Production of PEG-SWCNTs
Commercially available SWCNTs were purchased from Carbon Solution Inc. (Riverside, CA). They were purified by air oxidation. Amino-functionalized PEGylated SWCNTs (PEG-SWCNT) were fabricated through our published non-covalent protocol  based on the adsorption of PEG-modified phospholipids onto SWCNTs’ sidewalls. Briefly, 5 mg of pristine (non-modified) SWCNTs (Carbon Solutions, Riverside, CA) were oven dried at 160°C for 3 h, sonicated with 25 mg of phospholipids modified with amino-functionalized 2-kDa molecular weight (mw) linear PEG chains (DSPE-PEG(2 k)-NH2, Avanti Polar Lipids, Alabaster, AL) in PBS for 6 h in an ultrasonic bath (Ultrasonic Cleaner, Cole-Parmer, Vernon Hills, IL) and ultracentrifuged (Optima™ XL-80 K Ultracentrifuge, Beckman, Palo Alto, CA) at 20,000 × g for 6 h at 4°C. Approximately 80% of the supernatant fraction was collected and ultracentrifuged at 40,000 × g for 6 h at 4°C. Approximately 80% of this second supernatant fraction was collected and ultracentrifuged at 70,000 × g for 6 h at 4°C. The resulting pellet was dispersed in PBS and filtered through 100 kDa mw-cut off centrifugal devices (Vivaspin 500, Sartorius, Göttingen, Germany) to remove free phospholipids. All the fabrication steps were carried out under conditions of sterility. To carry out investigations about the accumulation profile of PEG-SWCNTs in living animals, fluorescent PEG-SWCNTs (PEG-SWCNT-750) were obtained by acylation of terminal PEG amino groups with N-hydroxysuccinimide ester (NHS)-modified near infrared (NIR)-emitting fluorochromes (Seta750-NHS, SETA BioMedicals, Urbana, IL). The fluorochrome had excitation and emission distribution maxima at 751 nm and 779 nm (in PBS, pH 7.4), respectively, and it was chosen in order to minimize the background noise arising from tissue auto-fluorescence.
PEG-SWCNTs were characterized through elemental analysis, atomic force microscopy (AFM) and matrix-assisted laser desorption/ionization (MALDI). Elemental analysis was performed on a samples containing 100 μg/ml of nanoparticles by Elemental Analysis Inc. (Lexington, KY) utilizing Proton Induced X-ray Emission (PIXE). In PEG-SWCNT samples the amount of metallic contaminants was under the detection limit of the technique (0.001 μg/cm2). Samples for AFM imaging were prepared as follow: first 10 μL of PEG-SWCNTs in PBS (concentration 2.5 μg/ml) were dropped onto a freshly cleaved mica substrate (Ted Pella, Redding, CA), next the droplet was allowed to stand for a couple of minutes at room temperature, and finally the mica surface was rinsed with water and dried under a gentle nitrogen stream. AFM images were recorded using a 5500 AFM (Agilent Technologies, Santa Clara, CA) in acoustic alternate current (AAC) mode. MALDI samples were prepared as follow: 1 μL of PEG-SWCNTs in PBS diluted 1:1 with matrix solution [10 mg/mL α-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid/50% (v/v) water] was dropped onto MALDI plate and allowed to dry at room temperature. MALDI spectra were recorded by means of an Autoflex™ II TOF/TOF (Bruker Daltonics Inc., Billerica, MA).
Dispersion and stability of PEG-SWCNTs
PEG-SWCNTs and PEG-SWCNT-Seta750 were dispersed in PBS (pH7.4) at room temperature (approximately 23°C). Stability of the suspensions was investigated by recording the UV–vis absorbance spectrum at various storage times. Both PEG-SWCNT and PEG-SWCNT-Seta750 freshly fabricated dispersions (150 μg/ml) showed UV–vis absorbance spectra characterized by distinct and sharp peaks corresponding to the van Hove singularity transitions; thus suggesting the presence of individually dispersed nanoparticles . The UV–vis spectra did not display any changes upon storing nanotube solutions for several months at room temperature in PBS, thus suggesting absence of aggregation.
For animal exposure experiments, PEG-SWCNT solutions that were not older than 1 month were used. The UV–vis absorbance spectrum of nanotube solutions was regularly checked before their use in order to confirm that they were formed by individual (not-aggregated) nanoparticles. Stability of the fluorescent tag (Seta750) conjugated to PEG-SWCNT-750 was also investigated. PEG-SWCNT-750 solution was stored for two weeks at room temperature in PBS, filtered once through 100 kDa-mw cut-off centrifugal filtering devices and the UV–vis absorbance spectrum of the eluate recorded. The latter showed a very faint peak centred at approximately 750 nm, thus suggesting that the release of the fluorescent tag from PEG-SWCNT-750 was minimal. For the determination of endotoxin content of PEG-SWCNT samples, the LAL Chromogenic Endotoxin Quantitation kit (Thermo Scientific, Rockford, IL, USA) was used.
In the present study pregnant and non-pregnant females of the CD1 strain were used; such outbred strain is considered a multipurpose model suitable for toxicological studies. Animals were housed and mated under standard laboratory conditions and treated using humane care in order to inflict the least possible pain. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and carried out according to the Italian and European rules (D.L.vo 116/92; C.E. 609/86; European Directive 2010/63/EU). A veterinary surgeon has been present during the injection and blood sample collection experiments. Animal handling, before and after experiment, has been carried out only by trained personnel.
In vivo analysis of fluorescently labeled PEGylated-SWCNT biodistribution
For the evaluation of nanoparticle distribution in maternal organs, placentas and fetuses, mice were administered with fluorescently labeled PEG-SWCNTs (PEG-SWCNT-750; 5 animals), or with the fluorochrome Seta750 only (5 females), at the same concentration as that conjugated to the nanotubes. The concentration of free Seta 750 in PBS was calculated by recording the absorbance spectrum of Seta750 in PBS and dividing the value of absorbance at 750 nm by the extinction coefficient furnished by the supplier. The concentration of Seta750 bound to the nanotubes was calculated by a two-step process. First, the absorbance spectrum of Seta750 bound to the nanotubes was calculated by subtracting the absorbance spectrum of fluorochrome-devoid nanotubes from the spectrum of nanotubes loaded with fluorochromes. Next, the obtained value of absorbance at 750 nm was divided by the extinction coefficient furnished by the supplier. Control animals (5 animals) received the same volume of the dispersant medium (PBS). Animals were intra-venously injected with 10 μg of nanoparticles in a volume of 100 μl at either day 5.5 or day 14.5 of gestation, via the retro-bulbar plexus, as previously reported . Such route of administration is considered an alternative to the tail vein injection and is recommended for small laboratory animals , since it is much less technically challenging, does not require warming preparation of the animal or anesthesia, and the whole procedure is only a matter of seconds. For nanoparticle administration, the mouse was immobilized on absorbent paper, keeping it motionless, and a volume of 100 μl was gently injected in the center of the retro-orbital sinus of the right eye, by using a 1 cc syringe equipped with a 27 gauge needle. No local complications related to the procedure, such as local edema or relevant bleeding, were ever observed. Biodistribution of fluorescence was analyzed using a Kodak Image Station In Vivo FX apparatus after 10 minutes (time 0), 1 hour (time 1) and 4 hours (time 2). In order to determine the detection limit of our system, we have evaluated fluorescence of PEG-SWCNT-750 samples of scalar concentrations and observed no fluorescence between 25 and 50 ng/ml. For comparing tissue distribution between pregnant and non pregnant animals, non-pregnant females were also exposed to nanoparticles. Recording of the fluorescence was obtained during anesthesia, (Avertin 250 mg/kg) with the animals lying in the prone and supine position. Anesthesia lasted up to about 4 hr, that was the duration of fluorescence recording in live animals. After recovering from anesthesia, animals were placed back in their cages. After 24 hr, animals were euthanized and immediately placed in the Kodak apparatus for further evaluation of fluorescence distribution. Maternal organs, placentas and fetuses were then isolated and their fluorescence recorded.
Evaluation of PEG-SWCNT embryotoxic potential
PEGylate-SWCNTs were intravenously administered to pregnant females as above reported. Briefly, six to eight week old CD1 females were used. The mean age in all groups ranged from 6.8 to 7.2 weeks. For mating, 2-3 females were distributed in each cage containing one male of proven fertility. For each experiment, 5 females were randomly allocated to the different exposure groups, each of which had a predetermined final size of at least 5 animals. The presence of a vaginal plug was checked every morning, and the day of the vaginal plug was considered day 0.5 of gestation.
Pregnant females were divided in two groups, depending on the type of analysis intended: group A received either PEGylate-SWCNTs or vehicle on day 5.5 of gestation (5.5 dpc). For this group, administered doses were either 0.1 (5 females), 10 (10 females) or 30 μg/mouse (10 females, Table 1 and Figure 1). Group B was administered with a total amount of nanoparticles of 30 μg/mouse (10 females), but in three refracted doses of 10 μg/mouse, on day 5.5, 8.5 and 11.5 of gestation. In this case, intra venous administration was performed alternating injections in the right and left retro-orbital plexus, so that administration through the same eye occurred after six days. Control animals (18 for single administration and 10 for repeated injections) were administered with the PBS, that was the medium in which nanoparticles were dispersed. No local complications secondary to the injection procedure were observed.
The doses of PEG-SWCNTs used in this study are in the lower range of those employed in in vivo studies, showing no toxic effect in adult animals .
All groups were sacrificed at 15.5 dpc using carbon dioxide, and their organs, placentas and fetuses collected for further analyses. Maternal organs from all animals, including liver, lung, kidney and spleen were fixed and processed for paraffin embedding. Spleens were weighted before fixation. Placentas and fetuses were carefully evaluated for the presence of malformations under a stereomicroscope. Fetuses that presented evident morphological abnormalities were photographed and then fixed with their placentas in 4% paraformaldehyde together with a morphologically normal sibling for subsequent histochemical and immuno-histochemical analysis. In parallel, fetuses and placentas from control mothers, which received the vehicle itself, were analyzed.
Biochemical analysis of maternal blood
All blood samples were collected by retro-orbital bleeding in SST microtainers (Serum Separator Tube, Becton, Dickinson and Company, USA), from animals anesthetized with a drop of local anesthetic (Novesina, Novartis Pharma S.p.A., Italy). All samples were centrifuged in a microcentrifuge (5415R model, Eppendorf s.r.l., Italy) at 13,000 rpm for 7 min to separate the serum. Serum levels of aspartate aminotransferase, alanine aminotransferase, creatinine, blood urea nitrogen and lactate dehydrogenase were measured using the automatic analyzer Keylab (BPC BioSed s.r.l., Rome, Italy).
Histochemical and immuno-histochemical analysis of maternal tissues
For histochemical analysis, tissues from all animals used in this study were collected, and processed for paraffin embedding. At least twenty sections (one every other 10) of each paraffin block have been routinely stained by H&E. Based on results of the H&E staining, selected paraffin blocks were used for immuno-histochemical analysis. Five micron sections were collected on slides and a part was stained with hematoxylin and eosin, a part used for immuno-histochemical analysis with the rat monoclonal antibody anti-CD31 (clone Mec13.3, BD Pharmingen, NJ, USA). Briefly, slides were de-paraffinized in xylene, rehydrated through the ethanol series and treated in 0.3% H2O2-methanol for 30 min at room temperature (RT) to block endogenous peroxidase activity. Following a 30 min pre-treatment at 37°C with 30 μg/ml proteinase K (in 0.2 M Tris–HCl, pH 7.2), each section was incubated with a blocking reagent (0.5%, TSA-Indirect Kit, NEN Life Sciences) for 30 min at RT, and finally incubated overnight at 4°C with the anti-CD31 antibody at a concentration of 2.5 μg/ml. For control slides the primary antibody was replaced by a non-specific rat IgG at the same concentration as the primary antibody. After 30 min incubation with secondary biotinylated anti-rat antibodies, staining was revealed using the Tyramide Amplification System (TSA-Indirect Kit, NEN Life Sciences). Slides were counterstained with Mayer’s hematoxylin for 5 min, dehydrated and mounted in Permount mounting medium.
Apoptosis was evaluated with the In Situ Cell Death Detection Kit from Roche (Roche Applied Science, IN, USA), following the manufacturer specifications.
When not otherwise stated, data are presented as mean ± standard error. A two-tailed value of P < 0.05 was considered statistically significant. Student’s t test was used for inter-group comparison of continuous variables (e.g. weight of dams and fetuses, number of resorptions, fetal size), whereas Fisher’s exact text was used for comparison of categorical variables (e.g. prevalence of dams with at least one malformed embryo, prevalence of malformed embryos, prevalence of liver damage). Analyses were performed by means of the software SPSS Statistics 19 (IBM Corporation, Armonk, NY).