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Carboplatin-Loaded, Raman-Encoded Chitosan-Coated Silver Nanotriangles as Multimodal Traceable Nanotherapeutic Delivery Systems and pH Reporters inside Human Ovarian Cancer Cells Monica Potara, Timea Nagy-Simon, Ana Maria Craciun, Sorina Suarasan, Emilia Licarete, Florica Imre-Lucaci, and Simion Astilean ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10075 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 5, 2017

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ACS Applied Materials & Interfaces

Carboplatin-Loaded, Raman-Encoded ChitosanCoated Silver Nanotriangles as Multimodal Traceable Nanotherapeutic Delivery Systems and pH Reporters inside Human Ovarian Cancer Cells Monica Potara*a, Timea Nagy-Simona, Ana Maria Craciuna, Sorina Suarasana, Emilia Licareteb, Florica Imre-Lucacic and Simion Astilean*a,d a

Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in Bio-Nano-Sciences, Babes-Bolyai University, T. Laurian Str. 42, 400271 Cluj-Napoca, Romania

b

Molecular Biology Center, Interdisciplinary Research Institute in Bio-Nano-Sciences,BabesBolyai University, T Laurian Str. 42, 400271 Cluj-Napoca, Romania c

Physico-Chemical Analysis Center, Interdisciplinary Research Institute in Bio-Nano-

Sciences, Babes-Bolyai University, T Laurian Str. 42, 400271 Cluj-Napoca, Romania d

Department of Biomolecular Physics, Faculty of Physics, Babes-Bolyai University, M Kogalniceanu Str. 1, 400084 Cluj-Napoca, Romania

KEYWORDS: NIR traceable drug nanocarriers; pH nanosensor; SERS imaging; two-photon excited FLIM imaging; carboplatin; ovarian cancer; theranostics 1 ACS Paragon Plus Environment


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ABSTRACT. Ovarian cancer is a common cause of cancer death in women and is associated with the highest mortality rates of all gynaecological malignancies. Carboplatin (CBP) is the most used cytotoxic agent in the treatment of ovarian cancer. Herein, we design and assess a CBP nanotherapeutic delivery system which allows combinatorial functionalities of chemotherapy, pH sensing and multimodal traceable properties inside live NIH:OVCAR-3 ovarian cancer cells. In our design, a pH sensitive Raman reporter, 4-mercaptobenzoic acid (4MBA) is anchored onto the surface of chitosan-coated silver nanotriangles (chit-AgNTs) to generate a robust surface-enhanced Raman scattering (SERS) traceable system. To endow this nanoplatform with chemotherapeutic abilities, carboplatin (CBP) is then loaded to 4MBA labeled chit-AgNTs (4MBA-chit-AgNTs) core under alkaline conditions. The uptake and tracking potential of CBP-4MBA-chit-AgNTs at different Z-depths inside live ovarian cancer cells is evaluated by dark-field and Differential Interference Contrast (DIC) microscopy. The ability of CBP-4MBA-chit-AgNTs to operate as near-infrared (NIR) responsive contrast agents is validated using two non-invasive techniques, two-photon excited Fluorescence Lifetime Imaging Microscopy (FLIM) and Confocal Raman Microscopy (CRM). The most informative data about the precise localization of nanocarriers inside cells correlated with intracellular pH sensing is provided by multivariate analysis of Raman spectra collected by scanning CRM. The in vitro cell proliferation assay clearly shows the effectiveness of the prepared nanocarriers in inhibiting the growth of NIH:OVCAR-3 cancer cells. We anticipate that this class of nanocarriers holds great promise for application in image-guided ovarian cancer chemotherapy.

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INTRODUCTION Ovarian cancer is a common cause of cancer death in women and is associated with the highest mortality rates of all gynaecological malignancies.1,2 Currently, the first-line ovarian cancer therapy consists of surgery followed by systemic chemotherapy to eliminate the residual disease. Despite of some progresses achieved in the treatment of the ovarian cancer by introducing the platinum-based chemotherapy, followed by the addition of taxanes, the efficiency of conventional chemotherapy in the management of ovarian cancer is still limited by several factors, including drug resistance mechanisms, the low drug levels at the diseased site and non-specific toxicity.2,3 The interdependence between the inadequate treatment strategies, the lack of effective screening methods and the asymptomatic nature of the disease determines a dismal prognosis in ovarian cancer treatment. Taking into consideration that the asymptomatic nature of ovarian cancer cannot be modified, the only available possibility of lowering the mortality rates related to this type of cancer and improving the quality of life is to develop new strategies for early diagnosis and more specific treatment. As an emerging approach, the development of smart, traceable drug nanocarriers which integrate optical imaging and improved therapeutic abilities into a single theranostic nanoparticle heralds a new paradigm in the fight against cancer.4–6 Owing to their biocompatibility, reduced size, feasible surface modification and unique optical properties, plasmonic nanoparticles (PNPs) appear as one of the most promising building blocks for fabrication of smart nanocarriers able to merge the controlled delivery and release of drug to the tumor sites with simultaneous drug localization and real-time monitoring of the therapeutic efficiency. Indeed, besides the generous surface for drug loading, the localized surface plasmon resonance (LSPR) of PNPs can be exploited for imaging and tracking of nanocarriers inside cells and tumors via a number of microscopic and microspectroscopic techniques.7,8 For instance, the size and shape



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dependent light scattering properties of PNPs facilitate real-time imaging of nanocarriers distribution inside cells through dark-field microscopy at a single-nanoparticle level.9 In addition to light scattering abilities, PNPs are known to be capable of generating their own intrinsic photoluminescence (PL) signal under excitation with light, which enables their tracking by fluorescence imaging without the need of additional fluorescent labels. In particular, imaging techniques based on the non-linear optical effect of two-photon (TP) excited fluorescence has grown in interest in recent years due to improved 3D spatial resolution owing to excitation with intense and extremely short laser pulses, as well as deep penetrability and less photodamage of living organisms due to excitation in near-infrared (NIR) region.10,11 PNPs are capable of generating strong PL signals under TP excitation, and, more significantly, the lifetime of their PL was found to be extremely short (few ps)12 and does not interfere with the cellular autofluorescence (few ns) being therefore easily differentiated within biological samples. Owing to all these, PNPs can be used as contrast agents in TP excited Fluorescence Lifetime Imaging Microscopy (FLIM), an investigation technique rapidly growing in interest nowadays. Among various techniques, surface-enhanced Raman spectroscopy (SERS) has emerged as a genuine tool to determine the accumulation of the nanocarriers at the tumor site, to monitor the release of the drug in a nondestructive and label-free manner and also to perform the diagnostic with ultrahigh sensitivity, specificity and multiplexing capability.13–15 Due to the weak Raman spectrum of water which does not interfere with the spectra of the investigated species and the possibility to perform the Raman measurements under NIR excitation this spectroscopic technique is readily applicable in vivo.16 Typically, a SERS traceable drug nanocarrier consists of a component for noninvasive imaging (a PNPs decorated with a Raman reporter molecule), an anticancer drug and a polymer coating to impart nanoparticle stability, biocompatibility and provide functional 4 ACS Paragon Plus Environment

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groups for bioconjugation. Owning to this particular architecture, the fabricated theranostic carriers operate as SERS imaging probes, whilst simultaneously modulate the delivery and drug release for cancer chemotherapy. Moreover, by selecting a pH sensitive molecule as Raman reporter, both the nanoparticle distribution and intracellular pH of the medium surrounding the particles can be successfully investigated. Despite the already demonstrated potential of SERS as diagnostic imaging tool, the fabrication of SERS traceable drug delivery systems is scarcely addressed. For instance, Zong et al. have used Raman-encoded Au@Ag nanorods as SERS active core and mesoporous silica as drug containing shell to fabricate a SERS traceable and stimuli-responsive nanocarriers.17 Later, Fang et al. have designed mesoporous silica nanoparticles as pH-controllable drug carriers that allow simultaneous SERS/fluorescence imaging and chemotherapy of specific cancer cells.18 Afterwards, Ramanencoded gold nanocages encapsulated into porous SiO2 shell, loaded with doxorubicin and functionalized with a Tat peptide were proposed by Zhang and co-workers as promising candidates for developing SERS-based theranostic agents.19 Recently, small gold nanoparticles with SERS reporters were distributed around silica nanoparticles and then loaded with doxorubicin leading to a SERS traceable plasmonic-based drug delivery system.20 Most of the reported theranostic nanoparticle constructs combining SERS imaging and chemotherapy rely on the use of doxorubicin as an anticancer drug. However, to date, almost 50% of all the clinically used chemotherapeutic agents are based on platinum-containing drugs.21,22 Moreover, the first-line ovarian cancer chemotherapy requires carboplatin (CBP), a clinical relevant platinum-based drug that is listed on the World Health Organization’s Model List of Essential Medicines.23 However, the chemotherapeutic performance of CBP is largely limited by the emergence of carboplatin-resistant tumor cells and side effects.24 A few years ago, we developed a new class of SERS imaging probes based on chitosancoated silver nanotriangles (chit-AgNTs) labeled with para-aminothiophenol Raman reporter 5 ACS Paragon Plus Environment


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molecules and demonstrated their ability to operate as biocompatible, highly sensitive platforms for noninvasive detection and imaging of lung cancer cells under a wide window of excitation wavelengths, ranging from visible to NIR region.25 In another study we have evaluated the proof of concept for theranostic applications through their combined ability to target and detect ovarian cancer cells with photothermal properties.26 Taking into consideration the multiwavelength-activatable properties of chit-AgNTs and the essential role of CBP in ovarian cancer chemotherapy, herein we set out to generate a biocompatible material endowed with multiple functionalities of chemotherapeutic effect combined with non-invasive sensing of intracellular pH and traceable properties inside live NIH:OVCAR-3 human epithelial ovarian cancer cells. Towards this aim, a pH sensitive Raman reporter, 4-mercaptobenzoic acid (4MBA) is anchored onto the surface of chit-AgNTs through silver–sulfur interactions to generate a robust and stable pH dependent Raman signal. To endow this nanoplatform with chemotherapeutic abilities, CBP was loaded to 4MBA labeled chit-AgNTs (4MBA-chit-AgNTs) core through electrostatic interaction between the positively charged drug molecules and COO- group of 4MBA in basic environment with simultaneous encapsulation of drug in the chitosan coating. Within this nanoarhitecture, the fabricated theranostic carriers facilitate their accurate localization inside NIH:OVCAR-3 cells at a 3D level through combined dark-field, Differential Interference Contrast (DIC) and TP excited FLIM imaging, whilst simultaneously modulate the delivery of CBP for ovarian cancer chemotherapy. Moreover, the Raman reporter molecule provides pH-dependent SERS features which allow the detection of the intracellular pH with precise localization of nanocarriers inside cells via multivariate analysis of Raman spectra collected by scanning Confocal Raman Microscopy (CRM). To our best knowledge, this is the first report integrating the chemotherapeutic abilities of CBP with SERS sensing of intracellular pH and multimodal traceable properties into a single theranostic nanoparticle construct. We anticipate 6 ACS Paragon Plus Environment

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that this class of nanocarriers holds great promise for application in ovarian cancer theranostics. MATERIALS AND METHODS Materials. Chitosan flakes (high molecular weight, > 75% deacetylated), 4mercaptobenzoic acid (4MBA) and carboplatin (CBP) were purchased from Aldrich. Silver nitrate (AgNO3), ascorbic acid, sodium borohydride (NaBH4), glacial acetic acid, trisodium citrate (C6H5Na3O7·2H2O), sodium hydroxide (NaOH) and hydrochloric acid were obtained from Merck. Platinum atomic spectroscopy standard (H2PtCl6) was purchased from LGC Standards GmbH. Glacial acetic acid was diluted to a 1% aqueous solution before use. Chitosan was dissolved in 1% acetic acid solution. All chemicals were used without further purification and the solutions were prepared using ultrapure water with a resistivity of at least 18 MΩcm. Nanoparticles preparation, SERS labeling and carboplatin loading. Chitosan-coated silver nanotriangles (chit-AgNTs) were prepared via seed-mediated growth according to our method described previously.

27

Briefly, the silver seeds were prepared through reduction

reaction of silver nitrate with sodium borohydride at 0 °C. In a typical procedure for the growth of silver nanotriangles, aqueous solution of seeds (200 µL), trisodium citrate (25 mM, 200 µL), ascorbic acid (0.1 M, 50 µL) and chitosan (2 mg/mL, 10 mL) were pre-combined and brought to 35 ± 2 °C. To this mixture, AgNO3 (0.01 M, 300 µL) was added dropwise under continuous stirring. Within these parameters the final product consists mainly on triangular and truncated-triangular silver nanoparticles with an average edge length of 50 nm. The colloidal solution was purified by centrifugation and re-suspended in ultrapure water. For SERS labeling, 990 µL colloidal suspension of chit-AgNTs were incubated at room temperature for several hours with 10 µL solution of 4MBA. The obtained 4MBA encoded chit-AgNTs (4MBA-chit-AgNTs) were centrifuged and re-suspended in ultrapure water. 7 ACS Paragon Plus Environment


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Carboplatin-loaded

4MBA-chit-AgNTs

(CBP-4MBA-chit-AgNTs) were

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prepared by

incubating the 4MBA-chit-AgNTs with CBP at pH 4. Then the pH of the mixture was changed to 8.4 by adding 1 M NaOH. The particles were subsequently washed to remove the free drug. The UV-vis-NIR spectrum of the colloidal particles was recorded at each step, to monitor the formation of CBP-4MBA-chit-AgNTs. The encapsulation was estimated using the following equation: Encapsulation efficiency % = where Mtotal

incubated CBP

M ! × 100% M" #$ !

represents the amount of CBP (expressed in micrograms) used for

loading in the initial colloidal solution and Mencapsulated

CBP

represents the amount of CBP

(expressed in micrograms) loaded/encapsulated onto the nanoparticles. The amount of CBP was measured by atomic absorption spectroscopy. Cell culture and incubation conditions. Human ovarian adenocarcinoma cells (NIH:OVCAR-3, Cell Line Service, Germany) were grown in RPMI-1640 culture medium (Sigma-Aldrich, Germany), supplemented with 2 mM L-glutamine, Penicillin/Streptomycin 100 U/mL (Sigma-Aldrich), 20% Fetal Calf Serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 0.01mg/mL bovine insulin (Sigma-Aldrich) and incubated in a humidified incubator (37 °C, 5% CO2). For dark field microscopy and FLIM assay, cells were grown on Ibidi µ-Dish (50 mm, low wall, ibiTreat coating). For Raman imaging cells were seeded on Ibidi µ-Dish with glass bottom (35 mm, high wall, uncoated). After 24 h of cultivation, the culture medium was replaced with a culture medium containing 4MBA-chitAgNTs or CBP-4MBA-chit-AgNTs and incubated for another 24 h. Control samples were prepared containing untreated cells. Cell proliferation assay. To determine the effects of the obtained theranostic nanoparticles on cell proliferation, NIH:OVCAR-3 cells (1,000 cells/well) were seeded in a 96-well plate 8 ACS Paragon Plus Environment

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for 24 h. Different concentrations of free CBP (ranging from 20 to 125 µM), 4MBA-chitAgNTs (0.6-3.75 µg/mL) and CBP-4MBA-chit-AgNTs with equivalent CBP and AgNTs concentrations were incubated for 24 h and evaluated in triplicate. Cells cultured in medium were used as controls. The proliferative activity of the cells following treatment administration was analyzed with an immunoassay (Cell Proliferation ELISA, BrdU (colorimetric); Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol. This method is based on the incorporation of the pyridine analogue bromo deoxyuridine (BrdU), instead of thymidine, into the DNA of proliferating cells.28 NIH:OVCAR-3 cells were incubated with a BrdU solution for 24 h, and the culture medium was then completely removed from each well. Following this step, the cells were fixed, and the DNA was denatured with FixDenat buffer provided in the kit. An anti-BrdU monoclonal antibody conjugated with peroxidase (anti-BrdU-POD, Cat. No. 11647229001, Roche Applied Science, diluted 1:100 part of Cell Proliferation ELISA, BrdU kit) was added in each well in order to detect the incorporated BrdU in the newly synthesized cellular DNA. The antibody was removed after 1 h of incubation at room temperature, and the cells were then washed three times with PBS. Next, a peroxidase substrate was added in each well, and the immune complexes were detected by measuring the absorbance of the reaction product at 450 nm with a reference wavelength of 655 nm. Experimental measurements. Optical extinction spectra of nanoparticles were measured in a 2 mm quartz cell using a Jasco V-670 spectrophotometer with 1 nm spectral resolution. The zeta potential of colloidal particles dispersed in ultrapure water was recorded at 25 °C using a Malvern Zetasizer Nano ZS-90 instrument. The concentration of silver in the colloidal suspension (µg/mL) was determined by atomic absorption spectroscopy (Avanta PM, GBCAustralia).The analysis was performed using Ag atomic spectroscopy standard1000 mg/L acidic solution (AgNO3 in 0.5 M HNO3) from LGC Standards GmbH (Wesel, Germany). For 9 ACS Paragon Plus Environment


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measurement, a hollow cathode Ag lamp (Model P851 HCL) was used. The concentration of CBP (µg/mL) was measured with the same atomic absorption spectrometer using a hollow cathode Pt lamp (Model P840 HCL) from Photron PTY Ltd (Victoria, Australia). The instrument operated at a lamp current of 4 mA, wavelength of 328.1 nm and slit width of 0.2 nm for silver measurements and at a lamp current of 14 mA, wavelength of 266 nm, and slit width of 0.5 nm for Pt analysis. A linear calibration curve was generated before each measurement using the “linear least squares” built-in method, an excellent correlation being obtained (R2 = 1.0) for the 0.25 to 4.0 mg/L domain in the case of silver determination and (r = 0.9990) for the 0.5 to 10 µg/mL domain in the case of Pt measurement. Each sample was measured in triplicate. Dark field and DIC microscopy was performed on cells cultured in 50 mm Ibidi µ-Dish using an inverted Zeiss Axio Observer Z1 microscope. For dark field imaging, a 100 W halogen lamp was used for illumination using a high numerical immersion dark field condenser (NA = 1.4). The scattered light was collected by a LD Plan-Neofluar 20× objective (NA = 0.4, Zeiss) or a Plan-Apochromat oil immersion 63× objective (NA = 0.7-1.4, Zeiss). DIC imaging was performed using a long distance condenser (NA = 0.8) with specific DIC prisms for illumination, and collection was performed with a Plan-Apochromat/DIC 63× oil immersion objective (NA = 1.4, Zeiss). Images were acquired using an AxioCamIcc Rev.4 CCD camera (1.4 megapixels, Zeiss) and processed by the ZEN 2012 software. TP excited FLIM assays were performed on cells cultured in 50 mm Ibidi µ-Dish and incubated with 4MBA-chit-AgNTs and CBP-4MBA-chit-AgNTs using a MicroTime200 time-resolved confocal fluorescence microscope system (PicoQuant) based on an inverted microscope (IX 71, Olympus) equipped with a PLAN Achromat 40×/NA = 0.65 objective. The excitation beam was provided by a tunable Coherent Mira 900 Titanium: Sapphire pulsed laser operating at 800 nm, at a power of 40 mW (0.8 mW on sample) and 76 MHz. For FLIM 10 ACS Paragon Plus Environment

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image acquisition the system uses a piezo x-y-scanning table and a PiFoc z-piezo actuator for microscope objective. The signal collected through the objective was spectrally filtered by a FF01-750SP (Semrock, USA) emission filter, before being focused on a PDM Single Photon Avalanche Diode (SPAD) from MPD. The detector signals were processed by a PicoHarp 300 Time-Correlated Single Photon Counting (TCSPC) data acquisition unit, from PicoQuant. Data were recorded and analyzed using the SymPhoTime software from PicoQuant. TP exited PL spectrum from selected region in the TPE - FLIM images was obtained with a SR-163 spectrograph equipped with a Newton 970 EMCCD camera from Andor Technology coupled to an exit port of the main optical unit of MicroTime200 through a 50 µm optical fiber. The integration time used for the acquisition of the TP excited PL spectrum was 30 s. Bright field and dark field images of the cells scanned by FLIM were obtained on the same microscope using Olympus IX-2 LW UCD (NA = 0.55) condenser and U-DCW cardioid immersion dark field condenser (NA = 1.40-1.20), respectively. Illumination was provided by a 100 W halogen lamp and the images were acquired with an Olympus CAM-XC30 digital camera with active Peltier cooling. Raman and SERS measurements were performed with a confocal Raman microscope (CRM alpha 300R from WITec GmbH, Germany). The cells grown on 35 mm glass bottom Ibidi µDish were mounted on a piezoelectric scanning stage. The spectroscopic imaging of the cells was performed using two laser excitation lines (532 nm and 785 nm) directed thorough a Wplan Apochromat 63× water immersion objective (NA = 1, WD = 2.1 mm, Zeiss). The Raman backscattered light collected through the objective was passed through a holographic edge filter, before being focused into a multimode optical fiber of 100 µm diameter which provides the optical pinhole for confocal measurement. The light emerging from the output optical fiber was analyzed by ultrahigh throughput spectrometer equipped with back-illuminated deep-depletion 1024 × 128 pixel CCD camera operating at -60 °C. The cellular imaging was 11 ACS Paragon Plus Environment


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conducted by choosing an integration time of 0.5 s and laser power of 15 mW under 532 nm excitation and 17 mW under 785 nm excitation for each spectrum. The reference SERS spectra of 4MBA-chit-AgNTs and CBP-4MBA-chit-AgNTs were recorded directly from the colloidal solutions through a 20× objective (NA = 0.4), employing for excitation the 532 nm wavelength (∼3 mW power on sample). The integration time was set at 10 s per spectrum. The WITec Project Four Plus software was used for spectral analysis and image processing. The SERS spectra in solution at NIR excitation were acquired with a portable Raman spectroscope (Raman Systems R3000 CN) equipped with a 785 nm diode laser coupled to a 200 µm optical fiber. The laser power was set to 200 mW and the spectra were measured with an integration time of 30 s.

RESULTS AND DISCUSSION Preparation and characterization of CBP-4MBA-chit-AgNTs. The platinum-containing drugs prevail in the ovarian cancer chemotherapy, with CBP being the most used anticancer drug, either as a single chemotherapeutic agent or in combination with other cytotoxic drugs.21–24 Although CBP have gained success in the management of ovarian cancer chemotherapy, there are still concerns associated with its clinical use, the development of drug-resistant tumor cells and side effects being the most important. In an attempt to improve the chemotherapeutic outcomes in ovarian cancer, several efforts have been recently invested in encapsulating the drug in smart, traceable nanocarriers that can preferentially deliver therapeutic concentration of CBP to the tumor sites with minimum side effects, whilst simultaneously allow real-time monitoring of therapeutic effect. Non-covalent conjugation (e.g. encapsulation, electrostatic interactions or using the loading space provided by the polymeric matrix) represents a low cost, easy strategy which allows the direct use of unmodified drug. To date, only one research group has reported the development of traceable 12 ACS Paragon Plus Environment

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CBP nanocarriers. Specifically, Ren et al. have designed a sensitive nanotheranostic platform for in vivo non-small-cell lung cancer recognition and treatment.29 The here reported theranostic agents are based on liposomes loaded with CBP and paclitaxel, conjugated with c(RGDyK), a targeted peptide that is recognized by αvβ3 integrin receptor and labeled with Gd-DTPA-BMA contrast agent. The designed theranostic carriers were proved to target and inhibit tumor progression, being readily detectable by Magnetic Resonance Imaging (MRI) and confocal microscopy. In our design, plasmonic nanoparticles were chosen as drug nanocarries because this class of nanoparticles offers the possibility to combine multiple imaging and therapeutic functionalities into a single nano-construct. For instance, the efficient light-to-heat conversion effect of PNPs not only that can induce tumor hyperthermia upon irradiation, but in combination with a drug delivery system the generated heat can facilitate the release of the drug payload when incorporated into thermo-responsive coating material, which can result in an improved or synergistic therapeutic efficiency.30 Another benefit of using PNPs in cancer therapy applications relies on the metal-enhanced singlet oxygen generation by photosensitizers conjugated to PNPs in view of improving the efficiency of photodynamic therapy.31 Moreover, it has been repeatedly shown that PNPs can act as radiosensitisers considerably enhancing the radiation dose at the tumor site therefore improve the efficacy of radiotherapy. 32 Due to the benefit of its unique biological and physicochemical characteristics such as excellent biocompatibility, biodegradability, nontoxicity, noncarcinogenic, nonimmunogenic, and antibacterial effect combined with pH dependent behavior, we selected chitosan biopolymer as the drug containing shell to fabricate a multimodal traceable and pH-sensitive nanocarriers.33 The preparation procedure of CBP-4MBA-chit-AgNTs is presented in Figure 1. In the first step, 4MBA Raman reporter molecules were attached onto Chit-AgNTs through a diffusion 13 ACS Paragon Plus Environment


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process at acidic pH environment to construct a robust and reproducible SERS traceable system. To endow this nanoplatform with chemotherapeutic abilities, CBP was then loaded to 4MBA-chit-AgNTs under alkaline conditions via cooperation of two mechanisms of drug entrapment in chitosan matrix. In our design the loading of CBP onto 4MBA-chit-AgNTs was realized by exploiting both the strong pH dependent swelling behavior of chitosan and the electrostatic interaction between 4MBA and drug molecules. It is known that, due to the presence of amino groups on its chain, chitosan exhibits a reversible solubility depending on pH.34,35 In particular, the positively charged amino groups in chitosan under acidic solution lead to a soluble state of chitosan, while their neutralization in basic environment transforms the biopolymer in a gel form, a state that allows the encapsulation of molecules. On the other hand, it was shown that the COOH end group of 4MBA is ionized to COO- group in basic environment, which subsequently provides attachment sites for the protonated NH4+ groups of CBP.36,37 In our system, the dual drug loading mechanism relies on the adsorption of CBP on 4MBA-chit-AgNTs core through electrostatic interaction between the positively charged drug molecules and COO- group of 4MBA in basic environment with simultaneous encapsulation of drug in the gelled chitosan coating.

Figure 1. Schematic illustration of the preparation procedure of CBP-4MBA-chit-AgNTs


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Several measurements were employed to monitor the formation of CBP-4MBA-chitAgNTs. Figure 2 illustrates the UV-vis-NIR extinction spectra of colloidal suspensions recorded before and after Raman encoding and drug loading. Relative to extinction spectrum of chit-AgNTs (curve a) which features the characteristic LSPR bands of silver nanotriangles at 594 nm (in-plane dipolar resonance), 401 nm (out-of-plane dipolar resonance), 451 nm (inplane quadrupolar resonance) and 339 nm (out-of-plane quadrupolar resonance), the plasmonic bands of 4MBA-chit-AgNTs (curve b) shifted to longer wavelengths (13 nm for the in-plane dipolar resonance) due to the local increase of the refractive index after the adsorption of 4MBA on silver nanoparticles. A red shift of 3 nm was observed for the inplane dipolar resonance after subsequent incubation of 4MBA-chit-AgNTs with CBP indicating the loading of drug molecules onto 4MBA-chit-AgNTs (Figure 2, curve c). Notably, chitosan shell acts as a biocompatible stabilizing agent, allowing the drug entrapment and the diffusion of reporter molecules through the polymeric matrix, while preserving the unique optical characteristics of AgNTs after the formation of the CBP-4MBAchit-AgNTs delivery system.

Figure 2. Normalized UV-vis-NIR extinction spectra of the colloidal nanoparticle solutions (a) before tagging with MBA (chit-AgNTs); (b) after tagging MBA (4MBA-chit-AgNTs); (c) 15 ACS Paragon Plus Environment


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after tagging MBA and loading with CBP (CBP-4MBA-chit-AgNTs). The inset shows a zoomed image of the dipolar plasmon resonances of the extinction spectra. The successful loading of CBP was further proved by atomic absorption spectroscopy. To infer the role of Raman reporter molecules on drug loading, a control sample without 4MBA (CBP-chit-AgNTs) was also prepared and tested in the same way. Compared with the encapsulation efficiency of CBP-chit-AgNTs (10.8%), CBP-4MBA-chit-AgNTs exhibited a significant enhancement of encapsulation efficiency (38%), clearly indicating that 4MBA provide an adjusting mechanism in the entrapment of CBP molecules in chitosan matrix. This result is consistent with a previous report which showed that chitosan biopolymer alone demonstrates a modest encapsulation efficiency of CBP.38 A higher drug content of 4MBAchit-AgNTs may lead to the increase of the dose delivered, thus resulting in an improved therapeutic effect.

pH-responsive SERS properties in solution. The approach of tagging the chit-AgNTs with 4MBA offers multiple advantages in view of constructing a drug loaded SERS traceable theranostic nanoplatform. First of all, 4MBA serves as Raman reporter molecule, providing the possibility to the theranostic agents to be tracked inside cells by its characteristic and intense Raman fingerprint. On the other hand, as shown before, conjugation with 4MBA considerably increased the loading efficiency of chit-AgNTs with CBP drug providing additional binding sites through electrostatic interactions in alkaline conditions when encapsulation of the drug molecules in the chitosan shell takes place. Moreover, 4MBA provides pH-dependent SERS features which offers particular benefits in monitoring the nanoparticles localization inside specific cellular compartments and information about the intravesicular pH which can trigger the drug release.37 Therefore, we performed step-by-step SERS measurements correlated with zeta-potential measurements during the preparation 16 ACS Paragon Plus Environment

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process of the nanoparticles and analyzed the pH-dependent SERS properties of 4MBA. The collected spectra are presented in Figure 3 together with Table 1 summarizing the Raman peak positions and the zeta-potentials of the nanoparticles at specific pH values. As one can see from Figure 3, the most intense peaks of 4MBA are located at 1073 and 1585 cm-1, assigned to the aromatic-ring vibrations (ring breathing and axial deformation modes, respectively).39 These peaks are pH independent, therefore are usually used as reference signal. The pH-sensitive vibration modes are related to the protonation or deprotonation to the carboxyl group of 4MBA upon modification of the pH and they are observed at around 1390 cm-1 (COO- stretching) and 1690 cm-1 (C=O stretching), which are well-documented in literature.40,41 As also observed by others, when the pH decreases to acidic values the intensity of the 1690 cm-1 band increases, whereas the increase of pH towards neutral and alkaline values causes the deprotonation of the COOH group resulting in an increase in the intensity of the COO- stretching mode along with its shifting from ca. 1370 to ca. 1425 cm-1.

Figure 3. pH-dependent SERS spectra of nanoparticles at different preparation steps (a-e). Excitation: 532 nm. 17 ACS Paragon Plus Environment


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TABLE 1. Peak positions of the pH-sensitive Raman bands of 4MBA and corresponding zeta-potentials at specific pH values pH

Zeta-potential

ν (COO- ) position

ν (C=O) position

(mV)

(cm-1)

(cm-1)

Sample 4MBA-chit-AgNT

4.6

58

1365

1690

4MBA-chit-AgNT

8.4

-27.6

1420

-

4MBA-chit-AgNT + CBP

4.6

58.9

1385

1695

CBP-4MBA-chit-AgNT

7.4

-17.8

1415

-

7.4

-

1426

-

4MBA-chit-AgNT (in cellular medium)

In particular, 4MBA-chit-AgNTs before loading with CBP at pH 4 presents clearly both bands at 1365 and 1690 cm-1 (spectrum a, Figure 3), meanwhile after changing the pH to alkaline (pH 8.4) causes the disappearance of the C=O stretching mode and shifting of the peak attributed to the COO- stretching to 1420 cm-1 featuring also a small shoulder at smaller wavenumbers (spectrum b, Figure 3). As a consequence of deprotonation of chitosan in basic conditions, the zeta-potential was also modified from +58 to -27.6 mV. After incubation with CBP at acidic pH, the zeta-potential of 4MBA-chit-AgNTs was not significantly modified suggesting that no drug molecules were attached at the outer chitosan shell of the nanoparticles. Changing the pH of the colloidal solution to alkaline in order to encapsulate the drug in the chitosan coating when gelation of chitosan takes place results again the loss of the peak at 1695 cm-1 and shift of COO- band from 1385 to 1420 cm-1 (spectrum d, Figure 3). This result indicates that loading of 4MBA-chit-AgNTs with CBP drug did not affect the pHsensitivity of the conjugated 4MBA reporter molecules. As expected, the zeta-potential of CBP-4MBA-chit-AgNTs dropped to negative values (-17.8 mV) in alkaline conditions. Next, 18 ACS Paragon Plus Environment

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the pH sensitivity of the nanoparticles was also tested in cell culture medium at pH 7.4, presenting only the COO- band shifted to 1426 cm-1 (spectrum e, Figure 3). As the ultimate goal of any image-guided drug delivery system targets the in vivo applicability, the pH sensitivity of the CBP-4MBA-chit-AgNTs was also investigated at NIR laser line excitation. NIR light is known to exhibit maximum penetration into biological tissue, therefore can also reach deeply situated tumors. SERS spectra of CBP-4MBA-chitAgNTs at acidic, neutral and basic pH values at 785 nm excitation are presented in Figure S1 in the Supporting Information. Similarly to the spectra measured at 532 nm excitation, the pH-sensitive spectral features of 4MBA could be also detected at NIR excitation. For instance, at pH of 4.6 both bands characteristic to the COOH and C=O stretching vibrations are present at 1395 and 1700 cm-1, respectively. Increasing the pH to 7.4 resulted in the disappearance of the C=O vibration peak, while the peak attributed to the COOH stretching shifted to 1420 cm-1, which further shifted to 1428 cm-1 at pH 8.4 suggesting the deprotonation of the COOH group.

Nanoparticle uptake studies by dark field and DIC microscopy. Owning to the surface plasmon resonances, silver nanoparticles are known to scatter light intensively; hence they can be visualized using dark field microscopy. This microscopic technique allows studying the internalization and distribution of nanoparticles in cells and also their interactions with cellular organelles.9 Since light scattering properties of plasmonic nanoparticles are dependent on their size, shape, aggregation as well as their surrounding medium, one can gain precious information about the state of nanoparticles and possible localization inside cells.42,43 As deviations of nanoparticles shapes from sphere to anisotropic forms significantly increase the scattering contribution to the total extinction, AgNTs are very adequate to be tracked by dark field imaging. Therefore, we employed this microscopic technique as the first evaluation tool 19 ACS Paragon Plus Environment


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of AgNTs internalization and distribution inside NIH:OVCAR-3 cells. Figure 4A shows a dark field image of control cells without nanoparticles presenting only a very weak intrinsic cellular scattering. On the contrary, cells incubated with both 4MBA-chit-AgNTs (Figure 4B) and CBP-4MBA-chit-AgNTs (Figure 4C) at a final nanoparticles concentration of 1µg/mL present a massive nanoparticles uptake represented by colored bright spots inside the cytoplasm of the cells. In both cases the nuclei can be seen as distinct dark object outlined by nanoparticles, suggesting that the nanoparticles were not able to penetrate the nuclear membrane. This is plausible as the size of the nanoparticles exceeds the pore size of the nuclear membrane. One can observe multicolor light scattering from internalized nanoparticles including blue, green orange and red colors, corresponding to the SPR of individual nanoparticles with different shapes and sizes. For instance, blue and green colors may be arising from mostly spherical nanoparticles which existed in a small number in the colloidal AgNTs solution. Orange-red colored spots, as majority of the nanoparticles found inside cells, correspond to the intact AgNTs owning LSPR centered at 594 nm as shown in the extinction spectrum in Figure 1, which presumably was further red-shifted after interacting with cellular components. These characteristic colors can be better visualized in Figure S2 A and B showing cells incubated at low nanoparticles concentrations (0.3 µg/mL), which resulted in a moderate nanoparticles uptake therefore their internalization as individual nanoparticles was dominant. Instead, at high nanoparticles concentration, larger nanoparticles aggregates are also visible as yellow-to-white spots, mostly gathered in the perinuclear region due to the very high local concentration of nanoparticles as a result of the massive uptake. Additional dark field images, presented in the Supporting Information file, focalized at different depth in the cell, show also nanoparticles at the surface of cell bound to the cell membrane (Figure S2 C and D for cells incubated with CBP-4MBA-chit-AgNTs and Figure S2 E and F for 4MBA-chit-AgNTs). It is worth mentioning that not all cells from a given cell 20 ACS Paragon Plus Environment

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culture present the same nanoparticle uptake and internalization pattern. As visible in the low magnification images from the Supporting Information (Figure S2 G and H for 4MBA-chitAgNTs and CBP-4MBA-chit-AgNTs, respectively), there are few cells with only a small amount of nanoparticles, meanwhile some cells display a massive nanoparticles internalization. This observation can be related to the different metabolism and varying cellular processes at different growth phases. The uptake mechanism of AgNTs includes the interaction between positively charged particles due to the presence of chitosan at their surface and negatively charged cell membrane.

Figure 4. Dark field images of control NIH:OCVAR-3 cells (A), incubated with 4MBA-chitAgNTs (B) and CBP-4MBA-chit-AgNTs (C) at a final nanoparticles concentration of 1 µg/mL. Thereafter, the time-dependent cellular uptake of nanoparticles by cells was also assessed by dark field imaging at 30 min, 2 h, 6 h, 14 h and 24 h incubation periods (Figure S3). Already after only 30 minutes of incubation, cells presented few internalized nanoparticles, however a large number of aggregated nanoparticles can be seen as bright large spots bound to the cell membrane. This pattern is maintained until 6 h incubation, however more and more nanoparticles can be observed inside cells, mostly internalized individually, in a nonaggregated form. Starting from 14 h incubation, massive nanoparticle internalization is 21 ACS Paragon Plus Environment


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visible, without large aggregates at the outer membrane. Contrarily, agglomeration of the nanoparticles inside cellular vesicles can be observed. Compared to 14 h incubation when nanoparticles are distributed in the whole cytoplasm, in the case of cells incubated for 24 h, nanoparticles are mostly gathered into the perinuclear region, probably in late endosomes and lysosomes. Taking into consideration that CBP after release has to reach the nucleus in order to exert its therapeutic effect, we consider that the perinuclear localization after 24 h incubation is the most advantageous in view of a better cytotoxic effect. Dark-field microscopy not only that allows the evaluation of nanoparticles uptake and localization by recording static images, but the intracellular movement of nanoparticles can be also captured in real-time, offering dynamic information about the intracellular transportation of nanocarriers. Movie 1 from SI shows a real-time video recording of two cells incubated with CBP-4MBA-chit-AgNTs for 24 h. Different types of nanoparticle motions can be identified as also shown by Liu et al

44

. For instance, Movie 2 si 3 from Supporting

Information (a magnified area from Movie 1) shows rapidly moving individual nanoparticles which after collision quickly separated and continued their trajectories individually. Others, after collision remained merged and continued to move together (Movie 4 from Supporting Information) with a well-observable color change due to the plasmonic coupling effect. These real-time movies help to gain insight into how the nanoparticles aggregation occurs inside cellular organelle and open new technical opportunities for analysis of drug delivery carriers or theranostic probes. For more accurate assessment of the localization and distribution of CBP-4MBA-chitAgNTs inside NIH:OVCAR-3 cells DIC microscopy imaging on the CBP-4MBA-chit-AgNTs incubated cells was performed. This microscopic technique relies on the contrast created by the refractive index gradients of different areas of a specimen and, similarly to confocal microscopy, allows visualization of a single focal plane of a cell which can be well exploited 22 ACS Paragon Plus Environment

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to investigate the intracellular distribution of nanoparticles. Herein, the same cells as presented in the dark field image from Figure 4C was visualized using DIC microscopy by focalizing to consecutive planes of the cell starting from the surface of the cell culture dish (considered as Z = 0 µm) and recording images from 2 to 2 µm until reaching the cell membrane (Supporting Information Figure S4). Similarly to dark field images, DIC images also reveal extranuclear localization of the nanoparticles distributed in the whole cytoplasm along the cells’ depth. The DIC image recorded from the top of the cell shows few nanoparticles presumably bound to the outer side of the cell membrane, which were not yet internalized at the moment of image recording.

Nanoparticle uptake studies by TP excited FLIM. Although PNPs are poor light emitters under one-photon excitation, they were found to exhibit enhanced PL under TP excitation, which was attributed to a combination of enhanced excitation efficiency due to the local electric field induced by LSPR with a serial process involving sequential absorption of photons and emission from the radiative recombination of electrons from sp-band with holes from d-band.45 Known to display stronger plasmon resonances compared to AuNPs, AgNPs are capable of providing larger enhancement of optical signals, such as TP excited PL in particular. More significantly, anisotropic NPs featuring high electromagnetic fields concentrated at their tips are considered more appealing. Another interesting feature of the intrinsic PL signal of PNPs is its extremely short lifetime. Typically, under TP excitation using NIR sources, the PL of PNPs does not interfere with the signal of biological tissue (few ns) which enables their use as contrast agents in bioimaging studies. Taking all these aspects into consideration we were interested in evaluating the performance of our samples as contrast agents in TP excited FLIM. As previously mentioned, the number of NPs internalized by OCVAR3 cells is very different from cell to cell due to different 23 ACS Paragon Plus Environment


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metabolism and varying cellular processes at different growth phases. As such, we first investigated a cell with a large number of internalized 4MBA-chit-AgNTs as shown in the FLIM images presented in Figure 5 (A-C), obtained under excitation at 800 nm.

Figure 5. TP excited FLIM images of aNIH:OVCAR-3 cell incubated with 4MBA-chitAgNTs, recorded at different Z levels (A, B and C), corresponding bright field (D) and dark field (E) images of the cell images by FLIM and TP excited PL spectrum (F) extracted from the region marked in C. Specifically, the three images, recorded at different depths of the cell, exhibit intense bright spots, with an extremely short lifetime,12 proving that the signal originates from the 4MBAchit-AgNTs internalized within the whole cytoplasm region. As compared to bright and dark field images of the same cell (Figure 5 D and E), there was not detected any signal from the cellular components which points towards the ability of employed samples to perform as reliable contrast agents for NIR imaging assays. According to previous studies, the formation of aggregated can significantly enhance the TP excited PL of AgNPs due to the formation of LSPR toward the NIR region allowing resonant excitation which greatly enhances the TP 24 ACS Paragon Plus Environment

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excitation efficiency and plasmonic coupling which amplifies the local-field intensity.46,47 Hence, we believe that the detection of 4MBA-chit-AgNTs inside NIH:OVCAR-3 cell was improved by the aggregation of NPs in the cellular compartments and cytoplasm. The TP excited PL spectrum extracted from the region marked in Figure 5C, presented in Figure 5F, exhibits a spectral profile close to the extinction spectrum of NPs displaying however an important red-shift which could indicate the aggregation process occurring inside cell. Going further, we were able to demonstrate that TP excited FLIM can be successfully employed to track even a reduced number of AgNPs inside NIH:OVCAR-3 cells. As shown in FLIM images from Figure S5 in the Supporting Information also obtained at 800 nm excitation, CBP-4MBA-chit-AgNTs are detectable inside live NIH:OVCAR-3 cell at different depths as dispersed short lived fluorescent spots. Nevertheless, the intensity of the signal is much more reduced than in the FLIM images from Figure 5 most probably to a reduced degree of aggregation and thereafter a lower strength of the PL signal.

Nanoparticle uptake studies by SERS imaging. Confocal Raman and SERS microscopy was proven to be a genuine, ultrasensitive,

non-invasive, non-photobleaching spectral

imaging tool for investigating the uptake of plasmonic nanocarriers inside cells and tumors with simultaneous mapping and analysis of intracellular components in terms of composition, incidence and their evolution in complex biochemical systems.14,15,48 In the following combined Raman and SERS spectral micro-imaging generated by multivariate K-means cluster analysis is used to perform the simultaneously Raman mapping of the whole NIH:OVCAR-3 cell and accurate SERS tracking of the internalized CBP-4MBA-chit-AgNTs. Taking into consideration the already demonstrated high SERS sensitivity of chit-AgNTs 25 a cell with a small amount of internalized CBP-4MBA-chit-AgNTs is selected to evaluate the performance of our samples as SERS traceable nanocarriers. In order to obtain in depth 25 ACS Paragon Plus Environment


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information about the localization of CBP-4MBA-chit-AgNTs inside cells we adjusted the laser power such as to be able to obtain both the Raman response from the cellular constituents and the SERS response from the particles. Figure 6 A and B shows representative multivariate Raman and SERS spectral images of a control cell (without nanoparticles) and a cell incubated with CBP-4MBA-chit-AgNTs for 24 h, obtained under excitation at 532 nm. The illustrated images enable the discrimination between the cellular membrane (green), cytoplasmatic region (blue), nucleus (yellow), cellular organelle (cyan), whilst simultaneously facilitate the visual localization of CBP-4MBA-chit-AgNTs (red) in the cytoplasmatic region. An interesting observation is that the cellular organelles are not longer discernible after the internalization of CBP-4MBA-chit-AgNTs (Figure 6B – the lack of cyan cluster). Moreover, the red cluster in Figure 6B associated with CBP-4MBA-chit-AgNTs shows a strong similarity with the cyan cluster in Figure 6A specific to cellular organelles. A likely explanation is the localization of CBP-4MBA-chit-AgNTs inside cellular organelles. The corresponding extracted spectra featuring the biochemical profile of cellular constituents are presented in Figure 6C and D. Specifically, the band at 1663 cm-1 corresponds to the vibration mode of amide I (CO stretching coupled with NH deformation), while the bands at 1453 and 1252 cm-1 originate from the CH3 and CH2 deformation modes of lipids.49 The prominent bands at 2885 and 2935 cm-1 are associated to the CH2 and CH stretching vibrations of the lipids and proteins, while the band at 1006 cm-1 is attributed to the ring breathing of phenylalanine.49 The bands at 1092 and 788 cm-1 attributed to the vibration of phosphate glycosidic link (PO2 stretching, CC stretching, COC stretching) and cytosine vibration, respectively are associated with the Raman signature of the genomic DNA.50 Composite bands of 4MBA Raman reporter molecules and of cellular components are observed in the extracted spectrum corresponding to the internalized CBP-4MBA-chit-AgNTs (Figure 6D, red spectrum). In particular, the prominent pH independent vibrational bands of 4MBA 26 ACS Paragon Plus Environment

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molecules located at 1073 and 1585 cm-1 are clearly identified in the spectrum together with the phospolipidic Raman fingerprint from cellular organelles at 2885 and 2935 cm-1. The spectral range of 1100-1500 cm-1 is dominated by the Raman signature of cellular constituents which makes rather hard the detection of the pH sensitive bands of 4MBA molecules. Specifically, the Raman vibrational bands of cellular components are clearly seen at 1453 and 1252 cm-1, while the vibrational band of the protonated COOH group of 4MBA at acidic pH is hardly identifiable ( * marked band at 1376 cm-1). Taken together, Raman analysis provides strong evidence of the efficient uptake of CBP-4MBA-chit-AgNTs and enables their localization inside cellular organelles.

Figure 6. Raman maps obtained by multivariate data analysis algorithm (K-means cluster analysis) of a single NIH:OVCAR-3 cell without nanoparticles (A) and incubated with CBP4MBA-chit-AgNTs for 24 h (B). Corresponding extracted spectra from distinctly colored



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areas in image A (C) and in image B (D). The band marked with * corresponds to the protonated COOH group of 4MBA at acidic pH. For in vivo applications is highly desirable to perform the SERS measurements under NIR excitation where the intrinsic tissue chromophores and water molecules exhibit minimal absorption and scattering for the excitation light, providing maximum irradiation penetration through tissue. Therefore, in the next step we focused our experiment on NIR laser excitation. As shown in SERS image from Figure S6 in the Supporting Information, CBP-4MBA-chitAgNTs are detectable inside live NIH:OVCAR-3 cell under 785 nm laser excitation. The extracted spectrum in Figure S6 in the Supporting Information features the chemical profile of 4MBA Raman reporter molecules, providing strong support that the fabricated nanotherapeutic system can be easily tracked inside living cells with multiple laser excitation wavelengths.

SERS sensing of intracellular pH. The chemotherapeutic performance of colloidal nanoparticles as pH-sensitive drug delivery systems demands their ability to control the release of drug usually as a response to the acidic medium of endosomes and lysosomes.51 Thus, the accurate localization of nanocarriers inside cells with information about the intravesicular pH which can trigger the drug release is crucial for designing efficient drug delivery systems with improved therapeutic abilities. As the uptake of CBP-4MBA-chitAgNTs inside live NIH:OVCAR-3 cells was clearly proven by combined dark field, DIC, TP excited FLIM and Raman imaging, next the basis Raman analysis is performed to determine the localization of nanocarriers inside live NIH:OVCAR-3 cells with simultaneous information regarding the pH that the particles are experiencing after their internalization. In order to avoid the overlay between the Raman signal of cellular constituents and the pH sensitive SERS bands of 4MBA molecules we reduced the laser intensity to the level where 28 ACS Paragon Plus Environment

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no Raman signal from cell could be detected in the spectral range of 1100-1500 cm-1. Figure 7 shows the Raman maps of a selected NIH:OVCAR-3 cell incubated with CBP-4MBA-chitAgNTs for 24 h.

Figure 7. (A) Raman map of a single NIH:OVCAR-3 cell incubated with CBP-4MBA-chitAgNTs. The map was generated using the Raman bands at 2800-3100 cm-1 attributed to the C–H stretching vibrations of the lipids. (B) The SERS pH map obtained with the pH-sensitive vibration bands at 1367 and 1428 cm-1 of the carboxyl group of 4MBA. (C) Overlay of the Raman image of the cell (A) and the SERS pH map (B). (D) Corresponding extracted average spectra from green and red spots in Figure B. Image A in Figure 7 was obtained by integrating the Raman intensity in the spectral range of 2800-3100 cm-1 originating from the C–H stretching vibrations of the lipids. The SERS pH map in Figure 7B was generated by basis analysis using the pH-sensitive vibration bands at 1367 and 1428 cm-1 related to the protonation or deprotonation of the carboxyl group of 4MBA. As one can see from the extracted spectra in Figure 7D, the green spots in image B 29 ACS Paragon Plus Environment


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are associated with the 1367 cm-1 band of COO- in acidic medium, while the red spots are related to the 1428 cm-1 band of COO- at neutral pH. By overlaying the SERS pH map (Figure 7B) and the corresponding Raman image of the cell (Figure 7A) sufficient contrast was obtained to observe the localization of CBP-4MBA-chit-AgNTs within the cell with visual discrimination between acidic (green spots) and neutral (red spots) intracellular pH (Figure 7C). For instance, red spots may be arising from nanoparticles retained in the neutral medium of endocytic vesicles or cytoplasm. Green color corresponds to CBP-4MBA-chit-AgNTs distributed within intracellular organelles, most probably in the acidic environment of late endosomes and lysosomes. This is a key result which demonstrates not only the efficient nanocarriers internalization and tracking inside live human ovarian cancer cells but also their localization in acidic compartments, which is an important prerequisite for pH-triggered release of drug. To the best of our knowledge, this is the first report on a drug nanocarrier integrating therapeutic abilities with simultaneous SERS monitoring of the local pH of the medium surrounding the nanoparticles after their internalization.

In vitro therapeutic potential. Next, we investigated the therapeutic effect of CBP-4MBAchit-AgNTs on the NIH:OVCAR-3 cell line. ELISA BrdU-colorimetric immunoassay was performed to compare the effects of CBP-4MBA- chit-AgNTs, 4MBA-chit-AgNTs and free CBP of similar concentrations on cell proliferation. As shown in Figure 8, 4MBA-chit-AgNTs exhibit no obvious effect on cell proliferation even at the highest concentration incubated. Contrarily, CBP-4MBA-chit-AgNTs influenced negatively the cell proliferation in a concentration dependent manner, by inducing an almost complete inhibition after 24 h of incubation at a drug concentration of 125 µM. However, compared to free CBP at similar concentration, the anti-proliferative effect induced by CBP-4MBA-chit-AgNTs is a somewhat higher at lower nanoparticle concentrations, meanwhile at higher nanoparticle concentrations, 30 ACS Paragon Plus Environment

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the effect is lower or similar. This different in vitro therapeutic effect between free and encapsulated drug, frequently reported also by other researchers,52 can be attributed to both the pH dependent behavior of chitosan and the different internalization pathways of free drug compared to loaded drug. Specifically, it is recognized that chitosan exhibits pH-dependent dissolution and swelling properties due to its amino groups, which are positively charged under acidic conditions leading to a soluble state of the chitosan.34,35 On the other hand, the pH-dependent coordination bond between CBP and 4MBA becomes also hydrolyzed when the [H+] increase.36 Consequently, at acidic pH both drug entrapment mechanism conditions are disengaged/unfastened, resulting the release the drug payload in its active form. As demonstrated by the intracellular pH mapping assays (green spots represent acidic and red spots neutral medium, after 24 h incubation, the nanocarrier is internalized in both acidic and neutral subcellular compartments. However, we presume that only those localized in the acidic environment of late endosomes and lysosomes have successfully released their CBP content. This may explain the somewhat above mentioned inferior therapeutic efficiency of CBP-4MBA-chit-AgNTs compared to the free drug. While the free drug is more rapidly uptaken and directly diffuses into the nuclei, the nanoparticles are internalized by the endocytotic pathway 53 and only when reach the compartments with acidic pH slowly release the drug, which afterwards has to reach the nuclei and exerts its effect. Taking into account that the use of such nanocarriers able to deliver the drug payload in a controlled manner could effectively reduce the side effects of the free drug and prevent the emergence of drug resistance mechanisms, we consider that the reported design of drug nanocarriers holds a great potential in improving the therapeutic efficacy of anticancer drugs.



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Figure 8. Concentration dependent anti-proliferative properties of 4MBA-chit-AgNTs, CBP4MBA-chit-AgNTs and free CBP on NIH:OVCAR-3 cell line.

CONCLUSIONS Herein, we have successfully developed a biocompatible nanotherapeutic platform based on CBP-4MBA-chit-AgNTs for simultaneous drug delivery, pH sensing and multimodal tracking inside live NIH:OVCAR-3 human epithelial ovarian cancer cells. The already demonstrated high SERS sensitivity of AgNTs was here combined with the unique biological and physicochemical characteristics of chitosan, and the chemotherapeutic performance of CBP to demonstrate a proof-of-concept for ovarian cancer theranostics. Notably, the 4MBA Raman reporter plays multiple roles here, from allowing the precise SERS tracking of the nanoconstruct inside live ovarian cancer cells, to sensing the intracellular pH and providing additional binding sites for drug loading. The intrinsic light scattering and photoluminescence properties of AgNTs enable the accurate localization of CBP-4MBA-chit-AgNTs inside NIH:OVCAR-3 cells at a 3D level through combined dark-field, DIC and TP excited FLIM imaging. Chitosan biopolymer acts as a biocompatible stabilizing shell, allowing the drug entrapment and the diffusion of reporter molecules through the polymeric matrix, while 32 ACS Paragon Plus Environment

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preserving the unique optical characteristics of AgNTs after the formation of the CBP-4MBAchit-AgNTs delivery system. The in vitro cell proliferation assay clearly shows the effectiveness of the prepared nanocarriers in inhibiting the growth of NIH:OVCAR-3 cancer cells. The outstanding physical, chemical and biological characteristics of these nanocomposites contribute all together to reveal a promising candidate for personalized medicine.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Monica Potara), [email protected] (Simion Astilean)

ACKNOWLEDGMENT This work was supported by CNCS-UEFISCDI, project number PN-II-RU-TE-2014-41988. Supporting Information Available: pH-dependent SERS spectra at 785 nm excitation; supplementary dark field images of NIH:OVCAR-3 cells incubated at low nanoparticles concentrations, at different Z-depths and at low magnification; dark field images recorded at different incubation periods, DIC images of NIH:OVCAR-3 cells incubated with CBP4MBA-chit-AgNTs; TP excited FLIM images of NIH:OVCAR-3 cell incubated with CBP4MBA-chit-AgNTs focalized at different depths; SERS map of NIH:OVCAR-3 cells incubated with CBP-4MBA-chit-AgNTs under 785 nm laser excitation and corresponding extracted SERS spectrum. Movies showing the dynamic intracellular behavior of nanocarriers 33 ACS Paragon Plus Environment


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recorded in real-time by dark field microscopy. Supporting Information is available free of charge from the ACS Nano home page (http://pubs.acs.org/journal/ancac3).

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Figure 1. Schematic illustration of the preparation procedure of CBP-4MBA-chit-AgNTs 150x63mm (300 x 300 DPI)

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Figure 2. Normalized UV-vis-NIR extinction spectra of the colloidal nanoparticle solutions (a) before tagging with MBA (chit-AgNTs); (b) after tagging MBA (4MBA-chit-AgNTs); (c) after tagging MBA and loading with CBP (CBP-4MBA-chit-AgNTs). The inset shows a zoomed image of the dipolar plasmon resonances of the extinction spectra. 85x68mm (300 x 300 DPI)


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Figure 3. pH-dependent SERS spectra of nanoparticles at different preparation steps (a-e). Excitation: 532 nm. 99x82mm (300 x 300 DPI)



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Figure 4. Dark field images of control NIH:OCVAR-3 cells (A), incubated with 4MBA-chit-AgNTs (B) and CBP4MBA-chit-AgNTs (C) at a final nanoparticles concentration of 1 µg/mL. 150x50mm (300 x 300 DPI)


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Figure 5. TP excited FLIM images of a NIH:OVCAR-3 cell incubated with 4MBA-chit-AgNTs, recorded at different Z levels (A, B and C), corresponding bright field (D) and dark field (E) images of the cell images by FLIM and TP excited PL spectrum (F) extracted from the region marked in C. 138x88mm (300 x 300 DPI)



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Figure 6. Raman maps obtained by multivariate data analysis algorithm (K-means cluster analysis) of a single NIH:OVCAR-3 cell without nanoparticles (A) and incubated with CBP-4MBA-chit-AgNTs for 24 h (B). Corresponding extracted spectra from distinctly colored areas in image A (C) and in image B (D). The band marked with * corresponds to the protonated COOH group of 4MBA at acidic pH. 109x109mm (300 x 300 DPI)


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Figure 7. (A) Raman map of a single NIH:OVCAR-3 cell incubated with CBP-4MBA-chit-AgNTs. The map was generated using the Raman bands at 2800-3100 cm-1 attributed to the C–H stretching vibrations of the lipids. (B) The SERS pH map obtained with the pH-sensitive vibration bands at 1367 and 1428 cm-1 of the carboxyl group of 4MBA. (C) Overlay of the Raman image of the cell (A) and the SERS pH map (B). (D) Corresponding extracted average spectra from green and red spots in Figure B. 99x96mm (300 x 300 DPI)



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Figure 8. Concentration dependent anti-proliferative properties of 4MBA-chit-AgNTs, CBP-4MBA-chit-AgNTs and free CBP on NIH:OVCAR-3 cell line. 85x67mm (300 x 300 DPI)


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