Journal of Colloid and Interface Science 451 (2015) 198–211

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Trastuzumab guided nanotheranostics: A lipid based multifunctional nanoformulation for targeted drug delivery and imaging in breast cancer therapy Priyambada Parhi, Sanjeeb Kumar Sahoo ⇑ Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 21 January 2015 Accepted 27 March 2015 Available online 3 April 2015 Keywords: Nanotheranostics Rapamycin Nanoparticles Quantum dots Trastuzumab

a b s t r a c t Nowadays, emerging aspects of cancer therapy involve both diagnostic and therapeutic modules in a single setting. Targeted theranostic nanoplatforms have emerged globally as frontier research for the improvement of cancer therapy. Trastuzumab (Tmab), a humanized monoclonal antibody is now being used to target human epidermal growth factor receptor-2 (HER 2) positive cancer cells. In the present study, we have analysed the imaging and theragnosis potentiality of Tmab functionalized lipid based nanoparticles (NPs) loaded with anticancer drug rapamycin and imaging agent (quantum dots) for targeted cancer therapy and imaging. The therapeutic evaluation of drug loaded NPs were evaluated through various in vitro cellular studies. The results showed enhanced therapeutic efficacy of targeted drug loaded NPs over native drug and unconjugated NPs in HER 2 positive SKBR 3 breast cancer cell line. Moreover, exploration of the therapeutic benefits of rapamycin loaded Tmab conjugated NPs (Tmabrapa-NPs) at molecular level, revealed augmented down regulation of mTOR signalling pathway thereby, inducing more cell death. Above all, our targeted multifunctional NPs have shown an excellent bio-imaging modality both in 2D monolayer and 3D tumor spheroid model. Thus, we can anticipate that such a multimodal nanotheranostic approach may be a useful tool for better cancer management in future. Ó 2015 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Nanomedicine Laboratory, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa 751 023, India. Fax: +91 674 230072. E-mail address: [email protected] (S.K. Sahoo). http://dx.doi.org/10.1016/j.jcis.2015.03.049 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.

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1. Introduction Chemotherapeutic regimens are at the upper edge for cancer management; however nonspecific cytotoxicity, poor aqueous solubility and bioavailability become the major hindrance for the success of cancer treatment [1]. In this regard, efficient drug delivery approaches are the prerequisite for site specific delivery of chemotherapeutic agents by surmounting above limitations to enhance their therapeutic potential. Currently, research is mostly centred on development of suitable carrier systems for site specific delivery of therapeutics and numerous studies indicate that, these drug delivery vehicles are capable of overcoming the lacunas of current chemotherapeutic agents by enhancing their bioavailability, aqueous solubility and limiting toxicity [2–4]. Furthermore, improvement of cancer therapy also involves monitoring the therapeutic response of drug after treatment. In this setting, theranostic platforms are emerging as a combinational approach for simultaneous cancer diagnosis and therapy and nowadays much effort is focussed around development of such an approach for successful cancer management [5–7]. Rapamycin is a potent anticancer drug which is effectively used for the treatment of different cancers. The anticancer activity of rapamycin is attributed to its binding with the immunophilin FK506-binding protein (FKBP12) to form a ternary complex, which is capable of inhibiting the mammalian target of rapamycin (mTOR) – a serine/threonine kinase recognized as a central controller of eukaryotic cell growth and proliferation. The mTOR pathway is frequently activated in many human cancers, including breast cancer [8,9]. Inhibition of mTOR pathway by rapamycin blocks the phosphorylation of its downstream targets like the 70 kDa, 40S ribosomal protein kinase (p70S6K1) and the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) leading to G1 arrest in most cancer cells and p53-independent apoptosis in some others [10–13]. Therefore, inhibiting the mTOR pathway is extensively considered as an effective approach for targeted cancer therapy. Though rapamycin represent a potent anticancer drug in preclinical settings, its clinical utility is floundered due to its poor aqueous solubility, low bioavailability, non-specificity and dose limiting toxicity [14]. In this context, nano-drug delivery systems are anticipated to overcome the drawbacks associated with native rapamycin. In relation to this, numerous research groups including us have developed different nanoparticulate systems with sustained release property to surmount the shortcomings associated with native rapamycin [15–17]. Among the various nanotechnology based platforms, lipid based NPs are considered as one of the most promising drug delivery vehicle owing to their small particle size, ability to cross the biological barriers and for accumulating at the targeted site for efficient delivery of chemotherapeutic agents [18,19]. In this regard, glyceryl monooleate (GMO), an amphiphilic lipid molecule approved by the food and drug administration (FDA) is well explored to form lipid based NPs [20]. In general, GMO is known to form different liquid crystalline phases like reverse micellar phase, lamellar phase, cubic phase, reverse hexagonal phase etc. depending upon their water content [21,22]. It is also known to form a three dimensional network of curved lipid bilayers where both water soluble and insoluble drugs can be entrapped and explored for controlled drug delivery [23–25]. Primary requirement of a targeted cancer therapeutic approach is to deliver chemotherapeutic drugs to the cancer cells in a site specific manner over a period of time without affecting surrounding noncancerous tissues. In this setting, a monoclonal therapeutic antibody Trastuzumab (Tmab), which has been FDA approved can be used for targeted therapy against HER 2 positive breast cancer cells and thus can act as an attractive tumor targeting ligand [26,27]. Recently, optical imaging has been explored extensively

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in biomedical research and semiconductor nanocrystals known as quantum dots (QDs) have emerged as unique biological imaging and labelling probe due to its superior optical properties [28,29]. To accomplish superlative cancer therapy using nanotheranostic approaches, current researches mostly focus on using quantum dots in combination with anticancer agents to intermingle both therapy and imaging [30,31]. Thus, in the present investigation, with an aim to formulate a targeted nanotheranostic system for improved breast cancer therapy and imaging we developed a multifunctional lipid based NPs by using Tmab as targeting ligand, rapamycin as anticancer drug and quantum dots as imaging probe. To substantiate our hypothesis that the developed Tmab functionalized GMO based lipid NPs may exhibit higher uptake and therapeutic efficiency through HER 2 receptor mediated targeting, we have evaluated the efficacy of our formulated rapamycin loaded Tmab conjugated NPs (Tmabrapa-NPs) in HER 2 positive and negative cell lines by cell cytotoxicity assay, uptake assay, apoptosis study, etc. The molecular mechanism related to apoptosis was investigated by western blot analysis. Further, the imaging potentiality of the targeted nanotheranostic system was validated in two dimensional (2D) monolayer culture and three dimensional (3D) tumor spheroid model in vitro. Thus, our preliminary studies suggest that such a theranostic nanocarrier may act as a multimodal vehicle for improved cancer therapeutics and imaging. 2. Materials and methods 2.1. Materials Rapamycin was purchased from Fujian Kerui Pharmaceutical Co., LTD., Fuzhou City, China. Pluronic F-127, potassium bromide (KBr), Tween-80, propidium iodide (PI), 6-coumarin, protease inhibitor cocktail, sodium dodecyl sulfate (SDS), glycine, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), p-Coumaric acid, luminol, glutaric acid, D-a-Tocopherol poly (ethylene glycol) 1000 succinate (TPGS), poly (ethylene glycol)10,000 (PEG-10,000), N, N0 -dicyclohexyl carbodiimide (DCC), N-hydroxy succinimide (NHS), uranyl acetate, Igepal CA-630 (NP-40), Sodium deoxycholate, Ethylene glycol-bis (2 amino ethyl ether)N, N, N0 ,N0 -tetra acetic acid (EGTA), Ethylene diamine tetra acetic acid (EDTA), agarose, DMSO-d6 (99.9 atom% deuterium-enriched) and dimethyl sulphoxide (DMSO) were procured from Sigma– Aldrich (St. Louis, MO). Glyceryl monooleate (GMO) was obtained from Eastman (Memphis, TN). Sodium chloride was procured from MP biomedicals (Cedex, France). Skimmed milk powder was obtained from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Acetonitrile was purchased from Spectrochem, Pvt. Ltd. (Mumbai, India) and Tris base was obtained from Promega (Promega Corporation, Madison, Wisconsin). Mitotracker Red (CMXRos) dye and QdotÒ 605 ITK™ amino (PEG) quantum dots (QD605) were purchased from Invitrogen Corp. (Carlsbad, CA). Trastuzumab (Tmab) was procured from Hoffmann-La Roche Ltd. (Basel, Switzerland). 2.2. Cell culture All the cell culture experiments were performed by taking HER 2 positive SKBR 3 and HER 2 negative MDA-MB-231 breast cancer cell lines obtained from National Centre for Cell Sciences (NCCS), Pune, India and cultured using DMEM (PAN BIOTECH GmbH, Aidenbach, Germany) with 10% fetal bovine serum (FBS) supplemented with 1% L-glutamine and 1% penicillin – streptomycin (Himedia Laboratories Pvt. Ltd., Mumbai, India). The cells were

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maintained at 37 °C in a humidified, 5% CO2 atmosphere in an incubator (Hera Cell, Thermo Scientific, Waltham, MA). 2.3. Synthesis of TPGS–Tmab conjugates TPGS–Tmab was synthesized using DCC–NHS coupling reaction by following the protocol of Zhang et al. with minor modifications as shown in Scheme 1 [32]. Briefly, carbonated TPGS was formulated by reacting TPGS: glutaric acid: DCC with 1:1:1 stoichiometric molar ratio in DMSO under N2 atmosphere for 24 hrs at the room temperature. The resultant solution was collected in a dialysis bag (Spectra/por 6, MWCO = 1 KDa, Spectrum Laboratories, Inc. Rancho Dominguez, CA) and dialyzed against 2 l of deionized water for a period of 24 hrs with a frequent change of dialysate in every 2 hrs. Finally, the dialyzed solution was freeze-dried using Labconco FreeZone 12 (Labconco Corporation, Kansas City, MO) maintained at 50 °C and 0.05 mBar. After that, the reaction of carbonated TPGS: NHS: DCC with 1:2:2 stoichiometric molar ratio were carried out for 6 hrs at 50 °C to form TPGS-NHS. Then Tmab was added to the above TPGS-NHS solution at molar ratio of 1:100 and allowed to react under N2 atmosphere for 48 hrs at

the room temperature. Dialysis of the resulted product was performed against DMSO and deionised water, as per the protocol of Zhang et al. mentioned earlier. The final product was collected by freeze-drying. 2.4. Characterization of TPGS–Tmab conjugates The characterization of TPGS–Tmab conjugate was done by FTIR and 1H NMR. The FT-IR spectral analysis of TPGS, Tmab, TPGS– Tmab conjugates and TPGS/Tmab mixture were performed by using a FT-IR spectrometer (Spectrum RX 1, Perkin Elmer, Waltham, MA) and 1H NMR spectra were recorded in Bruker BioSpin (Fallanden, Switzerland) Avance-III 400 MHz FT-NMR spectrometer as reported earlier [33]. In brief, different samples were crushed with KBr and pressed into pellets at a pressure of 150 kg cm2. The spectra were detected by averaging 32 interferograms with a resolution of 2 cm1 in the range 350–4000 cm1 through FT-IR spectrometer. The 1H NMR spectra were recorded for TPGS, Tmab, TPGS/Tmab mixture and TPGS–Tmab conjugates using DMSO-d6 as solvent in Bruker BioSpin FT-NMR spectrometer.

Scheme 1. Synthesis of TPGS–Tmab conjugate.

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2.5. Preparation of Tmab conjugated NPs The rapamycin loaded Tmab conjugated NPs (Tmab-rapa-NPs) were prepared by following our previously published protocol with slight modification [34]. Briefly, 20 mg of rapamycin was incorporated into the fluid phase of GMO (175 ll at 40 °C) and vortexed. The above mixture was emulsified with 1 ml of pluronic F-127 solution (10% w/v) by sonication, using a microtip probe sonicator (Model: VC 505, Vibracell Sonics, Newton, USA) set at amplitude 30% for 2 min over an ice bath. The resultant solution was further emulsified with 1 ml of TPGS (5% w/v of both free TPGS and TPGS– Tmab conjugates in the ratio 4:1) and sonicated for 2 min at amplitude 30% over an ice bath. The resultant nanoparticulate emulsion was centrifuged at 1000 rpm (Heraeus, Thermo Fisher Scientific, Germany) for 2 min to remove the unentrapped rapamycin and then 2% of PEG-10,000 (w/v) was added as lyoprotectant with a constant vortexing for 5 min and lyophilized by using Labconco FreeZone 12 (Labconco Corporation, Kansas City, MO) maintained at 50 °C and 0.05 mBar for six days to get the lyophilized powder for further use. To determine the cellular uptake of NPs, 6-coumarin loaded Tmab conjugated NPs (Tmab-6-coumarin-NPs) were formulated using the above procedure except that 100 lg of dye was added to GMO prior to emulsion instead of rapamycin. Further, rapamycin and QD605 loaded Tmab conjugated theranostic NPs (Tmab-QD-rapa-NPs) were prepared by incorporating rapamycin and QD605 into the fluid phase of GMO following the above protocol. 2.6. Physico-chemical characterization of NPs The particle size and size distribution and surface charge measurements were performed by using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) based on dynamic light scattering using our previously published protocol [34]. The shape and surface properties of Tmab-QD-rapa-NPs were observed by atomic force microscopy (JPK nanowizard II, JPK instrument, Bouchestrasse, Berlin, Germany). The size of the Tmab-QD-rapa-NPs was also observed by Transmission Electron Microscopy (TEM) (Philips 201/FE Inc, Barcliff, Manor, NY). The AFM and TEM analysis was performed by following our previously published protocol [35]. The drug content in NPs was estimated by using reverse phase isocratic mode of high performance liquid chromatography (RP-HPLC) system of Waters™ 600 (Waters Co., Milford, MA) [15]. In vitro release kinetics of rapamycin from Rapa-NPs and Tmab-rapa-NPs were carried out by using our previously published protocol [15]. 2.7. Cellular uptake study of Tmab conjugated NPs 2.7.1. Quantitative cellular uptake study For this study, we have used 6-coumarin dye as a fluorescent probe as it offers a sensitive method to determine their intracellular uptake and retention [36]. Briefly, SKBR 3 and MDA-MB231 breast cancer cells at a density of 1  105 cells per well of 24 well plate (Corning, NY, USA) were treated with either 40 ng/ml concentration of native 6-coumarin or equivalent concentration of 6-coumarin-NPs or Tmab-6-coumarin-NPs and incubated for 2 hrs at 37 °C in CO2 incubator. Then, the cells were trypsinized and washed thrice with phosphate buffer saline (PBS). Then cells were lysed with 2% of NP-40 solutions and kept in ice for 30 min and after that protein estimation was performed by taking 5 ll of the lysate through Bradford assay. The rest of the lysate was lyophilized and dissolved in methanol:chloroform (12.5:87.5 v/v) and then taken for fluorescence spectroscopic analysis (Perkin Elmer, Model No. LS 55, MA) to quantify the intracellular 6-coumarin uptake (Ex 420 nm, Em 495 nm). Further, to substantiate the fact that HER 2 receptor plays a vital role in enhanced uptake;

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competitive uptake study was performed in SKBR 3 cells. For this, cells were treated with different concentrations of native Tmab for 2 hrs prior to addition of Tmab-6-coumarin-NPs (40 ng/ml). After 2 hrs of incubation, cells were processed according to the above procedure to quantify the nanoparticle uptake. 2.7.2. Qualitative cellular uptake study For qualitative cellular uptake study, SKBR 3 and MDA-MB-231 cells at a density of 20,000 cells per BioptechÒ tissue culture plates (Bioptechs Inc. Butler, PA) were treated with 50 ng/ml concentration of either native 6-coumarin or equivalent concentration of 6-coumarin-NPs or Tmab-6-coumarin-NPs and incubated for 2 hrs. At the end of incubation time, the cells were washed thrice with PBS, fixed with 4% buffered formaldehyde for 15 min and after that cells were stained with PI for 40 min. The cells were washed three times with PBS and then visualized under a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Germany) equipped with an argon laser using FITC filter (Ex 488 nm, Em 525 nm) and PI filter (Ex 530 nm, Em 615 nm). The images were processed using Leica Application Suite software. 2.8. In vitro cytotoxicity of Tmab functionalized rapamycin loaded NPs To find out the cytotoxic effect of drug in native or in nanoformulation (Rapa-NPs or Tmab-rapa-NPs), MTT based colorimetric assay was carried out as described previously [35]. Briefly, SKBR 3 and MDA-MB-231 cells at a cell density of 2500 cells/well in 96 well plates (Corning, NY) were treated with different concentrations (0.1–200 ng/ml) of either native rapamycin (native rapa) or equivalent concentration of Rapa-NPs or Tmab-rapa-NPs. Equivalent concentration of non-loaded NPs (void NPs) was used to check the toxicity of void NPs. Medium treated cells were used as control for the experiment. MTT assay was carried out on 5th day by using our previously published protocol [35]. 2.9. Western blot analysis Western blot analysis was done to know the molecular mechanism of apoptosis and involvement of different signal transduction pathways modulated by rapamycin [37]. In brief, 2.5  105 SKBR 3 cells were treated with 50 ng/ml native rapa or Rapa-NPs or Tmabrapa-NPs for 24 hrs. Then, the cells were pellet down by scraping, washed with cold PBS, followed by protein isolation using lysis buffer. Western blot analysis of different proteins were performed using specific primary antibody for recognizing p-Stat-3, b-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and p-mTOR, pp70S6K1, p-4E-BP1, p-Akt, BCL-2 (Cell signalling Technology, Inc., Danvers, MA.) and c-Myc (IMGENEX India Pvt. Ltd., India) using our previously published protocol [38]. 2.10. Study of mitochondrial membrane potential loss The changes in the mitochondrial membrane potential (MMP) was evaluated by flow cytometry using Mitotracker Red (CMXRos) dye. The CMXRos, that passively diffuses across the plasma membrane and accumulates in the negatively charged mitochondrial matrix is generally used as a fixable probe for detection of loss of MMP [39,40]. In brief, the SKBR 3 cells were seeded at a density of 2  105 cells/well in 6-well plate (Corning, NY). Next day, the cells were treated with native rapa or Rapa-NPs or Tmabrapa-NPs at a concentration 50 ng/ml and incubated for 24 hrs. After incubation period, the cells were collected, washed twice with PBS and incubated with 100 nM of CMXRos at 37 °C for 1 hr in dark. After that, the cells were washed with PBS and then the cells were examined for each sample with respect to control in FL3-H channel by analyzing 10,000 gated cells using a FACScan

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flow cytometer (FACSCalibur; Becton-Dickinson, San Jose, CA) and FlowJo 4.5 software. 2.11. Apoptosis study by flow cytometry In brief, cells were seeded at density of 2  105 cells/well in 6well plate (Corning, NY) and kept overnight for attachment at 37 °C. Next day, cells were treated with 50 ng/ml concentration of either native rapa or Rapa-NPs or Tmab-rapa-NPs for 24 hrs and kept in CO2 incubator (Hera Cell, Thermo Scientific, Waltham, MA). The cells treated with media was taken as control. After incubation time, cells were collected and washed thrice with PBS, pelleted down and suspended with 500 ll of 1 X binding buffer, 1 ll of Annexin V-FITC (BD Biosciences Pharmingen, CA), 4 ll of 7-aminoactinomycin D (7-AAD) (BD Biosciences Pharmingen, CA) and incubated at room temperature in dark for 20 min. After incubation period, the samples were washed with PBS twice and analysed by FACScan flow cytometer using FlowJo 4.5 software [37]. 2.12. In vitro cellular imaging To explore the potency of the QD605 loaded NPs as imaging modalities; cellular imaging study was performed in SKBR 3 cells grown as 2D monolayer and 3D spheroid model by confocal microscopy. For 2D monolayer imaging, SKBR 3 cells at cell density of Ò 2  104 were seeded in Bioptech tissue culture plates (Bioptechs Inc. Butler, PA) and kept overnight in 37 °C incubator. Next day, 50 lg/ml concentration of QD605 and rapamycin loaded NPs (QD-rapa-NPs) and Tmab-QD-rapa-NPs was added and incubated for 4 hrs. At the end of incubation period, the cells were washed thrice with PBS and were imaged in confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems Gmbh, Germany). For the purpose of imaging in 3D tumor spheroid model, SKBR 3 cells at a cell density 12,000 cells/well were seeded with complete growth medium in 96 well plates precoated with 1% agarose for 10 days to form multi-cellular tumor spheroids [41]. After that, the spheroids were incubated with 500 lg/ml concentration of QD-rapa-NPs and Tmab-QD-rapa-NPs for 4 hrs. Following incubation period, the spheroids were washed with PBS thrice and visualized under confocal microscope. The images were processed using Leica Application Suite software. 2.13. Statistical analysis Two way analysis of variance (ANOVA) and student’s t test were performed to conduct statistical analysis. Data are expressed as mean ± standard error of mean (SEM) or standard deviation (SD) and values of p < 0.05 were indicative of significant differences. 3. Results

TPGS/Tmab mixture, though the peaks are present but are of less intense than that of conjugates. Further, 1H NMR spectral analysis was carried out to confirm the conjugation of TPGS with Tmab as shown in Fig. 1b. In 1H NMR analysis, the spectrum of TPGS– Tmab conjugate depicts the presence of signals as obtained in TPGS and Tmab, which may be attributed to the formation of covalent bond between TPGS and Tmab. Similar types of results are also observed by Zhang et al. [32].

3.2. Characterization of nanoparticles The diagrammatic representation of Tmab-QD-rapa-NPs is shown in Fig. 2a. Physico-chemical characterization of Tmab-QDrapa-NPs revealed that, the NPs and Tmab-NPs were of size 72.64 ± 5.07 and 72.04 ± 7.22 nm range (Fig. 2b) with a negative zeta potential of 17.5 ± 1.21 and 11.03 ± 0.602 respectively. The surface topology of Tmab-QD-rapa-NPs was smooth and spherical in nature as evident from AFM study (Fig. 2c). TEM analysis further validate that, the particles are of nanometer size range (Fig. 2d). HPLC analysis demonstrated that approximately 26 lg and 19 lg of rapamycin were entrapped per mg of Rapa-NPs and Tmab-rapa-NPs respectively. The entrapment efficiency was found to be 48% (in the case of rapa-NPs) and 34.67% (in the case of Tmab-rapa-NPs) with a loading of 6.35%. To investigate the sustained release behavior of rapamycin entrapped in Rapa-NPs and Tmab-rapa-NPs, in vitro drug release kinetics was performed. Result demonstrates a biphasic release pattern (an initial burst release for 24 hrs followed by sustained release of drug up to 21 days) of rapamycin from NPs (Fig. 2e). The initial rapid release may be due to the diffusion of drug present at the surface or just beneath the surface of NPs [15].

3.3. Cellular uptake study Cellular uptake analysis by fluorescence spectrophotometer reveals an augmented uptake of Tmab-6-coumarin-NPs compared to 6-coumarin-NPs in SKBR 3 cells, however no significant difference in uptake of above nanoformulations were found in MDAMB-231 cells (Fig. 3a (i) and a (ii)). The HER 2 receptor mediated uptake of Tmab-6-coumarin-NPs was validated by performing a competitive uptake study along with pre-treatment of different concentration of native Tmab in SKBR 3 cells. The results demonstrated the inhibition of uptake of Tmab-6-coumarin-NPs in cells pre-treated with increasing concentration of native Tmab (Fig. 3b), thus stressing the key role of HER 2 receptor in receptor mediated endocytosis of our targeted NPs. The uptake of Tmab6-coumarin-NPs compared to unconjugated NPs was further investigated by confocal microscopy. A similar trend in uptake of both the NPs was evident in SKBR 3 cells and MDA-MB-231 cells (Fig. 3c) corroborating our cellular uptake results as measured through fluorescence spectrophotometer.

3.1. Synthesis and characterization of TPGS–Tmab conjugates

3.4. In vitro cytotoxicity of Tmab functionalized rapamycin loaded NPs

The synthesis of TPGS–Tmab conjugates was performed by DCC–NHS conjugation chemistry as depicted in Scheme 1. Successful conjugation of Tmab to TPGS was confirmed by FT-IR and 1H NMR spectral analysis (Fig. 1). As evident from the FT-IR result (Fig. 1a) of TPGS–Tmab conjugates, presence of peak at 3420 cm1 which corresponds to NAH stretching, a high intense peak at 1653 cm1 corresponding to carbonyl (C@O) stretching of the amide bond (C@OANHA) and the peaks at 1539 cm1 and 669 cm1 illustrating NAH bending and out-of-plan NAH bending respectively, confirmed the conjugation of Tmab onto TPGS. In the

Cytotoxicity results depict an augmented cytotoxic effect of Tmab-rapa-NPs in comparison to unconjugated counterpart in SKBR 3 cells, while no significant difference in cytotoxicity was observed in MDA-MB-231 cells (Fig. 4a and b). The IC50 values of native rapa, Rapa-NPs and Tmab-rapa-NPs as obtained from the above study are shown in Table 1. Results demonstrated that Tmab-rapa-NPs and Rapa-NPs was 11 times and 4 times more effective than that of native rapamycin respectively as observed in SKBR 3 cells. Further, both Tmab-rapa-NPs and Rapa-NPs was 2 times effective than that of native rapamycin in MDA-MB-

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(a)

3431.68

2920.31

1108.50 1646.59 2359.39

1457.86 1299.89

1734.89

1351.371250.73

952.04 848.86

TPGS

526.38

2926.60

1449.47 3408.57 3024.59

698.71 1645.571494.00 1736.25 1379.631243.16

Absorbance

2361.83

1029.45

758.89

547.45

Tmab 3420.14 2925.00

1653.41 1539.56

2360.79 2343.52

1249.21 1350.72 1457.62

1108.52 953.02

1734.06

3839.09

669.02

502.20

848.62

TPGS-Tmab conjugate

1107.95

1351.21 1455.29 1643.79 1415.31 1250.26 1738.08 1537.77 1300.45

2924.22 3389.77

991.82 950.57 574.76 848.15

TPGS/Tmab Mixture

4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

350.0

Wave length (cm-1)

(b) TPGS

TPGS/Tmab mixture

Tmab

TPGS - Tmab conjugate

Fig. 1. Characterization of TPGS–Tmab conjugates (a) FT-IR spectral analysis of TPGS, Tmab, TPGS–Tmab conjugates and TPGS/Tmab mixture. (b) 1H NMR spectra of TPGS, Tmab, TPGS/Tmab mixture and TPGS–Tmab conjugates.

231 cells. Hence, the above results illustrated that Tmab-rapa-NPs can effectively inhibit the cell proliferation as compared to RapaNPs and native rapa treatments. It is also worth mentioning that

Tmab used in our study is non-toxic in nature (upto 500 ng/ml) and the concentration of void NPs used for this study was also found to be nontoxic (data not shown).

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O

(a)

NH

TPGS

TPGS - Tmab conjugate QD605 Rapamycin

(b)

12

Intensity (%)

10 8 6 4 2 0 1

10

100

1000

10000

Size (d.nm)

(c)

(d)

(e)

Fig. 2. Physico-chemical characterization of nanoformulations. (a) Diagrammatic representation of Tmab-QD-rapa-NPs. (b) Size distribution of Tmab-QD-rapa-NPs measured by zetasizer (n = 3). (c) The representative picture of Tmab-QD-rapa-NPs by atomic force microscopy. (d) The representative image of Tmab-QD-rapa-NPs by transmission electron microscopy (bar = 100 nm). (e) In vitro drug release kinetics study of Rapa-NPs and Tmab-rapa-NPs in PBS at 37 °C. Error bar shows mean ± SD (n = 3).

3.5. Western blot analysis Rapamycin has been shown to exhibit apoptotic cell death by inhibiting phosphorylation of Akt, mTOR, p70S6K1 and 4E-BP1 in

mTOR signalling pathway followed by inhibition of phosphorylation of Stat-3 [42,43]. To explore the mechanism of apoptotic cell death following treatment with either native rapamycin or rapamycin loaded different nanoformulations, expression of some

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a (ii)

a (i)

b

** ***

*** ***

c

SKBR 3 cells 6-coumarin

Propidium iodide

Bright Field 0

µm 250

0

µm 250

0

µm 250

Merged

Native 6-coumarin

6-coumarin-NPs

Tmab-6-coumarin-NPs

MDA-MB-231 cells 0

µm

75

0

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75

0

µm 75

Native 6-coumarin

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Tmab-6-coumarin-NPs

Fig. 3. Analysis of cellular uptake of different nanoformulations by fluorescence spectrophotometer and confocal microscopy. (a) Quantitative cellular uptake study of native 6-coumarin, 6-coumarin-NPs and Tmab-6-coumarin-NPs (40 ng/ml) in (i) SKBR 3 cells and (ii) MDA-MB-231 cells by fluorescence spectrophotometer after 2 hrs of incubation (The values are shown as mean ± SD, n = 4) (⁄⁄⁄)p < 0.0001 and (⁄⁄)p < 0.005, 6-coumarin-NPs and Tmab-6-coumarin-NPs verses native counterparts. (b) Competitive uptake study of Tmab-6-coumarin-NPs in SKBR 3 cells treated with different concentrations of native antibody for 2 hrs, followed by treatment with Tmab-6-coumarin-NPs (40 ng/ ml) for another 2 hrs. Then the uptake was measured by fluorescence spectrophotometer (values are shown as mean ± SD, n = 2). (c) Qualitative intracellular uptake of native 6-coumarin, 6-coumarin-NPs and Tmab-6-coumarin-NPs (50 ng/ml) in SKBR 3 cells and MDA-MB-231 cells by confocal microscopy after 2 hrs of incubation. Experiment was done three times and a representative figure has been provided.

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3.6. Study of mitochondrial membrane potential loss

SKBR 3 cells

(a)

* * * **

** **

*

During early apoptotic event disruption of active mitochondrion occurs, resulting in loss of mitochondrial membrane potential (MMP). Fig. 6a shows the loss of MMP following treatment with native rapa, Rapa-NPs and Tmab-rapa-NPs in SKBR 3 cells. It is important to note that, treatment with Tmab-rapa-NPs exhibited a greater percentage of cells with MMP loss as compared to that of native drug and unconjugated counterpart. 3.7. Apoptosis analysis by flow cytometry

*** The anticancer drug rapamycin is known to induce apoptosis by inhibiting mTOR pathway [13,44]. Apoptosis results demonstrated that Tmab-rapa-NPs treated cells showed higher percentage of apoptotic cell death than that of Rapa-NPs and native rapa treated cells (Fig. 6b). It is noteworthy to mention that, Tmab-rapa-NPs treated cells depicted augmented apoptotic cell death compared to all other treatments.

MDA-MB-231 cells

3.8. In vitro cellular imaging

(b)

* **

*** **

***

***

Fig. 4. Dose dependent cytotoxicity study of native rapa, Rapa-NPs and Tmab-rapaNPs in (a) SKBR 3 cell line and (b) MDA-MB-231 cell line after 5 days of drug treatment by MTT assay. Data are expressed as mean ± SEM, n = 3, (⁄⁄⁄)p < 0.001, (⁄⁄) p < 0.01 and (⁄)p < 0.05, rapa-NPs and Tmab-rapa-NPs verses native rapa.

key proteins were explored by western blot analysis. Results depict enhanced down regulation of p-Akt, p-mTOR, p-p70S6K1, p-4E-BP1 and p-Stat-3 in Tmab-rapa-NPs treated SKBR 3 cells than that of the unconjugated counterpart and native drug (Fig. 5). Apart from the above finding, enhanced down regulation of anti-apoptotic proteins like BCL-2 and c-Myc was observed in Tmab-rapa-NPs as compared to Rapa-NPs and native rapa treatment.

Table 1 IC50 values of native rapa, Rapa-NPs and Tmab-rapa-NPs in different cancer cells as observed by cell cytotoxicity assay. IC50 (ng/ml) Sample Native rapa Rapa-NPs Tmab-Rapa-NPs

SKBR 3 cells 111.07 ± 25.5 27.94 ± 9.6* 9.84 ± 1.58*

MDA-MB-231 cells 111.3 ± 18.93 50.55 ± 8.46* 58.41 ± 15.68

Data represented as mean ± SEM, n = 3. * p < 0.05 for native rapa vs Rapa-NPs or native rapa vs Tmab-rapa-NPs.

The current researches on semiconductor quantum dots as biological imaging agent have emerged due to its captivating optical properties over organic dyes [29]. Here, in this present study, we have investigated the imaging potentiality of Tmab conjugated or unconjugated QD605 loaded NPs through in vitro cellular imaging study in 2D monolayer and 3D spheroid model by confocal microscope. Confocal microscopic image of 2D cultured cells treated with Tmab-QD-rapa-NPs showed higher fluorescence intensity than that of the unconjugated counterpart (Fig. 7a), suggesting the imaging capability of the targeted NPs. The imaging modality was further validated in multi-cellular 3D spheroids as they mimic the in vivo tumor [41]. Confocal images clearly indicate higher fluorescence intensity in tumor spheroid treated with Tmab-QD-rapaNPs than that of unconjugated NPs treated case. The Z-stack images at the middle layer of the tumor spheroid (Z4 and Z5) showed higher fluorescence intensity than that of Z0 or Z9 which were located at top and bottom respectively in SKBR 3 spheroid model treated with Tmab-QD-rapa-NPs than that of QD-rapaNPs, demonstrating deeper penetration ability of targeted multifunctional NPs into the spheroid model which is a prerequisite for in vivo imaging (Fig. 7b). 4. Discussion Over the last few years, development of novel diagnostic and therapeutic agents has dramatically improved cancer diagnosis and treatment respectively. However, in the present scenario, molecularly targeted theranostic approach for simultaneous cancer detection, inhibition and therapeutic response evaluation has emerged as an encouraging therapeutic line of attack against cancer [45]. Importantly, recent advances in nanotechnology have enabled the development of different targeted nanotheranostic platforms [31,46,47]. In this context, with an objective of simultaneous delivery of therapeutic payload and diagnostic agent at tumor site, the present study involves developing Tmab functionalized rapamycin and QD605 loaded multifunctional NPs for breast cancer therapy and diagnosis. Physico-chemical characterization of nanoformulation is a prerequisite for developing an efficient drug delivery system, as it greatly influences the therapeutic response of the payload both in vitro and in vivo settings. We have formulated an efficient drug delivery system of nanometer size range having negative surface charge with sustained drug release profile (Fig. 2b–e) which could

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2

1

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p–Akt/ β-actin 0.395 0.497 0.304 0.045 2

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p-p70S6K1/ 0.639 0.659 0.569 0.129 β-actin

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0.752 0.505 0.331

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p-Stat-3 β-actin p-Stat-3/ β-actin

1.007 0.872 0.662 0.59

Fig. 5. Western blot analysis showing expression of signalling proteins following treatment with native rapa/Rapa-NPs/Tmab-rapa-NPs (50 ng/ml equivalent concentration of rapamycin) in SKBR 3 cells after 24 hrs of treatment. b-actin serves as loading control. Lane 1 – Control cells; Lane 2 – Native rapa treated cells; Lane 3 – Rapa-NPs treated cells; and Lane 4 – Tmab-rapa-NPs treated cells.

evade the reticuloendothelial system by escaping macrophage uptake resulting in enhanced drug accumulation at the targeted site for a longer period of time facilitating prolonged cytotoxicity [15,48,49]. The biological stability of NPs in serum represents a foremost requirement for its biomedical applications [50]. In the present study, the formulated NPs exhibited improved serum stability for a longer period of time, suggesting the potentiality of our system to be used as a delivery vehicle. Site specific targeting strategy has been the call of the hour for the present cancer therapeutic approaches for enhanced internalization of nanoformulations [32,51]. In our study, the uptake of Tmab-6-coumarin-NPs was considerably higher than that of unconjugated NPs in SKBR 3 cells but no significant difference in the cellular uptake pattern was evident in MDA-MB-231 cell line (Fig. 3). This may have resulted due to the higher expression of HER 2 in SKBR 3 breast cancer cells thereby facilitating more internalization of targeted NPs [52,53]. In this context, Steinhauser et al. have reported superior uptake of Tmab modified Human serum albumin (HSA) NPs in comparison to PEGylated HSA NPs in HER 2 positive SKBR 3 and BT-474 breast cancer cells, however no significant difference in uptake behavior of above NPs was observed in HER 2 negative MCF-7 cells [54]. The above findings corroborate our observations and support towards the role of HER 2 in the enhanced uptake of targeted NPs. The role of HER 2 in higher cellular internalization process was further confirmed by competitive uptake study. For validating the concept that the enhanced internalization of targeted NPs may be responsible for eliciting enhanced cellular toxicity, cytotoxicity assay was performed. The augmented cytotoxic response of Tmab-rapa-NPs than the unconjugated NPs in SKBR 3 cells and similar cytotoxicity of both Tmabrapa-NPs and Rapa-NPs in MDA-MB-231 cell line, accentuate the

key role of receptor mediated NPs binding and enhanced internalization in the augmentation of cytotoxicity (Fig. 4, Table 1). Rapamycin has been reported to block mTOR pathway by inhibiting phosphorylation of Akt, mTOR and its downstream targets p70S6K1 and 4E-BP1 to execute cytotoxic effect [8,11,37]. Further, Stat-3, a downstream target of mTOR is known to be activated in different type of tumors including breast cancer and rapamycin inhibits phosphorylation of Stat-3 causing cell growth inhibition [43]. The mechanism of cell death following rapamycin treatment was explored at the molecular level by studying some of the key regulatory proteins of mTOR signalling pathway. Western blot analysis reveals enhanced down regulation of the proteins like p-Akt, p-mTOR, p- p70S6K1, p-4E-BP1 and p-Stat-3 (Fig. 5) following treatment with Tmab-rapa-NPs in comparison to other treatments. Moreover, rapamycin is also reported to down regulate the expression of proteins like BCL-2 and c-Myc both of which play a major role in cell survival in different cancer types [55]. Our result showed enhanced down regulation in expression of BCL-2 and c-Myc following treatment with Tmab-rapa-NPs than other treatments (Fig. 5). Notably, in all cases Tmab-rapa-NPs showed greater efficacy in modulating the expression of above mentioned proteins compared to native rapamycin and unconjugated NPs following site specific drug delivery. The mechanisms of action of Tmab-NPs inducing apoptosis is presented schematically in Fig. 8. In the present study, an augmented loss of MMP was observed in Tmab-rapa-NPs treated SKBR 3 cells than the unconjugated counterparts and native drug (Fig. 6a). Loss of MMP may have elicited the apoptotic process as evident from higher apoptotic cell death in cells treated with Tmab-rapa-NPs as compared to unconjugated NPs and native drug (Fig. 6b). Superior cell death inducing ability of Tmab-NPs may have

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Control

Native rapa

a

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Tmab-rapa-NPs

Control

Native rapa

Rapa- NPs

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b

Fig. 6. Analysis of (a) mitochondrial membrane potential loss and (b) induction of apoptosis by flow cytometry. (a) SKBR 3 cells were treated with 50 ng/ml of native rapa, Rapa-NPs and Tmab-rapa-NPs for 24 hrs. After completion of incubation period, cells were treated with CMXRos (100 nM) for 1 hr in dark and then washed with PBS and MMP loss was measured by flow cytometry. Experiment was performed in triplicates and a representative picture has been given. (b) Induction of apoptosis in SKBR 3 cells treated with 50 ng/ml of native rapa, Rapa-NPs and Tmab-rapa-NPs for 24 hrs and apoptosis percentage was analyzed by Annexin-V FITC and 7-AAD staining using 10,000 gated cells through FACScan flow cytometer. Experiment was performed in triplicates and a representative picture has been given.

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Z1

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b QD-rapa-NPs

Tmab-QD-rapa-NPs

Fig. 7. In vitro bioimaging study was done by confocal microscopy in SKBR 3 cells grown as (a) 2D monolayer and (b) 3D spheroid model. The cells were treated with QD-rapaNPs and Tmab-QD-rapa-NPs (NPs concentration 50 lg/ml and 500 lg/ml for 2D and 3D culture respectively) for 4 hrs and then image were captured by confocal microscope. Experiment was performed for three times and a representative picture has been given.

resulted due to the higher accumulation of drug at the target site following enhanced cellular uptake through receptor mediated endocytosis with sustained drug release phenomenon [56,57]. Among a wide spectrum of imaging probes available, the unique optical properties of semiconductor quantum dots gives them an upper edge to be used as a new generation imaging regime both in vitro and in vivo settings [58,59]. In this context, in the present study we have formulated Tmab-QD-rapa-NPs for exploring its site specific imaging capability. The imaging results in SKBR 3 cells grown as 2D monolayer or tumor spheroid advocate towards the target specific imaging potentiality of Tmab-QD-rapa-NPs as compared to the unconjugated counterpart (Fig. 7). In a recent study

Lin et al. have developed daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS2 quantum dot (DNR–MUC1–QDs) conjugates and their results suggest the target specific internalization and imaging potentiality of above systems in MUC1 expressing PC-3M cells following receptor mediated endocytosis, thus corroborating our observation [60]. Further, in 3D tumor spheroid imaging study, our Tmab-QD-rapa-NPs have shown deeper penetrating capability compared to the unconjugated counterpart as shown from different z-scan confocal images (Fig. 7b). In this context, Savla et al. have demonstrated higher internalization and penetration ability of MUC1 aptamer conjugated QD in comparison to unconjugated QD in MUC1 expressing A2780/AD ovarian

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Fig. 8. Schematic representation of molecular mechanism of cell death induced by Tmab-rapa-NPs in SKBR 3 cells.

carcinoma cells grown in 3D spheroids [47]. Thus from the above study, it can be anticipated that, such a multifunctional theranostic nanosystem may be explored for achieving a nanotheranostic platform for better management of cancer. 5. Conclusion In spite of the considerable improvement on cancer diagnosis and treatment options available till date, there is an urgent need to improvise these approaches for complete remission of the disease. In this milieu, multifunctional Tmab conjugated NPs were developed for targeted co-delivery of a therapeutic agent rapamycin and an imaging agent QD605 for early detection and for improved therapeutic index of rapamycin. These targeted lipid based nanoparticulate systems are used to achieve better cellular uptake through receptor mediated targeting. The targeted NPs showed greater cytotoxic effect in HER 2 positive cells which were further confirmed by loss of MMP with enhanced apoptosis and by modulating different signalling molecules of mTOR pathway. In addition, the targeted multifunctional NPs also have revealed its effectiveness as imaging modality under in vitro system both in monolayer and spheroid culture. It is noteworthy to mention that the targeted multifunctional NPs can be used for targeted co-delivery of therapeutic and imaging agents through receptor mediated targeting. Here, we have also demonstrated that the targeted multifunctional NPs can be effective in 3D tumor spheroid model which mimics in vivo tumor. Thus, the above multifunctional NPs may be used as an effective vehicle for nanotheranostic approach in the current clinical settings for cancer treatment.

Bhubaneswar for use the NMR facility provided by Department of Science and Technology (DST), Mr. Bhabani Shankar Sahoo, Institute of Life Sciences, for his help during the confocal experiments, The Director, Institute of Life Sciences, Bhubaneswar for providing institutional fellowship to (P.P), Mr. Priyadarshi Roy for his help in taking images in Atomic force microscopy (AFM).

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