Abstract
We report the synthesis of 5-fluorouracil (5-FU) loaded biocompatible fluorescent zein nanoparticles. Zein is the storage protein in corn kernels that has a variety of unique characteristics and functionalities that makes zein valuable in various commercial applications. It is classified as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). We synthesized zein nanoparticles of around 800 nm in size and conjugated with quantum dot ZnS:Mn. The nanoparticle was in turn encapsulated with the drug 5-FU. The luminescent properties of these nanoparticles were studied by using fluorescence microscopy. The nanoparticles were characterized and the drug release profile was studied. The biocompatibility of zein nanoparticle and the cytotoxicity with drug-loaded nanoparticle was studied in L929 and MCF-7 cell lines. The nanoparticles were successfully employed for cellular imaging. In vitro drug release studies were also performed. The biocompatibility of the nanoparticle showed that nanoparticles at higher concentrations are compatible for cells and are expected to be promising agents for the targeted delivery of drugs in the near future.
Export citation and abstract BibTeX RIS
Content from this work may be used under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
1. Introduction
The application of natural polymers on colloidal systems facilitates the fabrication of nanomaterials with several attractive features such as high biocompatibility, biodegradability and low toxicity. There are numerous reports on the application of natural polymers such as chitosan, alginate, gelatin, cellulose, collagen, zein, etc as biomaterials [1–3]. Among these polymers, zein is the storage protein in corn kernels that has a variety of unique characteristics and functionalities that makes zein valuable in various applications. It is a hydrophobic protein of molecular weight of about 40 kDa and classified as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). Zein is considered as a biocompatible biopolymer that is gaining greater attention in the field of nanoscience, especially in the food, cosmetics and tissue engineering areas. Zein comprises of about one-third hydrophilic glutamine and two-thirds hydrophobic amino acid residues in its primary structure. Zein and its resins form a robust, lustrous, hydrophobic greaseproof coating that can provide resistance to microbial attack [4] and can be used as biodegradable films and plastics. In one study it was reported that zein exhibited antioxidative activity [5]. Previous studies proved that zein acts as a bioactive molecule. Upon hydrolysis of zein with the enzyme thermolysin, α-zein generates angiotensin-converting enzyme-inhibitory peptides [6] that can reduce blood pressure in hypertensive rats. Zein was studied as biomaterial for scaffold for tissue engineering in vitro and exhibited better biocompatibility [7]. Zein has been studied extensively as a microparticle drug delivery system to delay release of drugs until they reach the intestine, to protect the drugs from the acidic environment that prevails in the stomach [8, 9]. Although several synthetic biodegradable polymers have been extensively studied in controlled release technology, the bulk erosion of drug or bioactive component limits its wide-scale application [10]. Zein molecules exhibit distinctive brick-like shape and hence can effectively carry either drug or bioactive agents [11] and can also overcome the limitations of hydrophilic polymers for sustained drug release. Several reports have been published on the application of zein as a matrix for several food-grade antiseptics including lysozyme, nisin and thymol [12–18]. Recently, Ag nanoparticles were incorporated into zein matrix as an efficient antibacterial agent. All the above-described applications of zein suggest the potential of zein to be useful in the biomedical field. The biocompatibility of zein nanoparticles can be exploited for the development of fluorescent nanoparticles.
Quantum dots (QDs) are nanometer-sized inorganic semiconductor fluorophores with physical dimensions smaller than the exciton Bohr radius [19–24]. QDs absorb light in a particular wavelength and remit in another wavelength. Although other organic and inorganic materials exhibit a similar fluorescence phenomenon, ideal fluorophores should be bright, stable and non-photobleaching with narrow, symmetric emission spectra, with multiple resolvable colors that can be excited simultaneously using a single excitation wavelength [20]. They have been applied in biotechnology as optical labels in a variety of biomedical applications including immunoassays for proteins, nucleic acids, bacteria, and toxin analysis [25,26] and for cell labeling and imaging, particularly in cancer imaging studies. Although QDs have been used as excellent probes for bioimaging, the toxicity rendered by these nanoparticles limits the application in the wide range. Hence QDs have been coated with a variety of natural and synthetic biopolymers to render biocompatibility [27–29].
In our study we developed zein–QD nanoparticles for nanoscale delivery of 5-FU, a potent anticancer drug against cancer cells. These multifunctional nanoparticles could be used for delivery of drugs and imaging the cancer cells. Characterization of synthesized nanoparticles was carried out by evaluating the morphology, optical properties, molecular interactions and the drug release from nanoparticles. The biocompatibility and the cytotoxicity of drug-loaded nanoparticles were studied and fluorescence of the nanoparticles was used for cellular imaging.
2. Experimental
2.1. Materials
Zein was procured from Wako pure chemicals industries Ltd, Japan. Zinc acetate, and manganese sulfate were purchased from Kanto Chemicals, Japan. Sodium sulfide 9H 2 O was procured from Sigma-Aldrich and 2,4-dihydroxy-5-fluorpyrimidin (5-FU) was purchased from Nacalai tesque, Inc., Japan.
2.2. Synthesis of 5-FU encapsulated zein QD nanoparticles
Zein nanoparticles were prepared by slight modification from previously reported liquid–liquid dispersion method [30]. Briefly, 10 mg zein was dissolved in 15 ml of 90% isopropyl alcohol (isopropanol: water at 80:20 v/v) followed by the addition of 30 ml of deionized water under vigorous stirring in a magnetic stirrer at room temperature. The suspension was centrifuged at 10 000 rpm for 5 min, washed three times with water and freeze-dried.
ZnS:Mn QD was prepared based on a previously reported method [31]. Briefly, zinc acetate (0.1 M) was dissolved in 10 ml deionized water and N 2 was purged for 10 min. 1 ml manganese sulfate (0.01 M) was added drop wise and stirred for 10 min. 10 ml of 0.1 M sodium sulfide was added slowly and stirring continued for another 30 min. The fluorescence from the sample was observed by UV lamp at 365 nm excitation energy and the sample exhibited bright orangeish red emission. The QD suspension was centrifuged, washed with water and freeze-dried to get fine powder. 2 mg of as-synthesized QD was added along with zein in 90% aqueous isopropyl alcohol and the steps for zein nanoparticle preparation proceeded. 5-FU loaded zein–QD nanoparticles were prepared by dissolving 5-FU in methanol and added drop wise into the mixture of QD, zein and isopropyl alcohol under stirring, followed by the addition of deionized water. The resulting suspension was centrifuged and lyophilized to get fine yellow powder that was used for characterization studies.
2.3. Characterization
The scanning electron microscopy (SEM) images of zein nanoparticles and zein QD nanoparticles were acquired by scanning electron microscope (JEOL, JSPM-6490, Japan). Nanoparticle suspension was diluted with ultra pure water and dropped on silica substrate, dried under vacuum and images were acquired. Transmission electron microscopy (TEM) images of QD and zein QD nanoparticle were acquired by JEM-2200-FS field emission transmission electron microscope (JEOL, Japan) at an operating voltage of 200 kV. The grids were given hydrophilic treatment using a Joel Datum HDT-400 hydrophilic treatment device. TEM samples were prepared by dropping the diluted QDs and nanoparticle into the carbon coated hydrophilic copper TEM grids. The UV-Vis spectra of nanoparticles were measured using a Shimadzu UV-Vis spectrophotometer (UV-2100PC/3100 PC). The absorbance of QD, zein and zein QD nanoparticles was recorded. Fourier-transform infrared (FTIR) spectroscopic analysis of zein nanoparticles, 5-FU, QD, zein QD and 5-FU loaded zein QD was recorded on a Perkin Elmer spectrophotometer in the spectral range of 4000–400 cm −1 at room temperature. The data sets were averaged over 32 times. The thermogravimetric analysis (TGA) of zein, QD, and zein QD was carried out in a DTG-60H Shimadzu thermal analyzer. TGA of all the samples was performed up to a temperature of 1000 °C, starting from room temperature in nitrogen atmosphere. A heating rate of 10 °C min −1 was maintained for the samples.
2.4. Drug encapsulation efficiency and drug release studies
The 5-FU loaded zein QD in the reaction mixture was centrifuged at 10 000 rpm for 30 min. The supernatant with unencapsulated drug was collected separately and the absorbance was recorded at 265 nm. The concentration of free drug was calculated from the absorbance value, based on the standard curve for 5-FU. Encapsulation efficiency (%) was estimated as the ratio of the difference between total drug and the free drug in the supernatant to total drug. Drug release of 5-FU from nanoparticles was carried out in phosphate buffered saline (PBS) medium with pH 7.4. 10 mg of lyophilized nanoparticles dispersed in 200 ml of PBS, pH 7.4. The suspension was placed in a water bath shaker at 37 °C with a shaking speed of 120 rpm. 5 ml of supernatant from the sample was withdrawn for recording absorbance at 265 nm at fixed time intervals and the suspension was refilled with 5 ml fresh PBS. We define
where 5-FU Rel is the concentration of 5-FU released at collected time t and 5-FU Tot is the total amount of 5-FU encapsulated in the nanoparticles.
2.5. Cell culture and in vitro cytotoxicity studies
Human breast cancer cell line MCF-7 and mouse fibroblast cell line L929 were used for in vitro experimental studies. The cell lines were procured from Riken Culture collection Center, Japan. MCF-7 cell lines were routinely cultured in minimum essential medium supplemented with 5% heat inactivated fetal bovine serum, sodium pyruvate, non-essential amino acids, and penicillin/streptomycin (100 units ml −1). Mouse fibroblast cell line, L929 was cultured in Dulbecco's Modified Eagle Medium (DMEM) with 5% heat inactivated fetal bovine serum and penicillin/streptomycin (100 units ml −1). The cell lines were cultured at 37 °C in a humidified 5% CO 2 atmosphere. Nanoparticle samples were prepared and diluted to different concentrations (1, 2, 3, 4 mg ml −1) with PBS (pH 7.4) for treatment in 96 well tissue culture plates for cytotoxicity studies. The cell viability was assessed by Alamar blue assay.
2.6. Cellular imaging
Uptake of nanoparticles by MCF-7 and L929 were studied with cellular imaging. MCF-7 and L929 cell lines were routinely grown in their respective media at 37 °C under 5% CO 2 atmosphere. Cells were grown for 24 h prior to the addition of nanoparticles in cover slip glass plate. After 24 h the cells were washed with PBS and fresh culture medium with nanoparticles was added and was incubated for 4 h at 37 °C. Cells were again washed with PBS to remove excess nanoparticles and fresh medium was replaced. Nanoparticles labeled MCF-7 and L929 cell lines were imaged by an Olympus BX-51 fluorescent microscope equipped with a CCD camera and 20 × oil immersion objective lenses. The fluorescence of the zein QD nanoparticles was detected using band pass excitation and emission filters (BP 365/10 nm excitation, 400 nm emission, 400 nm dichromatic mirror).
3. Results and discussion
The solubility of zein in aqueous alcohol has been exploited for the formation of zein nanoparticles. The nanoparticles are formed as result of the shearing of zein solution in alcohol with the addition of bulk aqueous phase [30]. The drug or bioactive components can be encapsulated into zein nanoparticles efficiently by dissolving the component with zein in aqueous alcohol.
3.1. Characterization of nanoparticles
SEM images of zein nanoparticles and zein QD nanoparticle were found to be spherical in morphology. The average size of zein nanoparticles was around 600–700 nm (figure 1) and the average size of drug-loaded zein QD ranging from 800 to 900 nm in diameter was slightly larger than that of the zein nanoparticles (figure 2). This may be due to the encapsulation of drug and the adsorption of QDs in the nanoparticles. The TEM image of QD is shown in the figure 3, which shows its size to be around 5 nm in diameter and the adsorption of QDs on the surface of zein nanoparticle is clearly seen in figure 4.
Figure 1 SEM image of zein nanoparticles.
Figure 2 SEM image of zein QD nanoparticles.
Figure 3 TEM image of ZnS:Mn QD.
Figure 4 TEM of zein QD.
The UV-Vis spectra of the samples, QD, zein and zein QD are presented in figure 5. The spectrum of QD has two peaks, one at 225 nm and another at around 320 nm, and shows good absorption for light in the wavelength of 220–350 nm. Zein nanoparticles exhibit characteristic absorbance at 270 nm. The composite exhibits the typical absorbance of zein at 270 nm and another peak around 355 nm. This might be the peak of QD that could have shifted towards the red region owing to the interaction with the zein nanoparticle. The photograph of the zein nanoparticle, ZnS:Mn, and 5-FU loaded zein with QD under UV illumination at 365 nm is shown in figure 6.
Figure 5 UV-Vis absorbance of (a) QD, (b) zein nanoparticles and (c) zein QD.
Figure 6 Photograph of (a) zein nanoparticle, (b) QD and (c) zein QD under UV illumination.
The 5-FU, QD, zein nanoparticles, zein QD and 5-FU zein QD were characterized by FTIR spectroscopy technique to characterize the intermolecular interactions of complex. The representative spectrum of each component and the composites are shown in figure 7. The FTIR spectra of the zein nanoparticles exhibited the typical amides I and II peaks at 1650 and 1536 cm −1 that correspond to carbonyl stretching vibration and both C–N stretching and C–N–H in plane bending [32]. It was also reported that the amide I peak at 1650 cm −1 was documented to the α-helical secondary structure of zein [33]. The amide I peak related to α-helical structure of zein sustained at the same position in the FTIR spectra of zein QD and 5-FU zein QD suggesting the presence of α-helical structure of zein in the nanoparticles after binding with QD and drug. Although, after binding with QD and drug the amide I and II bands were broadened, indicating that the interactions between the QD and zein might be due to hydrogen bond and hydrophobic interactions [34]. The presence of a peak at 1348 cm −1 in 5-FU was assigned to vibration of the pyrimidine compound. The presence of a broad peak at 1396 cm −1 in the 5-FU zein QD can be attributed to the incorporation of 5-FU to zein QD. The presence of a peak at 1111 cm -1 in QD is the characteristic frequency of inorganic ions [35]. However, the peak is slightly shifted in zein QD and 5-FU zein QD to 1113 and 1117 cm −1, respectively. This suggests the prevalence of QD in both the composites. The peak characteristic for sulfides is present at 618 cm −1 for QD, zein QD and 5-FU zein QD which clearly indicates that QD has been incorporated in the zein nanoparticle [35].
Figure 7 FTIR spectra of (a) QD, (b) 5-FU zein QD, (c) zein QD, (d) zein nanoparticle and (e) 5-FU.
Thermogravimetric curves of zein nanoparticles, zein QD and plain QD are shown in figure 8. The rate of weight loss was more with the rise of temperature. The initial weight loss observed for all samples at around 100 °C was due to loss of moisture. The decrease of about 10% on mass for bare QD was observed around 100 °C and is probably due to the vaporization of water content that was present in QD. The decrease of mass observed for zein and zein QD nanoparticles in the same temperature range was lower than that for QD. This can be attributed to the lower hydrophilicity of zein nanoparticles. The second weight loss for zein occurs around 280 °C and can be attributed to the breakdown of the amino acid residues, as well as cleavage of the peptide bonds in the protein. The TGA curve of zein QD displayed a similar pattern to that of zein nanoparticles indicating the decomposition of zein around 280 °C and retaining the stability which was characteristic of QD. From 300 to 400 °C, the TGA curves for zein and zein QD nanoparticles presented significant mass loss. The loss of mass for zein nanoparticles was larger than that for zein QD. The mass loss in this range 200–400 °C for zein nanoparticles was around 65% and for zein QD the loss was around 50%. The residual weight of QD zein was more due to the thermal stability of QD.
Figure 8 TGA of (a) plain QD, (b) zein-QD and (c) zein nanoparticles.
The encapsulation efficiency of drug in zein nanoparticles in our study was 60% and the drug was released in a sustained manner over a period of 24 h (figure 9). The percentage of release of 5-FU from zein nanoparticle was based on the normal standard curve of 5-FU. We could observe 70% release of drug from the nanoparticle in about 8 h. The suggested mechanism for drug release from nanoparticles and microspheres is diffusion of drug from the polymer matrix and polymer matrix degradation [36]. Since the size of encapsulated drug molecule was smaller than the formed nanoparticles, diffusion of drug molecule from nanoparticle might have played a major role in release profile.
Figure 9 Drug release profile.
3.2. In vitro biocompatibility of zein nanoparticles and cytotoxicity of drug loaded zein nanoparticles
The cytotoxicity of zein nanoparticles, zein QD nanoparticles and 5-FU-zein QD nanoparticles was evaluated on the L929 cells and MCF-7 cancer cells at four different concentrations. More than 90% of cells were viable when incubated with the plain zein nanoparticles, signifying the same biocompatibility (figure 10). The viability was more than 80% when the cells were incubated with zein QD, indicating that the zein QD is also biocompatible. When the cells were treated with the 5-FU zein QD we observed a considerable drop in viability, and the percentage of viable cells decreased with the increase of drug-loaded zein QD concentrations, as shown in figure 11. Encapsulation of the drug in the zein nanoparticles resulted in significant increase in cytotoxicity on both cell lines.
Figure 10 Biocompatibility of zein nanoparticles and zein QD nanoparticles.
Figure 11 Cytotoxicity of 5-FU loaded zein QD nanoparticles.
3.3. In vitro cellular imaging with fluorescent zein nanoparticles
The internalization of nanoparticles by MCF-7 and L929 cells was studied by the fluorescence images. The nanoparticles are internalized into the cells that were observed by the fluorescence emitted by the zein QD nanoparticles, as shown in figures 12 and 13. From the results it is suggested that efficient targeting of nanoparticles with specific ligands can enhance the uptake of nanoparticles by specific cells.
Figure 12 Cellular imaging with zein QD nanoparticles (phase contrast and fluorescence image of MCF-7 cells treated with zein QD nanoparticles).
Figure 13 Cellular imaging with zein QD nanoparticles (phase contrast and fluorescence image of L929 cells treated with zein QD nanoparticles).
4. Conclusion
In summary, we demonstrated a simple liquid–liquid dispersion method for the efficient synthesis of zein nanoparticles. The nanoparticles were made fluorescent by the addition of ZnS:Mn QDs. Also, we encapsulated the nanoparticle with potent anticancer drug, 5-FU. We demonstrated the biocompatibility of zein and zein QD nanoparticles. The drug-loaded nanoparticles were efficient in destroying cancer cells. The fluorescence imparted for the nanoparticles by the QD was exploited for cellular imaging. Therefore, efficient targeting of zein nanoparticles with specific targeting moieties like antibodies, aptamers, etc, can be used for cell specific drug or bioactive component delivery to target cells.
Acknowledgments
R G Aswathy and D Brahatheeswaran would like to acknowledge MEXT, Japan and B Sivakumar thanks JASSO, Japan for providing fellowship for conducting their doctoral course.