Cerium oxide nanoparticles (CONPs) have unique surface chemistry allowing catalyst-like antioxidant properties, and are being investigated for several disease indications in medicine. Studies have utilized surface modified CONPs toward this application, but have been lacking in comprehensive biodistribution and pharmacokinetic data and a direct comparison to uncoated CONPs. We developed an enhanced single-pot synthesis of several coated CONPs and an efficient intrinsic core labeling of CONPs with the clinical PET isotope, zirconium-89, allowing detailed PET imaging and ex vivo biodistribution. All coated [89Zr]-CONPs showed benefit in terms of biodistribution compared to uncoated [89Zr]-CONPs, while retaining the intrinsic antioxidant properties. Among these, poly(acrylic acid) coated CONPs demonstrated excellent candidacy for clinical implementation due to their enhanced renal clearance and low reticuloendothelial system uptake. This work also demonstrates the value of intrinsic core labeling and PET imaging for evaluation of nanoparticle constructs to better inform future studies towards clinical use.

1085 Objectives The incorporation of radioisotopes within CuS nanoparticles (NPs) is an attractive approach, which can combine photothermal and localized radiation therapy; with quantitative image-guidance by PET and SPECT. In this work, we designed and synthesized Zr-89 doped CuS NPs and examined their biodistribution profile by in vivo PET and ex vivo analysis. The photoacoustic signal was assessed by multispectral optoacoustic tomography (MSOT). We manipulated reaction conditions to optimize size, absorption, surface property and impact on biodistribution profile of radiolabeled NPs. Methods Towards a future development of a tri-modal imaging agent, we additionally incorporated Manganese (Mn) into the synthesis design for MR imaging. A facile aqueous phase co-precipitation synthesis was developed to produce Zr-89 radiolabeled CuxMnyS, by reacting CuCl2 and MnCl2 with Na2S in a aqueous solution of organic ligand (Polyacrylic acid) for NP coating. To enable PET imaging, Zr-89, was doped into the crystal lattice of NPs during the synthesis. The effect of Mn doping on hydrodynamic size of NP was measured by dynamic light scattering (DLS), and the impact of size on NP accumulation and clearance profile, was quantitatively measured by PET and ex vivo gamma counting. MSOT signal was assessed as a function of NP concentration. For biodistribution experiments, [Zr-89]-CuS NPs ( Results Yield of aqueous phase co-precipitation synthesis of CuMnS NPs ranged from 80-90%, by ICP measurement. HD size of NPs decreased after doping with Mn, neat CuS (100% Cu) = 45 nm, CuMnS (91% Cu & 9% Mn) = 1-2 nm, by DLS measurement). Doping of Mn red-shifted the Abs max of CuS NPs, from 930 nm to 1050 nm. The radiolabeling yield of [Zr-89]-CuMnS NPs was > 63%. The photoacoustic signal response showed linearity with NPs concentration ranging from 0.11ug/ml to 10.2ug/ml of CuS. In vivo PET imaging of nude mice showed rapid renal clearance as early as 30 min post injection, due to the very small size of NPs. This pattern was also mirrored in a biodistribution study of a separate group of animals bearing A549 tumors. Compared with most nanoparticles, the RES uptake of PAA coated CuMnS NPs is low, probably due to its negative surface charge and small size. Conclusions We demonstrated a novel synthetic methodology to create [Zr-89]-labeled ultra-small CuMnS NPs, with high photoacoustic signal for dual PET/MSOT molecular imaging. Extremely small HD size and high negative surface charge of NPs facilitated rapid renal clearance and low uptake in RES organs.

Here we describe a novel strategy to incorporate indium-111 into near infrared (NIR) emitting Cu-In-Se quantum dots (CIS-QDs) to synthesize intrinsically radiolabeled QDs (rQDs), as a quantitative tool for in vivo SPECT/fluorescence imaging. Multidentate zwitterionic polymer ligands were used to functionalize and improve the stability of CIS-rQDs and reduce nonspecific binding with plasma proteins/cell membrane. CIS-rQDs were taken up by colorectal adenocarcinoma (COLO-205) and human epidermoid carcinoma (KB-3-1) cells at low uptake rate (∼0.4%, 2 × 105 QDs per cell at 24 h) and reduced nonspecific interaction of zwitterionic CIS-rQDs with cells was observed by fluorescence microscopy. The cytotoxicity of CIS-rQDs was reduced due to the low toxic inorganic composition of QDs and multidentate zwitterionic surface coating. In 5 out of 6 nude mice bearing either COLO-205 or KB-3-1 tumor, both SPECT and fluorescence imaging demonstrated passive localization of CIS-rQDs in the tumor as early as 6 h post-injection. In these mice the passive accumulation of CIS-rQDs in the tumor, due to leaky vasculature, ranged from ∼0.3% ID per g to ∼4.6% ID per g at 48 h post-injection (from region of interest analysis of SPECT imaging). This intrinsic radio-labeling strategy provides a nanoparticle platform which incorporates imaging and potentially therapeutic radionuclides with retention of fluorescence intensity. It also provides complimentary quantitative data capabilities for both in vivo SPECT imaging and radiotracer ex vivo analysis.

Towards the development of iron oxide nanoparticles with intrinsically incorporated radionuclides for dual Positron Emission Tomography/Magnetic Resonance Imaging (PET/MRI) and more recently of Single Photon Emission Computed Tomography/Magnetic Resonance Imaging (SPECT/MRI), we have developed intrinsically radiolabeled [(59)Fe]-superparamagnetic iron oxide nanoparticles ([(59)Fe]-SPIONs) as a proof of concept for an intrinsic dual probe strategy. (59)Fe was incorporated into Fe3O4 nanoparticle crystal lattice with 92±3% efficiency in thermal decomposition synthesis. Multidentate poly(acrylic acid)-dopamine-poly(ethylene-glycol-2000) (PAA-DOP-PEG) ligands were designed and synthesized based on facile EDC chemistry and utilized to functionalize the [(59)Fe]-SPIONs. The transverse relaxivity of [(59)Fe]-SPIONs (97±3 s(-1)mM(-1)) was characterized and found to be similar to non-radioactive SPIONs (72±10 s(-1)mM(-1)), indicating that (59)Fe incorporation does not alter the SPIONs' MRI contrast properties. [(59)Fe]-SPIONs were used to evaluate the nanoparticle biodistribution by ex vivo gamma counting and MRI. Nude mice (n=15) were injected with [(59)Fe]-SPIONs and imaged at various time points with 7T small animal MRI scanner. Ex vivo biodistribution was evaluated by tissue-based gamma counting. MRI signal contrast qualitatively correlates with the %ID/g of [(59)Fe]-SPIONs, with high contrast in liver (45±6%), medium contrast in kidneys (21±5%), and low contrast in brain (4±6%) at 24 hours. This work demonstrates the synthesis and in vivo application of intrinsically radiolabeled [(59)Fe]-SPIONs for bimodal detection and provides a proof of concept for incorporation of both gamma- and positron-emitting inorganic radionuclides into the core of metal based MRI contrast agent nanoparticles.

Abstract Nanomedicine is an emerging field with increasing applications in cancer diagnosis and therapy. However, these applications strongly depend on the nature of cellular and tissue interactions in vitro and in vivo, which in turn is strongly influenced by properties of nanomaterials. The effect of surface modification of nanoparticles on their interaction with biological compartments is not yet well understood. Here, a series of multidentate zwitterionic polymeric ligands were synthesized and applied to functionalize the surface of quantum dots (QDs), which have been extensively used for fluorescent imaging in vitro and in vivo. The structure of polymeric ligands was tuned by changing their composition, and QDs with different surface charge density were obtained using these polymers as coating ligands. Highly stable, compact and biocompatible hydrophilic QDs were obtained after ligand exchange and QDs were tuned to have zeta potentials from -25 mV to -55 mV. Compared with small zwitterionic ligand coated QDs, cell viability of multidentate zwitterionic polymer ligand coated QDs were improved due to their better bio-stability originating from the multiple chelation of polymer ligand with surface of nanoparticles. The interaction of QDs, having different surface charge density, with various tumor/normal cell lines (KB 3-1, KB 8-5, COLO 205, A-549, FaDu, SK-OV-3, and HEK-293) was investigated. The surface charge densities of the nanoparticles profoundly influence their interactions with most cells. High surface charge (-55 mV and -40 mV) density increased interaction of nanoparticles with some cell membranes of tumor cells such as COLO 205 and KB 3-1. However, other tumor cells showed less sensitivity to the surface charge density of nanoparticles and displayed reduced nonspecific binding (FaDu, A-549, and SK-OV-3). The normal prostate cell line HEK-293 showed lowest interaction with these zwitterionic QDs. The surface bioconjugation of QDs with folic acid increased the intracellular internalization of QDs in KB 3-1 cell lines (with high folate receptor expression) compared with COLO 205 cell lines (low folate receptor expression), most likely due to the folate receptor mediated endocytosis. From our initial investigations, the surface properties of nanoparticles influence the nature of their interactions at the cellular level. Such information could guide in vivo studies of these nanomaterials within an intact in vivo environment. Citation Format: Minghao Sun, Purnima Jose, Likun Yang, Gobalakrishnan Sundaresan, Li Wang, Jamal Zweit. Surface engineering of quantum dots with multidentate polymer ligands: surface charge density affect on interactions at the nano-bio interface in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4142. doi:10.1158/1538-7445.AM2013-4142

Cerium oxide nanoparticles (CONPs) have demonstrated protection properties against oxidation in various cells and tissues. The mechanism of this, however, is poorly understood. Monitoring the interaction of CONPs with biological compartments 'in situ' is crucial to understand their biochemical and physiological properties in vivo. In this paper, a multifunctional nanoparticle platform was obtained through an intrinsic radiolabeling strategy and extrinsic surface functionalization to combine dual imaging components (Single Photon Emission Computed Tomography/Optical Imaging, SPECT/OI) in one nanoparticle. The cell viability, cell uptake and overall in vivo biodistribution of CONPs were also manipulated through surface functionalization. The intrinsic radiolabeling strategy is demonstrated by incorporating radionuclides (141Ce, 111In or 65Zn) into CONPs and a radiolabeled CONP (rCONP) was coated with biocompatible polymers including Dextran T10 (DT10), poly(acrylic acid) (PAA), or functionalized DT10 (DT10-NH2, DT10-PEG and DT10-sulfobetaine). Fluorescent CONPs were obtained through conjugation of fluorescein isothiocyanate (FITC) with DT10-NH2 rCONP and used for cell imaging. The DT10 and DT10-NH2 rCONP did not show decreased viability up to 120 μg mL-1 whilst the PAA rCONP showed decreased viability beyond 40 μg mL-1. Variations in blood circulation and renal/hepatic clearance of rCONPs were demonstrated and were dependent on surface coating and the hydrodynamic size of nanoparticles. The ex vivo biodistribution results were reflected in SPECT imaging of 141Ce-rCONPs, showing accumulation in the liver and spleen of a living mouse over a one week period. The intrinsic radiolabeling and extrinsic surface modifications together determine the biophysical properties of CONPs and their potential applications for in vivo studies and biomedical imaging.

Surface functionalization of nanoparticles is an important determinant of their interactions with biological compartments at the nano-bio interface. In this paper, a series of multidentate zwitterionic polymeric ligands were synthesized and used to functionalize the surface of quantum dots (QDs). The structure of polymer ligands was designed by changing the molar ratio of reactants and precursors used in the reaction. A three-component micro-emulsion method was developed to improve the efficiency of ligand exchange and avoid cross-linking reactions. Highly stable, compact and biocompatible zwitterionic QDs with different surface charge densities were obtained after ligand exchange. Variation of the surface charge density of QDs was verified by zeta potential measurements. The interaction of zwitterionic QDs with different cancer and normal cell lines (KB 3-1, COLO 205 and HEK 293) was surface charge density dependent. From cell viability studies, it was shown that higher surface charge density resulted in lower cytotoxicity of zwitterionic QDs when incubated with both cancer and normal cell lines. Furthermore, the feasibility of conjugating functionalized QDs (coated with amine zwitterionic polymer ligands) with a biomolecule was demonstrated. This was exemplified by the conjugation of amine zwitterionic QDs with a cRGD peptide, which showed improved interaction of cRGD-QDs with ανβ3 integrin receptors expressed on U87MG glioblastoma tumor cells. Engineering the surface charge density and functionalization of nanoparticles, by multidentate zwitterionic ligands, provides a strategy to tune the surface properties of QDs, which impacts their cytotoxicity and cellular interaction at the nano-bio interface.

A novel approach was developed to synthesize radioactive quantum dots (r-QDs) thereby enabling both optical and radionuclide signals to be detected from the same intrinsic bimodal probe. This proof-of-concept is exemplified by the incorporation of the radionuclide (109)Cadmium into the core/shell of the nanoparticle. Green and near infrared (NIR) emission intrinsic r-QDs were synthesized and characterized. Zwitterionic and Poly-polyethlene glycol (PEGylated) ligands were synthesized and used to coat r-QDs. Zwitterionic NIR r-QDs (quantum yield = 11%) and PEGylated NIR r-QDs (quantum yield = 14%) with an average size of 13.8 nm and 16.8 nm were obtained respectively. The biodistribution of NIR zwitterionic and PEGylated r-QDs in nude mice was investigated and zwitterionic r-QDs showed longer blood circulation (t(1/2) = 21.4±1.1 hrs) than their PEGylated counterparts (t(1/2) = 6.4±0.5 min). Both zwitterionic and PEGylated r-QDs exhibited progressive accumulation in the liver and spleen, but the magnitude of the accumulation (%ID/g) was about 3-6 fold higher with the PEGylated r-QDs at all the time points. The results demonstrated the feasibility of r-QDs synthesis in quantitative yield and retention of fluorescence following incorporation of radioactivity into the core/shell of the nanoparticle. The gamma signal from the same fluorescent elemental material enabled quantitative and robust pharmacokinetic measurements and how these changed depended on the type of coating ligands used. This strategy for intrinsically radio-labeling the QDs is currently being implemented in our laboratory for the incorporation of other radiometals.

235 Objectives To quantitatively evaluate the biodistribution of novel CdTeSe/109CdZnSe radio quantum dots (rQDs). These rQDs are novel and would mimic the biodistribution of native QDs because the radioisotope is incorporated into the QD’s structure, as opposed to being chemically chelated. Methods We synthesized near-infrared rQDs, coated with a zwitterionic DHLA ligand, to demonstrate the feasibility of incorporating the radiolabel into the rQDs’ structure. We studied the in vivo biodistribution of the rQDs by i.v. injection into Colo-205 tumor bearing nude mice. Tissues were harvested at various time points, between 5 minutes and 168 hours (n=3 mice/time point). The percent injected dose per gram (%ID/g) value was calculated. The near-infrared fluorescent signal from the rQDs was characterized by multi-spectral imaging. Results The calculated blood half-life was 22±2 hours while notable accumulation of the rQDs was seen in the liver (31.9±3.1 %ID/g at 168 hours) and spleen (7.1±0.8 %ID/g at 168 hours). Even at 48 hours post injection, 9.0±1.2 %ID/g of rQDs was available in the blood, demonstrating bioavailability of these non-targeted rQDs. Notable retention of the rQDs was seen in the tumor (5.1±0.58 %ID/g) at 48 hours post injection, possibly due to the enhanced permeability and retention effect. Measurable fluorescent signal was achieved with non-toxic concentrations of the rQDs in mice. Conclusions This work demonstrates the concept of in vivo quantitation of rQDs incorporating a radioisotope in the nanopraticle lattice, thereby providing a more direct measurement of the core/shell structure. The 22 hour blood half-life indicates that zwitterionic coating effectively reduces scavenging by the mononuclear phagocyte system. Further investigation will characterize zwitterionic ligand coated probes with different Zeta potentials as well as incorporating different imaging/ therapeutic radioisotopes and targeting molecules

A new magnetic resonance imaging (MRI) contrast agent based on the trimetallic nitride templated (TNT) metallofullerene Gd(3)N@C(80) was synthesized by a facile method in high yield. The observed longitudinal and transverse relaxivities r(1) and r(2) for water hydrogens in the presence of the water-soluble gadofullerene 2 Gd(3)N@C(80)(OH)(approximately 26)(CH(2)CH(2)COOM)(approximately 16) (M = Na or H) are 207 and 282 mM(-1) s(-1) (per C(80) cage) at 2.4 T, respectively; these values are 50 times larger than those of Gd(3+) poly(aminocarboxylate) complexes, such as commercial Omniscan and Magnevist. This high (1)H relaxivity for this new hydroxylated and carboxylated gadofullerene derivative provides high signal enhancement at significantly lower Gd concentration as demonstrated by in vitro and in vivo MRI studies. Dynamic light scattering data reveal a unimodal size distribution with an average hydrodynamic radius of ca. 78 nm in pure water (pH = 7), which is significantly different from other hydroxylated or carboxylated fullerene and metallofullerene derivatives reported to date. Agarose gel infusion results indicate that the gadofullerene 2 displayed diffusion properties different from those of commercial Omniscan and those of PEG5000 modified Gd(3)N@C(80). The reactive carboxyl functionality present on this highly efficient contrast agent may also serve as a precursor for biomarker tissue-targeting purposes.