Protein Corona around Gold Nanorods as a Drug Carrier for

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Protein Corona around Gold Nanorods as a Drug Carrier for Multimodal Cancer Therapy Eugenia Li Ling Yeo, Joshua U-Jin Cheah, Bing Yi Lim, Patricia Soo Ping Thong, Khee Chee Soo, and James Chen Yong Kah ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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ACS Biomaterials Science & Engineering

Protein Corona around Gold Nanorods as a Drug Carrier for Multimodal Cancer Therapy Eugenia Li Ling Yeo1, Joshua U-Jin Cheah2, Bing Yi Lim1, Patricia Soo Ping Thong3, Khee Chee Soo3, James Chen Yong Kah1,2 1

Department of Biomedical Engineering, National University of Singapore 4 Engineering Drive 3, E4-04-08, Singapore 117583 2

NUS Graduate School for Integrative Sciences and Engineering

Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456 3

Division of Medical Sciences, National Cancer Centre Singapore 11 Hospital Drive, Singapore 169610

KEYWORDS Gold nanorods, photosensitizer, protein corona, multimodal therapy, photothermal therapy, photodynamic therapy, chemotherapy

ACS Paragon Plus Environment

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ABSTRACT A single nanodevice based on gold nanorods (NRs) co-loaded with a photosensitizer, Chlorin e6 (Ce6), and a chemotherapeutic, Doxorubicin (Dox) on its endogenously formed human serum (HS) protein corona i.e. NR-HS-Ce6-Dox was developed with the aim of performing multimodal cancer therapy: photodynamic (PDT), photothermal (PTT) and chemotherapy (CTX) simultaneously upon irradiation with a single 665 nm laser. Here, the excitation of NRs and Ce6 resulted in photothermal ablation (PTT), and production of reactive oxygen species (ROS) to kill Cal 27 oral squamous cell carcinoma (OSCC) cells by oxidative stress (PDT) respectively, while the laser-triggered release of Dox intercalated into the DNA of cancer cells to result in DNA damage and cell death (CTX). High laser-triggered Dox release efficiency of 71.5 % and strong plasmonic enhancement of ROS production by Ce6 (4.8-fold increase compared to free Ce6) was observed. Uptake of both Ce6 and Dox by Cal 27 cells was greatly enhanced, with 3.3 and 52 times higher intracellular Dox and Ce6 fluorescence observed respectively 6 h after dosing with NR-HS-Ce6-Dox compared to free drugs. The simultaneous trimodal therapy achieved a near complete eradication of cancer cells (98.7% cell death) with an extremely low dose of 15 pM NR-HS-Ce6-Dox loaded with just 1.26 nM Ce6 and 12.5 nM Dox due to strong synergistic enhancement in cancer cell kill compared to individual therapies performed separately. No dark toxicities were observed. These drug concentrations were far lower than any previously reported in vitro, thus eliminating any potential systemic toxicity of these agents.


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INTRODUCTION Multimodal cancer treatment involving the combination of two or more therapies shows an improved therapeutic efficacy over a single therapy. Often, plasmonic nanomaterials e.g. gold nanoparticles (NPs) capable of photothermal therapy (PTT) have been combined with photodynamic therapy (PDT) or chemotherapy (CTX) owing to their ease of surface modification that allows various chemotherapeutic drugs1-8 and photosensitizers9-13 to be easily conjugated onto their surface. These dual therapies have demonstrated synergistic improvement over individual therapy1, 2, 4-6 since the conjugated drugs exhibited less adverse side effects1-3, 6, better tumor selectivity and stability in physiological condition1, 3, and ability to escape multidrug resistance (MDR)8, 14-16 compared to their free form. These were due to a combination of high drug loading capacity17, enhanced permeability and retention (EPR) effect18, better internalization into cancer cells via endocytosis compared to passive diffusion of free drugs9, 17, and the evasion of glycoprotein P or ATP-binding cassette transporter-mediated drug efflux14-16. Collectively, these properties presented a higher bioavailability of therapeutics to cancers while significantly lowering the minimum effective drug concentration when loaded on NPs relative to free drugs, thereby reducing their systemic toxicity. While dual therapies of PTT+PDT and PTT+CTX have been widely reported, studies on trimodal therapies based on nanoscale systems that combine all three treatment modalities have been few and far between, with studies first emerging only in 201519-22. This was despite its promise to be even more efficacious compared to dual modality, and was likely due to limited capacity for loading multiple drugs (chemotherapeutic agent and photosensitizer) on NPs where competition between different drugs may result in significantly lower loading of each drug on


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NPs, and consequently poorer treatment efficacy. Amongst the few studies that demonstrated trimodal therapies, complex multistep processes were required to fabricate the nanoconstructs and load both the photosensitizer and chemotherapeutic on them. We have recently demonstrated the use of endogenous protein corona formed human serum (HS) around gold nanorods (NRs) to achieve loading and triggered release of drugs23, 24, and we applied it for combined PTT and PDT25. Here, the protein corona behaved like a “sponge” that absorbed and achieved high drug loading capacity of the photosensitizer chlorin e6 (Ce6), as well as conferred colloidal stability while mitigating the cytotoxicity of NRs26, 27. Using this system, we achieved almost complete eradiation of cancer cells at 50 pM NR dose containing 4.83 nM Ce6, which was much lower compared to other NP-based systems 1-4, 6, 8-12, 28, 29. A low dose was advantageous not just in reducing systemic toxicity, but also in minimizing accumulation in healthy organs such as liver, spleen and kidneys1, 3, 7, 9 which NP-based systems are prone towards. Encouraged by our results, we sought to further advance our system to investigate the coloading of a photosensitizer Ce6 and chemotherapeutic agent doxorubicin hydrochloride (Dox) on the high-capacity protein corona around NRs to form the novel nanodevice NR-HS-Ce6-Dox, capable of laser-triggered simultaneous trimodal PTT+PDT+CTX under a single 665 nm laser (see Table of Contents graphic). The work here represented for the first time that we were able to co-load more than one drug on the protein corona of NPs without any significant drop in the loaded amount of each drug compared to when it was loaded alone. This was rarely demonstrated in other types of drug delivery strategies. Compared to others, our nanodevice did not just present a large drug loading capacity, but also a simple one-pot process to fabricate. We achieved near-complete cell kill (98.7%) of Cal 27 oral squamous cell carcinoma cells (OSCCs)


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at an extremely low dose of 15 pM NR-HS-Ce6-Dox containing just 1.26 nM Ce6 and 12.5 nM Dox. Such a low dose has not been reported for trimodal therapy to date, and was mediated largely by a good cell uptake and synergy between the three therapies. This allowed the effective drug dose to be further minimized, thereby eliminating any systemic side effects.

MATERIALS AND METHODS All reagents were purchased from Sigma Aldrich unless specified otherwise. Milli-Q water with a resistivity of 18.2 MΩ cm was used for all experiments.

Synthesis of NR-CTAB A

previously

established

seed-mediated

method

was

adapted

to

synthesize

hexadecyltrimethylammonium bromide (CTAB) coated NRs (NR-CTAB) with a localized surface plasmon resonance peak at 665 nm30. Briefly, 250 µL of 10 mM chloroauric acid was added to 9.75 mL of 100 mM CTAB followed by 600 µL of ice-cold 10 mM sodium borohydride under constant stirring at 27 °C. Stirring was continued for 1 h to form a gold nanosphere seed solution. Separately, 4.75 mL of 100 mM CTAB was prepared, to which 250 µL of 10 mM chloroauric acid, 50 µL of 10 mM silver nitrate, 31 µL of 10 mM ascorbic acid and 6 µL of seed solution were added in the given order, swirling after each addition to ensure thorough mixing. The final mixture was left undisturbed in the dark at 27 °C for 4 h to allow NR-CTAB with a longitudinal surface plasmon resonance (LSPR) peak at 665 nm to form. The NR-CTAB colloid was centrifuged at 7,000 rpm for 15 min, redispersed in Milli-Q water, and stored at room temperature before further experiments.


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Formation of HS corona around NRs and co-loading of Ce6 and Dox The hydrophobicity of Ce6 (Frontier Scientific, U.S.) resulted in poor aqueous solubility and aggregation in water31. Hence, a 2 mM Ce6 stock solution was first prepared by dissolving Ce6 in HS under sonication, forming a clear, dark green, homogenous solution. The 2 mM Ce6 stock solution was mixed with 1 mM aqueous Dox, 100 mM pH 7.0 sodium phosphate buffer (PhB) (Axil Scientific, Singapore) and diluted in Milli-Q water to give a final Ce6+Dox solution in HS (HS-Ce6-Dox) containing 50% v/v HS, 1 mM Ce6, 200 µM Dox and 10 mM PhB. Here, 200 µM was used to prepare the HS-Ce6-Dox loading solution as higher Dox concentrations destabilized NR-HS-Ce6-Dox, as observed with an increase in zeta potential and DH of NR-HS-Ce6-Dox at [Dox] > 200 µM (see Supporting Information Figure S1A and B). NR-HS-Ce6-Dox was prepared by centrifuging 1 mL of 0.5 nM as-synthesized NR-CTAB at 7,000 rpm for 15 min. The supernatant was then aspirated before 1 mL of HS-Ce6-Dox loading solution was added to the soft pellet. The mixture was left to incubate at 37 °C for 20 h to allow spontaneous formation of HS protein corona around the NRs and simultaneous co-loading of Ce6 and Dox. The NR-HS-Ce6-Dox was then purified by three repeated centrifugation at 6,000 rpm for 20 min to remove excess HS, Ce6 and Dox, before re-dispersion in medium for subsequent experiments. To prepare NR-HS-Ce6 as a dual PTT+PDT therapy control, a Ce6-only solution in HS containing 50% v/v HS, 1 mM Ce6 and 10 mM PhB was prepared and incubated with NRCTAB as described above. Likewise, to prepare NR-HS-Dox as a dual PTT+CTX therapy control, a Dox-only solution in HS containing 50% v/v HS, 200 µM Dox and 10 mM PhB was prepared and incubated with NR-CTAB. Subsequent steps of centrifugal washing were performed as described above to purify our dual therapy controls.


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Characterization of NR-HS-Ce6-Dox and its controls UV-Vis spectroscopy was used to characterize the absorbance spectrum of NRs with and without the protein corona and drugs (Safire2TM, Tecan Group Ltd, Switzerland), and NR concentration was calculated from the absorbance spectrum and known extinction coefficients32. Colloidal stability was determined by calculating the ratio of the area under the longitudinal LSPR absorbance peak to the height of the peak. This ratio was termed as the aggregation index (AI)33. Transmission electron microscopy (TEM) (JEM-1220, JEOL Ltd., Japan) was used to characterize the morphology of the NRs, while zeta potential (ζ) and dynamic light scattering (DLS) measurements were acquired using a Zetasizer (Nano ZS, Malvern, UK) at 25°C to determine their surface charge and hydrodynamic diameter (DH) respectively.

Quantifying amount of Dox loaded in NR-HS-Ce6-Dox Due to quenching of Dox fluorescence by NRs, direct measurement of Dox fluorescence in NR-HS-Ce6-Dox to quantify the loaded amount against calibration standards containing the free drugs with known concentrations would result in inaccuracy8,

9, 11, 34-36

. Instead, we used a

previously established method of thermal treatment to release drug payloads from the protein corona24. Briefly, NR-HS-Ce6-Dox was heated in boiling water for 30 min which resulted in the denaturation of the protein corona, causing the hydrophilic and thermally stable Dox to be released readily into solution. On the other hand, the thermally labile Ce6 was degraded37-41. The solution was then centrifuged at 10,000 rpm for 15 min to remove the NR-HS-Ce6 from the released Dox, and the amount of Dox loaded was quantified by measuring its fluorescence


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(λex/λem = 480/600 nm) in the supernatant against a set of calibrating standards of known Dox concentrations using Tecan Safire2TM (Tecan Group Ltd., Switzerland). Here, interference from fluorescence of Ce6 was negligible since Ce6 was not excited at 480 nm and was previously thermally degraded in high temperature as the same thermal treatment on NR-HS-Ce6 in the absence of Dox resulted in negligible Ce6 fluorescence in the supernatant.

Quantifying amount of Ce6 loaded in NR-HS-Ce6-Dox Quantification of Ce6 by thermal release was not possible due to thermal degradation of Ce6 when heated. Direct absorbance measurement of the characteristic Ce6 absorbance peak at λ = 405 nm9, 28, 35, 42 was also not possible due its weak absorbance at concentrations < 100 nM coupled with interference by strong absorbance of NRs (Supporting Information Figure S1C). Therefore, no Ce6 peak was visible in the absorbance spectrum of NR-HS-Ce6-Dox after three centrifugal washes and removal of unbound Ce6. Hence, we quantified the amount of loaded Ce6 through measuring the Ce6 fluorescence directly from NR-HS-Ce6-Dox. Since Ce6 fluorescence could also be quenched by NRs, we first determined the extent of Ce6 fluorescence quenching by NRs as previously reported25. This was done by first measuring the Ce6 fluorescence (λex/λem = 405/665 nm) (Tecan Safire2TM, Tecan, Switzerland) in 0.5 nM NR-HS-Ce6-Dox loaded with 200 µM Dox and varying Ce6 concentrations to obtain a calibration curve of Ce6 in NR-HS-Ce6-Dox. A separate concentration calibration curve of Ce6 in HS-Ce6-Dox was also determined in the absence of NRs and at equivalent Dox and Ce6 concentrations. From the two sets of linear Ce6 concentration calibration curves, we determined the percentage fluorescence quenching at various Ce6 concentrations as follow,


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Percentage fluorescence quenching =

FlCe6 in HS-Ce6-Dox -FlCe6 in NR-HS-Ce6-Dox ×100% FlCe6 in HS-Ce6-Dox

From this, we showed that for Ce6 concentration < 500 nM, an average of 46.4 ± 0.5 % of Ce6 fluorescence was consistently quenched in the presence of NR-HS-Ce6-Dox. Since the range of Ce6 concentrations used in subsequent experiments did not exceed 500 nM, the Ce6 fluorescence quenching was corrected for by this constant when Ce6 fluorescence was measured directly from NR-HS-Ce6-Dox to quantify the amount of Ce6 loaded on each NR-HS-Ce6-Dox.

Photothermal heating, ROS production and Dox release from NR-HS-Ce6-Dox in buffer 200 µL of 0.2 nM NR-HS-Ce6-Dox in serum-free medium was added into a 96-well plate and irradiated with a 665 nm continuous wave (CW) laser (Photonitech, Singapore) at 500 mW/cm2 using an optical fiber to deliver the light dose directly above the plate for 15 min with a laser spot size of 12.57 mm2 in the center of the well, giving a total energy dose of 56.6 J. To determine the photothermal heating effect, the temperature of the colloid was measured before and after laser irradiation with a thermocouple probe (Fluke, USA). ROS production from NR-HS-Ce6-Dox was determined by adding 0.8 µL of 5 mM 3’-(paminophenyl) fluorescein (APF) (Invitrogen, U.S.) to 199.2 µL of 0.2 nM NR-HS-Ce6-Dox in a 96-well plate prior to irradiation as above. APF is a probe that reacts with ROS to produce a green fluorescence (λex/λem = 490/515 nm). Following irradiation, the samples were transferred to 1.5 mL tubes and centrifuged at 10,000 rpm for 15 min to remove the NR-HS-Ce6-Dox, and 100 µL of the supernatant was then transferred to a black 96-well plate for measuring APF fluorescence (Tecan Safire2TM, Tecan, Switzerland). We also characterized the amount of Dox released after laser irradiation of 200 µL of 0.2 nM NR-HS-Ce6-Dox. The samples were transferred to 1.5 mL tubes and centrifuged at 10,000 rpm


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for 15 min to remove the NR-HS-Ce6-Dox. 100 µL of supernatant was then transferred to a black 96-well plate and Dox fluorescence measured. An identical NR-HS-Ce6-Dox sample was kept in the dark as a control for passive leakage of Dox before being subjected to the same centrifugation and Dox fluorescence measurements. The amount of Dox released by laser activation was then quantified against calibrating standards after subtraction against the control. In the above studies, NR-HS-Ce6, NR-HS-Dox, NR-HS, NR-CTAB, free drugs alone and their combination (i.e. Ce6 alone, Dox alone, and Ce6+Dox) dissolved in HS, and medium alone at concentrations and volumes equivalent to that loaded on NR-HS-Ce6-Dox served as controls and were also subjected to the same irradiation, centrifugation and measurement conditions. Equivalent Ce6 and Dox concentrations were calculated from Ce6-to-NR and Dox-to-NR ratios determined earlier.

Cell culture Cal 27 oral squamous cell carcinoma (OSCC) cells (ATCC, USA) were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) (GE Healthcare, U.K.), 1% non-essential amino acids (Gibco, USA), 100 U/mL penicillin-streptomycin (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA) and 2 mM L-Glutamine, and incubated at 37 °C in a humidified atmosphere with 5% CO2.

Cell uptake of Ce6 and Dox We examined the cell uptake of Ce6 and Dox by Cal 27 cells dosed with NR-HS-Ce6-Dox and compared it against free Ce6 alone (HS-Ce6), Dox alone (HS-Dox) and Ce6+Dox dissolved in HS (HS-Ce6-Dox) in the absence of NRs using flow cytometry under varying dose


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concentrations and dosing time. To examine the cell uptake of the non-fluorescent NRs, dark field microscopy was used. Confocal laser scanning microscopy (CLSM) was also used to examine the localisation of NRs, Ce6 and Dox in cells. Cal 27 cells were seeded at a density of ~3 x 105 cells per well either on glass cover slips placed in a 6-well plate for microscopy, or directly on empty 6-well plates for flow cytometry before incubating for 24 to allow cell adhesion to occur. For flow cytometry, the cells were dosed with varying concentrations of NR-HS-Ce6-Dox, HS-Ce6, HS-Dox and HS-Ce6-Dox in serum-free medium for 6 h in a dose-dependent cell uptake study. In a time-dependent cell uptake study, dose concentrations were fixed at 0.2 nM NRs with an equivalent 16.8 nM Ce6 and 167 nM Dox concentrations while the dose time was varied. The cells from both studies were then rinsed twice with phosphate buffered saline (PBS) to remove excess NRs, Ce6 and Dox not taken up by cells. The cells were then trypsinized and transferred into a flow cytometry tube where the Ce6 and Dox fluorescence in cells were acquired with a FACSCanto flow cytometer with FACSDiva software (Becton, Dickinson, U.S.). The data was then analysed based on at least 1.5 x 104 cells with FlowJo version 7.2.2 (FlowJo, USA). For dark field microscopy and CLSM, cells on coverslips were fixed by 3.7% formaldehyde in PBS for 15 min. The cells were rinsed twice with PBS before the nuclei were stained with 4’,6diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher Scientific, USA). The cells were then rinsed twice with PBS again before being mounted on glass slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, USA). Dark field and fluorescence images were acquired using a Nikon Ci-L Fluorescence Upright microscope (Nikon Instruments, Japan) equipped with a CytoViva 150 Condenser (CytoViva, USA) and an sCMOS camera


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(pco.edge 4.2 M-USB-PCO, PCO, Germany). An oil immersion 60X objective was used in the imaging. CLSM images were obtained with a Leica TCS SP8 system (Leica, Germany) with an oil immersion 100X objective.

Therapeutic efficacy of NR-HS-Ce6-Dox The therapeutic efficacy of trimodal PDT+PTT+CTX by NR-HS-Ce6-Dox in vitro was studied in terms of the cell viability of Cal 27 cells. The cells were seeded in a 96-well plate at a density of 2.7 x 104 cells per well and left for 24 h at 37 °C to allow cell adhesion. The cells were then dosed with NR-HS-Ce6-Dox, NR-HS, HS-Ce6-Dox, HS-Ce6 and HS-Dox alone in serum-free medium for 6 h at varying concentrations up to 50 pM NRs, or an equivalent of 4.19 nM Ce6 and 41.7 nM Dox based on the amount of Ce6 and Dox loaded on NR-HS-Ce6-Dox. Thereafter, the cells were rinsed twice with PBS to remove excess drugs not taken up by cells, before reintroducing into serum-free medium. The cells were then irradiated with a 665 nm CW laser at 250 mW/cm2 for 15 min (Total energy dose = 28.3 J) and then left to incubate for another 24 h at 37°C before cell viability was determined using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA). Dark toxicities of NR-HS-Ce6-Dox and its respective controls were also assessed by dosing cells with the same concentration range for 6 h and keeping them in the dark before determining their viability.

RESULTS AND DISCUSSION Synthesis and characterization of NR-HS-Ce6-Dox The as-synthesized NR-CTAB was isolated and monodisperse with dimensions of 46.5 ± 1.2 nm by 19.0 ± 0.7 nm (aspect ratio = 2.45 ± 0.11), as observed from TEM (Figure 1A, inset). The


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NRs had an LSPR peak at 657.1 ± 3.2 nm which matched the excitation Q-band of Ce6 at 665 nm (Figure 1A), thus allowing a single 665 nm laser to excite both NRs and Ce6 simultaneously. There was no significant change in the LSPR peak following the formation of the HS corona and loading of Ce6 and Dox in NR-HS-Ce6-Dox, with λpeak = 659.0 ± 3.4 nm. The aggregation index (AI) of NR-HS-Ce6-Dox also remained similar to as-synthesized NRCTAB (Figure 1B). The absence of red-shift in the LSPR peak and change in AI after HS protein corona formation and drugs loading on NRs demonstrated the colloidal stability of NR-HS-Ce6Dox, which was important to ensure high uptake and therapeutic efficacy43. We also noted that the AI of NR-HS-Dox and NR-HS without Ce6 loaded were generally higher, suggesting that presence of Ce6 loaded on the protein corona could stabilize the NR-HS against centrifugationinduced aggregation.


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2 1 0 400

B

500

700

800

900

Wavelength (nm)

120

Aggregation Index (A.U.)

600

115 110 105

Intensity (%) Intensity (%)

3

10.0 7.5 5.0 2.5 0.0

Intensity (%)

NR-CTAB NR-HS NR-HS-Ce6 NR-HS-Dox NR-HS-Ce6-Dox

10.0 7.5 5.0 2.5 0.0

Intensity (%)

D

Normalized Absorbance (A.U.)

A

10.0 7.5 5.0 2.5 0.0

NR-HS-Dox

10.0 7.5 5.0 2.5 0.0

NR-HS-Ce6-Dox

NR-CTAB

10.0 7.5 5.0 2.5 0.0

NR-HS

1

10

100

1000

NR-HS-Ce6

100 40 20 0

SC

e6

-D ox

ox SD -H

-H N R

N

R

SC e6 -H

R -H N R

N

R -C N

S

-20

TA B

C

Zeta Potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Intensity (%)


1

10

100

Hydrodynamic diameter (nm)

1000

Figure 1. Physical characterization of NR-HS-Ce6-Dox and its controls. NR-HS-Ce6-Dox possessed good colloidal stability as observed by its (A) the sharp LSPR peak in the UV-Vis absorbance spectrum, similar to as-synthesized NR-CTAB, and absence of aggregation under the TEM, as well as its (B) low aggregation index. The formation of the HS protein corona around NRs and co-loading of Ce6 and Dox resulted in (C) a flip in the zeta potential from the positively charged CTAB surface to the negatively charged protein corona, and (D) an increase in DH, similar to other NR-HS corona controls.

The formation of the protein corona due to the non-specific adsorption of negatively charged HS proteins onto NRs capped with the positively charged CTAB ligand resulted in the zeta


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potential of NR-CTAB flipping from ζ = +41.6 ± 0.6 mV to -18.2 ± 0.4 mV for NR-HS (Figure 1C). This was consistent with observations by others where protein binding to NPs resulted in a net negative charge regardless of the initial NP charge44, 45. Further loading of the negatively charged Ce6 (ζNR-HS-Ce6 = -19.3 ± 1.2 mV) or positively charged Dox (ζNR-HS-Dox = -17.7 ± 0.8 mV), and co-loading of both Ce6 and Dox simultaneously (ζNR-HS-Ce6-Dox = -18.9 ± 0.6 mV) did not caused the zeta potential of NRs to change much since the surface of NRs predominantly comprised of HS proteins. We observed two peaks in the histogram of DH distribution of NR-CTAB under the DLS, typical of gold NRs46, 47 (Figure 1D). The smaller peak signified that the rotational diffusion coefficient of NRs was equal to the translational diffusion coefficient of a spherical gold NP with DH = 4.8 ± 0.1 nm, while the larger peak was indicative of NRs having the same translational diffusion coefficient as a spherical gold NP with DH = 57.8 ± 0.1 nm. The DH for both peaks of NR-HS-Ce6-Dox increased to 16.1 ± 0.1 nm and 88.2 ± 0.6 nm respectively. Corona formation and drug loading in NR-HS-Ce6, NR-HS-Dox and NR-HS also resulted in similar increase in DH, suggesting that the non-specific adsorption of HS protein corona around NRs played a major role in the final DH of NRs, with less significant contribution from the drugs loaded in the corona. Here, the final DH of NR-HS-Ce6-Dox would eventually affect its biodistribution and tumor selectivity in vivo. Passive targeting and accumulation of NPs in tumors is dependent on the EPR effect, which in turn is a result of leaky tumor vasculature and impaired lymphatic drainage17, 48, 49

. Larger NPs (60 – 100 nm) have been shown to accumulate better in tumors than smaller

NPs50, which may be attributed to reduced renal clearance and longer blood circulation time51.


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Hence, the increase in DH in NR-HS-Ce6-Dox could potentially result in more efficient tumor accumulation.

Quantifying Ce6 and Dox loading on NR-HS-Ce6-Dox The co-loading of Ce6 and Dox occurred simultaneously with the formation of the HS protein corona, which behaved like a “sponge” to hold the drug payload. This was possible due to the presence of several endogenous transport proteins in HS, e.g. human serum albumin (HSA)52-54 and lipoproteins55-57 that were able to interact strongly with small drugs and biomolecules. By releasing Dox from NR-HS-Ce6-Dox thermally through heating24 with Ce6 thermally degraded simultaneously, we determined from Dox fluorescence measurements that ~833.0 ± 91.6 Dox molecules were loaded on each NR (Figure 2A). This amount was less than that loaded on NR-HS-Dox (~1780 ± 99.9 Dox per NR) in the absence of Ce6 since Ce6 could undergo competitively binding with Dox on the HS corona. Here, the hydrophilic Dox interacted with the hydrophilic charged domains of serum proteins and remained stably bound in the protein corona with significantly less leakage on its own without the thermal release (Figure 2A).


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Figure 2. (A) Quantification of Dox loaded in NR-HS-Ce6-Dox and its controls by thermal release of Dox compared to the amount of Dox leaked from corona without heating. The thermally labile Ce6 was degraded from the heat and does not interfere with Dox quantification. (B) In quantifying Ce6, we observed that fluorescence of Ce6 at λex/λem = 405/665 nm increased with increasing concentration of free HS-Ce6 in solution (solid line). (B, inset: when excited at 405 nm, Dox exhibited negligible fluorescence while Ce6 exhibited a strong fluorescence peak at 665 nm). However, loading of Ce6 onto NR-HS-Ce6-Dox resulted in fluorescence quenching (dotted line). (C) A constant Ce6 fluorescence quenching of 46.4 ± 0.5 % was observed when Ce6 was loaded on NR-HS-Ce6 at 500 nM Ce6 and below compared to free Ce6. The amount of


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Ce6 loaded on NR-HS-Ce6 could thus be determined by measuring the fluorescence of NR-HSCe6-Dox directly and correcting for the constant Ce6 fluorescence quenching.

Since quantification of Ce6 by thermal release and direct measurement of its absorbance in NR-HS-Ce6-Dox were not possible due to its thermal degradation and negligible absorbance at 90% cell kill, thus offering less benefit over performing PDT on its own with the same photosensitizer concentration9-12, 28, 34, 42, 62, 72, 83-86. Likewise, higher Dox concentrations of 1 to 20 µM (~80 to 1,600 times higher concentration) have to be co-delivered with NPs to achieve >90% cell kill in dual modal PTT+CTX studies by


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others1-3, 5, 6, 8, 85, 87. In contrast, even our dual mode PTT+CTX with NR-HS-Dox could achieve 50% cell killing efficacy with 41.65 nM Dox loaded via the protein corona on NRs. A reduction in photosensitizer or chemotherapeutic concentrations in these aforementioned studies reported by others would result in considerable decrease in cell killing. There has been no report to date of complete cell kill at such a low dose of photosensitizer, chemotherapeutic and NPs as achieved with our NR-HS-Ce6-Dox involving the protein corona as the carrier. Furthermore, the light dose we administered to the cells was 28.3 J (250 mW/cm2 laser power density, 15 min irradiation time, 12.57 mm2 laser spot size), which was also comparable to other in vitro single-laser combined phototherapy studies. Such a low-dose and high efficacy trimodal therapy by NR-HS-Ce6-Dox would be crucial towards eliminating tumor recurrence with complete cell kill while simultaneously minimizing off-target systemic toxicity with the extremely low dose.


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Figure 7. Cell viability of Cal 27 cells when dosed with increasing concentrations of NR-HSCe6-Dox and different dual mode and single mode therapy controls with each axes showing the equivalent amount of drugs loaded and dosed to the cells. (A) Following irradiation with a 665 nm laser, trimodal PDT+PTT+CTX with NR-HS-Ce6-Dox (purple) achieved near complete cell kill of 98.7% with only 15 pM NR-HS-Ce6-Dox, while dual modal PTT+PDT by NR-HS-Ce6 (green), PTT+CTX by NR-HS-Dox (blue) and PDT+CTX by free Ce6+Dox (pink) achieved significantly less cell kill at the same equivalent concentrations. Individual therapies by NR-HS (PTT), free Ce6 (PDT) and Dox (CTX) were even less effective, with insignificant cell kill at the same equivalent concentrations. (B) In the absence of laser irradiation, cell viability remains high (>86%) for all the compounds across all concentrations used, indicating that NR-HS-Ce6-Dox


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and its various controls were non-toxic on their own, and that the trimodal therapy by NR-HSCe6-Dox is highly controllable by light.

Conversely, irradiation of cells dosed with a mixture of 1.26 nM Ce6 and 12.5 nM Dox in the absence of NRs for dual modal PDT+CTX resulted in much poorer cell kill of 19.4 % (Figure 7A, pink). Likewise, dual modal PTT+PDT with 15 pM NR-HS-Ce6 (Figure 7A, green) and PTT+CTX with 15 pM NR-HS-Dox (Figure 7A, blue) resulted in lower 63.0 % and 24.2 % cell kill respectively. While this may still be considered effective considering the very low dose of 15 pM used, it is still far less effective than the same concentration of NR-HS-Ce6-Dox used. Individual therapies by 15 pM NR-HS (PTT), 1.26 nM free Ce6 (PDT) and 12.5 nM free Dox (CTX) (Figure 7A, orange, brown, red respectively) separately achieved the weakest therapeutic efficacy on cells, with cell kill of only 7.70 %, 10.8 % and 16.5 % respectively. The high cell kill efficacy achieved by trimodal therapy with an extremely low dose of NRHS-Ce6-Dox was more than an additive effect that summed the cell killing efficacy of PDT, PTT and CTX individually. Several factors could have contributed to achieve this. First, the delivery of both Ce6 and Dox into cells was greatly enhanced by exploiting the HS protein corona around NRs as a carrier for loading and delivery, as observed in flow cytometry and fluorescence microscopy. Apart from affording a higher payload of biomolecules using the protein corona compared to covalent monolayer strategies of attaching biomolecules on the NPs

24

, the cancer

cells appeared to preferentially uptake the drugs loaded on the NR-HS compared to drugs in their free form. Hence, while the same amount of drugs between free Ce6, Dox and NR-HS-Ce6-Dox were dosed to the cells, the actual concentration of Ce6 and Dox in the cells were much higher with NR-HS-Ce6-Dox. Subsequent high Dox release efficiency (71.5 % release) and strong


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plasmonic enhancement of ROS production from NR-HS-Ce6-Dox (4.8-fold increase over free Ce6 at same concentration) upon laser irradiation would have in turn resulted in enhanced CTX and PDT efficacy respectively compared to using free Dox and Ce6 on their own. Secondly, synergy between simultaneous PDT, PTT and CTX in trimodal therapy could have occurred instead of an additive effect, leading to more effective treatment outcome. A purely additive effect of PTT, PDT and CTX (7.70 %, 10.8 % and 16.5 % cell kill respectively) should have resulted in a total cell kill of only 35.0%. The much higher 98.7 % cell kill that was realized by trimodal therapy using NR-HS-Ce6-Dox suggested a strong synergy between the three different therapies. In this case, ROS production from PDT was an oxygen-dependent process which was more effective at the start of the therapy. As irradiation proceeded, oxygen in the irradiated environment was depleted, resulting in a decrease in PDT efficacy12, 88, 89. The oxygenindependent treatment by PTT and CTX were then able to compensate for this decrease in PDT efficacy. Similarly, at the low dose of 12.5 nM Dox, CTX alone would be ineffective. However, the efficacy of CTX was enhanced by hyperthermia90-92, which was induced by PTT heating. In contrast to laser irradiation, cell viability of Cal 27 cells remained high at >86% in the absence of laser irradiation, when dosed with NR-HS-Ce6-Dox and the relevant controls at concentrations up to 50 pM (equivalent to 4.19 nM Ce6 and 41.7 nM Dox) (Figure 7B). Hence, we established low dark toxicity with NR-Hs-Ce6-Dox, likely due to both Dox and Ce6 remaining bound and inactive in the HS protein corona around NRs. Drug activation is thus highly controllable by laser irradiation, which would be important in localizing the treatment to the cancer site, thus ensuring low systemic off-target toxicity, a common problem associated with chemotherapeutics like Dox.


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CONCLUSION In this study, we demonstrated that the protein corona formed around NRs can be utilized to co-load two different types of drugs simultaneously, a photosensitizer Ce6 and the chemotherapeutic Dox to form the nanoconstruct NR-HS-Ce6-Dox capable of trimodal PTT+PDT+CTX simultaneously. We achieved near-complete eradication of cancer cells (98.7 %) with an extremely low dose of just 15 pM NRs loaded with 1.26 nM Ce6 and 12.5 nM Dox. We attributed this to the far greater cellular uptake of Ce6 and Dox aided by the protein corona, as well as synergistic effects of simultaneously performing PTT+PDT+CTX. In addition, the large plasmonic enhancement in Ce6 ROS production induced by NRs (4.8-fold increase) and high laser-triggered release efficiency of Dox (71.5 % release) also played a major role in the observed synergy. This trimodal therapy was highly controllable with optical excitation with potentially low systemic toxicity in the absence of laser irradiation. PTT heating by NRs was also unaffected by protein corona formation. While several other studies have shown dual modal PDT+PTT or CTX+PTT, very few have been able to expand their work in the same manner to allow multiple drug loading and trimodal therapy due to the use of direct conjugation and limitation in the available surface of NPs, or due to the use of complex and highly specific loading strategies. Here, we established how protein corona formation stood out among the large variety of drug loading strategies available due to its capability to easily expand into a multiple drug loading strategy with minimal modification in the loading process to allow trimodal therapy. In addition to this key advantage, other advantages including the endogenous biocompatible nature of the protein corona, the simplicity of the onepot loading process involved, and the possibility of using the proteins presented in the corona to


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perform site-directed targeting93 further differentiates the protein corona drug loading strategy from others in the field. We conclude that the formation of an endogenous protein corona on NPs may be exploited beneficially in cancer treatment instead of regarding it merely as an undesirable biological artefact to be eliminated. This paves the way for further in vivo studies where we can expect the same high therapeutic efficacy to be achieved using an extremely low drug dose. Our study has demonstrated the possibility of drawing inspiration from endogenous systems to embrace the “stickiness” of NPs and engineer them into useful functionalities for more effective therapeutic intervention and drug delivery applications in nanomedicine. To further enable discovery, we could now perhaps relook at certain natural phenomena from an opportunist’s perspective to adopt new innovative approaches in improving medicine instead of considering them as undesirable side effects

ASSOCIATED CONTENT Supporting Information Available. Additional data on the quantification of Dox and Ce6 loaded on NR-HS-Ce6-Dox is available as Supporting Information.

AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 2 Grant MOE2014-T2-2-147 (WBS 397-000-226-112).

ACKNOWLEDGMENT Funding was from the MOE AcRF Tier 2 Grant MOE2014-T2-2-147 (WBS 397-000-226-112).

ABBREVIATIONS AI, aggregation index; APF, 3’-(p-aminophenyl) fluorescein; Ce6, Chlorin e6; CLSM, confocal laser scanning microscopy; CT, chemotherapy; CTAB, cetyltrimethylammonium bromide; DH, hydrodynamic diameter; Dox, Doxorubicin; EPR, enhanced permeability and retention; FBS, fetal bovine serum; HS, human serum; LSPR, longitudinal surface plasmon resonance; NIR, near infrared; NP, nanoparticle; NR, gold nanorods; OSCC, oral squamous cell carcinoma; PDT, photodynamic therapy; PhB, phosphate buffer; PTT, photothermal therapy; ROS, reactive oxygen species; TEM, transmission electron microscopy.

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GRAPHICAL ABSTRACT FOR TOC

Schematic showing (a) the preparation of NR-HS-Ce6-Dox with Ce6 and Dox co-loaded simultaneously onto NRs using the protein corona, (b) its cell uptake, and (c) laser irradiation at 665 nm to (d) heat the NRs for PTT, generate reactive oxygen species (ROS) from Ce6 for PDT, and (e) laser-triggered release Dox to intercalate with DNA in the nucleus for CTX. Synergistic effects of simultaneous trimodal PTT+PDT+CT was observed to result in greatly enhanced cell killing efficacy compared to performing dual modal or individual therapies on their own.


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Figure 1. Physical characterization of NR-HS-Ce6-Dox and its controls. NR-HS-Ce6-Dox possessed good colloidal stability as observed by its (A) the sharp LSPR peak in the UV-Vis absorbance spectrum, similar to as-synthesized NR-CTAB, and absence of aggregation under the TEM, as well as its (B) low aggregation index. The formation of the HS protein corona around NRs and co-loading of Ce6 and Dox resulted in (C) a flip in the zeta potential from the positively charged CTAB surface to the negatively charged protein corona, and (D) an increase in DH, similar to other NR-HS corona controls. 267x200mm (72 x 72 DPI)


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Figure 2. (A) Quantification of Dox loaded in NR-HS-Ce6-Dox and its controls by thermal release of Dox compared to the amount of Dox leaked from corona without heating. The thermally labile Ce6 was degraded from the heat and does not interfere with Dox quantification. (B) In quantifying Ce6, we observed that fluorescence of Ce6 at λex/λem = 405/665 nm increased with increasing concentration of free HS-Ce6 in solution (solid line). (B, inset: when excited at 405 nm, Dox exhibited negligible fluorescence while Ce6 exhibited a strong fluorescence peak at 665 nm). However, loading of Ce6 onto NR-HS-Ce6-Dox resulted in fluorescence quenching (dotted line). (C) A constant Ce6 fluorescence quenching of 46.4 ± 0.5 % was observed when Ce6 was loaded on NR-HS-Ce6 at 500 nM Ce6 and below compared to free Ce6. The amount of Ce6 loaded on NR-HS-Ce6 could thus be determined by measuring the fluorescence of NR-HS-Ce6-Dox directly and correcting for the constant Ce6 fluorescence quenching. 232x174mm (72 x 72 DPI)



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Figure 3. Effect of irradiation with a 665 nm laser on NR-HS-Ce6-Dox and its respective controls. (A) Significant photothermal heating accompanied by a temperature rise was observed in all samples with NRs. The temperature rise was significantly lower in the absence of NRs. (B) Enhanced levels of ROS relative to free Ce6+Dox alone was generated by NR-HS-Ce6-Dox, similar to NR-HS-Ce6 versus Ce6 alone. (C) The amount of laser-triggered Dox release by NR-HS-Ce6-Dox was comparable to NR-HS-Dox, although (D) NRHS-Ce6-Dox demonstrated improved release efficiency against the amount of Dox loaded compared to NRHS-Dox. 246x184mm (72 x 72 DPI)


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Figure 4. Uptake of Ce6 and Dox by Cal 27 OSCC cells as measured by fluorescence of these drugs in cells under flow cytometry. When dose concentration was fixed at 0.2 nM NRs (equivalently 16.8 nM Ce6 and 167 nM Dox), NR-HS-Ce6-Dox showed greater uptake of both (A) Ce6, and (B) Dox over time, compared to dosing the cells with free drugs (Ce6+Dox, Ce6 alone and Dox alone). With the dose duration fixed at 6 h and dose concentration varied, cell uptake of (C) Ce6, and (D) Dox also increased significantly with increasing NR-HS-Ce6-Dox concentration, but remained negligible with an equivalent increase in the concentrations of the free drugs. 62x46mm (300 x 300 DPI)



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Figure 5. Uptake of NRs probed by darkfield microscopy. Strong light scattering was observed in cells dosed with 0.2 nM NR-HS-Ce6-Dox and NR-HS for 6 h due to the intracellular presence of NRs, whereas negligible light scatterings signal was detected when cells were dosed with the free Ce6+Dox alone (at an equivalent concentration of 16.8 nM Ce6 and 167 nM Dox as that loaded on NR-HS) in the absence of NRs. All scale bars are 50 µm. 339x128mm (72 x 72 DPI)


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Figure 6. Localisation of Ce6, Dox and NRs in Cal 27 OSCC cells probed by CLSM after 6 h of dosing. (A) An XY optical section and 2 orthogonal cuts (XZ and YZ, z0 = 0 µm indicating the base of the cells) of a sample after dosing with NR-HS-Ce6-Dox, as well as (B) Z-axis profiling over the selected cell showed that Ce6, Dox, and NRs were successfully internalised by the cells when dosed with NR-HS-Ce6-Dox, with colocalisation of all 3 active components within the cell cytoplasm observed. The NRs seemed to aggregate and localize near the endomembrane system surrounding the cell nucleus. (C) Comparing fluorescence intensities across different samples within a single optical slice (z = 1.2 µm), intracellular Ce6 and Dox fluorescence was much greater when dosed with NR-HS-Ce6-Dox than when dosed with free Ce6+Dox, which agreed with flow cytometry results. All scale bars are 20 µm. 24x22mm (300 x 300 DPI)



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Figure 7. Cell viability of Cal 27 cells when dosed with increasing concentrations of NR-HS-Ce6-Dox and different dual mode and single mode therapy controls with each axes showing the equivalent amount of drugs loaded and dosed to the cells. (A) Following irradiation with a 665 nm laser, trimodal PDT+PTT+CTX with NR-HS-Ce6-Dox (purple) achieved near complete cell kill of 98.7% with only 15 pM NR-HS-Ce6-Dox, while dual modal PTT+PDT by NR-HS-Ce6 (green), PTT+CTX by NR-HS-Dox (blue) and PDT+CTX by free Ce6+Dox (pink) achieved significantly less cell kill at the same equivalent concentrations. Individual therapies by NR-HS (PTT), free Ce6 (PDT) and Dox (CTX) were even less effective, with insignificant cell kill at the same equivalent concentrations. (B) In the absence of laser irradiation, cell viability remains high (>86%) for all the compounds across all concentrations used, indicating that NR-HS-Ce6-Dox and its various controls were non-toxic on their own, and that the trimodal therapy by NR-HS-Ce6-Dox is highly controllable by light. 20x15mm (300 x 300 DPI)


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Schematic showing (a) the preparation of NR-HS-Ce6-Dox with Ce6 and Dox co-loaded simultaneously onto NRs using the protein corona, (b) its cell uptake, and (c) laser irradiation at 665 nm to (d) heat the NRs for PTT, generate reactive oxygen species (ROS) from Ce6 for PDT, and (e) laser-triggered release Dox to intercalate with DNA in the nucleus for CTX. Synergistic effects of simultaneous trimodal PTT+PDT+CT was observed to result in greatly enhanced cell killing efficacy compared to performing dual modal or individual therapies on their own. 327x214mm (72 x 72 DPI)


Protein Corona around Gold Nanorods as a Drug Carrier for

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