Synthesis and characterization of CdO/GrO nanolayer for in vivo imaging

Introduction

The recent discovery of graphene has been accompanied with increasing research attention to explore this new material for drug delivery applications. Due to its unique structure and geometry, graphene possesses remarkable physical-chemical properties including high Young’s modulus, high fracture strength, excellent electrical and thermal conductivity, fast mobility of charge carriers, large specific surface area and biocompatibility [1-5].

The development of fluorescence and bioluminescence imaging approaches started in the 1990s with fluorescence reflectance imaging. These properties enable graphene to be considered as an ideal material for broad applications, ranging from quantum physics, nanoelectronics, energy research, catalysis and engineering of nanocomposites and biomaterials [6-9]. In the area of nanomedicine, graphene and its composites have emerged as new biomaterials which provide exciting opportunities for the development of broad applications including a new generation of biosensors, nano-carriers for drug delivery, and probes for cell and biological imaging [10-14]. Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties including size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans [15].

 Materials and physical measurements

All the chemical reagents used in our experiments were of analytical grade and were used as received without further purification. Graphite powders were used from Alfa (natural, briquetting grade, 10 meshes, 99.9995%) [16]. Graphene oxide powders were prepared following Staudenmaier’s method and reduced to graphene powders by annealing at 1050 ◦C under an argon atmosphere [17]. XRD patterns were recorded by a Rigaku D-max C III, X-ray diffractometer using Ni-filtered Cu Kα radiation of Kashan-Iran University. Microscopic morphology of products was visualized by SEM (LEO 1455VP). Transmission electron microscopy (TEM) images were obtained on a Philips EM208 transmission electron microscope with an accelerating voltage of 200 kV. UV-Vis diffuse reflectance spectroscopy analysis was carried out using Shimadzu UV-2600 UV–Vis spectrophotometer with an integrating sphere attachment and BaSO₄ was used as reference.

Synthesis of Cd (OAc)₂ Nanostructures

In this work, cadmium (II)­ acetate powder was used as the starting reagent. Cd(OAc)₂ nanostructure as precursor was prepared in a vertical quartz pipe set in vacuum condition. Each experiment was carried out by loading 1 g of cadmium (II)­ acetate powder, which was transferred to the external pipe of the set. Then, the system was vacuumed by a pump. Afterwards, water entered the inner pipe from one side and exited from the other side. Water was circulated in the system in order to solidify the product vapors. The resulting product was gradually heated to the desirable temperature 130 °C for 120 min. After the heating process, the precipitations at the external part of the inner pipe were collected.

Synthesis of CdO/GrO nanolayer

In a typical experiment, 1 g of the as-obtained Cd(OAc)₂ nanostructure and 0.08 g graphene were loaded into a silicon boat that was later put in a high-temperature tube furnace. The sample was heated in air at 330 °C for 120 min. After the thermal treatment, the system was allowed to cool to room temperature and the obtained precipitations were collected.

In vivo study

For observing the overall effects of optical properties CdO/GrO nanolayer on a living subject, 15 ppm, 30 ppm, and 45ppm of QDs was injected to rat-tail as cell ‘taggants’ for in vivo imaging. After that, we put rat-tail exposed to UV irradiation for investigation of In vivo study.

Result

Physicochemical properties of CdO/GrO nanolayer

The XRD results indicated that pure cubic CdO/GrO nanoparticles without any impurities could be obtained after thermal decomposition at 330 °C for 2 h. All reflection peaks of the XRD pattern for CdO/GrO nanoparticles are indexed well to orthorhombic phase with calculated cell parameters a = 3.3110˚A and b = 5.5260A˚.

XRD analysis is based on constructive interference of monochromatic X-rays and a crystalline sample. The characteristic x-ray diffraction pattern generated in a typical XRD analysis provides a unique “fingerprint” of the crystals present in the sample. XRD pattern of a) Cd (OAc)₂nanostructure and b) CdO/GrO nanolayer sublimated at 330 °C for 120 min and c) XRD pattern of graphene are shown in Figure 1.

SEM Images of samples show nanolayer structures have been produced. Transmission electron microscopy (TEM) image of CdO/GrO nanolayer structures are well correlated with the scanning electron microscopy image. Dynamic light scattering (DLS) diagram of CdO/GrO nanolayers shows size range between 95 to 100 nm. Photoluminescence (PL) spectroscopy shows the emission peak at 523 nm.

As shown in Figure 2a, highly aggregated particles have been obtained by sublimation at 130 °C and in many parts micro and agglomerated nanostructures have been formed. By increasing temperature to 330 °C for the synthesis of CdO/GrO nanostructures (Figure 2b), morphology changed and the nanolayer structures were produced. According to Figures 2a-b, it was found that sublimation temperature has significant effect on layers morphology. Calcinating the samples with different morphologies, produced  structure with different sizes and the finest particles. These results demonstrate that the morphology of the starting reagent has remarkable effect on product size (i.e. starting reagent with specific morphology leads to a product with specific morphology). To further investigate the details of the morphology, TEM images were taken. To prepare the TEM sample, the powder was dispersed in highly pure ethanol via ultrasonication for 20 min. TEM image of CdO/GrO nanoparticles synthesized from the Cd(OAc)₂ sublimated at 130 °C are shown in Figure 3. The AFM image of CdO/GrO nanostructures are presented in Figure 4 which clearly reveals the nanoparticles aggregates. Dynamic light scattering (DLS) was used to study the hydrodynamic size of nanolayers. DLS data of CdO/GrO nanostructures are shown in Figure 5. Optical property of the CdO/GrO nanoparticles under difference reaction conditions was investigated by photoluminescence (PL) spectroscopy and the results are given in Figure 6. The spectrum shows the emission peak at 523 nm. Importantly, the PL properties were the same irrespective of the different sizes of the CdO/GrO nanostructures. Such a large blue shift of excitonic absorption band can be attributed to the small crystallite size of the samples [19].

 In vivo imaging using CdO/GrO nanolayers in mice

For in vivo imaging study, we put rat-tail exposed to UV irradiation between 200nm-800nm.figure 7 shows the UV–Vis spectrum of CdO/GrO nanostructures and graphite structures, reflecting variation of % absorbance of CdO/GrO nanostructures as a function of wavelength. Broad peak around 398 nm indicates that the particle possesses quantum confinement.

One method for in vivo optical imaging employes a two-dimensional planar approach, akin to in vitro epifluorescence microscopy, which allows researchers to explore surface tissues and bones of an animal with fluorescence or bioluminescence. By using the in vivo imaging with CdO/GrO nanostructures, we investigated light emission of CdO/GrO nanostructures from the circulation within minutes after injection. The schematic of methods was displayed in   (Figure 8). It was found that CdO/GrO nanostructures had the ability to track and target cells by photoluminsence properties. QDs were found to stay in the blood circulation and for in vivo optical imaging for an extended period of time (half-life more than 3 h). This long-circulating can be explained by the exclusive  structural properties of QD nanoparticles. In vivo studies also confirmed the nontoxic nature of stably protected QDs [18]. Indeed, in vivo toxicity is haply  to be a key factor in characterize whether QD imaging probes would be approved by regulatory agencies for human clinical use.

The optical property of the produced nanoparticles was studied by measuring the % absorbance and band gap energy. The estimated optical band gap energy is an accepted value for application in هn vivo targeting and imaging. Gao [20] reported a new class of multifunctional QD probes for simultaneous targeting and imaging of tumors in live animals. Under in vivo conditions, QD probes can be delivered to tumors either by a passive targeting mechanism or through an active targeting mechanism. In the passive mode, macromolecules and nanometer-sized particles are accumulated preferentially at tumor sites through an enhanced permeability and retention effect [21-27].

 Conclusion

In this manuscript, CdO/GrO nanostructures have been successfully synthesized through a solvent-less solid-state thermal decomposition of cadmium (II) acetate nanostructures obtained by the sublimation of cadmium (II) acetate powder. We investigated the influence of the sublimation temperature on the morphology of Cd(OAc)₂ nanoparticles and consequently on the CdO/GrO nanoparticles. The characteristics of products were determined by using XRD, SEM, PL, AFM and TEM analysis. The XRD results indicated that pure cubic CdO/GrO nanoparticles without any impurities obtained after thermal decomposition at 330 °C for 2 h. Nanostructures injection to rats and their optical properties were investigation by UV–Vis spectroscopy. This study indicated that new composites developed in the current study are suitable materials for improved biocompatibility and circulation half-life.

 Acknowledgment

Authors are grateful to council of Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, for providing financial support to undertake this work.

 Conflict of Interest

The authors declare that they have no conflict of interest.