{'fraction_non_alphanumeric': 0.028738212842388863, 'fraction_numerical': 0.02240183605248715, 'mean_word_length': 4.831830084375909, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Pegylated Eu-enabled submicron alumina spheres as potential theranostics agent RD cell line as model

Objectives This study is aimed to synthesis and evaluate PEGylated Eu enabled spherical alumina submicron particles (s-Al2O3:Eu) for potential theranostic applications. Methods This study is bisected into two parts, a) synthesis of PEGylated Eu enabled spherical alumina submicron particles (s-Al2O3:Eu), and b) characterization of the synthesized particles to determine their efficacy for potential theranostic applications. The synthesis of the particles involved the following steps. In the first step, s-Al2O3:Eu is synthesized using solvothermal synthesis. In the next step, the particles undergo post synthesis water–ethanol treatment and calcination. The surface of the synthesized s-Al2O3:Eu particles is then coated by PEG to increase its biocompatibility. Once the particles are prepared, they are characterized using different techniques. The microstructure, composition and structure of the particles is characterized using SEM, EDX and XRD techniques. The detection of the functional groups is done using FTIR analysis. The photoluminescence emission spectrum of s-Al2O3:Eu is studied using Photoluminescence spectroscopy. And, finally, the biocompatibility is studied using MTT assay on RD cell lines. Results The microstructure analysis, from the micrographs obtained from SEM, shows that the spherical alumina particles have a submicron size with narrow size distribution. The compositional analysis, as per EDX, confirms the presence of Oxygen, Aluminum and Europium in the particles. While, XRD analysis of s-Al2O3:Eu confirms the formation of alpha alumina phase after calcination at 700 °C. Emission peaks, obtained by Photoluminescence emission spectroscopy, show that the optimum emission intensities correspond to the transition from 5D0 to 7Fj orbital of Eu+3. FTIR analysis confirms the successful coating of PEG. Finally, a cell viability of more than 86% is observed when the biocompatibility of the particles is studied, using MTT assay on RD cell lines. Conclusions s-Al2O3:Eu with narrow distribution are successfully synthesized. Structural and functional characterizations support the suitability of s-Al2O3:Eu as potential theranostic agent.


Introduction
The rapid research and development in the field of nanotechnology in recent years has transformed the scientific landscape of bioimaging and drug delivery. Hence, the investigation of novel biocompatible materials for application in theronastic has become a key area of interest for scientists. A theranostic agent is the one that has the capability of therapy as well as diagnosis (Xie et al., 2010). The combination of nanoparticle-based drug delivery systems with appropriate contrast agents for application in theranostic is a promising research domain with a lot of recent developments. The desired characteristics of nanoparticles-based theranostic agents include; enhanced blood circulation time, improved specific localization for drug delivery and superior imaging characteristics for diagnosis.
Research has been conducted on several nanomaterials in order to determine their efficacy as a potential theronastic. These nanomaterials include polymeric nanomaterials, metallic nanomaterials, ceramic nanomaterials and composites (Liechty et al., 2010, Huxford et al, 2010, Paul and Sharma, 2003. All these nanomaterials have their own sets of advantages and disadvantages, which are discussed briefly as follows. Polymeric nanomaterials have low toxic effects and high biocompatibility but they have low therapeutic life, which decreases their efficiency as a theranostic agent (Kwon and Furgeson, 2007). Metallic nanomaterials have high surface area, increased bioavailability and ease of surface functionalization, which makes them a viable option for drug delivery and bioimaging. However, their inadequate biodistribution, pharmacokinetics and possible toxicity remain a challenge (Arvizo et al., 2010, Mathur et al., 2018, Singh et al., 2010. Among metallic nanomaterials, silver, gold, nickel, titanium and iron-based nanoparticles are widely used for biomedicine applications (Patra et al., 2018). Ceramic nanomaterials are a suitable option as theranostic agents because of their high mechanical strength, low biodegradability, and good stability in the harsh bioenvironment which increases their bioavailability as drug carrier. The most widely researched bio-ceramics include silica and alumina (Subhapradha et al., 2018). Mesoporous silica has gained considerable importance, in the field of theranostic, owing to its high surface area and pore volume. It can serve as multifunctional drug carrier when combined with a suitable contrast agent (Xie et al., 2010). Despite all these benefits, its applicability to clinical use is still hindered because of the difficulty in scaling up its synthesis, under good manufacturing practices (GMP). The reproducibility of the desired silica based therapeutic agent is a challenge when dealing with a fine control of size, shape, charge and surface functionalization during large scale synthesis (Nigro et al., 2018) Among the class of inorganic porous materials for drug delivery, anodic alumina is a promising candidate because of its controlled pore size, and uniform size distribution. It also has high surface area and chemical stability. Ceramic alumina has high biocompatibility. Hence, it is being used in orthopedic proteases and dental implants. To develop therapeutic capabilities in anodic alumina, magnetic particles can be loaded on the pores. Iron based magnetic particles can act as T2 contrast agents for magnetic resonance (MR) imaging (Palanisamy and Wang, 2019). The initial work on anodic alumina as a drug carrier in anticancer treatment comprised of loading of Apo2L/TRAIL in the pores of anodic alumina. The drug loading capacity of 104 mg/mg was achieved with a sustained drug release profile (Wang et al., 2014). Anodic Alumina is a promising candidate for application in drug delivery and bio imaging, but the electrochemical process used for its synthesis yields high level of chemical waste (Stę pniowski and Bojar, 2011). A facile solvothermal synthesis of porous alumina is cost effective with the least chemical waste generated. Furthermore, the porous submicron alumina spheres obtained by this synthesis are found to be biocompatible with high drug loading capacity (Ali et al., 2019). It is also possible to incorporate imaging and diagnostic characteristics in a drug delivery system by doping them with suitable contrast agents. Several polymer, metallic and ceramic based theranostic agents are being explored for simultaneous application in drug delivery and imaging (Johnson et al., 2014, Luk and Zhang, 2014, Roy et al., 2003, Soica et al, 2018. Depending on the contrast agents being used, various imaging techniques are being explored and studied for diagnostic purposes, including magnetic resonance imaging (MRI), computerized tomography (CT), Fluorescence resonance energy transfer (FRET), photoluminescence imaging (PLI), positron emission tomography (PET) among others. Optical imaging like photoluminescence has different advantages like high resolution, better sensitivity, low cytotoxicity and portable instrumentation (Chen et al., 2020;Zhu et al., 2019) Rare earths (RE) doped biomarkers are well known for their sharp emissions, low cytotoxicity, and broad emission spectrum. The absorption behavior of RE dopants depend on the f-f transitions and is independent of particle size and morphology. Eu +3 , a lanthanide based phosphors exhibits good emission properties originating from the transition between the 5 D 0 to 7 F J (J = 0,1,2,3 and 4) comprising of the electric and magnetic dipole transitions at different wavelengths (Wang and Liu, 2009). The choice of a host material for doping of the rare earth doped is also critical, desired characteristics of host materials include high quantum yield, controlled emission profile, low phonon energy and lattice parameters with dimensions comparable to the dopant (Li, et al., 2009) In a study by Liu and coworkers, the doping of Eu in alpha alumina yields promising photoluminescence results. Photoluminescence properties of the Eu-doped alpha-Al 2 O 3 microspheres. Moreover, PEGylated alumina is also found to be biocompatible (Ali et al., 2019). The doping of Europium in porous alumina spheres yield encouraging photoluminescence properties, that is desired in bioimaging applications, while alumina acts as a drug delivery system. This naturally directs a new spherical Eu-enabled Alumina spheres with inherent water dispersibility and biocompatibility. The study of PEGylated Eu-enabled Alumina sub-micron is a novel study in this regard. Following this theme, a novel idea has been developed to fabricate Eu doped spherical alumina with photoluminescence properties as a potential theranostic agent. The submicron capsules are inherently water dispersible and show photoluminescence characteristics with good biocompatibility.

Synthesis of PEG coated Eu doped submicron sized alumina spheres
Submicron alumina microspheres were prepared by a modified solvothermal synthesis (Saptiama et al., 2018). In a typical experiment, Al(NO 3 ) 3 Á9H 2 O is added in 40 ml of 2-Propanol along with the addition of europium nitrate. After complete formation of solution, 16 ml of analytical grade glycerol was added in the mixture under vigorous stirring. The resultant solution was then placed in an autoclave at a temperature of 180°C for 16 h. The reaction results in the formation of aluminum alkoxide. In the second stage of synthesis europium doped monodispersed glycerated aluminum was partially converted into aluminum oxide hydroxide (Bohemite) by the post synthesis water-ethanol treatment. In a typical procedure 20 mg of europium doped glycerated aluminum was mildly stirred for 48 h in a mixture of 10 ml distilled water and 10 ml ethanol. Finally, mesoporous europium doped alumina was obtained by calcination of the resultant product at a temperature of 700 0 C for 2 h. PEG is then coated on the final product by stirring it in distilled water containing 20 mg of PEG. The synthesis strategy of Eu doped submicron alumina spheres is shown in Fig. 1.

Cytotoxicity evaluation on RD cell lines using MTT assay
The biocompatibility of the prepared PEG coated Eu doped alumina spheres, their solutions in PBS were made at various molarities. These solutions were then incubated in a 24 well plate for 24 h in RD cell lines (0.5 Â 10 6 cells/ml). The cell viability was then studied by reading the absorbance values of MTT assay using microplate reader. This method is in good agreement with previous reported studies (Alam et al., 2020.

Characterization
SEM is done on Mira TESCAN FESEM. The XRD analysis of the alumina samples is performed on Bruker D8 Discover X-ray Diffractometer with Cu-Ka radiation. FTIR analysis was performed by Nicolet 600 FTIR spectrometer operated in transmission mode. The photoluminescence emission spectrum was studied using photoluminescence spectrometer.

Results
The structural and morphological characteristics of s-Al 2 O 3 :Eu are studied by XRD, SEM and FTIR analysis. In order to analyze the phase and crystal structure of the s-Al 2 O 3 :Eu XRD is performed. Fig. 2(a). shows the XRD graph of s-Al 2 O 3 :Eu after calcination at 700 0 C, it is observed that a-alumina and c-alumina phase coexist. FTIR analysis of PEG coated s-Al 2 O 3 :Eu is shown in Fig. 2  (b). Multiple peaks observed in the PEG coated alumina at 956 cm À1 , 840 cm À1 which are absent in the sample without PEG addition validates successful coating of PEG on s-Al 2 O 3 :Eu parti-cles. SEM analysis is done to study the particle size and morphology.EDX analysis as shown in Fig. 3(a). confirms the presence of Al, O and Eu. The SEM images are shown in Fig. 3. Spherical morphology is observed in the SEM micrographs with average particle size of 440 nm. A narrow particle size distribution is observed and SEM images after calcination show compaction of powder.
The photoluminescence emission spectrum of s-Al 2 O 3 :Eu is given in Fig. 4(a). The emission peaks of Eu +3 originates from the transition between 5 D 0 to 7 F J orbitals (J = 0,1,2,3,4) in the range of 560 to 700 nm. Fig. 4(c). shows s-Al 2 O 3 :Eu under UV lamp of wavelength 375 nm, an orange red emission is observed. In order to study the biocompatibility, cytotoxicity evaluation is done on RD cell lines. Fig. 5. (a) % shows cell viability of s-Al 2 O 3 :Eu in PBS at various concentrations in RD cell line.High cell viability is observed with the minimum value being 86%.

Discussion
In order to study the phase transformation at various synthesis steps, it is important to recognize the chemical reaction occurring at the different stages of synthesis.The synthesis of s-Al 2 O 3 :Eu comprised of three steps. First in the solvothermal synthesis, aluminum nitrate converts to aluminum alkoxide after reaction with 2-propanol according to the equation.

Al(NO 3 ) 3 + 3C 3 H 7 OH ! Al(OC 3 H 7 ) 3 + 3HNO 3 ð1Þ
Afterwards, water-ethanol treatment is done which results in simultaneous removal of organic groups by the attack of hydroxyl ions and partial conversion of Al(OR) 3 to AlOOH as given in the following equation It is observed that this partial conversion results in the decrease of crystallization temperature for the formation of a-alumina and c-alumina phase and hence these phases co exists in the XRD pattern of s-Al 2 O 3 :Eu obtained after calcinations at 700 0 C as shown in Fig. 2(a). In order to enhance the biocompatibility of s-Al 2 O 3 :Eu particles, PEG is coated on their surface, PEG enhances the blood circulation time of Eu doped alumina particles and thus aids in passive targeting. To confirm whether PEG is coated on the surface of the particles, chemical bonding analysis using FTIR spectroscopy is done and is shown in Fig. 2(b). The transmittance band in the range of 3250-3690 cm À1 relates to the -OH stretching band. The 2883 cm À1 corresponds to the C-H symmetrical stretching vibration. Transmittance band at 1110 cm À1 is indicative of metal glycerates. However peaks observed in the range of 960 to 800 cm À1 are attributed to the functional groups present in the PEG molecule and hence confirm the successful coating of PEG. Morphology of the Eu doped alumina spheres at various stages of the synthesis techniques are shown by the SEM images given in    3. The solvothermal synthesis yields uniform sized alumina spheres with narrow size distribution. Glycerol acts as a nucleating agent and thus aids in the uniform sized nucleation of the alumina particles by forming a spherical quasi emulsion because of hydrogen bonding. Fig. 3(a). shows perfectly spherical glycerated aluminum particles formed after the solvothermal reaction. The average particle size of alumina spheres after this stage is found to be 440 nm. The later stage of synthesis that comprised of water-ethanol treatment results in enhanced surface roughness of the particles. It is during this stage that the organic groups attached to the surface of glycerated aluminum are removed because of the attack of hydroxyl groups present in the water molecule. The water-ethanol treatment thus results in size reduction which is evident From Fig. 3(b-c). A broken particle is also identified during this stage, it suggests that alumina spheres are not solid and comprises of porosity which is beneficial for high drug loading capacity. Fig. 3(d). shows the SEM images of s-Al 2 O 3 :Eu after calcinations at 700 0 C. Compaction of alumina spheres occurs during this stage. EDX pattern of s-Al 2 O 3 :Eu after calcinations also shows the presence of Eu along with O and Al.
The photoluminescence emission spectrum of s-Al 2 O 3 :Eu is given in Fig. 4(a). An excitation wavelength of 393 nm is used to perform the emission study. The emission peaks of Eu +3 are observedin the range of 560 to 700 nm. The emission peak at 593 nm ( 5 D 0 to 7 F 1 ) corresponds to the magnetic dipole transition whereas the 5 D 0 to 7 F 2 at 613 nm corresponds to the electric dipole transition. The emission peak at 580 nm is ascribed to 5 D 0 to 7 F o transition. According to the parity selection rule, when the Eu 3+ ions are located at the site with an inversion symmetric center, the 5 D 0 ? 7 F 1 magnetic dipole transition is permitted, which results in orange red emitting around 590 nm but if not located in the an inversion symmetric center then the forbidden f-f transitions occurred, resulting in red emission around 613 nm. The former scenario is dominated in our case.
In order for s-Al 2 O 3 :Eu particles to be an efficient theranostic agent, it is very vital to study their biocompatibility. For this purpose cytotoxicity evaluation is done on RD cell lines by making a dispersion of s-Al 2 O 3 :Eu particles in PBS at different concentrations. An incubation time of 24 h is set to study the percentage cell viability using MTT assay. All the concentrations showed excellent cell viability with lowest being 86% at 0.0625 mg/ml ( Fig. 5(a)). Percentage cell viability is reported to decrease as the concentration of particles is increased however in this study the lower concentration showed lower cell viability results than that taken at higher concentrations. This error is seen in the cell viability results at concentration of 0.125 mg/ml and 0.0625 mg/ml being lower than the cell viability at the particle concentration of 0.25 mg/ml. This unusual behavior can be attributed to the localized effect of poorly distributed of s-Al 2 O 3 :Eu in PBS. Due to the inadequate distribution of particles, clusters of particles may have formed in the PBS solution hence the localized concentration being higher than the documented value. It is observed that a cell viability of approximately 110% ± 5.5% is observed at a concentration of 0.03125 mg/ ml. The % cell viability at a particle concentration of 0.03125 mg/ml and 0.0625 mg/ml is 86.22 ± 6.1% and 88.75 ± 7.2% respectively. Similarly a cell viability of nearly 94.92 ± 5.7% is observed with concentration of 0.25 mg/ml of s-Al 2 O 3 :Eu. The high values of percentage cell viability support that the particles are biocompatible and PEG coating enhances the biocompatibility of our theranostic agent. These results are further complemented in Fig. 5(b-e). of cell micrographs showing good cell density after incubation for 24 h.
The high biocompatibility along with good photoluminescence properties make PEGylated Eu-enabled sub-micron alumina spheres a good candidate for a potential theranostics agent. The structural morphological, optical and functional properties have  been thoroughly investigated. The PL emission properties of Eu have been exploited for use in potential diagnostic purpose accompanied by detailed cell viability studies to confirm the biocompatibility. In the nut shell a photoluminescence enabled biocompatible alumina spheres reported here are good candidate as theranostics probe.

Conclusion
This study shows the suitability of s-Al 2 O 3 :Eu as potential theranostic agent. A uniformly distributed Eu doped alumina spheres with an average particle size of 440 nm are successfully synthesized as confirmed by the SEM images. The photoluminescence emission spectrum shows good emission intensities, hence proving the appropriateness of alumina as a suitable host material for Eu doping. Major emission peak is observed at 580 nm. Finally, the cytotoxicity evaluations reveal that s-Al 2 O 3 :Eu particles are highly biocompatible even at higher concentration of 0.25 mg/ml. These results advocate the potential of PEGylated photoluminescence alumina sub microspheres as a potential theranostic agent.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.