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Bovine Serum Albumin Conjugated Gold-198 Nanoparticles as Model to Evaluate the Damage Caused by Ionizing Radiation to Biomolecules Jonnatan J. Santos, Jessica Leal, Luis A. P. Dias, Sergio Hiroshi Toma, Paola Corio, Frederico A. Genezini, Kattesh V Katti, Koiti Araki, and Ademar B. Lugao ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01174 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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ACS Applied Nano Materials

Bovine Serum Albumin Conjugated Gold-198 Nanoparticles as Model to Evaluate the Damage Caused by Ionizing Radiation to Biomolecules Jonnatan J. Santos,*†‡‡ Jessica Leal,†‡ Luis A. P. Dias, † Sergio H. Toma,‡ Paola Corio,‡ Frederico A. Genezini, † Kattesh V. Katti, § Koiti Araki,‡ and Ademar B. Lugão† † Instituto de Pesquisas Energéticas e Nucleares (IPEN-CNEN/SP) - Av. Prof. Lineu Prestes, 2242, Cidade Universitária, 05508-000 São Paulo, SP, Brazil. ‡ Instituto de Química, Universidade de São Paulo (IQ-USP) - Av. Prof. Lineu Prestes, 748, Cidade Universitária, 05508-000 São Paulo, SP, Brazil. § Department of Radiology and Chemistry, University of Missouri, Columbia, MO 65212, USA. KEYWORDS: gold-198, gold nanoparticles, bovine albumin serum, ionizing radiation, chromophore destruction ABSTRACT: Gold nanoparticles (AuNPs) have several applications including in medicine. Considering cancer as one of the most common diseases for men and women, new treatments and more specific and effective drugs, which cause less side effects, have been actively pursued. Among them, gold-198 can be engineered as theranostic agents, working as contrast (exploiting gamma emission) and treatment agents (beta emission). Accordingly, a new procedure for the production of 14 nm diameter radioactive citrate protected gold-198 nanoparticles, which were then conjugated with bovine serum albumin utilizing 3-mercaptopropionic acid directly bound to AuNPs surface as anchoring groups, generating fully dispersible nanoparticles in aqueous media, are described. The effect of gamma and beta radiation on grafted BSA was evaluated by direct irradiation of the corresponding cold material and comparing with the damage caused on BSA grafted gold-198 nanoparticles prepared from a neutron activated gold foil. The investigation by fluorescence and Raman spectroscopy indicated that the damage to BSA chromophore groups is proportional to the dose (from 0.1 to 1 kGy) and that chromophores groups close to the particle surface are more prone to damage. Gold-198 nanoparticles conjugated with bovine serum albumin showed that process is much more localized next to nanoparticles surface since each gold core acts as a punctual radiation source. In short, AuNPs can enhance the damage caused by irradiation of cold nanoparticles and AuNPs@MPA-BSA is a suitable model to probe the effect of gamma and beta emitter on biomolecules. Furthermore, the strategy of diluting the gold-198 with cold gold atoms was shown to be suitable to control the activity of 198AuNPs aiming medical applications, since the damage to BSA was found to be proportional to the relative concentration of gold-198.

INTRODUCTION Cancer has become a major problem nowadays, and more than 1,700,000 new cases are expected to be diagnosed in 2018 only in the United States of America, according to the National Cancer Institute. Among women, breast cancer is the most common while prostate cancer is the most representative among men. Although treatments and diagnosis methods have been already developed for both, new more efficient methods that cause less damage to the patient are actively being searched.1 Nanoparticles are attractive in cancer therapy for many reasons including unique pharmacokinetic properties, minimal renal filtration, the possibility of using essentially inert and non-toxic core materials, and a high surface area for conjugation of molecular species, allowing the tuning of their interaction and biological properties. Gold

nanoparticles generally are modified by molecules with coordinating thiol groups, whose can be used to anchor specific targeting or chemotherapeutic agents with relative facility. In fact, new designs and strategies can be devised to obtain multifunctionalized nanoparticles with enhanced contrast efficiency for detection and diagnosis as well as therapy of tumors.2-6 Ionizing radiation is used on a large scale for the treatment of almost all types of solid tumors (radiotherapy). Unfortunately, it is non-selective and damage cells without discrimination, so that the dose must be strictly controlled to minimize the effect on healthy tissues while eradicating the tumor cells. Thus, the application of nanoparticles that specifically target the tumor can improve the radiotherapy efficiency by increasing the effect on tumor cells while lowering toxicity to normal cells, improving the selectivity of the treatment.7

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Blood proteins were shown to accumulate in tumor tissues.8 Among them, the serum albumin is one of most interesting since it is the most abundant and widely used as drug carrier and capable of transporting hydrophobic molecules in the body. In addition, its medical relevance is recognized and widely accepted in the pharmaceutical industry due to biocompatibility, ease of purification and, consequently, low cost.9-11 Gold nanoparticles are being explored in catalysis and electrocatalysis12, surface enhanced Raman spectroscopy (SERS)13 and surface enhanced Fluorescence spectroscopy, photothermal therapy, drug delivery, and as a contrast agent, among other applications. Radioactive gold-198 (198Au) nanoparticles have already been utilized in nuclear medicine as therapeutics and diagnostics agent.14 It is a beta (0.96 MeV) and gamma (0.412 MeV) emitter with a half-life of 2.7 days. The presence of radioactive gold atoms does not change their excellent stability and natural affinity for tumor tissues 15-20. Also, the possibility of manipulation of its core diameter and conjugated molecules can favor the uptake in tumor cells and tissues, resulting in efficient radiopharmaceuticals or labels for such tissues16. In addition, the penetration of beta radiation is limited to 3.8 mm in water, enough to act on but restrict its effects to tumor tissues. Katti et al.21 recently demonstrated by in vivo assays the effectiveness of gold-198 in the treatment of cancer. AuNPs with 85 nm diameters conjugated to glycoprotein (GA) demonstrated optimal penetration into prostate cancer cells, diminishing the tumor volume by up to 82% upon a single treatment with 198Au@GA. AuNPs are nontoxic to human and animal organisms20, 22, offering no harm to the vascular system and being extremely stable when conjugated to biomolecules in different media and pH conditions. Besides therapeutics properties of gold-198, it has also been utilized in the diagnosis of different types of tumors, the gamma radiation emitted with energy at 411 ev, higher than typical imaging agents like technetium-99m (140 keV) but with a unique energy, is very interesting in the point of view of penetration.23 The relative easy modification of 198AuNPs with biomolecules, such as human monoclonal antibody24 or folic acid25, made it highly selective and specific for detecting ovarian cancer and tumor cells, creating a system with high mobility and high contrast. Gold-198 was also successfully utilized to modify graphene, being applied in the imaging of rat bearing fibrosarcoma tumor.26 The radionuclide 198Au can be created from nonradioactive gold. One of the most prevalent methods uses neutron bombardment of the gold target in a reactor. The 197 Au is transformed into 198Au with shock section at 98.8 barns, and the decay of 198Au occurs by beta and gamma particle emissions, being converted to mercury-198. 27 In this work, the effect of gamma irradiation on bovine serum albumin (BSA) conjugated to cold gold nanoparticles were investigated by fluorescence spectroscopy and Raman Spectroscopy and compared to the damaged on

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BSA conjugated to radioactive 198AuNPs. Several compositions of gold-197 198/197AuNPs conjugated with bovine serum albumin (BSA) were also prepared and studied. Gold nanoparticles were shown to enhance the degree of damage of molecules next to the surface, and dilution of 198 AuNPs with 197Au was shown to be a suitable strategy to control the BSA damage, being a suitable strategy for adjusting the radiotherapy with 198AuNPs.

EXPERIMENTAL SECTION Chemicals. All aqueous solutions were prepared using deionized water (resistivity > 18.2 MΩ·cm) obtained from a MilliQ deionizer (Elix Millipore). Nitric acid (65%), hydrochloric acid (35-37%), trisodium citrate (99%), sodium phosphate dibasic (99%), sodium phosphate monobasic (99%), sodium hydroxide and sodium chloride (>99%) were purchased from Synth, Brazil. Gold foil (>99.99%), Bovine Serum Albumin Fraction V (98.5%), 3-mercaptopropionic acid (MPA) (>99%), N-hydroxysuccinimide (NHS) (98%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (>97%), Whatman paper 3MM were obtained from Sigma Aldrich, USA. Characterization. UV-Vis spectra in the 350 to 800 nm range were registered using a Varian Cary 1E UV-VIS Spectrophotometer. Fluorescence spectra were obtained on a Photon Technology International, INC model Fluorescence Master System. Dynamic light scattering measurements were performed on a Malvern Zetasizer Nanoseries, model NanoZS. Transmission electron microscopy images were obtained using a JEOL, model JEM 2100 microscope, operating at 200 kV. Scanning electron microscopy image was obtained in a JEOL, model 7401, microscope operating at 5 kV. Gamma irradiation experiments were performed in a gammacell 220 Irradiator (Atomic Energy of Canada Limited, Canada) at a dose rate of 1.03 kGy/h, established by alanine, using 60Co as a radioactive source, being irradiated 20 mL of AuNP@MPA-BSA suspensions ([BSA}= 3.75 10-6 mol/L) at different doses. The chromatographic analyses of the radioactive samples were performed in a well counter, model CR 643, Cobra II, Packard - Canberra (USA). The Raman spectra were acquired using an inVia Raman spectrometer microscope from Renishaw equipped with a 50× objective (N.A. = 0.45) and a 785 nm solid-state laser. Synthesis of Gold Nanoparticles using Au197 and Au198 A gold foil (10 mg) was transferred into a 125 mL round bottom flask and reacted with 150 μl of Aqua Regia. After complete dissolution of the gold foil, the excess of acids was removed using a vacuum system at a mean pressure of 160 mmHg, showed in the Scheme 1. Then, 100 mL of DI-water were added and the solution kept under reflux while stirred with a 1.5 cm magnetic bar at about 1000 revolutions per minute (RPM). Then, 4 mL of 0.034 mol/L sodium citrate solution was added into, Scheme 2.


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Scheme 1. The process of oxidation of gold foil with Aqua Regia and elimination of acid using the vacuum pump . Cold Finger

Round Bottom Double Neck Reaction Flask and Heating System

Vaccum Pump

Scheme 2. The system where the gold solution (yellow solution) is heated and become red, after citrate addition.

1. Heated to reflux 2. Sodium citrate added

In a few seconds, the pale-yellow color of the initial solution changed to blue and then to red, indicating the formation of gold nanoparticles. The suspension was cooled to room temperature, transferred to a clean, dry, tightly closed vial and stored in a refrigerator at 4 °C. The same procedure was used to produce 198AuNPs, except for the previous activation of 10 mg of 197Au foil by neutron irradiation in the IEA-R1 nuclear reactor, located at IPEN/SP, for 30 h at 4.5 MW. The activity measured after 30h was 32 mCi and the experiments were performed after sample activity decrease to 20 mCi.

Modification of Gold Nanoparticles (197Au and 198Au) by Bovine Serum Albumin In order to favor the cross-linking of AuNPs with biomolecules, they were functionalized by reaction with 500 μL of a 1mM solution of 3-mercaptopropionic acid (MPA). After 24 h, a phosphate buffer solution was added to the suspension adjusting the final concentration to 1 mM. The nanoparticles were further conjugated with BSA by adding 20 μL of a 1mM EDC/NHS mixture as the catalyst for the formation of amide bonds, to aid the binding of different concentrations of BSA on the AuNPs.

000 years31) can be generated precluding their future applications in medicine. Thus, this strategy requires the utilization of high purity (superior to 99.99 %) gold foil as the source in order to minimize the generation of unwished impurities. Considering this strategy, an efficient method for the preparation of gold nanoparticles starting from a gold foil was developed, as described below. Initially, a small piece of gold foil was neutron activated, then dissolved by Aqua Regia, a common method utilized since 700 AD to purify gold, and dried under vacuum. After the elimination of excess of acid by such a gentle procedure, a yellow solid formed by HAuCl4 was obtained. The solid was solubilized in deionized water, heated to reflux, then sodium citrate aqueous solution (1 %, w:w) was added, generating the AuNPs. Figure 1 shows a picture of the starting gold foil, the picture of the resulting gold nanoparticles suspension, and the corresponding extinction spectrum. The ruby red color of the suspension (Figure 1) is characteristic of AuNPs localized surface plasmon resonance (LSPR), as confirmed by the electronic spectrum with the extinction band at 521 nm, very similar to those found in the literature.32 The narrow and intense peak suggest the presence of essentially spherical monodisperse nanoparticles, as confirmed by scanning and transmission electron microscopy (Figure 2, and Figure S1). The AuNPs synthesized by citrate method are slightly oval shaped as consequence of the mechanism of nucleation and growth of nanoparticles, particularly in the pH 3.7 to 6.5 range, according to Ji et al.33 since a step of ripening of the particles from initially formed wire shaped seeds is involved. The morphology can be modified by increasing the pH to 7 or more but is not interesting when dealing with ionizing radiation emitters since demands an additional processing step. Summarizing, about 17 nm diameter high-quality monodisperse nanoparticles were obtained, as is shown in the size histogram in Figure S1.

RESULTS AND DISCUSSION Synthesis of Gold Nanoparticles from a Foil and Optimization of Modification Using Bovine Serum Albumin. There are several different methodologies to obtain gold nanoparticles, but most of them utilize the gold chloride salt or tetrachloroauric acid.28-30 However, both are not recommended as reactants to obtain gold-198 by direct activation of gold-197 with neutrons (typical method utilized to obtain gold-198) since species with longer half-lives than gold such as chlorine-36 (half-life of 300



Figure 1. Extinction spectrum of gold nanoparticles prepared from a gold foil (insert on the left) and the photo of the corresponding suspension (right).

Figure 2. Scanning electron microscopy (A) and transmission electron microscopy (B) of the AuNPs synthesized.

However, the previously prepared citrate protected AuNPs are not convenient considering our intention of using them for medical applications. It is fundamental to protect the surface with biocompatible molecules that also assure their proper dispersion in biological fluids. Among several possibilities, albumin was the molecule of choice since it is the most abundant biomolecule in the blood, and less prone to cause any rejection generating more biocompatible materials. BSA conjugated AuNPs were already prepared and shown to exhibit a significantly enhanced uptake by cells.34 Besides, albumin has several aromatic amino acids in its structure which can be used as convenient chromophores as well as fluorescence and Raman spectroscopy35 markers to monitor the effect of ionizing radiation on the integrity of that biomolecule. But, bovine serum albumin (BSA) was used instead of human serum albumin (HSA) given its availability and structural similarity. In fact, their isoelectric points are the same (4.7), and the molecular weight very similar (BSA ~66.4 kDa vs. HSA ~66.5 kDa).

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lecular species, and can be used to monitor that process qualitatively. In contrast, large changes are expected in the DLS measurements when BSA is conjugated to 17 nm (29.4 nm in DLS) diameter nanoparticles, as shown in Table 1 where the LSPR shift and the hydrodynamic size of AuNPs conjugated with increasing concentrations of BSA are compared. Considering that BSA is a prolate ellipsoid whose size is 14 x 4 x 4 nm, the lateral binding of one BSA molecule should increase from 29 nm the size of the conjugate to about 33 nm, while the binding with upright conformation will increase the size to about 44 nm. It is possible to see that the first significant shift of LSPR from 523 to 526 nm correspond to a change in the hydrodynamic size compatible with the binding of one up to a monolayer of BSA, as consequence of changes in the dielectric environment around the AuNPs. The morphology and composition of nanomaterials are generally characterized by electron microscopy techniques. However, the electron beam used to probe the sample in SEM and TEM techniques tend to damage sensitive species such as biomolecules, whose contrast is inherently much lower than the nanoparticle core, making them almost invisible and more appropriate to show the size and morphology of nanoparticles core. Table 1. LSPR band position and size (DLS) of AuNPs conjugated with different concentrations of BSA. BSA Concentration (mol/L) 0 -8 3.75 10 -7 3.75 10 -7 7.50 10 -6 3.75 10 -6 7.50 10 -5 1.5 10 -5 3.75 10

LSPRmax (nm)

Hydrodynamic size (nm)

523 523 526 526 526 527 527 528

29 30 33 45 53 53 73 78

Standard Deviation (nm) 10 10 10 22 27 25 43 44

However, controlling the colloidal stability and how a large molecule such as albumin anchors on nanoparticles surface is not a simple task. Accordingly, AuNPs were first functionalized with 3-mercaptopropanoic acid (MPA) to stabilize and generated well-defined anchoring sites to connect that biomolecule. MPA binds strongly on gold nanoparticles surface through the thiol group forming a monolayer, leaving the carboxylate group free to bind to an amine group in the biomolecule. This cross-coupling reaction generating an amide bond was convenient catalyzed in aqueous phosphate buffer media using the EDC/NHS method, to adjust the pH and avoid denaturation or loss of activity induced by conformation changes in the biomolecule.

On the other hand, DLS is a non-destructive technique quite sensitive to the whole particle, including the molecular functionalization layer and solvation layer, thus being more suitable to probe the conjugation of molecular species on AuNPs by the increase of the initial hydrodynamic radius upon binding of BSA on the surface, as shown in Table 1. Clearly, the hydrodynamic size of AuNPs tend to increase as the concentration of BSA used in the conjugation reaction is increased up to 3.75 10-5 mol/L, while the LSPR band position is quite insensitive.

As expected, the conjugation of BSA on the AuNPs@MPA did not change the LSPR band significantly, in the UV-Vis spectrum (Figure S2). In fact, only a small increase and redshift of the LSPR band (Table 1) was observed when reacted with increasing amounts of BSA until up to 3.75 10-6 mol/L. This is the typical behavior observed when plasmonic nanostructures/nanoparticles, immobilized or in solution36, 37, interact with or bind mo-

Gamma Effects over BSA modifying Gold Nanoparticles The effects of gamma radiation on BSA were already reported in the literature, where different techniques have been used to monitor their nature and extent as a function of energy and dose. Fluorescence, infrared and Raman spectroscopies are the main tools utilized to identify and quantify the alterations in the structure of BSA, how-


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ever, electrophoresis in a gel, dynamic light scattering, and circular dichroism spectroscopy have been employed also.38-41 Considering that the beta particles emitted by gold-198 have a low penetration in skin and others tissues, and the fact that one focus on the use for therapy and gamma radiation has a high penetration focusing on diagnosis, it is more interesting to keep the thickness of the BSA layer thick enough to fully protect the core, but transparent as possible to the radiation.

was already shown by gel electrophoresis that gamma irradiation tends to produce molecules with lower molecular weight. Accordingly, aromatic chromophores rich in electrons such as tyrosine (Tyr) and tryptophan (Trp) should be more easily damaged than other amino acid residues.43

Thus, the AuNPs conjugated with 3.75 10-6 mol/L of BSA were chosen for the experiments, and the effects monitored by fluorescence and Raman/SERS spectroscopy. The presence of radioactive gold-198 was simulated by irradiation of different samples of AuNPs@MPA-BSA conjugates with increasing doses of gamma radiation and monitoring the damage by fluorescence spectroscopy. BSA has an extinction band at 280 nm with the corresponding emission band at 341 nm, as shown in Figure 3. However, the intensity of the fluorescence bands decreased as a function of the gamma radiation dose, almost disappearing completely after irradiation with a dose of 1 kGy. Interestingly, the emission spectrum is very similar to that of tryptophan which exhibits a maximum at 348 nm.42 This small shift to higher energy can be explained by differences in the environment surrounding that amino acid, whereas the sharp band at 303 nm can be assigned to tyrosine residues present in the structure of BSA. This behavior is very similar to that observed when pure BSA is irradiated with gamma radiation and was attributed to changes in the chromophores local environment quenching the fluorescence.38

Figure 4. Raman/SERS spectra of non-irradiated -6 AuNPs@MPA-BSA ([BSA]= 3.75 10 mol/L, black line), and irradiated with 0.1 kGy (red line) and 1 kGy (blue line).

Raman spectroscopy was already utilized to characterize BSA vibrational modes, as well to investigate the interaction between BSA and plasmonic nanoparticles (silver or gold)40, 41, 44, and its vibrational modes were assigned. However, we were capable of monitoring more closely the cleavage of that biomolecule induced by gamma irradiation utilizing Raman/SERS spectroscopy, as shown in Figure 4. In fact, it is possible to see several Raman/SERS peaks in the spectrum of AuNPs@MPA-BSA, where the signals assigned to phenylalanine (Phe) at 500 and 925 cm-1, tyrosine (Tyr) at 570 and 695 cm-1, and for both amino acids at 1400 and 1495 cm-1, assigned to amide III group at 1130 cm-1 and to carboxylate groups at 1265 cm1 . Figure 3. Excitation spectrum of AuNP@MPA-BSA sample non-irradiated and a set of fluorescence emission spectra of -7 AuNPs@MPA-BSA (λexc = 280 nm, [BSA] = 3.75 10 mol/L, pH = 7.2 at room temperature) irradiated with increasing doses of γ radiation.

However, Gaber38 suggested this occurs only at doses lower than 1 kGy, whereas the cleavage of BSA chromophores is expected at doses higher than 1 kGy. In fact, it

The set of Raman/SERS spectra obtained for those samples exhibited an analogous tendency of signal decreasement, as found in fluorescence spectra when irradiated with 0.1 kGy to 1 kGy of gamma radiation dose. However, the signals associated with BSA chromophores disappeared, and only some low-intensity peaks related to the MPA ligands could be observed, contrast with the fluorescence in which the band intensity decayed much more slowly.



The SERS effect is enhancing selectively the scattering cross-section of the amino acid residues that are close to the AuNP surface, especially the more polarizable ones. In other words, it is important to note that SERS is a technique that probes just the molecules next to the gold nanoparticles surface, in contrast to fluorescence. Comparing the results of fluorescence and Raman spectroscopy after irradiation with 0.1 kGy of gamma radiation, the fluorescence spectrum indicated that only about 50% of chromophore species were destroyed, whereas SERS spectrum was consistent with almost complete decomposition since almost no signal associated with BSA chromophores could be detected. Accordingly, SERS is considered for analytical purposes but its limitations especially for samples with angular and/or spatial dependences.45-47 In our case, it indicates that chromophore groups next to surface are being destroyed preferentially to those farther from the surface of gold nanoparticles.

Synthesis of 198AuNPs and Effects of Radiation on BSA The sample containing gold-198 atoms was prepared in the IEA-R1 reactor which can generate a vast number of radioactive elements, by irradiating a 10 mg high purity gold-197 (99.99%) target with neutrons. The activation can be easily proved by gamma spectroscopy, as shown in Figure 5. Gamma spectrum exhibits a single peak at 411 keV assigned to the gold-198 decay to mercury-198. The presence of a single peak also strongly indicates the production of a single radioactive element, what is especially important for medicinal applications, once elements with longer lifetimes were not produced. In that decay process, less penetrating beta particles, which can cause local damages in tissue and molecules, are also emitted. Accordingly, beta and gamma emitters such as Au-198 can be used for diagnosis and treatment of tumors. The synthesis of activated gold nanoparticles was performed solubilizing the 198Au sample with Aqua Regia and evaporating the excess of acid using a vacuum pump, as described in the experimental section. The resultant salt was solubilized in deionized water and fractionated as follow. 25 mL were reserved (198Au3+) and analyzed via ascending paper chromatography method; 50 mL utilized directly to prepare 198AuNPs; and 25 mL were diluted to a quarter of the initial concentration of radioactive Au-198 by mixing with 75 mL of a solution of HAuCl4 with the same concentration of non-irradiated Au atoms and utilized to prepare the 198/197AuNPs.

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Figure 5. Gamma spectrum of the gold sample after irradiation in the IEA-R1 reactor.

The nanoparticles samples were analyzed by ascending paper chromatography method and the results shown in Figure S5 and Table 2. Visually, the chromatograms obtained with 198Au3+ solution are not informative since are almost colorless, but it is possible to see the presence of a red spot in the base of the 198AuNPs sample (Figure S5B) indicating that gold nanoparticles do not migrate and stay in origin. More detailed information relative to the behavior of the Au(III) solution and AuNPs suspension can be obtained by gamma counting analyses of the chromatograms sections, whose results are presented in Table 2. Table 2. Gamma spectroscopy analyses of the chromatogram sections presented in the Figure S5. 198

1(origin) 2 3 4 5 6 7 8 9 10 (front)

3+

Au solution (counts) 24757 18252 26827 34727 115258 108498 16302 4627 877 357

198

AuNPs suspension (counts) 415903 557 327 102 112 172 137 97 252 487

The Au(III) ions can migrate with the solvent front and distribute along the path but are more concentrated in the 5-6 sections. A similar tendency is observed for the 198 AuNPs dispersion but with a different distribution as expected for the presence of gold ions coordinated to different molecular species, probably generated during the nanoparticle formation reaction. Nevertheless, almost all radiation was measured in origin indicating that nanoparticles with strong metal-metal bonds connecting the gold atoms have very low capacity to migrate due to their stronger interaction with the paper. Considering a direct relation between the gamma counting and 198Au atoms, it is possible to estimate the yield of that reaction as being as high as 99.5% of AuNPs. This result is fascinating from the point of view of the basic understanding of synthesis since it is quite difficult


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to determine the yield of AuNPs in the synthesis using conventional non-radioactive atoms. We believe this was the first-time gold-198 was used to get the nanoparticles to yield with such precision. Then, the labile citrate protecting molecules bound on AuNPs and 198/197AuNPs were then replaced by strongly coordinating MPA to generate binding sites for conjugation with 3.75 10-6 mol/L of BSA. The suspensions were left to decay for one month (10 half-lives) in a refrigerator and analyzed by UV-Vis absorption, fluorescence and Raman spectroscopy. 198

The UV-Vis spectra of 198AuNPs and 198/197AuNPs respectively conjugated with MPA and BSA are shown in Figure S6. Both have similar spectral profile and behavior. The 198 AuNPs@MPA exhibited the LSPR band with the maximum at 524 nm which shifted to 528 nm after conjugation with BSA, whereas in the case of 198/197AuNp@MPA that band shifted from 523 to 528 nm. This result shows that both are electronically similar; however, Raman/SERS spectroscopy and fluorescence spectroscopy showed quite contrasting results.

the distance (∝ d-2). Accordingly, one can infer that the chromophores groups next to 198AuNPs surface are preferentially destroyed, and those farther away from nanoparticles surface should remain in the structure of the biomolecule, as confirmed by fluorescence spectroscopy (Figure 7). In this case, samples in three different conditions were studied: the AuNPs were conjugated by reacting with 3.75 10-6 mol/L BSA a) after modification with MPA and b) 2.7 days (one half-life of gold-198) after the modification of 198 AuNPs@cit with MPA (198AuNPs@MPA) and c) 198/197 AuNPs@MPA-BSA, nanoparticles prepared relatively with 25 % of gold-198, modified with MPA and BSA. The fluorescence spectra of 198/197AuNPs@MPA-BSA and 198 AuNPs@MPA-BSA, measured 10 half-lives of the syntheses and modifications, are presented in Figure 7. Such a difference reflects directly in the dose of radiation and, consequently, on the relative amounts of chromophores groups remaining in the BSA conjugated AuNPs, as confirmed by fluorescence spectroscopy. Accordingly, the quantity of BSA is inversely proportional to the concentration of radioactive atoms, making possible control of the degree of damage induced by gold-198 by dilution with a suitable concentration of cold Au atoms, as in 198/197AuNPs@MPA-BSA.

198/197

Figure 6. Raman/SERS spectra of AuNPs@MPA-BSA 198 -6 (black) and AuNPs@MPA-MPA (red). ([BSA]= 3.75 10 mol/L).

The Raman/SERS spectrum of 198AuNPs@MPA-BSA exhibited only peaks that can be assigned to the MPA ligand, whereas 198/197AuNPs@MPA-BSA presented more intense peaks characteristic of both BSA and MPA ligand (Figure 6). In fact, several peaks typical of chromophores groups of BSA were found at 700, 750, 1100 and 1500 cm-1. These peaks were also observed in the corresponding cold samples not irradiated with gamma radiation (Figure 4). These results indicate that the gamma and beta radiation emitted by gold-198 are destroying more rapidly the sensitive chromophore groups of BSA than the MPA ligands, as confirmed by the larger BSA/MPA peaks intensity ratio in 198/197AuNPs@MPA-BSA. It also suggests that the damage promoted by the gamma radiation probably is similar but not at the same extent. However, nanoparticles behave as punctual sources of radiation, and the dose should decay as a function of the inverse of the square of

Figure 7. Fluorescence emission spectrum of 198 ____ 198/197 AuNPs@MPA-BSA ( ) and of AuNPs@BSA-MPA (·· 198 · ·), and of AuNPs@MPA-BSA after one more half-life decay -7 (- - - -). (λexc = 280 nm, [BSA] = 3.75 10 mol/L, pH = 7.2 at room temperature).

The raw spectra obtained by fluorescence spectroscopy of gamma-irradiated samples or gold-198 samples correlate to the quantity of radiation dose or quantity of radiation emitted by radioactive atoms; however fluorescence intensity depends on several parameters intrinsic of technique, such intensity of the source, slits opening, the temperature of samples, etc. Most of these problems can be solved by adding an internal standard, but it is also a



problem, once it can interact with the others molecules quenching fluorescence or by modifying the surface of the nanoparticles. Fortunately, water fluorescence band can be used as an internal standard.48 Water has a fluorescence signal (also known as Raman scattering peak of water) which depends on excitation wavelength but appears around 25 nm higher than the wavelength on excitation. In our case, it is observed around 304 nm as a very well-defined band in AuNPs@MPA-BSA sample irradiated with 1.0 kGy (Figure 3) or as a shoulder to the sample non-irradiated (Figure 3). Thus, the intensity of fluorescence spectra can be normalized (as can be seen inserted in Figure 8 and Figure S7 and Figure S9) and an analytical curve plotted related to the internal signal of water, dividing the emission at 304 nm (water fluorescence) by 341 nm (BSA fluorescence), in function of gamma dose (Figure 8), linear regression of this curve can be seen in Figure S8. It is possible to see that AuNPs@MPA-BSA has a linear response to the gamma radiation dose (R2= 0.9998, inserted in Figure S8). Also, the fluorescence versus gamma dose-response curve can be used to estimate the equivalent dose of radiation produced by gold-198 nanoparticles. From this approach, the integrated radiation dose collected by BSA can be calculated as 0.026 kGy for 198/197 AuNPs@MPA-BSA (prepared at ~2 mCi), 0.078 kGy for 198AuNPs@MPA-BSA (prepared at ~4 mCi) and 0.211 kGy for 198AuNPs@MPA-BSA (prepared at ~8 mCi). These results show that BSA can be used as a model to evaluate the radiation dose- response caused by external sources of ionizing radiation, as well as radioactive nanoparticles. Also, It is possible to see that the irradiation dose can be fine-tuned by the addition of cold gold atoms, instead of waiting passively for radioactive decay.

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justed to the equation curve obtained from gamma samples( ) (inserted normalized fluorescence spectra of Figure 3, used to plot curve).

CONCLUSIONS A new procedure for the production of 14 nm diameter radioactive citrate protected gold-198 nanoparticles, starting with the neutron activation of a conventional gold foil, as well as the derivatives conjugated with MPA and BSA, is described. The effect of irradiation of cold AuNPs@MPA-BSA with gamma radiation was also investigated by fluorescence and Raman spectroscopy indicate that the damage to BSA chromophore groups is proportional to the dose (from 0.1 to 1 kGy) and that chromophores groups close to the nanoparticles surface are more prone to damage. Gold nanoparticles prepared from a gold-198 foil, obtained by neutron activation in a reactor, and conjugated with bovine serum albumin showed that its decay also could damage the BSA chromophores groups. However, that process is much more localized next to the nanoparticles surface where the doses are much larger since each one acts as a punctual radiation source. An analytical curve was plot using the emission of BSA (correct by water fluorescence) vs. gamma radiation doses and shows that BSA responds linearly to the damage, and it can be used to correlate damages caused by radioactive samples. Also, the degree of molecular damage was found to be proportional to the concentration of gold-198, such that dilution of the activated with cold atoms is a suitable way to control the activity of 198AuNPs aiming medical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures S1−S9, TEM additional image and histogram, extinction spectra of AuNPs@MPA modified with increasing concentrations BSA, dynamic light scattering measurement of AuNP@MPA-BSA with different concentrations of BSA, zeta potential AuNP@MPA, BSA and AuNP@MPA-BSA, ascending paper chromatograms of 198 3+ 198 Au solution and AuNPs and electronic spectra of 198 198/197 AuNPs@MPA, AuNPs@MPA before and after conjugation with BSA, normalized fluorescence spectra of AuNPs@MPA-BSA, presented in Figure 3, data points obtained from relative emission at 305 nm/ 341 nm of AuNPs@MPA-BSA of normalized spectra in Figure S8 vs. Gamma dose and linear regression and Normalized fluorescence spectra of AuNPs@MPA-BSA, presented in Figure 7. (PDF)

AUTHOR INFORMATION Figure 8. The analytical curve plotted using relative fluorescence signal at 304 nm and fluorescence signal at 341 nm of normalized AuNP-MPA-BSA samples irradiated with Gamma radiation ( ) and different gold-198 samples prepared, ad-

Corresponding Author

*Jonnatan J. Santos, E-mail: [email protected] Tel.: +55-11-2648-1376.


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ACS Applied Nano Materials ORCID Jonnatan J. Santos: 0000-0003-3789-6229 Sergio H. Toma: 0000-0002-3003-7889 Paola Corio: 0000-0002-0010-5131 Koiti Araki: 0000-0003-3485-4592

Author Contributions

‡These authors contributed equally. All authors have approved to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the financial support.

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