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shell nanoparticles for controlled drug release
Materials Science & Engineering B 243 (2019) 115–124
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Materials Science & Engineering B journal homepage: www.elsevier.com/locate/mseb
Pegylated CdSe/ZnS core/shell nanoparticles for controlled drug release a
b
c
Shivani Bharti , Gurvir Kaur , Shikha Gupta , S.K. Tripathi a b c
a,⁎
T
Centre of Advanced Study in Physics, Department of Physics, Panjab University, Chandigarh 160014, India Sri Guru Gobind Singh College, Chandigarh, India Goswami Ganesh Dutta Sanatan Dharma College, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: PEGylation Core/shell nanoparticles Flavonoids In vitro studies Drug delivery
A facile strategy to synthesize PEGylated CdSe/ZnS core/shell nanoparticles (NPs) loaded with a hydrophobic anticancer drug Quercetin is presented in this manuscript. Drug is loaded over PEGylated NPs by two different mechanisms; one by using organic solvent to dissolve Quercetin and second by directly dispersing Quercetin in Water. FTIR spectra of Quercetin loaded NPs predict the successful drug loading over spherical NPs having size ∼30 nm obtained from TEM. Quercetin loading over PEGylated NPs increases the size of NPs as obtained from TEM and dynamic light scattering. The emission spectra show enhancement in fluorescence intensity of Quercetin after conjugating with PEGylated NPs, making NPs as drug release indicators. Effect of pH on Quercetin conjugated NPs have been investigated on the basis of optical measurements. The results show that in vitro release of Quercetin from the PEGylated NPs is pH dependent. These NPs could be beneficial for long-term release of drug.
1. Introduction The rapid growth of nanotechnology and availability of various nanoparticles (NPs) with controlled size and tunable properties has generated widespread interest in their utilization in biological systems [1,2]. Water soluble NPs surface can be functionalized to produce useful systems that have found applications in tracking, labeling, sensing, imaging and drug delivery in biology [3–5]. The surface chemistry of NPs play an important role in active targeting because conventional NPs are uptake by macrophage system or reticulo-endothelial system (RES), when given intravenously by a process known as “opsonisation” [6]. The comparable size of NPs to biomolecules and their tunable properties are the main motivation for their use [7–9]. The major concern which hinders their use in biological system is their toxicity which is due to their increased surface reactivity [10]. However, photo toxicity of NPs is increasingly being employed for targeted cell killing by conjugating them to traditional photosensitizing drugs [11]. NPs have the advances in changing the distribution of the enfolded or adsorbed drug in the body, the release rate by increasing bioavailability and the permeability of the membrane. Polyethylene glycol (PEG) is a polymer of choice for conjugation due to its widespread acceptance and availability. The choice of PEG and strategy used for PEGylation affects various properties like protein adsorption, blood half life etc. PEG grafted to NPs surface has been proved to increase their
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biocompatibility, reduces immunogenicity and toxicity [12]. Quercetin (Qu) is a naturally available anticancer drugs, possesses various pharmacological properties such as antioxidation, antiulcer, antiviral, antiangiogenic, anti-allergic, anti-hypertensive, anti-inflammatory, cardiovascular protection, cataract prevention etc. Qu is a flavoinoid consisting of a core structure having 15 carbon atoms arranged in three rings (A, B and C) shown in Fig. 1a. There are various subclasses of flavonoids i.e. flavan-3-ol, flavonol, flavone, flavanonol and flavanone, depending upon the variation of C ring [13]. More than 4000 natural flavonoids distributed in plants are commonly consumed by humans as food or medicinal herbs since ancient time. However these are practically ineffective due to their low hydrophilicity, instability in physiological medium, low blood circulation, reduced drug clearance and other side effects and are the major challenges for drug adsorption [14]. The oxidative degradation of Qu is another issue which reduces its pharmacological efficiency. These difficulties can be overcome by formulating efficient drug delivery mechanism [15–18]. Different studies have been done to investigate the effect of flavonoids on the optical properties of QDs. It has also been reported that Qu can be used as ion chelating agent and prevent reaction between ions and DNA. The solubility and stability of Qu is the major concern during the studies of its properties and influence on the living organism. Quchitosan nanoconjugates have been used for the oral delivery of Doxorubicin by Mu et al. [19]. Nanoconjugate micelles enhance the cellular
Corresponding author. E-mail address: [email protected] (S.K. Tripathi).
https://doi.org/10.1016/j.mseb.2019.03.015 Received 19 February 2018; Received in revised form 8 February 2019; Accepted 21 March 2019 0921-5107/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. (a) Chemical Structure of Quercetin (b) Experimental setup used for synthesis.
procured from SD Fine-Chem. Ltd. Sodium hydroxide [NaOH], Borax and Hydrochloric acid were purchased from NICE Chemicals Pvt. Ltd. N-hydroxysuccinimide pure (NHS) and EDC Hydrochloride (EDC-HCl) extra pure, were purchased from HIMEDIA and SRL, respectively. Polyethylene glycol di-amine (PEG-diamine), Qu and Potassium chloride were purchased from SIGMA ALDRICH. Sodium acetate trihydrate and di-Potassium Hydrogen Orthophosphate were purchased from S.D. Fine and Qualigens, respectively. Acetic acid and Potassium dihydrogen phosphate were purchased from Central Drug House. Dimethyl sulfoxide (DMSO) was purchased from Merck. Dialysis Membrane-60 and Dialysis closure clips were purchased from HIMEDIA. All the reactions were performed in standard glassware, cleaned with chromic acid, rinsed with distilled water, ethanol, and acetone and dried before use.
uptake of doxorubicin by 2.2 folds higher than that of free doxorubicin. Kumar et al. [20] reported the synthesis of Qu conjugated with dextran coated Fe3O4 nanocarriers and concluded that these nanocarriers enhance the drug’s bioavailability. Vidal et al. [21] had synthesized polyhydroxybutyrate-co-hydroxyvalerate NPs loaded with Qu using a high-speed double-emulsion technique, where biocompatibility analysis of the NPs showed no cytotoxicity of drug loaded NPs. The solubility of Qu was increased by loading it on polymeric micelles (PEG-OCL) and via inducing cell cycle arrest in G2/M phase it inhibits the growth of cancer cells [22]. Clinical use of Qu requires an efficient carrier where PEGylated NPs can be a suitable option. PEGylation of delivery vehicle (drug carrier) leads to improvement in vivo performance and stability of various drugs and gene vectors [23,24]. Large number of biological obstacles for the transportation of NPs, such as mucus from healthy volunteers or patients with cystic fibrosis are being improved through PEGylation [25]. It also reduces adhesive interactions of colloids with intracellular components. Thus, drugs conjugated with PEGylated NPs offers many advantages for the better treatment and diagnosis of human diseases [26,27]. These properties of flavonoid Qu and PEG encouraged to synthesize Qu loaded PEGylated NPs and analyze their morphological and optical properties for drug delivery application. The hydrophobicity and stability in physiological medium are the two main hindrances for using Qu in biological applications. Therefore, in the present work, we report the simple and convenient method to conjugate the hydrophobic drug Qu with water dispersed PEGylated CdSe/ZnS NPs by chemical method using two different mechanisms. The optical and morphological properties have been investigated using UV–Visible absorption (UV–Vis), Photoluminescence (PL), Transmission electron microscopy (TEM), XRay Diffraction (XRD) and Fourier Transform Infra-Red spectroscopy (FT-IR). The optical properties of Qu loaded NPs have been investigated for different pH environments to explore the stability of Qu loaded NPs under physiological medium. Release profile of Qu from PEGylated NPs has also been studied under different pH environment. Overall the studies are novel and contribute to pH dependent drug delivery mechanism and thus the nanobiotechnology.
2.2. Characterization techniques All the synthesized samples were characterized by TEM, FT-IR, XRD, UV–Vis absorption, and PL spectroscopy. A Hitachi (H-7500) transmission electron microscope was operated at 120 kV to obtain high resolution images of the NPs. The samples for TEM were prepared by placing sample droplets onto a 300-mesh carbon film coated with Cu grid and wicking off the remaining solvent after 30 sec. FT-IR measurements were done using Perkin Elmer (Spectrum 400-FTIR) Spectrometer. The hydrodynamic size and polydispersity index of PEGylated NPs have been characterized using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) based on dynamic light scattering principle technique. The XRD spectra were collected on a MiniFlex Xray diffractometer with Cu Kα (1.54 Å) radiation. Absorption spectra were recorded on Double Beam Spectrophotometer (jasco v-630 spectrophotometer). PL spectra were recorded on Shimadzu Spectrofluorophotometer (RF-5301PC) using 1 cm × 1 cm quartz cuvette. XRD, FT-IR, UV–Vis absorption and PL spectra were taken at room temperature. De-ionized (DI) water was used as a reference for both UV–Vis and PL measurements. Digital pH-meter (Max electronics, India) equipped with a combined glass electrode was used for pH measurements.
2. Materials and methods 2.3. Experimental procedure 2.1. Materials 2.3.1. Preparation of buffer solutions Buffer solutions (pH 2, 4, 5, 6, 7.4, 8, 10) are prepared using DI water and following salts: pH 2 – (Potassium chloride/Hydrochloric
The reagents were of analytical grade and used without further purification. Mercaptoacetic acid extra pure (MAA) [HSCH2CO2H] was 116
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acid); pH 4, 5 – (Sodium acetate trihydrate/Acetic acid); pH 6, 7.4, 8 – (Potassium dihydrogen phosphate/Dipotassium hydrogen phosphate); pH 10 – (Borax/Sodium hydroxide).
% of Drug Released = (Concentration of drug released /Total amount of drug taken) × 100
Cumulative Percentage = (Volume of sample withdrawn (ml) 2.3.2. Synthesis of PEGylated CdSe/ZnS NPs The procedure to synthesize PEGylated NPs using PEG-diamine is same as adopted by Bharti et al. [28]. The synthesis of MAA capped CdSe QDs has been carried out using chemical route method. Cadmium acetate dihydrate and Sodium selenosulphate are used as precursors for Cd2+ and Se2− ions. The MAA capped CdSe QDs have been synthesized at 60 °C and followed by the growth of ZnS shell using Zinc Acetate dihydrate and sodium sulphide as precursors for Zinc and Sulfur, respectively, at room temperature. The precursors for shell growth are added alternatively and slowly to avoid the formation of alloys. PEGdiamine of Mn 2000 has been used for the PEGylation of CdSe/ZnS core/shell NPs. 10 ml of CdSe/ZnS core/shell NPs are taken in a round bottom flask. 0.7 M of NHS powder is added to core/shell NPs and stirred the solution to dissolve it completely. 10 mg PEG-diamine is added to the above mixture. The pH of the above mixture is maintained within 7.4–8 using 1 M NaOH solution for the stable carbodiimide reaction. Aqueous solution of EDC-HCl has been prepared separately and added to the mixture of NPs solution dropwise. The solution is left for stirring for 5 h at room temperature. The molar ratio of CdSe/ZnS NPs, EDC-HCl, NHS and PEG-diamine is 1:14:14:10.
/ Bath Volume) × PD (t− 1) + PD (t) where PD (t) = Percentage release of drug at (t − 1) = Percentage release of drug previous to ‘t’.
time
‘t’;
PD
2.3.5. Release kinetics The release data is evaluated kinetically using software KinetDS3.0 (revolution copy 2010) [29] to study the possible mechanism of drug release from PEGylated NPs. This software has several models which can be used to characterize the dissolution profile and release mechanism of drug release from PEGylated NPs. The model which gave highest value of coefficient of determination (R2) is considered to be the most suitable kinetic model for describing the release of Qu from PEGylated NPs. R2 is an important parameter in determining the degree of linear-correlation of variables (‘goodness of fit’) in regression analysis. 3. Results and discussion 3.1. TEM micrographs Image analysis using TEM has been performed on non-filtered samples. Fig. 3 shows the TEM micrographs of CZPD-I and CZPD-II NPs. The average size of PEGylated CdSe/ZnS NPs obtained from CdSe/ZnS NPs is 22 nm, with particle density of 1.28 × 109 particles/cm2 [28]. TEM images show that the size of PEGylated NPs slightly increased after conjugation with Qu. Also the drug loading leads to more agglomeration of NPs in comparison to the prior sample of PEGylated CdSe/ZnS NPs. The particles are nearly spherical in shape with average particle size of CZPD-I is ∼24 nm and CZPD-II is ∼26 nm. Overall the size of NPs obtained is suitable for drug delivery applications. The particle size along with particle size distribution determines the in vivo distribution and biological outcome of the NPs, which are important parameters to consider in the development of viable cancer treatments. In addition it influences the drug loading, drug release rate and drug stability of the nano drug carriers [30,31].
2.3.3. Synthesis of Qu loaded PEGylated CdSe/ZnS NPs The limited solubility and stability under physiological pH of Qu is the principal obstacle to its encapsulation. So two different mechanisms are adopted for loading Qu over the PEGylated CdSe/ZnS NPs; first using an organic solvent DMSO to dissolve Qu and second by dispersing Qu directly in water. Solution 1: In a round bottom flask, 5 ml PEGylated NPs have been taken and added 0.89 mM EDC-HCl and 0.9 mM NHS to it. Stir the mixture to completely dissolve EDC-HCl and NHS. Method I: 5 ml solution of Qu (1.2 mM) taking DMSO and water in 1:4 by volume is prepared. Add the prepared Qu solution to Solution 1. Stir the mixture for 2 h at room temperature. The resulting sample is named as CZPD-I in this paper. Method II: 1.2 mM solution of Qu is directly made in 5 ml water by sonicating the mixture for 15 min. Add this mixture of drug to Solution 1 and stir for 2 h at room temperature. The resulting sample is named as CZPD-II in this paper.
3.2. Dynamic light scattering The hydrodynamic size of the suspended NPs is determined by DLS, where 3 runs of sample, each for 3 min are taken. Fig. 4 shows the size distribution of Qu conjugated PEGylated CdSe/ZnS NPs (a) CZPD-I (b) CZPD-II NPs. The average hydrodynamic size of CZPD-I and CZPD-II NPs in colloidal solution is 213 and 260 nm, respectively. The size obtained from DLS is more than the size of NPs obtained from TEM. The reason behind the large particle size obtained from DLS is because it gives the hydrodynamic size of the particles, which include not just the particle itself, but the ionic and solvent layers associated with it in solution [32]. The obtained polydispersity index (PDI), which is measure of particle size distribution (mono/poly dispersity) is 0.268 and 0.581 for CZPD-I and CZPD-II NPs, respectively.
2.3.4. Drug release of Qu loaded PEGylated NPs Drug release studies have been carried out by measuring the absorption of released drug. First of all, a calibration curve is plotted for different concentration (µg/ml) of Qu vs. absorption of Qu, for two different pH i.e. 5 & 7.4, respectively. A stock solution of Qu has been prepared by weighing Qu accurately and transferred to a 100 ml volumetric flask and dissolved in buffer solutions (pH 5 & pH 7.4). Absorption spectra of drug solutions having different known concentrations (µg/ml) are acquired and plotted concentration vs. absorption. In vitro release profile of Qu loaded NPs is carried out in buffer solution of pH 5 and pH 7.4. 5 ml NPs are placed in a dialysis membrane-60. The layout of the experiment is shown in Fig. 2. The temperature of the chamber is maintained at 37 ± 1 °C and rpm of stirrer is set to 200. To estimate the release of Qu from NPs the samples are withdrawn at predefined time interval and absorbance is recorded at 240 nm. After removal of 3 ml sample the suspension is refilled with 3 ml fresh buffer solution of respective pH. The cumulative percentage of released drug is calculated using the following method:
3.3. UV–Visible absorption spectroscopy The absorption spectra provide valuable information regarding the loading of Qu over PEGylated CdSe/ZnS NPs. The absorption spectra of Qu dissolved in water is shown in Fig. 5a. All the samples used for taking the absorption spectra are prepared by dispersing the NPs in DI water. The molar concentration of the nanoconjugates in the solvent is maintained constant. The spectrum of Qu consists of two bands in 250–700 nm range [33]. Band I is called cinnamoyl band, it lies in the
Concentration of Drug (μ g/ml) = (Slope × Absorbance) ± Intercept 117
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Fig. 2. Layout of the Drug Release experiment using Dialysis membrane.
range of 300–380 nm having λmax around 350–370 nm. The absorption in this range can be attributed to ring C shown in Fig. 1a. Band II is called benzoyl band, it lies in the range 240–280 with λmax around 250–270 nm. This band is attributed to the two rings of Qu: Ring A and Ring B [34,35]. The absorption spectroscopic measurements of PEGylated NPs and Qu loaded PEGylated NPs are presented in Fig. 5b. The colloidal solution of PEGylated NPs shows band to band absorption (peak at 388 nm and absorption edge at 448 nm) in the UV–Vis spectrum. It is observed from the spectrum of Qu conjugated NPs that there is no formation of new peak. This may be due to the fact that Qu is adsorbed on the surface of NPs. For CZPD-I the absorption peak lies at 395 nm and absorption edge at 456 nm, so there is nearly ∼7 nm shift in peak and the edge in comparison to the PEGylated NPs. For CZPD-II the absorption peak lies at 390.5 nm and absorption edge at 452 nm with very small red shift. The red shift in the spectrum is due to the conjugation of PEGylated NPs to Quercetin molecule through hydrogen bonding. The formation of hydrogen bond between two molecules results in a shift of
absorption band towards higher wavelength without forming new peaks [36]. There is more shift in the absorption band towards higher wavelength in CZPD-I as compared to CZPD-II. This may be due to more agglomeration in first case. The absorption peaks are broader in drug loaded NPs as compared to PEGylated NPs which may be due to the increase in polydispersity of drug conjugated NPs. Amino eNH2 of PEGylated NPs can form a strong hydrogen bonding with eOH groups of Qu. 3.4. Photoluminescence spectroscopy Fluorescence is an excellent probe for looking at the electronic transitions of the NPs. Fig. 6a shows the emission spectra of Qu excited at 400 nm. The obtained spectrum shows that Qu is not a fluorescent molecule for excitation wavelength 400 nm. The spectroscopic properties of flavonoids are dependent on the number and position of OH groups over chemical structure. The structure of Qu contains 5 eOH group which belongs to special class of non-fluorescent molecules in
Fig. 3. TEM images of Qu loaded PEGylated CdSe/ZnS NPs (a) CZPD-I (b) CZPD-II. 118
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Fig. 4. Dynamic light scattering (DLS) size measurement of (a) CZPD-I (b) CZPD-II NPs.
emission intensity of NPs (i.e.) fluorescence quenching [41,42]. Quenching in CZPD-I is more as compared to CZPD-II. This may be due to the fact that CZPD-I contains DMSO and it has greater proton affinity than water. In DMSO, a hydrogen bonding interaction is established between the phenolic eOH groups of the phenols and the oxygen atom of DMSO (OeH⋯O), which polarize the OeH bond, making the proton transfer easier than other solvents [43].
aqueous solution. When dispersed in aqueous medium, the intermolecular hydrogen bond between the aqueous and polar groups of solute becomes much higher than intramolecular hydrogen bonds. Therefore, a large dihedral angle makes it difficult for excited state intramolecular charge transfer to occur. Thus, the formation of a distorted excited state will become very active in water and Qu shows no fluorescence emission [37,38]. Fig. 6 (b) shows the emission spectra of CdSe, CdSe/ZnS, PEGylated CdSe/ZnS, CZPD-I, and CZPD-II NPs, at excitation wavelengths λex: 400 nm. All the samples used for taking the emission spectra are prepared by dispersing NPs in DI water. The concentration of the synthesized nanoconjugates is maintained constant. PEGylated CdSe/ZnS NPs emits the strongest fluorescence as compared to CdSe and CdSe/ZnS NPs. PEG chain provides good adsorption over CdSe/ZnS NPs and protects them from fluorescence quenching. The enhancement in the emission intensity of PEGylated NPs is due to the reduction of surface defects [39]. The presence of Qu has quenched the luminescence of PEGylated NPs without affecting the position of their emission peak. Quenching of the emission spectra can be attributed to the electronic interactions between the drug molecule and the PEGylated CdSe/ZnS NPs. Qu can quench PEGylated NPs through photo-induced electron transfer (PIET) process [40]. The decreased fluorescence confirms that a large fraction of excited PEGylated CdSe/ZnS molecules are quenched by the Qu particles. This result suggests that Qu binding indeed causes energy transfer from PEGylated NPs to Qu, which quenches the fluorescence of NPs. On adding a hole acceptors i.e. Qu to PEGylated NPs the electron hole recombination process will be prevented which causes decrease in the
3.5. Fourier transform infra-red spectroscopy FT-IR analysis traces the fingerprints of the encapsulation of chemical molecules. Fig. 7 shows the FT-IR spectra of (a) Quercetin (b) PEGylated CdSe/ZnS (c) CZPD-I (d) CZPD-II NPs. The presence of characteristics band at ν ∼1654 cm−1 and 1578 cm−1 in PEGylated CdSe/ZnS IR spectra are attributed to the presence of NeH bond. The attachment of PEG-diamine to CdSe/ZnS core/shell NPs is confirmed by the presence of peak around ν ∼1162 cm−1 due to the ether bond (eCeOe) of PEG-diamine. The spectrum reveals that the basic structure of PEG does not change, except for the conversion of terminal groups. Free Qu has major peaks in the range 900–1700 cm−1 due to the hydroxyl, aromatic and carboxylic groups of Qu [20]. All the peaks in free Qu are very well matched with the literature. The broad absorption bands at 3382 cm−1, 3420 cm−1, 3385 cm−1 and 3347 cm−1 in Fig. 7 (a, b, c and d), respectively are due to OeH vibrations of absorbed hydroxyl molecule. The intensity of the peak at 1643 cm−1, 1641 cm−1 for CZPD-I and CZPD-II, respectively increases as compared to PEGylated CdSe/ZnS NPs due to the band C]O of Qu
Fig. 5. Room temperature absorption spectra of (a) Qu, (b) PEGylated CdSe/ZnS, CZPD-I and CZPD-II NPs dispersed in DI water with concentration maintained constant i.e. 1.42 mM. 119
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Fig. 6. Room temperature emission spectra of (a) Qu at λex = 400 nm (b) CdSe, CdSe/ZnS, PEGylated CdSe/ZnS, CZPD-I, CZPD-II NPs (λex = 400 nm) dispersed in DI water with concentration maintained constant i.e. 1.42 mM.
which is not intense in PEGylated CdSe/ZnS. In CZPD-I and CZPD-II the major characteristic peaks of Qu aromatic bending and stretching at 1100–1600 cm−1, and eOH phenolic bending at 1200–1400 cm−1 are present, which shows the presence of Qu. Peak at ∼903 cm−1 for CZPD-I and for CZPD-II at 906 cm−1 are due to the para di-substituted benzene of Qu. The band present at 705 cm−1 and 708 cm−1 for CZPD-I and CZPD-II, respectively represents the aromatic CeH out of plane blend of Qu. The presence of peaks at 1318 cm−1, 1010 cm−1 and
[44]. This gives the indirect confirmation of conjugation of Qu with PEGylated CdSe/ZnS NPs. The addition of Qu in PEGylated CdSe/ZnS causes the stretching of OeH bond with a slightly blue shift along with narrowing down the peak width [45]. For CZPD-I, peaks at 1439 cm−1, 1408 cm−1 and for CZPD-II peaks at 1440 cm−1, 1417 cm−1 may be due to the presence of aromatic ring of Qu. In CZPD-I and CZPD-II the intense peaks at 1010 cm−1, 951 cm−1 and 1014 cm−1, 950 cm−1 are well matched with free Qu
Fig. 7. FT-IR spectra of (a) Qu (b) PEGylated CdSe/ZnS (c) CZPD-I and (d) CZPD-II NPs. 120
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Fig. 8. Schematic diagram representing adsorption of Qu over PEGylated CdSe/ZnS NPs surface.
1319 cm−1, 1014 cm−1 for CZPD-I and CZPD-II cm−1, respectively are due to the presence of stretching at CeO of Qu. The FT-IR analysis confirms the conjugation of hydrophobic Qu molecules over PEGylated CdSe/ZnS NPs surface via the addition of water soluble EDC-HCl and NHS as illustrated in Fig. 8.
spectra of CdSe QDs and CdSe/ZnS core/shell NPs. The observed peaks for core/shell NPs are same as that of bare CdSe QDs with a little shift towards higher theta and no peak for free ZnS is observed. In the XRD spectrum of PEGylated CdSe/ZnS NPs there is a presence of both the peaks of PEG-diamine. The spectrum predicts that NPs retained their crystallinity on PEGylation. But after conjugation of PEGylated NPs with Qu it shows amorphous characteristics for both CZPD-I and CZPD-II drug loaded NPs. There is no peak characterizing the diffraction pattern of pure flavonoid in the XRD graph of Qu-loaded PEGylated NPs. It may be due to the dispersion of Qu in a noncrystalline state over the PEGylated NPs [46]. The amorphous forms of the drugs are always more acceptable as they possess higher energy and higher surface area which provides higher solubility to the material [47].
3.6. XRD spectra Fig. 9 (a–d) shows the XRD spectra of PEG-diamine, PEGylated CdSe/ZnS NPs, CZPD-I, CZPD-II NPs respectively. The XRD spectra of PEG-diamine contain only two peaks at 19° and 23.2° shown in Fig. 9 (a). As CdSe/ZnS NPs contains peaks around 23.45°, 25.75°, 26.65°, 33.95° and broad hump between 40° and 50° correspond to (1 0 0), (0 0 2), (1 0 1), (1 1 0), (1 0 3) and (2 0 0) planes, respectively [39]. The hexagonal (wurtzite) phase dominates during the formation of core CdSe QDs. The hexagonal state is the stable phase, while the sphalerite cubic (Zinc-Blende) is the metastable state. Hexagonal phase is thermodynamically more stable and is achieved at higher temperature, at which thermodynamics (rather than the reaction kinetics), governs the crystal growth. Fig. S1 [SI] shows the XRD
3.7. Effect of pH on absorption and luminescence properties of Qu loaded NPs Stability of synthesized NPs in solution is an important requirement for their therapeutic and biomedical applications. This feature is analyzed by monitoring the absorption and
Fig. 9. XRD spectra of (a) PEG-diamine (b) PEGylated CdSe/ZnS (c) CZPD-I (d) CZPD-II NPs. 121
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Fig. 10. Room temperature absorption spectra of (a) CZPD-I (b) CZPD-II NPs at different pH.
Fig. 11. Room temperature emission spectra of (a) CZPD-I and (b) CZPD-II NPs at different pH.
Fig. 12. In-vitro release profile of CZPD-I and CZPD-II NPs at (a) 7.4 and (b) 5 pH.
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Table 1 Represents the R2 values obtained after fitting the data using different models. Sample name
pH
Zero order
1st order
2nd order
Korsmeyer peppas
Weibull with lagtime
K.M peppas with lagtime
Hill equation
CZPD-I CZPD-II CZPD-I CZPD-II
7.4
0.4841 0.5113 0.2624 0.1819
0.0526 0.0554 0.0397 0.0384
0.0325 0.0334 0.0269 0.0270
0.9788 0.9768 0.9766 0.9764
0.9913 0.9899 0.9875 0.9868
0.9883 0.9871 0.9865 0.9862
0.9879 0.9853 0.9795 0.9786
5
result of dissolution process parameters, a is the scale parameter that describe the time dependence, b is the shape of dissolution curve. The obtained value of the parameter b lies between 0.69 and 0.75, which indicates the drug release by diffusion process [51].
luminescence spectra of drug loaded NPs at different pH environment. Fig. 10 shows the absorption spectra of (a) CZPD-I and (b) CZPD-II NPs dispersed in different pH solutions keeping the concentration of NPs same for both UV–Vis and PL spectroscopy. Absorption spectra for both samples reveal that there is no absorption at pH 2 i.e. high acidic medium. NPs degrade/agglomerate at highly acidic environment. The absorbance edge of Qu loaded PEGylated NPs shifts towards longer wavelength as pH decreases (from pH 10 to 2). The absorption of Qu loaded PEGylated NPs in the range 6–10 pH shows narrow absorbance band as compared to pH 4 and 2, i.e. highly acidic medium. It is well known that the behavior of NPs is greatly influenced by pH environment. It was reported earlier that PEGylated CdSe/ZnS NPs show maximum emission at pH 6 [28]. Fig. 11 shows the photoluminescence spectra of Qu conjugated NPs at different pH (2–10). Both CZPD-I and CZPD-II NPs show maximum emission at pH 10 i.e. alkaline medium, which shows that these synthesized NPs are stable at alkaline pH. In CZPD-I as pH changes from (2–10) emission increases. But in case of CZPD-II emission at pH 6 is more as compared to pH 8. The change in the emission spectra of Qu conjugated NPs at different pH may be due to the deprotonation of the molecule [48].
4. Conclusions This manuscript presents the successful synthesis of water dispersed core/shell CdSe/ZnS NPs functionalized with PEG-diamine and loaded with a hydrophobic drug molecule Qu using two different methods. The synthesized Qu loaded NPs using this method shows no agglomeration when dispersed in water. FT-IR, UV–Vis and TEM results show the successful loading of Qu over PEGylated NPs. Particle size obtained from TEM micrographs is 24 nm–26 nm, therefore these NPs can be suitable for drug delivery systems. As it is known that spherical particles bigger than 200 nm are efficiently filtered by liver, spleen and bone marrow, while particles smaller than 10 nm can be quickly cleared by the kidney or through extravasation, thus it makes 10–200 nm the ideal size range for the circulating spherical carriers. The obtained Qu loaded PEGylated CdSe/ZnS NPs are in non-crystalline state in both CZPD-I and CZPD-II. Thereby, one can expect an improvement of drug solubility. The effect of pH environment on the optical properties shows that Qu loaded NPs completely agglomerate in high acidic medium. Qu loaded PEGylated NPs show high emission in the pH range 6–10, shows the optical stability of synthesized NPs in the range 6–10 pH. Presented study has also demonstrated that the release of Qu is controlled pH dependent and liberating more Qu at neutral pH rather than mildly acidic medium. The amine group of PEG-diamine contributes to the delay of release of Qu. Reduction of drug release at low pH conditions is advantageous because it reduces possible toxicity to the stomach wall, avoids degradation or inactivation by stomach secretions such as hydrochloric acid or pepsin, and increases the amount capable of being released in the small intestine.
3.8. Drug release of Qu loaded PEGylated CdSe/ZnS NPs Fig. 12 shows the in-vitro release profile of Qu loaded over PEGylated NPs at (a) pH 7.4 and (b) pH 5. Inset figures show the burst release of Qu within first 200 min. Release behavior of Qu from both samples CZPD-I and CZPD-II is pH dependent. Release of Qu from both CZPD-I and CZPD-II is significantly more intense at neutral pH condition i.e. pH 7.4, than that of mildly acidic conditions i.e. pH 5. In first 30 min Qu release at pH 7.4 reaches 15.8% and 13% for CZPD-I and CZPD-II, respectively. Whereas at pH 5 its only 8.3% and 7% for CZPD-I and CZPDII, respectively. The amount of Qu release from PEGylated NPs is rapid in first 100 min i.e. burst release. Subsequently, the release of Qu becomes much slower and sustained. The maximum release for CZPD-I is 63.7% and 29% at pH 7.4 and 5, respectively. Similarly for CZPD-II maximum release is 57.8% and 30% at pH 7.4 and 5, respectively. Obtained results show that the Qu is not completely transferred from PEGylated NPs to buffer medium. As the functionalization plays important role in release of drugs, for instance, the functionalized-CdSe/ ZnS NPs with PEG-diamine is able to regulate drug release. Thus, the amino-groups in PEGylated CdSe/ZnS NPs contribute to delay of Qu release [49]. The results show that in-vitro release of Qu is not only dependent on the pH of the NPs but also depends on the physicochemical properties of PEGylated NPs. Reduction in the drug release at low pH is advantageous for various applications [50]. Seven different kinetic models are employed to evaluate the possible changes in the release mechanism. The obtained values of correlation coefficient for all the seven models have been shown in Table 1. The highest value for coefficient of determination (R2) is obtained for Weibull with lag time model. The empirical equation of this model is described by Weibull in 1951 given as:
Acknowledgments Miss. Shivani Bharti is grateful to Department of Science and Technology, India for providing the INSPIRE fellowship. We are also thankful to the chairperson of Department of Biochemistry, Panjab University Chandigarh for providing Shimadzu Spectrofluorophotometer (RF-5301PC) facility. Conflict of interest declaration Authors have no conflict of interest. Statement of data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
(t − T )b ⎤ ⎤ Q = Qo ⎡1 − exp ⎡− ⎥ ⎢ ⎢ ⎥ a ⎣ ⎦⎦ ⎣
Appendix A. Supplementary data
where, Q is the amount of dissolved drug as a function of time, t‟, Q0 is the total amount of drug being released, T is the lag time measured as a
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mseb.2019.03.015. 123
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References
0608611104. [26] S. Huo, S. Chen, N. Gong, J. Liu, X. Li, Y. Zhao, X. Liang, Ultrasmall gold nanoparticles behavior in vivo modulated by surface polyethylene glycol (PEG) grafting, Bioconjugate Chem. 28 (2017) 239–243, https://doi.org/10.1021/acs. bioconjchem.6b00488. [27] D. Dorniani, A.U. Kura, M.Z. Hussein, S. Fakurazi, A.S. Halim, A. Zalinah, Controlled-release formulation of perindopril erbumine loaded PEG-coated magnetite nanoparticles for biomedical applications, J. Mater. Sci. 49 (2014) 8487–8497, https://doi.org/10.1007/s10853-014-8559-7. [28] S. Bharti, G. Kaur, S. Gupta, S.K. Tripathi, PEGylation of CdSe/ZnS core/shell nanoparticles and its behavior at different pH, J. Lumin. 181 (2017) 459–466, https:// doi.org/10.1016/j.jlumin.2016.09.005. [29] R.K. Deshmukh, J.B. Naik, Optimization of spray-dried diclofenac sodium-loaded microspheres by screening design, Dry. Technol. 34 (2016) 1593–1603, https://doi. org/10.1080/07373937.2016.1138121. [30] B. Ankamwar, Biosynthesis of gold nanoparticles (green-gold) using leaf extract of terminalia catappa, E-J. Chem. 7 (2010) 1334–1339, https://doi.org/10.1155/ 2010/745120. [31] P. Bocchiaro, A. Zamperini, World’s largest Science, Technology & Medicine Open Access Book Publisher, RFID Technology, Security Vulnerabilities, and Countermeasures, 2016. [32] P. Eaton, P. Quaresma, C. Soares, C. Neves, M.P. de Almeida, E. Pereira, P. West, A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles, Ultramicroscopy 182 (2017) 179–190. [33] W. Wang, C. Sun, L. Mao, P. Ma, F. Liu, J. Yang, Y. Gao, The biological activities, chemical stability, metabolism and delivery systems of quercetin : a review, Trends Food Sci. Technol. 56 (2016) 21–38, https://doi.org/10.1016/j.tifs.2016.07.004. [34] P. Pinheiro, G. Justino, Structural analysis of flavonoids and related compounds—a review of spectroscopic applications, in: Phytochem. – A Glob. Perspect. Their Role Nutr. Heal., vol. 33, 2012, pp. 33–56. [35] M. Bancirova, Changes of the quercetin absorption spectra in dependence on solvent, Chem. J. 1 (2015) 31–34. [36] M.C.M. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size related properties and applications toward biology, catalysis and nanotechnology, Chem. Rev. 104 (2004) 293–346. [37] G.S. Pillai, K. Krishnakumar, B. Krishnan, Fluorescence properties of quercetin – a review, Asian J. Res. Biol. Pharm. Sci. 5 (2017) 44–49. [38] J.C.A. Ensastegui, M.P. Pardave, M.R. Romo, M.T. Ramırez-Silva, Quercetin spectrofluorometric quantification in aqueous media using different surfactants as fluorescence promoters, RSC Adv. 8 (2018) 10980–10986, https://doi.org/10. 1039/c8ra01213j. [39] G. Kaur, S.K. Tripathi, Probing photoluminescence dynamics of colloidal CdSe/ZnS Core/shell nanoparticles, J. Lumin. 155 (2014) 330–337, https://doi.org/10.1016/ j.jlumin.2014.06.052. [40] S. Daniel, G.A.G. Raj, Photoinduced electron-transfer reactions of tris(4,4’-dinonyl2,2’-bipyridyl) ruthenium (ii) cation with phenolate ions in aqueous acetonitrile, J. Chem. Pharm. Res. 5 (2013) 220–227. [41] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, D.A. Ananth, S. Jagadeeswari, R. Renganathan, Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic wistar rats, Colloids Surf. B 112 (2013) 255–263, https://doi.org/10.1016/ j.colsurfb.2013.07.065. [42] J.M. Asha, V. Ellappan, S. Raja, K.A. Kumar, R. Renganathan, Investigation on the photoinduced interaction between water soluble CdTe quantum dots and certain antioxidants, Adv. Sci. Lett. 4 (2011) 3490–3495, https://doi.org/10.1166/asl. 2011.1895. [43] D. Sheeba, A.G.R. George, Luminescence quenching of tris(4,4’-dinonyl-2,2’-bipyridyl) ruthenium(ii) cation with phenolate ions in DMSO, Arab. J. Chem. 10 (2017) 2429–2435, https://doi.org/10.1016/j.arabjc.2013.09.006. [44] M. Heneczkowski, M. Kopacz, D. Nowak, A. Kuzniar, infrared spectrum analysis of some flavonoids, Acta Pol. Pharm. 58 (6) (2001) 415–420. [45] K. Wang, M. Li, H. Li, F. Guan, D. Zhang, H. Feng, H. Fan, Synthesis and characterization of CdSe/CdS/N-Acetyl-L-cysteine/quercetin nano-composites and their antibacterial performance, J. Korean Chem. Soc. 59 (2015) 136–141, https://doi. org/10.5012/jkcs.2015.59.2.136. [46] H. Pool, D. Quintanar, J.D.D. Figueroa, C.M. Mano, J.E.H. Bechara, L.A. Godínez, S. Mendoza, Antioxidant effects of quercetin and catechin encapsulated into PLGA nanoparticles, J. Nanomater. 2012 (2012) 1–12, https://doi.org/10.1155/2012/ 145380. [47] P.N. Kendre, V.V. Pande, K.M. Chavan, Novel formulation strategy to enhance solubility of quercetin, Pharmacophore 5 (2014) 358–370. [48] Z. Jurasekova, C. Domingo, J.V.G. Ramos, S.S. Cortes, Effect of pH on the chemical modification of quercetin and structurally related flavonoids characterized by optical (UV-Visible and Raman) spectroscopy, Phys. Chem. Chem. Phys. 16 (2014) 12802–12811, https://doi.org/10.1039/C4CP00864B. [49] K.E.A. AbouAitah, A.A. Farghali, A.S. Sroda, W. Lojkowski, A.M. Razin, M.H. Khedr, Mesoporous silica materials in drug delivery system: pH/glutathione-responsive release of poorly water-soluble pro-drug quercetin from two and three-dimensional pore-structure nanoparticles, J. Nanomed. Nanotechnol. 7 (2016) 1–12, https://doi. org/10.4172/2157-7439.1000360. [50] C.A. Schoener, N. Hutson, N.A. Peppas, pH-responsive hydrogels with dispersed hydrophobic nanoparticles for the delivery of hydrophobic therapeutic agents, Polym. Int. 61 (2012) 874–879, https://doi.org/10.1002/pi.4219. [51] V. Papadopoulou, K. Kosmidis, M. Vlachou, P. Macheras, On the use of the weibull function for the disernment of drug release mechanisms, Int. J. Pharm. 309 (2006) 44–50, https://doi.org/10.1016/j.ijpharm.2005.10.044.
[1] G.F. Paciotti, J. Zhao, S. Cao, P.J. Brodie, L. Tamarkin, M. Huhta, L.D. Myer, J. Friedman, D.G.I. Kingston, Synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery, Bioconjug. Chem. 27 (2016) 2646–2657, https://doi.org/10.1021/acs.bioconjchem.6b00405. [2] Q. Liu, C. Zhan, D.S. Kohane, Phototriggered drug delivery using inorganic nanomaterials, Bioconjug. Chem. 28 (2017) 98–104, https://doi.org/10.1021/acs. bioconjchem.6b00448. [3] D. Yeo, C. Wiraja, Y.J. Chuah, Y. Gao, C. Xu, A nanoparticle-based sensor platform for cell tracking and status/function assessment, Sci. Rep. 5 (2015) 14768, https:// doi.org/10.1038/srep14768. [4] A. Rodzinski, R. Guduru, P. Liang, A. Hadjikhani, T. Stewart, E. Stimphil, C. Runowicz, R. Cote, N. Altman, R. Datar, S. Khizroev, Targeted and controlled anticancer drug delivery and release with magnetoelectric nanoparticles, Sci. Rep. 6 (2016) 20867, https://doi.org/10.1038/srep20867. [5] X. Gao, Z. Liu, Z. Lin, X. Su, CuInS2 quantum dots/poly((L)-glutamic acid)-drug conjugates for drug delivery and cell imaging, Analyst 139 (2014) 831–836, https://doi.org/10.1039/C3AN01134H. [6] J.M. Montenegro, V. Grazu, A. Sukhanova, S. Agarwal, J.M. de la Fuente, I. Nabiev, A. Greiner, W.J. Parak, Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. 65 (2013) 677–688, https:// doi.org/10.1016/j.addr.2012.12.003. [7] O.V. Salata, Applications of nanoparticles in biology and medicine, J. Nanobiotechnol. 2 (2004) 1–6, https://doi.org/10.1186/1477-3155-2-3. [8] I.Y. Kim, E. Joachim, H. Choi, K. Kim, Toxicity of silica nanoparticles depends on size, dose, and cell type, Nanomed. Nanotechnol., Biol. Med. 11 (2015) 1407–1416, https://doi.org/10.1016/j.nano.2015.03.004. [9] D. Sahu, G.M. Kannan, M. Tailang, R. Vijayaraghavan, In vitro cytotoxicity of nanoparticles : a comparison between particle size and cell type, J. Nanoscience 2016 (2016) 1–9, https://doi.org/10.1155/2016/4023852. [10] S. Bakand, A. Hayes, Toxicological considerations, toxicity assessment, and risk management of inhaled nanoparticles, Int. J. Mol. Sci. 17 (2016) 929, https://doi. org/10.3390/ijms17060929. [11] K.V. Chakravarthy, B.A. Davidson, J.D. Helinski, H. Ding, W.C. Law, K.T. Yong, P.N. Prasad, P.R. Knight, Doxorubicin-conjugated quantum dots to target alveolar macrophages and inflammation, Nanomed. Nanotechnol., Biol. Med. 7 (2011) 88–96, https://doi.org/10.1016/j.nano.2010.09.001. [12] D. Hutanu, M.D. Frishberg, L. Guo, C.C. Darie, Recent applications of polyethylene glycols (PEGs) and PEG derivatives, Mod. Chem. Appl. 2 (2014) 1–6, https://doi. org/10.4172/2329-6798.1000132. [13] J.S. Nam, A.R. Sharma, L.T. Nguyen, C. Chakraborty, G. Sharma, S.S. Lee, Application of bioactive quercetin in oncotherapy: from nutrition to nanomedicine, Molecules 21 (2016) 108, https://doi.org/10.3390/molecules21010108. [14] O.M. Andersen, K.R. Markham, Flavonoids. Chemistry, Biochemistry and Applications, CRC Press, 2005. [15] L. Gibellini, M. Pinti, M. Nasi, J.P. Montagna, S. De Biasi, E. Roat, L. Bertoncelli, E.L. Cooper, A. Cossarizza, Quercetin and cancer chemoprevention, Evid. Based Complementary Altern. Med. 2011 (2011), https://doi.org/10.1093/ecam/neq053. [16] Z. Xingyu, M. Peijie, P. Dan, W. Youg, W. Daojun, C. Xinzheng, Z. Xijun, S. Yangrong, Quercetin suppresses lung cancer growth by targeting aurora B kinase, Cancer Med. 5 (2016) 3156–3165, https://doi.org/10.1002/cam4.891. [17] J.H. Jeong, J. An, Y. Kwon, J.G. Rhee, Y.J. Lee, Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression, J. Cell. Biochem. 106 (2009) 73–82, https://doi.org/10.1002/jcb.21977. [18] L. Ma, J.M. Feugang, P. Konarski, J. Wang, J. Lu, S. Fu, B. Ma, B. Tian, C. Zou, Z. Wang, Growth inhibitory effects of quercetin on bladder cancer cell, Front. Biosci. 11 (2006) 2275–2285, https://doi.org/10.2741/1970. [19] Y. Mu, Y. Fu, J. Li, X. Yu, Y. Li, Y. Wang, X. Wu, K. Zhang, M. Kong, C. Feng, X. Chen, Multifunctional quercetin conjugated chitosan nano-micelles with P-gp inhibition and permeation enhancement of anticancer drug, Carbohydr. Polym. 203 (2019) 10–18, https://doi.org/10.1016/j.carbpol.2018.09.020. [20] S.R. Kumar, S. Priyatharshni, V.N. Babu, D. Mangalaraj, C. Viswanathan, S. Kannan, N. Ponpandian, Quercetin conjugated superparamagnetic magnetite nanoparticles for in-vitro analysis of breast cancer cell lines for chemotherapy applications, J. Colloid Interface Sci. 436 (2014) 234–242, https://doi.org/10.1016/j.jcis.2014.08. 064. [21] S.L. Vidal, C. Rojas, R.B. Padin, M.P. Rivera, A. Haensgen, M. Gonzalez, S.R. Llamazares, Synthesis and characterization of polyhydroxybutyrate-co-hydroxyvalerate nanoparticles for encapsulation of quercetin, J. Bioact. Compat. Polym. 31 (2016) 439–452, https://doi.org/10.1177/0883911516635839. [22] R. Khonkarn, S. Mankhetkorn, W.E. Hennink, S. Okonogi, PEG-OCL micelles for quercetin solubilization and inhibition of cancer cell growth, Eur. J. Pharm. Biopharm. 79 (2011) 268–275, https://doi.org/10.1016/j.ejpb.2011.04.011. [23] P. Muralidharan, E. Mallory, M. Malapit, H. Don, H.M. Mansour, Inhalable PEGylated phospholipid nanocarriers and PEGylated therapeutics for respiratory delivery as aerosolized colloidal dispersions and dry powder inhalers, Pharmaceutics 6 (2014) 333–353, https://doi.org/10.3390/ pharmaceutics6020333. [24] H. Otsuka, Y. Nagasaki, K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv. Drug Deliv. Rev. 64 (2012) 246–255, https:// doi.org/10.1016/j.addr.2012.09.022. [25] S.K. Lai, D.E. O’Hanlon, S. Harrold, S.T. Man, Y.-Y. Wang, R.A. Cone, J. Hanes, Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 1482–1487, https://doi.org/10.1073/pnas.
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Contents lists available at ScienceDirect
Materials Science & Engineering B journal homepage: www.elsevier.com/locate/mseb
Pegylated CdSe/ZnS core/shell nanoparticles for controlled drug release a
b
c
Shivani Bharti , Gurvir Kaur , Shikha Gupta , S.K. Tripathi a b c
a,⁎
T
Centre of Advanced Study in Physics, Department of Physics, Panjab University, Chandigarh 160014, India Sri Guru Gobind Singh College, Chandigarh, India Goswami Ganesh Dutta Sanatan Dharma College, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: PEGylation Core/shell nanoparticles Flavonoids In vitro studies Drug delivery
A facile strategy to synthesize PEGylated CdSe/ZnS core/shell nanoparticles (NPs) loaded with a hydrophobic anticancer drug Quercetin is presented in this manuscript. Drug is loaded over PEGylated NPs by two different mechanisms; one by using organic solvent to dissolve Quercetin and second by directly dispersing Quercetin in Water. FTIR spectra of Quercetin loaded NPs predict the successful drug loading over spherical NPs having size ∼30 nm obtained from TEM. Quercetin loading over PEGylated NPs increases the size of NPs as obtained from TEM and dynamic light scattering. The emission spectra show enhancement in fluorescence intensity of Quercetin after conjugating with PEGylated NPs, making NPs as drug release indicators. Effect of pH on Quercetin conjugated NPs have been investigated on the basis of optical measurements. The results show that in vitro release of Quercetin from the PEGylated NPs is pH dependent. These NPs could be beneficial for long-term release of drug.
1. Introduction The rapid growth of nanotechnology and availability of various nanoparticles (NPs) with controlled size and tunable properties has generated widespread interest in their utilization in biological systems [1,2]. Water soluble NPs surface can be functionalized to produce useful systems that have found applications in tracking, labeling, sensing, imaging and drug delivery in biology [3–5]. The surface chemistry of NPs play an important role in active targeting because conventional NPs are uptake by macrophage system or reticulo-endothelial system (RES), when given intravenously by a process known as “opsonisation” [6]. The comparable size of NPs to biomolecules and their tunable properties are the main motivation for their use [7–9]. The major concern which hinders their use in biological system is their toxicity which is due to their increased surface reactivity [10]. However, photo toxicity of NPs is increasingly being employed for targeted cell killing by conjugating them to traditional photosensitizing drugs [11]. NPs have the advances in changing the distribution of the enfolded or adsorbed drug in the body, the release rate by increasing bioavailability and the permeability of the membrane. Polyethylene glycol (PEG) is a polymer of choice for conjugation due to its widespread acceptance and availability. The choice of PEG and strategy used for PEGylation affects various properties like protein adsorption, blood half life etc. PEG grafted to NPs surface has been proved to increase their
⁎
biocompatibility, reduces immunogenicity and toxicity [12]. Quercetin (Qu) is a naturally available anticancer drugs, possesses various pharmacological properties such as antioxidation, antiulcer, antiviral, antiangiogenic, anti-allergic, anti-hypertensive, anti-inflammatory, cardiovascular protection, cataract prevention etc. Qu is a flavoinoid consisting of a core structure having 15 carbon atoms arranged in three rings (A, B and C) shown in Fig. 1a. There are various subclasses of flavonoids i.e. flavan-3-ol, flavonol, flavone, flavanonol and flavanone, depending upon the variation of C ring [13]. More than 4000 natural flavonoids distributed in plants are commonly consumed by humans as food or medicinal herbs since ancient time. However these are practically ineffective due to their low hydrophilicity, instability in physiological medium, low blood circulation, reduced drug clearance and other side effects and are the major challenges for drug adsorption [14]. The oxidative degradation of Qu is another issue which reduces its pharmacological efficiency. These difficulties can be overcome by formulating efficient drug delivery mechanism [15–18]. Different studies have been done to investigate the effect of flavonoids on the optical properties of QDs. It has also been reported that Qu can be used as ion chelating agent and prevent reaction between ions and DNA. The solubility and stability of Qu is the major concern during the studies of its properties and influence on the living organism. Quchitosan nanoconjugates have been used for the oral delivery of Doxorubicin by Mu et al. [19]. Nanoconjugate micelles enhance the cellular
Corresponding author. E-mail address: [email protected] (S.K. Tripathi).
https://doi.org/10.1016/j.mseb.2019.03.015 Received 19 February 2018; Received in revised form 8 February 2019; Accepted 21 March 2019 0921-5107/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. (a) Chemical Structure of Quercetin (b) Experimental setup used for synthesis.
procured from SD Fine-Chem. Ltd. Sodium hydroxide [NaOH], Borax and Hydrochloric acid were purchased from NICE Chemicals Pvt. Ltd. N-hydroxysuccinimide pure (NHS) and EDC Hydrochloride (EDC-HCl) extra pure, were purchased from HIMEDIA and SRL, respectively. Polyethylene glycol di-amine (PEG-diamine), Qu and Potassium chloride were purchased from SIGMA ALDRICH. Sodium acetate trihydrate and di-Potassium Hydrogen Orthophosphate were purchased from S.D. Fine and Qualigens, respectively. Acetic acid and Potassium dihydrogen phosphate were purchased from Central Drug House. Dimethyl sulfoxide (DMSO) was purchased from Merck. Dialysis Membrane-60 and Dialysis closure clips were purchased from HIMEDIA. All the reactions were performed in standard glassware, cleaned with chromic acid, rinsed with distilled water, ethanol, and acetone and dried before use.
uptake of doxorubicin by 2.2 folds higher than that of free doxorubicin. Kumar et al. [20] reported the synthesis of Qu conjugated with dextran coated Fe3O4 nanocarriers and concluded that these nanocarriers enhance the drug’s bioavailability. Vidal et al. [21] had synthesized polyhydroxybutyrate-co-hydroxyvalerate NPs loaded with Qu using a high-speed double-emulsion technique, where biocompatibility analysis of the NPs showed no cytotoxicity of drug loaded NPs. The solubility of Qu was increased by loading it on polymeric micelles (PEG-OCL) and via inducing cell cycle arrest in G2/M phase it inhibits the growth of cancer cells [22]. Clinical use of Qu requires an efficient carrier where PEGylated NPs can be a suitable option. PEGylation of delivery vehicle (drug carrier) leads to improvement in vivo performance and stability of various drugs and gene vectors [23,24]. Large number of biological obstacles for the transportation of NPs, such as mucus from healthy volunteers or patients with cystic fibrosis are being improved through PEGylation [25]. It also reduces adhesive interactions of colloids with intracellular components. Thus, drugs conjugated with PEGylated NPs offers many advantages for the better treatment and diagnosis of human diseases [26,27]. These properties of flavonoid Qu and PEG encouraged to synthesize Qu loaded PEGylated NPs and analyze their morphological and optical properties for drug delivery application. The hydrophobicity and stability in physiological medium are the two main hindrances for using Qu in biological applications. Therefore, in the present work, we report the simple and convenient method to conjugate the hydrophobic drug Qu with water dispersed PEGylated CdSe/ZnS NPs by chemical method using two different mechanisms. The optical and morphological properties have been investigated using UV–Visible absorption (UV–Vis), Photoluminescence (PL), Transmission electron microscopy (TEM), XRay Diffraction (XRD) and Fourier Transform Infra-Red spectroscopy (FT-IR). The optical properties of Qu loaded NPs have been investigated for different pH environments to explore the stability of Qu loaded NPs under physiological medium. Release profile of Qu from PEGylated NPs has also been studied under different pH environment. Overall the studies are novel and contribute to pH dependent drug delivery mechanism and thus the nanobiotechnology.
2.2. Characterization techniques All the synthesized samples were characterized by TEM, FT-IR, XRD, UV–Vis absorption, and PL spectroscopy. A Hitachi (H-7500) transmission electron microscope was operated at 120 kV to obtain high resolution images of the NPs. The samples for TEM were prepared by placing sample droplets onto a 300-mesh carbon film coated with Cu grid and wicking off the remaining solvent after 30 sec. FT-IR measurements were done using Perkin Elmer (Spectrum 400-FTIR) Spectrometer. The hydrodynamic size and polydispersity index of PEGylated NPs have been characterized using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) based on dynamic light scattering principle technique. The XRD spectra were collected on a MiniFlex Xray diffractometer with Cu Kα (1.54 Å) radiation. Absorption spectra were recorded on Double Beam Spectrophotometer (jasco v-630 spectrophotometer). PL spectra were recorded on Shimadzu Spectrofluorophotometer (RF-5301PC) using 1 cm × 1 cm quartz cuvette. XRD, FT-IR, UV–Vis absorption and PL spectra were taken at room temperature. De-ionized (DI) water was used as a reference for both UV–Vis and PL measurements. Digital pH-meter (Max electronics, India) equipped with a combined glass electrode was used for pH measurements.
2. Materials and methods 2.3. Experimental procedure 2.1. Materials 2.3.1. Preparation of buffer solutions Buffer solutions (pH 2, 4, 5, 6, 7.4, 8, 10) are prepared using DI water and following salts: pH 2 – (Potassium chloride/Hydrochloric
The reagents were of analytical grade and used without further purification. Mercaptoacetic acid extra pure (MAA) [HSCH2CO2H] was 116
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acid); pH 4, 5 – (Sodium acetate trihydrate/Acetic acid); pH 6, 7.4, 8 – (Potassium dihydrogen phosphate/Dipotassium hydrogen phosphate); pH 10 – (Borax/Sodium hydroxide).
% of Drug Released = (Concentration of drug released /Total amount of drug taken) × 100
Cumulative Percentage = (Volume of sample withdrawn (ml) 2.3.2. Synthesis of PEGylated CdSe/ZnS NPs The procedure to synthesize PEGylated NPs using PEG-diamine is same as adopted by Bharti et al. [28]. The synthesis of MAA capped CdSe QDs has been carried out using chemical route method. Cadmium acetate dihydrate and Sodium selenosulphate are used as precursors for Cd2+ and Se2− ions. The MAA capped CdSe QDs have been synthesized at 60 °C and followed by the growth of ZnS shell using Zinc Acetate dihydrate and sodium sulphide as precursors for Zinc and Sulfur, respectively, at room temperature. The precursors for shell growth are added alternatively and slowly to avoid the formation of alloys. PEGdiamine of Mn 2000 has been used for the PEGylation of CdSe/ZnS core/shell NPs. 10 ml of CdSe/ZnS core/shell NPs are taken in a round bottom flask. 0.7 M of NHS powder is added to core/shell NPs and stirred the solution to dissolve it completely. 10 mg PEG-diamine is added to the above mixture. The pH of the above mixture is maintained within 7.4–8 using 1 M NaOH solution for the stable carbodiimide reaction. Aqueous solution of EDC-HCl has been prepared separately and added to the mixture of NPs solution dropwise. The solution is left for stirring for 5 h at room temperature. The molar ratio of CdSe/ZnS NPs, EDC-HCl, NHS and PEG-diamine is 1:14:14:10.
/ Bath Volume) × PD (t− 1) + PD (t) where PD (t) = Percentage release of drug at (t − 1) = Percentage release of drug previous to ‘t’.
time
‘t’;
PD
2.3.5. Release kinetics The release data is evaluated kinetically using software KinetDS3.0 (revolution copy 2010) [29] to study the possible mechanism of drug release from PEGylated NPs. This software has several models which can be used to characterize the dissolution profile and release mechanism of drug release from PEGylated NPs. The model which gave highest value of coefficient of determination (R2) is considered to be the most suitable kinetic model for describing the release of Qu from PEGylated NPs. R2 is an important parameter in determining the degree of linear-correlation of variables (‘goodness of fit’) in regression analysis. 3. Results and discussion 3.1. TEM micrographs Image analysis using TEM has been performed on non-filtered samples. Fig. 3 shows the TEM micrographs of CZPD-I and CZPD-II NPs. The average size of PEGylated CdSe/ZnS NPs obtained from CdSe/ZnS NPs is 22 nm, with particle density of 1.28 × 109 particles/cm2 [28]. TEM images show that the size of PEGylated NPs slightly increased after conjugation with Qu. Also the drug loading leads to more agglomeration of NPs in comparison to the prior sample of PEGylated CdSe/ZnS NPs. The particles are nearly spherical in shape with average particle size of CZPD-I is ∼24 nm and CZPD-II is ∼26 nm. Overall the size of NPs obtained is suitable for drug delivery applications. The particle size along with particle size distribution determines the in vivo distribution and biological outcome of the NPs, which are important parameters to consider in the development of viable cancer treatments. In addition it influences the drug loading, drug release rate and drug stability of the nano drug carriers [30,31].
2.3.3. Synthesis of Qu loaded PEGylated CdSe/ZnS NPs The limited solubility and stability under physiological pH of Qu is the principal obstacle to its encapsulation. So two different mechanisms are adopted for loading Qu over the PEGylated CdSe/ZnS NPs; first using an organic solvent DMSO to dissolve Qu and second by dispersing Qu directly in water. Solution 1: In a round bottom flask, 5 ml PEGylated NPs have been taken and added 0.89 mM EDC-HCl and 0.9 mM NHS to it. Stir the mixture to completely dissolve EDC-HCl and NHS. Method I: 5 ml solution of Qu (1.2 mM) taking DMSO and water in 1:4 by volume is prepared. Add the prepared Qu solution to Solution 1. Stir the mixture for 2 h at room temperature. The resulting sample is named as CZPD-I in this paper. Method II: 1.2 mM solution of Qu is directly made in 5 ml water by sonicating the mixture for 15 min. Add this mixture of drug to Solution 1 and stir for 2 h at room temperature. The resulting sample is named as CZPD-II in this paper.
3.2. Dynamic light scattering The hydrodynamic size of the suspended NPs is determined by DLS, where 3 runs of sample, each for 3 min are taken. Fig. 4 shows the size distribution of Qu conjugated PEGylated CdSe/ZnS NPs (a) CZPD-I (b) CZPD-II NPs. The average hydrodynamic size of CZPD-I and CZPD-II NPs in colloidal solution is 213 and 260 nm, respectively. The size obtained from DLS is more than the size of NPs obtained from TEM. The reason behind the large particle size obtained from DLS is because it gives the hydrodynamic size of the particles, which include not just the particle itself, but the ionic and solvent layers associated with it in solution [32]. The obtained polydispersity index (PDI), which is measure of particle size distribution (mono/poly dispersity) is 0.268 and 0.581 for CZPD-I and CZPD-II NPs, respectively.
2.3.4. Drug release of Qu loaded PEGylated NPs Drug release studies have been carried out by measuring the absorption of released drug. First of all, a calibration curve is plotted for different concentration (µg/ml) of Qu vs. absorption of Qu, for two different pH i.e. 5 & 7.4, respectively. A stock solution of Qu has been prepared by weighing Qu accurately and transferred to a 100 ml volumetric flask and dissolved in buffer solutions (pH 5 & pH 7.4). Absorption spectra of drug solutions having different known concentrations (µg/ml) are acquired and plotted concentration vs. absorption. In vitro release profile of Qu loaded NPs is carried out in buffer solution of pH 5 and pH 7.4. 5 ml NPs are placed in a dialysis membrane-60. The layout of the experiment is shown in Fig. 2. The temperature of the chamber is maintained at 37 ± 1 °C and rpm of stirrer is set to 200. To estimate the release of Qu from NPs the samples are withdrawn at predefined time interval and absorbance is recorded at 240 nm. After removal of 3 ml sample the suspension is refilled with 3 ml fresh buffer solution of respective pH. The cumulative percentage of released drug is calculated using the following method:
3.3. UV–Visible absorption spectroscopy The absorption spectra provide valuable information regarding the loading of Qu over PEGylated CdSe/ZnS NPs. The absorption spectra of Qu dissolved in water is shown in Fig. 5a. All the samples used for taking the absorption spectra are prepared by dispersing the NPs in DI water. The molar concentration of the nanoconjugates in the solvent is maintained constant. The spectrum of Qu consists of two bands in 250–700 nm range [33]. Band I is called cinnamoyl band, it lies in the
Concentration of Drug (μ g/ml) = (Slope × Absorbance) ± Intercept 117
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Fig. 2. Layout of the Drug Release experiment using Dialysis membrane.
range of 300–380 nm having λmax around 350–370 nm. The absorption in this range can be attributed to ring C shown in Fig. 1a. Band II is called benzoyl band, it lies in the range 240–280 with λmax around 250–270 nm. This band is attributed to the two rings of Qu: Ring A and Ring B [34,35]. The absorption spectroscopic measurements of PEGylated NPs and Qu loaded PEGylated NPs are presented in Fig. 5b. The colloidal solution of PEGylated NPs shows band to band absorption (peak at 388 nm and absorption edge at 448 nm) in the UV–Vis spectrum. It is observed from the spectrum of Qu conjugated NPs that there is no formation of new peak. This may be due to the fact that Qu is adsorbed on the surface of NPs. For CZPD-I the absorption peak lies at 395 nm and absorption edge at 456 nm, so there is nearly ∼7 nm shift in peak and the edge in comparison to the PEGylated NPs. For CZPD-II the absorption peak lies at 390.5 nm and absorption edge at 452 nm with very small red shift. The red shift in the spectrum is due to the conjugation of PEGylated NPs to Quercetin molecule through hydrogen bonding. The formation of hydrogen bond between two molecules results in a shift of
absorption band towards higher wavelength without forming new peaks [36]. There is more shift in the absorption band towards higher wavelength in CZPD-I as compared to CZPD-II. This may be due to more agglomeration in first case. The absorption peaks are broader in drug loaded NPs as compared to PEGylated NPs which may be due to the increase in polydispersity of drug conjugated NPs. Amino eNH2 of PEGylated NPs can form a strong hydrogen bonding with eOH groups of Qu. 3.4. Photoluminescence spectroscopy Fluorescence is an excellent probe for looking at the electronic transitions of the NPs. Fig. 6a shows the emission spectra of Qu excited at 400 nm. The obtained spectrum shows that Qu is not a fluorescent molecule for excitation wavelength 400 nm. The spectroscopic properties of flavonoids are dependent on the number and position of OH groups over chemical structure. The structure of Qu contains 5 eOH group which belongs to special class of non-fluorescent molecules in
Fig. 3. TEM images of Qu loaded PEGylated CdSe/ZnS NPs (a) CZPD-I (b) CZPD-II. 118
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Fig. 4. Dynamic light scattering (DLS) size measurement of (a) CZPD-I (b) CZPD-II NPs.
emission intensity of NPs (i.e.) fluorescence quenching [41,42]. Quenching in CZPD-I is more as compared to CZPD-II. This may be due to the fact that CZPD-I contains DMSO and it has greater proton affinity than water. In DMSO, a hydrogen bonding interaction is established between the phenolic eOH groups of the phenols and the oxygen atom of DMSO (OeH⋯O), which polarize the OeH bond, making the proton transfer easier than other solvents [43].
aqueous solution. When dispersed in aqueous medium, the intermolecular hydrogen bond between the aqueous and polar groups of solute becomes much higher than intramolecular hydrogen bonds. Therefore, a large dihedral angle makes it difficult for excited state intramolecular charge transfer to occur. Thus, the formation of a distorted excited state will become very active in water and Qu shows no fluorescence emission [37,38]. Fig. 6 (b) shows the emission spectra of CdSe, CdSe/ZnS, PEGylated CdSe/ZnS, CZPD-I, and CZPD-II NPs, at excitation wavelengths λex: 400 nm. All the samples used for taking the emission spectra are prepared by dispersing NPs in DI water. The concentration of the synthesized nanoconjugates is maintained constant. PEGylated CdSe/ZnS NPs emits the strongest fluorescence as compared to CdSe and CdSe/ZnS NPs. PEG chain provides good adsorption over CdSe/ZnS NPs and protects them from fluorescence quenching. The enhancement in the emission intensity of PEGylated NPs is due to the reduction of surface defects [39]. The presence of Qu has quenched the luminescence of PEGylated NPs without affecting the position of their emission peak. Quenching of the emission spectra can be attributed to the electronic interactions between the drug molecule and the PEGylated CdSe/ZnS NPs. Qu can quench PEGylated NPs through photo-induced electron transfer (PIET) process [40]. The decreased fluorescence confirms that a large fraction of excited PEGylated CdSe/ZnS molecules are quenched by the Qu particles. This result suggests that Qu binding indeed causes energy transfer from PEGylated NPs to Qu, which quenches the fluorescence of NPs. On adding a hole acceptors i.e. Qu to PEGylated NPs the electron hole recombination process will be prevented which causes decrease in the
3.5. Fourier transform infra-red spectroscopy FT-IR analysis traces the fingerprints of the encapsulation of chemical molecules. Fig. 7 shows the FT-IR spectra of (a) Quercetin (b) PEGylated CdSe/ZnS (c) CZPD-I (d) CZPD-II NPs. The presence of characteristics band at ν ∼1654 cm−1 and 1578 cm−1 in PEGylated CdSe/ZnS IR spectra are attributed to the presence of NeH bond. The attachment of PEG-diamine to CdSe/ZnS core/shell NPs is confirmed by the presence of peak around ν ∼1162 cm−1 due to the ether bond (eCeOe) of PEG-diamine. The spectrum reveals that the basic structure of PEG does not change, except for the conversion of terminal groups. Free Qu has major peaks in the range 900–1700 cm−1 due to the hydroxyl, aromatic and carboxylic groups of Qu [20]. All the peaks in free Qu are very well matched with the literature. The broad absorption bands at 3382 cm−1, 3420 cm−1, 3385 cm−1 and 3347 cm−1 in Fig. 7 (a, b, c and d), respectively are due to OeH vibrations of absorbed hydroxyl molecule. The intensity of the peak at 1643 cm−1, 1641 cm−1 for CZPD-I and CZPD-II, respectively increases as compared to PEGylated CdSe/ZnS NPs due to the band C]O of Qu
Fig. 5. Room temperature absorption spectra of (a) Qu, (b) PEGylated CdSe/ZnS, CZPD-I and CZPD-II NPs dispersed in DI water with concentration maintained constant i.e. 1.42 mM. 119
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Fig. 6. Room temperature emission spectra of (a) Qu at λex = 400 nm (b) CdSe, CdSe/ZnS, PEGylated CdSe/ZnS, CZPD-I, CZPD-II NPs (λex = 400 nm) dispersed in DI water with concentration maintained constant i.e. 1.42 mM.
which is not intense in PEGylated CdSe/ZnS. In CZPD-I and CZPD-II the major characteristic peaks of Qu aromatic bending and stretching at 1100–1600 cm−1, and eOH phenolic bending at 1200–1400 cm−1 are present, which shows the presence of Qu. Peak at ∼903 cm−1 for CZPD-I and for CZPD-II at 906 cm−1 are due to the para di-substituted benzene of Qu. The band present at 705 cm−1 and 708 cm−1 for CZPD-I and CZPD-II, respectively represents the aromatic CeH out of plane blend of Qu. The presence of peaks at 1318 cm−1, 1010 cm−1 and
[44]. This gives the indirect confirmation of conjugation of Qu with PEGylated CdSe/ZnS NPs. The addition of Qu in PEGylated CdSe/ZnS causes the stretching of OeH bond with a slightly blue shift along with narrowing down the peak width [45]. For CZPD-I, peaks at 1439 cm−1, 1408 cm−1 and for CZPD-II peaks at 1440 cm−1, 1417 cm−1 may be due to the presence of aromatic ring of Qu. In CZPD-I and CZPD-II the intense peaks at 1010 cm−1, 951 cm−1 and 1014 cm−1, 950 cm−1 are well matched with free Qu
Fig. 7. FT-IR spectra of (a) Qu (b) PEGylated CdSe/ZnS (c) CZPD-I and (d) CZPD-II NPs. 120
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Fig. 8. Schematic diagram representing adsorption of Qu over PEGylated CdSe/ZnS NPs surface.
1319 cm−1, 1014 cm−1 for CZPD-I and CZPD-II cm−1, respectively are due to the presence of stretching at CeO of Qu. The FT-IR analysis confirms the conjugation of hydrophobic Qu molecules over PEGylated CdSe/ZnS NPs surface via the addition of water soluble EDC-HCl and NHS as illustrated in Fig. 8.
spectra of CdSe QDs and CdSe/ZnS core/shell NPs. The observed peaks for core/shell NPs are same as that of bare CdSe QDs with a little shift towards higher theta and no peak for free ZnS is observed. In the XRD spectrum of PEGylated CdSe/ZnS NPs there is a presence of both the peaks of PEG-diamine. The spectrum predicts that NPs retained their crystallinity on PEGylation. But after conjugation of PEGylated NPs with Qu it shows amorphous characteristics for both CZPD-I and CZPD-II drug loaded NPs. There is no peak characterizing the diffraction pattern of pure flavonoid in the XRD graph of Qu-loaded PEGylated NPs. It may be due to the dispersion of Qu in a noncrystalline state over the PEGylated NPs [46]. The amorphous forms of the drugs are always more acceptable as they possess higher energy and higher surface area which provides higher solubility to the material [47].
3.6. XRD spectra Fig. 9 (a–d) shows the XRD spectra of PEG-diamine, PEGylated CdSe/ZnS NPs, CZPD-I, CZPD-II NPs respectively. The XRD spectra of PEG-diamine contain only two peaks at 19° and 23.2° shown in Fig. 9 (a). As CdSe/ZnS NPs contains peaks around 23.45°, 25.75°, 26.65°, 33.95° and broad hump between 40° and 50° correspond to (1 0 0), (0 0 2), (1 0 1), (1 1 0), (1 0 3) and (2 0 0) planes, respectively [39]. The hexagonal (wurtzite) phase dominates during the formation of core CdSe QDs. The hexagonal state is the stable phase, while the sphalerite cubic (Zinc-Blende) is the metastable state. Hexagonal phase is thermodynamically more stable and is achieved at higher temperature, at which thermodynamics (rather than the reaction kinetics), governs the crystal growth. Fig. S1 [SI] shows the XRD
3.7. Effect of pH on absorption and luminescence properties of Qu loaded NPs Stability of synthesized NPs in solution is an important requirement for their therapeutic and biomedical applications. This feature is analyzed by monitoring the absorption and
Fig. 9. XRD spectra of (a) PEG-diamine (b) PEGylated CdSe/ZnS (c) CZPD-I (d) CZPD-II NPs. 121
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Fig. 10. Room temperature absorption spectra of (a) CZPD-I (b) CZPD-II NPs at different pH.
Fig. 11. Room temperature emission spectra of (a) CZPD-I and (b) CZPD-II NPs at different pH.
Fig. 12. In-vitro release profile of CZPD-I and CZPD-II NPs at (a) 7.4 and (b) 5 pH.
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Table 1 Represents the R2 values obtained after fitting the data using different models. Sample name
pH
Zero order
1st order
2nd order
Korsmeyer peppas
Weibull with lagtime
K.M peppas with lagtime
Hill equation
CZPD-I CZPD-II CZPD-I CZPD-II
7.4
0.4841 0.5113 0.2624 0.1819
0.0526 0.0554 0.0397 0.0384
0.0325 0.0334 0.0269 0.0270
0.9788 0.9768 0.9766 0.9764
0.9913 0.9899 0.9875 0.9868
0.9883 0.9871 0.9865 0.9862
0.9879 0.9853 0.9795 0.9786
5
result of dissolution process parameters, a is the scale parameter that describe the time dependence, b is the shape of dissolution curve. The obtained value of the parameter b lies between 0.69 and 0.75, which indicates the drug release by diffusion process [51].
luminescence spectra of drug loaded NPs at different pH environment. Fig. 10 shows the absorption spectra of (a) CZPD-I and (b) CZPD-II NPs dispersed in different pH solutions keeping the concentration of NPs same for both UV–Vis and PL spectroscopy. Absorption spectra for both samples reveal that there is no absorption at pH 2 i.e. high acidic medium. NPs degrade/agglomerate at highly acidic environment. The absorbance edge of Qu loaded PEGylated NPs shifts towards longer wavelength as pH decreases (from pH 10 to 2). The absorption of Qu loaded PEGylated NPs in the range 6–10 pH shows narrow absorbance band as compared to pH 4 and 2, i.e. highly acidic medium. It is well known that the behavior of NPs is greatly influenced by pH environment. It was reported earlier that PEGylated CdSe/ZnS NPs show maximum emission at pH 6 [28]. Fig. 11 shows the photoluminescence spectra of Qu conjugated NPs at different pH (2–10). Both CZPD-I and CZPD-II NPs show maximum emission at pH 10 i.e. alkaline medium, which shows that these synthesized NPs are stable at alkaline pH. In CZPD-I as pH changes from (2–10) emission increases. But in case of CZPD-II emission at pH 6 is more as compared to pH 8. The change in the emission spectra of Qu conjugated NPs at different pH may be due to the deprotonation of the molecule [48].
4. Conclusions This manuscript presents the successful synthesis of water dispersed core/shell CdSe/ZnS NPs functionalized with PEG-diamine and loaded with a hydrophobic drug molecule Qu using two different methods. The synthesized Qu loaded NPs using this method shows no agglomeration when dispersed in water. FT-IR, UV–Vis and TEM results show the successful loading of Qu over PEGylated NPs. Particle size obtained from TEM micrographs is 24 nm–26 nm, therefore these NPs can be suitable for drug delivery systems. As it is known that spherical particles bigger than 200 nm are efficiently filtered by liver, spleen and bone marrow, while particles smaller than 10 nm can be quickly cleared by the kidney or through extravasation, thus it makes 10–200 nm the ideal size range for the circulating spherical carriers. The obtained Qu loaded PEGylated CdSe/ZnS NPs are in non-crystalline state in both CZPD-I and CZPD-II. Thereby, one can expect an improvement of drug solubility. The effect of pH environment on the optical properties shows that Qu loaded NPs completely agglomerate in high acidic medium. Qu loaded PEGylated NPs show high emission in the pH range 6–10, shows the optical stability of synthesized NPs in the range 6–10 pH. Presented study has also demonstrated that the release of Qu is controlled pH dependent and liberating more Qu at neutral pH rather than mildly acidic medium. The amine group of PEG-diamine contributes to the delay of release of Qu. Reduction of drug release at low pH conditions is advantageous because it reduces possible toxicity to the stomach wall, avoids degradation or inactivation by stomach secretions such as hydrochloric acid or pepsin, and increases the amount capable of being released in the small intestine.
3.8. Drug release of Qu loaded PEGylated CdSe/ZnS NPs Fig. 12 shows the in-vitro release profile of Qu loaded over PEGylated NPs at (a) pH 7.4 and (b) pH 5. Inset figures show the burst release of Qu within first 200 min. Release behavior of Qu from both samples CZPD-I and CZPD-II is pH dependent. Release of Qu from both CZPD-I and CZPD-II is significantly more intense at neutral pH condition i.e. pH 7.4, than that of mildly acidic conditions i.e. pH 5. In first 30 min Qu release at pH 7.4 reaches 15.8% and 13% for CZPD-I and CZPD-II, respectively. Whereas at pH 5 its only 8.3% and 7% for CZPD-I and CZPDII, respectively. The amount of Qu release from PEGylated NPs is rapid in first 100 min i.e. burst release. Subsequently, the release of Qu becomes much slower and sustained. The maximum release for CZPD-I is 63.7% and 29% at pH 7.4 and 5, respectively. Similarly for CZPD-II maximum release is 57.8% and 30% at pH 7.4 and 5, respectively. Obtained results show that the Qu is not completely transferred from PEGylated NPs to buffer medium. As the functionalization plays important role in release of drugs, for instance, the functionalized-CdSe/ ZnS NPs with PEG-diamine is able to regulate drug release. Thus, the amino-groups in PEGylated CdSe/ZnS NPs contribute to delay of Qu release [49]. The results show that in-vitro release of Qu is not only dependent on the pH of the NPs but also depends on the physicochemical properties of PEGylated NPs. Reduction in the drug release at low pH is advantageous for various applications [50]. Seven different kinetic models are employed to evaluate the possible changes in the release mechanism. The obtained values of correlation coefficient for all the seven models have been shown in Table 1. The highest value for coefficient of determination (R2) is obtained for Weibull with lag time model. The empirical equation of this model is described by Weibull in 1951 given as:
Acknowledgments Miss. Shivani Bharti is grateful to Department of Science and Technology, India for providing the INSPIRE fellowship. We are also thankful to the chairperson of Department of Biochemistry, Panjab University Chandigarh for providing Shimadzu Spectrofluorophotometer (RF-5301PC) facility. Conflict of interest declaration Authors have no conflict of interest. Statement of data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
(t − T )b ⎤ ⎤ Q = Qo ⎡1 − exp ⎡− ⎥ ⎢ ⎢ ⎥ a ⎣ ⎦⎦ ⎣
Appendix A. Supplementary data
where, Q is the amount of dissolved drug as a function of time, t‟, Q0 is the total amount of drug being released, T is the lag time measured as a
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mseb.2019.03.015. 123
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References
0608611104. [26] S. Huo, S. Chen, N. Gong, J. Liu, X. Li, Y. Zhao, X. Liang, Ultrasmall gold nanoparticles behavior in vivo modulated by surface polyethylene glycol (PEG) grafting, Bioconjugate Chem. 28 (2017) 239–243, https://doi.org/10.1021/acs. bioconjchem.6b00488. [27] D. Dorniani, A.U. Kura, M.Z. Hussein, S. Fakurazi, A.S. Halim, A. Zalinah, Controlled-release formulation of perindopril erbumine loaded PEG-coated magnetite nanoparticles for biomedical applications, J. Mater. Sci. 49 (2014) 8487–8497, https://doi.org/10.1007/s10853-014-8559-7. [28] S. Bharti, G. Kaur, S. Gupta, S.K. Tripathi, PEGylation of CdSe/ZnS core/shell nanoparticles and its behavior at different pH, J. Lumin. 181 (2017) 459–466, https:// doi.org/10.1016/j.jlumin.2016.09.005. [29] R.K. Deshmukh, J.B. Naik, Optimization of spray-dried diclofenac sodium-loaded microspheres by screening design, Dry. Technol. 34 (2016) 1593–1603, https://doi. org/10.1080/07373937.2016.1138121. [30] B. Ankamwar, Biosynthesis of gold nanoparticles (green-gold) using leaf extract of terminalia catappa, E-J. Chem. 7 (2010) 1334–1339, https://doi.org/10.1155/ 2010/745120. [31] P. Bocchiaro, A. Zamperini, World’s largest Science, Technology & Medicine Open Access Book Publisher, RFID Technology, Security Vulnerabilities, and Countermeasures, 2016. [32] P. Eaton, P. Quaresma, C. Soares, C. Neves, M.P. de Almeida, E. Pereira, P. West, A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles, Ultramicroscopy 182 (2017) 179–190. [33] W. Wang, C. Sun, L. Mao, P. Ma, F. Liu, J. Yang, Y. Gao, The biological activities, chemical stability, metabolism and delivery systems of quercetin : a review, Trends Food Sci. Technol. 56 (2016) 21–38, https://doi.org/10.1016/j.tifs.2016.07.004. [34] P. Pinheiro, G. Justino, Structural analysis of flavonoids and related compounds—a review of spectroscopic applications, in: Phytochem. – A Glob. Perspect. Their Role Nutr. Heal., vol. 33, 2012, pp. 33–56. [35] M. Bancirova, Changes of the quercetin absorption spectra in dependence on solvent, Chem. J. 1 (2015) 31–34. [36] M.C.M. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size related properties and applications toward biology, catalysis and nanotechnology, Chem. Rev. 104 (2004) 293–346. [37] G.S. Pillai, K. Krishnakumar, B. Krishnan, Fluorescence properties of quercetin – a review, Asian J. Res. Biol. Pharm. Sci. 5 (2017) 44–49. [38] J.C.A. Ensastegui, M.P. Pardave, M.R. Romo, M.T. Ramırez-Silva, Quercetin spectrofluorometric quantification in aqueous media using different surfactants as fluorescence promoters, RSC Adv. 8 (2018) 10980–10986, https://doi.org/10. 1039/c8ra01213j. [39] G. Kaur, S.K. Tripathi, Probing photoluminescence dynamics of colloidal CdSe/ZnS Core/shell nanoparticles, J. Lumin. 155 (2014) 330–337, https://doi.org/10.1016/ j.jlumin.2014.06.052. [40] S. Daniel, G.A.G. Raj, Photoinduced electron-transfer reactions of tris(4,4’-dinonyl2,2’-bipyridyl) ruthenium (ii) cation with phenolate ions in aqueous acetonitrile, J. Chem. Pharm. Res. 5 (2013) 220–227. [41] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, D.A. Ananth, S. Jagadeeswari, R. Renganathan, Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic wistar rats, Colloids Surf. B 112 (2013) 255–263, https://doi.org/10.1016/ j.colsurfb.2013.07.065. [42] J.M. Asha, V. Ellappan, S. Raja, K.A. Kumar, R. Renganathan, Investigation on the photoinduced interaction between water soluble CdTe quantum dots and certain antioxidants, Adv. Sci. Lett. 4 (2011) 3490–3495, https://doi.org/10.1166/asl. 2011.1895. [43] D. Sheeba, A.G.R. George, Luminescence quenching of tris(4,4’-dinonyl-2,2’-bipyridyl) ruthenium(ii) cation with phenolate ions in DMSO, Arab. J. Chem. 10 (2017) 2429–2435, https://doi.org/10.1016/j.arabjc.2013.09.006. [44] M. Heneczkowski, M. Kopacz, D. Nowak, A. Kuzniar, infrared spectrum analysis of some flavonoids, Acta Pol. Pharm. 58 (6) (2001) 415–420. [45] K. Wang, M. Li, H. Li, F. Guan, D. Zhang, H. Feng, H. Fan, Synthesis and characterization of CdSe/CdS/N-Acetyl-L-cysteine/quercetin nano-composites and their antibacterial performance, J. Korean Chem. Soc. 59 (2015) 136–141, https://doi. org/10.5012/jkcs.2015.59.2.136. [46] H. Pool, D. Quintanar, J.D.D. Figueroa, C.M. Mano, J.E.H. Bechara, L.A. Godínez, S. Mendoza, Antioxidant effects of quercetin and catechin encapsulated into PLGA nanoparticles, J. Nanomater. 2012 (2012) 1–12, https://doi.org/10.1155/2012/ 145380. [47] P.N. Kendre, V.V. Pande, K.M. Chavan, Novel formulation strategy to enhance solubility of quercetin, Pharmacophore 5 (2014) 358–370. [48] Z. Jurasekova, C. Domingo, J.V.G. Ramos, S.S. Cortes, Effect of pH on the chemical modification of quercetin and structurally related flavonoids characterized by optical (UV-Visible and Raman) spectroscopy, Phys. Chem. Chem. Phys. 16 (2014) 12802–12811, https://doi.org/10.1039/C4CP00864B. [49] K.E.A. AbouAitah, A.A. Farghali, A.S. Sroda, W. Lojkowski, A.M. Razin, M.H. Khedr, Mesoporous silica materials in drug delivery system: pH/glutathione-responsive release of poorly water-soluble pro-drug quercetin from two and three-dimensional pore-structure nanoparticles, J. Nanomed. Nanotechnol. 7 (2016) 1–12, https://doi. org/10.4172/2157-7439.1000360. [50] C.A. Schoener, N. Hutson, N.A. Peppas, pH-responsive hydrogels with dispersed hydrophobic nanoparticles for the delivery of hydrophobic therapeutic agents, Polym. Int. 61 (2012) 874–879, https://doi.org/10.1002/pi.4219. [51] V. Papadopoulou, K. Kosmidis, M. Vlachou, P. Macheras, On the use of the weibull function for the disernment of drug release mechanisms, Int. J. Pharm. 309 (2006) 44–50, https://doi.org/10.1016/j.ijpharm.2005.10.044.
[1] G.F. Paciotti, J. Zhao, S. Cao, P.J. Brodie, L. Tamarkin, M. Huhta, L.D. Myer, J. Friedman, D.G.I. Kingston, Synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery, Bioconjug. Chem. 27 (2016) 2646–2657, https://doi.org/10.1021/acs.bioconjchem.6b00405. [2] Q. Liu, C. Zhan, D.S. Kohane, Phototriggered drug delivery using inorganic nanomaterials, Bioconjug. Chem. 28 (2017) 98–104, https://doi.org/10.1021/acs. bioconjchem.6b00448. [3] D. Yeo, C. Wiraja, Y.J. Chuah, Y. Gao, C. Xu, A nanoparticle-based sensor platform for cell tracking and status/function assessment, Sci. Rep. 5 (2015) 14768, https:// doi.org/10.1038/srep14768. [4] A. Rodzinski, R. Guduru, P. Liang, A. Hadjikhani, T. Stewart, E. Stimphil, C. Runowicz, R. Cote, N. Altman, R. Datar, S. Khizroev, Targeted and controlled anticancer drug delivery and release with magnetoelectric nanoparticles, Sci. Rep. 6 (2016) 20867, https://doi.org/10.1038/srep20867. [5] X. Gao, Z. Liu, Z. Lin, X. Su, CuInS2 quantum dots/poly((L)-glutamic acid)-drug conjugates for drug delivery and cell imaging, Analyst 139 (2014) 831–836, https://doi.org/10.1039/C3AN01134H. [6] J.M. Montenegro, V. Grazu, A. Sukhanova, S. Agarwal, J.M. de la Fuente, I. Nabiev, A. Greiner, W.J. Parak, Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. 65 (2013) 677–688, https:// doi.org/10.1016/j.addr.2012.12.003. [7] O.V. Salata, Applications of nanoparticles in biology and medicine, J. Nanobiotechnol. 2 (2004) 1–6, https://doi.org/10.1186/1477-3155-2-3. [8] I.Y. Kim, E. Joachim, H. Choi, K. Kim, Toxicity of silica nanoparticles depends on size, dose, and cell type, Nanomed. Nanotechnol., Biol. Med. 11 (2015) 1407–1416, https://doi.org/10.1016/j.nano.2015.03.004. [9] D. Sahu, G.M. Kannan, M. Tailang, R. Vijayaraghavan, In vitro cytotoxicity of nanoparticles : a comparison between particle size and cell type, J. Nanoscience 2016 (2016) 1–9, https://doi.org/10.1155/2016/4023852. [10] S. Bakand, A. Hayes, Toxicological considerations, toxicity assessment, and risk management of inhaled nanoparticles, Int. J. Mol. Sci. 17 (2016) 929, https://doi. org/10.3390/ijms17060929. [11] K.V. Chakravarthy, B.A. Davidson, J.D. Helinski, H. Ding, W.C. Law, K.T. Yong, P.N. Prasad, P.R. Knight, Doxorubicin-conjugated quantum dots to target alveolar macrophages and inflammation, Nanomed. Nanotechnol., Biol. Med. 7 (2011) 88–96, https://doi.org/10.1016/j.nano.2010.09.001. [12] D. Hutanu, M.D. Frishberg, L. Guo, C.C. Darie, Recent applications of polyethylene glycols (PEGs) and PEG derivatives, Mod. Chem. Appl. 2 (2014) 1–6, https://doi. org/10.4172/2329-6798.1000132. [13] J.S. Nam, A.R. Sharma, L.T. Nguyen, C. Chakraborty, G. Sharma, S.S. Lee, Application of bioactive quercetin in oncotherapy: from nutrition to nanomedicine, Molecules 21 (2016) 108, https://doi.org/10.3390/molecules21010108. [14] O.M. Andersen, K.R. Markham, Flavonoids. Chemistry, Biochemistry and Applications, CRC Press, 2005. [15] L. Gibellini, M. Pinti, M. Nasi, J.P. Montagna, S. De Biasi, E. Roat, L. Bertoncelli, E.L. Cooper, A. Cossarizza, Quercetin and cancer chemoprevention, Evid. Based Complementary Altern. Med. 2011 (2011), https://doi.org/10.1093/ecam/neq053. [16] Z. Xingyu, M. Peijie, P. Dan, W. Youg, W. Daojun, C. Xinzheng, Z. Xijun, S. Yangrong, Quercetin suppresses lung cancer growth by targeting aurora B kinase, Cancer Med. 5 (2016) 3156–3165, https://doi.org/10.1002/cam4.891. [17] J.H. Jeong, J. An, Y. Kwon, J.G. Rhee, Y.J. Lee, Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression, J. Cell. Biochem. 106 (2009) 73–82, https://doi.org/10.1002/jcb.21977. [18] L. Ma, J.M. Feugang, P. Konarski, J. Wang, J. Lu, S. Fu, B. Ma, B. Tian, C. Zou, Z. Wang, Growth inhibitory effects of quercetin on bladder cancer cell, Front. Biosci. 11 (2006) 2275–2285, https://doi.org/10.2741/1970. [19] Y. Mu, Y. Fu, J. Li, X. Yu, Y. Li, Y. Wang, X. Wu, K. Zhang, M. Kong, C. Feng, X. Chen, Multifunctional quercetin conjugated chitosan nano-micelles with P-gp inhibition and permeation enhancement of anticancer drug, Carbohydr. Polym. 203 (2019) 10–18, https://doi.org/10.1016/j.carbpol.2018.09.020. [20] S.R. Kumar, S. Priyatharshni, V.N. Babu, D. Mangalaraj, C. Viswanathan, S. Kannan, N. Ponpandian, Quercetin conjugated superparamagnetic magnetite nanoparticles for in-vitro analysis of breast cancer cell lines for chemotherapy applications, J. Colloid Interface Sci. 436 (2014) 234–242, https://doi.org/10.1016/j.jcis.2014.08. 064. [21] S.L. Vidal, C. Rojas, R.B. Padin, M.P. Rivera, A. Haensgen, M. Gonzalez, S.R. Llamazares, Synthesis and characterization of polyhydroxybutyrate-co-hydroxyvalerate nanoparticles for encapsulation of quercetin, J. Bioact. Compat. Polym. 31 (2016) 439–452, https://doi.org/10.1177/0883911516635839. [22] R. Khonkarn, S. Mankhetkorn, W.E. Hennink, S. Okonogi, PEG-OCL micelles for quercetin solubilization and inhibition of cancer cell growth, Eur. J. Pharm. Biopharm. 79 (2011) 268–275, https://doi.org/10.1016/j.ejpb.2011.04.011. [23] P. Muralidharan, E. Mallory, M. Malapit, H. Don, H.M. Mansour, Inhalable PEGylated phospholipid nanocarriers and PEGylated therapeutics for respiratory delivery as aerosolized colloidal dispersions and dry powder inhalers, Pharmaceutics 6 (2014) 333–353, https://doi.org/10.3390/ pharmaceutics6020333. [24] H. Otsuka, Y. Nagasaki, K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv. Drug Deliv. Rev. 64 (2012) 246–255, https:// doi.org/10.1016/j.addr.2012.09.022. [25] S.K. Lai, D.E. O’Hanlon, S. Harrold, S.T. Man, Y.-Y. Wang, R.A. Cone, J. Hanes, Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 1482–1487, https://doi.org/10.1073/pnas.
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