Preparation , characterization , cytotoxicity and biological evaluation of 99 mTc-Doxorubicin-Epigallocatechingallate functionalized gold nanoparticles as a new generation of theranostic radiopharmaceutical

Gold nanoparticles are currently used for the treatment of cancer through a myriad of modalities and delivery approaches. Conjugation of tumor imaging Single Photon Emitting Computed Tomographic (SPECT) radiopharmaceutical to gold nanoparticles will allow systemic targeting and imaging of cancer tissues simultaneously. In this study, gold nanoparticles (AuNPs) were prepared using Epigallocatechingallate (EGCG), loaded with doxorubicin (Dox), and characterized before and after doxorubicin conjugation. Cytotoxicity of EGCG-AuNPs and Dox-EGCG-AuNPs were evaluated against breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG-2) cell lines demonstrating high cytotoxic effects of Dox-EGCG-AuNPs against both cell lines. Doxorubicin was radiolabeled with 99mTc and our new approach has optimized various labeling conditions resulting in a radiochemical yield of 93.5 ± 2.04%. Biodistribution of 99mTc-Dox-EGCG-AuNPs was studied in normal and tumor bearing mice following I.V. and intratumoral injections at different time intervals. Results showed high uptake of the intravenously injected 99mTc-Dox-EGCG-AuNPs in tumor tissue (22.45 %ID/g at 2h). In addition, localized intratumoral injection of 99mTc-Dox-EGCG-AuNPs showed extremely high levels of uptake in tumor (80.22 %ID/g at 15min) with high retention for extended periods post injection. Our results present prospects for the utility of 99mTc-Dox-EGCG-AuNPs as a multiplexed theranostic agent through SPECT imaging of tumor tissue and therapy through photothermal destruction of cancer tissue through the application of exogenous laser lights as well as through tyrosine phosphatases inhibitor (through EGCG), and topoisomerase II inhibitor (through doxorubicin) effects.


Introduction
Design and development of functionalized nanoparticles continues to be a burgeoning area of unparalleled growth because of continued applications within the health care, materials science, energy and environmental sectors [1][2][3][4].Although a variety of nanoparticles encompass myriad of applications, certain types of nanoparticles with dual diagnostics and therapeutics, referred to as 'theranostics', have gained particular prominence in molecular imaging and therapy of cancers [1,[5][6][7].Gold nanoparticles (AuNPs) for example are ideal candidates for therapeutic applications due to their inherent properties to emit heat upon irradiation with lasers/X-rays.The large surface area of gold nanoparticles allows incorporation of therapeutic agents thus making functionalized AuNPs as multiplexed therapeutic agents [8][9][10][11].Katti et.al. have shown the application of AuNPs as X-ray contrast agents thus opening up new opportunities in the utility of functionalized AuNPs as theranostic agents [5,12].Katti et al have also demonstrated the development of radioactive gold nanoparticles (Au-198) with beta and gamma emission properties for theranostic applications in molecular imaging and therapy of cancers [1].Recent studies have also shown that gold nanoparticles functionalized with tumor receptor specific peptides or receptor-avid small biomolecules, when injected directly into tumors, are retained within the tumor in significant amounts.Such intratumoral delivery approaches would be ideally suited for treating solid tumors (prostate, hepatic, glioblastoma, etc) because of the potential to reduce damage to healthy cells [13,14].Nanotechnology appears to offer new avenues for the development of intratumorally injectable paradigm thus overcoming existing challenges of drug delivery, which suffer from difficulties of optimal uptake of drugs at the tumor site due to various physiological barriers.
Recent studies have shown that gold nanoparticles (AuNPs) are considered a clear choice in intratumroal drug delivery due to their ability to be functionalized with a variety of tumor-avid biomolecules, ideal hydrodynamic size for penetration across tumor cell membranes, and efficient matrix/receptors mediated active and passive internalization [9,15,16].Recent studies on the efficacy of functionalized gold nanoparticles in treating various cancers provide illustrious examples about the realistic scope of gold-based nanomedicine in tumor therapy [17][18][19][20][21]. Certain phytochemicals such as Epigallocatechingallate (EGCG) from tea are electron rich.Katti et.al. have demonstrated the extraordinary electron injection capability of EGCG to transform gold into gold nanoparticles, through innovative green nanotechnology, with concomitant encapsulation of tumor-avid phytochemical on the surface [22][23][24][25][26].
Highly reactive and the vast surface area of gold nanoparticles allows effective conjugations with the FDA approved chemotherapeutic agents including Doxorubicin (DOX) [27][28][29][30][31][32].Doxorubicin is a chemotherapeutic agent being currently used for treating multiple types of cancers including breast cancer, bladder cancer, ovarian cancer, lymphoma, and acute lymphocytic leukemia.Common side effects of doxorubicin include cardiotoxicity, hair loss, bone marrow suppression, vomiting, rash, and inflammation of the mouth [33,34].An important consideration is that conjugating doxorubicin with gold nanoparticles might reduce its side effects due to passive targeting of cancer tissue aided through enhanced permeability and retention effects (EPR) [35,36].In addition, attaching doxorubicin to gold nanoparticles provides a dual mode of treatment of cancer involving both the chemotherapeutic effects of doxorubicin as well as thermally-mediated cancer cell killing destructive effects of gold nanoparticles [37,38].Gold nanoparticulates functionalized with doxorubicin would also present attractive prospects for labeling with Single Photon Emitting Computed Tomographic (SPECT) radioisotopes such as 99m Tc. 99m Tc radiopharmaceuticals provide excellent SPECT images of tumors/lesions [39][40][41].Therefore, multiplexing of doxorubicin-loaded gold nanoparticles and subsequent labeling of such therapeutic conjugates with 99m Tc opens up new avenues in the design and development of theranostic agents with applications in oncology [42][43][44][45][46]. We, herein, report the development of gold nanoparticles functionalized doxorubicin and its subsequent labeling with 99m Tc to produce a new generation of a theranostic nano-radiopharmaceutical ( 99m Tc-Dox-EGCG-AuNPs).We also report full details of biodistribution of 99m Tc-Dox-EGCG-AuNPs in normal and tumor bearing mice following I.V and intratumoral injection at different time intervals.Our results provide new directions in oncology for the utility of 99m Tc-Dox-EGCG-AuNPs as a theranostic agent through SPECT imaging of tumor tissue and therapy through the conjugated Doxorubicin as well as through the photothermal destruction of cancer tissue through the application of exogenous laser lights.

Preparation of gold nanoparticles (EGCG-AuNPs)
EGCG-AuNPs were prepared following a protocol we have developed in our laboratory [20,58].This preparation was optimized by mixing 300 µL of EGCG solution with 60 µl NaAuCl 4 .2H 2 O solution until the formation of ruby red color indicating the formation of gold particles in the optimum nano size.Finally the prepared EGCG-AuNPs solution was diluted to 2 ml using physiological solution.

Preparation of EGCG-AuNPs loaded with doxorubicin
DOX is adsorbed onto EGCG-AuNPs mainly through electrostatic interactions between the positively charged amine group of DOX and the negatively charged polyphenolics groups of EGCG, used in the synthesis as a reducing and stabilizing agents [59,60].The hydrophobic forces drive the doxorubicin molecule to the surface of the EGCG-AuNPs which has a strong corona of negatively charged polyphenols of EGCG to produce a robust and stable electrostatic interaction between DOX-EGCG-AuNPs (Figure 1) [61].Several key features must be considered for nanoscale-drug conjugates to be successful in animal-based models.First, the size of the nanoparticle complex must remain in nano range of 10-100 nm for effective penetration of 99m Tc-DOX-EGCG-AuNPs mediated by EPR effects.In addition, the nanoparticle must be stable in the high ionic strength environments present invivo.Secondly, controlled release of the drug at the target site must be guaranteed.The effective adsorption and electrostatic interaction of DOX with EGCG corona onto gold nanoparticles appear to provide ideal size, stability for the controlled release of DOX at tumor sites.Upon adsorption of high concentration of DOX onto EGCG corona conjugated to EGCG-AuNPs, the overall in-vitro and the invivo stability is enhanced with limited or no aggregation.
Overall, we have succeeded in loading EGCG-AuNPs with 0.4 ml of doxorubicin solution, which is equivalent to 0.4 mg doxorubicin, without compromising its stability.

Characterization of EGCG-AuNPs and Dox-EGCG-AuNPs
Figure 2 shows the DLS, TEM and IR charts of EGCG-AuNPs and Dox-EGCG-AuNPs.The EGCG-AuNPs nanoparticles, as measured through dynamic light scattering (DLS) experiments, showed a mean diameter of 21.4±1 nm which is within the optimum nano-size allowing ready diffusion across the cancer leaky cancer cell vessels with minimal hindrance [62,63].Furthermore, the DLS analysis of the Dox-EGCG-AuNPs showed mean diameter of 58.9±2 nm indicating a significant increase in particle size suggesting Dox loading onto the EGCG-AuNPs.It is important to recognize that the overall size of Dox-EGCG-AuNPsis still within the optimum range, allowing efficient penetration, through receptor-mediated endocytosis as well as through EPR effects, to provide optimum doses of theranostic drugs to tumor cells.
TEM (transmission electron microscopy) analysis is a useful tool for examining the morphological characteristics of nanoparticles. Figure 2 showed non-aggregated EGCG-AuNPs and Dox-EGCG-AuNPs with a predominant spherical shape and narrow size distribution, besides; electronic microscopy clearly showed that the diameter of the particles observed through TEM measurements were in harmony with those measured through DLS analysis (Figure 2).
The IR chart revealed that similarity between EGCG-AuNPs and Dox-EGCG-AuNPs preparations.

Radiolabeling of doxorubicin using 99m Tc
Optimum radiochemical yield of 93.5 ± 2.04% was obtained using 1 mg doxorubicin and 100 µg stannous chloride at 15 min reaction time, room temperature and pH 7 (Figure 4).99m Tc-doxorubicin showed good in-vitro stability up to 6 h.

In-vitro stability of 99m Tc-Dox-EGCG-AuNPs
In vitro stability evaluations of 99m Tc-Dox-EGCG-AuNPs were carried out in saline and rat serum.99m Tc-Dox-EGCG-AuNPs showed excellent in-vitro stability in saline and in rat serum for 3 days.Radiochemical yields of 99m Tc-Dox-EGCG-AuNPs remained unchanged over three days period.The optimum in vitro stability of 99m Tc-Dox-EGCG-AuNPs provides compelling rationale for a detailed in vivo investigations of this theranostic agent in tumor bearing mice.

Biodistribution study of 99m Tc-Dox-EGCG-AuNPs
Results of biodistribution studies revealed substantial accumulation of 99m Tc-Dox-EGCG-AuNPs in tumor tissue (22.45 %ID/g at 2h) (Figure 5).This targeting efficacy selectively to tumor sites is attributed to active targeting enhanced by doxorubicin as well as gold nanoparticles-mediated passive targeting due to enhanced permeability and retention (EPR) effects [64][65][66][67].On the other hand, localized intratumoral injections of 99m Tc-Dox-EGCG-AuNPs showed extremely high level of radioactivity uptake in tumor tissue (80.22 %ID/g at 15min) and remained at that level for 2 hr post injection.These promising tumor targeting and retention data indicate the usefulness of 99m Tc-Dox-EGCG-AuNPs as a theranostic agent (Figure 5 and 6).The 99m Tc SPECT probe in 99m Tc-Dox-EGCG-AuNPs molecular imaging of lesions particularly for early diagnosis of tumors as well as for follow up in monitoring therapeutic response in cancer patients [68][69][70].Besides, our overall approach has multiplexed dimension due to the presence of gold nanoparticles which allow their use for photothermal destruction of cancer tissue when exogenous laser lights are applied [71,72].

Synergistic multiplexing therapeutic and imaging of 99m Tc-Dox-EGCG-AuNPs:
The ability of doxorubicin to exert its antitumor effects via inhibition of topoisomerase II continues to attract significant clinical interest for its applications as the most important anticancer drug in the clinic [28].Unfortunately, its potent antitumor activity is also accompanied by severe systemic short and long-term tissue toxicities including cardiotoxicity.In addition, shorter intracellular retention time, as noted in a number of clinical investigations of doxorubicin, has been the main vexing medical problem in exploiting the full clinical potential of this class of anthracycline compounds in tumor therapy.Limited tumor retention is often overcome by repeated doses causing dose-limiting side effects in the form of cardiotoxicity, leading to heart failure in the most severe cases.
Extensive preclinical studies have demonstrated that chronic administration of doxorubicin would result in a severe DRG (dorsal root ganglia) neuronopathy [33,34].Therefore, current and future clinical applications of doxorubicin, as a first line cancer therapy drug, requires development of delivery technologies that would not only result in selective delivery at tumor sites but also enhance retention of therapeutic doses with minimal/no systemic side effects.Biodistribution and tumor retention results of 99m Tc-Dox-EGCG-AuNPs provide conclusive evidence that conjugation of doxorubicin with tumor targeting EGCG provides selective delivery at the same time enhance the retention of optimal therapeutic doses of this drug (Figures 5 and 6).The gold nanoparticles in 99m Tc-Dox-EGCG-AuNPs, to which doxorubicin is tagged with, also provide synergistic advantages of both tumor targeting and retention of doxorubicin because of the well-known affinity of gold nanoparticulate surface to the leaky vasculature found on tumor cells.The selective affinity of EGCG to tumor cell receptors also provides selective affinity of the 99m Tc SPECT probe of 99m Tc-Dox-EGCG-AuNPs, thus providing for the first time, a functional doxorubicin-gold nanoparticulate-based SPECT theranostics agent for dual imaging and therapy applications in the treatment of a myriad of cancers.The well-established chemotherapeutic effects of EGCG are also put to advantage in the theranostics action of 99m Tc-Dox-EGCG-AuNPs It is well known that receptor tyrosine kinases (RTKs) are the tumor proliferation machinery as they are directly involved in cell proliferation, survival and angiogenesis.The fact that EGCG regulates activities of cell surface growth factor receptors, especially receptor tyrosine kinases (RTK), including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor receptor (IGFR), and the insulin receptor (InsR) [73][74][75][76] individually and collectively present unprecedented opportunities in the application of 99m Tc-Dox-EGCG-AuNPs agent as a multiplex tyrosine phosphatases inhibitor (through EGCG), and topoisomerase II inhibitor (through doxorubicin) [28].

Materials
Epigallocatechingallate (EGCG), Sodium tetrachloroaurate dehydrate and Doxorubicin are purchased from Sigma-Aldrich Company, USA.All other chemicals and solutions were of analytical grade and purchased from Merck Co. Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza, Whatman No.1 paper chromatography, Whatman International Ltd, Maidstone, Kent, UK. 99 Mo/ 99m Tc generator is purchased from Egyptian Atomic Energy Authority as a source of 99m TcO 4 -.

Cell lines
Mammalian cell lines: MCF-7 cell line (human breast cancer) and HepG-2cell line ((human hepatocellular carcinoma) were purchased from the American Type Culture Collection (ATCC, Rockville, MD).

Animals
Male Swiss albino mice (20-40 gm) were used for the biodistribution studies that confirmed to the ethical guidelines for animal care set by the EAEA.Ehrlich Ascites Carcinoma tumor line solution (12.5x10 6 cells/mL) was injected intramuscularly (0.2 mL) in the right thigh to produce a solid tumor.It took about 10-15 days for the solid tumor to be apparent in the right thigh muscle [47].

Preparation of gold nanoparticles (EGCG-AuNPs)
Two separate solutions were prepared by dissolving 10mg of EGCG in10 mL of absolute ethanol (Solution A) dissolving 10 mg of sodium tetrachloroaurate dehydrate in 10mL of double-distilled water (solution B).We choose EGCG due to its dual action as a reducing and also as a stabilizing agent in addition to its inherent property as an anticancer agent [23,[48][49][50].Gold nanoparticles were prepared by drop wise addition of different volumes of solution B to different volumes of solution A in a 10 mL vial placed on a magnetic hot plate with stirring for about 15 min until a ruby red color appeared.The EGCG-AuNPs preparation was optimized after studying the effect of EGCG amount, gold amount, reaction time and reaction temperature factors.Then the prepared EGCG-AuNPs solution was diluted with physiological solution.

Preparation of EGCG-AuNPs loaded with doxorubicin (Dox-EGCG-AuNPs)
After the formation of EGCG-AuNPs, using different amounts of doxorubicin solution in H 2 O (1 mg/mL) was added with magnetic stirring to evaluate the maximum amount of doxorubicin that can be loaded on EGCG-AuNPs.

In-vitro cytotoxicity study of gold nanoparticles and doxorubicin-gold nanoparticles cell line propagation
Medium of RPMI-1640 supplemented with 10% inactivated fetal calf serum and 50µg/mL gentamycin was used for cells growth.In a humidified atmosphere, cells were maintained at 37ºC and 5% CO2 and subculturing was done two to three times a week.

Cytotoxicity evaluation using viability assay
For antitumor assays, the tumor cell lines were suspended at concentration 5x10 4 cells/well in medium of Corning® 96-well tissue culture plates and incubated for 24 hr.The compounds to be examined were added into 96-well plates (three replicates) to achieve eight dilutions for each compound.The numbers of viable cells after 24 h incubation were determined by the MTT test.Media was removed from the 96 well plates and replaced with 100 µL of fresh culture RPMI 1640 medium without phenol red then 10 µL of 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) added to each well including the untreated controls.The 96 well plates were then incubated at 37°C and 5% CO2 for 4 hours.Then, 85 µl aliquot of the media was removed from the wells, and 50 µL of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37°C for 10 min.
Then, the optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [1-(ODt/ODc)]x100% where ODt is the mean optical density of wells treated with the sample and ODc is the mean optical density of untreated cells.The surviving cells percentage was plotted versus drug concentration to get the survival curve for each compound.The 50% inhibitory concentration (IC50) was estimated from graphic plots of the dose response curve for each concentration using Graphpad Prism software (San Diego, CA.USA) [51,52].

Radiolabeling of doxorubicin using 99m Tc
Radiolabeling of doxorubicin with 99m Tc was done by direct technique under reductive conditions using SnCl2.2H2O[53].Labeling reaction was done by adding 1 mL of doxorubicin solution in DMSO (0.5-2 mg doxorubicin) in an evacuated 10 mL penicillin vial.Then, 7.2 MBq of 99m TcO 4 -(100 µL of generator eluate) was added to each reaction.Initiation of the reaction was achieved by adding 0.5 ml stannous chloride solution (20-150 μg SnCl 2 .2H 2 O).Effect of reaction pH on radiochemical yield (RCY) was studied in range of 4-9 using different amounts of 0.1N sodium hydroxide or 0.1N hydrochloric acid solutions.Reaction time effect on radiolabeling yield was also studied (5-60 min).Ascending paper chromatography was used for analysis of RCY using acetone as a mobile phase to determine free 99m TcO 4 -% and water:ethanol:ammonia mixture (5:2:1 v:v:v) to determine colloidal impurities percentage [54,55].

Preparation of EGCG-AuNPs loaded with 99m Tc-doxorubicin ( 99m Tc-Dox-EGCG-AuNPs)
Various stoichiometric amounts of 99m Tc-doxorubicin and EGCG-AuNPs were mixed in order to arrive at an optimized preparation of 99m Tc-Dox-EGCG-AuNPs.The highest loading of Doxorubicin onto 99m Tc-Dox-EGCG-AuNPs was achieved by mixing 0.4 mL of the radiolabeled 99m Tc-doxorubicin solution with 1 mL of EGCG-AuNPs solution with magnetic stirring for 20 min.

In-vitro stability of 99m Tc-Dox-EGCG-AuNPs
Before going through the in-vivo evaluations, the in-vitro stability of the 99m Tc-Dox-EGCG-AuNPs was conducted in saline and rat serum to confirm its suitability for in-vivo injections.99m Tc-Dox-EGCG-AuNPs was examined for in-vitro stability in saline and rat serum by mixing 0.1 mL of each preparation with 0.9 mL of saline or rat serum for 1 week.

Biodistribution study of 99m Tc-Dox-EGCG-AuNPs
Mice were classified into three groups (9 mice/group) A, B and C. Line of Ehrlich Ascites Carcinoma was used to induce solid tumor in mice of groups B and C while group A is considered as normal group.The parent tumor line was withdrawn from 7 days old donor female Swiss Albino mice and diluted with sterile physiological saline solution to give 12.5 X 10 6 cells/ml.Exactly 0.2 mL solution was then injected intramuscularly in the right thigh muscle to produce a solid tumor.The animals were maintained till the tumor development was apparent (10-15 day) [47].
On the experiment day, nearly 6.2 MBq of 99m Tc-Dox-EGCG-AuNPs (150 μl) were injected intravenously in group A mice, intravenously in group B mice and finally intra tumor injection in group C mice at 15, 60 and 120 minutes (three mice/time interval).Each mouse was weighed, anaesthetized by chloroform, euthanized and then dissected.Different body organs were separated, weighed and rinsed with saline.Uptake of radioactivity in each organ was measured using a gamma counter and expressed as percent injected dose per gram (% ID/gram ±

Figure 6 :
Figure 6: Tumor uptake of 99m Tc-Dox-EGCG-AuNPs in tumor bearing mice following I.V. and intratumor injection at different time intervals