Facile One-Pot Synthesis of Intrinsically Radiolabeled and Cyclic RGD

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Applied Chemistry

Facile One-Pot Synthesis of Intrinsically Radiolabeled and Cyclic RGD Conjugated 199Au Nanoparticles for Potential Use in Nanoscale Brachytherapy Rubel Chakravarty, Sudipta Chakraborty, Apurav Guleria, Rakesh Shukla, Chandan Kumar, K.V. Vimalnath Nair, Haladhar Dev Sarma, Avesh Kumar Tyagi, and Ashutosh Dash Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02526 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

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Industrial & Engineering Chemistry Research

Facile One-Pot Synthesis of Intrinsically Radiolabeled and Cyclic RGD Conjugated 199

Au Nanoparticles for Potential Use in Nanoscale Brachytherapy

Rubel Chakravarty,1,5,* Sudipta Chakraborty,1,5 Apurav Guleria,2 Rakesh Shukla,3 Chandan Kumar,1 K.V. Vimalnath Nair,1 Haladhar Dev Sarma,4 Avesh Kumar Tyagi3,5 and Ashutosh Dash1,5 1

Radiopharmaceuticals Division, 2Radiation and Photochemistry Division, 3Chemistry

Division, 4Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. 5

Homi Bhabha National Institute, Anushaktinagar, Mumbai 400 094, India.

*To whom all correspondences must be addressed: Rubel Chakravarty, Ph.D. E-mail: [email protected], [email protected] Phone: +91-22-25590624 Fax: +91-22-25505151

1

ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Synthesis of intrinsically radiolabeled nanoparticles has shown tremendous prospective in offering an easier, faster, stable, and more specific radiolabeling technique for development of advanced radionanomedicine agents for cancer treatment. In this study, a facile one-pot synthesis protocol for preparation of cyclic arginine-glycine-aspartate (RGD) conjugated and intrinsically radiolabeled

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Au (t½ = 3.14 d; β1 = 462 keV, 6.0%; β2 = 296

keV, 71.6%; β3 = 250 keV, 22.4%; γ = 159 keV, 37 %) nanoparticles has been developed targeting integrin αvβ3 receptors for potential use in neoadjuvant brachytherapy. The nanoparticles synthesized by this method were characterized by numerous analytical methods to determine the identity, particle size, in vitro stability, biocompatibility and amenability for clinical use. Large-scale synthesis of intrinsically radiolabeled

199

Au nanoparticles could be

achieved with excellent yield and it met all the requirements for clinical use. The biological efficacy of the intrinsically radiolabeled 199Au nanoparticles was confirmed by biodistribution studies in C57BL/6 mice having melanoma tumors after intratumoral injection. The results of the biodistribution studies demonstrated high tumor radioactivity concentration of integrin αvβ3 targeted

199

Au nanoparticles which reduced only marginally over the period of 1 week.

Intratumoral administration of 5 MBq and 10 MBq doses of radiolabeled nanoparticles resulted in significant regression of tumor growth in melanoma tumor bearing mice. Apparent body weight loss was not observed in all the treated mice. Based on the encouraging results acquired in this study, it is envisaged that this approach would be useful toward clinical translation of this innovative class of radionanomedicine agents for neoadjuvant brachytherapy of selected cancer patients.

Keywords:

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Au, cancer, integrin αvβ3, intrinsically radiolabeled nanoparticles, nanoscale

brachytherapy, RGD peptide 2


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INTRODUCTION Clinical brachytherapy using low dose rate permanent radioactive source implantation is an established treatment option for select cancer patients. 1-3 The benefits of this treatment modality include favourable scheduling logistics, low initial capital equipment costs, no need for a shielded room, completion in a single implant, and the possibility of obtaining robust data from clinical trials. 4 However, the conventional brachytherapy sources are typically of few millimetre sizes and therefore this technique often suffers from heterogeneous distribution in the tumor resulting in sub optimal therapeutic efficacy. 5 With the advances in nanotechnology, nanosized radiation sources for brachytherapy have recently been introduced which demonstrated larger therapeutic indices compared to the conventional sources and this new advanced therapeutic modality has come to be known as ‘nanoscale brachytherapy’ 5, 6 The integral component of nanoscale brachytherapy involves design of functionalized nanoplatforms and development of suitable methodologies for radiolabeling of the same. In this regard, synthesis of intrinsically radiolabeled inorganic nanoparticles is an emerging concept that has shown attractive potential to offer facile, rapid and more specific radiolabeling possibilities for the development of new generation radionanomedicine agents. 7-10

This novel approach overcomes inherent limitations of the classical radiolabeling strategy

involving use of exogenous chelators that could coordinate with specific radioisotopes to form complexes.

7-9

In addition to providing enhanced in vitro and in vivo stabilities,

intrinsically radiolabeled nanoparticles would potentially retain the native biodistribution and pharmacokinetics of the nanomaterial, thereby accurately reflecting its real in vivo behavior in living subjects.

7

Moreover, this approach is easily executable in a hot cell facility under

current good manufacturing practices (cGMP) compliant conditions, thus exhibiting tremendous prospective for future clinical translation. Despite excellent attributes, this radiolabeling approach has hardly been explored for preparation of radioactive sources for 3



nanoscale brachytherapy mainly due to unavailability of suitable protocols for synthesis of clinically relevant doses of intrinsically radiolabeled nanoparticles. With the rapidly growing interest in using radioisotopes for nanooncology, a broad spectrum of radioactive sources involving nanomaterials has been developed in the recent times. 11-15 Gold-199 is a relatively new radioisotope whose potential is yet to be explored for preparation of nanoscale brachytherapy sources. added (NCA) form by neutron activation of separation.

16

16

Gold-199 can be obtained in no-carrier-

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Pt target followed by radiochemical

Owing to complex coordination chemistry of Au, it cannot be utilized for

radiolabeling nanoplatforms by standard coordination chemistry based approaches. Therefore, synthesis of intrinsically radiolabeled nanoparticles is the most viable option towards effective utilization of this excellent radioisotope for clinical benefits. Herein, it is reported for the first time a facile method for large scale synthesis of integrin αvβ3 targeted and intrinsically radiolabeled

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Au nanoparticles for potential

application in nanoscale brachytherapy. The procedure for large-scale synthesis of intrinsically radiolabeled

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Au nanoparticles could be straightforwardly performed in a

shielded facility operated with tongs. Different analytical methods were used for structural characterization of the synthesized Au nanoparticles. The usability of the intrinsically radiolabeled nanoparticles as nanoscale brachytherapy agent was demonstrated in C57BL/6 mice having melanoma tumors after intratumoral injection.

EXPERIMENTAL SECTION Materials Platinum metal sponge (natural isotopic composition, Specpure grade) used as target for neutron irradiation in the reactor was procured from Roviur International, Singapore. Hydrochloric acid, ammonium hydroxide, ethyl acetate, HAuCl4.3H2O and other reagents 4


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and chemicals were of analytical grade and procured Sigma-Aldrich, India. Cyclic (arginineglycine-aspartate-phenylalanine-lysine) [c(RGDfK)] peptide was procured from ABX Advanced Biochemical Compounds, Germany. PD-10 desalting columns were procured from G.E. Healthcare, Germany. Flexible silica plates (coating thickness = 0.25 mm), from J.T. Baker Chemical Company, United States were used for thin layer chromatography (TLC) studies. Synthesis and characterization of Au nanoparticles In the present study, integrin αvβ3 targeted Au nanoparticles conjugated with cyclic (arginine-glycine-aspartate-phenylalanine-lysine) [c(RGDfK)] peptide (Figure S1) was synthesized adopting a method similar to that reported by Yin et al.

17

For this purpose,

aqueous solution of Au ions (as HAuCl4; 1 mM, 1 mL) was added dropwise into the aqueous solution of the peptide (5.0 mM, 1 mL) under continuous magnetic stirring. The pH of the resultant solution was adjusted to ~ 10 with 1 M NaOH. The reaction was continued for about 30 min at 100 °C in a water bath (Figure 1). The color of the reaction mixture changed from light yellow to wine red, indicating the formation of Au nanoparticles. As-synthesized, Auc(RGDfK) nanoparticles were purified from free Au(III) ions by ultrafiltration using 10 kDa centrifugal filter tube (Merck). Adopting the same procedure, non-targeted Au nanoparticles conjugated with a similar peptide with scrambled RGD sequence [cyclic (arginine-glycinelysine-phenylalanine aspartic acid [c(RGKfD)]] was also synthesized for control studies (Figure S1). UV-visible absorption spectra of Au-c(RGDfK) nanoparticles were recorded on a Thermo-Fisher UV-1800 spectrophotometer. Circular dichroism (CD) measurements were done on a MOS-450 circular dichroism spectrometer using a 0.01 cm quartz cell. The infrared spectra were recorded in the range 400–4000 cm−1 on a Fourier transform infrared spectrometer (FTIR, Bomen Hartman and Braun, MB series). The identity and crystallinity of 5



the Au-c(RGDfK) nanoparticles were confirmed by XRD measurements (Phillips PW1710 diffractometer with Cu Kα radiation). For transmission electron microscopy (TEM) studies, a drop of Au nanoparticles was carefully put onto ultrathin carbon-coated copper grids and dried. The TEM image was recorded using a JEM-2100 transmission electron microscope. The hydrodynamic diameter of Au-c(RGDfK) nanoparticles was determined by dynamic light scattering (DLS) measurements (Malvern 4800 Autosizer employing a 7132 digital correlator). The zeta potential of Au-c(RGDfK) nanoparticles was determined using a zetasizer (Zetasizer nano series, Malvern Instruments). The stability of Au-c(RGDfK) nanoparticles preserved at 4 °C was observed up to a period of 6 months by visual inspection. Also, the stability of Au-c(RGDfK) nanoparticles was investigated by incubating the solution in 20-times excess of PBS or mouse serum (v/v) at 25 °C and determining the hydrodynamic diameters by DLS measurements over a period of 7 days. In vitro toxicity study of Au-c(RGDfK) nanoparticles The in vitro cytotoxicity of Au-c(RGDfK) nanoparticles was assessed by a standard 3[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in cells (CHO, Chinese Hamster Ovary epithelial cells; MCF-7, human breast adenocarcinoma cells and B16F10, murine melanoma cells). Firstly, cells were seeded in 96-well plates at (5 × 103) cells per well and then cultured at 37 ºC and 5 % CO2 for 24 h. Different concentrations of Au-c(RGDfK) nanoparticles (0-2 mg/mL, diluted in RPMI Medium 1640) were added to the wells. The cells were incubated for 48 h at 37 ºC under 5 % CO2. Subsequently, MTT (100 µL, 5 mg/mL) was added to each well, and incubated for another 4 h under 5 % CO2. Dimethyl sulfoxide (100 µL / well) was then added, and the optical density at 490 nm of each well was recorded.

6


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Synthesis of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles No-carrier-added (NCA) 199Au was produced through neutron activation of natural Pt target in Dhruva reactor at Bhabha Atomic Research Centre, Mumbai followed by radiochemical separation of

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Au employing solvent extraction technique.

regarding production and radiochemical separation of

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The details

Au are provided in the Supporting

Information. Synthesis of intrinsically radiolabeled and integrin αvβ3

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Au-c(RGDfK)

nanoparticles was carried out in a hot cell equipped with remotely operable tongs. The synthesis procedure was essentially the same as reported above for synthesis of nonradioactive nanoparticles, except that

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Au solution was added to the non-radioactive Au

solution prior to synthesis. After the radiosynthesis procedure, the radiolabeled formulation was transferred to a 10 kDa centrifugal filter tube and centrifuged at 5000 rotations per minute (rpm) for 15 min in order to remove unreacted peptide and Au(III) ions. This procedure was repeated once and

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Au-c(RGDfK) nanoparticles left on the membrane was

re-dispersed in 5 mL of phosphate buffered saline (PBS) solution. Subsequently,

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Au-

c(RGDfK) nanoparticle solution was passed through 0.22 µm filter, collected in a sterile vial. The radionuclidic purity, radiochemical purity, sterility and apyrogenicity of intrinsically radiolabeled

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Au-c(RGDfK) nanoparticles were analyzed by standard techniques as

described in the Supporting Information. The same protocol as described above for synthesis of integrin αvβ3 targeted

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Au-c(RGDfK) nanoparticles was also used for synthesis of non-

targeted 199Au-c(RGKfD) nanoparticles for control studies. The radiochemical stability of the radiolabeled nanoparticles was determined in vitro in PBS and mouse serum media by radio-TLC assay. For this, tracer level of radiolabeled nanoformulation was added to excess volume of PBS or mouse serum and incubated at 37°C for up to 7 d. The radio-TLC pattern was developed using saline in 0.02 M HCl as the eluting medium. The stability of radiolabeled nanoparticles in PBS or mouse serum (at 37 ◦C) media 7



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over a prolonged period of time was analyzed by radio-thin layer chromatography (radioTLC) assays at different time intervals. As a control, tracer level of 199Au-HAuCl4 was added to excess volume of PBS or mouse serum and radio-TLC pattern was developed under the same conditions. In vitro cell binding and inhibition studies Cell binding assay was performed in triplicate using melanoma cells, which are known to overexpress integrin αvβ3 receptors. 18, 19 In a typical experiment, 2 × 107 cells were used to study the binding of 198Au-RGD nanoparticles. Different amounts of 199Au-c(RGDfK) nanoparticles (KBq) was added in cells and incubated with or without excess of c(RGDfK) for 1 h. Cells were washed twice with ice cold buffer. Radioactivity associated with the cell pellets and corresponding total radioactivity were measured in NaI(Tl) gamma counter and percent binding and inhibition were calculated. Biodistribution studies All animal studies were conducted following the protocol approved by the institutional animal ethics committee. Biodistribution studies were performed in C57BL/6 mice having melanoma tumors after intratumoral injection of the radiolabeled

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Au-

c(RGDfK) nanoparticles, as per the details provided in the Supporting Information. At 24, 72, and 192 h post-injection (p.i.), the mice were sacrificed and the tumor and samples of normal tissues including blood were collected. The radioactivity in each organ and tissue was measured in a flat-type NaI (Tl) counter and expressed as percentage injected dose per gram (%ID/g). As a control, non-targeted

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Au-c(RGfKD) nanoparticles were also intratumorally

administered in another set of C57BL/6 mice bearing melanoma tumors and biodistribution studies were carried out.

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Treatment studies Tumor therapy studies were performed in C57BL/6 mice having melanoma tumors. The studies started when the tumor volume reached about 150 mm3 (~ 10 days after inoculation of melanoma cells). Mice were randomly divided into 5 groups (5 mice per group) to receive single intratumoral injection of saline (control), Au-c(RGDfK) nanoparticles (control), 2 MBq

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Au-c(RGDfK) nanoparticles, 5 MBq

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Au-c(RGDfK)

nanoparticles, or 10 MBq 199Au-c(RGDfK) nanoparticles. Tumor volume and body weight of the mice were monitored for 15 days. Tumor volume was calculated by (length × width2) /2. 5, 20

The tumor growth index (TGI) for treated and control mice was calculated by dividing

the tumor volume at each time point by the initial tumor volume prior to treatment. Similarly, body weight index (BWI) was calculated by dividing the body weight at each time point by the initial body weight. The mean TGI and BWI were plotted versus the time post-treatment.

RESULTS Synthesis and characterization of Au nanoparticles A single step synthesis of Au-c(RGDfK) nanoparticles was achieved using c(RGDfK) peptide which played the role of both reducing as well as stabilizing agent (Figure 1A). The UV–visible absorption spectra showed the existence of the surface plasmon resonance (SPR) band (with maxima at 535 nm), confirming the formation of Au nanoparticles (Figure 1B).21 In the CD spectroscopy, the c(RGDfK) peptide displayed characteristic CD responses in the UV range with negative ellipticity peak at 220 nm (Figure 1C), and the absence of CD responses in the visible range. Unfunctionalized Au nanoparticles do not exhibit any chiroptical features and are considered achiral.

22

Interestingly, in the Au-c(RGDfK)

nanoparticles, a moderate and reproducible CD response was observed in the visible region of the spectrum. The negative ellipticity of the Au-c(RGDfK) nanoparticle complex in the 9



visible range correspond to the surface plasmon resonance of the Au nanoparticle (~ 535 nm), that is presumably induced by the dipole of the peptide interacting with the plasmon resonance of the Au nanoparticles. 22 Also observed were minor changes in the CD spectra in UV region between c(RGDfK) peptide and Au-c(RGDfK) nanoparticles (Figure 1C). In order to gain further insight into the peptide conjugated on the surface of Au nanoparticles, the Fourier Transform Infrared Spectroscopy (FTIR) spectra of c(RGDfK) peptide and the corresponding Au-c(RGDfK) nanoparticles were recorded (Figure S2). The characteristic IR absorption peaks of c(RGDfK) peptide at 1654 cm-1 (assigned to amide C=O stretching vibrations) and 1636 cm-1 (assigned to the guanidine group, C-N stretching vibrations),

23

were also found in the spectra of the corresponding Au-c(RGDfK)

nanoparticles, indicating the successful binding of c(RGDfK) peptide molecules to the Au nanoparticles. The indexed XRD pattern of Au-c(RGDfK) nanoparticles is shown in Figure S3. The high background of XRD pattern can be attributed to the low concentration of gold and also the organic content coating on the sample. XRD peaks matched well with standard gold (metal; space group: Fm3m) JCPDF card (04-0784). From the XRD plot, it can be inferred that the Au is present in the nanostructured form, which was further corroborated from the TEM studies. The TEM image illustrated that Au-c(RGDfK) nanoparticles were well dispersed and the average particle size was estimated to be 11 ± 1 nm with a polydispersity of only ~ 9 %. (Figure 2A). The hydrodynamic Au-c(RGDfK) nanoparticles, as determined by DLS was 30.2 ± 0.6 nm (Figure 2B). The significantly higher hydrodynamic diameters of Auc(RGDfK) nanoparticles compared to their particles sizes determined by TEM was due to the presence of hydrated layers in aqueous medium. The zeta potential of Au-c(RGDfK) nanoparticles was -34.7 ± 3.4 mV in PBS medium, which is responsible for the high stability of Au nanoparticles in this medium. 10


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As visibly observed, Au-c(RGDfK) nanoparticles could resist agglomeration tendency when kept at 4 °C even 6 months after synthesis (Figure S4). Further, the colloidal stability of Au-c(RGDfK) nanoparticles was also analyzed by mixing the nanoparticles in mouse serum and PBS media and determining the hydrodynamic diameters by DLS measurements over a period of 7 d (Figure S5). The Au-c(RGDfK) nanoparticles displayed near constant hydrodynamic diameters over this period of time, establishing its high colloidal stability in mouse serum and PBS media. The details regarding characterization of non-targeted Au-c(RGKfD) nanoparticles are provided in the Supporting Information (Table S1, Figure S6). In vitro toxicity study of Au-c(RGDfK) nanoparticles After 48 h of incubation, no obvious toxicity of Au-c(RGDfK) nanoparticles to CHO, MCF-7 and melanoma cells was observed (Figure 3), establishing the appreciable biocompatibility of Au-c(RGDfK) nanoparticles. Synthesis of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles On irradiation of 100 mg of natural Pt target at a flux of 8 × 1013 n.cm-2.s-1 for 7 d, 3774 ± 185 MBq of

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Au was produced with > 99.9 % radionuclidic purity (Table S2,

Figure S7). Clinically relevant doses (> 3500 MBq in a typical batch) of intrinsically radiolabeled

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Au-c(RGDfK) nanoparticles was synthesized following the same protocol

which was adopted for synthesis of inactive Au-c(RGDfK) nanoparticles (Table 1). The overall yield of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles was > 90 % and the results were quite reproducible in all the batches. Quality control of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles The radiochemical purity of

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Au-c(RGDfK) nanoparticles, as determined from the

radio-TLC patterns, was > 99 % (Figure 4A, Figure S8). The size exclusion chromatography

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pattern developed using PD-10 column as shown in Figure S9, further confirmed the high (> 99 %) radiochemical purity of the intrinsically radiolabeled nanoparticles. After passing through 0.22 µm membrane filter, the nanoparticles were established to be sterile. The level of endotoxins in the synthesized nanoparticles was < 4 endotoxin unit (EU)/mL, which is within the acceptable limit for clinical use. 24 In vitro radiochemical stability of 199Au-c(RGDfK) nanoparticles Intrinsically radiolabeled stable (intact

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Au in

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Au-c(RGDfK) nanoparticles were found to be highly

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Au-c(RGDfK) nanoparticles was > 98%) in mouse serum and PBS

media over 7 d time period (Figure 4B). In vitro cell binding and inhibition studies 199

The binding affinity and specificity of

Au-c(RGDfK) nanoparticles were tested in

melanoma cells, the results of which are shown in Figure 5. It is evident from the figure that 199

Au-c(RGDfK) nanoparticles have good affinity for the melanoma cell line. Also, inhibition

studies confirmed the specificity of the radiolabeled nanoparticles towards melanoma cell line. Biodistribution studies Biodistribution studies revealed that intratumoral injection of targeted c(RGDfK) nanoparticles and non-targeted

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Au-

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Au-c(RGKfD) nanoparticles resulted in high

tumor accumulation of radioactivity at 24 h p.i. (Figure 6). However, the tumor uptake of targeted

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Au-c(RGDfK) nanoparticles was slightly higher that of non-targeted

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Au-

c(RGKfD) nanoparticles (497 ± 56 %ID/g vs. 400 ± 67 %ID/g) at this time point (Figure 7). Tumor radioactivity decreased gradually from 24 to 192 h p.i. for both targeted c(RGDfK) nanoparticles as well as non-targeted

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but there was 2-fold-greater retention of targeted targeted

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Au-

Au-c(RGKfD) nanoparticles (Figure 7),

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Au-c(RGDfK) nanoparticles than non-

Au-c(RGKfD) nanoparticles even at 192 h p.i. (375 ± 78 %ID/g vs. 182 ± 23 12


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%ID/g). The radioactivity in the blood was lower for targeted nanoparticles compared to the non-targeted nanoparticles indicating leakage of non-targeted 199Au-c(RGKfD) nanoparticles from the tumor (Figure 7). Liver and kidney uptake for targeted

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Au-c(RGDfK)

nanoparticles was significantly lower than that of non-targeted

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Au-c(RGKfD)

nanoparticles during the entire duration of the study (Figure 7), indicating stronger and more specific binding of targeted of both targeted

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Au-c(RGDfK) nanoparticles to the tumor. The spleen uptake

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Au-c(RGDfK) nanoparticles and non-targeted

Au-c(RGKfD)

nanoparticles were almost comparable at all time points. Radioactivity in all other organs was less than 1 %ID/g at all time points (Figure 6). Treatment studies The treatment effect of the intratumoral injection of 199Au-c(RGDfK) nanoparticles on the TGI in mice having melanoma tumors, over a period of 15 d is shown in Figure 8. One time injection of different doses (2 MBq, 5 MBq or 10 MBq) of

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Au-c(RGDfK)

nanoparticles resulted in significant tumor growth delay as compared with mice injected with saline or unlabeled Au-c(RGDfK) nanoparticles (Figure 8A). For mice treated with 5 MBq or 10 MBq of

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Au-c(RGDfK) nanoparticles, significant tumor volume reduction was

observed. Over this period of time, there was no significant change in BWI of mice treated with different doses of 199Au-c(RGDfK) nanoparticles (Figure 8B).

DISCUSSION Nanoscale brachytherapy is poised to play a pivotal role in cancer care because intrinsically radiolabeled nanoparticles can be synthesized to match the sizes of tumor vasculature so that optimal therapeutic payloads with minimum leakage away from target sites can be achieved. There are several potential advantages of using intrinsically radiolabeled

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Au nanoparticles for nanoscale brachytherapy. 13


5, 6

Firstly, since the


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radiolabeled nanoparticles are microscopically dispersed in aqueous medium, they can be easily administered intra or peritumorally by syringe, which is a less invasive procedure than placing of millimetre sized solid brachytherapy sources into the tumor using catheters. Several small injections could be used to achieve a more homogeneous intra or peritumoral distribution of intrinsically radiolabeled 199Au nanoparticles. Secondly, the nanometre size of the brachytherapy agent may permit local diffusion from the injection site, thereby, further homogenizing the radiation dose deposition in the tumor. Thirdly, the intrinsically radiolabeled nanoparticles could be synthesized with very high radiochemical stability and therefore the possibility of leakage of radioactivity from the nanoparticles leading to undesirable dose deposition in healthy organ is negligible. Fourthly, because of the moderateenergy β- particles emitted by

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Au (β1 = 462 keV, 6.0%; β2 = 296 keV, 71.6%; β3 = 250

keV, 22.4%) with 0.8 mm maximum tissue range, a conformal radiation field is provided around the injection site leading to local cross-fire effect.16 Furthermore,

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Au emits an

imageable γ-ray of 159 keV (37%) that not only allows initial dosimetric experiments prior to administering therapeutic doses but also aids in analyzing the in vivo localization of the radioactive source and monitoring the therapeutic efficacy by molecular imaging strategies.16 An essential prerequisite for utilization of

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Au for nanoscale brachytherapy in

clinical context is to develop a facile method for large scale synthesis of intrinsically radiolabeled nanoparticles. Though several procedures for synthesis of functionalized and targeted Au nanoparticles have been reported over the last decade,

25-29

a majority of them

share common limitations such as multistep synthesis, relatively complicated procedures, time consuming and harsh synthetic conditions. In order to circumvent these limitations, a facile single step method for synthesis of intrinsically radiolabeled

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Au nanoparticles

conjugated with cyclic RGD peptide was developed. Cyclic RGDfK was chosen for the synthesis because the tripeptide Arg-Gly-Asp (RGD) amino acid sequence has high affinity 14


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for integrin αvβ3 receptor which is upregulated on tumor cells and tumor endothelial cells of varying cancer types. and metastases.

30-32

30, 31

The integrin αvβ3 receptor plays important roles in angiogenesis

Thus, cyclic RGD conjugated

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Au nanoparticles holds relevance in

antiangiogenic therapeutics for personalized cancer management. In this procedure for synthesis of Au nanoparticles, the cyclic RGDfK peptide acted as both reducing as well as stabilizing agent.

17

Basically, cyclic RGDfK contains two free

amino and one free carboxyl groups. The amino groups reduced Au(III) ions to Au(0) to form Au nanoparticles and the carboxyl group stabilized the Au nanoparticles.

17

Cyclic RGDfK

peptide binding to the Au nanoparticles was confirmed by FTIR spectroscopy. This was further corroborated by UV-visible and CD spectrometry. As evident from the absorption spectra, Au nanoparticles exhibited strong absorption in the visible wavelength range due to surface plasmon resonance.

21

As such, Au nanoparticles are achiral with no inherent

chiroptical properties. On synthesis of cyclic RGDfK conjugated Au nanoparticles, the chiral c(RGDfK) peptide interacts with the Au nanoparticle due to electronic interaction between the chiral peptide moiety and metal electrons.

22

Thus, the peptide imparted chirality to the

nanomaterial and artificially created a plasmon-induced CD signal in the visible spectral region. Minor changes in the UV region of the CD spectra between c(RGDfK) peptide and Au-c(RGDfK) nanoparticles can be due to conformational change from the peptide native state in solution upon binding to the nanoparticle surface, and/or energy transfer from the peptide to the metal nanoparticle. Overall, these observations prove that Au nanoparticles are embedded in RGD peptide scaffold. Intrinsically radiolabeled

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Au nanoparticles exhibited very good radiochemical

stability in mouse serum and PBS media over a time period of 7 d. Further, the nanoparticles met all the purity requirements for preclinical studies. As a proof of concept, the radiolabeled nanoparticles (both targeted and non-targeted) were intratumorally administered in C57BL/6 15



Page 16 of 33

mice having melanoma tumors. Biodistribution studies revealed very high retention of radioactivity of both targeted and non-targeted nanoparticles at initial time point (24 h p.i.). However, in case of non-targeted nanoparticles, the tumor uptake decreased significantly at later time points due to leaching out of radiolabeled nanoparticles from the tumor vasculature. The targeted nanoparticles were retained in the tumor over a prolonged period of 1 week, corroborating the high integrin αvβ3 binding affinity and specificity of c(RGDfK) conjugated 199

Au nanoparticles. Because integrin αvβ3 targeting provided greater tumor retention and

thereby possibly leading to higher tumor radiation-absorbed dose, the targeted

199

Au-

c(RGDfK) nanoparticles were found to be more effective for arresting tumor growth compared to non-targeted nanoparticles. The absence of cellular toxicity of cyclic RGDfK conjugated Au nanoparticles suggested that application of radiolabeled nanoparticles could also be combined with neoadjuvant chemotherapy for treatment of cancer in order to achieve better patient outcomes.

CONCLUSIONS A single step method for synthesis of intrinsically radiolabeled

199

Au nanoparticles

conjugated with cyclic RGDfK peptide was developed for potential use in nanoscale brachytherapy. The cyclic RGDfK peptide conjugated to the Au nanoparticles performed the triple roles of reducing, stabilizing and targeting agents. The intrinsically radiolabeled nanoparticles demonstrated excellent stability and reasonably good biocompatibility for in vivo use. Biodistribution studies established the superiority of integrin αvβ3 targeted nanoparticles compared to non-targeted nanoparticles, in terms of greater tumor retention over a period of 1 week. This study amply demonstrates that nanoscale brachytherapy using integrin αvβ3 targeted and intrinsically radiolabeled

199

Au nanoparticles has the potential to

meet the pressing clinical demands for effective cancer therapy. 16


Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGEMENTS Research at Bhabha Atomic Research Centre is an ongoing activity of the Department of Atomic Energy, India and is fully supported by government funding. The authors are grateful to Dr. P.K. Pujari, Associate Director, Radiochemistry and Isotope Group, Bhabha Atomic Research Centre for his valuable support to the isotope program. Thanks are due to Dr. Kanhu Charan Barick, Chemistry Division, Bhabha Atomic Research Centre for dynamic light scattering studies. The authors are also thankful to Dr. Nilotpal Barua and Dr. Amit Kunwar of Radiation and Photochemistry Division, Bhabha Atomic Research Centre for circular dichroism and toxicity studies with Au nanoparticles, respectively.

SUPPORTING INFORMATION Synthesis and characterization of non-targeted Au-c(RGKfD) nanoparticles, production of

199

Au, quality control of intrinsically radiolabeled

biodistribution studies, Tables S1 and S2, Figures S1-S9.

17


199

Au nanoparticles,


Page 18 of 33

Table 1: Production of 199Au-c(RGDfK) nanoparticles in 5 different batches

Batch No.

Activity of 199

Au taken (MBq)

Amount of non-

Activity of 199

Au-

Yield of 199

Au-

Radiochemical purity of 199

radioactive

c(RGDfK)

c(RGDfK)

Au carried

nanoparticles

nanoparticles

c(RGDfK)

added

obtained after

(%)

nanoparticles

(µg)

radiochemical

Au-

(%)

purification (MBq) 1

3737

200

3513

94

99.6 ± 0.2

2

3682

200

3387

92

99.8 ± 0.1

3

3700

200

3515

95

99.3 ± 0.3

4

3885

200

3613

93

99.5 ± 0.2

5

3774

200

3547

94

99.7 ± 0.1

18


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure captions: Figure 1: (A) A schematic diagram showing synthesis of intrinsically radiolabeled

199

Au

nanoparticles by single step reaction of Au (III) solution with c(RGDfK) peptide. (B) Absorption spectra of Au-c(RGDfK) nanoparticles and c(RGDfK) peptide. Spectrum of Auc(RGDfK) nanoparticles clearly show the SPR band with maxima at 535 nm. (C) CD spectra of c(RGDfK) and Au-c(RGDfK) nanoparticles in the wavelength range of 200-800 nm. The amplified view of the CD spectra in the wavelength range of 450-600 nm is shown in the inset.

Figure 2: (A) TEM image of Au-c(RGDfK) nanoparticles. (B) DLS size distribution of Auc(RGDfK) nanoparticles.

Figure 3: Determination of cell viability of Au-c(RGDfK) nanoparticles by MTT assay. The analysis was performed after incubation of CHO, MCF-7 and melanoma cells with Auc(RGDfK) nanoparticles (concentration range: 0 – 2 mg / mL) for 48 h. Values shown represent the mean ± SD (n = 5).

Figure 4: (A) Radio-TLC pattern of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles developed in saline in 0.02 M HCl medium. The chromatography pattern for

199

Au-HAuCl4

(as control) is shown in the inset. (B) In vitro stability of intrinsically radiolabeled

199

Au-

c(RGDfK) nanoparticles in PBS and mouse serum media over a period of 7 d. Values shown represent the mean ± SD (n = 5).

Figure 5: In vitro cell binding and inhibition studies with

199

melanoma cells. 19


Au-c(RGDfK) nanoparticles in


Page 20 of 33

Figure 6: Biodistribution study in C57BL/6 mice bearing melanoma tumors after administration of (A) integrin αvβ3 targeted intrinsically radiolabeled nanoparticles. (B) non-targeted intrinsically radiolabeled

199

199

Au-c(RGDfK)

Au-c(RGKfD) nanoparticles.

Values shown represent the mean ± SD (n = 5).

Figure 7: The uptake of radioactivity upon intratumoral injection of integrin αvβ3 targeted 199

Au-c(RGDfK) nanoparticles and non-targeted

199

Au-c(RGKfD) nanoparticles in (A)

tumor, (B) blood, (C) kidney, (D) liver, at different time points p.i. Values shown represent the mean ± SD (n = 5).

Figure 8: (A) Effect of intratumoral injection of increasing doses (2 MBq, 5 MBq and 10 MBq) of

199

Au-c(RGDfK) nanoparticles, unlabeled Au-c(RGDfK) nanoparticles or normal

saline on the TGI for melanoma tumor bearing mice. (B) Effect of these treatments on the BWI. Values shown represent the mean ± SD (n = 5).

20


Page 21 of 33

A

NH H 2N

O N H

O

H2N

199Hg

200Hg

201Hg

16.8 % σ = 2150 b

23.1 % σ = 60 b

13.2 % σ = 7.80 b

cRGDfK

NH

N H HN

NH

HN

O OH O

O

HN

Tumor

OH

β198Au 2.7 d

199Au

3.1 d

200Au

Water bath

48 m

β(n,γ) 199Pt

197Pt

198Pt

20 h

7.163 % σ = 0.35 b

199Au

solution Stirrer

31 m

Mixing

B 0.6

C

199Au-(cRGDfK)

nanoparticles

10

cRGDfK only Au-cRGDfK

Au-c(RGDfK) nanoparticles c(RGDfK) 0

535 nm

0.2

3 2

Ellipticity (mdeg)

0.4

Ellipticity (mdeg)

Absorbance (A. U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-10

-20

1 0 -1 -2 -3 450

500

550

600

Wavelength (nm)

0.0 300

400

500

600

700

Wavelength (nm)

800

-30 200

300

400

500

600

700

800

Wavelength (nm)

Figure 1: (A) A schematic diagram showing synthesis of intrinsically radiolabeled 199Au nanoparticles by single-step reaction of Au (III) solution with c(RGDfK) peptide. (B) Absorption spectra of Au-c(RGDfK) nanoparticles and c(RGDfK) peptide. Spectrum of Au-c(RGDfK) nanoparticles clearly show the SPR band with maxima at 535 nm. (C) CD spectra of c(RGDfK) and Au-c(RGDfK) nanoparticles in the wavelength range of 200-800 nm. The amplified view of the CD spectra in the wavelength range of 450-600 nm is shown in the inset.

21



A

B 70 60 50

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 22 of 33

40 30 20 10 0 0

20

40

60

Size (nm)

Figure 2: (A) TEM image of Au-c(RGDfK) nanoparticles. (B) DLS size distribution of Au-c(RGDfK) nanoparticles.

22


80

100

Page 23 of 33

130 CHO MCF7 melanoma

120 110 100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 80 70 60 50 40 30 20 10 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Concentration (mg/mL)

Figure 3: Determination of cell viability of Au-c(RGDfK) nanoparticles by MTT assay. The analysis was performed after incubation of CHO, MCF-7 and melanoma cells with Au-c(RGDfK) nanoparticles (concentration range: 0 – 2 mg / mL) for 48 h. Values shown represent the mean ± SD (n = 5).

23



A

B 100

Radioactivity (%)

80

80

60

199Au-AuCl

4

199

Au-(cRGDfK) in PBS Au-(cRGDfK) in serum 199 Au-HAuCl4 in PBS (control)

60

199

80 40

20

0 0.0

0.2

0.4

0.6

0.8

1.0

Rf

40

Intact 199Au (%)

100

Radioactivity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 24 of 33

20

199

Au-HAuCl4 in serum (control)

60

40

20

199

Au-c(RGDfK)

0

0 0.0

0.2

0.4

0.6

0.8

1.0

0

Rf

24

48

72

96

120 144

168 192

Time (h)

Figure 4: (A) Radio-TLC pattern of intrinsically radiolabeled 199Au-c(RGDfK) nanoparticles developed in saline in 0.02 M HCl medium. The chromatography pattern for 199Au-HAuCl4 (as control) is shown in the inset. (B) In vitro stability of intrinsically radiolabeled 199Auc(RGDfK) nanoparticles in PBS and mouse serum media over a period of 7 d. Values shown represent the mean ± SD (n = 5). 24


Page 25 of 33

30

% Binding in melanoma cells

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Binding Binding in excess of RGD

25 20 15 10 5 0 4.4 KBq

Activity of

11 KBq

17.6 KBq

199

Au-(cRGDfK) nanoparticles

Figure 5: In vitro cell binding and inhibition studies with 199Au-c(RGDfK) nanoparticles in melanoma cells.

25



A

B

600

24 h 72 h 192 h

400

600

24 h 72 h 192 h

199Au-c(RGDfK)

400

200

199Au-c(RGKfD)

200

%ID/g

%ID/g

6

12 4

8 4

0

0

lo od Li ve r G K IT i St dn om ey ac H h e Lu art ng Ti s M bi us a c Sp les le Tu en m or

lo od Li ve r G K IT i St dn om ey ac H h e Lu art ng T s M ibi us a c Sp les le Tu en m or

2

B

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 26 of 33

Figure 6: Biodistribution study in C57BL/6 mice bearing melanoma tumors after administration of (A) integrin αvβ3 targeted intrinsically radiolabeled

199

Au-c(RGDfK) nanoparticles. (B) non-targeted intrinsically radiolabeled

Values shown represent the mean ± SD (n = 5). 26


199

Au-c(RGKfD) nanoparticles.

Page 27 of 33

B

A 600

199

Tumor

199

500

199

Au-c(RGDfK) Au-c(RGFkD)

Blood

199

Au-c(RGfKD)

2.5

2.0

%ID/g

%ID/g

3.0

Au-c(RGDfK)

400

300

1.5

200

1.0

100

0.5

0.0

0 0

23

46

69

92

0

115 138 161 184

24

48

14

D 12

199

Au-c(RGDfK) 199 Au-c(RGKfD)

12

72

96

120

144

168

192

Time (h)

Time (h) C

199

Kidney

199

10

10

Au-c(RGDfK) Au-c(RGfKD)

Liver

8

8

%ID/g

%ID/g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

6

4

4 2

2 0

0

0

24

48

72

96

0

120 144 168 192

24

48

72

96

120

144

168

192

Time (h)

Time (h)

Figure 7: The uptake of radioactivity upon intratumoral injection of integrin αvβ3 targeted

199

Au-c(RGDfK)

nanoparticles

and

non-targeted

199

Au-c(RGKfD)

nanoparticles in (A) tumor, (B) blood, (C) kidney, (D) liver, at different time points p.i. Values shown represent the mean ± SD (n = 5).

27



B

6

Normal Saline Au-(cRGDfK) nanoparticles 199 Au-(cRGDfK) nanoparticles (2 MBq) 199 Au-(cRGDfK) nanoparticles (5 MBq) 199 Au-(cRGDfK) nanoparticles (10MBq)

5

4

3

2

1

Body Weight Index (BWI)

A

Tumor Growth Index (TGI)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

0

1.3

Page 28 of 33

Normal Saline Au-(cRGDfK) nanoparticles 199 Au-(cRGDfK) nanoparticles (2 MBq) 199 Au-(cRGDfK) nanoparticles (5 MBq) 199 Au-(cRGDfK) nanoparticles (10MBq)

1.2

1.1

1.0

0.9

0.8

0

3

6

9

12

15

0

Time (d)

3

6

9

12

15

Time (d)

Figure 8: (A) Effect of intratumoral injection of increasing doses (2 MBq, 5 MBq and 10 MBq) of

199

Au-c(RGDfK) nanoparticles,

unlabeled Au-c(RGDfK) nanoparticles or normal saline on the TGI for melanoma tumor bearing mice. (B) Effect of these treatments on the BWI. Values shown represent the mean ± SD (n = 5).

28


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract NH H2 N

O N H

H2 N

199 Hg

200Hg

201Hg

16.8 % σ = 2150 b

23.1 % σ = 60 b

13.2 % σ = 7.80 b

cRGDfK

O

NH

N H HN

NH

HN

O OH O

O

HN

OH

β198Au

199Au

2.7 d

3.1 d

197Pt 20 h

200Au 48 m

β198Pt (n,γ) 199Pt 7.163 % σ = 0.35 b

199Au

31 m

199 Au-(cRGDfK)

production

Intrinsically radiolabeled

199

nanoparticles

Brachytherapy

Au nanoparticles conjugated with cyclic RGD peptide was

synthesized and biodistribution studies were carried out in C57BL6 mice to demonstrate their biological efficacy as nanoscale brachytherapy agent.

29



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Facile One-Pot Synthesis of Intrinsically Radiolabeled and Cyclic RGD

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