Engineered magnetic hybrid nanoparticles with enhanced relaxivity for tumor imaging

Biomaterials 34 (2013) 7725e7732

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Engineered magnetic hybrid nanoparticles with enhanced relaxivity for tumor imaging Santosh Aryal a, b, Jaehong Key a, b, Cinzia Stigliano a, b, Jeyarama S. Ananta a, b, Meng Zhong a, b, Paolo Decuzzi a, b, c, * a

Department of Translational Imaging, The Methodist Hospital Research Institute, Houston, TX 77030, USA Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX 77030, USA BioNEM e Center for BioNanotechnology and Engineering, Department of Experimental and Clinical Medicine, University of Magna Graecia, Catanzaro 88100, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 June 2013 Accepted 1 July 2013 Available online 17 July 2013

Clinically used contrast agents for magnetic resonance imaging (MRI) suffer by the lack of specificity; short circulation time; and insufficient relaxivity. Here, a one-step combinatorial approach is described for the synthesis of magnetic lipidepolymer (hybrid) nanoparticles (MHNPs) encapsulating 5 nm ultrasmall super-paramagnetic iron oxide particles (USPIOs) and decorated with Gd3þ ions. The MHNPs comprise a hydrophobic poly(lactic acid-co-glycolic acid) (PLGA) core, containing up to w5% USPIOs (w/ w), stabilized by lipid and polyethylene glycol (PEG). Gd3þ ions are directly chelated to the external lipid monolayer. Three different nanoparticle configurations are presented including Gd3þ chelates only (Gd-MHNPs); USPIOs only (Fe-MHNPs); and the combination thereof (MHNPs). All three MHNPs exhibit a hydrodynamic diameter of about 150 nm. The Gd-MHNPs present a longitudinal relaxivity (r1 ¼ 12.95  0.53 (mM s)1) about four times larger than conventional Gd-based contrast agents (r1 ¼ 3.4 (mM s)1); MHNPs have a transversal relaxivity of r2 ¼ 164.07  7.0 (mM s)1, which is three to four times larger than most conventional systems (r2 w 50 (mM s)1). In melanoma bearing mice, elemental analysis for Gd shows about 3% of the injected MHNPs accumulating in the tumor and 2% still circulating in the blood, at 24 h post-injection. In a clinical 3T MRI scanner, MHNPs provide significant contrast confirming the observed tumor deposition. This approach can also accommodate the co-loading of hydrophobic therapeutic compounds in the MHNP core, paving the way for theranostic systems. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Contrast agents Gd-DOTA SPIO Polymeric particles MRI

1. Introduction Nanoparticles have been proposed for the intravascular administration of imaging and therapeutic agents. As compared to freely administered molecules, nanoparticle can provide multiple functionalities simultaneously, such as imaging, therapy, sensing and targeting [1e3]. In this regard, the design and synthesis of magnetic nanomaterials have attracted the interest of numerous investigators for improving the performance of contrast agents in magnetic resonance imaging (MRI) [4e8], in vitro cell separation and manipulation [9]; controlled and triggered release of therapeutic agents [10,11]; and thermal ablation-based therapies via alternating magnetic fields [12,13]. Super-paramagnetic iron oxide

* Corresponding author. Department of Translational Imaging, The Methodist Hospital Research Institute, Houston, TX 77030, USA. E-mail addresses: [email protected], [email protected] (P. Decuzzi). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.07.003

nanoparticles (SPIOs) and gadolinium chelates (Gd-chelates) are the most clinically successful and safe contrast agents for T1- and T2-weigthed MR imaging, respectively [4,6,14e22]. Sufficiently small SPIOs are decomposed by the acidic endo/lisosomal environment and the resulting iron is assimilated by the body for the synthesis of metalloproteins such as hemoglobin [17]; whereas Gdchelates firmly sequester the metal ions (Gd3þ) limiting transmetallation and the consequent possible occurrence of nephrogenic systemic fibrosis [23]. The major limitations of the currently clinically used contrast agents for MR imaging reside in the lack of tissue specificity, inevitably affecting the signal-to-noise ratio; the short circulation time, due to their rapid excretion thought the kidneys and non-specific sequestration by reticulo-endothelial system (RES); and the insufficient relaxivity. Systemically injected SPIOs are easily recognized and sequestered by macrophages residing within the RES organs, primarily the liver and spleen, so that their circulation half-life is limited to a few minutes and the portion of the injected dose reaching the biological

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target may be insufficient to induce any significant contrast enhancement. A quite successful and well-known technology to minimize RES uptake consists in coating SPIOs with hydrophilic polymers, such as polyethylene glycol (PEG) and dextran [24e27]. For instance FeridexÒ (r2 ¼ 120 (mM s)1) and SupravistÒ (r2 ¼ 57 (mM s)1) have their magnetic cores coated with hydrophilic polymeric chains. This has shown to prolong the half-life in the circulation up to w2 h [28e30], but it also may modulate the relaxation performance [31]. Different nanoparticle formulations engulfing multiple SPIOs have been recently developed in the attempt of extending the circulation half-life and, possibly, enhancing the relaxivity. For instance, 40 nm PEG coated iron oxide nanoparticles induced, at 24 post-injection, a 30% enhancement in contrast as compared to immediately post-injection [32]. This confirms that long circulating and sufficiently small nanoparticles can take advantage of the hyperpermeability of the tumor vessels and accumulate progressively therein [33]. Clusters of SPIOs selfassembled with block copolymers of poly(ethyleneimine) (PEI), poly(caprolactone) (PCL) and PEG have shown in vitro relaxivities up to 300 (mM s)1 at 1.41 T, for complexes of about 80 nm in diameter [6]. Similarly, magnetic micelles with high transversal relaxivity and long circulation half-life were demonstrated by assembling together SPIOs, molecules of paclitaxel and PEG for a total average size of w180 nm [7]. In this work, we demonstrate the synthesis of lipid coated poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles encapsulating hydrophobic ultra-small SPIOs (USPIOs; with a magnetic core of 5 nm) and directly conjugated to Gd-DOTA (Gd-1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) through its superficial lipid coating. The resulting magnetic lipidepolymer (hybrid) nanoparticles (MHNPs) are characterized for their physicochemical properties and stability under physiological conditions. Three different nanoparticles are synthesized including Gd-DOTA only (Gd-MHNPs); USPIOs only (Fe-MHNPs); and the combination thereof (MHNPs). The loading efficiency and in vitro MRI relaxometric properties are quantified for all three different systems. In mice developing a melanoma tumor on their flank, the MHNPs have been systemically injected and their specific organ accumulation has been measured via inductively coupled plasma mass spectroscopy (ICP-MS) on the element Gd. A clinical 3T MRI scanner has been used for imaging the malignant mass. 2. Materials and methods 2.1. Materials DSPE-PEG-COOH (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-(polyethyleneglycol)-2000), egg PC (L-a-phosphatidylcholine, egg, chicken), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), (L-a-phosphatidyl ethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), PE-NBD) were purchase from Avanti Polar Lipid Inc. and used as received. PLGA (50:50) was purchased from Lactate Absorbable Polymers e DURECT Corporation. N-Hydroxysuccimidyl ester activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was purchased from MACROCYCLICS. All other chemicals and solvents were purchased from SigmaeAldrich and used as received. 2.2. Synthesis of Gdelipid Gdelipid was synthesized as illustrated in Supporting information Fig. S1 in two step preparation. In the first step, DSPE was conjugated with DOTA-NHS to form Lipid-DOTA which when reacted with gadolinium nitrate yield Gdelipid. Briefly, 100 mg (0.13 mmol) of DSPE was hydrated with PBS for 5 h and 100.3 mg (0.2 mmol) of solution of DOTA-NHS in PBS was added and the reaction was carried for 6 h. After the completion of the reaction, the reaction mixture was extensively dialyzed (Mw cutoff ¼ 3.0k) with water to remove excess of DOTA and any water soluble byproducts (Yield 70% by weight). Thus formed lipid-DOTA was further treated with an excess of gadolinium nitrate dissolved in acetate buffer (pH 6.0) for 3 days at 50  C. Resulting Gdelipid complex was purified by extensive dialysis against water and was freeze dried to get dry powder. Sample was analyzed by ICP-OES to determine the reaction yield (75% by weight).

2.3. Synthesis and characterization of lipidepolymereinorganic hybrid nanoparticles (MHNPs) MHNPs with polymeric cores consisting of the 5 nm sized hydrophobic USPIOs and Gdelipid shell were prepared through a single step nanoprecipitation method. In a typical experiment, 200 mg of Gdelipid and 260 mg DSPE-PEG-COOH were dissolved in 4% ethanol at 68  C, to this solution, PLGA (1 mg, Mn w 50 kDa) and a calculated amount of USPIOs dissolved in acetonitrile were added drop wise while heating and stirring. Then the vial was vortexes for 3 min followed by the addition of water (1 mL). The solution mixture was stirred at room temperature for 2 h, washed using an Amicon Ultra centrifugal filter (Millipore, Billerica, MA) with a molecular-weight cutoff of 10 kDa. Finally, the purified MHNPs were collected in 1 mL water or phosphate buffer saline and stored at 4  C. MHNP-Gd, MHNP-Fe and control particles were prepared similarly by replacing necessary constituents with bare PLGA, Egg-PC and PLGA/Egg-PC (for control NPs, Gdelipid was replaced by EggPC). The nanoparticle size and surface zeta potential were obtained from nine measurements (n ¼ 9) by dynamic light scattering (DLS) (Malvern Zetasizer, ZEN 3600) with a backscattering angle of 173 . The morphology of the particles was characterized by scanning electron microscopy (SEM) (ZEISS NEON 40). Samples for SEM were prepared by dropping nanoparticle suspension (5 mL) onto a polished silicon wafer. After drying the droplet at room temperature overnight, the sample was coated with platinum and then imaged by SEM. Further to understand the internal structure and the distribution of USPIOs into the polymeric core, the TEM measurements were performed. Samples for TEM were prepared by drop casting method over cupper grid using phosphotungustic acid as a negative stain. The quantitative amount of USPIOs per particle was determined by ICP-OES analysis. Time dependent stability of MHNPs was carried for the period of one by measuring the size and PDI using DLS. 2.4. In vitro cytotoxicity and cellular internalization studies First, cells were seeded at 8  103 per well in 96-well plates and incubated for 24 h to reach 50% confluence. Then the culture media were replaced with 150 mL of fresh media and cells were incubated with different concentrations of Fe in MHNPs, cells with out MHNP treatments were taken as a control. After 24 h of incubation, the cells were washed with PBS and incubated in fresh media for 48 h. Cellular viability was then determined using the XTT assay following a protocol provided by the manufacturer. Results were presented in relative viability with respect of control cells. For cellular internalization study, PE-NBD labeled MHNPs were incubated with J-774 cells for 1 h. After 1 h of incubation, cells were washed with PBS three times, nucleus was stained with DAPI and fixed with 4% formalin. Fixed cells were imaged under confocal microscopy. 2.5. Relaxometric analysis In vitro relaxation times were measured in a Bruker Minispec (mq 60) bench-top relaxometer (B0 ¼ 1.41 T) operating at 60 MHz and 37  C. The longitudinal (T1) relaxation times were obtained using inversion recovery pulse sequence. The transverse (T2) relaxation times were measured using CarrePurcelleMeiboomeGill (CPMG) sequence. In vitro T2-weighted MR phantom studies were performed in a clinical 3T scanner (Philisp IngeniaÒ) using turbo spin echo (TSE) sequence with TR ¼ 2500 ms, TE ¼ 100 ms and a slice thickness of 400 mm. Phosphate buffer saline (PBS) suspension of MHNPs was used for phantom study. 2.6. In vivo studies In vivo studies were carried out in accordance with IACUC approved protocol. A male nu/nu mouse (8e10 weeks old) was used. Before the imaging procedure and contrast agent administration, mice were anesthetized with isoflurane and kept under its influence for the duration of the experiment (maximum 4 h) and euthanized by CO2 overdose and cervical dislocation at the end of the imaging session. The contrast agents used were injected intravenously through the tail vein using 0.5 mL insulin syringes. Mice were kept on a 12 h lightedark cycle with food and water ad libitum. All animal experiments in this study were approved by the Institutional Animal Care & Use Committee (IACUC) of The Methodist Hospital Research Institute. 2.7. Cell line and tumor model B16eF10 cells (from ATCC, Rockville, MD, USA) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin, and maintained at 37  C in a 5% CO2 incubator. All the cell culture products were purchased from Invitrogen (Carlsbad, CA, USA). For the tumor model, 106 B16eF10 cells in 200 mL PBS were injected subcutaneously into the flank of 12-weeks old male Nude mice (Nu/Nu) purchased from Charles River (Wilmington, MA, USA). Mice were kept on a 12 h lightedark cycle with food and water ad libitum. All animal experiments in this study were approved by the Institutional Animal Care & Use Committee (IACUC) of The Methodist Hospital Research Institute.

S. Aryal et al. / Biomaterials 34 (2013) 7725e7732 2.8. Biodistribution and in vivo MR imaging 10e15 days after tumor implantation, the mice were injected intravenously with MHNPs (6 mg/kg of Fe and 0.78 mg/kg Gd). After 24 h of injection mice were euthanized by CO2 overdose followed by cervical dislocation and organs were collected. The collected organs were digested by nitric acid and hydrogen peroxide for ICP-MS analysis. MR imaging were performed at pre-injection and 4 h postinjection. T2-weighted MR images were acquired in a 3T clinical scanner (Philips IngeniaÒ) using spin echo sequence with TR ¼ 3000 ms, TE ¼ 100 ms, and a slice thickness of 400 mM. MRI images were analyzed using OsiriX imaging software e DICOM viewer (http://www.osirix-viewer.com/). All animal experiments performed were in line with the institutional guidelines on the ethical use of animals.

3. Results and discussion 3.1. Synthesis and characterization of Gdelipid complex The proposed magnetic hybrid nanoparticles (MHNPs) comprise three distinct compartments with specific functions (Fig. 1A): i) a solid PLGA polymeric core, acting as a cytoskeleton and providing mechanical stability, and encapsulating poorly water-soluble payloads, such as the hydrophobic USPIOs or drug molecules; ii) a lipid

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shell wrapped around the core, acting as a cell membrane, where the Gd-DOTA molecules are anchored; and iii) a hydrophilic polymer stealth layer outside the lipid shell, enhancing nanoparticle stability and circulation lifetime [27,34]. In generating lipidepolymer MRI nanoconstruct, the first step was to form Gdelipid complex (Fig. 1A), beginning with the synthesis of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as demonstrated in synthetic scheme (Supporting information Fig. S1). DOTA is a macrocyclic chelator that chelates Gd3þ-ions in its cyclic core and protects the rapid diffusion of Gd3þions thereby reducing toxicity and the possible occurrence of nephrogenic systemic fibrosis. Briefly, phosphate buffered saline, pH 7.4, hydrated DSPE is reacted with DOTA-N-hydroxysuccinimide ester (DOTA-NHS) for 6 h, which results in the formation of DSPEDOTA (lipid-DOTA, m/z: 1134.8 [M þ H]þ) conjugates (Supporting information Fig. S2). The formed lipid-DOTA was further reacted with Gd(NO3)3 at pH 6.0 under mild heating at 50  C for three days, so that the Gd3þ ions could be chelated with the carboxylate ions of the DOTA moiety forming a stable Gdelipid complex. These

Fig. 1. Geometrical configuration and physiochemical characterization of the magnetic hybrid nanoparticles (MHNPs). (A) Schematic representation of the engineered MHNPs showing a lipid coated poly(lactic acid-co-glycolic acid) (PLGA) core containing 5 nm USPIOs and conjugated on the surface with Gdelipid complexes (see inset). The MHNPs were synthesized by a single step nanoprecipitation process. (B) MHNP hydrodynamic size and surface x potential characterized by dynamic light scattering measurements and electrophoretic measurement in aqueous suspension. (C) Scanning electron measurement (SEM) of MHNPs. The inset shows a representative TEM image showing densely packed USPIOs within the MHNP core (scale bar 100 nm). (D) Time dependent stability of MHNPs assessed in PBS at 37  C over a period of 7 days. Each day, the MHNPs were measured by DLS to study the hydrodynamic size and stability over time.

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complexes were extensively dialyzed against deionized water to carefully remove unchelated Gd3þ ions and the conjugation yield was assayed using ICP-OES analysis. 3.2. Synthesis and characterization of magnetic hybrid nanoparticles (MHNPs) After their synthesis, Gdelipid complexes were used along with PLGA and USPIOs for assembling the actual magnetic lipidepolymer nanoparticles (MHNPs) (Fig. 1A), following previously described protocols [27,35]. Three types of nanoparticles were fabricated: Gd-MHNP, Fe-MHNP and Gd/Fe-MHNP, simply referred to as MHNP. The Gd-MHNP contains Gdelipid only, Fe-MHNP encapsulates USPIOs only, and the MHNP is a combinatorial system containing both Gdelipid and USPIOs. The control nanoparticles had no contrast agents. The physico-chemical properties and morphological characterization of MHNPs were determined using dynamic light scattering (DLS), scanning electron microscopy

Fig. 2. Determination of Gd and Fe content in MHNPs using inductively coupled plasma-optical emission spectroscopy (ICP-OES). (A) Determination of encapsulation efficiency (Ee% ¼ output/initial input  100). (B) Optimization of USPIOs loading into the MHNPs. Fe yield was determined by using ICP-OES analysis per mg of PLGA.

(SEM) and transmission electron microscopy (TEM), as shown in Fig. 1BeD, respectively. The hydrodynamic size of the MHNPs showed a diameter ranging between 140 nm and 150 nm, depending on the payload, and with a very low polydispersity index (PDI) varying between 0.1 and 0.15. Importantly, the DLS data showed a single nanoparticles population with no secondary peaks, thus excluding the presence of PEGelipid micelles in the suspension (Fig. S3). This demonstrates that the MHNPs are highly monodispersed and do not form any clusters. Note that the narrow variation in overall diameter observed with the different configuration should possibly be ascribed to the coating of the MHNPs. These nanoparticles are coated with DSPE-PEG-COOH of fixed molecular-weight providing similar hydration condition and PLGA amount for all the MHNPs. As confirmed by SEM analysis (Fig. 1C),

Fig. 3. Cytotoxicity and cellular colocalization of MHNPs in J-774 cells. (A) Cytotoxicity profile of MHNPs, containing both Gd3þ-ions and USPIOs, in J-774 cells assessed by XTT assay at 24 h post-incubation. Results were presented as percentage cell viability (mean  SD) from 6 independent experiments and normalized to cells incubated in the absence of any MHNPs. (B) Fluorescent microscopy micrographs showing the subcellular localization of MHNPs. MHNPs were labeled with green fluorescent dye (L-aphosphatidylethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), PE-NBD) and nucleii were stained with 40 ,6-diamidino-2-phenylindole (DAPI).

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the MHNPs are highly monodispersed in agreement with DLS measurement (Fig. S3), spherical in shape; and, under dry conditions, exhibit a diameter ranging between 100 and 120 nm. The inset of Fig. 1C shows a representative TEM image of Fe/Gd-MHNPs, clearly showing the USPIOs loading (darker spots) into the core of the nanoparticles. The measurement of the MHNP surface x potential revealed a net charge 25.0  3.0 mV in aqueous suspension, in agreement with the presence of carboxylate terminated group on the surface (i.e. the DSPE-PEG-COOH moiety). Furthermore, the MHNP stability in phosphate buffer saline (PBS), at pH 7.4, was studied over a period of 1 week. No appreciable change in size was observed as documented in Fig. 1D. 3.3. Contrast agent loading and cytotoxicity studies We next examined the USPIOs loading efficiency keeping the Gdelipid concentration constant for all formulations. As shown in Fig. 2A, we have precisely loaded USPIOs and Gd into the MHNPs with initial input of 10% USPIOs and 20% of Gdelipid with respect to the weight of PLGA and observed 40% and 30% encapsulation efficiency, respectively. In this system, the Gd3þ-ions are part of the lipid monolayer stabilizing the PLGA core, as such the Gdelipid concentration was kept constant throughout the synthesis and the weight percentage of USPIOs was varied solely. A series of experiments were conducted with initial input of USPIOs varying from 0.1% to 10%. As shown in Fig. 2B, at 5% and 10% initial input of USPIOs, the formulation reached 40% encapsulation efficiency. From the loading experiment, it was revealed that the 40% loading is the maximum loading under these experimental conditions. We assessed the cytotoxicity of the MHNP, as a representative particle containing both Gd and Fe, against J-774 macrophage cells using a XTT [3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide] assay. Fig. 3A shows that cell viability was largely unaffected by the presence of MHNP, leading to no measurable toxicity. Some MHNPs are internalized into the phagocytic J-774 cells within 1 h post incubation, as shown by the green fluorescent dye labeling the MHNPs (Fig. 3B). Note that 5 nm USPIOs have been used for the magnetic hybrid nanoparticles, in that they would be more easily degraded, metabolized, and possibly rapidly cleared through the kidneys, thus improving more the level of safety and tolerability of the overall system.

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3.4. Magnetic properties The relaxometric properties of the MHNPs were studied using a bench-top relaxometer (1.41 T), whereas phantom images and the in vivo experiments were conducted using 3T clinical scanners (Philips IngeniaÒ), as in Fig. 4. The longitudinal r1 and transversal r2 relaxivities of all the three nanoparticles (Gd-MHNP, Fe-MHNP and MHNP) were estimated and compared with standard iron oxidebased contrast agents, as shown in Fig. 4A and Table 1. For the Gd-MHNP, it is measured as r1 ¼ 12.95  0.53 (mM s)1 and r2 ¼ 18.8  0.46 (mM s)1, with a ratio r2/r1 ¼ 1.45. These nanoparticles show a longitudinal relaxation about 4 times larger than the clinically used Gd-DTPA (MagnevistÒ) and Gd-DOTA moleucles (DotaremÒ). The MHNP, loaded with USPIOs and labeled with GdDOTA, presented a r1 ¼ 2.09  0.2 (mM s)1 and r2 ¼ 164.07  7.0 (mM s)1, with r2/r1 ¼ 78.5. This clearly indicates that the MHNP are T2 MRI contrast agents and their transversal relaxation is higher than FeridexÒ (r2 ¼ 120 (mM s)1) and at least 3 times larger than conventional USPIO-based contrast agents (r2 w 50 (mM s)1). On the other hand, Fe-MHNP retuned a r1 ¼ 4.66  0.5 (mM s)1 and r2 ¼ 67.4  5.0 (mM s)1, with a r2/r1 ¼ 14.46. Although, the FeMHNP have a transversal relaxation comparable with most commercially available systems, all the proposed magnetic hybrid nanoparticles present higher stability, monodispersity, and longer half-life in circulation. From the data in Fig. 4A, it can be derived that the inclusion of the USPIOs in the Gd-MHNPs leads to an expected increase in r2, followed also by a significant decrease in r1 (w6-folds, from 12.95  0.53 to 2.09  0.2 (mM s)1). Also, the inclusion of the Gd-DOTA in the Fe-MHNPs leads to an increase in r2 (w2.5-folds, from 67.4  5.0 to 164.0  7.0 (mM s)1), and a moderate reduction in r1. The observed decrease in r1 is likely due to the close proximity of the weakly paramagnetic Gd-DOTA molecules and the strong super-paramagnetic USPIO nanocrystals [36]. It should be noted that the r1 and r2 relaxivities for the MHNPs were calculated considering the total concentrations of Gd and Fe, in 0 Þ=½Gd þ Fe (Fig. S4). From the other words r1;2 ¼ ð1=T1;2  1=T1;2 data on Gd-MHNP, we realize that Gd can significantly contribute on the transversal relaxivity of the MHNPs; whereas the contribution of Fe to the longitudinal relaxavity of the MHNPs is quite negligible. Therefore, the r1 of the MHNPs could also be computed considering the sole contribution of Gd, leading to a much higher

Fig. 4. Relaxometric and magnetic properties of MHNPs. (A) Assessment of the magnetic relaxations for MHNPs using a bench-top magnetic relaxometer (Burker Minispec, B ¼ 1.41 T). Magnetic relaxation times were measured in PBS suspension at pH 7.4 and at different MHNPs concentration. (B) Time dependent magnetic dragging. (C) Phantom images for different concentrations of the MHNPs (the reported concentration in mM corresponds to USPIOs) generated using a 3T Philips MRI clinical scanner.

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Table 1 The comparative study of USPIOs and Gd-based contrast agents and their corresponding nanoparticles’s hydrodyanamic size (DH), surface coating, and relaxation properties. Sample

DH (nm)

Coatings

r1 (mM1 s1)

r2 (mM1 s1)

r2/r1

Ref

MHNP-Gd MHNP-Fe MHNP Ferucarbotran (SupravistÒ) Ferumoxytol (C7228) Ferrumoxtran-10 (SineremÒ) FeridexÒ MagnevistÒ DOTARAMÒ

140 145 145 21 35 34 1120e180 e e

PLGA þ lipid þ PEG PLGA þ lipid þ PEG PLGA þ lipid þ PEG Carboxy-dextran Carboxymethyl-dextran Dextran Dextran-P10 Gd-DTPA Gd-DOTA

12.95  0.53 4.66  0.5 2.09  0.2 7.3 7.5 9.9 10.1 3.4 3.4

18.8  0.46 67  5 164  7 57.0 92.0 66.0 120 3.8 4.8

1.45 14.4 78.5 7.81 12.27 6.6 11.8 1.1 1.4

This work This work This work Ref [37,38] Ref [39]

r1 ¼ 42.01  4.07 (mM s)1. Note that the r2/r1 ratio would still be much larger than unity (r2/r1 ¼ 4.1). Similarly, if in calculating the transversal relaxivity, we only consider the contribution of the Fe ions, the resulting r2 would be slightly higher as 172.66  6.0 (mM s)1. In any case, the encapsulation of USPIOs and incorporation of Gdelipid lead eventually to enhancement in MRI relaxivity. The properties of the MHNPs were further characterized in terms of

Ref [40,41] Ref [42]

magnetic dragging under the effect of an external static field. As shown in Fig. 4B, within 10 min of exposure, most of the MHNPs were displaced towards the magnet. The magnetic behavior of MHNPs was further demonstrated by T2-weighted MR phantoms generated for different nanoparticle concentrations. As shown in Fig. 4C, the phantom images become darker as the USPIO concentration increases.

Fig. 5. In vivo performance of the MHNPs. (A) Biodistribution of MHNPs. MHNPs were administered intravenously into nu/nu mice bearing melanoma tumor. After 24 h of postinjection mice (n ¼ 6) were sacrificed and selected organs were harvested and the Gd content was analyzed using ICP-MS. For ICP-MS analysis, the organs were digested using 70% nitric acid and hydrogen peroxide alternatively, the digested colorless organs residues were dissolved in 2% nitric acid and analyzed under ICP-MS. (B) In vivo T2 MR imaging of the MHNPs accumulating in melanoma bearing mice (n ¼ 3). A 3T Philips MRI clinical scanner was used. Tumors were grown in the right flank of a mouse. (C) Intensity ratios for three regions of interest in the tumor and muscles were estimated using Image-J software and results were presented in percentage darkening with respect to the control (pre-injection). Inset circles in the pre-injection MRI micrographs are the region of interest taken for image analysis using image J (NIH) software.

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3.5. Biodistribution

Acknowledgment

In addition to imaging, the presence of Gdelipid can be effectively used to assess the organ biodistribution of the MHNPs using inductively-coupled plasma mass spectrometry on the element Gd, whose basal level in normal animals is zero. The MHNPs biodistribution was assess in mice bearing melanoma tumor cells, implanted in the flank, and the percentage of the injected dose of MHNPs per gram organ (ID/g) was quantified at 24 h post-injection (Fig. 5A). As expected, the highest uptake is observed in the RES organs, with the liver at 14.0  4.0% ID/g and the spleen at 4.0  0.15% ID/g. About 2% ID/g was observed in the tumor (2.1  0.35% ID/g). The amount of Fe accumulating within the malignant mass was estimated to be w3.0 mg. Notably after 24 h, almost 2% of the injected MHNPs were still in the blood (1.0  0.15% ID/mL). The surface decoration of the MHNPs and the size (w150 nm) support the long circulation and progressive accumulation of these nanoparticles in the malignant mass via the well-known enhanced permeation and retention effect.

This work is partially supported by the Cancer Prevention Research Institute of Texas through the grant CPRIT RP110262 and The Methodist Hospital Research Institute.

3.6. In-vivo magnetic resonance imaging Finally, the potential of MHNPs in MR imaging of tumors was demonstrated using a 3T clinical scanners (Philips IngeniaÒ). This scanner was first used for phantom imaging. Fig. 4C presents T2 weighted MR images of MHNPs phantoms at different concentrations. Clearly, as the local MHNP concentration increases, the phantom images become darker as expected for a T2 MRI contrast agent. After intravenous injection, we observed a time-dependent increase in T2 contrast at the tumor site (n ¼ 3). At 4 h post-intravenous injection, large darker areas were visible within the tumor mass (Fig. 5B, post-injection and Fig. S7). These images were analyzed (Image-J by NIH) for the relative contrast enhancement, pre- and post-injection, by measuring the intensity ratio between regions within the tumor tissue and the muscles in the mice (Fig. 5C and Fig. S6). A 50% increase in contrast is clearly shown in three different regions of interest within the malignant mass, supporting the ICP data documenting the MHNP accumulation in the melanoma tumor. Note that spatial heterogeneities in vascular permeability could explain the observed non uniform darkening across the malignant mass. 4. Conclusions Magnetic lipidepolymer nanoparticles (MHNPs) were synthesized encapsulating 5 nm USPIOs within their hydrophobic PLGA core and decorated with Gdelipid complexes. Under physiological conditions, they showed high stability and monodispersity with an average hydrodynamic diameter of w150 nm. The Gd-MHNPs, hybrid nanoparticles carrying only Gdelipid complexes, showed longitudinal relaxation about 4 times higher than conventional Gd-based contrast agents. On the other hand, hybrid nanoparticles loaded with USPIOs and carrying Gdelipid complexes (MHNPs) showed a marked T2 MRI behavior with a transversal relaxivity at least 3 times larger than super-paramagnetic iron oxide nanoparticles used in the clinic. In mice bearing melanoma, at 24 h post-intravenous injection, MHNPs were observed to accumulate within the malignant tissue (2% ID/g) and still circulate in the blood at the level of 1% ID/mL. The MHNPs produced also significant darkening of the tumor tissue in T2-weighted MR imaging, performed with a 3T clinical MRI scanner. Since the PLGA core of these nanoparticles can contained also hydrophobic therapeutic compounds, the MHNPs could be further developed in theranostic systems.

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