulbecco's modified eagle medium (DMEM), and 0.25% trypsin were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biological Laboratories (Kibutz Beit Haemek, Israel). Instrumentation. The laser generator used was a Laser7855HS0 model, purchased from Ocean Optics, with a center wavelength of 784.802 nm, output power of 516mW and a FWHM of 0.0525 nm. The spectrometer used was a MAYP112511 model, purchased from Ocean Optics with a linearity of 99.74045. The integrated probe’s elements, including fibers, filters, and fiber collimation, were all purchased from Thorlabs. The interior structure of the integrated probe is shown in Figure S1. Operating conditions for these studies were setting the laser power density at 168.4 mW/cm2 and setting the distance of the probe from the samples at 4.6 cm using a spacer. The signal collection time was set at 5 s. Cell Line. A mouse mammary gland cancer cell line, 4T1, was purchased from ATCC (Manassas, VA, USA). Cultures were maintained in DMEM medium supplemented with 10% FBS, 80 U/mL penicillin and 80μg/mL streptomycin. Cells were cultured at 370C in a humidified environment at a 5% CO2 atmosphere. Mouse Tumor Model. Female nude BALB/c mice (5-6 weeks, 18±2 g) were provided by Jiesijie lab-animal company in Shanghai (Animal License No. SCXK (HU) 2013-0006). The mice were kept in a standard laboratory environment and given water and food freely. All experimental procedures were performed in strict conformance with guidelines of the Committee on the Care and Use of Laboratory Animals and the related ethical regulation of Nanjing University. To generate the 4T1 tumor model, 5x106 4T1 cells in a 100 μL solution was subcutaneously injected into the flank area of each nude mouse. Animals were used for in vivo experiments when the

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tumor size reached approximately 500 mm3 after tumor-cell inoculation. Measurement of Mass Density. Female nude mice bearing 4T1 tumors were fully anesthetized with pentobarbital sodium salt (1.5 % m/v diluted in PBS) and sacrificed. Tumors, livers and thigh muscles were harvested from these mice, and were washed with PBS twice, then wiped clean by medical absorbent cotton. Tumors, livers and thigh muscles were weighed and the volumes of each were measured by the displacement method using PBS. Calibration Curves to Determine Concentration of ICG Based on the Intensities of Homogenate Tissues. First, we harvested 0.5 – 1.0 g of tumor, liver, and thigh muscle tissues from mice, without any ICG administration, then homogenized the excised tissues diluted in a 1:9 mass by volume ratio using a 4% albumin phosphate buffered saline solution. Then, homogenate solutions with known ICG concentrations of 1 x 10-8, 5 x 10-9, 2.5 x 10-9, 1.25 x 10-9, 6.25 x 10-10, 3.13 x 10-10, 1.56 x 10-10, 7.81 x 10-11, 3.91 x 10-11, and 0 M were prepared, where the fluorescence intensity of the mixtures was examined upon a black 96 well plate (270 µL per well) to establish the relationship between the measured fluorescence intensity to the applied ICG concentration. Determining the ICG Concentration Based on Fluorescence Intensity Levels Observed from Intact Excised Tissues. Here, mice were injected with ICG doses of 1, 2, 4, 8, and 16 mg/kg and sacrificed at either 1 or 24 hours after administration to allow varying concentration levels of dye within the tissues of interest. Tissues of the muscle, liver, and tumor were then excised, and the fluorescence intensities of each were recorded. Then the tissues were homogenized following the same procedure described previously. The fluorescence intensity of the homogenate samples was examined upon a black 96 well plate (270 µL per well). To calculate the concentration of ICG within these tissues we used the following relations: The total amount of ICG in the excised tissue sample and the homogenate are the same. Therefore, the following equation holds,

(1) where

is the ICG concentration to be determined of the intact tissue sample,

is the volume of the tissue sample, is the concentration of ICG in the homogenate, and

is the volume of the added saline solution. Since the tissue samples were diluted in a 1:9 mass by volume ratio to create the homogenate, the following equation is established.

9

/ 9

/ (2) Here,

is the mass of the tissue sample, is 1 g/cm3 for unit conversion, and

is the mass density of the tissue sample. Combining equations (1) and (2) leads to the following equation for the ICG concentration in the tissue.


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1 9

/

(3) The ICG concentration from these homogenized samples were determined by the fluorescence intensity calibration curves presented in the main manuscript. Now knowing the ICG content of the tissues based on fluorescent measurements once they are homogenized, calibration curves were then established based on the measured intensities of the intact tissues. Relationship between Fluorescence Intensity of Single Layer Skin, Pinched-double Layer of Skin, and Underlying Tissues. First, our mouse tumor models were injected with ICG doses of 2, 4, or 8 mg/kg and sacrificed after 2 to 4 hours to allow varying concentrations levels of the dye within the skin and underlying tissues of interest. Next, the fluorescence intensity of tissues the muscle, liver, and tumor covered by skin of intact mice was examined by the device. After recording these values, mice were sacrificed, then tissues consisting of the skin, tumor, liver, and muscle were then excised individually and examined upon the bottom of a black 96 well plate. Finally, the fluorescence intensity of two layers of skin were obtained while illuminating through a pinched (i.e. folded) region of skin. The model for skin subtraction of the recorded intensity of the intact tissue through skin is as follows: The skin samples having various ICG content were examined where we found a linear relationship between the fluorescence intensity recorded from a single layer of skin to that of a folded, or pinched, double layer of skin. The linear relation is written below.

4.0247 # 161.76482 (4) Here, is the ICG fluorescence intensity recorded for the folded, or pinched, double layer of skin, and # is the corresponding intensity recorded by a single layer of skin (i.e. the double layer laid flat). This relationship allowed us to determine the fluorescence contribution from a single layer of skin based upon recorded values of pinched, here called double layer, skin. Next, we placed single layers of skin over intact tissues, each having varying levels of fluorescence intensity. Here we also found a linear relationship between the merged tissue intensity ( &# ) consisting of the combined emissions from the intact tissue (muscle, tumor, or liver) with a layer of skin laid flat on top and the individual emissions of the single layer skin and intact tissue ( # and

, respectively).

&# 0.94858

0.87149 # − 609.91 (5) By combining (4) and (5), we created a model between &# , and

.

1.0542 &# − 0.22827 679.89 (6)

We found that this relationship held well regardless of the underlying tissue type (muscle, liver, and tumor) and had low specimen-to-specimen variability (Absolute value of variance from calculated ones = 8 %). This subtraction reduces the variance of measured intensity levels over time (Figure S2). All values of C.V. with or without this subtraction did not follow a normal distribution (p < 0.005, by one-sample KolmogorovSmirnov 2-tailed test), so a two-related-samples nonparametric test was done to test if this subtraction made any difference to the variance of the measured tissue intensity. This subtraction was proven to reduce the variance of measured tissue intensity (p < 0.001, two-tailed). We find that a linear model well represents the fluorescence data with adjusted R-squared values of 0.980. Fitting to higher order polynomial models only increases the adjusted R-squared value by a few tenths of a percent (Table S1). Test of the Quantification Model. First, our mouse tumor models were injected with ICG doses of 2, 4, or 8 mg/kg and sacrificed after 0.5 to 4 hours to allow varying concentrations levels of the dye within the skin and underlying tissues of interest. Next, the fluorescence intensity of tissues the muscle, liver, and tumor covered by skin was examined by the probe. Then we obtained the signal from two layers of skin by recording the fluorescence while illuminating through a pinched region of skin. Finally, we determined the signal of the underlying tissue of interest based on our model, then excised the tissue to compare our calculated value to the measured value of the excised tissue. To test for time dependence of our model, we compared tissues with similar ICG intensities, but accumulated the dye under different dose and time conditions. Here, we obtained 2 skin samples that had similar intensity levels using a 2 mg/kg dose at 24 h (skin-1) and an 8 mg/kg dose at 72 h (skin-2). 2 muscle tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 26 h (muscle-1) and at an 8 mg/kg dose collected at 74 h (muscle-2). 2 liver tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 25 h (liver-1) and at an 8 mg/kg dose collected at 73 h (liver-2). 2 tumor tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 24 h (tumor-1) and at an 8 mg/kg dose collected at 72 h (tumor-2). We combined either skin-1 or skin-2 with either of the 2 samples of a particular tissue type (tissue-type-1 or tissue-type-2) to confirm the combined intensity yielded similar values (skin1 + tissue-type-1 = skin-1 + tissue-type-2 = skin-2 + tissuetype-1 = skin-2 + tissue-type-2). Real-time Measurements of ICG in Vivo with Various Dose. Here, we intravenously administered ICG doses ranging from 1, 2, 4 and 8 mg/kg via tail vein injection and monitored the fluorescence intensity levels of tumor, liver and muscle at time points of 1, 2, 4, 8, 12, 24, 48, and 72 hours. Intensity levels were translated to concentrations using our established model presented herein. Statistical Analysis. The fluorescence intensity and ICG concentration of each timing were expressed as mean ± SD (standard deviation) of n = 9 (3 individual mice each with 3 separate measurements at different spatial locations) independent experiments. Linear fittings and nonlinear fittings were assessed by the Pearson correlation coefficient (Pearson’s r) or coefficient of determination (COD/R-Square). Confidence intervals of fluorescence intensity at each timing were obtained by One-Sample T-test compared with 0. One-sample



Kolmogorov-Smirnov 2-tailed test was taken to test if all the values of coefficients of variation (C.V.) of target tissue intensity (with/without skin interference) followed a normal distribution. The difference in measured tissue intensity made by skin interference subtraction was tested by two-relatedsamples nonparametric test. We conducted all analyses in OriginPro 2016 Version 9.3.226 (OriginLab Corporation) and IBM SPSS Statistics Version 22 (IBM Corporation).

RESULTS AND DISCUSSION Quantification of ICG within the Underlying Tissues. In this process we used mice models bearing 4T1 breast cancer cell line tumors, where ICG signals were collected using a hand-held spectroscopic device (Figure 1). For the mouse models, we used BALB/c mice with tumors derived from the 4T1 breast cancer cell line. The tumors were grown subcutaneously in the flank area and allowed to reach a size of 500 mm3. The construction of the device followed a previously reported procedure.32 In addition to our hand-held spectroscopic device, we added a 4.6 cm spacer that maintains a constant working distance while imaging across complex topographies of tissue samples (Figures 2a and b, and S1). Our quantification of ICG within the underlying tissues involved a three-step process: First, calibration curves were developed using homogenates of excised tissues with known applied concentrations of ICG. Second, we recorded the fluorescence of excised tissues, then homogenized them to develop a relationship between the measured ICG fluorescence intensity of intact tissues with their homogenized values. From this, we can infer the ICG concentrations based on the measured fluorescence intensity from non-homogenized excised tissues. Third, we developed a relationship of the skin contribution to the overall ICG signal obtained when illuminating underlying tissues of interest through the skin of the intact mouse model. From this relationship, we were then able to subtract the skin interference and determine the ICG content of the underlying tissues. To develop calibration curves to determine ICG concentrations from intensities of homogenate tissues, we performed the following procedures: First, we harvested 0.5 – 1.0 g of tumor, liver, and thigh muscle tissues from mice and homogenate the excised tissues diluted in a 1:9 mass by volume ratio using a 4% albumin phosphate buffered saline solution. Then, homogenate solutions with known ICG concentrations ranging from 3.91 x 10-11 – 1 x 10-8 M were prepared, and the fluorescence intensity of the mixtures were examined upon a black 96 well plate to establish the relationship between the measured fluorescence intensity to the ICG concentration. A separate calibration curve is generated for each tissue type inspected (Figure 2c). Though each tissue displayed a nonlinear response of fluorescence intensity with ICG concentration, the responses were quite similar among the tissues inspected over nanomolar concentration ranges. Such concentration ranges are commonly encountered within the tissues during image guided surgeries using ICG. We then progressed to determining the ICG concentration based on fluorescence intensity levels observed from intact (non-homogenized) excised tissues. Here, the mice were injected with ICG doses of 1, 2, 4, 8, and 16 mg/kg and sacrificed after 1 to 24 hours after administration to allow varying concentrations of the dye to accumulate within tissues of interest. Tissues of the muscle, liver, and tumor were then ex-

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cised, and the fluorescence intensities of each were recorded. Once the fluorescence intensity of the intact sample is recorded, the tissue is then homogenized following the same procedure described in the previous paragraph. The ICG content from these homogenized samples were determined from the calibration curves of Figure 2c. Now knowing the ICG content of the homogenized samples, calibration curves were then established based on the intensities of the intact tissues from which the homogenate samples were obtained (Figure 2d). Finally, we established a model to subtract the skin interference from the intensity obtained when illuminating underlying tissues through the skin of an intact mouse. Here, our mouse tumor models were injected with ICG doses of 2, 4, or 8 mg/kg and sacrificed after 2 to 4 hours to allow varying concentrations levels of the dye within the skin and underlying tissues of interest. Tissues consisting of the skin, tumor, liver, and muscle were then excised for further analysis. First the skin was examined where we found a linear relationship between the fluorescence intensity recorded from a single layer of skin to that of a folded double layer of skin. This relationship allowed us to determine the fluorescence contribution from a single layer of skin based upon recorded values of pinched, here called double layer, skin

Figure 2. a) A depiction of the process of excising and inspecting the intact and later homogenized tissue samples of the tumor, liver, and muscle from a mouse that has been injected with a fluorescent ICG dye solution. b) A depiction of the procedure to remove the skin interference from the underlying tissue signal. c) Calibration curves determining the ICG concentration based on the fluorescence intensity levels obtained from muscle, liver, and tumor homogenate samples. d) Calibration curves determining the


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concentration of ICG based on recorded fluorescence intensities observed within intact excised tissue of the muscle, liver, and tumor. e) Plot of the linear relation found between the fluorescence signal intensities observed when the laser passes through a folded double layer of skin and it’s unfolded single layer counterpart. f) Plot showing the relationship between the merged signal intensity and the intensities of its individual constituents consisting of the skin and underlying tissue.

(Figure 2e). Next, we then placed single layers of skin over intact tissues, each having varying levels of fluorescence intensity. The signal of the tissues combined were recorded and compared to the intensities obtained when illuminated individually. From the measurements, we established a relationship between the merged signal (single layer of skin overlying tissue of interest) and the double layer skin signal to extract the tissue of interest signal (Figure 2f). We found that this relationship holds well regardless of the underlying tissue type (muscle, liver, and tumor) With this relationship, one can simply record the intensity of the merged signal consisting of the skin and underlying tissue, then record the signal obtained from illuminating a pinched region of skin, to obtain the intensity originating from the underlying tissue (i.e. skin contribution to the merged signal is subtracted). With our model completed, we were then able to obtain ICG intensities and concentration levels from underlying tissues in live mouse models, in real-time. We note photobleaching of dyes are a concern when performing quantitative fluorescence measurements. However, we find that under our experimental conditions (illuminating with 785 nm excitation at 168.4 mW/cm2 for 5 s), hardly any effects of photobleaching on the recorded intensity levels are observed (Figure S3). Test of the Quantification Model. We then tested our model using mice that have been administered varying levels of ICG under the same conditions described previously. Here, the merged (skin and underlying tissue) and pinched skin signals are obtained, and the ICG content of the underlying tissue is determined based on our model. Then, we excise the tissue of interest, record the ICG content, and compare this value to that determined by our model. As seen from Figure 3, our model determining the ICG fluorescence from the merged signal represented well the intensity obtained directly from the excised tissue. Again, our test of the model showed no dependence of the underlying tissue type (liver, muscle, and tumor) on the merged value. To test for time dependence of our model, we compared tissues with similar ICG intensities, but accumulated the dye under different dose and time conditions. Here, we obtained 2 skin samples that had similar intensity levels using a 2 mg/kg dose at 24 h (skin-1) and an 8 mg/kg dose at 72 h (skin-2). 2 muscle tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 26 h (muscle-1) and at an 8 mg/kg dose collected at 74 h (muscle-2). 2 liver tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 25 h (liver-1) and at an 8 mg/kg dose collected at 73 h (liver-2). 2 tumor tissues having similar ICG intensity levels were obtained at a 2 mg/kg dose collected at 24 h (tumor-1) and at an 8 mg/kg dose collected at 72 h (tumor-2). Combining either of the 2 skin and either of the 2 liver, muscle, or tumor tissues, the collective intensity levels yielded similar values (Figure 4, skin-1 + tissue-type-1 = skin-1 + tissue-type-2 = skin-2 + tissue-type-1 = skin-2 + tissue-type2). Additionally, each pair of liver, muscle, and tumor tissues also had similar intensities after homogenization. Thus, the

intensity measured does not depend on the history of accumulation. We believe that within the time period of our experiments, ICG levels within tissues become perfused. Any large gradients in concentration that can possibly cause time dependencies in our model have subsided. Real-time Measurements of ICG in Vivo. Now having a model that well represents the tissue fluorescence intensities and concentrations of the tumor, muscle, and liver by simply measuring the merged and pinched skin intensities, we then wanted to apply this method to demonstrate the effects of ICG dose and preoperative period on the specimen-to-specimen variability of tissue intensity levels in real-time. Here, we intravenously administered ICG doses ranging from 1, 2, 4, 6, and 8 mg/kg via tail vein injection and monitored the fluorescence intensity levels at time periods extending to 72 hours.

Figure 3. a) Here we tested the model presented in Figure 2f where the test points are in blue, and the original model points are in red. The test points here are used to calculate the signal intensity of the underlying tissue of interest based on the measured merged signal (skin and underlying tissue) and measured signal through a region of pinched skin. b) The calculated signal intensities of the test points in (a) of the underlying tissue are compared to the observed intensities of the excised tissue illuminated directly.



Figure 4. 2 samples each of the muscle (a), liver (b), and tumor (c) having similar intensity levels were obtained and their intensities were measured in combination with 2 skin samples having similar intensity levels. Combining either of the 2 skin and either of the 2 liver, muscle, or tumor tissues, the collective intensity levels yielded similar values (skin-1 + tissue-type-1 = skin-1 + tissue-type-2 = skin-2 + tissue-type-1 = skin-2 + tissue-type-2). Here the sample obtained at a lower dose and shorter time is labeled tissue-type-1 and the sample obtained at a larger dose and longer time is labeled tissue-type-2. d) Each pair of liver, muscle, and tumor tissues presented in figures a-c also have similar intensities after homogenization.

As can be seen from Figures 5a, b, and c, at all dose ranges tested, the ICG clears from tissues closely following an exponential decay. Though each tissue exhibited close to exponential decay behavior of ICG intensity overtime, tumors are observed to have longer typical decay times and tend to have higher ICG concentration values compared to normal tissues with the average tumor ICG half-life being 11.3 hours compared to 2.87 hours and 1.28 hours for the muscle and liver tissues, respectively. The current accepted mechanism for the greater retention of ICG within tumors arises as a result from the molecules first binding to blood proteins and the subsequent enhanced permeation and retention (EPR) effect. In this case, the association ICG to the various blood proteins increases the dye’s effective hydrodynamic diameter to the nanometer scale (~ 5-10 nm).33-36 Nano-sized objects, including ICG-protein complexes,37 are observed to have preferential accumulation in tumors due to their leaky vasculature and impaired lymphatics, otherwise known as the EPR effect.38,39

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Figure 5. ICG fluorescence intensity and estimated concentration level in tissues of the liver, muscle, and tumor (a, b, and c, respectively) d) Tumor to background ratio (TBR) consisting of ICG intensities of the tumor to muscle. e) Coefficient of variation of the mean TBR over time. All observations are from a 12 to 72hour periods using i.v. administered doses of 1, 2, 4, and 8 mg/kg.

Interestingly, we found that at doses of 4mg/kg and 8mg/kg, there are larger specimen-to-specimen variabilities in the signal intensities among the various tissues at observed time points less than 48 hours, which are typical preoperative time periods. In general, we observe that the coefficient of variation (C.V.) of the TBRs, TBR being defined by the tumor intensity divided by the normal muscle intensity (Figure 5d), increases at these shorter time periods with increasing the applied dose (Figure 5e). Such high variabilities limit the ability for one to standardize device operation procedures in determining what TBR level is appropriate to determine between tumor and normal tissues among most, if not all, patients. We also observe quite large variabilities in specimen-to-specimen ICG concentration in the tissues at all measured time points shorter than 12 hours at all applied doses (Figure S4). However, preoperative time periods less than 12 hours after ICG administration is hardly used in a clinical setting. The trend of variances being larger at shorter time points were consistent in each of the tissues examined (muscle, liver, and tumor). In general practice of NIRFGS, a preoperative timing too short will have low TBR due to higher backgrounds whereas too long of a period of time is commonly associated with too much clearance of the contrast agent.28 Nevertheless, we feel the variability among the ICG clearance kinetics among tissues has not been properly addressed by previous reports. The larger variabilities at periods less than 48 hours can be a result of several compounding factors. One is the difference in the hepatic metabolism variances between specimens. Clearance of ICG from the blood is mainly determined by the hepatocyte uptake of the dye,40 where different kinetics of clearance rates may occur due to differing liver pathologies and disease.40,41 Our results show there are noticeable specimen-to-specimen variabilities in ICG accumulation within the liver among mice which are deemed healthy. Subjects with poorer hepatic clearance of ICG will have greater blood circulation times, thus


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having an increased concentration gradient and greater ICG accumulation towards both normal and tumor tissues. Much of the ICG concentration within the tissues will be extracellular and eventually be cleared out of normal tissues via the lymphatic system, leaving the tumor tissues lacking functional lymphatics with higher ICG levels. Though tumors will still have ICG clearance as a result of the concentration gradient becoming more favorable to move the dye to the blood stream at longer time points. However, in a report by Onda et al.,42 tumor cells have higher uptake rates of ICG within membrane trafficking systems inside cells than normal cells and this process has a slow turnover rate ( > 24 hours). Thus, there may be more uniformity in the longer-term cellular clearance mechanisms of ICG in normal and tumor tissues than the shorterterm vascular permeation and lymphatic drainage processes. Smaller doses, not being driven as strongly by concentration gradients, may rely more on this cellular uptake mechanism for retention and avoid the higher variances at shorter preoperative time periods. However, this will rely on the device sensitivity, since variability will start to increase when signal intensities start to merge with noise levels. Seeing that either lower doses or longer preoperative times decrease the C.V. of TBRs, we wanted to see at what time points for each dose used offers a clear distinction of signal between tumor to normal tissues among all specimens tested. Here using a 99.9 % confidence interval, we took the upper bound of the variance of the muscle intensity and the lower bound of the variance of the tumor intensity, then divided the tumor lower bound by the muscle upper bound and sought points where values were greater than one (Table 1). Points where the value is greater than one indicates that with high confidence tumors can be detected without recalibrating the machine each time a new subject is examined. In our analysis, we found 1 mg/kg was unable to achieve a value over one due to the muscles and tumor being depleted of dye, reaching similar levels. At 2 mg/kg dose, at times between 12-24 hours are optimal. Beyond 48 hours, we again see concentrations becoming similar among tumor and normal tissues. The higher doses of 4 mg/kg and 8 mg/kg for the time periods observed, the great variances of tissue intensities at times less than 24 hour can sometimes lead to negative values, indicating a propensity for false positive generation under these conditions. However, once a 48-hour preoperative time period has been reached, we can consistently detect tumor over background with high accuracy. Table 1. For doses of 1, 2, 4, and 8 mg/kg at observed time points of 12, 25, 48, and 72 hours, we examined at what dosing and time points are optimal for a consistent tumor to background (TBR) level. Here we used a confidence interval of 99.9% using the lower bound of the mean for the tumor and the upper bound of the mean for the muscle (background) tissue intensities.

TBRLEAST 0.12 TLOWER 820.02 72h MUPPER 924.18 TBRLEAST 0.89

0.65 641.65 898.44 0.71

1.55 1.76 837.21 1867.55 1039.39 1236.05 0.81 1.51

CONCLUSIONS In conclusion, our observations indicate that the highest TBRs and lowest C.V. of these values occur at greater doses and longer preoperative periods. However, we acknowledge that there are limits to the time and dose that can be allowed within a clinical setting. Doses are typically rendered at values less than 2 mg/kg in a clinical setting, a level where ICG is deemed virtually nontoxic unless the patient has an iodide allergy.43 Due to the dire situation of cancer, higher dosing of ICG may be considered. Extending the preoperative time by 12 days we feel is much less of an issue. Though lower doses have lower variabilities in TBRs at typical preoperative periods, they also result in lower TBR values. We caution that tumor detection in this case depends more heavily on the sensitivity and the limit of quantitation of the NIRFGS device. As more devices achieving nanomolar sensitivity (or greater) of ICG within tissues come to market, this may become less of an issue. Since this has yet to be confirmed, we deem a clinical investigation into maximum tolerable ICG doses and prolonging preoperative periods in NIRFGS is warranted. In either case, minimizing the variabilities in TBRs will allow standardization of device operation between patients, and as a result can increase the clinical workflow and increase the number of surgeons willing to adopt NIRFGS.

ASSOCIATED CONTENT Supporting Information Figures showing optical beam paths of the probe with a spacer; time-dependent C.V. of fluorescence intensity of liver, tumor, muscle tissues, and TBRs prior and after subtraction of skin interference; dynamic intensity plots of ICG in solution and tissues under continuous illumination with a 785 nm laser at 168.4 mW/cm2 intensity; time-dependent fluorescence intensity and ICG concentration of liver, tumor and muscle tissues at periods between 1 – 8 hours; table comparing fitting parameters using either a linear, quadratic, or cubic model to fit the fluorescence signals of merged and individual tissues.

AUTHOR INFORMATION Corresponding Author *

Email: [email protected], [email protected], [email protected] Author Contributions §

1mg/kg 2mg/kg 4mg/kg 8mg/kg

TLOWER 12h MUPPER TBRLEAST TLOWER 24h MUPPER TBRLEAST TLOWER 48h MUPPER

350.17 1632.77 0.21 436.98 1451.36 0.30 153.82 1238.74

2005.05 1127.07 1.78 1420.62 1146.36 1.24 662.78 1011.89

-1648.56 3840.95 -0.43 -438.63 6067.16 -0.07 1565.83 1012.53

4350.58 5908.86 0.74 3320.09 2583.84 1.28 2462.81 1400.18

Z.W. and K.N. contributed equally to this work. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from the State Commission of Science & Technology of China (grant no. 2016YFC0104100), the Jiangsu Province Science & Technology Department (grant no. SBE2016750057), and the Fundamental Research Funds for



the Central University (021314380081). L.A.L. acknowledges support from the 1000 Global Talents Recruitment Program of China, the Research Fellowship for International Young Scientists from the National Natural Science Foundation of China (NSFC no. 21750110440), and startup funds provided by Nanjing University. Y.W. acknowledges and the "Jiangsu SpeciallyAppointed Professor" award.

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Method for Real-Time Tissue Quantification of Indocyanine Green

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