Effects of Surface Charge of Hyperbranched Polymers on Cytotoxicity

Nov 8, 2017 - All cells were kept in a humidified, 37 °C incubator in an atmosphere of 5% CO2 (v/v) in air and used between passages 5 and 15. The Ce...
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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX-XXX

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Effects of Surface Charge of Hyperbranched Polymers on Cytotoxicity, Dynamic Cellular Uptake and Localization, Hemotoxicity, and Pharmacokinetics in Mice Liyu Chen,†,‡,§ Joshua D. Simpson,†,‡,§ Adrian V. Fuchs,†,‡,§ Barbara E. Rolfe,*,† and Kristofer J. Thurecht*,†,‡,§ †

Australian Institute for Bioengineering and Nanotechnology and ‡Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia § ARC Centre of Excellence in Convergent BioNano Science and Technology, Canberra Australian Capital Territory 2601, Australia S Supporting Information *

ABSTRACT: Nanoscaled polymeric materials are increasingly being investigated as pharmaceutical products, drug/gene delivery vectors, or health-monitoring devices. Surface charge is one of the dominant parameters that regulates nanomaterial behavior in vivo. In this paper, we demonstrated how control over chemical synthesis allowed manipulation of nanoparticle surface charge, which in turn greatly influenced the in vivo behavior. Three methacrylate/methacrylamide-based monomers were used to synthesize well-defined hyperbranched polymers (HBP) by reversible addition−fragmentation chain transfer (RAFT) polymerization. Each HBP had a hydrodynamic diameter of approximately 5 nm as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Incorporation of a fluorescent moiety within the polymeric nanoparticles allowed determination of how charge affected the in vivo pharmacokinetic behavior of the nanomaterials and the biological response to them. A direct correlation between surface charge, cellular uptake, and cytotoxicity was observed, with cationic HBPs exhibiting higher cellular uptake and cytotoxicity than their neutral and anionic counterparts. Evaluation of the distribution of the differently charged HBPs within macrophages showed that all HBPs accumulated in the cytoplasm, but cationic HBPs also trafficked to, and accumulated within, the nucleus. Although cationic HBPs caused slight hemolysis, this was generally below accepted levels for in vivo safety. Analysis of pharmacokinetic behavior showed that cationic and anionic HBPs had short blood half-lives of 1.82 ± 0.51 and 2.34 ± 0.93 h respectively, compared with 5.99 ± 2.30 h for neutral HBPs. This was attributed to the fact that positively charged surfaces are more readily covered with opsonin proteins and thus more visible to phagocytic cells. This was supported by in vitro flow cytometric and qualitative live cell imaging studies, which showed that cationic HBPs tended to be taken up by macrophages more effectively and rapidly than neutral and anionic particles. KEYWORDS: hyperbranched polymer, surface charge, biological fate, nanomedicine

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INTRODUCTION

performed to ensure their safety and effectiveness for clinical application? To date, several types of particle-based therapeutics have been approved by the U.S. Food and Drug Administration (FDA) including liposomes (e.g., doxorubicin-loaded PEGylated liposomes)4 and polymeric nanoparticles (e.g., Copaxone, Renagel, PEGylated alpha-interferons.).5,6 While lipid-based materials have had significant success in translation to the clinical setting, the outlook for polymeric MNPs is promising due to their biocompatibility, simple and cheap production methods, flexible structure, and functionality.7 A variety of biomedical applications have been investigated for polymeric nanoparticles including cancer targeting8 and

One of the major aims in nanomedicine is to develop versatile and advanced forms of macromolecular nanocarriers with desired pharmacokinetic parameters, enhanced therapeutic efficacy, and negligible systemic toxicity following intravenous administration.1,2 Accordingly, multifunctional nanoparticles (MNPs) have been explored as vehicles for drug or gene delivery or as smart medical devices to detect, diagnose, and treat diseases through loading of molecules for molecular imaging and therapy.3 These nanostructures are typically composed of (a) a therapeutic molecule, (b) an imaging molecule or agent, and (c) a cellular targeting ligand. However, questions around the design of the MNPs have arisen: (1) what are the most suitable materials to fabricate these nanostructures?; (2) what properties should they possess so that they can escape detection and clearance via the mononuclear phagocyte system?; and (3) what preclinical evaluation(s) should be © XXXX American Chemical Society

Received: July 16, 2017 Revised: October 19, 2017 Accepted: October 24, 2017

A

DOI: 10.1021/acs.molpharmaceut.7b00611 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics effective protein and gene delivery.9 Polymeric materials can also incorporate different contrast agents or fluorescent molecules within a single matrix, making them an attractive platform for highly sensitive molecular imaging agents.10−13 In general, their robust synthetic and postmodification chemistry makes polymeric nanoparticless amenable to flexible tailoring, in regards to architecture, molecular weight, number and types of drugs per polymer, targeting moieties, and even bioresponsive elements, enabling simultaneous drug/gene delivery and molecular imaging.14,15 Despite these inherent advantages of nanoparticles, there is still a disproportionately low number of clinically approved polymeric nanoparticles. One reason for the relatively low number of particles approved for clinical application arises from the lack of guidelines for relevant biological testing.16 Indeed, while a great deal of research has focused on ways to develop new therapeutics that specifically target cancer cells through exploitation of the differences between neoplastic and normal tissues, a significant knowledge gap still exists regarding how their physicochemical properties ultimately influence the biological fate (in vitro, ex vivo, in vivo) of these materials. In particular, it is important to understand how specific physicochemical properties of nanoparticles impact cells and tissues.17 Exploring these interactions will provide beneficial information regarding the relationships between nanomaterial properties (size, shape, surface charge, and surface coating) and biological responses to them, thus allowing more efficient and safer translation of synthetic identity into clinical application.18,19 Furthermore, over the past decades, the concerns over the potential risks of nanomaterials to human health and safety have risen dramatically, due to the increased application of nanomaterials in the treatment of cancer, diabetes, infection, and inflammation, from disease diagnostics to theranostics.20,21 Recently, scientists have been particularly interested in developing and applying nanoparticles as safely as possible to avoid the pitfalls previously encountered following the commercial introduction of new chemicals and chemical processes.22 The hugely variable physicochemical characteristics of nanomaterials are well recognized to play a critical role in assessing potential toxicity of a particular nanomaterial. Previous studies have investigated relationships between nanomaterial hazard and their particular parameters such as morphology,23 particle size,24,25 and surface charge.26 Hyperbranched polymers (HBPs) can be developed as multifunctional nanoplatforms that are capable of delivering therapeutic drug molecules or DNA to cancer cells or other diseased cells.27−29 Due to pioneering studies in the field, the great improvement in synthesis, analysis, and bioapplications of hyperbranched polymers has led to the development of methods to precisely manipulate the physicochemical properties of HBPs, thereby allowing us to carry out a systematic safety evaluation, in particular how this is influenced by a particular parameter. Until now, research involving these unique branched macromolecules has been limited to a specific disease application, with little development of a fundamental understand the influence of surface properties on in vitro and in vivo behavior. In general, the performance of nanoparticles as therapeutic and diagnostic carriers in nanomedicine is dependent upon their size and surface functional groups. This is particularly true for HBPs, where their numerous surface functional groups can produce a highly localized charge, which may significantly influence interactions with the cell membrane and subsequent uptake into cells. An understanding of the way

in which surface charge can alter the local environment and influence cellular interactions and intracellular fate is essential for rational design of HBP-based carriers to target drugs to specific intracellular compartments. With this concept in mind, this paper investigates the effect of HBP surface charge within physiological settings. Specifically, we synthesized three generic HPBs with similar hydrodynamic radii, but with different surface charges, by incorporating selected monomer units into the structure. We then investigated the effect of surface charge on cellular responses (cytotoxicity, cellular uptake, intracellular distribution, and hemolysis) and in vivo pharmacokinetic behavior. The data provide an insight into how such properties might influence the safety and efficacy of HBPs for nanomedical application.

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EXPERIMENTAL SECTION

Materials and Reagents. Unless otherwise stated, all reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) at the highest available purity. Poly(ethylene glycol monomethyl ether methacrylate) (PEGMA, Mw = 475 g mol−1), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 98%), t-butyl methacrylate (tBMA, 98%), and ethylene glycol dimethacrylate (EGDMA, 98%) were passed through an alumina column to remove inhibitor before use. Prop-2-yn-1yl4-cyano-4(((ethylthio)carbonothioyl)thio)pentanoate (PCEPA) was employed as chain transfer agent (CTA) and synthesized as previously reported.30 Tetrahydrofuran (THF) was purified by a MB-SPS800 solvent purification system before use. Distilled water with a resistivity of 18.2 MΩ cm was obtained from an Elga ultra-pure water system. The mouse leukemic macrophage cell line, RAW264.7 (ATCC TIB-71), was grown in complete cell culture medium consisting of RPMI (Life Technologies, Carlsbad, CA) containing 10% (v v−1) fetal bovine serum (Moregate, Australia), 100 U mL−1 penicillin-G and 100 mg mL−1 streptomycin. The 3T3 mouse fibroblast cell line was cultured in high-glucose DMEM (Life Technologies, USA) supplemented with 10% (v v−1) fetal bovine serum, 100 U mL−1 penicillin-G, and 100 mg mL−1 streptomycin. All cells were kept in a humidified, 37 °C incubator in an atmosphere of 5% CO2 (v/v) in air and used between passages 5 and 15. The CellTiter 96 AQueous One Solution Reagent was purchased from Promega Corporation (Madison, WI). Techniques. 1H nuclear magnetic resonance (NMR) spectra were measured on a Bruker AC 300 or 400 MHz spectrometer in CDCl3 or DMSO-d6 at 25 °C. Owing to the different solubilities of the polymers, two different gel permeation chromatography (GPC) systems were employed to investigate the total molecular weight of HBPs. GPC equipped with multiangle laser light scattering (GPC-MALLS) using HPLC-grade THF was used for analysis of poly(PEGMA) and poly(tBMA) and GPC equipped with MALLS was used for analysis of the poly(DMAEMA) using DMAc with 0.3 wt % of LiCl as eluent. Both GPC systems utilized a flow rate of 1 mL min−1. All samples were freshly prepared before analysis and filtered through a 0.45 μm filter to eliminate large aggregates. The hydrodynamic diameters and surface charges of HBPs were explored on a Nanoseries zetasizer (Malvern 90, UK). The scattering angle and temperature were 90 °C and 298 K, respectively. For transmission electron microscope (TEM) imaging, particles were dissolved in water, dropped onto copper grids, and air-dried before imaging. B

DOI: 10.1021/acs.molpharmaceut.7b00611 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

of the fluorophore during this process (as measured by UV− vis). In Vitro Cytotoxicity 3-(4,5-Dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay. The in vitro cytotoxicity of HBPs against RAW264.7 macrophage cells and 3T3 fibroblast cell lines was determined by MTS assay. Polymer solutions were prepared in serum supplemented tissue culture medium to final concentrations of 2000 μg mL−1 and 200 μg mL−1, respectively. Cells without any HBP treatment were used as control. Briefly, cells were seeded in a 96-well plate at a density of 104 per well and left in incubator overnight. The following day, cells were washed twice using PBS, then treated with freshly prepared HBPs solution. After 24 h incubation period, tissue culture medium was replaced by 100 μL of MTS solution (20 μL of CellTiter 96 Aqueous One Solution Reagent and 80 μL of tissue culture medium). Cells were left in incubator for another 1 h, and then the absorbance was measured using a plate reader (BioTek, Winooski, VT) at 490 nm. The relative cell viability (%) of each HBPs sample related to control well was calculated as follows: [A]test/[A]control × 100%, where [A]test is the cell viability percentage of the cells following incubation with the HBPs, while [V]control is the cell viability percentage of control sample. All samples have five repeats. In Vitro Cell Uptake Assessed via Flow Cytometry. Studies were undertaken to quantify the HBP internalization in RAW264.7 macrophages and 3T3 fibroblast cells. Briefly, cells in 2 mL of growth medium were seeded in a six-well plate at a density of 2 × 105 cells per well and allowed to adhere overnight. On the second day, the tissue culture medium was replaced with freshly prepared Cy5-HBPs in the same tissue culture medium with the final concentration at 10 μg mL−1. To study the uptake kinetics of HBPs, the Cy5-labeled HBPs were incubated with cells for time periods ranging from 30 min to 4 h. At each time point, cells were washed twice with chilled PBS. 3T3 fibroblast were harvested with 0.05% Trypsin-EDTA, while RAW264.7 macrophages were collected by cell scraper before fluorescent activated cell sorting (FACS) analysis using an Accuri C6 Flow Cytometer (BD Bioscoience, USA). For each time-point, the internalization of each HBP was determined as the percentage of Cy5-positive cells. Time-Lapse Live Cell Uptake and Cytoplasmic Distribution via Confocal Microscopy. RAW264.7 macrophages were plated into glass-bottom μ-dishes (Ibidi, Martinsried, Germany) and allowed to reach confluence overnight. Then 30 min prior to imaging, the cell growth medium was replaced with 2 mL of phenol red free medium containing 2 μg mL−1 Hoechst 33342 (Life Technologies, Carlsbad, CA). Samples were imaged using a Zeiss 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) housed within the Australian Nanofabrication Facility Queensland node. The health of the cells was examined using bright field setting to proceeding. The medium was then exchanged for phenol red free RPMI containing 100 μg mL−1 of Cy5-HBPs, and images taken at 15, 30, 45, 60 min post exposure. To investigate the distribution and accumulation information on our three differently charged polymers, we analyzed the fluorescence intensities for specific areas within the cellular environment using the region of interest tool within the Zen Blue Zeiss Lite software. Among the areas examined were the nucleus, nucleolus, vesicles, background, and cytoplasm (which was divided into peripheral and inner components using the bright field overlay to avoid user bias). The inner cytoplasm is

Methods. Synthesis of Charge Varied HBPs. The hyperbranched polymers consisting of major components including poly(PEGMA), poly(DMAEMA), or poly(methacrylic acid) (poly(MAA)) were synthesized via the RAFT process27 with PCEPA employed as RAFT agent. A typical polymerization procedure was as follows: PEGMA (2.000 g, 4.21 mmol), DMAEMA (0.662 g, 4.21 mmol), or tBMA (0.599 g, 4.21 mmol) was mixed with EGDMA (0.042 g, 0.211 mmol), PCEPA (0.063 g, 0.221 mmol), initiator AIBN (0.00693 g, 0.0422 mmol), and THF (4 mL) in a round-bottom flask. The mixture was purged with argon in an ice-water bath for 25 min to remove oxygen. Then the flask was sealed and the polymerization was carried out in oil bath at 70 °C. After 24 h, the reaction was quenched by immersion in an ice-water bath and air was introduced. Excess THF was gently removed under nitrogen, and the residue mixture was precipitated three times through 10-times volume hexane to remove unreacted monomer. The precipitate was dried under vacuum at room temperature (RT) overnight. For further purification, the poly(PEGMA) and poly(DMAEMA) polymers were dialyzed in deionized water which was changed regularly for 72 h and collected after lyophilizing. To produce HBPs with a negatively charged surface, an additional step was required to remove the t-butyl group of poly(tBMA). Generally, poly(tBMA) was dissolved in THF, then trifluoroacetic acid (TFA) (five times equivalent to the tert-butyl ester) was added into the reaction flask to remove isobutene as the elimination product.31 After a period of 12 h stirring at RT, excess THF was removed through gently bubbling nitrogen. For purification, the polymer was precipitated through hexane twice, followed by vacuum drying overnight. Then the resulting residue was dissolved in water and dialyzed in deionized water, which was changed regularly for 72 h. The final product (poly(MAA)) was obtained by freeze-drying. All polymers were analyzed by a variety of techniques including GPC, NMR, UV−vis, and DLS as described in the Discussion and Supporting Information. Synthesis of Charge Varied Cy5-HBP. To understand the complex behavior of HBPs in biological systems from in vitro to in vivo, we synthesized fluorescent Cy5-HBPs by incorporating Cy5 methacrylamide monomer. Cy5 was chosen as fluorophore as it could overcome some of the photon attenuation in living tissue and has been intensively investigated for monitoring polymer behavior in vivo in mouse models.15 A typical polymerization process for incorporation of fluorophore into poly(PEGMA) was as follows: PEGMA (0.1 g, 0.211 mmol), Cy5 methacrylamide monomer (1.04 mg, 0.00152 mmol), EGDMA (2.09 mg, 0.01 mmol), PCEPA (3 mg, 0.0105 mmol), initiator AIBN (0.3 mg, 0.0021 mmol), and THF (0.2 mL) were placed in a 4 mL vial equipped with a magnetic stirrer bar. The vial was sealed with a silicon septum followed by reinforcing with a cable tie, and then the vial was placed in an oil bath and stirred at 70 °C for 24 h. The resultant polymer was precipitated into an excess of n-hexane twice to remove unreacted monomers. Then the precipitate was dried under vacuum at RT overnight, followed by further purification through dialysis in Milli-Q water of which was changed regularly for 48 h. The final product was obtained after lyophilizing. The Cy5-poly(DMAEMA) and Cy5-poly(tBMA) were synthesized as described above. Similarly, we utilized TFA to remove the t-butyl group of Cy5-poly(tBMA) to obtain the negatively charged Cy5-HBPs and noticed minimal degradation C

DOI: 10.1021/acs.molpharmaceut.7b00611 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Scheme 1. Synthetic Methodology for Differently Charged Hyperbranched Polymers, Their Relative Charge As Measured by Zeta Potential, and Size Analysis by Dynamic Light Scattering and Electron Microscopy

stock solution was prepared by mixing 1 mL of washed erythrocytes with 19 mL of PBS buffer. HBP solutions (of various concentrations prepared in the same PBS buffer) were added to the erythrocyte suspensions and the mixture incubated at RT for 3 h with the HBPs (at final concentrations ranging from 12.5−400 μg mL−1). RBCs incubated with MilliQ water or PBS were used as positive and negative controls, respectively. After centrifugation (600g, 10 min) to remove RBCs, the release of hemoglobin was measured by photometric analysis of the supernatant under the Infinite M200 Microplate

the location for the endomembranous system, which houses important organelles, trafficking components and processing machinery. Cells (n = 5) from each treatment were analyzed and fluorescence intensity data exported to Microsoft Excel and normalized prior to statistical analysis. Hemolysis Evaluation. The hemolytic activity of each class of HBP was studied according to the work of Lin.23 Whole blood was freshly obtained from CD1 mice, erythrocytes collected by centrifugation at 600g for 10 min, and then washed three times with phosphate buffered saline (PBS) buffer. A D

DOI: 10.1021/acs.molpharmaceut.7b00611 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Physical Properties of Hyperbranched Polymers arms/HBP

a

polymer

Mnd 1H NMR/kDa

Mn GPC-MALLS/kDa (ĐM)

poly(PEGMA)a poly(DMAEMA)b poly(tBMA)c,d poly(MAA)

10.7 5.2 7.0

47.1 (1.31) 23.9 (1.33) 25.7 (1.26)

b

4.3 4.6 3.7 c

in water TEM size/nm

number size/nm

zeta potential/mV

4.4 ± 0.3 4.8 ± 0.4

4.8 ± 0.2 4.9 ± 0.7

3.5 ± 0.2 +37.6 ± 0.4

4.9 ± 0.3

5.3 ± 0.2

−33.1 ± 0.3

d

dn/dc Poly(PEGMA) = 0.042. dn/dc Poly(DMAEMA) = 0.052. dn/dc Poly(tBMA) = 0.069. Mn of each arm length in the HBP. eDLS was not conducted for poly(tBMA) due to its insolubility in water.

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RESULTS AND DISCUSSION Correlating the physicochemical properties of hyperbranched polymers with the biological response to them is challenging as particle surface characteristics, concentration, and solubility all affect the in vitro cellular response and subsequent pharmacokinetic behavior. The purpose of this study was to understand the influence of surface charge on physiological behavior of nanoparticles both in vitro and in vivo. While many studies have investigated a specific nanomaterial (e.g., drug delivery vehicle or imaging agent) for a specific purpose,11,33 we synthesized and evaluated three generic hyperbranched polymers (HBPs) of similar size and degree of branching to inform on how charge affects their biological distribution. HBPs were chosen because these materials can be readily synthesized with reproducible and tunable properties in terms of morphology, physical size, surface charge, etc.11,15,34,12,35 These HBPs were synthesized by RAFT polymerization as this method allows control over polymer architecture, particle size, and molecular weight dispersity.13,28,36−38 Synthesis of Charge Varied HBPs. The general synthetic methodology and basic physical properties are shown in Scheme 1 and Table 1. Three different monomers were utilized in this approach. PEGMA was utilized as a “neutral” polymer, as it has been used extensively in the literature as a biologically inert material. DMAEMA contains tertiary amino groups that are protonated under physiological conditions in which the pH is lower than its pKb (8.4 at 25 °C).39 Poly(methacrylic acid) contains negatively charged species due to the low pKa (4.7 at 25 °C) of the acid group.40 All three HBPs were synthesized using an alkyne-terminated RAFT agent, which provides a unique handle for NMR analysis (labeled as “a” in Supporting Information, Figures S1−S3) and potential for further postsynthetic modifications. The experimental composition and molar mass (Mn) of each arm of the HBP was determined by 1H NMR (Supporting Information, Figures S1−S3) through comparison of the integrals between the main chain protons (peaks b and c in Supporting Information Figure S1) and chain end protons (methylene adjacent to the alkyne group of the RAFT agent; peak a in Supporting Information Figure S1). The absolute molecular weights were obtained using GPC-MALLS. The refractive index increment, dn/dc, was calculated after measuring the refractive indices of a series of polymer/THF or DMAc solutions with a range of concentrations at 25 °C. The number of arms per HBP was calculated through the following equation: Nend group = Mn (GPC‑MALLS)/Mn (1H NMR), assuming the hyperbranched structure was formed via intermolecular rather than intramolecular reaction during RAFT polymerization (i.e., cyclization is negligible). In addition, 1H NMR was used to monitor the successful formation of the polyanion (Supporting Information, Figure S3), through loss of the t-butyl protecting group at 1.4 ppm. As shown in Table 1, poly(PEGMA),

reader (Tecan, Switzerland), at 570 nm (test) and 655 nm (reference). The percentage of RBC hemolysis was calculated using the following formula: percentage of hemolysis = ((sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance)) × 100. All hemolysis data are presented as mean ± standard deviation (SD) (n = 4/group). In Vivo Pharmacokinetics Studies. Animal experiments were undertaken according to the University of Queensland Animal Ethics Committee Guidelines and conformed to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). All mice were housed in the animal facility of the Centre for Advanced Imaging, with free access to water and food. CD1 mice (male, 6−8 weeks old) were provided by University of Queensland Biological Resources. Nine CD1 mice were randomly assigned to three groups. Neutral, cationic, or anionic Cy5-HBPs solution (5 mg mL−1) was freshly dissolved in sterile PBS before i.v. injection via the retinal vein plexus into each mouse in a single dose of 20 mg kg−1. Mice were monitored closely for adverse effects (altered gait, chills, lethargy, or gross manifestation of stress) after administration. Blood was withdrawn at designated time points (at each time point, blood was collected from 3 mice, 0.5, 2.5, 4.5, 7.5, and at the cull-point 24 h post injection) via the tail vein. Plasma was then collected by centrifugation (3300g, 10 min) and stored at −80 °C until analysis. For analysis, 10 μL aliquots of plasma were mixed with 40 μL of n-hexane, vortexed for 5 min, followed by centrifugation at 14 000g for 10 min at 4 °C to remove the blood proteins. The separated lower aqueous layer was collected, and a 5 μL aliquot of each sample solution was diluted using Milli-Q water before transfer to a 96-well plate for fluorescence intensity analysis (ex 647 nm, em 665 nm, slit width 4 nm) to quantify the plasma HBP concentration at each time-point. Fluorescence data were fitted to a double exponential using SigmaPlot. The measurement methodology was constructed and validated prior to the experiments using freshly prepared Cy5-HBP samples in plasma. A standard curve was constructed using Cy5-HBPs at five concentrations, Cy5 fluorescence measured on a Horbia FluoroMax-4, and plotted as concentration versus fluorescence intensity (Supporting Information, Figure S9−S11). Linearity in fluorescence was obtained for mass concentrations ranging from 3.12−20 μg mL−1.32 All calibration curves had correlation values of at least 0.98 with a recovery above 70%. Statistical Analysis. Data were expressed as mean ± standard deviation and analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test; a p value