Potential Nanomedicine Applications of Multifunctional Carbon

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Potential nanomedicine applications of multifunctional carbon nanoparticles developed using green technology Jadi Praveen Kumar, Rocktotpal Konwarh, Manishekhar Kumar, Ankit Gangrade, and Biman B. Mandal ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03557 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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ACS Sustainable Chemistry & Engineering

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Potential nanomedicine applications of multifunctional carbon nanoparticles developed using green technology

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Jadi Praveen Kumar, Rocktotpal Konwarh, Manishekhar Kumar, Ankit Gangrade and Biman B. Mandal*

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Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering,

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Indian Institute of Technology Guwahati (IITG), Guwahati-781039, Assam, India.

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*Author for correspondence: [email protected], [email protected], Phone: +91

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3612582225

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Abstract

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Carbon nanonmaterial development through green technology is gaining pace owing to their

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biocompatibility, inertness, modifiability and photoluminescence. These smart nanomaterials

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are much sought after and have great potential in bioimaging and drug delivery. In this study,

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we focused on the preparation of carbon nanoparticles (CNPs) using edible yogurt drink

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(lassi) by microwave irradiation. The physicochemical properties of synthesized CNPs were

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extensively studied. Results demonstrated that CNPs had average size of 12.58 ± 0.60 nm

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with zeta potential of -24.62 ± 0.15 mV. Cytocompatibility of CNPs assessed using L929 and

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rat primary VSMCs, demonstrated enhanced viability after 48 h incubation. At lower

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concentrations of CNP, intracellular calcium levels remain unaffected in VSMCs.

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Doxorubicin (Dox) was used as model molecule to evaluate sythesized CNPs for their

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efficacy in drug delivery. Dox-loaded CNPs (Dox-CNPs) showed pH-dependent (pH 4.6 and

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7.4) drug release. Toxicity of Dox-CNPs assessed with MCF-7 and SAS cell lines indicated

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IC50 values at 0.25 µg/mL. Cell cycle arrest, elevation of reactive oxygen species and loss of

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inner mitochondrial membrane potential corroborated efficient delivery of Dox to the nuclei

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with enhanced activity. The successful delivery of drug into the nuclei and its subsequent pH-

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dependent release projects CNPs as promising drug delivery vehicles in nanomedicine

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approach.

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Keywords: Carbon nanoparticles, Drug delivery, Doxorubicin, ROS, Anti-cancer,

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Membrane potential.

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Introduction

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Cancer is one of the most aggressive diseases in the world that kills millions of people every

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year.1 Cancer represents uncontrolled growth of cells, which lacks senescence mechanism

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through genetic and phenotypic changes. These changes lead to

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therapeutic resistances.2 There are several treatment modalities such as chemotherapy,

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radiotherapy, immunotherapy, hormonal therapy and surgery to treat cancer.3-4 Chemotherapy

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is the most common and effective method wherein anticancer drugs are mainly administered

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intravenously.1 The administration of such anticancer drugs result in adverse side effects such

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as cardiac toxicity, neutropenia, vomiting and nausea.5 These side effects are majorly due to

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the non-specificity of the drugs.6 Delivery of chemotherapeutic drugs to the targeted site is

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reported to reduces side effects, enhances activity with lower dose administration.6

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Nanocarriers are gaining much interest in the field of drug delivery due to their ability to

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carry therapeutic molecules to the target site with enhanced activity.1 In the field of drug

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delivery, liposomes, microspheres, emulsions and cyclodextrins have been extensively

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proposed and studied.1 In comparison with polymer-based nanoparticles or organic lipids,

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inorganic carbon nanomaterials exhibit unique assets like chemical inertness, stability and

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ease of modifications.1 Different formats of carbon nanomaterials like carbon nanotubes,

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nanodiamonds, graphene, and fullerenes have been developed and used for bio-imaging and

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drug/gene delivery.7 Of late, carbon based nanomaterials have received considerable impetus

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as drug immobilization vehicles.8-10 Previously, the efficacy of C60+ doxorubicin (Dox)

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composite was documented as a new pharmacological agent that effectively kills tumour cells

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in vitro while preventing the toxic side effects of the free form of Dox on normal cells.8

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Similarly, highly mesoporous carbon nanospheres have been reported for Dox delivery and

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cancer therapy10 while supramolecular interactions have been exploited to functionalize

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carbon dots with Dox for pH dependent drug release.9

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In this context, our current work is directed towards investigating the prospects of carbon

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nanoparticles (CNPs) prepared using an affordable bioresource and via green chemistry

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approach as an effective Dox ferry system. At this juncture, it is relevant to mention that

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numerous research articles and reviews have projected carbon-based quantum dots (CD/C-

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dots) as rising star in the niche of material science.11-12 High photostability, tunable emission,

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non-blinking fluorescence, large two-photon excitation and even multiphoton imaging have

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endowed special impetus to carbon dots for novel applications.13 Traditionally, the

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preparation protocol comprises of synthesis of raw carbon dots, purification, passivation and

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clinical diversity and

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functionalization.12 The omission of the critical steps of passivation and functionalization

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often results in low quantum yield and consequently limits the use of C-dots.12 These

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bottlenecks necessitate endeavours to devise a simpler strategy with easier functionalization

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approach for high quantum efficiency.12

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Selecting an appropriate starting material is an important aspect and in this direction the use

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of readily available and affordable biomass for the preparation of carbon nanomaterials has

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been a recent trend. In this context, carbon dots have been prepared using banana, egg shell

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ash, orange juice, soya-milk, pomelo-peel, glycerol etc.11 Furthermore, carbon nanoparticles

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have been reported in different carbohydrate based food caramels, including bread, jiggery,

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biscuits etc., and the preparation protocol of which involved heating of the starting material.14

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Recently, hydrothermal heating of milk at 180 °C for 2 h has been reported to generate

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fluorescent carbon dots for prospective application in bioimaging.15 We have probed into the

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prospects of using lassi (a popular, traditional, yoghurt-based drink, from the Indian

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subcontinent) as the starting material for the preparation of CNPs based on Maillard reaction

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(Figure S1) using the microwave as a fabrication approach. Microwave is a form of

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electromagnetic energy16 and material processing using microwave as a green methodology

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is drawing much attention.17 When compared with the conventional industrial processes this

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technique conserves energy and improves efficiency.17 Microwave-assisted material

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processing

avoids the use of harsh chemical (strong acid/alkali) and is relatively cost

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effective.18

Processing or cooking of food items using microwave results in chemical reaction

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between amino acids and reducing sugars leading to formation of Maillard reaction products

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(MRPs), which may be beneficial or toxic to the health, depending on the processing

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conditions.19 It has also been noted that diverse MRPs act as antioxidants, bactericidal, anti-

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browning, anti-allergenic and pro-oxidants agent.20

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In this study, we have assessed the prospects of microwave irradiation to prepare carbon

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nanoparticles (CNPs) from lassi. The various physicochemical characterization of the CNPs

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with high quantum yield have been complemented by the assessments of their free radical

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scavenging activity, cytocompatibility, and immunocompatibility along with their effect on

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store operated calcium entry. The CNPs have been projected as a suitable vehicle for the non-

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covalent loading and efficient delivery of the chemotherapeutic agent, Dox. We have

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assessed the potency of the pH-responsive Dox-CNP hybrid system against two cancer cell

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lines, MCF-7 (breast cancer cell line) and SAS (tongue cancer cell line) in the context of

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alteration in membrane potential, cytotoxicity, cell-cycle arrest and nuclear localization. The

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various experimental evidences attested the immense prospects of the system for possible

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biomedical translation.

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Materials and Methods

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Materials. Potassium bromide (KBr), quinine sulphate, antibiotic-antimycotic

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solution and trypsin-EDTA were procured from Himedia, India. Sulphuric acid (H2SO4),

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dimethyl sulfoxide (DMSO) and acetic acid were sourced from Merck, India. 2, 2-Diphenyl-

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1-picrylhydrazyl (DPPH), ascorbic acid, doxorubicin (Dox), lipopolysaccharide (LPS) from

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Escherichia coli, sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor

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thapsigargin, thiazolyl blue tetrazolium bromide, RNase A, neutral buffered formalin (NBF),

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Hoechst33342 and dichloro-dihydro-fluorescein-diacetate (DCFH-DA) were obtained from

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Sigma, USA. Dulbecco’s modified eagle media (DMEM), fetal bovine serum (FBS) were

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procured from (Gibco, USA) and Mouse TNF α ELISA kit, fura-2 AM, pluronic acid,

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propidium iodide (PI), JC-1 assay kit was supplied from Invitrogen, USA.

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Preparation of carbon nanoparticles (CNPs). The lassi (Amul India Pvt. Ltd.) was

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procured from the local market. The nutritional composition of lassi is shown in Table S1

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(Supporting information). Ultra-pure water (18.2 MΩ cm-1, Milli-Q, Millipore) was used in

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all the experiments. The preparation of the CNPs was performed in the household microwave

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system (LG, model: MS-2349EB, India). The reaction was carried out in a 50 mL glass vessel

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using 20 mL of the lassi. The reaction vessel was placed in the microwave and heated for 6.5

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min at 800 W.

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During the heating process, brown coloured nitrogenous polymers were produced. Post de-

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plugging, the reaction vessel was cooled to room temperature and kept in a desiccator for

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about 15 min to absorb the volatile low molecular weight products. The formed carbonaceous

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products were solubilized in 20 mL water, followed by centrifugation (5000 rpm, 15 min).

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The carbonaceous particles were re-dispersed in 20 mL water, filter-sterilized and used for

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further characterization or kept at 4 °C until further use.

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Fourier transform infrared (FTIR). The IR spectra of CNPs were recorded by

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FTIR spectrophotometer (Spectrum two FTIR spectroscopy, PerkinElmer) in the region of

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4000-600 cm-1. The sample was prepared as pellets using spectroscopic grade KBr and

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spectrum was attained by accumulation of 32 scans with a resolution of 4 cm-1.

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Dynamic light scattering (DLS). The size and surface charge of the CNPs were

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determined using dynamic light scattering (DLS) (Nano ZS Zetasizer, Malvern). The

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measurement was carried out at 25 °C and the scattering angle was fixed at 90°.

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High resolution transmission electron microscope (HRTEM). The morphological

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and microstructural visualization of the CNPs were performed in HRTEM (JEOL, JEMCXII,

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operating voltage 200 kV).

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Fluorescence studies of carbon nanoparticles. Absorbance spectra of CNPs in the

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ultraviolet-visible (UV-visible) region was recorded on a UV-visible spectrophotometer

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(Lambda 750, PerkinElmer). Fluorescence measurements were carried out using Fluoromax-

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4 spectrofluorometer (HORIBA Scientific).

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The quantum yield of the CNPs was assessed through a comparative approach using the

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following equation

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QCNP= Qref × ICNP/Iref × Aref/ACNP× ηCNP 2/ ηref 2

……….(1)

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where ‘Q’ is the quantum yield, ‘I’ is the intensity of luminescent spectra, ‘A’ is the

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absorbance at excited wavelength and ‘η’ is the refractive index of the solvent being used

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while quinine sulfate (quantum yield 54%) in 0.1 M H2SO4 solution served as the reference.

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The subscripts used in this equation are CNPs and ‘ref’ to indicate our test sample and the

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reference, respectively.

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Fluorescence decay times of the CNPs were measured on an Edinburgh Instruments, UK,

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FSP920 equipped with the light emitting diodes (excitation wavelengths 375 nm) at room

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temperature.

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DPPH scavenging activity. DPPH scavenging activity of CNPs was determined

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using the protocol reported by Kumar et al.21 Briefly, 100 µL of different concentration (5-50

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μg/mL) of CNPs were added to 100 μL of 0.2 mM of DPPH prepared in methanol (Merck,

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India). Post incubation for 1 h under dark, absorbance was measured at 520 nm using a

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multiplate reader (Tecan Infinite M200). Ascorbic acid (Sigma, USA) was used as the

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positive control.

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Biophysical characterization of doxorubicin loaded carbon nanoparticles (Dox-

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CNPs). Dox was loaded on CNPs by adsorption. In brief, 1:2 (w/w) of CNPs and Dox was

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kept on constant shaking for 12 h at room temperature. Post 12 h, the solution was subjected

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to centrifugation at 14,000 rpm for 30 min followed by dissolution of the pellet using 1 mL

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Millipore water. For uniform distribution, the Dox loaded CNPs (Dox-CNPs) were sonicated

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(SONICS, VC-505, 20 kHz, acoustic power density 500 W/cm2, USA) for 10 min (with 5 s/5

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s ON/OFF pulse cycle) at 20% amplitude kept at 4 °C. Post sonication, the size and zeta

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potential (ζ-potential) of the Dox-CNPs was measured using Nano ZS Zetasizer (Malvern

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Instruments). Fabricated Dox-CNPs were stored at 4 °C until used further.

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The collected supernatant was used for analysis of free drug in order to determine the loading

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capacity (LC) (%). Subtracting the amount of drug left in the supernatant from the initial

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amount of drug allowed us to determine the nanoparticle drug loading. The loading capacity

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(%) was determined using the following equation:

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LC (%) = (Total Dox – Free Dox/Nanoparticle weight) × 100

………….(2)

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To quantify the in vitro drug release in solution, small aliquots (200 μL) of Dox−CNPs were

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rapidly added to equal volumes (3.8 mL) of PBS thermostated at 37 °C and gently shaken.

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The release behaviour of Dox from the complex was studied at two different pH levels (pH

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4.6 and pH 7.4). At defined time intervals, the fluorescence emission intensities of the

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solutions were measured at 593 nm to determine the amount of released Dox. The percentage

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of Dox released was calculated according to the following equation 22 % Dox release = (If/It) × 100 %

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………….(3)

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Where If is the fluorescence emission intensity of free (released) Dox, measured at specific

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time intervals (between 0 and 24 h), and It is the fluorescence emission intensity of total Dox

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loaded onto CNPs.

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Cell culture. Mouse fibroblast (L929), human breast cancer (MCF-7), murine

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macrophages (Raw 264.7) (procured from NCCS, Pune), tongue cancer (SAS) cell lines and

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vascular smooth muscle cells (VSMCs) (primary cells, procured from Gauhati University)

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were cultured in DMEM with high glucose, supplemented with 10% FBS, 1X antibiotic-

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antimycotic solution.

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Cytocompatibility of the CNPs. Cytocompatibility of the CNPs was evaluated by

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MTT assay. L929 and VSMCs were plated at a density of 1×104 cells/well in 96 well plate

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and incubated for 24 h at 37 °C in 5% CO2 atmosphere. Post incubation, wells were

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replenished with fresh media containing different concentration (10, 25, 50 and 75 μg/mL) of

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the CNPs (filtered through sterile 0.22 µm sterile filter) and incubated for another 24 h. Post

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incubations, 20 µL of MTT solution (5 mg/mL in phosphate buffered saline, PBS, pH 7.4)

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was added to each well. Post 4 h of incubation, MTT solution was removed and formazan

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crystals were solubilized with 100 µL DMSO. Absorbance was recorded in multiplate reader

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(Tecan Infinite 200) at 570 nm.

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Tumour necrosis factor (TNF) α release study. Immunogenicity of CNPs was

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evaluated by the release of TNF α when co-incubated with RAW 264.7. For TNF α study,

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cells were seeded at a density of 5×104 cells/well in 12-well cell culture plate and left

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overnight. Further, CNPs were added to culture wells. TCP wells with similar cell density

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(without CNPs) were taken as negative control. Plates containing 500 ng/mL LPS were taken

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as positive control. Post 12 h and 24 h of incubation, media was collected and stored at -20

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°C. The released TNF α was determined with ELISA kit (mouse TNF α ELISA kit,

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Invitrogen, USA) as per manufacturer's instructions. The TNF α release by macrophage was

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calculated from standard curve and plotted accordingly.

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Intracellular Ca+2 measurement studies. In order to study the effect of the CNPs on

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store operated calcium entry (SOCE), 1×104 VSMCs were seeded per well in 96 well plates.

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Post 24 h, cells were treated with CNPs of different concentrations (5, 10, 15, 25 and 50

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μg/mL) and incubated for 24 h in 5% CO2 incubator at 37 °C. Fura-2 AM stock solution

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(1mM) was prepared in DMSO. VSMCs were incubated with 2 µM Fura-2 AM and 0.01%

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pluronic acid in standard bath solution (SBS) (NaCl 135 mM, KCl 5 mM, MgCl2 1.2 mM,

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Glucose 8 mM, HEPES 10 mM and CaCl2 1.5 mM) for 1 h at 25 °C. The cells were then

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incubated with sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor

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thapsigargin in calcium free SBS for 30 min at 25 °C. Data were recorded immediately post

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addition of SBS containing 3 mM Ca2+. Intracellular Ca2+ ([Ca2+]i) concentration was

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measured fluorometrically as ratio of emission intensities for the two excitation wavelengths,

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340 nm and 380 nm for emission recorded at 510 nm in Novostar micro plate reader.23

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In vitro cytotoxicity study. For assessment of in vitro cytotoxicity of Dox and Dox-

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CNPs, MCF-7 and SAS cell lines were used through MTT assay. MCF-7 and SAS cells were

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plated at a density of 1×104 cells/well in 96 well plate and incubated for 24 h. Post

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incubation, spent media within wells were replaced with fresh media containing different

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concentration (0.125, 0.25, 0.5, 0.75 and 1 μg/mL) of free Dox and Dox-CNPs followed by

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incubation for 24 h. After incubation, 20 µL of MTT solution (5 mg/mL in PBS at pH 7.4)

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was added to each well. Post 4 h of incubation, MTT solution was removed and formazan

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crystals were solubilized in DMSO. Absorbance was recorded in multiplate reader (Tecan

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Infinite 200) at 570 nm.

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Nuclear localization of doxorubicin loaded CNPs. In order to study the

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internalization of the complex, cultured cells (2 × 105 cells/well) were treated with 0.25

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µg/mL of Dox and Dox-CNPs for 24 h. Intracellular fluorescence was determined post

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processing of the cells for analysis by flow cytometry (FACS, Caliber). Furthermore, nuclear

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localization of Dox and Dox-CNPs was assessed using Hoechst33342. Post treatment of the

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cells with 0.25 µg/ mL of Dox and Dox-CNPs for 24 h, spent media was removed followed

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by PBS washing and fixing with neutral buffered formalin (NBF) for 10 min. Cell membrane

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was permeabilized using 3:1 ratio of NBF and acetic acid followed by Hoechst33342

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staining. Post staining, cells were visualized for nuclear localization of Dox and Dox-CNPs

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using fluorescence microscopy (EVOS FLC, Life technologies).

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Cell cycle analysis. Dox and Dox-CNPs induced cell cycle arrest was studied using

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PI. 2 × 105 cells/well were seeded in 6 well plate and incubated at 37 °C for 24 h. Cultured

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cells were treated with 0.25 µg/mL free Dox and Dox-CNPs for 24 h. Post treatment, cells

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were harvested by trypsin-EDTA. The effect of trypsin was neutralized by addition of 6 mL

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complete media and followed by centrifugation at 1500 rpm for 5 min. The cell pellet was re-

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suspended in cold PBS (pH 7.4) and centrifuged for 5 min at 1500 rpm. The cell pellet was

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vortexed in 70 % (v/v) chilled ethanol, followed by incubation at -20 °C for 30 min. After

243

incubation, the pellet was centrifuged at 1500 rpm for 5 min and re-suspended in PBS,

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followed by centrifugation. This was followed by re-suspension of the cell pellet in 200 µL

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PBS containing 0.1 mg/mL RNase A and incubation for 30 min at 37 °C. Post incubation,

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800 μL PBS containing 20 μL PI solution (1 mg/mL) was added and incubated for 20 min in

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the dark at 4 °C. After 20 min of incubation, the resulting suspension was analysed with a

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flow cytometer (FACS Calibur, BD).

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Determination of intracellular reactive oxygen species (ROS). For the

250

measurement of intracellular reactive oxygen species (ROS), cultured cells (2×105 cells/well)

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were treated with Dox and Dox-CNPs for 12 h. After treatment, cells were incubated with 10

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μM DCFH-DA for 1 h at 37 °C. Fluorescence resulting from the hydrolysis of DCFH-DA to

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DCHF was measured by flow cytometry.

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Mitochondrial membrane potential (ψ ψ) study. Modulation in inner mitochondrial

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membrane potential of tumour cells after Dox-CNPs and Dox treatment was evaluated by

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using JC-1 assay kit. Cultured cells were treated with 0.25 µg/mL of Dox for 12 h. After

257

treatment, cells were harvested and suspended in 1 mL PBS. According to the manufacturer’s

258

protocol, the suspended single cells were treated with the JC-1 dye and incubated for 30 min

259

at 37 °C. Post-incubation, 2 mL of PBS was added and the cells were centrifuged at 1500 rpm

260

for 5 min. This was followed by re-suspension in 1 mL PBS. Mitochondrial membrane

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potential was assessed by flow cytometry (FACS Calibur, BD).

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Statistical analysis. All quantitative experiments were carried out atleast in triplicate

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(n=3). Results are conveyed as a mean ± standard deviation. Statistical analysis was carried

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out using one-way ANOVA with Holm-Sidak method using Sigma-plot software. Statistical

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difference between groups in the range of #p≤0.05 was considered statistically significant and

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values in the range of ##p≤0.001 as highly significant.

267 268

Results

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Dynamic light scattering microscopy (DLS). CNPs were characterized for their size

270

and surface charge using DLS. The DLS result (Figure S2) indicated that the average

271

hydrodynamic radius of the CNPs was 12.58 ± 0.60 nm with a negative zeta potential (ζ)

272

value of -24.62 ± 0.15 mV, vouching for the stability of the nanoparticles (without any

273

aggregation) in aqueous solution.

274

High resolution transmission electron microscope (HRTEM). Size and

275

morphology of the CNPs were assessed using HRTEM. The HRTEM images of the CNPs

276

were depicted in Figure 1. They appeared spherical in shape, clustered nearby (Figure 1A)

277

and non-homogeneously distributed. From the inverse fast Fourier transform (IFFT) image

278

(Figure 1C) the interplanar distance was found to be ~0.3 nm while the diameter was 11.57 ±

279

1.12 nm.

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Figure 1. (A) HRTEM micrograph of CNPs clustered nearby, (B and C) IFFT image of

282

representative CNP.

283 284

Fourier transform infrared (FTIR) spectroscopy. Structural conformation of CNPs

285

was recorded using FTIR. The FTIR analysis of CNPs (Figure 2) displayed the stretching of

286

C-OH and N-H at 3395 cm-1, stretching of C-H at 2923 cm-1 and 2853 cm-1, C-N vibration at

287

1237 cm-1, the vibrational peak of C=O at 1634 cm-1, furthermore, the peaks at about 1405

288

cm-1, 1237 cm-1 and 1056 cm-1 were also indicative of the presence of C-N, S=O and S=C

289

group. 9



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Figure 2. FTIR spectra of CNPs.

292 293

Fluorescence study of carbon nanoparticles. The UV-visible absorption spectrum

294

of CNPs in water is depicted in Figure 3A. The maximum absorption peak of the CNPs was

295

observed in the UV region at 282 nm and a tail was extended till visible region. The

296

photoluminescence (PL) spectra of CNPs fluorescence spectra of the CNPs (Figure 3B)

297

showed maximum fluorescence intensity at λex 360 and λem 460 nm. The quantum yield of the

298

prepared CNPs was found to be 45.51%. Single photon timing technique was used to collect

299

the fluorescence decay trace of the prepared CNPs in water. The decay curve for the YG

300

based CNPs in water could be best fitted with a double-exponential function. The fast

301

component, τ1 was 0.648 ns while τ2 was 3.189 ns for λex = 375 nm (Figure 3C).

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Figure 3. (A) UV-visible spectrum, (B) Dependence of PL emission peak of the CNPs on the

305

excitation wavelength; (C) Data and fitted curve showing the PL decay time of the CNPs (λex

306

= 375 nm).

307 308

Biophysical characterization Dox loaded CNPs (Dox-CNPs). The change in the

309

size and surface charge of the CNPs after Dox loading was determined by DLS (Figure S3).

310

Dox-CNPs showed an average hydrodynamic radius of 13.08 ± 0.90 nm with a zeta potential

311

of 7.24 ± 0.21 mV. 8% Dox loading capacity was achieved using CNPs. Further, Dox release

312

was assessed at two different pH levels of 4.6 and 7.4. This was to mimic the physiological

313

pH and the acidic environment of the tumour tissue, respectively. The rate and amount of

314

Dox released from the nano-hybrid system is depicted in Figure 4. In the first 2 h, ~13% of

315

the initial Dox content was released at pH 7.4. After 24 h of incubation, the amount of

316

released Dox reached 21% at pH 7.4, which proved the stability of the nanochemotherapeutic

317

system at physiological pH. On the contrary, a much faster release (~38% during the first 2 h)

318

was observed when the Dox-CNPs were incubated at pH 4.6. Further, a sustained release

319

trend was recorded post 24 h of incubation. The cumulative Dox released amount reached 11



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320

approximately ~58% at pH 4.6 (pH of tumours). In comparison with physiological pH 7.4,

321

significantly higher release of Dox was observed pH 4.6 (p≤0.001) indicating pH-

322

responsiveness of the Dox CNP system.

323 324

Figure 4. Effect of pH on DOX release from CNPs at 37 °C (##p≤0.001 in comparison to

325

drug release at pH 7.4).

326 327

Cytocompatibility study. Cytocompatibility of the CNPs was analysed by MTT

328

assay using VSMCs and L929 cells. Figure 5 (A) and (B) represents cellular viability of L929

329

and VSM cells after incubation with CNPs at various concentrations for 24 and 48 h. Post 24

330

h of CNPs treatment L929 cells showed lesser viability in comparison to control (p≤0.05).

331

Whereas, VSMCs remained unaffected for low concentration of CNPs. However, on

332

increasing dosage of CNPs (50 and 75 µL/mL) lowered their viability (p≤0.05). Further, post

333

48 h CNPs treatment, both VSMCs and L929 cells exhibited comparable cell growth with

334

control.

12


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335 336

Figure 5. Effect on (A) L929 and (B) VSMC cell viability after treatment with different

337

concentration of CNPs for 24 and 48 h. (#p≤0.05 and ##p≤0.001 in comparison to control).

338 339

Intracellular Ca+2 [Ca2+]i measurement. Cellular uptake of nanoparticles might

340

affect the cascade of signalling pathways. The effect of CNPs on signalling pathways was

341

assessed by analysing the store operated calcium entry (SOCE), which is an important

342

physiological pathway to maintain intracellular calcium level. Modulation in the [Ca2+]i level

343

after CNPs treatment were detected using fura 2-AM. The magnitude of SOCE recorded in

344

primary rat VSMCs remained unaffected at lower concentrations (up to 15 μg/mL) of CNPs

345

post 24 h treatment. However, at higher concentrations (25 μg/mL and 50 μg/mL) SOCE was

346

significantly reduced (Figure 6).

347 348 349

Figure 6. (A) SOCE amplitudes in rat primary VSMCs treated under the indicated

350

conditions. (B) Measurement of SOCE in rat primary VSMCs treated for 24 h with CNPs at

13



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351

different concentrations. Traces recorded post exposure to thapsigargin in the absence of

352

extracellular calcium, followed by the reintroduction of calcium. (#p ≤ 0.05 in comparison to

353

control).

354 355

TNF α release study. Macrophages are the primary source of inflammatory responses

356

in the body. On activation, they secrete cytokine mediators and regulate immune

357

responses. TNF α is an important pro-inflammatory cytokine that plays a pivotal role in

358

regulating inflammation and immune response 24. We assessed the impact of the CNPs on the

359

immunological response in terms of TNF α release by murine macrophages (RAW 264.7).

360

TNF α released by RAW 264.7 cells after CNPs treatments are depicted in Figure 7. RAW

361

264.7 cells treated with CNPs displayed significantly low release of TNF α in comparison

362

with LPS treated cells.

363 364

Figure 7. TNF α production by RAW 264.7 murine macrophages in response to stimulation

365

by the CNPs. (##p≤0.001 in comparison to LPS treated cells).

366 367

Cytotoxicity study of Dox-CNPs. In vitro cytotoxicity of Dox and Dox-CNPs at

368

different concentrations against two different cancer cell lines, MCF-7 and SAS were

369

evaluated using MTT assay. It is relevant to note that the CNPs alone did not show any

370

cytotoxic effect on these cell lines (data not shown). Percentage of cell viability after Dox and

371

Dox-CNPs treatment is presented in Figure 8. In comparison to the control (untreated), Dox

372

and Dox-CNPs treated MCF-7 and SAS cells showed significantly low percentage of cell

373

viability (p≤0.001). 0.25 µg/mL of Dox-CNPs showed 50% (IC50) killing for both MCF-7 and

374

SAS cells. 14


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375 376

Figure 8. In vitro cytotoxicity assessment. Percent (%) viability of (A) MCF-7 and (B) SAS

377

cells after treatment with different concentrations of free Dox and Dox-CNP for 24 h. Data

378

are expressed as mean ± S.D (n=3). (##p≤0.001 in comparison to control).

379 380

Nuclear localization of doxorubicin loaded carbon nanoparticles. Cellular uptake

381

of Dox and Dox-CNPs was evaluated by using flow cytometry. Intracellular fluorescence

382

intensity of Dox is shown in Figure S4. Dox-CNPs treated MCF-7 and SAS cells showed

383

enhanced fluorescence intensity than free Dox treated cells and control, respectively. Nuclear

384

distribution of Dox was qualitatively examined by fluorescence microscopy. MCF-7 and SAS

385

cells (Figure 9) treated with free Dox showed faint red fluorescence signals. On the contrary,

386

strong red fluorescence signals were observed from the nucleus of MCF-7 and SAS cells

387

treated with Dox-CNPs. At this juncture it is pertinent to note that Hoechst 33342 dye

388

(emitting blue-cyan fluorescent light) binds to the AT-rich minor grooves of double-stranded

389

DNA. The merged panels, (D+H) in each case vouched for the localization of Dox in the

390

nucleus.

15



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391

16


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392

Figure 9. Localization of Dox in the nucleus after treating (A) MCF-7 and (B) SAS cells

393

with Dox and Dox-CNPs for 24 h. (i) control (untreated) cells, (ii) Dox and (iii) Dox-CNPs

394

treated cells. (Scale 200 µm)

395 396

Cell cycle analysis. The effect of free Dox and Dox delivered by CNPs on the cell

397

cycle of MCF-7 and SAS was analysed using PI. Figure 10 depicts the gated percentage of

398

MCF-7 and SAS cells indicating their presence in different stages of cell division. Gated cell

399

population represented in G1 (where DNA is present in uniform size) and sub-G1 (where

400

DNA is chopped into smaller fragments), correspond to cell cycle arrest and cell death by

401

apoptosis, respectively. In comparison with the control (untreated cells), Dox and Dox-CNPs

402

treated MCF-7 cells showed significantly high (p≤0.001) percentage cell population in sub-

403

G1 phases. Dox-CNPs treated MCF-7 cells showed significantly lower percentage of cell

404

population in G1, S and G2 phases in comparison with the control.

405

On the other hand, Dox and Dox-CNPs treated SAS cells showed significantly higher gated

406

cell population in sub-G1 phase in comparison with the control (p≤0.001). With respect to the

407

control, the G1 phase of Dox-CNPs treated SAS cells showed enhanced cell population.

408

Lower percentage of gated cells population was seen in G2. Furthermore, Dox treated SAS

409

cells showed significantly low percentage of the gated cell population in G1 and G2 phases

410

when compared with the control (p≤0.001). However, Dox treated SAS cells showed

411

enhanced cell population in S phase.

412

413

17



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Page 18 of 42

414

Figure 10. Cell cycle analysis of (A) MCF-7 and (B) SAS cells: Where (a) control

415

(untreated), (b) Dox treated and (c) Dox-CNPs treated cells. Data are expressed as mean ±

416

S.D (n=3).

417 418

Intracellular ROS measurement. Oxidative stress induced by Dox elevates the ROS

419

in cancer cells.25 The elevated levels of ROS was assessed using

420

fluorescent dye, which is activated in presence of ROS and becomes fluorescent.26

421

Intracellular ROS levels of MCF-7 and SAS cells are depicted in Figure 11. MCF-7 cells

422

treated with Dox-CNPs showed enhanced fluorescence intensity in comparison with Dox-

423

treated and control (untreated) cells, respectively. SAS cells treated with Dox and Dox-CNPs

424

showed negligible change in the fluorescence intensity when compared to control.

DCFH-DA, a non-

425 426

Figure 11. Representative flow cytometer profile for intracellular ROS production by (A)

427

MCF-7 and (B) SAS cells after treatment with (a) control (untreated) cells (b) Dox and (c)

428

Dox-CNPs.

429 430

Mitochondrial membrane potential (ψ ψ) study. The change in mitochondrial inner

431

membrane potential is a sensitive marker of early mitochondrial damage during apoptosis.27

432

The membrane permeant JC-1 dye shows potential-dependent accumulation in mitochondria,

433

as revealed by fluorescence emission shift from green to red.27 A dip in the red/green

434

fluorescence intensity ratio is indicative of mitochondrial depolarization. Analysis of such

435

fluorescence ratio assists in making comparative quantification of membrane potential.

436

Carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial un-coupler was used

437

for compensation. The change in mitochondrial membrane potential in MCF-7 and SAS cells

438

post Dox and Dox-CNPs treatment are presented in Figure 12. In comparison with the control

18


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439

(untreated cells), Dox-CNPs-treated MCF-7 cells showed 10 fold depletion in red

440

fluorescence. Whereas, Dox treated MCF-7 cells displayed 1.42 fold depletion in red

441

fluorescence. SAS cells treated with Dox-CNPs showed 3.18 fold depletion in red

442

fluorescence when compared to control. On the other hand, Dox treated cells showed 1.29

443

fold depletion in red fluorescence.

444 445 446

Figure 12. Mitochondrial membrane potential studies of (A) MCF-7 and (B) SAS cells. (a)

447

control cells stained with JC-1 dye, (b) control cells treated with CCCP and stained with JC-1

448

dye, (c) Dox treated cells stained with JC-1 dye, and (d) Dox-CNPs treated cells stained with

449

JC-1 dye.

450 451

Discussion

452

In cancer treatment, nanotechnology based interventions much attention due to their unique

453

applications in drug delivery, imaging, diagnosis and therapeutics.28 Nanocarriers are known

454

to reduce the adverse side effects of chemotherapeutic drugs and enhance their activity by

455

delivering them to the target site.28 Several nanocarriers such as liposomes, polymeric

456

micelles and albumin nanoparticles are approved in many countries for cancer treatment.28 In

457

recent times, carbon nanocarriers developed through nanotechnology are attaining much

458

impetus due to their inertness, stability and cytocompatibility.29 Carbon nanocarriers are

459

prepared using different methods to obtain specific properties.18 Laser ablation and high-

460

energy ion beam radiations are two common methods for preparation of carbon nanocarriers,

461

however these methods use expensive precursors and energy systems.30-31 In order to

462

minimize the cost, chemical methods are adopted to prepare carbon nanocarriers. However, 19



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463

the use of strong acids for preparation of carbon nanocarriers are undesirable

464

hazardous.18 In this direction, microwave radiation mediated carbon nanocarrier preparation

465

and modification is a promising technology due to the strong interaction of microwave

466

radiation with carbon species.32 The main focus of the present study was to prepare carbon

467

nanoparticles (CNPs) using an edible drink product (lassi) through microwave irradiation

468

pursuing a green technology. The fabricated CNPs were physico-chemically characterized

469

and further assessed for in vitro cytotoxicity of Dox-loaded CNPs (Dox-CNPs).

470

Milk contains proteins (casein and whey), carbohydrates (lactose and glucose), fat (saturated

471

and unsaturated fatty acids) minerals and water.33-34 Yogurt drink (lassi) is a milk-based

472

product and lysine is the second major amino acid of casein and whey protein of milk.33

473

Lysine and reducing sugar (lactose and glucose) of lassi undergoes aldol condensation after

474

irradiation with microwave and forms lactulosyllysine (Maillards reaction products).35 Fat

475

and other solid residues are removed by centrifugation and filtration. The size, surface

476

charge, fluorescence and antioxidant properties of the carbon nanoparticles (CNPs) depend

477

on the chemical interactions formed between lysine and reducing sugars (lactose and

478

glucose).36 HRTEM study showed the aggregated CNPs, which might be due to the presence

479

of added sugar.37 FTIR study showed the condensation between amino acids of proteins and

480

reducing sugars.36 UV/Vis absorption spectra further confirmed absorbance maxima at 282

481

nm which attributes to the aromatic amino acids and π–π* transition of the C=C band.36, 38

482

Change in the fluorescence intensity at different wavelength depends on their size (a quantum

483

effect) and different energy traps on the CNPs.38 MRPs have been shown to possess

484

antioxidant activity by scavenging free radicals.19 DPPH scavenging activity of CNPs (Figure

485

S5) is attributed to the sulfhydryl group of cysteine amino acids residues of MRPs.19

486

Most of the MRPs elevates the reactive oxygen species (ROS) levels which lead to depletion

487

of energy, inhibition of store operated calcium entry (SOCE) and oxidative cell death.39-40

488

Further change in the redox balance upregulates the release of pro-inflammatory cytokines.39

489

Cytocompatibility studies of CNPs displayed low cell viability after 24 h treatment, which

490

might be due to oxidative stress generated by CNPs. However, cells recovered from the

491

oxidative stress post 48 h treatment and regained viability similar to controls. At higher

492

concentrations (25 and 50 µg/mL) of CNPs might have elevated the oxidative stress in the

493

VSMCs that leads to inhibition of SOCE. Whereas, changes in redox balance after CNPs

494

treatment might not have attained optimum levels to stimulate pro-inflammatory cytokines

495

(TNF α) release by murine macrophages (Raw264.7). A lower concentration (

Potential Nanomedicine Applications of Multifunctional Carbon

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