Introduction

Breast cancer is one of the leading causes of death among women worldwide [1]. Over decade several imaging techniques have been developed, improved and used in the field of bio-imaging to combat again cancer. Well known cancer cell imaging techniques, such as magnetic resonance imaging (MRI), computed tomography (CT) scans, ultrasound scans, positron emission tomography (PET) scans, and optical imaging are widely used for visualising the tumours macroscopically. Unfortunately, these techniques have certain limitations to detect cancer cell abnormalities at macroscopic level [2]. Recently, Cadmium based quantum dots combined with optical imaging based technology is used in imaging of cancer cells. These techniques have been successfully demonstrated to be useful for detecting tumours [3–5], but these cadmium based quantum dots possess toxicity towards normal cells [6]. Hence, biological applications of these molecules are very limited. To avoid this and to obtain compatible properties, the quantum dots surface were to be modified with capping agents such as Silica [3], D, L-lactide [7], Glutathione [8], nonetheless they pose long term side effects. To overcome these issues, less cytotoxic fluroprobe with specific cancer targeting molecules are required with features such as water dispersibility, low cytotoxicity, higher stability in aqueous environment and intense optical properties [9–11].

Surface modified nano materials also called semiconducting Nanoparticles (Nps) have been receiving increasing interest among researchers in the field of fluorescence cell imaging due to its low toxicity and unique optical properties. Over the decade numbers of methods have been reported in literature about conjugation of biomolecules on Nps surface. Mostly, these modifications are based on silanization of Nps [12] and using cross linking molecules such as N,N methylene bis acrylamide, N-hydroxysuccinimidyl-3-(2-pyridyldithio)-propionate to introduce targeting moieties [13]. However these methods involve much complicated steps to achieve successful conjugation. Furthermore, it is difficult to achieve biocompatible fluoroprobe characteristic like high conjugation ability, high photo stability, water dispersibility and more consistent conjugation. Among Nps, ZnO Nps are envisaged as an important building block to fabricate biocompatible fluoro probe because ZnO Nps has a wide band gap (3.37 eV) with high exciton binding energy (60 meV). This high value of exciton energy is responsible for recombination of exciton at room temperature and can exhibit ultra violet luminescence [14]. It is well known that luminescence efficiency of ZnO Nps mainly depends on its size and shape. Due to size dependant properties of ZnO Nps, we can use this material for imaging applications. ZnO Nps with diameter less than 100 nm are by far the most promising agents with good biocompatibility [15]. In addition, the surfaces of ZnO Nps could be modified with a number of capping agents which are suitable for conjugation with various cancers targeting moieties such as transferrin, peptides and vitamins [16–18].

In this work, we have chosen N, N-dimethyl-N-2-propenyl-2-propene-1-ammonium chloride-2-propenamide (polyquaternium-7(PQ7)), a biocompatible cationic surfactant to modify the surface of ZnO Nps. For instance PQ7 is frequently used in skin care products [19], since it is a cationic surfactant; we speculated that it can easily conjugate with ZnO NPs electrostatically. Moreover PQ7 also has an amino group which can be bioconjugated with folic acid through N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) chemistry. It is well known that folic acid receptors are over expressed in tumor cells [20]. Modification of ZnO with PQ7 and folic acid has not been reported earlier. Notably this method employs simple steps to obtain fluoroprobe with high photo stability, low cytotoxicity and free from aggregation and agglomeration. This nano fluoroprobe could be an advanced material in the field of cancer cell imaging.

Materials and Methods

Materials

N,N-dimethyl-N-2-propenyl-2-propene-1-ammonium chloride-2-propenamide solution, 10 wt.% in H2O, zinc acetate dehydrate (Zn(OAc)2.2H2O), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride(EDC), anhydrous dimethyl sulfoxide (DMSO), N-Hydroxysuccinamide (NHS), Folicacid (FA), NaOH, were obtained from Sigma–Aldrich, USA. All other chemicals and solvents used in this experiment were of analytical grade.

Synthesis of ZnO and ZnO-PQ-7 Nanoparticles

The pure ZnO and PQ7 modified ZnO was prepared by simple wet chemical method. Zn(OAc)2.2H2O was dissolved in double distilled water to prepare 0.2 M solution. In a typical synthesis, 0.2 % PQ7 was added to the 0.2 M Zn(OAc)2.2H2O solution with vigorous stirring at room temperature. The mixture was then stirred on a hot plate for another 30 min at 60 °C. The pH of this mixture was adjusted to 10 using 1 M NaOH. The temperature was increased from 60 °C to 80 °C and stirred about 2 h to achieve nucleation of the nanocrystals. Subsequently, the ZnO Nps were brought down to room temperature. The obtained material was thoroughly washed 10 times with distilled water, Isopropyl alcohol and centrifuged at 25,218 g. Same procedure was followed for the synthesis of ZnO nanoparticles without PQ7. Finally the collected sample was vacuum dried for 24 h. PQ7 modified ZnO Nps with different PQ7 concentrations (0.4 %, 0.8 % and 1.6 %) were synthesized by adding stoichiometric amount of PQ7 to the zinc acetate precursor solution keeping the total initial concentration of metal ions constant.

Functionalization of ZnO-PQ7 Nano Disks with Folic Acid

Folate conjugated ZnO-PQ7 nanodisk was prepared according to the procedure reported elsewhere [21] with slight modification. 0.10 mM of Folic acid in PBS, 0.10 mM of EDC in PBS (pH 7.4), 0.10 mM of NHS solution in PBS were added, resulting solution was stirred until the folic acid had dissolved (1 h), Then 5 mg of ZnO-PQ7 Nps was dispersed in 10 ml of Phosphate-buffered saline (PBS) (pH 7.4) was added. The resulting solution was then allowed to stir for 2.5 h in dark under inert atmosphere. Folic acid-functionalized ZnO-PQ7 Nps were precipitated by adding ethanol to the reaction mixture. The obtained product (ZnO-PQ7-FA) was separated by centrifugation at 25,218 g, washed twice with ethanol, dried under vacuum and finally dispersed in double distilled water.

Characterization

X-ray powder diffraction analysis of the samples was performed using Bruker D-8 Advance X-ray diffractometer (Cu Kα (1.54 °A)). UV-Visible spectra were recorded using Agilent 8453 Diode Array Spectrophotometer. Fluorescence was measured on an Agilent Varian Cary Eclipse spectrophotometer (FL1201M002). 10 mm path –length quartz cuvette was used for fluorescence and UV-Visible spectrum measurements. The surface modified ZnO-PQ7 Nps morphology and average particle sizes was characterized by High resolution transmission electron microscopy (HRTEM) (FEI-TECHNAI, G2-Model (T-30 S TWIN)) with an accelerating voltage 250kv andelemental analysis was done by EDAX. To confirm the folic acid conjugation on ZnO-PQ7 Nps, we have carried out Fourier transform infrared spectroscopy (FTIR) in the spectral range of 400–4000 cm−1 (Perkin Elmer) using the pressed KBr pellet technique.

Cell Culture

MCF-7 (Human breast cancer cell line) was procured from National Centre for Cell Sciences (NCCS, Pune, India). Cells were cultured in DMEM supplemented with 10 % Fetal bovine serum (FBS) in the presence of penicillin and streptomycin in a humidified incubator with 5 % CO2 at 37 °C.

In Vitro Cytotoxicity (MTT Assay)

Cell viability was assessed by MTT assay as reported previously with slight modifications [22]. Briefly 1 × 104 cells were seeded onto 96 well plates. Cells were treated with different concentrations (10 μg/ml to125 μg/ml) of ZnO-PQ-7-FA prepared in DMSO for 24 and 48 h. After incubation 20 μl of MTT (5 mg/ml) was added to each well. After 4 h of incubation the media was removed and the purple colored formazan crystals were dissolved in 150 μl of DMSO and read on a micro plate reader at 570 nm. The cell viability is expressed as percentage of live cells. All the values are expressed as the mean ± standard deviation.

Confocal Microscopy Imaging

For confocal imaging cells were seeded on sterile cover slips in a culture dish with DMEM and allowed for attachment overnight. Cells were washed with PBS and treated with 76 μg/ml of ZnO-PQ7-FA in DMEM for 2 h at 37o C in a humidified incubator. Before imaging, cells were washed with PBS to remove the unbound probes. The cover slip was inverted, fixed onto a clean glass slide and imaged using Carl Zeiss (LSM 710) confocal laser scanning microscope. The excitation and emission wavelengths used were 405 nm and 450 nm respectively.

Results and Discussion

XRD patterns of unmodified ZnO and PQ7 modified ZnO with different PQ7 concentrations are given in Fig. 1. Pure hexagonal phase was found and indexed with the help of standard JCPDS data (JCPDS, 75–0576). It can be clearly seen from the patterns that the samples showed a single-phase nature with a wurtzite structure. No diffraction peaks from any other impurities were detected and the sharpness of the peaks indicates the high crystallite of prepared Nps. The bigger particle size of the sample prepared without PQ7 indicates faster growth of the particles in the absence of surfactant. The XRD patterns of the products obtained using different concentrations of PQ7 are shown in Fig. 1 b, c, d and e. The crystallinity of ZnO-PQ7 at concentration 1.6 % was decreased as compared with 0.2 %, 0.4 % and 0.8 %. Crystallite size was calculated using scherrer formula. The crystallite sizes were found to be 85 nm for pure and 65 nm, 58 nm, 48 nm, 41 nm, 30 nm for PQ7 modified ZnO. With increasing PQ7 concentration the crystallite size and intensity were decreased which indicates that the PQ7 play an important role in modifying the ZnO surface. The reduced crystallite of ZnO-PQ7 may be due to the steric hindrance arising from the soluble PQ7 molecules which inhibits crystal growth rate and hence the reduction of crystallite of ZnO-PQ7.

Fig. 1
figure 1

XRD patterns of the (a). Pure ZnO, (b). 0.2 % PQ7 modified ZnO, (c) 0.4 % PQ7 modified ZnO, (d) 0.8 % PQ7 modified ZnO, and (e) 1.6 % PQ7 modified ZnO

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UV-Vis absorption maxima and absorption intensity occur mainly by quantum confinement effect [23]. Figure 2 depicts the UV-Vis absorption spectra of pure ZnO and along with different concentration of PQ7 capped ZnO. Pure ZnO shows an absorption peak at 373 nm. ZnO-PQ7 (0.2 %, 0.4 % and 0.8 %) also shows the absorption at 373 nm. But the intensity at 373 nm increases with increase in PQ7 concentration. The decreased size arises due to the strong interactions between ZnO and PQ7 at lower concentrations of PQ7 (0.2 %, 0.4 % and 0.8 %). Further increasing concentration of PQ7 (1.6 %), the smaller particles apparently agglomerated to form larger nano particles and hence slight shift in absorption maxima towards longer wavelength (377 nm). The formation of larger ZnO-PQ7 Nps perhaps decreased interaction between the PQ7 and ZnO NPs. This indicates PQ7 plays an important role in controlling the size of ZnO-PQ7 Nps.

Fig. 2
figure 2

UV–Vis absorption spectra of (a) Pure ZnO (b) 0.2 % PQ7 modified ZnO (c) 0.4 % PQ7 modified ZnO (d) 0.8 % PQ7 modified ZnO, and (e) 1.6 % PQ7 modified ZnO

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Figure 3 shows the Fluorescence emission spectrum of the pure ZnO and PQ7 modified ZnO with different PQ7 concentration. Unmodified ZnO exhibited blue emission centered at about 443 nm when excited at a wavelength of 350 nm. Interestingly, with increasing concentration of PQ7, there is a significant enhancement in fluorescence intensity. When PQ7 concentration was increased to 0.8 %, the fluorescence intensity approximately achieved its maximum. With further increase in PQ7 concentration a significant decrease in fluorescence intensity was observed. So far it is well known that the visible emission of ZnO Nps was derived from oxygen vacancies [24–26]. In the present study the enhanced emission intensity of modified ZnO Nps could be attributed to the fact that more oxygen vacancies are present in ZnO-PQ7. More oxygen vacancies could be a result of decreased ZnO-PQ7 particle size when compared with unmodified ZnO [27].

Fig. 3
figure 3

Fluorescence spectrum of the (a) pure ZnO, (b) 0.2 % PQ7 modified ZnO, (c) 0.4 % PQ7 modified ZnO, (d) 0.8 % PQ7 modified ZnO, and (e) 1.6 % PQ7 modified ZnO. The inset shows a photograph of (a) ZnO-PQ7 Nps dispersed in water under visible light and (b) the ZnO-PQ7 Nps dispersed in water and after illuminated with an ultraviolet lamp at 360 nm

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Varying fluorescence intensity of ZnO-PQ7 with respect to increasing PQ7 concentration is presented in Fig. 4. It can be seen that the intensity of ZnO-PQ7 increased with increasing cationic polymer PQ7 concentrations. On the contrary, at 1.6 % the fluorescence intensity declined. This may be due to the steric hindrance arising from the soluble PQ7 molecules which reduces the intensity. Therefore the best possible percentage of PQ7 for modifying ZnO surface is determined from this study. The optimized concentration of PQ7 was found to be 0.8 %. The same is used for further studies like material characterization and in vitro experiments.

Fig. 4
figure 4

Fluorescence intensity of ZnO-PQ7 with respect to varying concentration of PQ7

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For biological cell imaging application, photo stability is considered as one of the important factors. The photostability of the ZnO-PQ7 compared with organic dyes such as, fluorescein isothiocyanate (FITC) and Rhodamine B is presented in Fig. 5. To determine photostability we irradiated ZnO-PQ7 nanodisks, fluorescein isothiocyanate (FITC), and Rhodamine B at 350 nm for different time period ranging from 0 to 60 min. It can be seen in Fig. 4, that the fluorescence of FITC, sharply quenched within 5 min of excitation and further quenched with increase in time due to photo bleaching, inferring that the molecule is less photostable. However Rhodamine B is found to be more photostable, retaining ~48 % of the original PL intensity even after 40 min of excitation. Interestingly, ZnO-PQ7 preserved ~80 % of its initial intensity even after 45 min of exposure. It is concluded that ZnO-PQ7 conjugates possess photostability, and there was no obvious dissociation or aggregation of ZnO-PQ7 conjugates Furthermore blue fluorescence can be detected by the naked eye under UV lamp at excitation wavelength of 365 nm (Fig. 2 inset) which is promising for the applications in bioimaging.

Fig. 5
figure 5

Photostability of the ZnO-PQ7 compared with FITC and Rhodamine B

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Figure 6a shows the TEM image and corresponding selected-area electron diffraction (SAED) pattern of the ZnO-PQ7 Nps. TEM image confirms the formation of ZnO-PQ7 hexagonal nanodisks with an average size of 30-45 nm. Figure 6b shows the diffraction rings of SAED pattern of ZnO-PQ7 hexagonal nanodisks and their corresponding planes (200), (103), (110), (102), (101), (002) were identified.

Fig. 6
figure 6

(a) TEM image of ZnO-PQ7 (b) Magnified Image of ZnO-PQ7 and (c) corresponding selected-area electron diffraction (SAED) pattern of the ZnO-PQ7 nanodisks

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The EDAX spectrum of the ZnO-PQ7 nanodisks is presented in Fig. 7, which shows the peaks of zinc and oxygen elements as 55.92 % and 44.08 % proves that ZnO-PQ7 nanodisks prepared are essentially free from impurities.

Fig. 7
figure 7

EDAX spectrum of the ZnO-PQ7 nanodisks

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Figure 8a shows the FT-IR spectrum of the ZnO-PQ7. Peak at 3339 cm−1 is a characteristic of primary amine group (N-H stretching vibration). The peak at 3212 cm−1 are assigned to the OH stretching vibration may be due to the presence of adsorbed moisture on the ZnO Nps surface. The bands at 2948 cm−1 corresponds to CH stretching. Peaks at 1666 cm−1 show the presence of Amide C = O stretching vibration and the peaks at 1460 cm−1 indicate C-H bending vibration. This confirms that PQ7 molecules are bound onto the ZnO surface. The hydroxyl group present on the surface confers good water stability.

Fig. 8
figure 8

FTIR spectra of ZnO-PQ7-FA fluoroprobe (a) ZnO-PQ7, (b) Folic acid and (c) ZnO-PQ7-FA

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Figure 8b shows FTIR spectrum of pure folic acid. The peaks at 3543 indicate O-Hstretching vibration. The peaks at 3416 cm−1, 3324 cm−1 indicate N-H2 stretching vibration. The characteristic peaks at 2959 cm−1, 2924 cm−1, 2844 cm−1indicate the symmetric (C-H), and asymmetric stretching (C-H) of -CH2. The characteristic absorption peak at 1694cm−1 corresponds to C = O stretching from –COOH. The bands at 1640 indicate C = O stretching from -CONH2. Peak at 1605 cm−1 corresponds to bending vibration of NH2. The band at 1484 cm−1indicates a Phenyl ring. The peak at 1411 cm−1 relates to O-H deformation of phenyl skeleton.

Figure 8c shows the characteristic IR absorption peaks of FA and ZnO-PQ7, which confirms that folic acid, is successfully conjugated with ZnO-PQ7 nanodisks.

Figure 9 illustrates schematic representation of ZnO-PQ7-FA preparation. First, the ZnO-PQ7 modification was achieved by electrostatic interaction. Secondly, the γ-carboxyl group of FA was activated by EDC and then the conjugation with the amino groups on the surface of ZnO-PQ7 was achieved.

Fig. 9
figure 9

Schematic illustration of ZnO-PQ7-FA nanodisks preparation

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In Vitro Cytotoxicity and Imaging Studies

The cell viability studies by means of Methyl thiazolyltetrazolium(MTT) assay is shown in Fig. 10. Cell toxicity assay shows that even at a concentration of 80 μg/ml, more than 80 % of cells are viable. Hence this can be used as a fluoroprobe in cancer cell imaging and as a carrier molecule in drug delivery in the coming days.

Fig. 10
figure 10

Cell viability of MCF-7 cells incubated with various concentrations of ZnO-PQ7-FA for 24 h and 48 h

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Based on the MTT assay, the ZnO-PQ7-FA shows significant biocompatibility. The ZnO-PQ7-FA conjugates were used for imaging of MCF-7 breast cancer cells. Fig. 11 (d-f) shows the confocal microscopic images of MCF-7 grown in DMEM containing 76 μg/ml of ZnO-PQ-7-FA. Fig. 11(a-c) shows MCF-7 control cells fluorescence, dark field, and merged image. Fig. 11(d-f) shows MCF-7 cells co cultured with ZnO-PQ7-FA for 2 h and Fig. 11(g-i) shows MCF-7 cells co cultured with ZnO-PQ7-FA for 6 h. We have already reported that folic acid conjugated Nps plays a vital role in uptake by cancer cells [28]. The fluorescence field and the merged images clearly confirm that the bright luminescence is localized in the cytoplasmic region. It can be clearly seen that the particles are not merely staining the membrane surface, but internalized into the cells possibly by endocytosis. From the confocal images, it can be clearly seen that there is no morphological damage to the cells even after co-incubation for 6 h and more uptake of the conjugates were observed. This suggests significant and better biocompatibility of the ZnO-PQ7-FA conjugates. Since FA is conjugated onto ZnO-PQ-7 this can be used in drug delivery to specifically target cancer cells in future. Receptor mediated endocytosis mechanism of cells will help in uptake of ZnO-PQ-7-FA.

Fig. 11
figure 11

(a-c) shows untreated MCF-7 cells. (d-f) shows MCF-7 cells co cultured with ZnO-PQ7-FA for 2 h and (g-i) shows MCF-7 cells co cultured with ZnO-PQ7-FA for 6 h.

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Conclusions

For the first time, a simple fluoroprobe based on ZnO-PQ7 nanodisks conjugated with folic acid for imaging MCF-7 cells was successfully assembled. We have reported a simple method to prepare ZnO-PQ7-FA in aqueous medium. PQ7 is used as a capping agent for modifying ZnO surface for the first time. PQ7 played an important role to control the size, shape and fluorescence properties of ZnO Nps. The optimized PQ7 concentration was found as 0.8 % to modify ZnO surface. ZnO-PQ7-FA conjugates showed effective fluorescence performance for MCF-7 breast cancer cell imaging by in vitro. The prepared ZnO-PQ7-FA possesses low cytotoxicity, photostability and well dispersibility in water. This ZnO-PQ7-FA material can play an important role in biomedical cancer imaging and drug delivery applications in the coming days.