In Vivo Evaluation of Porous Silicon and Porous Silicon Solid Lipid

Article pubs.acs.org/molecularpharmaceutics

In Vivo Evaluation of Porous Silicon and Porous Silicon Solid Lipid Nanocomposites for Passive Targeting and Imaging Annukka M. Kallinen,† Mirkka P. Sarparanta,†,∥ Dongfei Liu,‡ Ermei M. Mak̈ ila,̈ ‡,§ Jarno J. Salonen,§ Jouni T. Hirvonen,‡ Hélder A. Santos,*,‡ and Anu J. Airaksinen*,† †

Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, FI-00014 Helsinki, Finland Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland § Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland ‡

ABSTRACT: The use of nanoparticle carriers for the sustained release of cytotoxic drugs in cancer therapy can result in fewer adverse effects and can thus be of great benefit for the patient. Recently, a novel nanocomposite, prepared by the encapsulation of THCPSi nanoparticles within solid lipids (SLN), was developed and characterized as a promising drug delivery carrier in vitro. The present study describes the in vivo evaluation of unmodified THCPSi nanoparticles and THCPSi−solid lipid nanocomposites (THCPSi−SLNCs) as potential drug delivery carriers for cancer therapy by using 18F radiolabeling for the detection of the particle biodistribution in mice. Passive tumor targeting of 18F-THCPSis and 18F-THCPSi−SLNCs by the enhanced permeation and retention effect was investigated in a murine breast cancer model. Encapsulation of THCPSi nanoparticles with solid lipids improved their accumulation in tumors at a 7 week time point (tumor-to-liver ratio 0.10 ± 0.08 and 0.24 ± 0.09% for 18F-THCPSis and 18F-THCPSi−SLNCs, respectively). KEYWORDS: porous silicon, solid lipid nanocomposite, 18F radiolabeling, passive targeting, EPR effect, cancer

1. INTRODUCTION

because PSi particles degrade to silicic acid that is excreted in the urine in vivo.14−18 Much of the biological fate of the nanoparticles is dictated by their surface chemistry.22 In plasma, nanoparticles are instantaneously coated with proteins, such as opsonins that promote the fast clearance of the nanoparticles by the mononuclear phagocyte system (MPS). The hydrophobic or highly charged particles are usually opsonized faster than neutral or hydrophilic particles.23 In order to reach the target efficiently, it is highly desirable for the nanoparticles to avoid fast uptake by MPS and to display prolonged circulation time. Modification of the particle surface with brush-like polymers, such as poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), has been shown to impede their protein adsorption by creating steric hindrance for the adsorption of proteins onto the nanoparticle surface.24−26 It has been observed that by increasing the molecular weight of PEG coating the PSi surface, the degradation rate of the particles could be decreased under simulated physiological conditions.27 In addition, an agarose hydrogel coating of PSi particles has also been shown to protect the payload from biodegradation.28 Coating PSi nanoparticles with dextran has been shown to slow the

Cancer is a complex disease hallmarked by heterogeneity arising from a combination of genetic and environmental factors. However, independent of the type of the cancer, it is desirable to develop therapeutics that can target the tumor cells, either passively or by tumor-specific ligands, while sparing the surrounding tissues. Nanoparticles have shown promise for passive tumor targeting, relying on the enhanced permeation and retention effect (EPR).1 The EPR effect arises from the pathophysiological changes encountered in tumor vasculature, such as fenestrated, leaky endothelium and impaired lymphatic drainage that results in increased extravasation of macromolecules from the circulation to the tumor tissue.2 Usually, a small size of ∼10−150 nm is a prerequisite for the EPR effect;3,4 however, the shape of the nanoparticles may also significantly affect passive targeting.5,6 Porous silicon (PSi) is a promising carrier material for targeted and drug delivery applications.7,8 Several inherent properties of PSi nanoparticles, such as large surface area and pore volume,9−12 high loading efficiency,9−11 biocompatibility,13−16 and biodegradability,14−18 make them especially interesting for drug delivery. In addition, surface chemistry modifications can be attained during fabrication of the particles, making the fine tuning of the material for tumor targeting and tumor imaging applications convenient.19−21 The material is well-warranted for chronic administration in cancer therapy © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2876

March 26, 2014 June 23, 2014 June 30, 2014 June 30, 2014 dx.doi.org/10.1021/mp500225b | Mol. Pharmaceutics 2014, 11, 2876−2886

Molecular Pharmaceutics

Article

degradation of the particles in the liver.4 Recently, we also reported the effect of a fungal hydrophobin protein coating on the biodistribution, plasma protein adsorption, and cellular interactions of THCPSi particles.29,30 In these studies, the hydrophobin functionalization was found to increase the retention of orally administered THCPSi particles in the gastric mucosa. When administered intravenously, the hydrophobin coating altered the composition of the adsorbed plasma protein corona, resulting in a change in the sequestration of the nanoparticles to the liver and the spleen.30 Despite all of the progress in the development of PSi-based drug delivery systems, most of the studied PSi drug carriers have shown fast clearance from circulation by the MPS organs. Recently, we have reported a PSi-based drug delivery system, solid lipid encapsulated thermally hydrocarbonized porous silicon nanocomposite (THCPSi−SLNC), and evaluated the particles in vitro.31 The use of solid lipid nanoparticles (SLNs) for drug delivery via parenteral routes has been reviewed earlier.32,33 These colloidal particles, within the size range of 100−400 nm, are made of a lipid matrix that is solid at body temperature.32 The so-called protocell is a very similar drug carrier system that is formed by the fusion of liposomes with mesoporous silica nanoparticles, and it has been described as a promising drug delivery vehicle elsewhere.34,35 In this drug carrier system, the lipid coating consists of a thin lipid bilayer resembling a cell membrane. SLNs have shown improved bioavailability and targeting as well as enhanced cytotoxicity against multidrug-resistant cancer cells when administered parenterally.32 By adding PEG on the surface of SLNs, the fast uptake of the particles in MPS organs can be avoided, thus resulting in longer circulating, stealth SLNs.33 In our recent study, THCPSi−SLNCs showed better stability against aggregation in aqueous solutions, increased smoothness and cytocompatibility, and prolonged drug delivery over a longer period of time compared to that of bare THCPSi particles.31 Interestingly, the size of the SLN-encapsulated THCPSi particles did not increase in human plasma, suggesting the possibility for prolonged circulation time. Few studies on the tumor targeting of PSi nanoparticles have been reported thus far. It has been observed that the shape of the passively targeted PSi particles affects the tumor uptake of the particles in a murine breast cancer model.5 Discoidal PSi particles were reported to accumulate in tumor tissue more efficiently than spherical PSi particles having a similar diameter. To the best of our knowledge, only one study on the utility of PSi particles on tumor imaging has been reported so far, specifically on luminescent PSi nanoparticles for nontargeted tumor imaging.4 To assess critically the suitability of THCPSi nanoparticles and THCPSi−SLNCs for carrier-mediated therapy and tumor imaging, in vivo evaluation is needed. A high-affinity radiolabeling method based on 18F and Si was recently developed for the investigation of the biodistribution of PSi materials in our laboratory.36 18F is a positron emission tomography (PET) compliant radionuclide widely used in clinical practice. 18Flabeled PSi-based theranostic systems would have higher translational potential than systems based on optical detection, because PET does not have the same limitations with signal attenuation in tissue. In this study, 18F-radiolabeled THCPSi and THCPSi−SLNC particles were prepared using the methods developed previously,30,31 and the overall biodistribution of the nanocomposites was studied in healthy female mice (Crl:CD1). The potential of THCPSi nanoparticles and

THCPSi−SLNCs for passive tumor targeting by the EPR effect was investigated in athymic female mice (Crl:CD1Foxn1nu) bearing orthotopic human MDA-MB-231 breast cancer xenografts. The orthotopic breast cancer model was chosen over the subcutaneous model because it exhibits faster tumor development and more consistently generates permeable vasculature for observation of the EPR effect.37

2. MATERIALS AND METHODS 2.1. Synthesis of the 18F-THCPSi−SLNCs. THCPSi nanoparticles were prepared as described previously from monocrystalline p+-type boron-doped Si⟨100⟩ wafers anodized in hydrofluoric acid (38%)/ethanol (1:1, v/v) and treated with N2/acetylene at 500 °C. The SLNC coating was adapted to 18Fradiolabeled THCPSi nanoparticles from a method developed for the THCPSi nanoparticles in our previous study.31 18 F radiolabeling of THCPSi nanoparticles was carried out as described previously.36 All reagents were purchased from Sigma-Aldrich (USA) and used without further purification. All solutions used for nanoparticle synthesis, purification, and formulation were sterile-filtered (through a Millex-GV filter, 0.22 μm, PVDF, Millipore, USA). 18F was produced with 18 O(p,n)18F reaction using 18O enriched water as a target on an IBA Cyclone 10/5 cyclotron. 18F was trapped in an anionexchange cartridge (Sep-Pak QMA Light Plus, Waters Corporation) and eluted as a 18F−/Kryptofix2.2.2/K+ complex. After azeotropic distillation, the dried complex was dissolved in anhydrous dimethylformamide (DMF, 0.4 mL). The solution was added to 1 mg of THCPSi nanoparticles suspended in 0.1 mL of DMF with 4% (v/v) acetic acid and heated for 10 min at 120 °C. The radioactivity of the reaction mixture was measured before particle purification and was used for calculating the radiolabeling efficiency. Particles were separated from the reaction mixture with centrifugation at 15 000g for 10 min and washed with 10 min sonications in 1 mL of absolute ethanol (Altia Corporation, Finland) followed by 1 mL of ultrapure water. For the uncoated 18F-THCPSi nanoparticles that were used as control, a third wash in 1 mL of phosphate buffered saline (1× PBS, pH 7.4) was included. The radiolabeling efficiency after the purification was 54 ± 13% (n = 12). The nanoparticles were collected with centrifugation between the washes and resuspended carefully with sonication. Finally, the washed THCPSi nanoparticles were resuspended either in formulation solution (0.3% Tween-80 in 0.9% NaCl) for the in vivo studies with 18F-THCPSi or in ethanol (0.5 mL) and forwarded to the SLN coating step. The concentration of 18FTHCPSi was 98 ± 35 MBq/mL (4 ± 3 mg/mL) in the final formulated solution. Specific radioactivity was determined from a 0.08−1.36 mL aliquot of the formulated solution washed with 3 × 1 mL of ultrapure water, freeze-dried overnight, and weighed. In the coating procedure, the ethanolic 18F-THCPSi nanoparticle suspension (0.5 mL) was added to a solution of glyceryl monostearate (100 mg; VWR International Ltd., UK) and L-α-phosphatidyl choline (40 mg; Sigma-Aldrich, USA) in absolute ethanol (4 mL) at 80 °C. After 5 min, the solution containing 18F-THCPSi nanoparticles was added dropwise to a hot aqueous phase (12 mL, 80 °C) consisting of 1% PVA (31− 50 kDa; 1%, w/v) and 1% PEG (6000 Da, Sigma-Aldrich, USA; 1%, w/v) under vigorous stirring (1200 rpm) over 10 min. The hot nanoparticle emulsion was mixed for 10 min, after which the emulsion was added gradually to a cooled aqueous phase (20 mL, 0 °C) of PVA and PEG (1%, w/v) under vigorous 2877

dx.doi.org/10.1021/mp500225b | Mol. Pharmaceutics 2014, 11, 2876−2886


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stirring (1200 rpm) over 15 min. The final dispersion was stirred for another 15 min at 0 °C. The 18F-THCPSi−SLNCs were isolated from the solution with centrifugation at 15 000g for 10 min. The supernatant was discharged, and the 18FTHCPSi−SLNC pellet was dispersed in the formulation solution (0.3% Tween-80 in 0.9% NaCl) by vortexing. The formulated 18F-THCPSi−SLNCs were tip-sonicated using a 3 mm microtip (400 W, power 65%) for 5 s out of every 30 s for 3 min. The concentration of 18F-THCPSi−SLNCs was 68 ± 12 MBq/mL (6 ± 2 mg/mL) in the formulated solution. Specific radioactivity was determined as described above. 2.2. Characterization of 18F-THCPSi−SLNCs. For size and zeta (ζ)-potential measurements, 0.05 mL samples were drawn from the formulated solution. For the size distribution measurements, the sample was directly diluted in 1 mL of ultrapure water, and the size distribution was measured with dynamic light scattering (DLS). In the ζ-potential samples, the particles were freed from NaCl by washing with 3 × 1 mL of ultrapure water and suspended in 1 mL of ultrapure water. The ζ-potential was calculated from the electrophoretic mobility using the Smoluchowski relation. The measurements were conducted on a ZetaSizer Nano instrument (Malvern Ltd., UK). The structure of the fabricated nanocomposites was evaluated by transmission electron microscopy (TEM; Tecnai 12, FEI Company, USA) at an acceleration voltage of 120 kV. The TEM sample was prepared by depositing of the nanoparticle suspension (20 μg/mL; 2 μL) onto carbon-coated copper grids (300 mesh, Electron Microscopy Sciences, USA). Samples were blotted away after a 5 min incubation and airdried prior to imaging. 2.3. Synthesis of 125I-Radiolabeled Bovine Serum Albumin (125I-BSA). To the bottom of a Pierce Iodogen tube (Thermo Scientific, USA), 50 μL of PBS (1× , pH 7.4) and 50 μL of a freshly prepared bovine serum albumin (BSA, >96%, fatty acid free) stock solution (1 mg/mL in 1× PBS, pH 7.4) were added. Twenty-five megabecquerels of no-carrieradded 125I as sodium iodide (PerkinElmer, USA) was added as a slightly basic solution (10 μL, 0.01 mM NaOH) to the tube. The solution was left to react for 15 min at room temperature, after which Na2S2O5 (50 μL, 3.8 mg/mL) was added to quench the reaction. The reaction mixture was diluted with 1 mL of PBS (1×, pH 7.4) and purified by size-exclusion chromatography on a PD-10 desalting column (GE Healthcare, UK) using PBS (1×, pH 7.4) as eluent. Radiochemical purity of the collected fractions was analyzed on Whatman 1 chromatography paper (Millipore, USA) using an eluent system of methanol/H2O (1:1) (albumin Rf = 0.0; 125I Rf = 1.0). The chromatography paper was exposed to a digital imaging plate (Fujifilm Corporation, Japan) for 10 min and scanned on a Fujifilm FLA-5100 scanner. The autoradiograph was analyzed with AIDA 2.0 imaging software (Raytest Isotopenmessgeräte GmbH, Germany). The radiochemical purity of the combined 125 I-BSA fractions was analyzed by a radio-HPLC system consisting of two LC-20AD pumps, a Shimadzu SPD-M20A diode array detector (Shimadzu Corporation, Japan), and an external NaI(Tl) radiodetector (Ortec, Oak Ridge, USA). The analysis was performed on Agilent Zorbax Eclipse XDB-C8 column (4.6 × 150 mm, 3.5 μm particle size) using eluents A (0.1% trifluoroacetic acid (TFA) in H2O) and B (0.1% TFA in acetonitrile) at 1.8 mL/min with UV detection at 208 nm. A linear gradient of B from 20 to 68% was applied over 10 min. The combined fractions were diluted with PBS (1×, pH 7.4) to the activity concentration of 2 MBq/100 μL for administration.

The isolated radiochemical yield and the radiochemical purity of 125I-BSA were 54 ± 2% and 96 ± 2%, respectively (n = 3). 2.4. Biodistribution Studies. All animal experiments were performed according to European Community Guidelines for animal experimentation and approved by the Finnish National Board of Animal Experiments (license no. ESAVI/6735/ 04.10.03/2012). Female Crl:CD1 mice (25−30 g, 8−10 weeks, Charles River) were used for studying the biodistribution in healthy mice. Female athymic Crl:CD1-Foxn1nu mice (25−29 g, 8−10 weeks, Charles River) were used for studying the biodistribution in mice bearing orthotopic human breast carcinoma xenografts (see below). Crl:CD1 mice were group-housed in standard polycarbonate cages and nude mice were group-housed in filter-top cages with aspen bedding and with food (Harlan Teklad Global 16% protein rodent diet 2916) and tap water available ad libitum. Environmental conditions of a 12:12 light/dark cycle, temperature of 23.5 ± 1.5 °C, and relative humidity of 62.5 ± 1.5% were maintained throughout the study. Crl:CD1 mice received either 18FTHCPSi−SLNCs (5.2 ± 1.2 MBq) or uncoated 18F-THCPSi nanoparticles (4.6 ± 1.3 MBq) in a sterile solution of 0.3% Tween-80 in 0.9% NaCl (0.1 mL) intravenously (i.v.) in the lateral tail vein via a temporary catheter fabricated out of a 30G needle and PE10 polyethylene tubing (AgnTho’s, Sweden). Similarly, either 18F-THCPSi−SLNCs (4.5 ± 1.0 MBq) or 18FTHCPSi nanoparticles (4.8 ± 0.6 MBq) in a sterile solution of 0.3% Tween-80 in 0.9% NaCl (0.1 mL) were administered to the lateral tail vein of Crl:CD1-Foxn1nu mice via the temporary catheter. Additionally, all Crl:CD1-Foxn1nu mice received 125IBSA (0.9 ± 0.3 MBq) in 0.1 mL of PBS (1×, pH 7.4., sterile filtered) as EPR control to the lateral tail vein via the temporary catheter 24 h prior to the nanoparticle administration. During the injections, the mice were kept under 1.5−2.0% isoflurane (IsoFlo Vet, Orion Pharma, Finland) anesthesia in O2 or air carrier at 0.5 L/min. Animals (n = 3−4 per time point) were sacrificed at 15, 30, 90, and 240 min after administration with CO2 asphyxiation followed by cervical dislocation. Samples from blood, urine, and major organs were collected. The gamma activities arising from 18F were measured for 60 s on the day that the nanoparticles were administered. The gamma activities arising from 125I were measured after 48 h of the nanoparticle administration, when 18F had decayed. Measurements were performed with gamma counter Wizard 3 (PerkinElmer, USA). 2.5. Orthotopic Human Breast Carcinoma Model. Human breast adenocarcinoma MDA-MB-231 cells (American Type Culture Collection, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, USA), 1% nonessential amino acids, 1% L-glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) (all from EuroClone S.p.A., Italy). For tumor implantation, the cells were suspended at a 1 × 106/ 50 μL concentration in 1:1 RPMI/Matrigel (BD Biosciences, USA). The cells were injected unilaterally (1 × 106 cells/site) to the fourth mammary fat pad in Crl:CD1-Foxn1nu mice under isoflurane anesthesia (1.5% in 100% air at 0.5 L/min) after it was surgically exposed through a 5 mm incision. The animals received buprenorphine (Temgesic, RB Pharmaceuticals Ltd., UK) at a dose of 0.1 mg/kg subcutaneously for perioperative analgesia. A second dose of buprenorphine was given 12 h after the first one if the animal was exhibiting signs of pain or discomfort. The tumors were allowed to develop for 4 and 7 weeks before dosing the 18F-radiolabeled THCPSi−SLNCs and 2878



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Figure 1. TEM pictures of 18F-THCPSi (a) and 18F-THCPSi−SLNC (c) nanoparticles formulated in 0.3% Tween-80 in saline. The particle size distributions based on TEM measurements are given on the right: 18F-THCPSi (b) and 18F-THCPSi−SLNC (d).

ethanol concentration and finally 100% xylene. The sections were air-dried, mounted in DPX, and imaged with an Axioplan 2 microscope fitted with Axiocam HRc camera and AxioVision 4.6 software (all from Carl Zeiss Microimaging GmbH, Germany). The human cells in the sections were stained for immunofluorescence with a Alexa Fluor 488-conjugated primary mouse monoclonal anti-human nuclei antibody clone 235-1 (Millipore, USA) at a 1:500 dilution using standard methods. The sections were counterstained with Hoechst (1:5000) and imaged as described above. 2.8. Statistical Analysis. Statistical analysis of the results was carried out with Student’s t-test. P values < 0.05 between THCPSi and THCPSi−SLNC were considered to be statistically significant.

THCPSi nanoparticles. During that time, the animals developed palpable tumors with a maximum dimension (either width or length) of 2−3 mm (4 weeks) and 5−9 mm (7 weeks). Additionally, the weight of the animals was measured every third day starting from tumor implantation for the entire duration of the experiment. Body condition scoring was used as an aid for monitoring the welfare of the animals.38 No animals needed to be sacrificed during the course of the study because of marked weight loss (>20% maintained for 72 h), tumor size reaching the recommended limit, or other signs of distress as listed for humane end points elsewhere.39 2.6. Ex Vivo Autoradiography. The excised tumors were snap-frozen in liquid N2 and sectioned to 30 μm thickness on a cryostat microtome (Leica CM1950, Leica Microsystems, Germany). The sections were thaw-mounted on SuperFrost Plus slides (VWR Collection, USA) and allowed to air dry until exposure. The slides were exposed to a Fujifilm SR2040 digital imaging plate on the day of the 18F-radiolabeled nanoparticle administration for 16 h, resulting in an autoradiographic image of both 18F and 125I signals. A second 16 h exposure was performed 7 days after the first one (after 18F decay) to visualize the distribution of 125I-BSA in the tumor sections. Digital autoradiography of tissue sections was based on photostimulated luminescence detection on a Fujifilm FLA5100 system with Image Reader FLA-5000 series software (version 1.0, Fujifilm, Japan). The autoradiography data was processed using AIDA Image Analyzer program (version 4.00, Raytest Isotopen messgeräte GmbH, Germany). The autoradiographs were quantified using 18F (0.1−50.0 kBq) and 125I (0.005−5.0 kBq) standards exposed to the imaging plate at the same time with the tissue sections. 2.7. Immunostaining. The 30 μm cryosections of the tumors were stained with hematoxylin and eosin. Briefly, sections were circled with a hydrophobic barrier pen (ImmEdge, Vector Laboratories, USA) and rinsed in running tap water. The sections were dipped in ultrapure water for 1 min, after which they were stained with Harris’ hematoxylin (Sigma-Aldrich, USA) for 10 s and rinsed in running tap water for 5 min with two changes. The sections were then stained with eosin (Sigma-Aldrich, USA) for 10 s and rinsed briefly in running ultrapure water. After air drying the slides, the sections were dehydrated by passing them through a series of increasing

3. RESULTS 3.1. Synthesis of 18F-THCPSi−SLNCs. Coating of the 18FTHCPSi nanoparticles with solid lipids was performed by using a solid-in-oil-in-water (S/O/W) method as described elsewhere.31,40 The radiolabeling and purification of THCPSi nanoparticles were performed with a radiolabeling efficiency of 54 ± 13% in 134 ± 16 min (from the end of bombardment). The subsequent solid lipid coating of 18F-THCPSi particles and formulation were performed in 108 ± 7 min, and the particles were isolated with a radiochemical yield of 29 ± 8% (decaycorrected yield). The specific activity of the 18F-THCPSi− SLNCs was 12 ± 2 MBq/mg (68 ± 12 MBq/mL, particle concentration 6 ± 2 mg/mL), and that of 18F-THCPSi particles was 39 ± 29 MBq/mg (98 ± 35 MBq/mL, 4 ± 3 mg/mL). Both particles were formulated in a sterile solution of 0.3% Tween-80 in saline (0.9%). 3.2. Characterization of the Nanoparticles. The zaverage size and the ζ-potential of the untreated THCPSi nanoparticles before radiolabeling were 170 ± 2 nm (polydispersity index (PDI) 0.153) and −36 ± 4 mV, respectively. The z-average size of uncoated 18F-THCPSi nanoparticles was 179 ± 8 nm (PDI 0.155), and the average ζ-potential was −28.2 ± 6.3 mV, demonstrating that the 18F radiolabel can be introduced without significant alterations to the nanoparticle properties. The z-average size of 18FTHCPSi−SLNC in formulated batches was 193 ± 29 nm (PDI 0.255), and the value of ζ-potential was −39.6 ± 4.7 mV. 2879



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Figure 2. Biodistribution of 18F-THCPSi nanoparticles and 18F-THCPSi−SLNCs in the blood and heart at 15, 30, 90, and 240 min after i.v. administration of the nanoparticles to Crl:CD1 female mice. 18F-THCPSi t = 15−90 min, n = 3; t = 240 min, n = 2. 18F-THCPSi−SLNC t = 15−30 min, n = 4; t = 90−240 min, n = 3. Student’s t-test was used to assess the statistical significance of the difference in the uptake of the two particle types in different organs: in blood, p > 0.17; in heart, p > 0.20.

In our early study, the value of the ζ-potential was ca. −15 mV.31 The lipid sheath around the dense PSi core was visible in the representative TEM image of a 18F-THCPSi (Figure 1A) and 18F-THCPSi−SLNC particle (Figure 1C). The morphology of the particle is in accordance to the structural data reported elsewhere.31 The particle size distribution measured with TEM (Figures 1B and 1D) was found to be broader than the size distribution based on DLS measurements. 3.3. Biodistribution of the Nanoparticles in Healthy Mice after Intravenous Administration. The biodistribution of 18F-THCPSi−SLNCs was investigated in healthy female mice (Crl:CD1) by measuring the radioactivity of selected excised organs at 15, 30, 90, and 240 min postinjection. Uncoated 18F-THCPSi nanoparticles served as a control. At the 15 min time point, the observed amount of 18F-THCPSi− SLNCs in the blood and heart (1.06 ± 0.56 and 5.61 ± 5.70% ID/g, respectively) appeared to be higher than the amount of 18 F-THCPSi particles in the blood and heart (0.53 ± 0.29 and 0.95 ± 0.80% ID/g, respectively) (Figure 2). However, we observed a large variation between the animals; hence, the apparent difference was not statistically significant (blood, p = 0.17; heart, p = 0.20). The amount of both particle types in the circulation quickly decreased after 15 min. The uncoated 18FTHCPSi nanoparticles (Figure 3a) and 18F-THCPSi−SLNCs (Figure 3b) showed similar, high uptake in the MPS organs, liver and spleen. The spleenic uptake of the 18F-THCPSi particles in mice was noted to be only 21% of the spleenic uptake reported in rats, 13 which can result from the physiological and anatomical differences in the MPS between species.41 The lung uptake of 18F-THCPSi−SLNCs remained constant and low over time (Figure 3), whereas some of the animals that received 18F-THCPSi nanoparticles exhibited high accumulation of particles in the lungs. However, the interindividual variation was high, which has also been seen in the lung uptake of 18F-THCPSi nanoparticles in rats.13 Because of this, the observed difference in the lung uptake between the coated and the uncoated nanoparticles was not statistically significant (p =

0.2). The observed lung uptake of 18F-THCPSi also cleared over time, analogous to what has been observed in rats. The amount of the detached, free 18F was determined by measuring the radioactivity in bone, where free 18F is known to accumulate. The uptake of the radioactivity in the skull was minimal ( 0.24; in gall bladder, p > 0.41; in kidney, p > 0.28; in spleen, p > 0.09; in bone, p > 0.33.

SLNCs (0.56 ± 0.21% ID/g) in tumor bearing mice at this time point (p = 0.01). The observed difference is statistically significant and corresponds to our earlier observation of the higher lung uptake of THCPSi nanoparticles as compared to that of THCPSi−SLNCs in healthy mice. The uptake of 18FTHCPSi and 18F-THCPSi−SLNC particles was found to be minimal in the stomach and intestine (data not shown, < 0.4% ID/g). The uptake of 18F in the skull was found to be slightly higher when uncoated 18F-THCPSi nanoparticles were administered (1.72 ± 0.41% ID/g) compared to that of 18FTHCPSi−SLNCs (0.86 ± 0.37% ID/g) (p = 0.02). The level of 18 F-fluoride uptake in the skull was, however, in the same range as that observed in healthy animals. The tumor uptake values and tumor-to-liver uptake ratios of the nanoparticles at 4 and 7 weeks are presented in Table 1. In addition, the ratio of the 18F-nanoparticles uptake to 125I-BSA uptake in tumor at 4 and 7 weeks is also given in Table 1. The tumor uptake, tumor-to-liver ratio, and nanoparticle-to-albumin ratio were observed to be similar for both 18F-THCPSi

nanoparticles and 18F-THCPSi−SLNCs at the 4 week time point (Table 1). The accumulation of 18F-THCPSi nanoparticles in the tumor did not change from 4 weeks (0.028 ± 0.015% ID/g) to 7 weeks after tumor cell inoculation (0.039 ± 0.029% ID/g). However, the tumor uptake of 18F-THCPSi− SLNCs was noted to increase from 0.027 ± 0.014% ID/g (4 weeks) to 0.081 ± 0.036% ID/g (7 weeks) (p = 0.01). Accordingly, a similar increase could be seen in the tumor-toliver accumulation ratio of the 18F-THCPSi−SLNC particles: from 0.04 ± 0.02% (4 weeks) to 0.24 ± 0.09% (7 weeks) (p = 0.003). At 7 weeks postinoculation, by comparing the tumor accumulation values described in Table 1, a trend of higher accumulation of 18F-THCPSi−SLNCs compared to that of 18FTHCPSi nanoparticles in the tumor was observed. A statistically significant difference (p = 0.04) was seen in the tumor-to-liver uptake ratios between 18F-THCPSi−SLNCs (0.24 ± 0.09%) and 18F-THCPSi nanoparticles (0.10 ± 0.08%) at the 7 week time point. Tumor uptake of the 18F2881



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Figure 4. Biodistribution of 18F-THCPSi nanoparticles and 18F-THCPSi−SLNCs in Crl:CD1-Foxn1nu female mice at 90 min after i.v. administration of the nanoparticles 4 weeks after inoculation of the MDA-MB-231 breast cancer cells. Student’s t-test was used to assess the statistical significance of the difference in the uptake of two particle types in different organs: in blood, p = 0.08; in lung, p = 0.01; in heart, p = 0.12; in liver, p = 0.04; in gall bladder, p = 0.31; in kidney, p = 0.37; in spleen, p = 0.15; in bone, p = 0.02.

Table 1. Tumor Accumulation of 18F-THCPSi Nanoparticles and 18F-THCPSi−SLNCs in Crl:CD1-Foxn1nu Female Mice at 90 min after i.v. Administration of the Nanoparticles at 4 and 7 Weeks after Tumor Cell Inoculationa week 4 4 7 7

particle type 18

F-THCPSi 18 F-THCPSi−SLNC 18 F-THCPSi 18 F-THCPSi−SLNC

tumor uptake (% ID/g) 0.028 0.027 0.039 0.081

± ± ± ±

0.015 0.014 0.029 0.036

(n (n (n (n

= = = =

8) 8) 4) 6)

tumor-to-liver (%) 0.060 0.040 0.099 0.235

± ± ± ±

0.035 0.020 0.082 0.092

particle-to-albumin (%) 5.1 3.8 5.0 10.6

± ± ± ±

1.3 1.0 4.8 7.6

a

Student’s t-test was used to assess the statistical significance of the difference in the particle uptake values. Tumor uptake (% ID/g) and tumor-toliver accumulation ratio of 18F-THCPSi−SLNCs increased from 4 to 7 weeks (p = 0.01 and p = 0.003, respectively). At 7 weeks, 18F-THCPSi− SLNCs exhibited a higher tumor-to-liver ratio than that of 18F-THCPSi nanoparticles (p = 0.04).

THCPSi−SLNCs correlated moderately with the observed albumin uptake in tumor (Pearson correlation, r = 0.53); however, a negative correlation (r = −0.79) was observed between the 18F-THCPSi and albumin control tumor uptake at this time point. This indicates that accumulation of the uncoated THCPSi nanoparticles in the tumor by the EPR effect is not possible and that surface modification of the particles is needed for passive targeting. 3.5. Ex Vivo Autoradiography and Immunohistochemistry. Histological staining of the tumors revealed two different tumor populations by morphology: diffuse and solid. In this study, the nanoparticles were radiolabeled only with 18F, and the resolution of the digital autoradiography (Figure 5A) does not permit the detection of individual nanoparticles at the cellular level in the tissue cryosections. The tumor sections show a generally uniform pattern of nanoparticle distribution in both 4 and 7 week old tumors despite clear morphological differences between the two tumor types, with a slight concentration of the radioactivity signal at the tumor periphery in the older solid tumors. This is typical for i.v. administered nanoparticles, as the tumor’s vascular network is often the richest at the tumor boundary. It was confirmed by immunohistochemical staining that human MDA-MB-231 cells were present in both the diffuse and solid tumor types (Figure 5B). The solid tumor mass primarily consisted of human MDA-MB-231 cells distributed homogeneously in the tumor tissue. In diffuse tumors, however, the MDA-MB-231 tumor cells formed distinct patches surrounded by adipose and connective tissue. However, ex vivo autoradiography of the tumor sections revealed homogeneous albumin and nano-

particle distributions, with no clear distinction between the diffuse and solid forms.

4. DISCUSSION The previously reported procedure for coating THCPSi nanoparticles with SLNs by the S/O/W method was successfully modified to comply with the radiation safety demands and the time restrictions of working with short-lived positron emitters. Methodwise, the 18F label was introduced to the PSi core in a rather early phase of the procedure. However, we were able to produce the 18F-labeled THCPSi nanoparticles coated with SLNs with high enough activities for animal studies with a reasonable radiochemical yield. The encapsulation of the THCPSi particles in SLNs decreased the numerical value of the ζ-potential (from −28.2 ± 6.3 to −39.6 ± 4.7 mV), i.e., increasing the ζ-potential of the particles, which may be indicative of improved stability of the 18 F-THCPSi−SLNCs suspension compared to that of the uncoated THCPSi nanoparticles. Phosphatidylcholine and glyceryl monostearate are likely to arrange on the surface of the THCPSi nanoparticles, with the hydrophobic ends pointing toward the hydrophobic surface of THCPSi, whereas the hydrophilic moieties face the aqueous medium.42 This arrangement is known to result in enhanced dispersion and stability of 18F-THCPSi−SLNCs against aggregation in aqueous solutions.31Also, the addition of PVA to the particle dispersion is known to increase the particle’s stability.43 Compared to that of 18F-THCPSi, the observed low lung uptake of 18F-THCPSi−SLNCs might indicate an enhanced stability of the dispersion immediately after injection, avoiding aggregation and subsequent entrapment of the 18F-THCPSi− 2882



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Figure 5. (a) Autoradiography of tumor cross-sections showing nanoparticles (18F) and albumin control (125I) in the tumor at 4 and 7 week time points. Histological staining of the corresponding tumor section is given as a reference. Dashed arrow indicates a diffuse tumor, and solid arrow indicates a solid tumor. (b) Immunohistochemical staining of MDA-MB-231 cancer cells (HuNu) in diffuse and solid tumors illustrates the presence of cancer cells in both.

biodistribution of 18F-THCPSi and 18F-THCPSi−SLNC particles were found to be very similar. The average particle size of the 18F-THCPSi−SLNCs (193 ± 29 nm, DLS) was in the middle of the size range reported for SLNs that have been used as drug carrier systems in the literature.33 However, TEM images indicated a broader particle size distribution that may have given rise to the observed biodistribution. Nonstealth SLNs are mainly taken up in Kupffer cells in the liver, and the recognition of the SLNs by macrophages is known to be due to surface hydrophobicity and charge as well as particle size.33 In our study, PEG was added on the solid lipid coating to improve the stealth properties of the particles; however, the uptake of THCPSi−SLNCs by MPS was not extensively reduced. The SLN coating slightly improved the tumor uptake of the THCPSi particles. It was observed that the tumors used for the study exhibited variable morphology, from diffuse to solid tumors even at 7 weeks postinoculation. It is thus plausible that these structural differences are conferred to differences in vascular permeability and hence to the observed inconsistent tumor targeting by the EPR effect. Albumin is known to accumulate in inflamed and tumor tissue by the EPR effect and can be used as a macromolecular control for assessing tumor permeability and the extent of vascularization.45 Both mouse and bovine serum albumins have been used as radiotracers to quantify the EPR effect in vivo previously,45−47 yielding comparable values for maximum tumor uptake (5−7% ID/g) and time (20−24 h) to attain maximal tumor-to-background ratios despite the differ-

SLNC particles in the lung capillaries. In our previous studies, 18 F-THCPSi has been noted to accumulate in the lung in rats, presumably because of particle aggregates formation in plasma after administration.13 This was also observed for the uncoated 18 F-THCPSi nanoparticles in the present study. The circulation time of the 18F-THCPSi nanoparticles and 18 F-THCPSi−SLNCs in blood was, however, found to be short, and the particles were quickly taken up in the liver and spleen. Because of the rapid accumulation of the particles to the MPS organs, the concentration of the particles in the blood may have been insufficient for efficient passive tumor targeting to take place. THCPSi−SLNCs have been reported to be highly biocompatible and noncytotoxic in concentrations up to 250 μg/mL in the murine RAW 264.7 macrophages in vitro,31 suggesting that the observed uptake in MPS organs was not likely associated with the cytotoxicity of the particles. Previously, it has been noted that the particle size and ζpotential of THCPSi−SLNCs remained unchanged after a 2 h incubation in human plasma, whereas the average size of THCPSi particles increased under the same conditions (from 150 to 400 nm).31 However, protein adsorption on the nanoparticles takes place in 30 s and is a dynamic process;44 hence, the observed unchanged particle size and ζ-potential of THCPSi−SLNCs refer to the dynamic state of the protein adsorption and desorption. Hence, the protein corona of the THCPSi and THCPSi−SLNC particles is most likely different. Given that the composition of the protein corona is expected to affect the biodistribution of the particles, it is surprising that the 2883



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Notes

ence in the biological half-lives of the two in mice: 60 min for BSA versus 180 min for mouse serum albumin.47 We observed lower uptake of 125I-radiolabeled bovine serum albumin in the tumors (1.14 ± 0.35 and 0.94 ± 0.35% ID/g) at 25 h after administration at 4 and 7 weeks after tumor inoculation, respectively, compared to the previously reported values. This suggests that there might be differences in the tumor microenvironment and neovascular architecture between the orthotopic breast carcinoma model used in this study and the ectopic prostate carcinoma xenografts reported earlier. However, the short circulation time of the developed nanoparticle tracers is the most plausible cause for the low tumor uptake observed in this study. For the passive targeting of the THCPSi nanoparticles in tumors, the particles require a coating to be stably dispersed under physiological conditions and to increase their circulation time in vivo. However, unlike the case of the SLNs, the PEG solid lipid coating on the surface of the THCPSi nanoparticles was not enough to prevent the immune recognition and to increase the circulation time of the developed nanocomposite. A possible explanation for this is that despite the uniform encapsulation of the nanoparticle to the solid lipid sheath observed, the THCPSi surface becomes exposed after i.v. administration because of a possible disintegration of the SLN coating under physiological conditions, resulting in the loss of the potential stealth effects imposed by the coating. This possibility requires further studies on the stability of the SLN coating in vivo in the future.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS FinPharma Doctoral Program (FPDP, Drug Discovery section) is acknowledged for the financial support of the Ph.D. studies of A.M.K. H. A. Santos acknowledges University of Helsinki Funds, the Academy of Finland (decision nos. 252215 and 256394), Biocentrum Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013; grant no. 310892) for financial support. A.J.A acknowledges the Academy of Finland (decision nos. 136805 and 272908) for financial support.

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5. CONCLUSIONS Biological evaluation is essential for the assessment of the suitability of new nanomaterials for drug delivery and imaging. By using a highly stable 18F-Si-labeling for the detection of PSi particles, the passive targeting of uncoated 18F-THCPSi nanoparticles and 18F-THCPSi−SLNCs was reliably evaluated in an orthotopic human breast cancer model in mice. The solid lipid nanocoating improved the dispersibility of the THCPSi nanoparticles, resulting in a lower uptake of the particles in the lung. Tumor uptake of 18F-THCPSi−SLNCs was found to be higher than that of 18F-THCPSi nanoparticles at 7 weeks after tumor inoculation. However, the tumor accumulation still remained insufficient for imaging and therapeutic purposes. Both 18F-THCPSi and 18F-THCPSi−SLNCs were found to be cleared quickly from the circulation and to accumulate in the liver and spleen. Consequently, the low amount of particles available for extravasation is likely to account for the low tumor uptake observed. Given the extremely promising drug delivery carrier properties of the developed nanocomposite for sustained delivery of cancer therapeutics, optimization of the material properties for improved circulation and active tumor targeting is warranted.

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AUTHOR INFORMATION

Corresponding Authors

*(H.A.S.) E-mail: helder.santos@helsinki.fi; Fax: +358-294159138; Tel.: +358-2941-59661. *(A.J.A.) E-mail: anu.airaksinen@helsinki.fi; Fax: +358-294150121; Tel.: +358-2941-50124. Present Address ∥

(M.P.S.) Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, United States. 2884



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In Vivo Evaluation of Porous Silicon and Porous Silicon Solid Lipid

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