- Email: [email protected]
PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications
Accepted Manuscript Title: PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications. Authors: Shailja Kumar, Virendra Kumar Meena, Puja Panwar Hazari, Rakesh Kumar Sharma PII: DOI: Reference:
S0378-5173(17)30439-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.05.027 IJP 16675
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
13-3-2017 9-5-2017 11-5-2017
Please cite this article as: Kumar, Shailja, Meena, Virendra Kumar, Hazari, Puja Panwar, Sharma, Rakesh Kumar, PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications. Shailja Kumara, Virendra Kumar Meenaa,b, Puja Panwar Hazarib, Rakesh Kumar Sharma a* a
Nanotechnology and Drug Delivery Research Lab, Department of Chemistry, University of Delhi, Delhi-110007, India.
b
Institute of nuclear medicine and allied sciences, DRDO, Ministry of defense, Delhi Corresponding author (*): Dr. Rakesh Kumar Sharma ([email protected]) Graphical abstract
Gd-PEG-dox NPs
Drug delivery and optical imaging
TEM image of NPs
Cellular uptake of NPs
Magnetic Resonance Imaging
1
H-NMR spectrum of D2O (a) in the absence and (b) presence of Gd-PEG-dox NPs
Ph 91 9310050453
Abstract We report water-in-oil microemulsion mediated synthesis of polyethylene glycol (PEG1) coated Gd2O3 nanoparticles (NPs2) loaded with fluorescent anti-cancer drug doxorubicin (dox3) for synchronous drug delivery, optical and magnetic resonance (MR4) imaging applications. These PEG covered Gd 2O3 NPs loaded with dox (Gd-PEG-dox NPs) were found to possess spherical morphology with 13nm size as measured from TEM and the hydrodynamic diameter comes out to be 37nm as determined from DLS. Fluorescence spectra and fluorescence microscopy images confirmed optical activity of the NPs. The paramagnetic nature of NPs was affirmed by NMR line broadening effect on the spectrum of surrounding
1
Polyethylene glycol
2
Nanoparticles
3
Doxorubicin
4
Magnetic resonance
water protons. Therefore, these particles can be efficiently used as contrast agent (CA5) in MR imaging. In vitro analysis showed significant cellular uptake of particles by A-549 cells. A pH dependent drug release pattern was observed for the NPs. Cell viability assay performed on A-549, PANC-1 and U-87 cancerous cell lines revealed that Gd-PEG-dox NPs are cytotoxic. On the basis of these observations, it can be concluded that these multi-modal paramagnetic NPs promise potential cancer therapy along with optical and MR imaging applications. Keywords - Gadolinium oxide nanoparticles, reverse micelle, doxorubicin, contrast agent, drug delivery, optical imaging. 1. Introduction In biomedical science ample of attention is devoted, over the last few decades, on development of multifunctional NPs which can efficiently act as drug-delivery vehicles in biological system. Substantial efforts have been made to incorporate nanotechnology in drug delivery to have site-specific and sustained delivery of drug molecules. Mostly, efficacy of a drug is hampered by its high toxicity, poor solubility, high dose, non-specific distribution and in vivo decomposition [1]. Such limitations faced by naked drug are overcome by NPs based drug-delivery system which increases safety of drug molecule against chemical or enzymatic decomposition, amends extended bioavailability of drug to pathological site and allows targeted delivery of drug to disease site [2][3][4]. Numerous kinds of nano structures have been used as drug-delivery vehicles namely nanotubes, NPs, nanocapsules, microemulsion and liposomes [5]. Collaboration of therapy along with imaging in a single entity is an emerging trend in nanomedicine. Such drug delivery system having both therapeutic and diagnostic imaging moieties offer dual advantage of targeted drug delivery and real-time monitoring of drug response through imaging. These therapeutic NPs are called theranostic (therapeutic + diagnostic) NPs [6][7]. Chemotherapy remains one of the best treatments for various types of cancer but it is usually hindered by the unfavorable distribution of the drug which results in severe side effects. Therefore, nanosize drug delivery systems carrying therapeutic agent along with diagnostic imaging moiety are desirable [8][9]. Dox is an anthracycline anti-tumor drug which intercalates DNA and inhibits macromolecular biosynthesis [10]. Upon direct administration of dox in body, it shows low cellular uptake and distributes unevenly resulting in undesirable side-effects and toxicity to normal cells [11]. This concern led to the development of alternative routes for administering dox like entrapping or attaching it with nano materials. Missirlis et al. [12] prepared polymeric nanoparticles of PEG and poloxamer 407 encapsulating dox in inverse emulsion as controlled drug delivery system. Furthermore, they showed that encapsulation of drug delayed it degradation significantly. Dreis et al [13] prepared dox loaded human serum albumin NPs and cell viability study done on two neuroblastoma cell lines showed that anti-cancer activity of the NPs was more as compared to dox solution.
5
Contrast agent
Magnetic NPs, such as iron oxide and gadolinium oxide, exhibit potential biomedical applications such as contrast enhancing ability in MR imaging [14], in drug targeting [15] and hyperthermia [16], to name a few. Particularly, Gadolinium based magnetic NPs have drawn tremendous interest to be used as positive contrast enhancing agent in MRI. Numerous studies have shown that gadolinium oxide (Gd2O3) NPs can be efficiently used as CA [17][18][19]. The major advantage of using paramagnetic metal oxide NP as CA is the high surface to volume ratio which maximizes the interaction of paramagnetic metal ion with water protons giving high longitudinal relaxivities [20][21]. Maeng et al. [22] synthesized polymeric NPs made up of folate, poly (ethylene oxide)-trimellitic anhydride chloride-folate, dox and superparamagnetic iron oxide for chemotherapy and MR imaging in liver cancer. They showed that relative tumor volume decreased 2 and 4 fold in comparison to free dox and doxil drug in rat and rabbit models, respectively. Also, NPs showed MR imaging sensitivity which was as good as conventional MR imaging CA Resovist. Fang et al. [23] prepared superparamagnetic iron oxide NPs and poly (beta-amino ester) copolymer preloaded with dox, was assembled to its surface. They reported that these NPs could serve as smart theranostic system for sensitive detection via MR imaging and for chemotherapy through controlled drug release. Bridot et al. [24] synthesized luminescent hybrid NPs which consisted of paramagnetic Gd2O3 core and polysiloxane shell which had fluorophores and carboxylated PEG covalently attached to the surface. It was shown that the NPs had higher longitudinal proton relaxivities as compared to positive CAs like Gd-DOTA and are well suited for MR and fluorescence imaging. Keeping the dual purpose of therapy and imaging in mind, herein, we report synthesis of PEG coated paramagnetic Gd2O3 NPs loaded with dox (Gd-PEG-dox) for simultaneous contrast enhancement in MR imaging and drug delivery applications. The Gd2O3 nanoscale core was prepared in water-in-oil microemulsion of water/AOT/hexane which was followed by PEG coating and lastly dox loading via conjugation on surface. We have chosen PEG as coating material of Gd 2O3 core because of its biocompatibility [25][26], non-toxic nature [27] and it can be functionalized easily. The ensuing Gd-PEGdox NPs were subsequently characterized for their size, morphology, composition, surface, crystallinity, magnetic behavior and optical activity. After characterization, in vitro biological studies of the NPs were conducted which included their cellular uptake study and cytotoxicity evaluation by SRB cell viability assay on cancerous cells namely A549, PANC-1 and U-87. Such multifunctional NPs are anticipated to be explored profoundly in near future and such efficient systems will alter the way of cancer treatment in future. 2. Experimental section 2.1. Materials Sodium bis-(2-ethylhexyl)sulfosuccinate (AOT; 96%), hexane, gadolinium nitrate pentahydrate, ethanol and ammonia were purchased from Acrōs Organics (New Jersey, USA), Spectrochem Pvt. Ltd Mumbai (India), Central Drug House(Mumbai, India), Merck (Darmstadt, Germany), and Rankem (Delhi, India), respectively. Doxorubicin hydrochloride and deuterium oxide (D2O) were procured from Alfa Aesar (Heysham, England). Polyethylene glycol (6000 KD), citric acid anhydrous and sodium phosphate dibasic anhydrous (Na2HPO4) were procured from s.d.fine-chem limited, spectrochem pvt. Ltd. (India) and SRL Ltd (India) respectively. Phosphate buffer saline (PBS), Sulforhodamine B (SRB) and Hoechst Stain were incurred from Sigma Aldrich (St Louis, MO). Tris base and paraformaldehyde were purchased from Merck. The lung carcinoma cell line (A-549), pancreatic cancer cell line (PANC-1) and U-87 were
procured from National Centre for Cell Sciences, Pune India. All chemicals were used as such without any further purification. 2.2. Synthesis of PEGylated Gd2O3 NPs loaded with doxorubicin (Gd-PEG-dox). For the synthesis of Gd-PEG-dox NPs, we prepared microemulsion ‘A’ by adding 300 µl of aqueous gadolinium nitrate (Gd(NO3)3. 5H2O, 0.1 M) solution to 15 ml of 0.1 M AOT in hexane. Microemulsion ‘B’ was obtained in the similar manner in which 300 µl of 1M NH3 was added to 15 ml of 0.1 M AOT in hexane. Both the microemulsons were stirred till they were optically clear. Molar ratio of water to surfactant (Wo) was maintained at 11.11 in both the microemulsions [28][29]. Now microemulsion ‘B’ was added dropwise to microemulsion ‘A’ with constant stirring at room temperature. After complete transfer, the resultant solution was stirred for another 24 hours which resulted in the formation of Gd2O3 NPs. The ensuing particles were extracted and washed several times with hexane and alcohol. The washed Gd2O3 NPs were dispersed in 5 mL double-distilled water and 200 µL of 1% PEG solution was added to it. The solution was further stirred for another 24 hours. The PEG coated Gd2O3 particles (Gd-PEG) were separated and redispersed in 5 mL DDW to which 40 µL of 0.1% dox solution was added. The solution was stirred for another 48 hours which resulted in the formation of dox loaded NPs (Gd-PEG-dox NPs). The resulting Gd-PEG-dox NPs were separated and finally dispersed in 1 mL of DDW for characterization and further use. 2.3. Characterization 2.3.1. Ultraviolet-visible (UV-Vis) spectroscopy UV-Vis spectra of all the samples were recorded on Shimadzu-1601 UV-vis spectrophotometer in the range of 190 nm to 1100 nm. 2.3.2. Transmission electron microscopy (TEM) TEM images of the prepared NPs were taken using TECNAI G2-30 U TWIN instrument (FEI, Eindhoven, Netherlands) which operates at acceleration voltage of 300 KV. A drop of dilute aqueous NP dispersion was deposited on copper grid. TEM images were taken after completely drying the grid at room temperature. 2.3.3. Dynamic Light Scattering (DLS) Hydrodynamic diameter of NPs was determined by DLS analysis performed on Zetasizer Nano ZS-90 analyzer from Malvern having He-Ne laser (λ= 633 nm, power 4 mV) as light source and recorded at a backscattering angle of 90 0 using an avalanche photodiode detector. The hydrodynamic diameter (d) of NPs was determined from diffusion of the NPs using Stokes–Einstein equation. 2.3.4. Fourier Transform Infrared (FT-IR) Spectroscopy FT-IR spectrums of the samples were taken using RXIFT IR (Perkin Elmer) instrument which has nichrome wire coated with alloy as source. Dry samples were dispersed into KBr powder and pressed into a pellet. The pellets were scanned from 400 cm-1 to 4000 cm−1 at room temperature. 2.3.5. X-ray diffraction (XRD) analysis.
XRD analysis of dry NP sample was carried out on Rigaku miniflex desktop XRD instrument at room temperature. Scanning of the sample was done in the 2θ range of 15–70 0. 2.3.6. Fluorescence Spectroscopy The fluorescence emission spectra of various samples were taken, at room temperature, on Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA). The optical activity of various samples was determined by scanning in the range of 500 nm to 800 nm. 2.3.7. Thermo gravimetric analysis (TGA). TGA of powered samples was carried out in nitrogen atmosphere on Perkin Elmer DTA/TGA/DSC instrument. The sample was placed on the sample holder of the instrument and scanned in the temperature range of 460C to 9720C. Variation in the weight of the sample with increase in temperature was studied. 2.3.8. 1H-Nuclear magnetic resonance (1H-NMR). 1
H-NMR spectrum of prepared NPs was recorded on 400 MHz spectrometer (JNM-ECX-400P, JEOL, Tokyo, Japan) in D2O as solvent. 2.4. Estimation of Attachment efficiency of doxorubicin. The attachment efficiency of multimodal Gd-PEG-dox NPs was calculated by an indirect approach using UV-Vis spectrophotometer. After preparation of Gd-PEG-dox NPs, the NPs were separated from supernatant by centrifugation. The amount of unentrapped drug present in the supernatant was determined spectrophotometrically by measuring its absorbance at 496 nm (characteristic peak of dox). On subtracting the amount of dox left in supernatant from the total drug added, attachment efficiency was calculated. 2.5. Release kinetics of doxorubicin at pH= 5.0 and 7.4. Release kinetics of dox from Gd-PEG-dox NPs was carried out at a pH of 5.0 and 7.4. For this, 5.5 mg of Gd-PEG-dox NPs were dispersed in 10 ml of citric acid-Na2HPO4 buffer (pH=5.0) and the solution was put on gentle stirring in a glass vial at room temperature. At regular intervals of time, 3ml of the NP dispersion was withdrawn and centrifuged at 10,000 rpm. The amount of drug released was estimated by measuring the absorbance of the drug in supernatant at 496 nm. After recording the absorbance, the solution was transferred back to the vial cautiously. The same procedure was repeated with buffer of pH 7.4. 2.6. In vitro cytotoxicity by SRB assay In vitro cytotoxicity of the NPs was evaluated by using Sulforhodamine (SRB) dye based assay. In a 96 well microtitre plate, exponentially growing cells were plated at a uniform cell density of 10,000 cells/well for 24 hours before treating with NPs. Cell viability was assessed after treating the cells with varying concentrations (55 ng/mL – 0.55 mg/mL) of NPs for 24 hours. Treated cells and negative control were incubated at 4 0C with trichloroacetic acid solution (10%) for 1 hour. At the end of the treatment, the cells were washed under running tap water to get rid of excess of solution and were kept at room temperature for air drying the wells overnight. Next day, 0.057% solution of SRB dye was used to stain the cells for 1 hour. Then, plates were washed under running tap water to remove excess dye and were allowed to air dry
overnight. Next day, 100µL of 10mM Tris buffer (pH 10.5) was added to solubilize the dye attached to the cells. Optical density of 100 µL of extracts was measured at 510 nm [30]. 2.7. In vitro cellular uptake Cellular uptake analysis was carried out on A-549 lung carcinoma cells. Firstly A-549 cells were seeded onto coverslips in a 6-well plate at a density of 5000 cells/well. After 24 hours, the cells were incubated with the Gd-PEG and Gd-PEG-dox NPs at concentration of 0.55mg/mL in culture media for 3 hrs. After the incubation, the media was carefully removed and cells were washed with PBS (pH-7.2) twice and further fixed with 1% formaldehyde solution (pH-7.0) for 5 mins. Later cells were again washed with PBS and incubated with DAPI solution (20mg/mL) for 2 mins. Then the cells were again washed twice with PBS gently to remove excess of DAPI stain, further coverslip containing the cells were picked and placed on glass slide with DPX mounting media and images were taken using fluorescence microscope (UV filter for DAPI and Green filter for Gd-PEG-dox NPs images). 3. Result and discussion Our primary motive was to develop multi-modal drug loaded magnetic NPs which could be efficiently used for various biomedical applications simultaneously. Paramagnetic Gd2O3 NPs were prepared via uncomplicated and soft chemical route using the aqueous core of the reverse micellar system of water/AOT/hexane [19][31]. These Gd2O3 NPs were then coated with PEG and subsequently attached with dox. Surface coating of Gd 2O3 NPs was done with PEG in order to impart biocompatibility to NPs, deter them from getting aggregated, for long circulation and for conjugating dox molecules to the particles. UV-Vis spectra of dox, dox conjugated NPs and NPs without dox were recorded and given in fig.1(a). The UV-Vis spectrum of free dox shows characteristic absorption peak at 496 nm which is also observed for Gd-PEG-dox NPs which depicts the presence of dox in the fabricated NPs. On the other hand, spectrum of Gd-PEG NPs without dox does not show absorption near 496nm which proves that the peak at 496 nm arises due to dox and not because of the nanoparticles or any other entity. Dox is a fluorescent anti-cancer drug which can be tracked optically [32]. The optical activity of the NPs was studied using fluorescence emission spectroscopy. The fluorescence spectra of aqueous solution of pure dox, Gd-PEG-dox NPs and Gd-PEG NPs without dox were taken and are shown in fig.1(b). From the UV-Vis absorption spectrum and fluorescence study, we observed a slight red-shift in the absorption spectrum of particles and a fluorescence quenching in Gd-PEG-dox NPs. This can be ascribed to an increased local concentration of dox which leads to dox-dox interaction through π-π stacking [12][33]. The particle diameter determined using TEM image, as shown in fig.2(a), comes out to be around 13 nm with spherical morphology and high degree of monodispersity. The size of the NPs, measured from dynamic light scattering (DLS) experiment, was found to be 37nm (fig.2(b)). This difference in size observed by DLS and TEM lies in the fact that in TEM we use dry sample for analysis and DLS is carried out in aqueous dispersion of NPs. Therefore, in DLS hydration layer surrounding the NPs also contribute to the size. The EDAX analysis (not shown) confirms the presence of expected elements in the NP sample. The crystallinity of particles was studied using X-ray diffraction (XRD) analysis. The XRD pattern, as shown in fig.3, exhibits sharp 2θ peaks at 29.30, 33.9 0, 35.50, 47.260, 59.60 and 69.20 which corresponds to (222), (400), (411), (440), (444) and (800) planes of cubic Gd2O3 NPs [34][35]. These values give the evidence for the crystalline nature of the fabricated NPs due to the presence of paramagnetic Gd 2O3 in them.
Thermogravimetric analysis (TGA) was carried out from 300C to 9550C in N2 atmosphere to probe the thermal properties of NPs. The thermograph of PEG and Gd-PEG-dox NPs is given in fig.4. TGA curve of PEG shows that its degradation started at 3500C after which a sudden drop in weight is observed up to 4280C. At 4280C, PEG is 97.5% decomposed and get completely decomposed at 523 0C. In the case of Gd-PEG-dox NPs, a gradual decrease in weight was observed over the entire temperature range. Total weight loss of 49% was observed for the NPs. Manocha et al. [36] showed that in the case of pure dox weight loss of 10% was observed from 25 0C to 600 0C. Therefore, the observed decrease in the weight of NPs can be attributed to the decomposition of PEG on the surface and the NPs are more thermally stable than pure PEG. The efficacy of these nanoparticles to be used as CA in MRI was exemplified by taking the 1H-NMR of pure D2O and D2O in presence of GD-PEG-dox NPs as shown in fig.5. 1H-NMR of D2O shows a single sharp peak corresponding to water protons (fig.5(a)). The 7 unpaired electrons in Gd3+ ion of Gd2O3 gives rise to high magnetic moment of 7.94 μB. Gadolinium has long electron relaxation time thereby enhancing dipole-dipole interaction between electrons and (water) protons. Such interactions tend to enhance the proton relaxation which increases the contrast in MR image. In the absence of the NPs, a sharp peak is observed in the NMR spectrum of D2O (fig.5(a)) but presence of NPs causes line broadening effect on the NMR spectrum of H2O protons present in its vicinity (fig.5(b))[19][21][37]. The conjugation of PEG and subsequently of dox on the surface of Gd2O3 nanoparticles was probed with the help of FT-IR spectra of PEG, Gd-PEG NPs and Gd-PEG-dox NPs as shown in fig.6. In the case of PEG, strong peaks at 2885 and 1110 cm-1 corresponds to C-H symmetric and C-O-C asymmetric stretching vibrations [38]. Two sharp peaks at 1460 cm-1 and 1346 cm-1 are assigned to scissoring, and wagging CH2 vibrational modes respectively [39]. Strong peak at 3432 cm-1 arises due to O-H stretching vibration of PEG [40]. FT-IR spectrum of Gd-PEG NPs shows two important peaks at 3420 cm-1 and 1100 cm-1 corresponding to O-H stretch of alcohol and asymmetrical C-O-C stretch, respectively, which confirms the attachment of PEG on to the surface of Gd2O3 NPs. Rana et al [41] reported that the FT-IR spectrum of pure dox exhibits a peak at 3410 cm-1 due to stretching frequencies of O-H and N–H of primary amine in dox. However, in the case of Gd-PEG-dox NPs, peak arising due to N-H and O-H stretching vibrations is broadened and shifted to lower frequency value (~ 3406 cm-1). The characteristic bands of dox located at 2932 cm-1 and 1730 cm-1 due to C–H and C=O stretching vibrations, are also observed for dox loaded NPs [42]. Two peaks located at 1521 cm-1 and 1043 cm-1 corresponding to bending of NH2 on aromatic ring and C-O stretch of alcohol group, respectively, were also observed in Gd-PEG-dox NPs [43][40][44]. The bands in pure dox which are observed at 870 cm−1 and 805 cm−1 due to N–H wagging have diminished in Gd-PEG-dox NPs. Taking into consideration this data, we can interpret that the attachment of dox to the surface of PEG coated Gd2O3 nanomaterial is occurring through hydrogen bonding between -OH and –NH2 groups of dox with the –OH groups of PEG which is also reported earlier by kayal et al. [42] and rana et al. [40]. The attachment efficiency (A%) of anti-cancer drug loaded particles was determined via UV-visible spectroscopy by measuring the difference of the total drug added at the time of synthesis and the amount of drug not attaching to the particle surface. The A% was calculated by using the formulae given below and it comes out to be 87%. Attachment efficiency (A%) =
× 100
To ascertain the utility of these particles for drug delivery, we probed the time-dependent release profile of the drug from the NPs. Release kinetic study was carried out in pH of 5.0 and 7.4 buffers and the release behavior is shown in fig.7. A sustained release pattern of the drug was observed in both the pH. In pH 7.4, a release of 35% and ~48% was observed after 24 hrs and 48 hrs respectively. It reached to ~68% after 165 hrs. However, in pH 5.0 buffer, 48% and 69.7% release of dox was noticed after 24 h and 46 h respectively, which reached to 90% after 167 h. As compared to neutral pH, release of dox was found to be higher in acidic pH which may be attributed to the dissociation of hydrogen bonds between PEG and dox due to complete protonation of –NH2 groups of dox which is consistent with earlier report [45]. Moreover, another probable reason for enhanced drug release in acidic medium could be that the cationic drug has higher affinity for the acidic microenvironment which acts as driving force for its enhanced release [46]. Further the in-vitro interaction of these NPs with cells was evaluated. The cellular uptake study of the NPs on A-549 cancerous cells was carried out using fluorescence microscopy. Dox is a fluorescent anti-cancer drug which can be used for optical imaging in cellular environment. The fluorescence of dox loaded NPs can be optically tracked within the treated cells. The fluorescence images of the A-549 cells treated with Gd-PEG NPs and Gd-PEG-dox NPs are shown in fig. 8. In the control experiment, the Gd-PEG treated cells did not show any fluorescence emission (fig.8(a)). On the other hand, treatment of cells with 0.55 mg/mL of Gd-PEG-dox NPs for 3hours resulted in strong fluorescence (fig.8(b)) which clearly demonstrates efficient uptake of the Gd-PEG-dox NPs by the cells. Additionally, the fluorescence images of the aqueous Gd-PEG NPs dispersion, dox solution and aqueous Gd-PEG-dox NPs dispersion were taken and are shown in fig. 9. The images clearly demonstrate that dox solution and dox loaded magnetic NPs show fluorescence whereas Gd-PEG NPs without dox do not exhibit any fluorescence. After affirming cellular uptake and fluorescence emission of the particles, we checked their anti-cancer activity by treating three different cancerous cell lines namely A-549, PANC-1 and U-87 with varying concentrations of dox solution, Gd-PEG NPs and Gd-PEG-dox NPs for 24 hours after which cell viability was analyzed. The results are summarized in fig.10. It was found that dox loaded NPs are effectively cytotoxic than other comparative test sets. In cytotoxicity curve, data point of concentration 0.55mg/ml, dox is showing minimum cytotoxicity as shown in all the graphs. But as the concentration of dox decreases, the viability of the cells increases significantly. But Gd-PEG NPs are more biocompatible and less toxic than Gd-PEG-dox NPs. At a concentration of 55ng/mL of Gd-PEG-dox NPs, surviving fraction was found to be 92.03%, 84.03% and 70.3% for U-87, A-549 and PANC-1 cell lines respectively and for Gd-PEG NPs of same concentration, surviving fraction was found to be almost 100% for all three types of cells. As the dose of dox loaded NPs was increased to 55µg/mL, surviving fractions reduced to 19.43%, 5.176% and 25.6% for U-87, A-549 and PANC-1 cell lines respectively as compared to Gd-PEG NPs of same concentration which gave 41.51%, 50.2% and 21.9% surviving fraction with U-87, A-549 and PANC-1 cell lines respectively. At all concentrations and in all the three cell lines, cytotoxicity of dox loaded NPs was found to be higher than GD-PEG NPs and dox solution. So these NPs can be potentially used for anti-cancer activity. From the above mentioned data, it can be concluded that these multimodal NPs can be efficiently used as drug delivery vehicle and for efficient optical and MR imaging applications. 4. Conclusion
Here, we have demonstrated synthesis of multi-functional PEGylated Gd2O3 NPs loaded with fluorescent anti-cancer drug dox for drug-delivery, optical and MR imaging applications. Optical activity of the NPs is evident from the fluorescence microscopy images, fluorescence emission spectrum and UV-Vis spectrum. The particles were efficiently taken up by the cells and in vitro cytotoxicity assay performed on U-87, A549 and PANC-1 cell lines demonstrated efficient drug delivery and cytotoxicity on these cells. Lastly, line broadening effect on neighboring heavy water protons in 1H-NMR spectrum proves that these Gd-PEG-dox NPs can work as efficient CA for MR imaging. These observations make these NPs an exciting drug delivery vehicle and imaging probe. We anticipate that further modulation of such nanomaterials for different biomedical applications will give new dimension to diagnosis and treatment in future. Acknowledgement We would like to thank University Science Instrumentation Centre (USIC), University of Delhi, for allowing us to use various instruments. We are very thankful to INMAS, DRDO Delhi for allowing us to use the necessary instrumentation facilities. Funding: This work was supported by Council of scientific and industrial research (CSIR) [Scheme No.: 02(0153)/13/EMR-II] New Delhi, India.
References References
1 Parveen, S., Misra, R., Sahoo, S.K., 2012. Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging. Nanomedicine 8, 147–166. 2 Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., Rudzinski, W.E., 2001. Biodegradable polymeric nanoparticles as drug delivery devices. J. Control. Release 70, 1–20. 3 Hamidi, M., Azadi, A., Rafiei, P., 2008. Hydrogel nanoparticles in drug delivery. Adv. Drug Deliv. Rev. 60, 1638–1649. 4 Moghimi, S.M., Hunter, A.C., Murray,J.C., 2001. Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol. Rev. 53, 283–318. 5 Wu, L., Zhang, J., Watanabe, W., 2011. Physical and chemical stability of drug nanoparticles, Adv. Drug Deliv. Rev. 63, 456–469. 6 Fang, C., Zhang, M., 2010. Nanoparticle-based theragnostics: integrating diagnostic and therapeutic potentials in nanomedicine. J. Control. Release. 146(1), 2–5. 7 Lammers, T., Kiessling, F., Hennink, W.E., Storm, G., 2010. Nanotheranostics and Image-Guided Drug Delivery: Current Concepts and Future Directions. Mol. Pharm. 7(6), 1899–1912. 8 Hong, G., Yuan, R., Liang, B., Shen, J., Yang, X., Shuai, X., 2008. Folate-functionalized polymeric micelle as hepatic carcinoma-targeted, MRI-ultrasensitive delivery system of antitumor drugs Biomed. Microdevices. 10, 693–700.
9 Shuai, X., Ai, H., Nasongkla, N., Kim, S., Gao, J., 2004. Micellar carriers based on block copolymers of poly(q-caprolactone) and poly(ethylene glycol) for doxorubicin delivery. J. Control. Release 98 415– 426. 10 Benyettou, F., Rezgui, R., Ravaux, F., Jaber, T., Blumer, K., Jouiad, M., L. Motte, L., Olsen, J.-C., Platas-Iglesias, C., Magzoub, M., Trabolsi, A., 2015. Synthesis of silver nanoparticles for the dual delivery of doxorubicin and alendronate to cancer cells. J. Mater. Chem. B 3, 7237-7245. DOI: 10.1039/c5tb00994d 11 Arya, S., Grailer, J.J., Pilla, S., Steeber, D.A., Gong, S., 2009. Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J. Mater. Chem. 19, 7879–7884. DOI: 10.1039/b914071a 12 Missirlis, D., Kawamura, R., Tirelli, N., Hubbell, J.A., 2006. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur. J. Pharm. Sci. 29 120–129. DOI:10.1016/j.ejps.2006.06.003 13 Dreis, S., Rothweiler, F., Michaelis, M., Cinatl Jr., J., Kreuter, J., Langer, K., 2007. Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles. Int. J. Pharm. 341, 207–214. DOI:10.1016/j.ijpharm.2007.03.036 14 S.A. Corr, S.J. Byrne, R. Tekoriute, C.J. Meledandri, D.F. Brougham, M. Lynch, C. Kerskens, L. O'Dwyer, Y.K. Gun'ko, J. Am. Chem. Soc. 130(13) (2008) 4214–4215. 15 Hans, M.L., Lowman, A.M., 2002, Linear Assemblies of Magnetic Nanoparticles as MRI Contrast Agents. Curr. Opin. Solid State Mater. Sci. 6, 319–327. 16 Fortin, J., Wilhelm, C., Servais, J., Me´nager, C., Bacri, J., Gazeau, F., 2007. Size-Sorted Anionic Iron Oxide Nanomagnets as Colloidal Mediators for Magnetic Hyperthermia. J. Am. Chem. Soc. 129, 2628-2635. 17 Ahmad, M.W., Xu, W., Kim, S.J., Baeck, J.S., Chang, Y., Bae, J.E., Chae, K.S., Park, J.A., Kim, T.J., Lee, G.H., 2015. Potential dual imaging nanoparticle: Gd2O3 nanoparticle. Sci. Rep. 5, 8549-8560. 18 Bhakta, G., Sharma, R.K., Gupta, N., Cool, S., Nurcombe, V., Maitra, A., 2011. Multifunctional silica nanoparticles with potentials of imaging and gene delivery. Nanomedicine 7, 472–479. DOI:10.1016/j.nano.2010.12.008 19 Gupta, N., Shrivastava, A., Sharma, R.K., 2012. Silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase for imaging and therapeutic applications. Int. J. Nanomedicine 7, 5491–5500. 20 Faucher, L., Tremblay, M., Lagueux, J., Gossuin, Y., Fortin, M., 2012. Rapid synthesis of PEGylated ultrasmall gadolinium oxide nanoparticles for cell labeling and tracking with MRI. ACS Appl. Mater. Interfaces 4, 4506–4515. 21 Singh, H.P., Mitra, S., Sharma, R.K., 2014. Surface modified silica nanoparticles for synchronous magnetic resonance imaging and drug delivery applications. RSC Adv. 4, 61028–61035.
22 Maeng, J.H., Lee, D., Jung, K.H., Bae, Y., Park, I., Jeong, S., Jeon, Y., Shim, C., Kim, W., Kim, J., Lee, J., Lee, Y., Kim, J., Kim, W., Hong, S., 2010. Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer. Biomaterials 31, 4995-5006. 23 Fang, C., Kievit, F.M., Veiseh, O., Stephen, Z.R., Wang, T., Lee, D., Ellenbogen, R.G., Zhang, M., 2012. Fabrication of magnetic nanoparticles with controllable drug loading and release through a simple assembly approach. J. Control. Release 162, 233–241. 24 Bridot, J., Faure, A., Laurent, S., Rivie‘re, C., Billotey, C., Hiba, B., Janier, M., Josserand, V., Coll, J., Elst, L.V., Muller, R., Roux, S., Perriat, P., Tillement, O., 2007. Hybrid gadolinium oxide nanoparticles: multimodal contrast agents for in vivo imaging. J. Am. Chem. Soc. 129, 5076–5084. 25 Oyewumi, M.O., Yokel, R.A., Jay,M., Coakley, T., Mumper, R.J., 2004. Comparison of cell uptake, biodistribution and tumor retention of folate-coated and PEG-coated gadolinium nanoparticles in tumorbearing mice. J. Control. Release 95, 613– 626. DOI:10.1016/j.jconrel.2004.01.002 26 Zhang, Y., Kohler, N., Zhang, M., 2002. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 23, 1553–1561. 27 Klapshina, L.G., Douglas, W.E., Grigoryev, I.S., Ladilina, E.Y., Shirmanova, M.V., Mysyagin, S.A., Balalaeva, I.V., Zagaynova, E.V., 2010. Novel PEG-organized biocompatible fluorescent nanoparticles doped with an ytterbium cyanoporphyrazine complex for biophotonic applications. Chem. Commun. 46 8398–8400. DOI: 10.1039/c0cc02842h 28 Lo’pez-Quintela, M.A., Tojo, C., Blanco, M.C., Garcı’a Rio, L., Leisa, J.R., 2004. Microemulsion dynamics and reactions in microemulsions. Curr. Opin. Colloid Interface Sci. 9, 264–278. DOI:10.1016/j.cocis.2004.05.029 29 Sharma, R.K., Sharma, P., Maitra, A.N., 2003. Size-dependent catalytic behavior of platinum nanoparticles on the hexacyanoferrate(III)/thiosulfate redox reaction. J. Colloid Int. Sci. 265, 134–140. 30 Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116. 31 Kumar, S., Meena, V.K., Hazari, P.P., Sharma, R.K., 2016. FITC-Dextran entrapped and silica coated gadolinium oxide nanoparticles for synchronous optical and magnetic resonance imaging applications. Int. J. Pharm. 506, 242–252. 32 Roy, I., Kumar, P., Kumar, R., Ohulchanskyy, T.Y., Yong, K., Prasad, P.N., 2014. Ormosil nanoparticles as a sustained-release drug delivery vehicle. RSC Adv. 4, 53498–53504. DOI: 10.1039/c4ra10293b 33 Reisch, A., Didier, P., Richert, L., Oncul, S., Arntz, Y., Me´ly, Y., Klymchenko, A.S., 2014. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat. Commun. 5, 1-9. DOI: 10.1038/ncomms5089|
34 Yang, J., Li, C., Cheng, Z., Zhang, X., Quan, Z., Zhang, C., Lin, J., 2007. Size-Tailored Synthesis and Luminescent Properties of One-Dimensional Gd2O3:Eu3+ Nanorods and Microrods. J. Phys. Chem. C 111, 18148-18154. 35 Cao, Y.C., 2004. Synthesis of Square Gadolinium-Oxide Nanoplates. J. Am. Chem. Soc. 126, 74567457. 36 Manocha, B., Margaritis, A., 2010. Controlled Release of Doxorubicin from Doxorubicin/γPolyglutamic Acid Ionic Complex. J. Nanomat. 2010, 1-9. DOI:10.1155/2010/780171 37 Hendrick, R.E., Haacke, E.M., 1993. Basic Physics of MR Contrast Agents and Maximization of Image Contrast. J. Magn. Reson. Imaging 3, 137–148. DOI: 10.1002/jmri.1880030126 38 Mahmoudi, M., Simchi, A., Imani, M., Häfeli, U.O., 2009. Superparamagnetic Iron Oxide Nanoparticles with Rigid Cross-linked Polyethylene Glycol Fumarate Coating for Application in Imaging and Drug Delivery. J. Phys. Chem. C 113(19), 8124–8131. DOI: 10.1021/jp900798r 39 Li, J., You, J., Dai, Y., Shi, M., Han, C., Xu, K., 2014. Gadolinium Oxide Nanoparticles and Aptamer-Functionalized Silver Nanoclusters-Based Multimodal Molecular Imaging Nanoprobe for Optical/Magnetic Resonance Cancer Cell Imaging. Anal. Chem. 86(22), 11306–11311. DOI: 10.1021/ac503026d 40 J.Y. Park, P. Daksha, G.H. Lee, S. Woo, Y. Chang, 2008. Highly water-dispersible PEG surface modified ultra small superparamagnetic iron oxide nanoparticles useful for target-specific biomedical applications. Nanotechnology 19, 365603. 41 Rana, S., Gallo, A., Srivastava, R.S., Misra, R.D.K., 2007. On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: Functionalization, conjugation and drug release kinetics. Acta Biomater. 3, 233–242. DOI:10.1016/j.actbio.2006.10.006 42 Unsoy, G., Khodadust, R., Yalcin, S., Mutlu, P., Gunduz, U., 2014. Synthesis of Doxorubicin loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery. Eur. J. Pharm. Sci. 62, 243– 250. 43 S. Kayal, R.V. Ramanujan, 2010. Doxorubicin loaded PVA coated iron oxide nanoparticles for targeted drug delivery. Mater. Sci. Eng. C 30, 484–490. DOI:10.1016/j.msec.2010.01.006 44 S. Wang, Y. Wu, R. Guo, Y. Huang, S. Wen, M. Shen, J. Wang, X. Shi, Langmuir 29(16) 2013 5030–5036. DOI: 10.1021/la4001363 45 Sanson, C., Schatz, C., Meins, J.L., Soum, A., Thévenot, J., Garanger, E., Lecommandoux, S., 2010. A simple method to achieve high doxorubicin loading in biodegradable polymersomes. J. Control. Release 147(3), 428–435. 46 Sethi, K., Roy, I., 2015. Organically modified titania nanoparticles for sustained drug release applications. J. Colloid Interface Sci. 456, 59–65.
FIGURES
Figure captions Fig.1 (a) UV-Vis absorbance spectrum and (b) Fluorescence emission spectrum of dox solution, Gd-PEG NPs and Gd-PEG-dox NPs. Fig.2 (a) TEM image of Gd-PEG-dox NPs revealed that the size of the NPs is 13nm and (b) DLS analysis revealed the hydrodynamic diameter of Gd-PEG-dox particles is 37nm. Fig.3 X-Ray Diffraction pattern of the prepared Gd-PEG-dox NPs confirms the cubic crystalline structure of particles. Fig.4 TGA curves of PEG and Gd-PEG-dox NPs showing that the prepared NPs are more thermally stable as compared to pure PEG which completely decomposes at 4270C. Fig.5 1H-NMR spectrum of (a) D2O and (b) Gd-PEG-dox NPs dispersed in D2O. Fig.6 FT-IR spectra of PEG, Gd-PEG NPs and GD-PEG-dox NPs. Fig.7 Release kinetics of dox from drug loaded NPs at pH, 7.4 and 5.0. Fig.8 Fluorescence images of A-549 cancerous cells taken (a) without treatment with NPs and (b) with treatment with Gd-PEG-dox NPs for 3 hours. Cellular uptake was studied on A-549 cancerous cell lines after treating with Gd-PEG-dox NPs for 3 hours. Fig.9 Fluorescence images of (a) aqueous dispersion of Gd-PEG NPs, (b) dox and (c) aqueous dispersion of Gd-PEG-dox in fluorescent light, taken by fluorescence microscopy. Fig.10 Cell viability (SRB) assay performed on PANC-1, U-87 and A-549 cell lines.
Fig.1 (
(
λ =
Fig.2 (
( Average
Fig.3
Fig.4
Fig.5
Chemical
Chemical
Fig.6
Fig.7
Fig.8
(
Bright
D
Bright
D
With Gd-PEG
(
With Gd-
Fig.9 (
(
(
Fig.10 do
U-
Gd-PEG Gd-PEG-
A-
PA
S0378-5173(17)30439-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.05.027 IJP 16675
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
13-3-2017 9-5-2017 11-5-2017
Please cite this article as: Kumar, Shailja, Meena, Virendra Kumar, Hazari, Puja Panwar, Sharma, Rakesh Kumar, PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PEG coated and doxorubicin loaded multimodal Gadolinium oxide nanoparticles for simultaneous drug delivery and imaging applications. Shailja Kumara, Virendra Kumar Meenaa,b, Puja Panwar Hazarib, Rakesh Kumar Sharma a* a
Nanotechnology and Drug Delivery Research Lab, Department of Chemistry, University of Delhi, Delhi-110007, India.
b
Institute of nuclear medicine and allied sciences, DRDO, Ministry of defense, Delhi Corresponding author (*): Dr. Rakesh Kumar Sharma ([email protected]) Graphical abstract
Gd-PEG-dox NPs
Drug delivery and optical imaging
TEM image of NPs
Cellular uptake of NPs
Magnetic Resonance Imaging
1
H-NMR spectrum of D2O (a) in the absence and (b) presence of Gd-PEG-dox NPs
Ph 91 9310050453
Abstract We report water-in-oil microemulsion mediated synthesis of polyethylene glycol (PEG1) coated Gd2O3 nanoparticles (NPs2) loaded with fluorescent anti-cancer drug doxorubicin (dox3) for synchronous drug delivery, optical and magnetic resonance (MR4) imaging applications. These PEG covered Gd 2O3 NPs loaded with dox (Gd-PEG-dox NPs) were found to possess spherical morphology with 13nm size as measured from TEM and the hydrodynamic diameter comes out to be 37nm as determined from DLS. Fluorescence spectra and fluorescence microscopy images confirmed optical activity of the NPs. The paramagnetic nature of NPs was affirmed by NMR line broadening effect on the spectrum of surrounding
1
Polyethylene glycol
2
Nanoparticles
3
Doxorubicin
4
Magnetic resonance
water protons. Therefore, these particles can be efficiently used as contrast agent (CA5) in MR imaging. In vitro analysis showed significant cellular uptake of particles by A-549 cells. A pH dependent drug release pattern was observed for the NPs. Cell viability assay performed on A-549, PANC-1 and U-87 cancerous cell lines revealed that Gd-PEG-dox NPs are cytotoxic. On the basis of these observations, it can be concluded that these multi-modal paramagnetic NPs promise potential cancer therapy along with optical and MR imaging applications. Keywords - Gadolinium oxide nanoparticles, reverse micelle, doxorubicin, contrast agent, drug delivery, optical imaging. 1. Introduction In biomedical science ample of attention is devoted, over the last few decades, on development of multifunctional NPs which can efficiently act as drug-delivery vehicles in biological system. Substantial efforts have been made to incorporate nanotechnology in drug delivery to have site-specific and sustained delivery of drug molecules. Mostly, efficacy of a drug is hampered by its high toxicity, poor solubility, high dose, non-specific distribution and in vivo decomposition [1]. Such limitations faced by naked drug are overcome by NPs based drug-delivery system which increases safety of drug molecule against chemical or enzymatic decomposition, amends extended bioavailability of drug to pathological site and allows targeted delivery of drug to disease site [2][3][4]. Numerous kinds of nano structures have been used as drug-delivery vehicles namely nanotubes, NPs, nanocapsules, microemulsion and liposomes [5]. Collaboration of therapy along with imaging in a single entity is an emerging trend in nanomedicine. Such drug delivery system having both therapeutic and diagnostic imaging moieties offer dual advantage of targeted drug delivery and real-time monitoring of drug response through imaging. These therapeutic NPs are called theranostic (therapeutic + diagnostic) NPs [6][7]. Chemotherapy remains one of the best treatments for various types of cancer but it is usually hindered by the unfavorable distribution of the drug which results in severe side effects. Therefore, nanosize drug delivery systems carrying therapeutic agent along with diagnostic imaging moiety are desirable [8][9]. Dox is an anthracycline anti-tumor drug which intercalates DNA and inhibits macromolecular biosynthesis [10]. Upon direct administration of dox in body, it shows low cellular uptake and distributes unevenly resulting in undesirable side-effects and toxicity to normal cells [11]. This concern led to the development of alternative routes for administering dox like entrapping or attaching it with nano materials. Missirlis et al. [12] prepared polymeric nanoparticles of PEG and poloxamer 407 encapsulating dox in inverse emulsion as controlled drug delivery system. Furthermore, they showed that encapsulation of drug delayed it degradation significantly. Dreis et al [13] prepared dox loaded human serum albumin NPs and cell viability study done on two neuroblastoma cell lines showed that anti-cancer activity of the NPs was more as compared to dox solution.
5
Contrast agent
Magnetic NPs, such as iron oxide and gadolinium oxide, exhibit potential biomedical applications such as contrast enhancing ability in MR imaging [14], in drug targeting [15] and hyperthermia [16], to name a few. Particularly, Gadolinium based magnetic NPs have drawn tremendous interest to be used as positive contrast enhancing agent in MRI. Numerous studies have shown that gadolinium oxide (Gd2O3) NPs can be efficiently used as CA [17][18][19]. The major advantage of using paramagnetic metal oxide NP as CA is the high surface to volume ratio which maximizes the interaction of paramagnetic metal ion with water protons giving high longitudinal relaxivities [20][21]. Maeng et al. [22] synthesized polymeric NPs made up of folate, poly (ethylene oxide)-trimellitic anhydride chloride-folate, dox and superparamagnetic iron oxide for chemotherapy and MR imaging in liver cancer. They showed that relative tumor volume decreased 2 and 4 fold in comparison to free dox and doxil drug in rat and rabbit models, respectively. Also, NPs showed MR imaging sensitivity which was as good as conventional MR imaging CA Resovist. Fang et al. [23] prepared superparamagnetic iron oxide NPs and poly (beta-amino ester) copolymer preloaded with dox, was assembled to its surface. They reported that these NPs could serve as smart theranostic system for sensitive detection via MR imaging and for chemotherapy through controlled drug release. Bridot et al. [24] synthesized luminescent hybrid NPs which consisted of paramagnetic Gd2O3 core and polysiloxane shell which had fluorophores and carboxylated PEG covalently attached to the surface. It was shown that the NPs had higher longitudinal proton relaxivities as compared to positive CAs like Gd-DOTA and are well suited for MR and fluorescence imaging. Keeping the dual purpose of therapy and imaging in mind, herein, we report synthesis of PEG coated paramagnetic Gd2O3 NPs loaded with dox (Gd-PEG-dox) for simultaneous contrast enhancement in MR imaging and drug delivery applications. The Gd2O3 nanoscale core was prepared in water-in-oil microemulsion of water/AOT/hexane which was followed by PEG coating and lastly dox loading via conjugation on surface. We have chosen PEG as coating material of Gd 2O3 core because of its biocompatibility [25][26], non-toxic nature [27] and it can be functionalized easily. The ensuing Gd-PEGdox NPs were subsequently characterized for their size, morphology, composition, surface, crystallinity, magnetic behavior and optical activity. After characterization, in vitro biological studies of the NPs were conducted which included their cellular uptake study and cytotoxicity evaluation by SRB cell viability assay on cancerous cells namely A549, PANC-1 and U-87. Such multifunctional NPs are anticipated to be explored profoundly in near future and such efficient systems will alter the way of cancer treatment in future. 2. Experimental section 2.1. Materials Sodium bis-(2-ethylhexyl)sulfosuccinate (AOT; 96%), hexane, gadolinium nitrate pentahydrate, ethanol and ammonia were purchased from Acrōs Organics (New Jersey, USA), Spectrochem Pvt. Ltd Mumbai (India), Central Drug House(Mumbai, India), Merck (Darmstadt, Germany), and Rankem (Delhi, India), respectively. Doxorubicin hydrochloride and deuterium oxide (D2O) were procured from Alfa Aesar (Heysham, England). Polyethylene glycol (6000 KD), citric acid anhydrous and sodium phosphate dibasic anhydrous (Na2HPO4) were procured from s.d.fine-chem limited, spectrochem pvt. Ltd. (India) and SRL Ltd (India) respectively. Phosphate buffer saline (PBS), Sulforhodamine B (SRB) and Hoechst Stain were incurred from Sigma Aldrich (St Louis, MO). Tris base and paraformaldehyde were purchased from Merck. The lung carcinoma cell line (A-549), pancreatic cancer cell line (PANC-1) and U-87 were
procured from National Centre for Cell Sciences, Pune India. All chemicals were used as such without any further purification. 2.2. Synthesis of PEGylated Gd2O3 NPs loaded with doxorubicin (Gd-PEG-dox). For the synthesis of Gd-PEG-dox NPs, we prepared microemulsion ‘A’ by adding 300 µl of aqueous gadolinium nitrate (Gd(NO3)3. 5H2O, 0.1 M) solution to 15 ml of 0.1 M AOT in hexane. Microemulsion ‘B’ was obtained in the similar manner in which 300 µl of 1M NH3 was added to 15 ml of 0.1 M AOT in hexane. Both the microemulsons were stirred till they were optically clear. Molar ratio of water to surfactant (Wo) was maintained at 11.11 in both the microemulsions [28][29]. Now microemulsion ‘B’ was added dropwise to microemulsion ‘A’ with constant stirring at room temperature. After complete transfer, the resultant solution was stirred for another 24 hours which resulted in the formation of Gd2O3 NPs. The ensuing particles were extracted and washed several times with hexane and alcohol. The washed Gd2O3 NPs were dispersed in 5 mL double-distilled water and 200 µL of 1% PEG solution was added to it. The solution was further stirred for another 24 hours. The PEG coated Gd2O3 particles (Gd-PEG) were separated and redispersed in 5 mL DDW to which 40 µL of 0.1% dox solution was added. The solution was stirred for another 48 hours which resulted in the formation of dox loaded NPs (Gd-PEG-dox NPs). The resulting Gd-PEG-dox NPs were separated and finally dispersed in 1 mL of DDW for characterization and further use. 2.3. Characterization 2.3.1. Ultraviolet-visible (UV-Vis) spectroscopy UV-Vis spectra of all the samples were recorded on Shimadzu-1601 UV-vis spectrophotometer in the range of 190 nm to 1100 nm. 2.3.2. Transmission electron microscopy (TEM) TEM images of the prepared NPs were taken using TECNAI G2-30 U TWIN instrument (FEI, Eindhoven, Netherlands) which operates at acceleration voltage of 300 KV. A drop of dilute aqueous NP dispersion was deposited on copper grid. TEM images were taken after completely drying the grid at room temperature. 2.3.3. Dynamic Light Scattering (DLS) Hydrodynamic diameter of NPs was determined by DLS analysis performed on Zetasizer Nano ZS-90 analyzer from Malvern having He-Ne laser (λ= 633 nm, power 4 mV) as light source and recorded at a backscattering angle of 90 0 using an avalanche photodiode detector. The hydrodynamic diameter (d) of NPs was determined from diffusion of the NPs using Stokes–Einstein equation. 2.3.4. Fourier Transform Infrared (FT-IR) Spectroscopy FT-IR spectrums of the samples were taken using RXIFT IR (Perkin Elmer) instrument which has nichrome wire coated with alloy as source. Dry samples were dispersed into KBr powder and pressed into a pellet. The pellets were scanned from 400 cm-1 to 4000 cm−1 at room temperature. 2.3.5. X-ray diffraction (XRD) analysis.
XRD analysis of dry NP sample was carried out on Rigaku miniflex desktop XRD instrument at room temperature. Scanning of the sample was done in the 2θ range of 15–70 0. 2.3.6. Fluorescence Spectroscopy The fluorescence emission spectra of various samples were taken, at room temperature, on Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA). The optical activity of various samples was determined by scanning in the range of 500 nm to 800 nm. 2.3.7. Thermo gravimetric analysis (TGA). TGA of powered samples was carried out in nitrogen atmosphere on Perkin Elmer DTA/TGA/DSC instrument. The sample was placed on the sample holder of the instrument and scanned in the temperature range of 460C to 9720C. Variation in the weight of the sample with increase in temperature was studied. 2.3.8. 1H-Nuclear magnetic resonance (1H-NMR). 1
H-NMR spectrum of prepared NPs was recorded on 400 MHz spectrometer (JNM-ECX-400P, JEOL, Tokyo, Japan) in D2O as solvent. 2.4. Estimation of Attachment efficiency of doxorubicin. The attachment efficiency of multimodal Gd-PEG-dox NPs was calculated by an indirect approach using UV-Vis spectrophotometer. After preparation of Gd-PEG-dox NPs, the NPs were separated from supernatant by centrifugation. The amount of unentrapped drug present in the supernatant was determined spectrophotometrically by measuring its absorbance at 496 nm (characteristic peak of dox). On subtracting the amount of dox left in supernatant from the total drug added, attachment efficiency was calculated. 2.5. Release kinetics of doxorubicin at pH= 5.0 and 7.4. Release kinetics of dox from Gd-PEG-dox NPs was carried out at a pH of 5.0 and 7.4. For this, 5.5 mg of Gd-PEG-dox NPs were dispersed in 10 ml of citric acid-Na2HPO4 buffer (pH=5.0) and the solution was put on gentle stirring in a glass vial at room temperature. At regular intervals of time, 3ml of the NP dispersion was withdrawn and centrifuged at 10,000 rpm. The amount of drug released was estimated by measuring the absorbance of the drug in supernatant at 496 nm. After recording the absorbance, the solution was transferred back to the vial cautiously. The same procedure was repeated with buffer of pH 7.4. 2.6. In vitro cytotoxicity by SRB assay In vitro cytotoxicity of the NPs was evaluated by using Sulforhodamine (SRB) dye based assay. In a 96 well microtitre plate, exponentially growing cells were plated at a uniform cell density of 10,000 cells/well for 24 hours before treating with NPs. Cell viability was assessed after treating the cells with varying concentrations (55 ng/mL – 0.55 mg/mL) of NPs for 24 hours. Treated cells and negative control were incubated at 4 0C with trichloroacetic acid solution (10%) for 1 hour. At the end of the treatment, the cells were washed under running tap water to get rid of excess of solution and were kept at room temperature for air drying the wells overnight. Next day, 0.057% solution of SRB dye was used to stain the cells for 1 hour. Then, plates were washed under running tap water to remove excess dye and were allowed to air dry
overnight. Next day, 100µL of 10mM Tris buffer (pH 10.5) was added to solubilize the dye attached to the cells. Optical density of 100 µL of extracts was measured at 510 nm [30]. 2.7. In vitro cellular uptake Cellular uptake analysis was carried out on A-549 lung carcinoma cells. Firstly A-549 cells were seeded onto coverslips in a 6-well plate at a density of 5000 cells/well. After 24 hours, the cells were incubated with the Gd-PEG and Gd-PEG-dox NPs at concentration of 0.55mg/mL in culture media for 3 hrs. After the incubation, the media was carefully removed and cells were washed with PBS (pH-7.2) twice and further fixed with 1% formaldehyde solution (pH-7.0) for 5 mins. Later cells were again washed with PBS and incubated with DAPI solution (20mg/mL) for 2 mins. Then the cells were again washed twice with PBS gently to remove excess of DAPI stain, further coverslip containing the cells were picked and placed on glass slide with DPX mounting media and images were taken using fluorescence microscope (UV filter for DAPI and Green filter for Gd-PEG-dox NPs images). 3. Result and discussion Our primary motive was to develop multi-modal drug loaded magnetic NPs which could be efficiently used for various biomedical applications simultaneously. Paramagnetic Gd2O3 NPs were prepared via uncomplicated and soft chemical route using the aqueous core of the reverse micellar system of water/AOT/hexane [19][31]. These Gd2O3 NPs were then coated with PEG and subsequently attached with dox. Surface coating of Gd 2O3 NPs was done with PEG in order to impart biocompatibility to NPs, deter them from getting aggregated, for long circulation and for conjugating dox molecules to the particles. UV-Vis spectra of dox, dox conjugated NPs and NPs without dox were recorded and given in fig.1(a). The UV-Vis spectrum of free dox shows characteristic absorption peak at 496 nm which is also observed for Gd-PEG-dox NPs which depicts the presence of dox in the fabricated NPs. On the other hand, spectrum of Gd-PEG NPs without dox does not show absorption near 496nm which proves that the peak at 496 nm arises due to dox and not because of the nanoparticles or any other entity. Dox is a fluorescent anti-cancer drug which can be tracked optically [32]. The optical activity of the NPs was studied using fluorescence emission spectroscopy. The fluorescence spectra of aqueous solution of pure dox, Gd-PEG-dox NPs and Gd-PEG NPs without dox were taken and are shown in fig.1(b). From the UV-Vis absorption spectrum and fluorescence study, we observed a slight red-shift in the absorption spectrum of particles and a fluorescence quenching in Gd-PEG-dox NPs. This can be ascribed to an increased local concentration of dox which leads to dox-dox interaction through π-π stacking [12][33]. The particle diameter determined using TEM image, as shown in fig.2(a), comes out to be around 13 nm with spherical morphology and high degree of monodispersity. The size of the NPs, measured from dynamic light scattering (DLS) experiment, was found to be 37nm (fig.2(b)). This difference in size observed by DLS and TEM lies in the fact that in TEM we use dry sample for analysis and DLS is carried out in aqueous dispersion of NPs. Therefore, in DLS hydration layer surrounding the NPs also contribute to the size. The EDAX analysis (not shown) confirms the presence of expected elements in the NP sample. The crystallinity of particles was studied using X-ray diffraction (XRD) analysis. The XRD pattern, as shown in fig.3, exhibits sharp 2θ peaks at 29.30, 33.9 0, 35.50, 47.260, 59.60 and 69.20 which corresponds to (222), (400), (411), (440), (444) and (800) planes of cubic Gd2O3 NPs [34][35]. These values give the evidence for the crystalline nature of the fabricated NPs due to the presence of paramagnetic Gd 2O3 in them.
Thermogravimetric analysis (TGA) was carried out from 300C to 9550C in N2 atmosphere to probe the thermal properties of NPs. The thermograph of PEG and Gd-PEG-dox NPs is given in fig.4. TGA curve of PEG shows that its degradation started at 3500C after which a sudden drop in weight is observed up to 4280C. At 4280C, PEG is 97.5% decomposed and get completely decomposed at 523 0C. In the case of Gd-PEG-dox NPs, a gradual decrease in weight was observed over the entire temperature range. Total weight loss of 49% was observed for the NPs. Manocha et al. [36] showed that in the case of pure dox weight loss of 10% was observed from 25 0C to 600 0C. Therefore, the observed decrease in the weight of NPs can be attributed to the decomposition of PEG on the surface and the NPs are more thermally stable than pure PEG. The efficacy of these nanoparticles to be used as CA in MRI was exemplified by taking the 1H-NMR of pure D2O and D2O in presence of GD-PEG-dox NPs as shown in fig.5. 1H-NMR of D2O shows a single sharp peak corresponding to water protons (fig.5(a)). The 7 unpaired electrons in Gd3+ ion of Gd2O3 gives rise to high magnetic moment of 7.94 μB. Gadolinium has long electron relaxation time thereby enhancing dipole-dipole interaction between electrons and (water) protons. Such interactions tend to enhance the proton relaxation which increases the contrast in MR image. In the absence of the NPs, a sharp peak is observed in the NMR spectrum of D2O (fig.5(a)) but presence of NPs causes line broadening effect on the NMR spectrum of H2O protons present in its vicinity (fig.5(b))[19][21][37]. The conjugation of PEG and subsequently of dox on the surface of Gd2O3 nanoparticles was probed with the help of FT-IR spectra of PEG, Gd-PEG NPs and Gd-PEG-dox NPs as shown in fig.6. In the case of PEG, strong peaks at 2885 and 1110 cm-1 corresponds to C-H symmetric and C-O-C asymmetric stretching vibrations [38]. Two sharp peaks at 1460 cm-1 and 1346 cm-1 are assigned to scissoring, and wagging CH2 vibrational modes respectively [39]. Strong peak at 3432 cm-1 arises due to O-H stretching vibration of PEG [40]. FT-IR spectrum of Gd-PEG NPs shows two important peaks at 3420 cm-1 and 1100 cm-1 corresponding to O-H stretch of alcohol and asymmetrical C-O-C stretch, respectively, which confirms the attachment of PEG on to the surface of Gd2O3 NPs. Rana et al [41] reported that the FT-IR spectrum of pure dox exhibits a peak at 3410 cm-1 due to stretching frequencies of O-H and N–H of primary amine in dox. However, in the case of Gd-PEG-dox NPs, peak arising due to N-H and O-H stretching vibrations is broadened and shifted to lower frequency value (~ 3406 cm-1). The characteristic bands of dox located at 2932 cm-1 and 1730 cm-1 due to C–H and C=O stretching vibrations, are also observed for dox loaded NPs [42]. Two peaks located at 1521 cm-1 and 1043 cm-1 corresponding to bending of NH2 on aromatic ring and C-O stretch of alcohol group, respectively, were also observed in Gd-PEG-dox NPs [43][40][44]. The bands in pure dox which are observed at 870 cm−1 and 805 cm−1 due to N–H wagging have diminished in Gd-PEG-dox NPs. Taking into consideration this data, we can interpret that the attachment of dox to the surface of PEG coated Gd2O3 nanomaterial is occurring through hydrogen bonding between -OH and –NH2 groups of dox with the –OH groups of PEG which is also reported earlier by kayal et al. [42] and rana et al. [40]. The attachment efficiency (A%) of anti-cancer drug loaded particles was determined via UV-visible spectroscopy by measuring the difference of the total drug added at the time of synthesis and the amount of drug not attaching to the particle surface. The A% was calculated by using the formulae given below and it comes out to be 87%. Attachment efficiency (A%) =
× 100
To ascertain the utility of these particles for drug delivery, we probed the time-dependent release profile of the drug from the NPs. Release kinetic study was carried out in pH of 5.0 and 7.4 buffers and the release behavior is shown in fig.7. A sustained release pattern of the drug was observed in both the pH. In pH 7.4, a release of 35% and ~48% was observed after 24 hrs and 48 hrs respectively. It reached to ~68% after 165 hrs. However, in pH 5.0 buffer, 48% and 69.7% release of dox was noticed after 24 h and 46 h respectively, which reached to 90% after 167 h. As compared to neutral pH, release of dox was found to be higher in acidic pH which may be attributed to the dissociation of hydrogen bonds between PEG and dox due to complete protonation of –NH2 groups of dox which is consistent with earlier report [45]. Moreover, another probable reason for enhanced drug release in acidic medium could be that the cationic drug has higher affinity for the acidic microenvironment which acts as driving force for its enhanced release [46]. Further the in-vitro interaction of these NPs with cells was evaluated. The cellular uptake study of the NPs on A-549 cancerous cells was carried out using fluorescence microscopy. Dox is a fluorescent anti-cancer drug which can be used for optical imaging in cellular environment. The fluorescence of dox loaded NPs can be optically tracked within the treated cells. The fluorescence images of the A-549 cells treated with Gd-PEG NPs and Gd-PEG-dox NPs are shown in fig. 8. In the control experiment, the Gd-PEG treated cells did not show any fluorescence emission (fig.8(a)). On the other hand, treatment of cells with 0.55 mg/mL of Gd-PEG-dox NPs for 3hours resulted in strong fluorescence (fig.8(b)) which clearly demonstrates efficient uptake of the Gd-PEG-dox NPs by the cells. Additionally, the fluorescence images of the aqueous Gd-PEG NPs dispersion, dox solution and aqueous Gd-PEG-dox NPs dispersion were taken and are shown in fig. 9. The images clearly demonstrate that dox solution and dox loaded magnetic NPs show fluorescence whereas Gd-PEG NPs without dox do not exhibit any fluorescence. After affirming cellular uptake and fluorescence emission of the particles, we checked their anti-cancer activity by treating three different cancerous cell lines namely A-549, PANC-1 and U-87 with varying concentrations of dox solution, Gd-PEG NPs and Gd-PEG-dox NPs for 24 hours after which cell viability was analyzed. The results are summarized in fig.10. It was found that dox loaded NPs are effectively cytotoxic than other comparative test sets. In cytotoxicity curve, data point of concentration 0.55mg/ml, dox is showing minimum cytotoxicity as shown in all the graphs. But as the concentration of dox decreases, the viability of the cells increases significantly. But Gd-PEG NPs are more biocompatible and less toxic than Gd-PEG-dox NPs. At a concentration of 55ng/mL of Gd-PEG-dox NPs, surviving fraction was found to be 92.03%, 84.03% and 70.3% for U-87, A-549 and PANC-1 cell lines respectively and for Gd-PEG NPs of same concentration, surviving fraction was found to be almost 100% for all three types of cells. As the dose of dox loaded NPs was increased to 55µg/mL, surviving fractions reduced to 19.43%, 5.176% and 25.6% for U-87, A-549 and PANC-1 cell lines respectively as compared to Gd-PEG NPs of same concentration which gave 41.51%, 50.2% and 21.9% surviving fraction with U-87, A-549 and PANC-1 cell lines respectively. At all concentrations and in all the three cell lines, cytotoxicity of dox loaded NPs was found to be higher than GD-PEG NPs and dox solution. So these NPs can be potentially used for anti-cancer activity. From the above mentioned data, it can be concluded that these multimodal NPs can be efficiently used as drug delivery vehicle and for efficient optical and MR imaging applications. 4. Conclusion
Here, we have demonstrated synthesis of multi-functional PEGylated Gd2O3 NPs loaded with fluorescent anti-cancer drug dox for drug-delivery, optical and MR imaging applications. Optical activity of the NPs is evident from the fluorescence microscopy images, fluorescence emission spectrum and UV-Vis spectrum. The particles were efficiently taken up by the cells and in vitro cytotoxicity assay performed on U-87, A549 and PANC-1 cell lines demonstrated efficient drug delivery and cytotoxicity on these cells. Lastly, line broadening effect on neighboring heavy water protons in 1H-NMR spectrum proves that these Gd-PEG-dox NPs can work as efficient CA for MR imaging. These observations make these NPs an exciting drug delivery vehicle and imaging probe. We anticipate that further modulation of such nanomaterials for different biomedical applications will give new dimension to diagnosis and treatment in future. Acknowledgement We would like to thank University Science Instrumentation Centre (USIC), University of Delhi, for allowing us to use various instruments. We are very thankful to INMAS, DRDO Delhi for allowing us to use the necessary instrumentation facilities. Funding: This work was supported by Council of scientific and industrial research (CSIR) [Scheme No.: 02(0153)/13/EMR-II] New Delhi, India.
References References
1 Parveen, S., Misra, R., Sahoo, S.K., 2012. Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging. Nanomedicine 8, 147–166. 2 Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., Rudzinski, W.E., 2001. Biodegradable polymeric nanoparticles as drug delivery devices. J. Control. Release 70, 1–20. 3 Hamidi, M., Azadi, A., Rafiei, P., 2008. Hydrogel nanoparticles in drug delivery. Adv. Drug Deliv. Rev. 60, 1638–1649. 4 Moghimi, S.M., Hunter, A.C., Murray,J.C., 2001. Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol. Rev. 53, 283–318. 5 Wu, L., Zhang, J., Watanabe, W., 2011. Physical and chemical stability of drug nanoparticles, Adv. Drug Deliv. Rev. 63, 456–469. 6 Fang, C., Zhang, M., 2010. Nanoparticle-based theragnostics: integrating diagnostic and therapeutic potentials in nanomedicine. J. Control. Release. 146(1), 2–5. 7 Lammers, T., Kiessling, F., Hennink, W.E., Storm, G., 2010. Nanotheranostics and Image-Guided Drug Delivery: Current Concepts and Future Directions. Mol. Pharm. 7(6), 1899–1912. 8 Hong, G., Yuan, R., Liang, B., Shen, J., Yang, X., Shuai, X., 2008. Folate-functionalized polymeric micelle as hepatic carcinoma-targeted, MRI-ultrasensitive delivery system of antitumor drugs Biomed. Microdevices. 10, 693–700.
9 Shuai, X., Ai, H., Nasongkla, N., Kim, S., Gao, J., 2004. Micellar carriers based on block copolymers of poly(q-caprolactone) and poly(ethylene glycol) for doxorubicin delivery. J. Control. Release 98 415– 426. 10 Benyettou, F., Rezgui, R., Ravaux, F., Jaber, T., Blumer, K., Jouiad, M., L. Motte, L., Olsen, J.-C., Platas-Iglesias, C., Magzoub, M., Trabolsi, A., 2015. Synthesis of silver nanoparticles for the dual delivery of doxorubicin and alendronate to cancer cells. J. Mater. Chem. B 3, 7237-7245. DOI: 10.1039/c5tb00994d 11 Arya, S., Grailer, J.J., Pilla, S., Steeber, D.A., Gong, S., 2009. Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J. Mater. Chem. 19, 7879–7884. DOI: 10.1039/b914071a 12 Missirlis, D., Kawamura, R., Tirelli, N., Hubbell, J.A., 2006. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur. J. Pharm. Sci. 29 120–129. DOI:10.1016/j.ejps.2006.06.003 13 Dreis, S., Rothweiler, F., Michaelis, M., Cinatl Jr., J., Kreuter, J., Langer, K., 2007. Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles. Int. J. Pharm. 341, 207–214. DOI:10.1016/j.ijpharm.2007.03.036 14 S.A. Corr, S.J. Byrne, R. Tekoriute, C.J. Meledandri, D.F. Brougham, M. Lynch, C. Kerskens, L. O'Dwyer, Y.K. Gun'ko, J. Am. Chem. Soc. 130(13) (2008) 4214–4215. 15 Hans, M.L., Lowman, A.M., 2002, Linear Assemblies of Magnetic Nanoparticles as MRI Contrast Agents. Curr. Opin. Solid State Mater. Sci. 6, 319–327. 16 Fortin, J., Wilhelm, C., Servais, J., Me´nager, C., Bacri, J., Gazeau, F., 2007. Size-Sorted Anionic Iron Oxide Nanomagnets as Colloidal Mediators for Magnetic Hyperthermia. J. Am. Chem. Soc. 129, 2628-2635. 17 Ahmad, M.W., Xu, W., Kim, S.J., Baeck, J.S., Chang, Y., Bae, J.E., Chae, K.S., Park, J.A., Kim, T.J., Lee, G.H., 2015. Potential dual imaging nanoparticle: Gd2O3 nanoparticle. Sci. Rep. 5, 8549-8560. 18 Bhakta, G., Sharma, R.K., Gupta, N., Cool, S., Nurcombe, V., Maitra, A., 2011. Multifunctional silica nanoparticles with potentials of imaging and gene delivery. Nanomedicine 7, 472–479. DOI:10.1016/j.nano.2010.12.008 19 Gupta, N., Shrivastava, A., Sharma, R.K., 2012. Silica nanoparticles coencapsulating gadolinium oxide and horseradish peroxidase for imaging and therapeutic applications. Int. J. Nanomedicine 7, 5491–5500. 20 Faucher, L., Tremblay, M., Lagueux, J., Gossuin, Y., Fortin, M., 2012. Rapid synthesis of PEGylated ultrasmall gadolinium oxide nanoparticles for cell labeling and tracking with MRI. ACS Appl. Mater. Interfaces 4, 4506–4515. 21 Singh, H.P., Mitra, S., Sharma, R.K., 2014. Surface modified silica nanoparticles for synchronous magnetic resonance imaging and drug delivery applications. RSC Adv. 4, 61028–61035.
22 Maeng, J.H., Lee, D., Jung, K.H., Bae, Y., Park, I., Jeong, S., Jeon, Y., Shim, C., Kim, W., Kim, J., Lee, J., Lee, Y., Kim, J., Kim, W., Hong, S., 2010. Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer. Biomaterials 31, 4995-5006. 23 Fang, C., Kievit, F.M., Veiseh, O., Stephen, Z.R., Wang, T., Lee, D., Ellenbogen, R.G., Zhang, M., 2012. Fabrication of magnetic nanoparticles with controllable drug loading and release through a simple assembly approach. J. Control. Release 162, 233–241. 24 Bridot, J., Faure, A., Laurent, S., Rivie‘re, C., Billotey, C., Hiba, B., Janier, M., Josserand, V., Coll, J., Elst, L.V., Muller, R., Roux, S., Perriat, P., Tillement, O., 2007. Hybrid gadolinium oxide nanoparticles: multimodal contrast agents for in vivo imaging. J. Am. Chem. Soc. 129, 5076–5084. 25 Oyewumi, M.O., Yokel, R.A., Jay,M., Coakley, T., Mumper, R.J., 2004. Comparison of cell uptake, biodistribution and tumor retention of folate-coated and PEG-coated gadolinium nanoparticles in tumorbearing mice. J. Control. Release 95, 613– 626. DOI:10.1016/j.jconrel.2004.01.002 26 Zhang, Y., Kohler, N., Zhang, M., 2002. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 23, 1553–1561. 27 Klapshina, L.G., Douglas, W.E., Grigoryev, I.S., Ladilina, E.Y., Shirmanova, M.V., Mysyagin, S.A., Balalaeva, I.V., Zagaynova, E.V., 2010. Novel PEG-organized biocompatible fluorescent nanoparticles doped with an ytterbium cyanoporphyrazine complex for biophotonic applications. Chem. Commun. 46 8398–8400. DOI: 10.1039/c0cc02842h 28 Lo’pez-Quintela, M.A., Tojo, C., Blanco, M.C., Garcı’a Rio, L., Leisa, J.R., 2004. Microemulsion dynamics and reactions in microemulsions. Curr. Opin. Colloid Interface Sci. 9, 264–278. DOI:10.1016/j.cocis.2004.05.029 29 Sharma, R.K., Sharma, P., Maitra, A.N., 2003. Size-dependent catalytic behavior of platinum nanoparticles on the hexacyanoferrate(III)/thiosulfate redox reaction. J. Colloid Int. Sci. 265, 134–140. 30 Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116. 31 Kumar, S., Meena, V.K., Hazari, P.P., Sharma, R.K., 2016. FITC-Dextran entrapped and silica coated gadolinium oxide nanoparticles for synchronous optical and magnetic resonance imaging applications. Int. J. Pharm. 506, 242–252. 32 Roy, I., Kumar, P., Kumar, R., Ohulchanskyy, T.Y., Yong, K., Prasad, P.N., 2014. Ormosil nanoparticles as a sustained-release drug delivery vehicle. RSC Adv. 4, 53498–53504. DOI: 10.1039/c4ra10293b 33 Reisch, A., Didier, P., Richert, L., Oncul, S., Arntz, Y., Me´ly, Y., Klymchenko, A.S., 2014. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat. Commun. 5, 1-9. DOI: 10.1038/ncomms5089|
34 Yang, J., Li, C., Cheng, Z., Zhang, X., Quan, Z., Zhang, C., Lin, J., 2007. Size-Tailored Synthesis and Luminescent Properties of One-Dimensional Gd2O3:Eu3+ Nanorods and Microrods. J. Phys. Chem. C 111, 18148-18154. 35 Cao, Y.C., 2004. Synthesis of Square Gadolinium-Oxide Nanoplates. J. Am. Chem. Soc. 126, 74567457. 36 Manocha, B., Margaritis, A., 2010. Controlled Release of Doxorubicin from Doxorubicin/γPolyglutamic Acid Ionic Complex. J. Nanomat. 2010, 1-9. DOI:10.1155/2010/780171 37 Hendrick, R.E., Haacke, E.M., 1993. Basic Physics of MR Contrast Agents and Maximization of Image Contrast. J. Magn. Reson. Imaging 3, 137–148. DOI: 10.1002/jmri.1880030126 38 Mahmoudi, M., Simchi, A., Imani, M., Häfeli, U.O., 2009. Superparamagnetic Iron Oxide Nanoparticles with Rigid Cross-linked Polyethylene Glycol Fumarate Coating for Application in Imaging and Drug Delivery. J. Phys. Chem. C 113(19), 8124–8131. DOI: 10.1021/jp900798r 39 Li, J., You, J., Dai, Y., Shi, M., Han, C., Xu, K., 2014. Gadolinium Oxide Nanoparticles and Aptamer-Functionalized Silver Nanoclusters-Based Multimodal Molecular Imaging Nanoprobe for Optical/Magnetic Resonance Cancer Cell Imaging. Anal. Chem. 86(22), 11306–11311. DOI: 10.1021/ac503026d 40 J.Y. Park, P. Daksha, G.H. Lee, S. Woo, Y. Chang, 2008. Highly water-dispersible PEG surface modified ultra small superparamagnetic iron oxide nanoparticles useful for target-specific biomedical applications. Nanotechnology 19, 365603. 41 Rana, S., Gallo, A., Srivastava, R.S., Misra, R.D.K., 2007. On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: Functionalization, conjugation and drug release kinetics. Acta Biomater. 3, 233–242. DOI:10.1016/j.actbio.2006.10.006 42 Unsoy, G., Khodadust, R., Yalcin, S., Mutlu, P., Gunduz, U., 2014. Synthesis of Doxorubicin loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery. Eur. J. Pharm. Sci. 62, 243– 250. 43 S. Kayal, R.V. Ramanujan, 2010. Doxorubicin loaded PVA coated iron oxide nanoparticles for targeted drug delivery. Mater. Sci. Eng. C 30, 484–490. DOI:10.1016/j.msec.2010.01.006 44 S. Wang, Y. Wu, R. Guo, Y. Huang, S. Wen, M. Shen, J. Wang, X. Shi, Langmuir 29(16) 2013 5030–5036. DOI: 10.1021/la4001363 45 Sanson, C., Schatz, C., Meins, J.L., Soum, A., Thévenot, J., Garanger, E., Lecommandoux, S., 2010. A simple method to achieve high doxorubicin loading in biodegradable polymersomes. J. Control. Release 147(3), 428–435. 46 Sethi, K., Roy, I., 2015. Organically modified titania nanoparticles for sustained drug release applications. J. Colloid Interface Sci. 456, 59–65.
FIGURES
Figure captions Fig.1 (a) UV-Vis absorbance spectrum and (b) Fluorescence emission spectrum of dox solution, Gd-PEG NPs and Gd-PEG-dox NPs. Fig.2 (a) TEM image of Gd-PEG-dox NPs revealed that the size of the NPs is 13nm and (b) DLS analysis revealed the hydrodynamic diameter of Gd-PEG-dox particles is 37nm. Fig.3 X-Ray Diffraction pattern of the prepared Gd-PEG-dox NPs confirms the cubic crystalline structure of particles. Fig.4 TGA curves of PEG and Gd-PEG-dox NPs showing that the prepared NPs are more thermally stable as compared to pure PEG which completely decomposes at 4270C. Fig.5 1H-NMR spectrum of (a) D2O and (b) Gd-PEG-dox NPs dispersed in D2O. Fig.6 FT-IR spectra of PEG, Gd-PEG NPs and GD-PEG-dox NPs. Fig.7 Release kinetics of dox from drug loaded NPs at pH, 7.4 and 5.0. Fig.8 Fluorescence images of A-549 cancerous cells taken (a) without treatment with NPs and (b) with treatment with Gd-PEG-dox NPs for 3 hours. Cellular uptake was studied on A-549 cancerous cell lines after treating with Gd-PEG-dox NPs for 3 hours. Fig.9 Fluorescence images of (a) aqueous dispersion of Gd-PEG NPs, (b) dox and (c) aqueous dispersion of Gd-PEG-dox in fluorescent light, taken by fluorescence microscopy. Fig.10 Cell viability (SRB) assay performed on PANC-1, U-87 and A-549 cell lines.
Fig.1 (
(
λ =
Fig.2 (
( Average
Fig.3
Fig.4
Fig.5
Chemical
Chemical
Fig.6
Fig.7
Fig.8
(
Bright
D
Bright
D
With Gd-PEG
(
With Gd-
Fig.9 (
(
(
Fig.10 do
U-
Gd-PEG Gd-PEG-
A-
PA