Gelatin stabilization of quantum dots for improved stability and biocompatibility

Accepted Manuscript Title: Gelatin stabilization of quantum dots for improved stability and biocompatibility Authors: Sundararajan Parani, Kannaiyan Pandian, Oluwatobi Samuel Oluwafemi PII: DOI: Reference:

S0141-8130(17)32991-4 http://dx.doi.org/10.1016/j.ijbiomac.2017.09.039 BIOMAC 8216

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

10-8-2017 11-9-2017 13-9-2017

Please cite this article as: Sundararajan Parani, Kannaiyan Pandian, Oluwatobi Samuel Oluwafemi, Gelatin stabilization of quantum dots for improved stability and biocompatibility, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.09.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gelatin stabilization of quantum dots for improved stability and biocompatibility Sundararajan Parani,a,b Kannaiyan Pandian,c and Oluwatobi Samuel Oluwafemi,*a,b a

Department of Applied Chemistry, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa b Centre for Nanomaterials Science Research, University of Johannesburg, Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa c Department of Inorganic Chemistry, University of Madras, Maraimalai (Guindy) Campus, Chennai–600025, India *Corresponding author: Oluwatobi Samuel Oluwafemi E-mail: [email protected] Phone/Fax: +27115599060 Highlights   

Gelatin stabilized CdTe/CdS/ZnS core/double shell quantum dots (QDs) were prepared The core/double shell structure and gelatin protection were confirmed

The QDs were highly luminescent with excellent stability over a period of one year  Gelatin stabilization reduced the cytotoxicity of QDs by about 50 % 

The QDs were served as fluorescent probe for in vitro imaging of HeLa cells

Abstract We herein report an aqueous synthesis of gelatin stabilized CdTe/CdS/ZnS (CSSG) core/double shell quantum dots (QDs) with improved biocompatibility. The as-synthesized QDs were characterized by ultraviolet-visible (UV-Vis) and photoluminescence (PL) spectroscopic techniques, x-ray diffraction technique (XRD), x-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The CSSG QDs revealed high photoluminescence quantum yield (PLQY) with excellent stability over a period of one year and retained 90 % of its initial PLQY without any aggregation or precipitation under

1

ambient condition. The cell viability study conducted on HeLa, cervical cancer cell lines indicated that the gelatin stabilization effectively decreased the QDs cytotoxicity by about 50 %. The CSSG QDs were conjugated with transferrin (Tf) for the efficient delivery to the cancer cells followed by fluorescence imaging. The results showed that the CSSG QDs illuminates the entire cell which renders the QDs as cell labeling markers. The gelatin stabilized core/double shell QDs are potential candidates for long time fluorescent bioimaging. Keywords: Quantum dots; Gelatin; Cytotoxicity 1.

Introduction Polymer nanocomposites have gained many interests in recent years since they offer

added advantages such as enhanced stabilizing, thermal and mechanical properties along with their specific functional properties [1-4]. In this regard, natural biopolymers are of special interest compared to synthetic polymers, due to their abundance in nature, hydrophilicity [5, 6], bio-degradability, bio-compatibility etc. Long-chain polymer molecules can cover the nanomaterials, interact with the capping ligands and surface metal atoms, stabilizes the material and preserves their core properties for long time. Over the past two decades, semiconductor fluorescent nanoparticles also called quantum dots (QDs) have been in the lime light due to their unique properties which greatly differ from those of bulk counterpart and their application has been explored in diverse areas such as biomedical field, sensors, catalysis, drug delivery [7-9]. Especially, they have been emerging as fluorescent markers for biomolecular and cellular imaging [10-13]. Compared with traditional organic fluorophores, QDs exhibit excellent properties such as size-dependent optical properties, wide absorption spectra, narrow photoluminescence spectra and good photo- and chemical stability. Several techniques have been reported for the synthesis of QDs and these can be broadly divided into organic phase and aqueous phase approaches [14]. The QDs prepared in the organic medium 2

typically have hydrophobic capping ligands which are not suitable for their biological applications and hence the need for ligand exchange with hydrophilic ligands. During this process, the hydrophobic capping agent is replaced with simple bifunctional ligands such as mercapto-carboxylic acids, in which the mercapto group binds onto the QD surface while the carboxyl group makes QDs water soluble [15,16]. However, the QDs after the ligand exchange are usually characterized with decreased PLQY compared to the parent QDs. Alternatively, direct aqueous method has been developed to synthesize water soluble QDs using water soluble ligands such as mercapto-carboxylic acids, mercapto-amines as capping agent [17, 18]. Compared to organic phase synthesis, aqueous synthesis involves less toxic greener approach, inexpensive and the products have good water solubility and biocompatibility. However, the as-prepared QDs often still exhibit low PLQY due to the presence of surface trap states which act as nonradiative de-excitation channels for the photogenerated charge carriers [19, 20]. A general approach to increase the PLQY of QDs is growing epitaxial layers of wide bandgap semiconductor over the core QDs to obtain a core/shell QDs structure [21, 22]. Earlier, we have shown the dramatic increase (70 %) in PLQY of QDs by growing multishells (CdS & ZnS) over the core CdTe QDs [23]. This enhancement in the PLQY is attributed to the suppression of surface defects of parent QDs by the shells. Even though the core is protected with shells, the simple capping ligands are still the one that acts as the stabilizing agents for the core/shell QDs. These small ligands can undergo dynamic binding and unbinding with the QD surface which causes them to get desorbed from the surface [24]. This can occur through excessive washing, irradiation, storage under open atmosphere. Furthermore, changes in pH, high salt concentration or induced dipole interaction also results in the agglomeration of the particles. In our previous report [25], the core/double shell QDs (CdTe/CdS/ZnS) lost 50 % of initial PL intensity during the storage over 10 months. 3

Therefore, simple ligands are insufficient in stabilizing the QDs. In such a case, polymers can offer the stability. As the QDs are sterically kept apart within the matrices of polymer, their aggregation is prevented [25]. The reports on the stabilization of QDs by the combination of inorganic shell protection and polymer material for biological applications is rather limited. Wang et al [26], synthesized hexadecyalamine (HDA) capped CdSe/CdS/ZnS QDs in organic medium and replaced the HDA by hydrophilic diblock copolymers to make water soluble, polymer stabilized core/double shell QDs. Nie et. al [27], prepared trioctylphosphine oxide (TOPO) capped CdSe/ZnS QDs in organic medium followed by exchanging the TOPO ligand with MPA. The MPA capped CdSe/CdS core/shell QDs were then embedded in chitosan polymer using coupling reaction. However, these QDs were synthesized in organic medium, employed toxic and expensive materials like TOPO, HDA etc which are not ecofriendly. The synthesis also needs high temperature and inert conditions and the QD material must be subjected to the ligand exchange process for biological applications which is a tedious process. Aswathy et. al [28], reported aqueous synthesis of thioglycolic acid (TGA) capped CdTe/CdSe QDs embedded in gelatin for bio-imaging application. These QDs are green emitting which often encounters problem of auto-fluorescence caused by biological samples. In addition, they tend to aggregate after 30 days of storage in the dark demanding additional stabilization. Hence, there is a need to synthesize red emitting QDs with high stability, biocompatibility via greener method for bio-imaging applications. Natural biopolymers such as gelatin, dextran, alginate, chitosan etc, exhibit excellent biocompatibility and biodegradability. They have been broadly examined for drug delivery and tissue engineering applications for decades. Among them, gelatin is widely used in food and pharmaceutical industries as a binder because of its gelling nature [29]. It is a denatured protein obtained from collagen and has been used as a stabilizing agent for the nanoparticles 4

of Ag [30] Au [31] and QDs [32, 33]. In this work, we report, for the first time, the aqueous synthesis of gelatin stabilized CdTe/CdS/ZnS (CSSG) core/double shell QDs. The assynthesized CSSG QDs were characterized by UV–Vis, PL, FT–IR spectroscopic techniques, TEM, XRD, and XPS techniques. Throughout the work, we demonstrated the collective advantage of gelatin stabilization and double shell protection of core CdTe QDs. These CSSG QDs are red emitting, exhibits high PLQY, excellent biocompatibility and exceptional stability (> 1 year) compared to core/shell QDs or polymer stabilized QDs alone. The CSSG QDs were conjugated with transferrin and utilized for in vitro fluorescence imaging of HeLa cancer cell lines.

2.

Experimental

2.1.

Materials All chemicals were of analytical grade and used as received. Cadmium chloride hemi

pentahydrate (CdCl2· 2.5H2O), potassium tellurite trihydrate (K2TeO3· 3H2O), mercaptosuccinic acid (MSA), sodium borohydride (NaBH4), anhydrous zinc chloride (ZnCl2) and thioacetamide were procured from Sigma-Aldrich, India. Gelatin was purchased from Fluka, India. N-(3-di-methyl-aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), n-hydroxy-succinimide (NHS), 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT salt) and transferrin were obtained from Merck. Cell culture medium, fetal bovine serum (FBS), were from Invitrogen, India. HeLa cell lines were obtained from National Centre for Cell Science (NCCS), India. All solutions were prepared using doubly distilled (DD) water.

2.2.

Synthesis of gelatin stabilized CdTe (CG) QDs 5

The gelatin stabilized CdTe QDs was synthesized based on our previous report [23]. Briefly, 1 mmol (0.228 g) of CdCl2 and 3 mmol (0.450 g) of MSA were mixed in a 40 mL of boratecitrate buffer followed by adjusting the pH to 7.2 using 1 M NaOH to form cadmium thiol complex. Subsequently gelatin (0.040 g) dissolved in 10 ml of hot DD water was added. A fresh solution of NaHTe prepared by the reduction of potassium tellurite (0.0769 g) using NaBH4 (0.020 g) was then quickly injected into the above mixture and heated to reflux for 6 h. The mole ratio of Cd:Te:MPA was 1:0.25:3. The as-prepared gelatin stabilized CdTe (CG) QDs were precipitated from the reaction solution by adding ethanol and collected via centrifugation.

2.3.

Growth of CdS and ZnS shells

1 mmol (0.228 g) of CdCl2 and 3 mmol (0.450 g) of MSA were added to the buffer solution of purified CG QDs and the pH of the solution was adjusted to 7.2. This was followed by the dropwise addition of 0.25 mmol (0.0188 g) of aqueous solution of thioacetamide. The reaction solution was heated at ~75 °C to obtain gelatin stabilized CdTe/CdS core/shell (CSG) QDs. ZnS shell was further grown on the purified CSG QDs in a similar way using 1 mmol (0.136 g) of zinc chloride, 3 mmol (0.450 g) of MSA, and 0.25 mmol (0.0188 g) thioacetamide to obtain gelatin stabilized CdTe/CdS/ZnS (CSSG) QDs. The CSSG QDs were purified as above and dispersed in DD water. 2.4.

Preparation of CSSG-Tf bioconjugates

CSSG QDs were conjugated to transferrin using EDC and NHC coupling method [34]. Typically, 2 mg of transferrin was added to 1 mL of CSSG QDs under stirring, followed by addition of EDC (1 mg) and NHS (0.1 mg) at room temperature. The stirring was continued

6

for overnight. The CSSG-Tf conjugates were collected using ultracentrifuge for 30 min at 50,000 rpm and redispersed in phosphate buffer solution (PBS, pH =7.4). 2.5.

Characterization

UV-Vis absorption spectra were obtained with a Shimadzu (UV-1800) spectrophotometer. PL measurements were performed using a Perkin Elmer LS5B spectrofluorimeter. The PLQY (Φ) of the QDs was calculated according to the following formula.

Φ(%) = ΦR ×

I × AR × 100 IR × A

where Φ and ΦR are the PLQY of sample and standard, I and IR are the integrated intensities of the sample and standard, A and AR are the absorbance of sample and the standard. The reference standard was an ethanolic solution of rhodamine 6G with the quantum yield, ΦR = 0.95. XRD patterns of the samples were obtained on Brucker D8 Advance diffractometer with monochromatic Cu-Kα1 radiation (λ = 1.5418 Å). TEM images were taken from a FEI Technai G2 (T-30). Fluorescence imaging experiments were carried out on standard fluorescent microscope at excitation wavelength of 428 nm. The amount of cadmium present in the aqueous solution of QDs was determined by inductively coupled plasma optical emission spectrometry (ICP- OES) technique using a Varian Vista AX spectrometer. X-ray photoelectron spectra (XPS) of the samples were performed on Omicron Nanotechnology spectrophotometer with monochromatic Al radiation (Kα = 1486 eV). 2.6.

Cytotoxicity assessment

The MTT assay was used to evaluate the viability of HeLa cells treated with QDs [35]. The cells were seeded in 96-well plates at a density of approximately 1×104 cells/well and kept at 37 °C for 24 hours in a 5 % CO2 incubator. This was followed by the addition of QDs

7

dissolved in the culture medium. After 48 hours, the medium was discarded and the cells were rinsed with PBS. A fresh medium containing MTT (5 mg/mL) was then added to each well and the microplates were incubated for overnight. The formazon crystals formed were dissolved by dimethyl sulfoxide (DMSO) and the absorbance of each well was recorded at 540 nm using ELISA microplate reader (Bio-Rad 680). A control experiment was conducted similarly in the absence of QDs. All measurements were made in quadruplicate. The percentage of cell viability was calculated by following equation:

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 % =

Absorbance of test cells × 100 Absorbance of control cells

3.

Results and discussion

3.1.

Synthesis and Characterization

The synthesis of gelatin stabilized, thiol capped core/double shell QDs involves three stages viz; (i) synthesis of gelatin stabilized CdTe QDs followed by successive growth of (ii) CdS and (iii) ZnS shells as shown in Fig. 1. Initially, cadmium chloride is made to react with mercaptosuccinic acid followed by gelatin at pH 7.2 to form a gelatinated cadmium thiolate complex. The polypeptide and protein functional groups of gelatin facilitate the dispersion of this complex within its matrices. The borate-citrate buffer medium is utilized to maintain the pH of the solution. After the quick injection of NaHTe, the colorless reaction solution immediately turns into light brown indicating the formation of CdTe clusters which serve as seeds for nucleation and growth of CdTe QDs. The CdS and ZnS shell growth were carried out by the addition of the corresponding precursors to the purified parent QDs solution

8

followed by heating at reduced temperature at which the growth of core CSG QDs is minimized. Fig.1 The UV-Vis absorption and PL spectra of the gelatin stabilized QDs shown in Fig. 2a and 2b indicates the stepwise formation of CdTe/CdS/ZnS core/double shell structured QDs. The absorption bands related to CdS and ZnS were not detected in the spectra of CSG and CSSG QDs. Instead a redshift was observed during the growth of the shells suggesting the formation of core/shell structured QDs [36,37]. In a core/shell type QDs, there is a reduction in quantum confinement due to the delocalization of charge carriers across the core/shell barrier through tunneling effect and this results in the red-shifting of the PL spectra. The fluorescence image of the gelatin stabilized core-shell QDs samples on excitation with the UV lamp (λexc = 352 nm) is shown in the Fig. 2b inset. The QDs emit color from yellowish green to reddish orange with gradual increase in brightness indicating the effective surface protection by the shells. The CSSG QDs emit in the red region with the PL maximum at 611 nm. It is found that PLQY of CSG (42.1 %) and CSSG (56.9 %) QDs are higher than that of CG QDs (32.3 %). It is also found that the QY of all gelatin stabilized QD samples is higher than that of QDs without gelatin stabilization This enhanced PLQY can be accredited to the elimination of the surface defects by the effective stabilization of gelatin. Fig.2 Figure 2d illustrates the stability of the integrated PL intensity of CSSG QDs at ambient condition. The gelatin stabilized CSSG QDs is exceptionally stable and retains its PL intensity (~90 %) for more than a year while the QDs without gelatin stabilization lost 50% stability within a year. Because of its random coil structure, gelatin can diffuse into and around the capping layer, and interact directly with both the capping agent and QD surface 9

[31, 38]. This prevents the aggregation of QDs and reduces the number of non-radiative centres thereby increasing PLQY. The formation of core/double shell structure was further investigated by analyzing the crystal nature of the QDs. Figure 3a shows the XRD patterns of the gelatin stabilized QDs and that of gelatin derived from hot gelatin solution. Three broad peaks centered at 2θ = 14°, 30° and 42° were observed for gelatin which are attributed to its amorphous nature. These peaks also appeared in all QDs indicating the presence of gelatin in all the samples. In all cases, these broad peaks predominate which diminishes the diffraction patterns by the QDs. Hence for a comparison, XRD of the CdTe/CdS/ZnS (CSS) core/double shell QDs synthesized without gelatin stabilization was examined which exhibited the cubic zinc blende structure (Fig S1). Thus, it is presumed that gelatin stabilized QDs also have cubic zincblende crystal structure. The (111) peak of CG is shifted towards ZnS crystal phase accompanied with the increase in its intensity after sequential CdS and ZnS shell growth which indicates the preferential formation of core/shell (CSG) and core/shell/shell (CSSG) QDs. Fig.3 The morphology of CSSG QDs analyzed by TEM is shown in Fig. 3b. From the TEM image it is seen that the size of the QD is ~ 5 nm. A selected area electron diffraction (SAED) pattern from CSSG QDs is shown in the Fig. 3b inset. Ring patterns indicate the cubic phase of CdTe and can be indexed to the (111), (220) and (311) planes. The corresponding energy dispersive x-ray spectrum (EDS) spectrum in Fig. 3c displays the signals of C, O, Cd, Te, S and Zn elements, indicating the presence of QDs and the gelatin. Gelatin stabilized QDs were further examined by XPS and the corresponding survey scan is presented in Fig. 4a. The survey XPS shows different signals corresponding to C, O, 10

Cd, Te, S, and Zn for the CSSG QDs. This is similar to that of the CSS QDs without gelatin stabilization (Fig S2). However, the intensity of C 1s and O 1s peaks are as high as that of Cd for CSSG QDs. Such high intensity peaks were not observed for QDs without gelatin stabilization Hence the strong intensity of these peaks can be majorly attributed to the gelatin polymer. In addition, the presence of N 1s peak can be seen along with Cd 3d peak energy levels (Fig. 4b). These results clearly indicate the presence of gelatin in the QDs. It is found that the cadmium intensity is increased after the CdS shell growth and it remains the same for the ZnS shell. In the high-resolution spectrum of Te 3d5/2, two peaks around 572.5 and 576 eV are seen attributed to Te bonded to Cd (Te-Cd) and oxidized Te (TeOx) respectively. It can be seen from this figure that the integrated area ratio of Te-Cd to Te-Ox is higher for CSSG when compared to that of CSS. This indicates that gelatin stabilization effectively prevents the QDs surface from oxidation. In the S 2p spectrum of gelatin stabilized QDs (Fig.4b), a weak broad band is observed for CG. Addition of CdS and ZnS shells over the CG QDs generates clear spectrum with the peak centered at 161.5 eV representing its sulfide form [39]. All these results suggest the proposed gelatin stabilized core/double shell structure of QDs. Fig. 4 3.2.

Cytotoxicity assessment

The effect of gelatin stabilization on the biocompatibility of QDs was analyzed by MTT assay using HeLa cells and the results are shown in Fig. 5a. The cell viability is shown against the amount of cadmium present in each QDs [Cd]QD, as determined by ICP-OES analysis according to previous reports [23, 40]. It is observed that more than 80 % of the HeLa cells treated with all three types (CG, CSG and CSSG) of QDs are alive upto the [Cd]QD =10 µg mL−1 where CSSG QDs showing ~100 % cell viability. Also, at any given 11

concentration, HeLa cells treated with CSSG QDs exhibited higher cell viability compared with either CG or CSG QDs. This can be clearly seen at [Cd]QD = 100 µg mL-1, for which, the viability is ~ 80 % for [Cd]CSSG, whereas it is 55% for [Cd]CSG and 35% for [Cd]CG QDs. Fig. 5. Half maximal inhibitory concentration, IC50, of [Cd]CG QDs is measured to be 40 µg mL−1, whereas that of [Cd]CSG and [Cd]CSSG QDs increased to 97 and 224 µg mL−1 respectively. From the above results, it is found that CSSG QDs are more biocompatible than both the CSG and CG QDs which can be attributed to the combined passivation of gelatin and the double shell. As expected, at any given concentration, all gelatin stabilized QDs have a higher number of viable cells compared to QDs without gelatin stabilization. The IC50 values of gelatin stabilized QDs are relatively two times (50%) higher compared to QDs without gelatin stabilization (Fig. 5b). This can be attributed to the use of gelatin as an additional stabilizing agent. 3.3.Cancer cell imaging For active labelling of cancer cells, the QDs were conjugated to transferrin (Tf), using EDC coupling reaction as shown in Fig. S3. Figure 6 illustrates the fluorescent microscopic images of HeLa cells incubated with CSSG-Tf bioconjugates in the bright field mode, dark field fluorescent mode and the overlapped images. From the bright-field image of the cells (Fig. 6a), it is seen that the cells incubated with CSSG QDs maintain their normal morphology. This indicates their good biocompatibility with CSSG QDs. In the fluorescent image (Fig. 6b), it is found that most of bright spots occupy the cytoplasm of the cells and some are found even in the nucleus. The overlap of bright field and dark field fluorescent images shown their well correlation (Fig.6c). The CSSG QDs illuminate almost the entire cell, rendering the QDs as cell-labeling markers. 12

Fig. 6 4. Conclusion Water soluble gelatin stabilized CdTe/CdS/ZnS core/double shell QDs were successfully synthesized in aqueous medium. The core/shell nature of the QDs was confirmed by UV-Vis, XPS and XRD techniques. The gelatin stabilized core/double shell QDs exhibited long lasting fluorescence over one year in ambient conditions. According to the MTT cell viability assay, we have shown that the cytotoxicity of QDs can be effectively reduced by almost 50% with gelatin stabilization. The CSSG QDs display excellent biocompatibility than their parent core/shell and core QDs as well as those QDs without gelatin stabilization. The gelatin stabilized core/double shell QDs can be readily conjugated with transferrin for targeted imaging. We established, in vitro, the application of CSSG-Tf QD bioconjugates as fluorescence markers for cell imaging. Thus, the gelatin stabilized CdTe/CdS/ZnS core/double shell QDs can be suggested for long time fluorescent bio-imaging and targeted drug delivery.

Conflicts of Interest The authors declare that they have no conflict of interest.

Acknowledgements The part of this work was financially supported by Department of Biotechnology (DBTNanomedicine), Government of India (Grant No: BT/PR10085/NNT/28/99/2007). The authors acknowledge the assistance from National Centre for Nanoscience and Nanotechnology (NCNSNT), University of Madras for TEM and XPS analyses and Kings

13

Institute of Preventive Medicine and Research (KIPMR), Chennai-600 032, for cytotoxic studies.

14

References

[1]

G. Armstrong, An introduction to polymer nanocomposites, Eur. J. Phys. 36 (2015) 063001.

[2]

P.Sundarrajan, P. Eswaran, A. Marimuthu, L.B. Subahadra, P. Kannaiyan, One Pot Synthesis and Characterization of Alginate Stabilized Semiconductor Nanoparticles, Bull. Korean Chem. Soc. 33 (2012) 3218- 3224

[3]

A. Manikandan, M. P. Mani, S. K. Jaganathan, R. Rajasekar, M. Jagannath, Formation of functional nanofibrous electrospun polyurethane and murivenna oil with improved haemocompatibility for wound healing, Polym. Test. 61 (2017) 106-113.

[4]

K. Thanigai Arul, E. Manikandan, P.P Murmu, J Kennedy, M. Henini, Enhanced magnetic properties of polymer-magnetic nanostructures synthesized by ultrasonication, J. Alloys Compd. 720 (2017) 395-420

[5]

K. Goda, M. S. Sreekala, S. K. Malhotra, K. Joseph, S. Thomas, Advances in Polymer Composites: Biocomposites–State of the Art, New Challenges, and Opportunities, Poly. Composites, (2013) 1-10.

[6]

D. Feldman, Polymer nanocomposites in medicine, J. Macromol Sci. A. (2012) 55-62.

[7]

Q. Maa, X. Su, Recent advances and applications in QDs-based sensors, Analyst, 136 (2011) 4883-4893.

[8]

S. Parani, B. S. Lakshmi, K. Pandian, Biopolymer Encapsulation of CdTe Quantum Dot for In Vitro Controlled Drug Delivery Release of 6-Mercaptopurine, Adv. Mater. Research, 584 (2012) 258-262

[9]

D. K. Manimegalai, A. Manikandan, S. Moortheswaran, S. A. Arul Antony, Adv. Sci. Eng. Med. 7 (2015) 722-727

[10]

R. Bilan, I. Nabiev, A. Sukhanova, Quantum dot-based nanotools for bio-imaging, diagnostics, and drug delivery, Chem. Biochem. 17 (2016) 2103-2114.

[11]

C. T Matea, T. Mocan, F. Tabaran, T. Pop, O. Mosteanu, C. Puia, C. Iancu, L. Mocan, Quantum dots in imaging, drug delivery and sensor applications, Int. J. Nanomedicine. 12 (2017) 5421–5431.

[12]

D. Depan, R.D.K. Misra, Structural and physicochemical aspects of silica encapsulated ZnO quantum dots with high quantum yield and their natural uptake in HeLa cells, J. Biomed. Mater. Res. A. 102 (2015) 2934-2941.

15

[13]

V. Ncapayi, S. Parani, S. P. Songca, T. Kodama, O.S. Oluwafemi, Simple green synthesis of amino acid functionalised CdTe/CdSe/ZnSe core-multi shell with improved cell viability for cellular imaging, Mater. Lett. 189 (2017) 168-171.

[14]

M.Z. Hu, T. Zhu, Semiconductor Nanocrystal Quantum Dot Synthesis Approaches Towards Large-Scale Industrial Production for Energy Applications, Nanoscale Res. Lett. 10 (2015) 469.

[15]

B-K. Pong, B.L. Trout, J-Y. Lee, Modified Ligand-exchange for efficient solubilization of CdSe/ZnS quantum dots in water: A procedure guided by computational studies, Langmuir 24 (2008) 5270–5276.

[16]

X. Wang, J. Wu, F. Li, H. Li, Synthesis of water-soluble CdSe quantum dots by ligand exchange with p-sulfonatocalix(n)arene (n = 4, 6) as fluorescent probes for amino acids. Nanotechnol. 19 (2008) 205501-205510.

[17]

D. Zhou, M. Lin, Z. Chen, H. Sun, H. Zhang, H. Sun, Yang B, Simple synthesis of highly luminescent water-soluble CdTe quantum dots with controllable surface functionality, Chem. Mater. 23 (2011) 4857–4862.

[18]

Q. Wang, T. Fang, P. Liu, B. Deng, X. Min, X. Li X, Direct synthesis of high-quality water-soluble CdTe:Zn2+ quantum dots, Inorg. Chem. 51 (2012) 9208–9213.

[19]

J. Zhang, X. Hu, Q. Gong, Surface defect states of CdTexS1−x quantum dots, Phys Lett A. 373 (2009) 4475–4478.

[20]

J. Ma, J.Y. Chen, J. Guo, C.C. Wang, W.L. Yang, L. Xu, P.N. Wang, Photostability of thiol-capped CdTe quantum dots in living cells: the effect of photo-oxidation, Nanotechnol 17 (2006) 2083.

[21]

M. Ulusoy, A. Lavrentieva, J. Walter, F. Sambale, M. Green, F. Stahl, T. Scheper, Evaluation of CdTe/CdS/ZnS core/shell/ shell quantum dot toxicity on three-dimensional spheroid cultures, Toxicol. Res. 5 (2016) 126–135.

[22]

D. Vasudevan, R.R. Gaddam, A. Trinchi, I. Cole, Core/shell quantum dots: properties and applications, J. Alloy. Comp. 636 (2015) 395-404.

[23]

S. Parani, G. Bupesh, E. Manikandan, K. Pandian, O.S. Oluwafemi, Facile synthesis of mercaptosuccinic acid-capped CdTe/CdS/ZnS core/double shell quantum dots with improved cell viability on different cancer cells and normal cells, J. Nanopart. Res. 18 (2016) 347.

16

[24]

X. Ji, D. Copenhaver, C. Sichmeller, X. Peng, Ligand bonding and dynamics on colloidal nanocrystals at room temperature: The case of alkylamines on CdSe nanocrystals, J. Am. Chem. Soc. 130 (2008) 5726-5735.

[25]

A.A.P. Mansur, F.G. de Carvalho, R.L. Mansur, S.M. Carvalho, L.S. de Oliveira, H.S. Mansur, Carboxymethylcellulose/ZnCdS fluorescent quantum dot nanoconjugates for cancer cell bio-imaging, Int. J. Biol. Macromolec. 96 (2017) 675-686.

[26]

M. Wang, M. Zhang, J. Qian, F. Zhao, L. Shen, G.D. Scholes, M.A. Winnik, Enhancing the photoluminescence of polymer-stabilized CdSe/CdS/ZnS Core/Shell/Shell and CdSe/ZnS core/shell quantum dots in water through a chemical-activation approach, Langmuir 25 (2009) 11732-11740.

[27]

Q. Nie, W.B. Tan, Y. Zhang, Synthesis and characterization of monodisperse chitosan nanoparticles with embedded quantum dots, Nanotechnol. 17 (2006) 140.

[28]

R.G. Aswathy, B. Sivakumar, D. Brahatheeshwaran, T. Ukai, Y. Yoshida, T. Maekawa, D.S. Kumar, Biocompatible fluorescent jelly quantum dots for bio-imaging, Mater Express. 1 (2011) 291–298.

[29]

V.B. Djagny, Z. Wang, S. Xu, Gelatin: a valuable protein for food and pharmaceutical industries, review, Crit. Rev. Food. Sci Nutr. 41 (2001) 481-492.

[30]

B. Manjula, K. Varaprasad, R. Sadiku, K. Ramam, G.V.S. Reddy, K.M. Raju, Development of microbial resistant thermosensitive Ag nanocomposite (gelatin) hydrogels via green process, J. Biomed. Mater. Res. A. 102 (2014) 928-934.

[31]

C. Lee, P. Zhang, Highly stable gelatin layer-protected gold nanoparticles as surfaceenhanced Raman scattering substrates, J. Nanosci. Nanotechnol. 14 (2014) 4325-4330.

[32]

S. Byrne, Y. Williams, A. Davies, S. Corr, A. Rakovich, Y. Gunko, Y. Rakovich, J. Donegan, Y. Volkov, “Jelly Dots”: Synthesis and cytotoxicity studies of CdTe quantum dot-gelatin nanocomposites, Small 3 (2007) 1152-1156.

[33]

L. Chen, A. Willoughby, J. Zhang, Luminescent gelatin nanospheres by encapsulating CdSe quantum dots, Luminescence 29 (2014) 74-78.

[34]

W. Jiang, S. Mardyani, H. Fischer, W.C.W. Chan, Design and characterization of lysine cross-linked mercapto-acid biocompatible quantum dots, Chem. Mater. 18 (2006) 872878.

[35]

S. Sathya, S. Sudhagar, M. Vidhya Priya, R. B. Raja, V.S. Muthusamy, S.N. Devaraj, B.S. Lakshmi, 3β-hydroxylup-20(29)-ene-27,28-dioic acid dimethyl ester, a novel natural

17

product from Plumbago zeylanica inhibits the proliferation and migration of MDA-MB231 cells, Chem. Biol. Interact. 188 (2010) 412-420. [36]

Z. Gu, L. Zou, Z. Fang, W. Zhu, X. Zhong, One-pot synthesis of highly luminescent CdTe/CdS core/shell nanocrystals in aqueous phase, Nanotechnol. 19 (2008) 135604 (7pp).

[37]

X. Zhong, S. Liu, Z. Zhang, L. Li, Z. Wei, W. Knoll, Synthesis of high-quality CdS, ZnS, and ZnxCd1-xS nanocrystals using metal salts and elemental sulfur, J. Mater. Chem. 14 (2004) 2790-2794.

[38]

Y. Wang, C. Ye, L. Wu, Y. Hu, Synthesis and characterization of self-assembled CdHgTe/gelatin nanospheres as stable near infrared fluorescent probes in vivo, J. Pharm. Biomed. Anal. 53 (2010) 235-242.

[39]

H. Peng, L. Zhang, C. Soeller, J. Travas-Sejdic, Preparation of water-soluble CdTe/CdS core/shell quantum dots with enhanced photostability, J. Lumin. 127 (2007) 721-726.

[40]

S. Deka, A. Quarta, M.G. Lupo, A. Falqui, S. Boninelli, C. Giannini, G. Morello, M.D. Giorgi, G. Lanzani, C. Spinella, R. Cingolani, T. Pellegrino, L. Manna, CdSe/CdS/ZnS double shell nanorods with high photoluminescence efficiency and their exploitation as biolabeling probes, J. Am. Chem. Soc. 131 (2009) 2948-2958.

18

Figure captions

Fig. 1

Schematic preparation of CSSG QDs

Fig. 2

(a) Absorption and (b) PL spectra of CG, CSG and CSSG gelatin stabilized QDs; inset (b) corresponding fluorescence under UV lamp (λexc =352 nm). (c) Evolution of integrated PL intensity of CSSG under storage at ambient conditions.

Fig. 3

(a) XRD patterns of gelatin and gelatin-stabilized QDs. (b) TEM image of CSSG QDs; scale bar is 50 nm, inset: corresponding SAED pattern (c) Corresponding EDS spectrum.

Fig. 4

(a) Survey XPS spectrum CSSG QDs. Corresponding high resolution spectra of (b) S 2p, N 1s, Cd 3d, Te 3d energy levels.

Fig. 5

(a) Viability of HeLa cells after 48 h of incubation with CG, CSG and CSSG QDs at different Cd[QD] concentrations; (control, [Cd]QD = 0). (b) Effect of gelatin stabilization on the IC50 values of QDs.

Fig. 6

Fluorescence microscopic images of HeLa cells incubated with CSSG-Tf bioconjugates. (a) Bright field (b) Dark field fluorescence images. (c) Overlap of bright and dark field images.

19

Fig.1

Fig. 2

Fig. 3 20

Fig. 4

Fig. 5

21

Fig. 6

Fig. 7

22