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Stability and Biodistribution of Thiol-Functionalized and 177LuLabeled Metal Chelating Polymers (MCP) Bound to Gold Nanoparticles Simmyung Yook, Yijie Lu, Jenny Jooyoung Jeong, Zhongli Cai, Lemuel Tong, Ramina Alwarda, Jean-Philippe Pignol, Mitchell A. Winnik, and Raymond M. Reilly Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01642 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016

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Stability and Biodistribution of Thiol-Functionalized and 177

Lu-Labeled Metal Chelating Polymers (MCP) Bound to

Gold Nanoparticles Simmyung Yook,†,# Yijie Lu, ‡,# Jenny Jooyoung Jeong,† Zhongli Cai,† Lemuel Tong,‡ Ramina Alwarda,‡ Jean-Philippe Pignol, §,¶ Mitchell A. Winnik,‡* and Raymond M. Reilly †,⊥, ¥*

†

Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada

‡

Department of Chemistry, University of Toronto, Toronto, Ontario, Canada

§

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

¶

Department of Radiation Oncology, Erasmus MC Cancer Institute, Rotterdam, The

Netherlands ⊥ Department

¥

of Medical Imaging, University of Toronto, Toronto, Ontario, Canada

Toronto General Research Institute, and Joint Department of Medical Imaging, University

Health Network, Toronto, Ontario, Canada #

These two authors contributed equally to this work.

* Corresponding authors.

ACS Paragon Plus Environment

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ABSTRACT: We are studying a novel radiation nanomedicine approach to treatment of breast cancer using 30 nm gold nanoparticles (AuNP) modified with polyethylene glycol (PEG) metal-chelating polymers (MCP) that incorporate 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA) chelators for complexing the β-particle emitter, 177Lu. Our objective was to compare the stability of AuNP conjugated to MCP via a single thiol [DOTA-PEG-ortho-pyridyl disulfide (OPSS)], a di-thiol [DOTA-PEG-lipoic acid (LA)] or multi-thiol end-group [PEG-pGlu(DOTA)8-LA4] and determine the elimination and biodistribution of these

177

Lu-labeled MCP-AuNP in mice. Stability to aggregation in the

presence of thiol-containing dithiothreitol (DTT), L-cysteine or glutathione was assessed and dissociation of

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Lu-MCP from AuNP in human plasma measured. Elimination of

radioactivity from the body of athymic mice and excretion into the urine and feces was measured up to 168 h post-intravenous (i.v.) injection of

177

Lu-MCP-AuNP and normal

tissue uptake was determined. ICP-AES was used to quantify Au in the liver and spleen and these were compared to 177Lu. Our results showed that PEG-pGlu(DOTA)8-LA4-AuNP were more stable to aggregation in vitro than DOTA-PEG-LA-AuNP and both forms of AuNP were more stable to thiol challenge than DOTA-PEG-OPSS-AuNP. PEG-pGlu(177LuDOTA)8-LA4 was the most stable in plasma. Whole body elimination of rapid for mice injected with for >90% of eliminated

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177

177

Lu was most

Lu-DOTA-PEG-OPSS-AuNP. Urinary excretion accounted

Lu. All

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Lu-MCP-AuNP accumulated in the liver and spleen.

Liver uptake was lowest for PEG-pGlu(177Lu-DOTA)8-LA4-AuNP but these AuNP exhibited the greatest spleen uptake. There were differences in Au and

177

Lu in the liver for PEG-

pGlu(177Lu-DOTA)8-LA4-AuNP. These differences were not correlated with in vitro stability of the

177

Lu-MCP-AuNP. We conclude that conjugation of AuNP with PEG-pGlu(177Lu-


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DOTA)8-LA4 via a multi-thiol functional group provided the greatest stability in vitro and lowest liver uptake in vivo, and is therefore the most promising for constructing 177Lu-MCPAuNP for radiation treatment of breast cancer.

Keywords: gold nanoparticles; gold-thiol bond; metal-chelating polymers (MCP);

177

Lu;

stability; radiation nanomedicine


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TOC Abstract


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INTRODUCTION There is great interest in the application of gold nanoparticles (AuNP) for cancer treatment due to their physico-chemical properties which permit photothermal therapy 1 as well as their ability to enhance the radiobiological effects of X-radiation treatment of tumors.

2, 3

In addition, chemotherapeutic drugs (e.g. paclitaxel or doxorubicin) may be

linked in high capacity to AuNP to increase their delivery to tumors. 4 The surface of AuNP may also be modified with peptides 5, antibodies

6

or aptamers

7

for specific binding to

tumor-associated receptors or antigens in order to route AuNP to tumors. In addition, AuNP passively accumulate in tumors due to the enhanced permeability and retention (EPR) effect which is mediated by “leaky” tumor vasculature and poor lymphatic drainage.8 AuNP may be modified with chelators that complex radiometals to enable tumor imaging by positronemission tomography (PET) 9, 10 or single photon emission computed tomography (SPECT). 6, 11

To stabilize AuNP to aggregation in vitro and provide “stealth” properties in vivo to

evade uptake by the reticuloendothelial system (RES), AuNP are surface-coated with polyethyleneglycol (PEG) polymers.

12-14

The most common approach for AuNP

modifications has been to react AuNP with PEG chains that presents a terminal free thiol (SH) to form a Au-thiol bond.

4

However the binding of mono-thiol containing ligands to

AuNP is unstable when exposed to heat, oxidizing agents or other thiol-containing molecules such as glutathione (GSH) or dithiothreitol (DTT).

15-17

GSH is a source of free

thiol that is naturally present at high concentrations in many tissues, and especially in the liver.

18

This may cause instability in vivo of AuNP constructs that are formed from mono-

thiol containing PEG ligands, particularly since the liver readily accumulates AuNP.6 One strategy to enhance the stability of thiol-functionalized ligands bound to AuNP


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is to increase the number of thiols per ligand to provide more avid multivalent binding to the gold surface. Mei et al.

19

demonstrated that AuNP modified with di-thiol functionalized

PEG polymers exhibited enhanced stability to aggregation in 1 M DTT compared to AuNP modified with mono-thiol PEG ligands. In a separate study, AuNP modified with PEGbased ligands providing two lipoic acid (LA) anchoring groups which permit multi-thiol conjugation were highly resistant to aggregation, even in the presence of 1.5 M DTT and in harsh conditions such as extreme pH (1.1-13.9) or high salt concentrations (2 M NaCl).20 Zopes et al.

21

reported that AuNP linked to PEG presenting a di- or tri-valent thiol group

exhibited improved stability to high salt concentrations (2 M NaCl) in contrast to PEG presenting only a single thiol for conjugation to AuNP. 21 Zhang et al. studied mono- or dithiol end-functionalized PEG polymers as well as different PEG molecular weight (2 kDa vs. 5 kDa) and AuNP size (20 to 80 nm) on the stability to DTT challenge, and on the in vivo biodistribution of 111In-labeled AuNP in athymic mice with A431 squamous cell carcinoma tumors.

22

The most stable PEG-AuNP constructs were formed using 5 kDa PEG end-

functionalized with LA presenting a di-thiol for conjugation to AuNP. The lowest uptake by the RES, longest circulation time and highest tumor uptake were observed for 20 nm AuNP modified with 5 kDa LA-PEG. Since these AuNP were not modified with targeting ligands, tumour uptake was mediated by the EPR effect. Our group

23

and others 5 have recently proposed the application of AuNP labeled

with the β-particle emitter,

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Lu [Eβ max=0.50 MeV (78.6%); 0.38 MeV (9.1%); 0.18 MeV

(12.2%), half-life (t1/2) = 6.7 d] for radiation treatment of cancer (radiation nanomedicine). We modified 30 nm AuNP with metal chelating polymers (MCP) composed of PEG (4 kDa) end-functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)


6

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chelator for complexing

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Lu, and a terminal ortho-pyridyl disulfide (OPSS) moiety that

provides a single thiol for AuNP conjugation to AuNP.

23

In some cases, AuNP were also

modified with PEG-OPSS (5 kDa) linked to panitumumab for targeting epidermal growth factor receptors (EGFR) on breast cancer cells.

23

These AuNP were finally coated with

shorter thiol-containing PEG chains (2 kDa) to stabilize them against aggregation. It is important for

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Lu-labeled PEG polymers to be stably linked to these AuNP for radiation

treatment of breast cancer. Thus, in the current study, we synthesized two new MCP with potentially greater stability than 177Lu-DOTA-PEG-OPSS: i) a heterobifunctional PEG (3.54 kDa) functionalized with DOTA for complexing 177Lu and a terminal LA group (DOTAPEG-LA) and ii) a di-block copolymer with a PEG (2 kDa) block and a block of polyglutamide with 8 pendant DOTA and 4 terminal LA groups [PEG-pGlu(DOTA)8-LA4]. We compared the stability of

177

Lu-MCP-AuNP constructed from these PEG polymers that

provide a di-thiol or multi-thiol for conjugation to AuNP with

177

Lu-DOTA-PEG-OPSS.

Stability was assessed by studying the aggregation in vitro of MCP-AuNP when challenged with thiol-containing molecules (DTT, L-cysteine and GSH) and by measuring the loss of 177

Lu-MCP from AuNP in human plasma. In addition, we compared the elimination and

excretion of these

177

Lu-MCP-AuNP in vivo in athymic mice commonly used to establish

tumor xenografts, and determined their tissue biodistribution including comparisons of 177Lu and Au in the liver and spleen.


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EXPERIMENTAL SECTION Materials. All reagents and solvents were used without further purification unless otherwise noted. Water was purified through a Milli-Q water purification system (18 MΩ.cm). Spectra/Pro dialysis membranes (MWCO 1 kDa) were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). Other supplies were purchased from Fischer Scientific (Ottawa, ON, Canada) and included 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMTMM, Acros Organics, 99+%), Millipore Amicon spin filters (15 mL, 3 kDa or 10 kDa MWCO and 4 mL, 3 kDa or 10 kDa MWCO) and 0.45 µm pore size polyvinylidene fluoride (PVDF) membrane syringe filters. 177LuCl3 was purchased from Perkin Elmer (Waltham, MA, USA). Vivaspin 20 GE Healthcare MWCO (5 k), and Aristar® Ultra, trace metal grade Au standard were purchased from VWR (Mississauga, ON, Canada). 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was purchased from Macrocyclics (Dallas, TX, USA). Methoxy-PEG-Amine (Mn 2 kDa) and ortho-pyridyl disulfide polyethylene glycol (OPSS)-PEG-Amine, HCl Salt (Mn = 3.5 kDa) were purchased from Jenkem (Plano, TX, USA). Lipoic-acid (LA)-PEG-amine (Mn = 3.5 kDa) was purchased from Nanocs (New York, NY, USA). 2-pyridone (2HP), anhydrous N,Ndimethylformamide (DMF), ethylenediamine (EDA), succinic acid monoamide (SUC), human plasma, glutathione (GSH), dithiothreitol (DTT), sodium bicarbonate (NaHCO3), 70% (w/w) nitric acid (>99.999% trace metal-free) and ReagentPlus® ≥99% hydrogen chloride (HCl) were purchased from Sigma-Aldrich (St. Louis. MO, USA). L-cysteine was obtained from BioShop (Burlington, ON, Canada). AuNP (30 nm diameter with 8% coefficient of variation; 2 × 1011 particles/ml) were purchased from Ted Pella (Redding, CA, USA). Low binding microcentrifuge tubes, UV-Star 96-well microplate and Bio-Gel P6


8

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chromatography resin were purchased respectively from Axygen Scientific Inc. (Union City, CA, USA), Greiner Bio One (Frickenhausen, Germany) and BioRad (Mississauga, ON, Canada). The synthesis of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) was was reported previously 24.

Animals. 5-6 week old female athymic mice were purchased from Harlan Laboratories, (Indianopolis, IN, USA). All animal studies were conducted in compliance with Canadian Council on Animal Care (CCAC) regulations under a protocol approved by the Animal Care Committee at the University Health Network (Protocol No. 2780).

Design and Synthesis of Multi-Thiol MCP. We synthesized two polymers that incorporated either a single lipoic acid (LA-PEG-DOTA) (Figure 1A) or multi-lipoic acid end group [PEG-pGlu(DOTA)8-LA4] (Figure 1B) for divalent or multivalent Au-thiol conjugation to AuNP. The details of the synthesis and characterization of these polymers are described in the Supporting Information (SI).


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Figure 1. Synthesis scheme of LA-PEG-DOTA and PEG-pGlu(DOTA)8-LA4. (A) LAPEG-DOTA was synthesized by monoamide formation between DOTA and LA-PEG-amine using DMTMM as a coupling reagent. (B). PEG-pGlu(DOTA)8-LA4 was based on a methoxy-PEG polyglutamide diblock copolymer modified with DOTA monoamide groups as 177Lu chelators and lipoic amide groups for anchoring to AuNP. Polymer synthesis started with ring opening polymerization of γ-benzyl-L-glutamate-N-carboxyanhydride (Glu-NCA) using methoxy-PEG-amine (PEG 2K-amine) as the initiator. Aminolysis converted the benzyl esters to aminoethylamide groups. Then the LA groups and DOTA chelators were installed to the free amino pendant groups using DMTMM as a coupling reagent to generate the polymer PEG-pGlu(DOTA)8-LA4.


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Construction of

177

Lu-MCP-AuNP. OPSS-PEG-DOTA, LA-PEG-DOTA and PEG-

pGlu(DOTA)8-LA4 were labeled by incubation at 80°C for 20 min with

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LuCl3 in 0.1 M

sodium acetate buffer (pH 6.0). Radiolabeling efficiency was measured as described in the SI. 177Lu-MCP-AuNP were assembled as previously reported 23 by incubating 177Lu-DOTAPEG-OPSS (25 µg in 500 µL of ddH2O),

177

Lu-DOTA-PEG-LA (25 µg in 500 µL of

ddH2O) or PEG-pGlu(177Lu-DOTA)8-LA4 (75 µg in 500 µL of ddH2O) with 2 × 1011 AuNP (30 nm diameter, Ted Pella, Redding, CA) in low binding microcentrifuge tubes for 16 h at 4oC (Figure 2). 177Lu-MCP-AuNP were purified by ultracentrifugation at 10,000 × g for 15 min at 4oC, repeated once. After each centrifugation, the supernatant was carefully removed from the AuNP pellet to separate

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Lu-MCP-AuNP from excess unconjugated

177

Lu-MCP.

To determine the PEGylation density, the AuNP-bound radioactivity was measured in a γcounter (Model 1480 Wizard 3, PerkinElmer, Turku, Finland) and the counts were converted into moles, then multiplied by Avogadro’s number to calculate the total number of 177Lu-MCP molecules bound to AuNP. The average number of 177Lu-MCP bound to each AuNP was determined by dividing the total number of

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Lu-MCP by the number of AuNP

used in the reaction (2 × 1011 particles). The method for determination of AuNP size using dynamic light scattering (DLS) is described in the SI.


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Figure 2.

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Lu-MCP-AuNP were assembled by incubating 30 nm diameter AuNP for 16 h

at 4 oC with

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Lu-DOTA-PEG-OPSS,

177

Lu-DOTA-PEG-LA or a di-block copolymer,

PEG-pGlu(177Lu-DOTA)8-LA4 which respectively provide mono-thiol, di-thiol or multithiol end-groups for conjugation to the Au surface.

MCP-AuNP Aggregation Assay. The stability against competitive ligand displacement in vitro was evaluated by challenging unlabeled MCP-AuNP (2 × 1011 particles/mL) with 10


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mM or 100 mM DTT in phosphate buffered saline (PBS, pH 7.5) or biologically relevant concentrations of L-cysteine (1 mM) or GSH (1 mM) in ddH2O or 0.02 M NaHCO3 (pH 9.5). The reaction mixture (200 µL) was transferred in triplicate into wells in a UV-Star 96well microplate for absorbance measurements using a microplate reader (BioTek Synergy 2, Fisher Scientific, Waltham, MA, USA). An aggregation factor (AF) was calculated by measuring the ratio of the absorbance at 615 and 524 nm [surface plasmon resonance band (SPB)] every 2 min up to 10 min, then every 10 min up to 1 h, and finally at 3, 6, 24, 48, 72 h post incubation. The ratio of absorbance at these two wavelengths was selected because it was the most sensitive for detecting changes resulting from aggregation.

19, 25

The stability

of each PEG-MCP bound to AuNP was determined from the area under the curve (AUC) estimated from the AF vs. time plot and calculated for 0 to 72 h (AUC0-72h; AF × h) using Prism Version 6.0 software (GraphPad Software Inc., San Diego, CA, USA). This change in absorbance was also noted visually by a progressive change in solution color from red to purple associated with AuNP aggregation.

MCP Displacement in Human Plasma. The displacement of 177Lu-MCP from AuNP was determined by incubating 177Lu-DOTA-PEG-OPSS-AuNP, 177Lu-DOTA-PEG-LA-AuNP or PEG-pGlu(177Lu-DOTA)8-LA4-AuNP (150 µg AuNP, 6 × 1011 particles, 1-2 MBq) with 1 mL of human plasma at 37oC for 0.2, 0.5, 1, 3, 5, 24, 48 and 72 h in low binding microcentrifuge tubes. At each time point, the samples were centrifuged at 10,000 × g for 15 min and the supernatant was removed. The radioactivity in the supernatant and AuNP pellet was measured in a γ-counter and expressed as the percentage remaining bound to AuNP, then plotted vs. the time of incubation.


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Whole Body Elimination and Excretion Study. The whole body elimination and urinary 177

or fecal excretion of MBq (250 µg) of

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Lu-MCP-AuNP were studied after i.v. injection (tail vein) of 2-3

Lu-DOTA-PEG-OPSS-AuNP,

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Lu-DOTA-PEG-LA-AuNP, or PEG-

pGlu(177Lu-DOTA)8-LA4-AuNP in 200 µL of phosphate buffered saline (PBS, pH 7.5) in groups of 4 female athymic mice. Mice were housed in disposable cages for 168 h and the urine and feces were collected daily. The whole body radioactivity and

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Lu excreted into

the urine or feces [percent injected dose (%ID)] were measured in a radioisotope dose calibrator (CRC-15R, Capintec Inc., Ramsey, NJ, USA) up to 168 h post-injection (p.i.). An equivalent amount of

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Lu-MCP not bound to AuNP was injected into separate groups of

mice and the whole body elimination and excretion of radioactivity similarly measured for comparison with 177Lu-MCP-AuNP.

Biodistribution Studies. Mice injected with 177Lu-DOTA-PEG-OPSS-AuNP, 177Lu-DOTAPEG-LA-AuNP

or

PEG-pGlu(177Lu-DOTA)8-LA4-AuNP

to

evaluate

whole

body

elimination and excretion were sacrificed by cervical dislocation under anesthesia using isoflurane in O2 and samples of blood and normal tissues were collected, weighed and the radioactivity in each measured in a γ-counter. Tissue radioactivity was calculated as percent injected dose/g (%ID/g), and then converted to % ID/organ using previously determined standard organ weights. model.

27

26

Organ weights for muscle were obtained from a standard mouse

Tissue samples were then stored at -20oC for 70 days to allow radioactive decay.

The concentration of Au in each organ was then measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). For this analysis, samples were prepared as described by Fischer et al.

28

Briefly, tissues were digested in 70% (w/w) HNO3 in 20 mL


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glass scintillation vials at 100oC for 1 h. Any undissolved particles were removed by filtration through a 0.45 µm pore size PVDF membrane syringe filter. Digested liver samples were diluted ~125 fold and other tissue samples were diluted ~50 fold in 4% high purity, trace metal-free concentrated HCl/2% HNO3 for ICP-AES analysis. ICP-AES was performed using an Optima 73100 (PerkinElmer, Waltham, MA) instrument and the concentration (mg/L) of Au in the tissues was obtained by reference to a standard curve. The operating conditions for ICP-AES analysis are described in the SI. The total AuNP mass in each organ was obtained by multiplying the total mass of Au in the diluted tissue sample by the dilution factor. The total mass in each organ was then divided by the total mass of AuNP injected per mouse to calculate the %ID/organ values. Statistical Analysis. All results were expressed as mean ± standard deviation (SD; n=3-4). A two-sided Student’s t test was used for statistical analysis and P85%

(SI, Figure S7) and the final radiochemical purity was >99%. The hydrodynamic diameter measured by DLS of unmodified AuNP was 37.8 ± 4.2 nm and the diameters of DOTAPEG-OPSS-AuNP, DOTA-PEG-LA-AuNP and PEG-pGlu(DOTA)8-LA4-AuNP were 56.6 ± 14.2, 54.2 ± 14.0 and 46.0 ± 8.4 nm, respectively. The hydrodynamic diameter of PEGpGlu(DOTA)8-LA4-AuNP was smaller than AuNP modified with DOTA-PEG-OPSS or DOTA-PEG-LA, due to the 2 kDa PEG chain used in PEG-pGlu(DOTA)8-LA4-AuNP and the 3.5 kDa PEG chain used in DOTA-PEG-OPSS and DOTA-PEG-LA. Based on AuNP bound radioactivity following reaction with 177Lu-MCP, there were 965 ± 96, 578 ± 63 and 847 ± 74 molecules of 177Lu-DOTA-PEG-OPSS, 177Lu-DOTA-PEGLA and PEG-pGlu(177Lu-DOTA)8-LA4 per AuNP, respectively. The mean surface area on AuNP occupied by AuNP,

177

177

Lu-MCP was 3.1, 5.0 and 3.6 nm2 for

Lu-DOTA-PEG-LA-AuNP

and

177

Lu-DOTA-PEG-OPSS-

PEG-pGlu(177Lu-DOTA)8-LA4-AuNP,

respectively. Note that the radius of gyration (Rg) of PEG 2 kDa and 3.5 kDa are 1.8 nm and 2.4 nm, respectively with an occupied area on the AuNP surface (πRg2) of 10 nm2 and 19 nm2 which are larger than the mean area occupied by the

177

Lu-MCP

31

. Thus the PEG

chains on the AuNP surface are likely extended and in the “brush” regime conformation. 32

MCP-AuNP Aggregation Assay. Displacement of MCP from AuNP causes AuNP aggregation resulting in a change in the UV-visible absorption spectrum. We took advantage of this phenomenon to evaluate the strength of binding of each MCP to AuNP when challenged with thiol-containing molecules DTT, L-cysteine or GSH. Similar assays have


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been used by others to compare different PEG ligands for stabilizing AuNP. 19, 20, 33 An AF was defined as the ratio between the absorbance at 615 nm and 524 nm. Changes in the UVvisible absorption spectrum over 60 min for AuNP bound to MCP in 10 mM DTT are shown in the SI, Figure S8. There was a rapid increase in the absorbance at 615 nm over 1 h for AuNP conjugated to DOTA-PEG-OPSS (SI, Figure S8A), whereas AuNP conjugated to DOTA-PEG-LA (SI, Figure S8B) and PEG-pGlu(DOTA)8-LA4 (SI, Figure S8C) exhibited stable spectroscopic properties. The time-dependent changes in AF in ddH2O, PBS pH 7.5, or 10 mM or 100 mM DTT, respectively, are shown in Figures 3A-D. The AF was integrated from 0 to 72 h to estimate the area under the curve (AUC0-72h = AF × h) which was then used to compare the stability of the MCP-AuNP constructs. All MCP-AuNP constructs were stable in ddH2O, exhibiting no significant difference in AUC0-72h (13.5 ± 0.3, 13.7 ± 0.1 and 13.5 ± 0.0 AF × h for DOTA-PEG-OPSS-AuNP, DOTA-PEG-LA-AuNP or PEG-pGlu(DOTA)8-LA4-AuNP, respectively; Figure 3A). All MCP-AuNP constructs exhibited an increase in AUC0-72h (23.3 ± 1.3, 20.8 ± 0.9 and 22.3 ± 2.1 AF × h for DOTAPEG-OPSS-AuNP,

DOTA-PEG-LA-AuNP

and

PEG-pGlu(DOTA)8-LA4-AuNP,

respectively) in PBS (pH 7.5; Figure 3B) but there were no significant differences between the constructs. Based on the AUC0-72h in 10 mM DTT, AuNP conjugated to DOTA-PEG-LA (23.3 ± 0.7 AF × h) or PEG-pGlu(DOTA)8-LA4 (25.2 ± 0.7 AF × h) exhibited significantly greater stability than AuNP bound to DOTA-PEG-OPSS (66.9 ± 0.2 AF × h; P

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