Optimizing Radiolabeling Amine-Functionalized Silica Nanoparticles

Apr 15, 2013 - ... Laurence M. Morandeau , Sun Chan , Charmaine M. Jeffery , Roger I. ... Karine Julienne , Patricia Le Saec , Sébastien G. Gouin , J...
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Optimizing Radiolabeling Amine-Functionalized Silica Nanoparticles Using SarAr-NCS for Applications in Imaging and Radiotherapy Linggen Kong,*,† Eskender Mume,‡,§ Gerry Triani,† and Suzanne V. Smith*,‡,§,∥ †

Institute of Materials Engineering and ‡LifeSciences, Australian Nuclear Science and Technology Organization (ANSTO), Locked Bag 2001, Kirrawee DC NSW 2232, Australia § Center of Excellence in Anti-matter Matter Studies (CAMS), Research School of Physics and Engineering, The Australian National University, Canberra, ACT 0200, Australia ∥ Collider−Accelerator Department, Brookhaven National Laboratory, Upton, New York 11973, United States S Supporting Information *

ABSTRACT: Silica nanoparticles functionalized with amine groups and in the size range of approximately 60−94 nm were produced by combining sol−gel processing and emulsion technology. Hexa-aza cage ligand SarAr-NCS was conjugated to the silica nanoparticles and subsequently radiolabeled with a solution of 57Co2+-doped carrier Co2+. The number of Co2+ ions bound to the silica particles at pH 7 was used to determine the average number of available SarAr-NCS ligands conjugated to a silica particle. For organically modified silica particles of 94.0 and 59.5 nm diameter, the maximum number of metal binding sites was determined to be 11700 and 3270 sites per particle, respectively. For silica particles (63.5 nm peak diameter) produced using an water-in-oil emulsion, the calculated average was 4480 on the particle surface. The number of SarAr-NCS conjugated on the particles was easily controlled, potentially providing for a range of products for applications in the risk assessment of particles and theranostic imaging or radiotherapy when radiolabeled with a suitable radioisotope such as 64Cu or 67Cu.

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INTRODUCTION Ceramic nanoparticles, such as silica, have attracted significant interest for biomedical applications recently.1−3 Silica particles are biologically inert, have a hydrophilic surface, and are highly biocompatible when the particle surface is properly functionalized. A number of strategies have been employed to synthesize silica particles and modify the particle surface.4 The functional groups can not only improve the dispersibility of the particles but also provide a site of attachment for radioisotopes and drugs for use in imaging and therapy. By employing sol−gel processing, we can synthesize silica nanoparticles at low temperature, protecting both the particle surface morphology and any drugs that might be encapsulated during the process.5 A combination of sol−gel processing and emulsion chemistry are the main synthesis strategies used to prepare silica nanoparticles at room temperature. One approach involves water-in-oil reverse micelles or a microemulsion using silicon alkoxide as the precursor.6−8 The other, an oil-in-water emulsion, uses organosilanes as starting materials, yielding organically modified silica (ORMOSIL) nanoparticles.9−14 Nuclear techniques offer extremely high sensitivity and accuracy for probing the properties of porous and functionalized nanoparticles.15,16 Hexaazamacrobicyclic cage ligand 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane1,8-diamine (SarAr) is efficient and accurate for determining the available carboxyl groups and possesses wider utility in the optimization of particle preparation and quality control of batches.17 Furthermore, this bifunctional ligand has remarkable stability for complexing a range of transition-metal ions rapidly.18,19 It has also been used to study porous nanomaterials © 2013 American Chemical Society

in an effort to understand solid−liquid interfaces and reactions.16,20 SarAr was designed with an amino group for conjugation to carboxylic group in proteins or other material surfaces. The conjugation of SarAr to carboxylic groups is readily achieved at mild pH 5 in the presence of excess activating agent such as 1-ethyl-3-(3-dimethy aminopropyl) carbodiimide hydrochloride (EDC). Once the reaction is complete, the byproducts are removed by simple centrifugation, and the resultant SarArconjugate (protein or particle) is radiolabeled with the radioisotope of choice. Radioactivity associated with the protein or the particles can be measured with a gamma counter. The new hexaaza cage (Figure 1), N1-(4-isothiocyanatobenzyl)3,6,10,13,16,19-hexaaza bicyclo[6.6.6]icosane-1,8-diamine (SarArNCS), has been synthesized for reaction with amino groups present in proteins and particle surfaces. The reactions between −NH2 and the −NCS on SarAr-NCS are efficient at pH >8. There are few references in the literature describing the radiolabeling of silica particles and matrices for biomedical applications. The radioisotopes used vary from the direct attachment of iodine radioisotopes, such as 125I (t1/2 = 59.408 days)21 and 124I (t1/2 = 4.1760 days ),22 to nonspecifically bound melanin on the surface of the particle for binding 188Re (t1/2 = 17.005 h) under reducing conditions23,24 and 32P (t1/2 = 14.262 days)25 using mobilized 32P-radiolabeled DNA. None of these strategies are likely Received: February 17, 2013 Revised: April 14, 2013 Published: April 15, 2013 5609

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Figure 1. Molecular structure of SarAr-isothiocyanate (SarAr-NCS). viewing chamber (model 782, Gatan, Pleasanton, CA). Samples for TEM were prepared by suspending particles in ethanol and depositing several drops onto a carbon-coated 200-mesh copper grid. The radioactivity of each sample was measured with a Wizard 1470 gamma counter (Wallac Oy, Turku, Finland). Measurements were made on triplicate samples, and gamma counting was the average of two readings after correction for background. Particle surface area and pore volume analyses were performed using a Micromeritics ASAP 2020 adsorption analyzer (Norcross, GA). Prior to measurement, the powder sample was degassed at 130 °C overnight to remove adsorbed species. The density functional theory (DFT) model was used to estimate the particle surface area and pore volume in the micropore range. The regularization value was set to 0.005 (little smoothing), with a cylindrical model selected for the analysis of the pores on the oxide surface. The densities of the powder samples were measured with a Quanta-Chrome MVP-1 multipyconometer. Synthesis of SarAr-isothiocyanate (SarAr-NCS). SarAr-NCS (p-isothiocynatobenzyl-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane1,8-diamine) was synthesized in a similar manner to that reported elsewhere.32 Typically, SarAr was synthesized using a similar procedure to that previously described.18,20 SarAr (60 mg, 0.14 mmol) was added to a round-bottomed flask, followed by the addition of both 9 mL of water and 360 μL of thiophosgene in chloroform (9 mL) to the flask with vigorous stirring at room temperature. The reaction was allowed to proceed for 4 h. Excess thiophosgene was extracted carefully from the aqueous phase with chloroform (4 × 10 mL or until a light-brown color was extracted), and the resulting aqueous phase containing the product was lyophilized to produce 63 mg of SarAr-NCS (white powder) in 94% yield. The compound was further purified by using HPLC Eclipse XDB-C18 (Agilent) with a 5 μm particle size (250 mm × 9.4 mm) with a linear gradient (0 → 10% CH3CN in water over 30 min). The compound was eluted at 20.9 min. 1H NMR: 3.12 (s, 12H, NCH2CH2N), 3.36 (s, 6H, NCCH2N), 3.50 (s, 6H, NCCH2NCCH2), 4.16 (s, 2H, ArCH2), 7.37 (d, 2H, Ar-H), 7.46 (d, 2H, Ar-H). ESI/MS: displayed a major signal at m/z = 462.4 [M + H]+, and the FT-IR showed an −NCS stretch at 2260 cm−1. Particle Synthesis. Briefly, the ORMOSIL particles were produced by an oil-in-water emulsion using NP-9 as the surfactant. The mixture of PTMS and TEOS was used as the silicon precursor; APTES was the catalyst and the source of the amine groups on the particle surface. Silica particles were synthesized by an water-in-oil microemulsion system. Triton X-100 and 1-hexanol were used as the surfactant and cosurfactant, and cyclohexane was the oil continuous phase. Silica nanospheres were produced by adding TEOS to the microemulsion, and then APTES was introduced to functionalize the particle surface with amines. The flowcharts and the detailed particle syntheses procedures are summarized in Supporting Information (SI). Procedures for Conjugating SarAr-NCS to Silica Particles and Subsequent Radiolabeling with 57/natCo. A mixture consisting of 1.454 mg of silica particles was added to a 1.5 mL eppendorf tube, and then the fresh prepared SarAr-NCS aqueous solution was added at varying molar ratios. SarAr-NCS was stored in a freezer, and freshly prepared solution was made for each conjugation study. The pH of the reaction mixture was adjusted to 9 using a buffer solution. The total volume of the final reaction mixture was 1.0 mL. The mixture was vigorously agitated using a vortex mixer for 2 min and then rotated on a RotaMix (60 rpm, 360° continuous rotation) for over 24 h at room temperature (23 ± 2 °C). The suspension was then centrifuged for 15 min at 2400 rpm before the supernatant was

to provide accurate information on the available functional sites on the surface of particles because they do not form stable covalent bonds or the radiolabel is attached via the formation of a radiolabeled complex of mixed oxidation states or indirectly via the binding of a protein to the surface of the particle. Nanoscale spherical particles (10−100 nm) have been reported to be distributed selectively to tumors within 1 to 2 days after intravenous injection.26 This occurs by virtue of the abnormal microvasculature within many tumors. Experimental data from animal studies have indicated that particles of ≤150 nm diameter, either neutral or slightly charged, are likely to be distributed within the tumor cells.27−31 We are interested to develop biodegradable silica particles of approximately 50−100 nm with a positively charge surface. In this study, we conjugated the SarAr-NCS ligand with two types of −NH2-functionalized silica nanoparticles prepared by waterin-oil and oil-in-water emulsion processes. The effect of the pH and concentration of SarAr-NCS on the conjugation chemistry is presented. A solution of natCo2+ doped with 57Co2+ (t1/2 = 271.8 days) was used to determine quantitatively the number of available SarAr-NCS ligands conjugated to the surface of the particles. The stability of these radiolabeled particles was also assessed at different pH values. The potential of SarAr-NCS as an agent for characterizing batch preparations as well as the optimization of SarAr-NCS silica particle conjugates for use in biomedical imaging and radiotherapy will be discussed.

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EXPERIMENTAL METHODS

Materials. All chemicals were A. R. grade, with Milli-Q water used in all experimental procedures. For silica particle synthesis, nonionic surfactants nonylphenoxypolyethoxyethanol [NP-9: C9H19C6H4(OCH2CH2)nOH, n = 8, 9, Sigma] and octylphenoxypolyethoxyethanol (Triton X-100: C8H17C6H4(OCH2CH2)xOH, x = 9, 10, Sigma) were used as received. Tetraethylorthosilicate (TEOS, Aldrich, 99+%), trimethoxyphenylsilane (PTMS, Aldrich, 98+%), and 3-aminopropyl triethoxysilane (APTES, Sigma-Aldrich, 98+%) were used as received. High specific activity (SA) radioisotope 57Co2+ (SA = 2.59 × 5 10 GBq/g) was obtained from PerkinElmer as CoCl2 in 0.1 M HCl. The radionuclidic purity was >99%. An analytical standard of CoCl2 (0.01 M) was supplied by Sigma-Aldrich. All solutions were prepared with analytical-grade buffer salts using high-purity Milli-Q water (with the resistance being 18.2 MΩ·cm at 25 °C), and their pH values were adjusted with the addition of concentrated sodium hydroxide or hydrochloric acid and calibrated with a pH meter. They are listed below: pH 1−3 (0.1 M sodium chloride + 0.1 M glycine); pH 4−5 (0.1 M sodium acetate); pH 6−9 (0.1 M sodium phosphate: PPB); and pH 9.5−10 (0.1 M sodium chloride + 0.1 M glycine). Characterization. The size and morphology of silica nanoparticles were analyzed on a JEOL JEM 2010F (JEOL Ltd., Akishima, Tokyo, Japan) transmission electron microscope (TEM) equipped with a field-emission gun (FEG) electron source operated at 200 kV. Brightfield TEM images were used to estimate the particle size by recording with a CCD camera mounted in the 35 mm port above the TEM 5610

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removed. The conjugated particles were washed to remove any unreacted SarAr-NCS with 1.0 mL of 0.1 M phosphate buffer (pH 7) and then centrifuged in a similar manner. The process was repeated two times, and the supernatants were discarded. A clean pellet of SarAr-NCS particles was then suspended in a buffer solution (PPB, pH 7) for radiolabeling. For the radiolabeling experiments a solution of known concentration of natCo2+ in 0.1 M PPB at pH 7 doped with 57Co2+ was prepared. The initial radioactivity added to each reaction mixture was kept within 100 000−200 000 CPM (count per minute). The washed SarAr-NCS−silica particles were suspended in 0.1 M PPB at pH 7, and 57/natCo2+ solution was added. The final volume of the reaction mixtures was kept at 1 mL. The Co2+ added to the reaction mixture was equivalent to the initial SarAr-NCS added to the conjugation reaction. The mixture was vortex mixed and then left to rotate gently using a RotaMix (60 rpm, 360° continuous rotation) for over 150 min at room temperature. The 57/natCo-SarAr-NCS-silica particle mixture was centrifuged at 2400 rpm for 15 min, and the supernatant from each sample tube was removed and counted on a gamma counter. The radiolabeled particles were washed an additional four times with 1.0 mL 0.1 M PPB at pH 7. On each occasion, the supernatant was monitored by gamma counting for the presence of uncomplexed 57/natCo2+. The associated radioactivity of the resultant radiolabeled silica particles (samples in triplicate) was determined using a gamma counter. All values were corrected for background and radioactivity associated with nonspecific controls.

the precursor and sodium bis(2-ethylhexyl)-sulfosuccinate (AOT) or Tween 80 as the surfactant. Ottenbrite14 synthesized 100−500 nm ORMOSIL using vinyltrimethoxysilane (VTMS) with Triton N-101 as the surfactant. These particles were positively charged at neutral pH because of the amine groups on the surface of the particles but not at the ideal size to target tumor tissue. The ORMOSIL nanoparticles in this study involved a different approach using an oil-in-water emulsion. The PTMS (an organosilane) was dispersed as the oil phase in a continuous phase of water in the presence of the surfactant, NP-9. APTES is generally used as a catalyst; however, in this preparation it was also used as a reactant to create amine functional groups on the particle surface. A range of conditions were investigated in the synthesis of the silica particles. In their preparation, it was noted that the use of 40 mol % TEOS increased the hydrophilicity of the particles and helped to achieve the smaller sizes required, although the use of 100 mol % PTMS resulted in the production of the largerdiameter particles. The use of small quantities of chloroform enhanced the production of the smaller particles, and >40 mol % TEOS encouraged the formation of a polydisperse material. The conditions for the synthesis of differently sized particles and their physical properties and morphology are summarized in Table 1 and Figure 2.

%radiolabeling efficiency ⎛ ⎞ activityparticles ⎟ × 100 = ⎜⎜ ⎟ ⎝ activityparticles + activitysupernatant ⎠

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Table 1. Composition, Size, and N2 Sorption Properties of Silica Nanoparticles samples PTMS (mmol) TEOS (mmol) chloroform (mL) APTES (mmol) size range: d5%−d95% (nm) peak diameter (nm) total surface area (m2/g) surface area (1.2− 38.7 nm) (m2/g) pore volume (≤38.7 nm) (cm3/g) calculated surface area (m2/g)

moles of Co2 + bound = Co2 +initial × radiolabeling efficiency

RESULTS AND DISCUSSION The application of nanotechnology to medicine enables the development of nanoparticle therapeutic carriers. These drug carriers capable of delivering cancer therapies are passively targeted to tumors through the enhanced permeability and retention effect, so they are ideally suited for the delivery of chemotherapeutics in cancer treatment.33−35 Although clinically approved nanoparticles have consistently shown value in reducing drug toxicity, their use has not always translated into improved clinical outcomes. This has led to the development of “multifunctional” nanoparticles, where additional capabilities such as targeting and image contrast enhancement are added to the nanoparticles.36 As a result, there has been considerable effort expended to incorporate both diagnostic and therapeutic functions into multifunctional nanoparticles37,38 and also to apply nanomedicine to chemoradiotherapy39 for the more effective treatment of cancer. Our work focuses on silica nanoparticles for potential biomedical applications.1−5,40 Silica Particle Synthesis. As previously mentioned, nanoscale spherical particles (10−100 nm) have been reported to distribute selectively to tumors within 1 to 2 days after intravenous injection.26 A combination of charge and size can significantly influence the likely distribution throughout tumor tissue.27−30 For this work, we were interested in developing biodegradable silica particles of approximately 50−100 nm with a positively charged surface at neutral pH. A number of groups have reported the synthesis of ORMOSIL silica particles by an oil-in-water emulsion system. In particular, Prasad et al.9−13 produced ORMOSIL particles of less than 30 nm diameter using vinyltriethoxysilane (VTES) as

O/W-1

O/W-2

12 8

12 8 1.0 12 46−73 59.5 72.9 36.5 0.055 77.6

12 67−121 94.0 47.2 23.6 0.036 49.1

W/O 3.2 0.8 55−72 63.5 72.9 32.7 0.112 72.7

Two ORMOSIL particles were prepared by oil-in-water emulsion. TEM imaging was used to determine the average diameter of the particles (sampling >300 particles), and their size distribution was reported with d5% and d95% in Table 1. One preparation had a peak diameter of 94.0 nm with diameters ranging from 67 to 121 nm (sample O/W-1), and the second yielded particles with a peak diameter of 59.5 nm, ranging from 46 to 73 nm (sample O/W-2) (Figure 2). Pure silica particles were prepared using a water-in-oil emulsion process. In this method, cyclohexane was used as the oil continuous phase, and Triton X-100 and 1-hexanol were the surfactant and cosurfactant, respectively. A TEM image of isolated particles is given in Figure 2 (sample W/O). The particles were functionalized with amine groups using APTES. The mean size of the isolated particles was 63.5 nm in diameter and ranged from 55 to 72 nm. Conjugation of SarAr-NCS to Particles and Radiolabeling with 57Co. Isothiocyanate groups (R−NCS) react with amine groups to form the relatively stable thiourea bond, (R−HN−CS−NH−R′). Reactions are typically undertaken in buffer solutions within a pH range of 8.0−9.5 over 24 h.41,42 For this study, the conjugation of SarAr-NCS to the amino groups on the particles was optimized over a pH range of 8−10 (Figure 3). The SarAr-NCS particles were separated 5611

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Figure 2. TEM images of silica particles of samples (a) O/W-1, (b) O/W-2, and (c) W/O.

Figure 4. Radiolabeling efficiency at different conjugation times for sample O/W-1 (mean ± SEM, N = 3). Experimental conditions: silica particles = 1.454 mg, SarAr-NCS = 300 nmol, [SarAr-NCS]/[Co2+] mole ratio = 1, conjugation at pH 9, and radiolabeling at pH 7. Particles were rotated 360° with 60 rpm at room temperature (23 ± 2 °C).

Figure 3. Radiolabeling efficiency at different conjugation pH values for sample O/W-1 (mean ± SEM, N = 3). Experimental conditions: silica particles = 1.454 mg, SarAr-NCS = 100 nmol, [SarAr-NCS]/ [Co2+] mole ratio = 1, and radiolabeling at pH 7.

the temperature was fixed at 23 °C. All reaction mixtures were incubated for 24 h. The SarAr-NCS conjugated particles were washed and radiolabeled with a known concentration of Co2+ solution doped with 57Co. Figure 5 illustrates the radiolabeling efficiency curve versus SarAr-NCS concentration for each particle preparation. The data show that the saturation of the available metal binding sites on all three particle types is almost reached by reacting approximately 800 nmol of SarAr-NCS with 1.454 mg of particle of various sizes. For ORMOSIL spheres of 94.0 and 59.5 nm average diameter (Figure 5a,b), we see that the curve for radiolabeling efficiency is similar, both reaching saturation at approximately 400 nmol. The total number of moles of Co2+ complexed by the particles is given by the right y axis. Although the concentration of Co2+ attached to batch of particles is similar, the calculated number of Co2+ ions attached to each particle will be dependent on the size and thus the surface area of the particles. The radiolabeling efficiency curve for the silica particles (average diameter 63.5 nm) prepared via a water-in-oil emulsion is quite different, indicating that the reaction is slower and requires a 150% increase in the SarAr-NCS reactant (up to 600 nmol) to reach saturation. The effective surface area and textural porosity of the silica nanoparticles after freeze drying were also investigated using the nitrogen adsorption/desorption technique. The N2 adsorption−desorption isotherm of sample O/W-1 in Figure S3 (SI) have been designated as type II using the IUPAC classification scheme.43 The isotherms of samples O/W-1 and W/O were similar to that of O/W-1 material (figures not shown). The reversible type II isotherm is the normal form of the isotherm obtained with a nonporous or macroporous adsorbent and represents unrestricted monolayer− multilayer adsorption. The DFT model was employed to assess the

by centrifugation and then washed with phosphate buffer at pH 7. The SarAr-NCS conjugated particles were then exposed to known concentrations of Co2+ doped with 57Co for up to 150 min. The final radiolabeled particles were washed twice with buffer to remove any nonspecifically bound Co2+. The radioactivity associated with the SarAr-NCS particles was measured using a gamma counter. A plot of the radiolabeling efficiency of the particles is illustrated in Figure 3. The data clearly show that the optimum pH for this conjugation for SarAr-NCS to silica particles is between pH 8 and 9.5. Conditions for the conjugation of SarAr-NCS to the silica particles were optimized for the reaction time, concentration of SarAr-NCS, temperature, and modes of mixing. Fixed quantities of particles were incubated with fixed concentrations of SarArNCS at room temperature over 24 h (Figure 4). Figure 4 shows a typical plot of the radiolabeling efficiency versus time of incubation. The fastest rate of conjugation occurs within 4 h, decreasing 5-fold over the next 24 h until completion. The conjugation was assessed over a range of temperature from 23 to 40 °C. Approximately a 3-fold increase in radiolabeling efficiency could be achieved at 35 °C compared to that at room temperature. No further improvement could be achieved by raising the temperature to 40 °C, which is mostly likely due to ligand degradation at high temperature. In the establishment of optimum reaction conditions, the use of different agitation modes, stationary agitation in a single plane, and complete 360° rotation were investigated. The latter was the most effective. The effect of SarAr-NCS concentration on the conjugation was also assessed. For these studies, the particle weight was fixed at 1.454 mg, the reaction volume was fixed at 1.0 mL, and 5612

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Figure 5. Radiolabeling efficiency and amount of Co2+ associated with particles for different amounts of SarAr-NCS for samples (a) O/W-1, (b) O/W-2, and (c) W/O (mean ± SEM, N = 3). Experimental conditions: silica particles = 1.454 mg, [SarAr-NCS]/[Co2+] mole ratio = 1, conjugation at pH 9, radiolabeling at pH 7.

Using data from Figure 5 and the measured surface areas, it is possible to calculate the number of active sites (available conjugated SarAr-NCS) on the surface of each particle (Table 2). For the ORMOSIL nanospheres with size diameters of 94.0 and 59.5 nm, it is estimated that there were 11 700 and 3270 SarAr-NCS conjugated to the surface of each particle, respectively. For silica particles prepared with a water-in-oil emulsion with a nominal size of 63.5 nm diameter, the available SarAr-NCS sites were approximately 4480. A plot of available sites versus particle average diameter is plotted in Figure 6. The data clearly show a linear relationship between

microporous structure of the matrix, and the data are given in Table 1. The total surface area and surface area for pore sizes from 1.2 to 38.7 nm in diameter were calculated. The pore volume was also derived for pores ≤38.7 nm. Results show that freeze-dried powders are nonporous or macroporous materials with medium pore volume and surface area and that smaller particles generally have higher surface areas. The pore volume measured in this study suggests that particles are aggregating or agglomerating during drying and probably represent the voids or interspaces between particles. The measured surface areas are consistent with the calculated geometric surface areas based on particle diameters (assuming that the particles are rigid, spherical, and nonporous) and the measured bulk density of silica powder, given in Table 2, which indirectly supports the finding that the particles are nonporous. Table 2. Estimation of the Number of Active Sites on Silica Nanoparticles samples mass (mg) density (g/cm3) diameter (nm) Co2+ at saturation (nmol) SarAr-NCS at saturation (× 1016) volume of total particles (cm3) (× 10−3) volume of each particle (cm3) (× 10−16) number of particles (× 1012) number of SarAr-NCS per particle (×103) surface area per particle (nm2) (×104) area occupied by one ligand (nm2) number of ligands per milligram of particles (× 1016)

O/W-1 O/W-2 W/O 1.454 1.30 94.0 50.0 3.01 1.12 4.35 2.57 11.7 2.78 2.37 2.07

1.454 1.30 59.5 55.0 3.31 1.12 1.10 10.1 3.27 1.11 3.41 2.28

1.454 1.30 63.5 62.0 3.73 1.12 1.34 8.34 4.48 1.27 2.83 2.57

Figure 6. Relationship between the number of active sites vs particle size.

the two components. In addition, from the calculation, one Co-SarAr-NCS complex occupies a 2.37 nm2 particle surface area on the 94.0 nm particles, and the estimated surface area for the same complex on the smaller particle (59.5 nm diameter) is higher at 5613

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3.41 nm2. This finding suggests that the number of ligands conjugated to the particle surface is most probably dependent on the curvature of the spheres resulting from the steric effect of the ligands. The smaller particles have a higher curvature, and thus one ligand occupies a larger surface area. Further analysis of the radiolabeling efficiency of the particles shows that the amount of radioactivity on the particles can be controlled for application in imaging and potentially in radiotherapy. A maximum 16.6 mCi of 57Co per milligram of 94.0 nm particles to 18.3 mCi per milligram of 59.5 nm particles is theoretically achievable. Because the Co2+ complexation is equivalent to Cu2+ complexation with SarAr-NCS, one can assume equivalent radiolabeling with carrier-free 64Cu or 67Cu; theoretically, approximately 5.3 Ci and up to 9.3 Ci of 64Cu per milligram can be loaded onto the 94.0- and 59.5-nm-diameter particles, respectively, at saturation. For 67Cu the values would be on the order of 1.7 and 1.8 Ci per milligram of these same particles, respectively. A 1000-fold decrease in the specific activity of 64Cu or 67Cu would still make the radiolabeling efficiencies for these SarAr-NCS conjugated particles very attractive for applications in radiotherapy. The calculation is explained in the Supporting Information. Figure 7 shows high-resolution TEM images of sample O/W-1. Before conjugation with a ligand, a rough surface texture was

Figure 8. Stability of radiolabeled silica particles for sample O/W-1. Experimental conditions: silica particles = 1.454 mg, SarAr-NCS = 300 nmol, [SarAr-NCS]/[Co2+] mole ratio = 1, conjugation at pH 9, radiolabeling at pH 7. Particles were rotated 360° (60 rpm) at room temperature (23 ± 2 °C). (Setting: T = 0 min as 100%).

or the breakdown of the particles in basic solution. In general, silica particles can be dissolved under basic conditions and are stable under acidic conditions.44 Therefore, it is most likely that the loss of radioactivity is associated with the Co-SarAr-NCS complex because the Co-SarAr-NCS complex is known to be stable under such conditions investigated. Further investigations are warranted to ascertain the exact byproducts.

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CONCLUSIONS Two types of silica nanoparticles, organically modified silica and normal silica functionalized with amine groups, were prepared via oil-in-water and water-in-oil emulsions by sol−gel processing. The bifunctional SarAr-isothiocyanate (SarAr-NCS) ligand was conjugated to the silica particles and radiolabeled with 57/natCo2+. After the conjugation conditions were optimized between SarAr-NCS and the silica particles, suspensions were efficiently labeled with 57 Co2+ and made stable for several days. We observed a linear relationship between the quantity of active sites and particle diameter, regardless of the synthesis method used to prepare nanoscale silica particles. Radiolabeling was found to be an effective tool for the quantitative measurement of functional groups on particulate surfaces. Further work continues to explore the potential of SarAr-NCS ligand conjugated particles in theranostic imaging and radiotherapeutic applications.

Figure 7. High-resolution TEM images of sample O/W-1 (a) before conjugation, (b) after conjugation with SarAr-NCS, and (c) after conjugation and complexing with SarAr-NCS-Co2+.

observed on the particles (Figure 7a). Following conjugation with SarAr-NCS (Figure 7b) and subsequent complexation of the Co2+ ions (Figure 7c), the particle surface appears to be smooth. Unfortunately, the concentration of Co2+ was too low to be detected by the energy dispersion spectrum (EDS). The stability of radiolabeled silica particles was also performed for sample O/W-1 (Figure 8). For this study, the silica particles (1.454 mg) were reacted with 300 nmol of SarAr-NCS and then radiolabeled with a solution of 300 nmol of Co2+ doped with 57Co. After being radiolabeling, silica particles were thoroughly washed and suspended in 1.0 mL of different pH buffer solutions and subsequently left at room temperature (23 ± 2 °C) for up to 7 days. At specific time intervals, particles were centrifuged at 2400 rpm for 15 min, and an aliquot of 0.70 mL of the supernatant (in triplicate) was extracted for gamma counting and then returned to the reaction mixture. The relative loss of radioactivity at each pH value versus time is plotted in Figure 8. The most stable condition was pH 7, with only a 3% loss of radioactivity in 24 h and up to 9% at day 7. Under acidic conditions (pH 1, 2, 3, and 5), the loss after 1 day was approximately 6% whereas at day 7 the loss was as high as 15%. For basic conditions (pH 9 and 10), the lost was 9% at day 1 and approximately 17% at day 7. The radioactivity analysis of the supernatant after the 7 day test by thin layer chromatography (TLC) showed no evidence of free Co2+, suggesting either the breakage of the SarAr-NCS−particle bond in an acidic environment

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ASSOCIATED CONTENT

S Supporting Information *

Explanation of calculations. Flowcharts and procedures for particle synthesis. Adsorption/desorption isotherm of the freeze-dried powder. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. Mark Blackford for high-resolution TEM imaging and Ms. Ilkay Chironi for particle surface area measurements. 5614

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