Tumor-Targeting NIRF NanoGUMBOS with Cyclodextrin-Enhanced

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Biological and Medical Applications of Materials and Interfaces

A Tumor Targeting NIRF NanoGUMBOS with Cyclodextrin Enhanced Chemo/Photothermal Antitumor Activities Mi Chen, Rocio Perez, Pu Du, Nimisha Bhattarai, Karen McDonough, Sudhir Ravula, Revati Kumar, J. Michael Mathis, and Isiah M Warner ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08047 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Applied Materials & Interfaces

A Tumor Targeting NIRF NanoGUMBOS with Cyclodextrin Enhanced Chemo/Photothermal Antitumor Activities

Mi Chen†, Rocío L. Pérez†, Pu Du†, Nimisha Bhattarai†⊥, Karen C. McDonough‡, Sudhir Ravula†, Revati Kumar†, J. Michael Mathis*§║, and Isiah M. Warner*†

†Department ‡AgCenter

of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States

Biotechnology Labs, Louisiana State University, Baton Rouge, LA 70803, United

States §Department

of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana

State University, Baton Rouge, LA 70803, United States *Corresponding

Authors

Present Adresses ⊥Department

of Biochemistry & Molecular Biology, School of Medicine, Tulane University,

New Orleans, LA 70112, United States ║Graduate

School of Biomedical Sciences, The University of North Texas Health Science

Center, Fort Worth, TX 76107, United States

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ABSTRACT

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The near-infrared fluorescent (NIRF) dye, IR780, is recognized as a promising theranostic

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agent and has been widely investigated for imaging, chemotherapeutic, and phototherapeutic

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applications. However, its poor photostability and non-selective toxicities towards both cancer and

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normal cells limit its biological applications. Herein, we introduce the use of GUMBOS (a group

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of uniform materials based on organic salts) developed through counter-anion exchange with

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IR780, and subsequent nanomaterials (nanoGUMBOS) formed by complexation with cyclodextrin

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(CD) for enhanced chemo/photothermal therapy. Such CD-based nanoGUMBOS display

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improved aqueous stability, photostability, and photothermal effects relative to traditional IR780.

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Examination of in vitro cytotoxicity reveals that CD-based nanoGUMBOS are selectively toxic

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towards cancer cells, and exhibit synergistically enhanced cytotoxicity towards cancer cells upon

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NIR laser irradiation. Additionally, in vivo NIRF imaging demonstrated selective accumulation of

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these nanoGUMBOS within the tumor site, indicating tumor targeting properties. Further in vivo

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therapeutic study of these CD-based nanoGUMBOS suggests excellent chemo/photothermal

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antitumor effects. Using these studies, we herein demonstrate a promising strategy, via conversion

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of IR780 into nanoGUMBOS, that can be used for improved theranostic cancer treatment.

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KEYWORDS

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NIRF, IR780, NanoGUMBOS, Cyclodextrin, Theranostic, Chemo/photothermal applications


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INTRODUCTION

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Recent developments in cancer therapy have resulted in a tremendous increase in

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investigations using theranostic agents, i.e., materials integrating both therapeutic and diagnostic

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imaging modalities. Such theranostics allow delivery of therapeutic drugs and diagnostic imaging

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agents simultaneously via a single dose. This overall strategy results in accelerated drug

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development, reduced side effects, and reduced cost relative to use of separate materials for

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theranostic applications.1-3

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NIRF dyes have emerged as promising theranostic agents since such drugs concomitantly

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serve as imaging agents and as agents for photothermal therapy (PTT) and photodynamic therapy

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(PDT). Several advantages accrue from the use of NIRF dyes for biomedical applications including

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low auto-fluorescence, deep tissue penetration, and minor invasiveness for phototherapy. Thus,

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the combined advantages of such dyes result in high sensitivity for imaging and high therapeutic

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efficacy.4-6 IR780 is a NIRF heptamethine dye with peak absorption at 780 nm and is recognized

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as an excellent theranostic agent. It has been widely studied for applications in imaging, PTT, and

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PDT due to its strong fluorescence intensity and superior tumor targeting properties.7-11 As a

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lipophilic cationic dye, IR780 has also been shown to preferentially accumulate in the

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mitochondria of cancer cells, resulting in cell death.11 As a result of differences from most

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lipophilic cations relying on mitochondria membrane potential, the mitochondrial targeting of

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IR780 is mediated by the mitochondria inner membrane transporter, i.e. ABCB10,12 thus making

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IR780 a unique mitochondrial probe for future study of mitochondrial medicine. Since the

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mitochondria is known to be highly sensitive to hyperthermia and excessive reactive oxygen

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species (ROS),13-14 these mitochondrial properties have also been exploited to design drugs with

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enhanced PTT and PDT effects.15-18 In this regard, the mitochondrial targeting property of IR780



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also makes it a favorable drug for PTT and PDT applications. Despite these attractive properties,

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these auxiliary application of IR780 for cancer treatment is still hindered by poor aqueous stability,

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photobleaching, severe toxicity at high doses, and rapid elimination from the body.19 In order to

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address these issues, considerable effort has been expended to encapsulate IR780 into various

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nanoscale delivery vehicles including micelles, proteins, or polymeric nanoparticles.5, 7, 20 In this

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context, the hydrophobic IR780 can be delivered to tumor tissues and retained for extended periods

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by use of passive targeting mechanisms such as enhanced permeation and retention (EPR) effects

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due to the hyper vasculature of tumor tissues.21 However, delivery of IR780 using these

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nanocarriers remains a challenge due to the complexity of fabrication, limited drug-loading

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capacity, and diminished chemotherapeutic activity.2, 7, 9, 20, 22

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Our research has focused on developing a new class of nanomaterials called

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nanoGUMBOS, derived from a group of uniform materials based on organic salts (GUMBOS),

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for various biomedical applications.23-25 GUMBOS are solid phase organic salts with highly

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tunable physicochemical properties achieved via counter-ion variation. NanoGUMBOS are

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nanomaterials derived from GUMBOS and combine the versatility of GUMBOS while displaying

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enhanced physicochemical properties at the nanoscale level. More importantly, using

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nanoGUMBOS as nanodrugs allows ultra-high drug loading and selective cytotoxicity as observed

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in one of our previous studies.23 The size and aqueous stability of nanoGUMBOS can also be

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easily tailored for further optimization of drug efficacy through EPR effects. Several investigations

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have been performed in our laboratory for controlled synthesis of nanoGUMBOS. In these studies,

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templated synthesis of nanoGUMBOS using cyclodextrin (CD) has been shown to produce

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uniform and stable nanoparticles.26 Moreover, CDs possess a unique cone structure with a

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hydrophilic exterior and a hydrophobic cavity, that may allow encapsulation of hydrophobic drugs


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within the cavity for enhanced water solubility and protection of the active drugs from light-,

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thermal- and oxidative-degradation.27

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In the study reported here, we have designed and synthesized an IR780-based GUMBOS

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as a prodrug, followed by conversion to nanoGUMBOS for enhanced in vitro and in vivo

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chemo/photothermal antitumor effects. Optimization of nanoGUMBOS fabrication with regard to

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size and aqueous stability is achieved by use of a reprecipitation method in the presence of various

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forms of β-CD. The cavity size of β-CD is preferred over other CDs for encapsulation of typical

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drug molecules.28-29 Following fabrication of CD-based nanoGUMBOS, characterizations

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regarding size, morphology, stability, and photophysical properties were performed. Subsequently,

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in vitro cytotoxicity (with and without NIR irradiation), cellular uptake, and subcellular

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localization were evaluated using human breast cancer cell lines. Tumor targeting studies were

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also performed using in vivo NIRF imaging. Finally, the antitumor efficacy of chemo/photothermal

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treatment was evaluated using an MDA-MB-231 tumor xenograft mouse model to explore the

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potential use of CD-based nanoGUMBOS as theranostic cancer therapeutic agents.

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RESULTS AND DISCUSSION

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Synthesis and characterization of GUMBOS. The IR780-based GUMBOS reported in this study

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was synthesized by pairing cationic IR780 with the lipophilic tetraphenylborate (TPB) using a

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metathesis reaction (Fig. 1a). This counter-anion is chosen to facilitate the transfer of IR780 cation

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into mitochondria for enhanced PTT effects, as previously demonstrated in other literature.30

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Details are provided in Supplemental Information (SI). Formation of the IR780 GUMBOS was

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confirmed using electrospray ionization mass spectrometry (ESI-MS). A high percentage yield of

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97% was achieved (Fig. S1). The [IR780][TPB] GUMBOS synthesized displayed a larger

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octanol/water partition coefficient (log P = 0.18) as compared to the parent compound [IR780][I]



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(log P = 0.09), indicating enhanced hydrophobicity upon anion exchange with TPB. This enhanced

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hydrophobicity should be a favorable characteristic for interacting with cell membranes, leading

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to higher cellular uptake as well as mitochondrial accumulation.31 Additionally, such tunable

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hydrophobicity of GUMBOS may aid in the formation of nanoGUMBOS in an aqueous system.32

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The absorption and fluorescence behaviors of GUMBOS were also evaluated in acetonitrile. As

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shown in Figure S2, these GUMBOS displayed similar absorption and emission spectra as the

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parent compound due to the IR780 cationic backbone. The absorption maximum was observed at

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780 nm with a shoulder at 710 nm, while the emission maximum was at 800 nm.

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Figure 1. (a) Depiction of a metathesis reaction for synthesis of [IR780][TPB] GUMBOS with a stoichiometry of 1 to 1. (b) Possible schematic representation of formation of CD-based nanoGUMBOS using HP-β-CD and [IR780][TPB] GUMBOS.

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Preparation and characterization of nanoGUMBOS. To examine application of [IR780][TPB]

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GUMBOS in a biological system, size-controllable nanoGUMBOS and CD-based nanoGUMBOS

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were prepared by use of a facile and green method with and without CD, respectively. Owing to 6 ACS Paragon Plus Environment

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the hydrophobic properties of [IR780][TPB] GUMBOS, they can be directly converted into

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nanoGUMBOS through self-assembly in an aqueous medium.10,

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nanoGUMBOS were firstly prepared using a reprecipitation method without CD.10 The formation

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of such nanoGUMBOS was confirmed using transmission electron microscopy (TEM) and

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dynamic light scattering (DLS). As indicated by TEM measurements, spherical nanoGUMBOS

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were obtained with approximately average size of 88 ± 17 nm under dried conditions (Figs. 2a

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and 2b). In contrast, the hydrodynamic size of these nanoparticles was 169 ± 2 nm with a

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polydispersity index (PDI) of 0.12 in DLS (Fig. S7). Variation in size analyses using TEM and

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DLS could be attributed to aggregation in the solution system, resulting in a larger size than in the

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dried state. The zeta potential of nanoGUMBOS at pH 7.4 was measured to be -27 mV, indicative

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of an overall negative surface charge under physiological condition. However, the hydrodynamic

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size of the control nanoGUMBOS in 0.01M phosphate buffer saline (PBS) determined by DLS

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increased significantly from 169 nm to 320 nm after 24 h. A large PDI of 0.5 was also obtained,

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indicating aggregation of nanoGUMBOS in PBS as a result of hydrophobicity of GUMBOS.

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In this regard, control

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To further improve the stability of nanoGUMBOS in an aqueous system, CD-based

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nanoGUMBOS were fabricated by use of ultrasonication in conjunction with freeze-drying. A

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possible schematic representation of the formation of such nanoparticles is shown in Figure 1b. In

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this scenario, CD serves as a drug carrier for incorporation of lipophilic GUMBOS into the

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hydrophobic cavity. In the presence of ulstrasonication, CD would molecularly cross-link with each other

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via hydrogen bonding between hydroxyl groups, ultimately forming CD-based nanoGUMBOS. Simialr

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observeation of such cross-linking has been validated by Xu et al.34 In order to optimize fabrication of

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these nanoparticles for chemo/photothermal therapeutic applications, different molar ratios of

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GUMBOS and CD including β-CD and HP-β-CD were used. The loading capacity of the resulting

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nanoparticles was characterized. As shown in Table S1, increasing the ratio of CD resulted in a 7 ACS Paragon Plus Environment


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slight reduction in loading capacity of nanoparticles, which is also in agreement with observations

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reported in previous literature.35 Statistical comparisons of all nanoparticles indicated that the

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loading capacity maximum was achieved when 1 to 1 molar ratio of [IR780][TPB] GUMBOS and

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HP-β-CD was used for nanoparticle preparation. This observation is also consistent with results

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from our in vitro cytotoxicity studies, in which nanoparticles prepared in this manner demonstrated

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the greatest toxicity towards breast cancer cells (Table S1). Therefore, this optimized CD-

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[IR780][TPB] nanoGUMBOS prepared using HP-β-CD was used for further studies. Examination

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of TEM data indicated that CD-[IR780][TPB] nanoGUMBOS had a spherical core shell structure

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with an approximate size of 132 ± 23 nm, in which an inner core with a diameter of approximately

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88 nm was observed with a 14 nm thin shell. Such a shell could be due to the HP-β-CD surface

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coating (Fig. 2d & Fig. S3); this possibility was further validated through characterization of CD-

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based nanoGUMBOS using Fourier-transformed infrared spectroscopy (FTIR). As shown in

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Figure S4, the characteristic peak at 3363 cm-1 attributed to O-H stretching from HP-β-CD was

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observed for these nanoparticles.

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(b)

(a)

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d = 88  17 nm Frequency

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0 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255

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(c)

Diameter (nm)

(d)

d = 132  23 nm

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0 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255

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Diameter (nm)

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Figure 2. Characterization of particle sizes for [IR780][TPB] nanoGUMBOS and CD[IR780][TPB] nanoGUMBOS using TEM. Each histogram constitutes 200 individual nanoparticles with a distribution curve overlay as well as a representative portion of a TEMn micrograph (all scale bars represent 500 nm): (a) and (b) [IR780][TPB], (c) and (d) CD[IR780][TPB].

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Evaluation of DLS measurements revealed a narrow size distribution with an average

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hydrodynamic size of 142 nm and PDI of 0.11(Fig. S7), which is in a good agreement with TEM

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analysis. The zeta potential was also examined in PBS at pH 7.4. These CD-based nanoGUMBOS

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displayed a zeta potential of -21 mV, which is similar to control nanoGUMBOS. Subsequently,

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the stability of CD-[IR780][TPB] nanoGUMBOS was examined by monitoring their

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hydrodynamic size change in 0.01M PBS. As presented in Figure S5, the size and PDI had minimal

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variation with time, concentration and temperature, indicating good stability of CD-[IR780][TPB]

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nanoGUMBOS under physiological conditions. For possible applications using in vivo studies,

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stability in PBS containing 10% mouse serum was also evaluated by monitoring the absorbance 9 ACS Paragon Plus Environment


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of the nanoparticles over a 120 h period at 37° C. Note in Figure S6, little or no change of the

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spectrum was identified with time, further indicating favorable stability of CD-based

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nanoGUMBOS under physiological conditions.

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Complexation study for formation of CD-based nanoGUMBOS. CD has been widely

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demonstrated to accommodate various hydrophobic drugs into their central cavity and form drug-

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inclusion complexes for improved drug stability in a biological environment.36-37 Thus, it is

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reasonable to hypothesize that our hydrophobic GUMBOS form complexes with HP-β-CD before

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further self-assembly into nanoGUMBOS. In order to confirm this hypothesis, differential

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scanning calorimetry (DSC) measurements and computational simulations were both performed

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to study the complexation between HP-β-CD and hydrophobic GUMBOS. Figure 3a shows the

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DSC thermograms of [IR780][TPB]GUMBOS, HP-β-CD, physical mixture of GUMBOS and

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HP-β-CD, and CD-[IR780][TPB] nanoGUMBOS. A small characteristic endothermic peak at 76.1

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oC

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to dehydration of adsorbed water molecules. The thermogram of their physical mixture displayed

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two well resolved endothermic peaks. One peak at 76.9 oC is similar to that of pure GUMBOS,

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while another at 38.5 oC is slightly shifted from the peak corresponding to HP-β-CD. In contrast,

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no clear endothermic peak was observed for CD-based nanoGUMBOS, indicating strong

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interactions between the GUMBOS and HP-β-CD or formation of the inclusion complex. Similar

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results were also obtained with other previously reported CD complexes.38-39

was observed for [IR780][TPB] GUMBOS, while HP-β-CD showed a broad peak at 54 °C due

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Docking and molecular dynamics (MD) simulations were performed to study possible

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complexation of each counter-ion of [IR780][TPB] GUMBOS with HP-β-CD due to their

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lipophilicities. Such data may provide further insight into the interactions of GUMBOS and HP-

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β-CD. The stable binding complexes (Figs. 3b(i) and 3b(iii)) with the highest fitting score from


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docking studies were used as initial structures in MD simulations. Figure 3b(iv) shows a separation

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of TPB from HP-β-CD during MD simulations, indicating an unstable state of the complex. Weak

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binding of a similar system (TPB and β-CD) was also observed in the experiment as previously

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reported.40 In contrast, the complex of IR780 and HP-β-CD was found to be stable during MD

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simulations (Fig. 3b(ii)). The free energy of binding for the IR780 and HP-β-CD complex was

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calculated to be -6.70 kcal/mol by using a MM/GBSA approach (Table S2).41-42 Details of

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computational methods are presented in SI. Using both experimental and computation approaches,

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we have demonstrated the formation of a stable complex of HP-β-CD and GUMBOS, which thus

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elucidates the enhanced stability of CD-based nanoGUMBOS in aqueous medium as compared to

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control nanoGUMBOS.



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Figure 3. (a) DSC thermograms of [IR780][TPB]GUMBOS, HP-𝛽-CD, their physical mixture, and CD-[IR780][TPB] nanoGUMBOS. (b) Initial structures of (i) IR780/HP-𝛽-CD complex and (iii) TPB/ HP-𝛽-CD complex from docking studies and their typical snapshots (ii &iv) from production of MD simulation trajectories. Water molecules and counter ions are omitted for clarity. Gray sticks represent HP-𝛽-CD molecules, green for IR780 and red for TPB.


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Photophysical properties of CD-based nanoGUMBOS. Photophysical properties of CD-

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[IR780][TPB] nanoGUMBOS in water were also investigated. As shown in Figure 4a, the CD-

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[IR780][TPB] nanoGUMBOS displayed a broad absorption spectrum in the near infrared (NIR)

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region from 600 nm to 900 nm, indicating a potential PTT effect with NIR irradiation. A similar

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spectrum was also obtained for the control nanoGUMBOS. In comparison with that of free IR780,

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red-shifted absorption spectra of both nanoGUMBOS were observed, which was most likely

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attributed to the formation of J-aggregation.43 For example, the absorption maximum of CD-

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[IR780][TPB] nanoGUMBOS was at 790 nm with a shoulder at 728 nm. In contrast, free IR780

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displayed an absorption spectrum with a peak at 774 nm and a shoulder at 710nm. Moreover, the

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fluorescence emission spectrum showed an intense fluorescence signal in the NIR region with peak

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emission at 800 nm (Fig. 4b). This wavelength would allow deep tissue NIRF imaging in vivo and

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provide real-time fluorescence guidance for PTT applications.44-45 Interestingly, the photo-

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stabilities of both nanoGUMBOS were found to be significantly improved in comparison with the

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parent compound. As shown in Figure 4c, a significant reduction in emission maxima was

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observed for IR780 with irradiation time. In contrast, no change of emission maxima was observed

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for either [IR780][TPB] nanoGUMBOS or CD-[IR780][TPB] nanoGUMBOS. This observation

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is important when considering the use of these nanoGUMBOS as theranostic agents. Photothermal

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properties of these nanoGUMBOS were also studied by measuring the temperature increase of

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nanoparticle solution under 808 nm NIR laser irradiation, and results were compared with the

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parent dye. As observed in Figure 4d, the parent dye displayed a rapid increase in temperature by

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approximately 9.4 °C and reached a temperature maximum after 150 seconds of irradiation. In

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contrast, [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS displayed a

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significantly higher temperature increase by approximately 16.8 °C and 18.0 °C respectively after



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10 min of irradiation. This enhanced photothermal property of nanoGUMBOS can be correlated

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with their enhanced photo-stabilities as described above. These results revealed that

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nanoGUMBOS could be superior to IR780 and may serve as more effective photothermal agents.

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In addition, examination of the size of both nanoGUMBOS before and after irradiation was

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performed using DLS measurements. For example, size distributions of both nanomaterials were

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narrow with PDI, i.e. less than 0.2 before laser irradiation. In contrast, size distribution became

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broader, and multiple peaks appeared after laser irradiation (Fig. S7). The calculated PDI for

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[IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS following laser irradiation

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were 0.36 and 0.47, respectively. These observations suggest dissociation of nanoGUMBOS upon

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laser irradiation; this effect may help to facilitate drug release, thus activating the

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chemotherapeutic activity of IR780 at the tumor site. A similar phenomenon was observed in our

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previous study, in which dissociation of rhodamine-based nanoGUMBOS in the acidic pH

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environment of lysosomes activated the toxicity of rhodamine in cancer cells.25


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Figure 4. (a) Absorption spectra, (b) emission spectra, and (c) photostability of [IR780][I], [IR780][TPB] nanoGUMBOS, and CD-[IR780][TPB] nanoGUMBOS in deionized water. Spectra were normalized by dividing each spectrum by its maxima of absorbance or emission. The normalized emission for photostability was determined by dividing the maximum emission intensity after the time of irradiation by the initial maximum emission. (d) Thermal curves of [IR780][I], [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] (100 M IR780 equivalent) after 808 nm laser irradiation (0.5 W/cm2).

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In vitro chemo/photothermal therapeutic effects of nanoGUMBOS. In vitro chemotherapeutic

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effects of the nanoGUMBOS were firstly studied by assessment of their cytotoxicity towards three

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breast cancer cell lines (MDA-MB-231, MCF-7, and Hs578T) as well as a normal breast epithelial

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cells (HMEC) and a myoepithelial cell line (Hs578Bst). As determined by MTT assay, significant

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cytotoxicity towards the breast cancer cell lines was observed upon treatments of [IR780][TPB]

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nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS while minimum cytotoxicity towards

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normal cell lines was observed (Figs. 5b and 5c). For example, treatment of MDA-MB-231 and

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Hs578T cancer cells with [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS

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led to almost eradication of cancer cells at a concentration at 50 g/mL, while greater than 75%

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cell viability of normal cells (HMEC and Hs578Bst) was still maintained. However, it was

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observed that the parent dye IR780 inhibited cell proliferation of both normal and breast cancer

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cell lines in a dose-dependent manner (Fig. 5a). Similar results were observed for the IR780 dye

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in prostate cancer and normal cells as reported in previous literature.8 In support of their potential

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chemotherapeutic application, analyses of these results demonstrated that nanoGUMBOS

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formulation greatly improved the selective toxicity towards cancer cells with reduced toxicity

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towards normal cells as compared with the parent dye. In addition, the IC50 data were calculated

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and compared (Fig. 5e). It is interesting to note that the two metastatic triple-negative (ER-, PR-,

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HER2-) breast cancer cell lines (MDA-MB-231 and Hs578T) were more sensitive towards the

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nanomaterials than the estrogen-responsive MCF-7 cancer cell line, as indicated by the smaller

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IC50 values. This result also suggests that our nanoGUMBOS can be further investigated as a

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potential chemotherapeutic agent for treating triple-negative breast cancer.


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Figure 5. Cell viability of breast cancer cell lines (MDA-MB-231, Hs578T, and MCF-7) and breast normal cell lines (HMEC and Hs578Bst) upon treatment with (a)[IR780][I], (b) [IR780][TPB] nanoGUMBOS, and (c) CD-[IR780][TPB] nanoGUMBOS for 48 h. (d) Colocalization of [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS with MitoTracker Green dye imaged by use of a ﬂuorescence microscope. All scale bars on the ﬂuorescence microscopy images represent 25 m. (e) IC50 values of all compounds for cultured cancer cell lines and normal cell lines. “---” represent not calculated due to the minimum toxicity.



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Cell viability studies of these nanomaterials under laser irradiation were conducted using

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two breast cancer cell lines (MDA-MB-231 and Hs578T) to examine in vitro PTT effects. As

279

observed in Figure 6a, no significant difference in the cytotoxicity of the parent dye was observed

280

between treatments with irradiation and without irradiation. This can be attributed to the low

281

uptake of parent dye into the cells. In contrast, both [IR780][TPB] nanoGUMBOS and CD-

282

[IR780][TPB] nanoGUMBOS displayed significantly higher cytotoxicity even at low

283

concentrations in MDA-MB-231 cancer cells (Figs. 6b and 6c ) and Hs578T cancer cells (Fig. S8)

284

with 808 nm laser irradiation relative to cytotoxicity without laser irradiation. Moreover, laser

285

irradiation treatment with CD-[IR780][TPB] nanoGUMBOS led to the highest cytotoxicity

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towards cancer cells among all materials tested. For instance, treatment with 0.39 g/mL of CD-

287

[IR780][TPB] nanoGUMBOS with laser irradiation resulted in a decreased cell viability of 49.5%,

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whereas [IR780][TPB] nanoGUMBOS and the parent dye yielded cell viabilities of 84.6% and 88%

289

respectively at the same concentration (Fig. 6). In this case, enhanced cancer-killing activity of

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CD-[IR780][TPB] nanoGUMBOS may be attributed to synergetic effects from their enhanced

291

chemotherapeutic behaviors and more efficient photothermal properties as compared to

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[IR780][TPB] nanoGUMBOS and the parent dye.


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293 294 295 296 297

Figure 6. Cell viability of MDA-MB-231 cancer cell lines after incubation with (a) [IR780][I], (b) [IR780][TPB], and (c) CD-[IR780][TPB] for 24 h with and without laser irradiation. Statistical significance was assessed using a two-way ANOVA test; (**P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001) 19 ACS Paragon Plus Environment


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Cellular uptake and localization. In order to further understand enhanced chemo/photothermal

299

activities of CD-[IR780][TPB] nanoGUMBOS, cellular uptake was examined and determined

300

using a previously reported method.10 As observed in Figure S9, significantly enhanced uptakes

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of [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS were observed as

302

compared to the parent dye. This result indicates the role of counter-ion variation on facilitating

303

internalization of these nanomaterials. Similar results were observed in our previously reported

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nanoGUMBOS.10 Moreover, the examination of these results indicated that the presence of CD

305

further promoted internalization of CD-[IR780][TPB] nanoGUMBOS with the greatest uptake

306

relative to the other two materials. This correlates well with the enhanced chemo/photothermal

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activities of CD-[IR780][TPB] nanoGUMBOS that were observed.

308

Given that cationic IR780 has been shown to be a mitochondrial toxin, the effect of using

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counter-ion variations of [IR780][TPB] and CD complexing of [IR780][TPB] on mitochondrial

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localization was studied. These studies may aid in further elucidation of the observed therapeutic

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behavior since the mitochondria plays an important role in the regulation of cell death.46 Co-

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localization of fluorescence from nanoGUMBOS and MitoTracker Green are displayed in Figure

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5(d). It is important to note a significant yellow overlay resulting from the red fluorescence of

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nanoGUMBOS and green fluorescence of MitoTracker Green for both [IR780][TPB]

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nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS in the merged images. The yellow overlay

316

indicates their mitochondria co-localization. From these results, we concluded that subcellular

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localization of [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS were similar

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for counter-ion variation and CD complexing.

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In vivo NIRF imaging and tumor targeting behavior. In vivo NIRF imaging of both control

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nanoGUMBOS and CD-based nanoGUMBOS was performed using an MDA-MB-231 tumor


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xenograft model. This allowed for the investigation of their biodistribution, as well as the

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determination of tumor localization, which is an important criterion for PTT applications. All

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compounds were intravenously injected into tumor-bearing mice and imaged at several time points.

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As shown in Figure 7a, mice treated with [IR780][TPB] nanoGUMBOS displayed a gradual

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increase in fluorescence signal over the entire body with time. At 24 h post-injection, a preferential

326

accumulation at the tumor site was observed in contrast to other organ sites. This tumor

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accumulation further increased with time, up to 72 h, which demonstrated long-term maintenance

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of these nanoGUMBOS in vivo. In mice treated with CD-[IR780][TPB] nanoGUMBOS (Fig. 7b),

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these nanoparticles preferentially accumulated at the tumor site after 6 h, indicating more rapid

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delivery into tumor tissues compared to [IR780][TPB] nanoGUMBOS (Fig. 7a). This observation

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was also consistent with in vitro cellular uptake kinetics, in which CD-[IR780][TPB]

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nanoGUMBOS displayed more rapid uptake than the control nanoGUMBOS (Fig. S10).

333

Subsequently, the fluorescence signal at the tumor site exhibited a continual increase up to 72 h,

334

with a slight reduction afterward, possibly due to metabolism and excretion from the body. In

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comparison with most of the IR780 injected doses in reported literature19 that displayed a rapid

336

clearance within 24 h after intravenous injection, both [IR780][TPB] and CD-[IR780][TPB]

337

nanoGUMBOS displayed prolonged drug retention at the tumor site. Thus, our nanoGUMBOS

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allowed tumor monitoring using NIRF imaging as well as show great potential for drug delivery.

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Ex vivo evaluation of organs excised at 120 h post-injection was also performed to gain

340

better insight into the biodistribution of nanoparticles (Figs. 7b and 7c). For the nanoGUMBOS

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treated mice, significantly higher fluorescence intensity was observed at the tumor tissue compared

342

to other organs. Interestingly, CD-[IR780][TPB] nanoGUMBOS also displayed enhanced tumor

343

accumulation as compared to [IR780][TPB] nanoGUMBOS, as demonstrated by significantly



344

stronger fluorescence intensity in tumor tissue (Fig. 7c). Analyses of these results further showed

345

that the use of CD for nanoGUMBOS preparation could effectively enhance drug bioavailability

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in vivo as well as promote the targeted accumulation of nanoGUMBOS in the tumor region without

347

the introduction of a targeting moiety.

348

Since some accumulation at other organs was also observed in the ex vivo images, the

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potential toxic side effect of both nanoGUMBOS was evaluated. At 14 days after injection of

350

[IR780][TPB] and CD-[IR780][TPB] nanoGUMBOS with two doses (2 mg/kg [IR780][TPB]

351

equivalent per dose), the mice were euthanized and major organs were collected for histological

352

analysis using hematoxylin and eosin (H&E) staining. As observed in Figure 7d, no histological

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changes in any of the major organs of mice were observed as compared with organs from PBS

354

treated mice, suggesting that nanoGUMBOS are nontoxic towards normal tissues at the doses used.

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These results are also in agreement with their selective toxicity in vitro as described earlier. Thus,

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in comparison with the parent dye, which displayed severe and acute toxicity at a dose of 2 mg/kg

357

as previously reported,20 our nanoparticles could be safer theranostic agents than traditional IR780

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in imaging-guided PTT experiments.


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359 360 361 362 363

Figure 7. (a) In vivo NIRF imaging of [IR780][TPB] nanoGUMBOS and CD-[IR780][TPB] nanoGUMBOS at different time points. (b) Ex vivo images of major organs and tumors after postinjection of nanoGUMBOS for 120 h. (c) Fluorescence intensities measured from ex vivo images of major organs and tumors. (d) H&E staining images of major organs for histology analysis. 23 ACS Paragon Plus Environment


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In vivo chemo/photothermal therapeutic effects of CD-[IR780][TPB] nanoGUMBOS.

365

Motivated by highly selective accumulation, long-term retention within tumor tissue, and

366

biocompatibility of CD-[IR780][TPB] nanoGUMBOS, we further evaluated in vivo

367

chemo/photothermal therapeutic efficacy of this nanoGUMBOS. The MDA-MB-231 tumor-

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bearing mice were randomly divided into four groups with six per group, subsequently treated with

369

PBS, PBS plus laser, CD-[IR780][TPB] nanoGUMBOS, and CD-[IR780][TPB] nanoGUMBOS

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plus laser. In these studies, laser irradiation was applied for 2 mins at a power density of 2W/cm2

371

using an 808 NIR laser. Upon laser irradiation, photothermal effects were examined using a

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thermal infrared camera. As shown in Figures 8a and 8b, the temperature of tumors treated with

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PBS only increased by approximately 5 oC and reached a temperature plateau at 42.6 oC. In

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comparison, tumors treated with nanoGUMBOS displayed a significantly enhanced temperature

375

increase by 16 oC on average, and maximum temperature reached was 55.5 oC, which is beyond

376

the threshold that induces damage of tumor vessels and tumor cells.47 Such a significant

377

temperature increase confirmed the high PTT effects of CD-based nanoGUMBOS in vivo.

378

In additon, tumor volumes from all groups were monitored and recorded for the

379

examination of the therapeutic effects of CD-[IR780][TPB] nanoGUMBOS. As shown in Figure

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8c, the tumor volume from the PBS-treated control group grew rapidly and reached nearly 6 times

381

their initial size by day 18. In comparison, tumors treated with CD-[IR780][TPB] nanoGUMBOS

382

displayed a significantly slower growth from day 10 to day 18 than the PBS-treated control group,

383

indicating the intrinsic antitumor effects of CD-[IR780][TPB] nanoGUMBOS, consistent with our

384

in vitro studies. More importantly, the tumor volume of mice treated with CD-[IR780][TPB] plus

385

laser treatment was significantly reduced as compared to that of the other three groups. This


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386

observation can be explained via a synergetic chemo/photothermal therapeutic effect of CD-

387

[IR780][TPB] upon laser irradiation.

388

389 390 391 392 393 394 395 396 397 398

Figure 8. In vivo photothermal therapy. (a) Infrared thermographs of tumor-bearing mice under 808 nm laser irradiation at a power density of 2W/cm2. (b)Temperature profiles of irradiated tumors as a function of irradiation time. (c) Relative tumor volume (RTV) of four groups of tumorbearing mice with six mice per group after various treatments. RTV was calculated using the formula: RTV= Vx/V0, where Vx and V0 is the volume measured at day x and injection day, respectively. Laser irradiation for 2 min at a power density of 2W/cm2 was applied at 72 h of postinjection of PBS and CD-[IR780][TPB] (3mg/kg), designated as day 3. Data were shown as mean ± SD.*P < 0.05.

399 25 ACS Paragon Plus Environment


400

CONCLUSIONS

401

In this study, we report fabrication, characterizations, and multifunctional applications of

402

NIR nanoGUMBOS as theranostic agents for enhanced chemo/photothermal anticancer effects.

403

Using counter-ion variations of a NIR dye, IR780, and CD complexing strategies, CD-based NIR

404

nanoGUMBOS were successfully fabricated to achieve an average size of approximately 132 nm.

405

The resulting CD-[IR780][TPB] nanoGUMBOS displayed a core-shell structure with improved

406

stability in comparison to [IR780][TPB] nanoGUMBOS without CD, providing several

407

advantages for biological applications. In addition, CD-based nanoGUMBOS showed excellent

408

photo-stability and photo-thermal properties under NIR irradiation, and allowed for enhanced PTT

409

application. Additional in vivo NIRF imaging results demonstrated preferential accumulation of

410

CD-[IR780][TPB] nanoGUMBOS at the tumor site with excellent tumor retention ability due to

411

EPR effects. Most importantly, remarkable chemo/photothermal therapeutic effects were achieved

412

using CD-[IR780][TPB] nanoGUMBOS for cancer treatment both in vitro and in vivo, without

413

inducing undesirable toxicity for the test dose. Thus, based on these results, we conclude that CD-

414

based nanoGUMBOS show great potential for use as cancer theranostic agents.


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415

ASSOCIATED CONTENT

416

Supporting Information: Detailed experimental methods, figures, and tables that support the

417

discussion. The supporting information is available free of charge.

418 419

AUTHOR INFORMATION

420

Corresponding Authors

421

J. Michael Mathis: [email protected]

422

Isiah M. Warner: [email protected]

423

Funding Sources

424

National Aeronautics and Space Administration (NASA)/Louisiana Board of Regents

425

National Science Foundation

426

Conflict of Interest

427

The authors declare no competing financial interest.

428 429

ACKNOWLEDGMENTS

430

The authors gratefully acknowledge partial financial support under NASA cooperative

431

agreement NNX 16AQ93A under and contract number NASA/LEQSF (2016-19)-Phase 3-10 and

432

the National Science Foundation under Grant No. CHE-1508726. Any opinions, findings, and

433

conclusions or recommendations expressed in this material are those of the author(s) and do not

434

necessarily reflect the views of the granting agencies. We are grateful for Dr. Dorin Boldor from

435

the Department of Biological and Agricultural engineering at LSU for use of the FLIR thermal

436

camera and constructing a laser set up. The use of LSU Shared Instrumentation Facility is also

437

acknowledged.



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functionalized gold nanoparticle photothermal therapy in melanoma cells. Sci. Rep. 2018, 8 (1), 8720.

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Abstract Graphic

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Near infrared fluorescent CD-based nanoGUMBOS displayed selective tumor

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accumulation and promising chemo/photothermal therapeutic effects in vivo.


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Tumor-Targeting NIRF NanoGUMBOS with Cyclodextrin-Enhanced

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