In Vivo Fate of Carbon Nanotubes with Different Physicochemical

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In vivo fate of carbon nanotubes with different physicochemical properties for gene delivery applications Anna Cifuentes-Rius, Nathan R.B. Boase, Ines Font, Núria Coronas, Victor Ramos-Perez, Kristofer James Thurecht, and Salvador Borros ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00677 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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ACS Applied Materials & Interfaces

In vivo fate of carbon nanotubes with different physicochemical properties for gene delivery applications Anna Cifuentes-Rius§,#,†, Nathan R.B. Boase#,‡, Ines Font§, Nuria Coronas§, Victor RamosPerez§, Kristofer J Thurecht#, Salvador Borrós§,‡*

§

Grup D’Enginyeria de Materials (GEMAT), IQS School of Engineering, Ramon Llull

University, Via Augusta 390, Barcelona 08022, Spain #

Australian Institute for Bioengineering and Nanotechnology (AIBN), Centre for Advanced

Imaging (CAI), Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, Brisbane, QLD 4072, Australia ‡

Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina

(CIBER-BBN), Zaragoza, Spain KEYWORDS. carbon nanotubes, carbon nanotube rigidity, biodistribution, cytotoxicity, gene transfection

ABSTRACT. Gene therapy has arisen as a pioneering technique to treat disease by direct employment of nucleic acids as medicine. The major historical problem is to develop efficient

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and safe systems for the delivery of therapeutic genes into the target cells. Carbon nanotubes (CNTs) have demonstrated considerable promise as delivery vectors due to their (i) high aspect ratio and (ii) capacity to translocate through plasma membranes as nanoneedle. To leverage these advantages, close attention needs to be paid on the physicochemical characteristics of CNTs used. CNTs with different diameters (thinner and thicker) were treated by chemical oxidation to produce shorter fragments. Rigid (thick) and flexible (thin) CNTs, and their shortened versions, were coated with polyallylamine (ppAA) by plasma enhanced chemical vapor deposition. The ppAA coating leads to a positively charged CNT surface able to electrostatically bind GFP plasmid reporter. This study shows how rigidity and length can affect their (i) behavior in biological media, (ii) ability to transfect in vitro, and (iii) biodistribution in vivo. This generate set of basic design rules for the development of more efficient CNT-based gene delivery vectors.

1. Introduction Carbon nanotubes (CNTs) are an interesting vector for gene delivery as they are capable of entering the cell directly through the plasma membrane, due to their favorable lipophilicity and size, without the use of endocytic pathways.1-4 Direct intracellular delivery, also called the nanoneedle effect, is an attractive approach, since conventional methods can be inefficient at overcoming biological membranes.5 Moreover, the nanoneedle behavior of CNT favors their diffusion into the cytosol and subsequently missing the endosomal cycle, which is a key requirement for gene delivery in order to avoid nucleic acid degradation by enzymes.6 However, clinical implementation of CNT gene delivery vectors has been hampered by solubility limitations and spontaneous aggregation in vivo. This low biocompatibility can induce toxicity at a cellular level and the recognition and clearance by the mononuclear phagocyte system (MPS).7 Thus, the phagocytosis and cytotoxicity of CNTs has been intensively investigated;. A collection


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of conflicting results has arisen as a result of these studies.7-12 Some studies show low or insignificant cellular responses when CNTs are exposed to several cell types, while others report toxic effects after their use.8, 13-14 Most of these studies highlight that there are certain common parameters that play a crucial role in the internalization process, biodistribution and cytotoxicity of CNTs. Not surprisingly, these parameters are based on the physicochemical properties of the CNTs (Scheme 1), such as their size, stiffness, surface functionality and aqueous dispersibility. Nevertheless, the effect of such features on the cellular and immune recognition cannot be directly compared from the results of these investigations due to the lack of standardized CNT samples and protocols. Thus, the aim of our work was to determine how rigidity, which is determined by the diameter, and length of CNTs affect their performance in vitro and in vivo as gene delivery vectors.

Scheme 1. Effect of the physicochemical properties of CNTs in their interaction with cells. Factors influencing the (A) cell internalization, viability and biodistribution of the CNTs: (B) size, (C) chemical functionalization, (D) stiffness and (E) dispersibility. The physical characteristics of CNTs can influence their behavior and final properties.15 This is also applicable in biological system, where the nanotube length and rigidity may be key to the penetration capability and play a significant role on the degree of toxicity of the system.9, 16-17 In


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the case of multi-walled CNTs (MWNTs), such as those studied in this work, the diameter mainly depends on the number of graphene layers forming the nanotubular structure, which in turn affects the MWNT rigidity. Both parameters, length and rigidity, have been implicated in enhanced CNT toxicity.7,

9, 11

Kostarelos suggested that short or tangled MWNTs could be

successfully engulfed by macrophages and thus cleared via the lymphatic system.9 On the other hand, long and rigid MWNTs were not completely phagocytized and therefore would be accumulated in tissues promoting carcinogenesis. Dispersion of CNTs is particularly difficult to achieve as a consequence of their hydrophobic nature, leading to the formation of bundles or aggregates due to strong cohesive forces between individual tubes. As such, chemical surface modification of CNTs is a prerequisite to achieving homogeneous dispersion in an aqueous phase. Ultimately, this leads to longer blood circulation times, improved biocompatibility and more efficient internalization into cells and tissues.1, 4, 18-22 Full exploitation of the benefit of CNTs in biological environments requires their appropriate surface modification with biocompatible materials in order to facilitate stabilization of individual nanotubes in aqueous dispersions. Different procedures for CNTs fabrication are available, producing nanotubes with a variety of lengths and diameters.23-25 Moreover, there are many strategies for improving CNTs dispersion in aqueous media, including covalent and non-covalent modifications as discussed in our previous work.26 However, the production of CNTs with the desired mechanical and physicochemical properties that will allow the nanoneedle-like internalization process with a reduced level of toxicity is still an open question. In addition, existing limitations in CNT fabrication may cause intrinsic differences, compromising the size and shape homogeneity within the same type of CNT.7-8,

27

Other differences, such as subtle variations in local and


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overall charge, the remains of catalyst residue used during fabrication (typically Fe, Co and Ni) or several structural defects, are prone to occur and may present an issue in terms of toxicity.8, 28 These impurities and flaws present in the inner structure of individual CNTs are difficult to remove after production and subsequent purification. CNT shortening via oxidative methods has been presented as an interesting tool to control both the length of CNTs and the presence of defects and metal impurities.23, 25, 29 Other approaches, including mechanical cutting via sonication of the CNTs, have been developed.30-32 Nevertheless, all these procedures require the application of harsh conditions to the nanotubes, damaging their sidewalls, increasing the amount of structural defects and ultimately altering their morphology. On the contrary, mild chemical oxidation of CNTs utilizes the already existing defects in their structure, cutting the nanotube in a more controlled way, and reducing the impact on their chemical and physical properties.23,

25, 29, 33 34-35

To further develop this technique requires

evaluating the level of control the cutting methodology has on the CNT morphology. Knowing that the physicochemical properties of the CNT will affect their behavior in biological systems, this report seeks to bring us closer to the optimized form of CNT for gene therapy. In our previous study we demonstrated that CNTs could be covalently modified by plasma enhanced chemical vapor deposition (PECVD) techniques with two polymers capable of condensing DNA, and we identified these vectors as promising gene carriers for gene delivery applications.26 The polymerization of one of these monomers, allylamine (AA), leads to a polymer coating with high density of amines, which is especially interesting given its ability to wrap DNA along the CNT surface. In this work, we use this system to explore the gene transfection efficiency when using CNT with different mechanical properties. We employed two different types of nanotubes with differences in their intrinsic physicochemical traits: we call


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them rigid (rCNTs) and flexible (fCNTs). These two types of CNTs were also shortened with an oxidizing agent (s-rCNTs and s-fCNTs) and their change in length was monitored. We examined and compared their properties in terms of in vitro cytotoxicity and in vivo biodistribution, after being polymerized with allylamine by PECVD (ppAA). Here, we present the key factors to control in order to develop successful CNT-based gene delivery vectors. We show that CNT rigidity has a major impact on its physiological behavior and thus, we can strategically use it to our advantage to preferentially target a specific organ with minimal cytotoxic effects.

2. Experimental section 2.1. Materials and reagents. Rigid CNTs (rCNT, 110-170 nm in diameter and 5-9 µm long) were purchased from Sigma-Aldrich. Flexible CNTs (fCNT, 15 nm in diameter and 1-5 µm long) were purchased from NanoLab Inc. Potassium permanganate (KMnO4, BioUltra 99.0%) was purchased from Fluka. Sodium hydroxide (NaOH), sodium sulfite (Na2SO3, 99%), sulfuric acid (H2SO4) were purchased from Panreac. All other chemicals were purchased from SigmaAldrich unless otherwise stated. 2.2. Shortening process of CNTs. rCNTs and fCNTs were heated to 590oC for 3 h in a porcelain flask in order to oxidize and remove the amorphous carbon. Approximately, 25 mg of heat-treated carbon nanotubes were dispersed in 30 mL of 0.2 M solution of KMnO4. A few drops of sodium hydroxide NaOH 0.2 M were added until the pH of the dispersion was 10. The mixture was placed in an ultrasonic bath for 20 min and then refluxed for 40 min at 100oC. The mixture was agitated for 20 h after adding 0.7 g of Na2SO3 and 7 mL of concentrated H2SO4. Milli-Q water was added to the mixture to stop the reaction until the volume reached approximately 100 mL and it was centrifuged (Hettich EBA 21) for 20 min at 1000 rcf. Part of


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the supernatant was removed and the pellet and the rest of aqueous phase was filtered through a polytetrafluoroethylene membrane (Millipore, FGLP 00 of 047, 0.22 µm of pore size). During filtration, several washes were carried out; first, with a solution of 0.4 g/L NaOH, second with Milli-Q water, and finally with concentrated HCl to remove the manganese oxide (MnO2) formed due to the reduction of the potassium permanganate. The washing procedure was continued until the typical brown color of manganese oxide was not observed on the filter. The membrane with the shortened nanotubes (s-rCNTs) was vacuum dried for 2-3 days. 2.3. Functionalization of CNTs by PECVD. CNTs were placed into the chamber of the home-built reactor as previously reported.26,

36-37

The AA monomer was fed into the reactor

chamber and the polymerization parameters were set at an input power of 15 W and a duty cycle of 10/20. The polymerization process was carried out for 5 min and repeated two more times after shaking the CNT powder to ensure homogeneous functionalization. ppAA-CNTs were dissolved in acetate buffer (NaOAc, 25 mM and pH 5, Merck Millipore) obtaining the same final CNT concentration of 1 mg/mL in solution. Samples were dispersed in an ultrasonic bath for 2 h. 2.4. Characterization of the CNTs. All types of CNTs (rCNTs and fCNTs) and their shortened versions were analyzed prior functionalization by HRTEM (JEOL LTD. 200 KV) equipped with an energy dispersive X-ray Spectroscopy (EDS) detector (Oxford LINCA). From the HRTEM images, 10-20 CNTs were measured and the average length was calculated. Solutions of around 10 µg/mL of ppAA-coated and non-functionalized CNTs were prepared in NaOAc (25 mM, pH 5). Zeta Potential and Dynamic Light Scattering (DLS) were performed in a Zetasizer Nano ZS (Malvern Instruments). Pictures of the vials with the CNTs solutions (1 mg/mL) were taken right after sonication and also being left at RT for 1 h.


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2.5. Dispersibility studies. The 1 mg/mL CNT solutions were placed in a 96-well plate (flat bottom, Nunc) and the absorption at 808 nm was measured with time using a microplate reader (Infinite M200, Tecan Group Ltd). The acquired absorbance at this wavelength and a calibration curve were used to determine the concentration of CNTs in solution. 2.6. Formation of the CNT-based gene delivery vectors. CNT-ppAA systems were complexed with a reporter gene (3300 base pair) encoding the production of green fluorescent protein (GFP), which can be easily determined by the fluorescent microscope. The plasmid (pGFP) was expressed, isolated and purified using the PureLink® HiPure Plasmid Miniprep Kit (Life Technologies) according to the manufacturer’s instructions. Freshly ppAA-modified CNTs were dissolved in NaOAc (25 mM, pH 5) leading to the protonation of the amines. Different ratios (w/w) of ppAA-modified CNTs and pGFP (from 1:1 to 200:1) at a fixed quantity of plasmid were prepared and incubated for 1 h at 37oC. The complexation ratio was analyzed by agarose

gel

electrophoresis.

Agarose

was

dissolved

at

0.8%

in

Tris-acetate-

ethylenediaminetretraacetic acid (TAE) buffer 1x and 6 µL of ethidium bromide. TAE buffer was prepared from mixing 2.42 g of Trizma® base, 0.57 mL of acetic acid, 0.185 g of ethylenediaminetretraacetic acid (EDTA) and 500 mL of Milli-Q water. The gel was loaded with the different complexes and was run at 65 V for 1 h. After that time, the resulting gel was imaged. 2.7. In vitro testing of the CNTs. To study the cellular uptake and cell viability of the CNTmediated pGFP delivery systems, 3T3 cells (ATCC) were used. Cells were plated into a 96-well plate at a concentration of 10000 cells/well (150 µl/well) in supplemented medium formed by DMEM/F-12 high glucose 4.5 mg/L (GLUTAMAXTM), 1% penicillin-streptomycin (Invitrogen)


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and 10% fetal bovine serum (Gibco®). After 24 h at 37oC and 5% CO2, CNT-ppAA+pGFP solutions in DMEM (4, 10 and 30 µl/mL) were added to the cells. Cells were washed after 24 h. 2.8. Cell viability. Cytotoxicity of the CNT-based delivery systems were measured using a 3(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyletrazoliumbromide (MTT) cell viability assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay) from Promega. The MTS reagent (diluted at 20% in fresh DMEM as specified by the supplier) was added 24 h after washing the cells from the CNTs. Cells were left for 40 min at 37oC and 5% CO2, and the absorbance at 490 nm was measured. Prior to adding the MTS solution, cells were suspended with 100 µl PBS and the background absorbance at 490 nm corresponding to the CNTs was determined for further calculations. The absorbance of a positive control with only 3T3 cells (considered 100% of viability for further quantification) and a negative one with the MTS reagent alone were also determined. 2.9. Cell transfection. Cells were imaged in a fluorescent microscope (Olympus IX51) 24 h after washing the cells with fresh media to evaluate the gene transfection efficiency of the different CNTs. A positive control was used (GeneJuice® Transfection Reagent, Merk Millipore; GJ) and prepared by mixing 2.7 µL of GJ with 300 µL of DMEM. After 5 min at room temperature, 60 µL of pGFP (18 µg/mL) were added and left for 15 min at room temperature. The resulting mixture was diluted 6 times in order to ensure that 50 ng/well (100 µL) of pGFP were incubated with the cells. Cell viability was also determined by the MTS assay previously explained (data not shown). The same procedure was carried out with cos-7 cells (ATCC) with both rCNTs and s-rCNTs modified with ppAA (4 µg/mL). GFP production was confirmed by fluorescence microscopy (Leica UK Ltd, Milton Keynes, UK).


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2.10. Preparation of fluorescently labeled CNT complexes for in vivo biodistribution studies. An infrared (IR) dye (IRDye® 680 Dye NHS Ester) purchased from Li-Cor was covalently immobilized on the ppAA-modified CNT surface. All types of ppAA-coated CNTs (rCNTs, s-rCNTs, fCNTs and s-fCNTs) were dissolved in phosphate salt (10 mM, pH 9) to a concentration of 500 µg/mL. 50 µL of dye solution in DMSO anhydrous (1 mg/mL) were added to the CNT solution and the mixture was left overnight at room temperature and protected from light. The fluorescently labeled CNTs were washed twice in PBS 1x centrifuging at 13,500 rcf. 2.11. In vivo and ex vivo biodistribution in mice. All animal experiments were performed in compliance with the local ethics committee of the Australian Institute for Bioengineering and Nanotechnology (AIBN) and the Centre for Advanced Imaging (CAI) at the University of Queensland (UQ), Australia (AIBN/251/12). In this study, 150 µL of the fluorescently labeled CNTs (200 µg/mL) in PBS 1x were intravenously injected by the tail vein to each C57BL/6J mouse (n=3). Biodistribution of the fluorescent CNTs was monitored in vivo over a period of 1 h, 8 h and 24 h after injection using fluorescence imaging (In Vivo MS FX Pro instrument, now supplied by Bruker Corporation). Fluorescence images were collected using parameters optimized for in vivo signal, with a 720 ± 10 nm excitation and 790 nm ± 17.5 nm emission filter set (f-stop 2.80, 4 × 4 binning, 120 mm FOV, 60 s exposure time). To provide anatomical context, fluorescence images were coregistered with an X-ray image (f-stop 2.80, 0.2 mm aluminum filter, 120 mm FOV, 30 s acquisition time). All mice were sacrificed 24 h post-injection and their main tissue organs (liver, gut, lung, kidneys, spleen and heart) were immediately dissected and imaged ex vivo. All images were batch exported as 16-bit TIFF images and image processing was completed using Image-J (National Institutes of Health, USA).38 All fluorescence images were normalized


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for injected dose, using the image math tool on Image-J, and false colored with a LUT to represent pixel intensity, and overlaid onto corresponding X-ray images. Fluorescence of each organ was evaluated by measuring the raw integrated density of the pixel gray values for the fluorescence image for each organ, and normalizing against the ROI area for that organ, measured from the X-ray image (reported as total normalized fluorescence). All imaging data was plotted and analysed using GraphPad Prism 7.0 (GraphPad Software, La Jolla California USA, www.graphpad.com). Comparisons of the differences in fluorescence signal between the different CNTs and between organs for the same CNT was performed using a two-way ANOVA of the mean, followed by Tukey’s multiple comparisons test (n=3, α=0.05).

3. Results and Discussion

3.1. Shortening of carbon nanotubes Commercially available CNTs, so-called rigid (rCNTs) and flexible (fCNTs) CNTs, were oxidized with KMNO4 to produce short rigid (s-rCNTs) and short flexible (s-fCNTs) CNTs. Their change in size and morphology was evaluated. High-resolution transmission electron microscopy (HR-TEM) images confirm the shortening of both types of CNTs without altering their structural integrity (Figure 1). By measuring 10-20 individual of shortened CNTs (both types), the length reduction is calculated to be approximately 60% (Figure S1). However, HRTEM images show a high polydispersity in overall length, in both types of CNTs. This can be explained by the interplay of two factors: (i) the lack of control during the CNTs synthesis, leading to the production of differently-sized nanotubes; and (ii) the unequal cutting procedure


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applied to all CNTs present in the solution due to their structural differences. Both issues lead to the high dispersity in CNTs length.

Figure 1. TEM images of (A) rigid CNTs and (B) flexible CNTs (1) as purchased (2) after shortening (s-rCNT). Scale bar: 200 nm. Insets: zoom-in of each corresponding CNT type; scale bar: 200 nm. As purchased rCNT were 110-170 nm in diameter and 5-9 µm long; fCNTs were 15 nm in diameter and 1-5 µm long. TEM-EDS analysis determined the chemical composition of the treated and bare CNTs (Table S1). Interestingly, several elements (Si, S, Fe) were only detected on the fCNTs, which were not detected on the rCNTs. These impurities are typically metal catalyst residues, most likely introduced during the commercial synthesis and purification of the fCNTs. The amount of such elements present in the original fCNT decreased after oxidative shortening. Some researchers have confirmed that these residues are responsible for inducing toxicity.7-8,

28, 39

Hence, these

results demonstrate that shortening CNTs by an oxidizing treatment with KMnO4 can lead to a significant reduction of impurities found on the original fCNTs, which should enhance their biocompatibility.


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3.2. Effect of chemical surface modification on CNT dispersibility While CNTs length and the presence of metal impurities may have a remarkable effect on toxicity issues, the degree of dispersion is also a crucial parameter to be taken into account.7-8, 12 This factor will govern the CNT-cell interactions, having a direct impact on their cellular internalization.20,

22

Highly dispersed CNTs are therefore desirable to successfully penetrate

through the cell membrane and can be obtained by chemical surface modification. 3.2.1. Functionalization of CNTs with ppAA by PECVD CNTs were coated with AA by PECVD, to improve dispersibility in aqueous media, and to allow DNA complexation through electrostatic interactions.26 Zeta-potential measurements confirmed surface functionalization (Figure 2A). As-purchased CNTs (rCNTs and fCNTs) showed negative zeta potential in water. The negative charge was increased after oxidation because of the formation of hydroxyl, carbonyl, and in particular carboxyl groups along the surface.34 When the CNT were modified with ppAA they presented positive zeta potential, due to the presence of the protonated amine species of ppAA.

Figure 2. (A) Zeta potential and (B) dispersibility of rCNTs, s-rCNTs, fCNTs and s-fCNTs in acetate buffer (25 mM, pH 5) before (untreated) and after functionalization with ppAA by PECVD. Images taken after sonication for 1 h at room temperature.


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HR-TEM images were also collected before and after ppAA polymerization for rigid and flexible CNTs (Figure S2 and Figure S3 respectively). These images show that the morphology along the wall or the tip of CNTs was not altered due to the PECVD treatment. For both rCNTs and s-rCNTs the ppAA film was observed to have a thickness of around 5 nm, which is consistent with what we have identified previously (Figure S2).26 On the other hand, the ppAA layer on the flexible CNTs (fCNTs and s-fCNTs) was barely detectable, probably due to the low contrast (Figure S2). However, zeta potential results confirm the presence of the ppAA coating. 3.2.2. Dispersibility of ppAA-coated CNTs Figure 2B shows the colloidal stability of CNTs in water, before and after ppAA polymerization. Untreated s-fCNTs are more stable in solution than s-rCNTs. The shortening process has caused a higher oxidation degree on the thinner fCNTs compared to the thicker rCNTs, as confirmed by the increased negative zeta potential due to the creation of oxidized species on the CNT wall (Figure 2A). The difference in their wall thickness is likely to change their resistance to oxidation – thinner CNTs are most easily oxidized. Once polymerized, rCNTs and their shortened version exhibit similar behavior in solution, whereas differences are observed between the flexible tubes (Figure 2B). After ppAA deposition, both types of rigid CNTs and s-fCNTs exhibit a better dispersion degree than that observed for fCNTs. For the latter, the combination of their intrinsic hydrophobicity and tangling behavior may cause a higher aggregation and lower stability in solution. However, when fCNTs are shortened (s-fCNTs), both hydrophobicity and length are reduced leading to an improved dispersibility when coated with ppAA. We suspect that a synergetic effect is produced between the shortening treatment and the plasma polymerization that increases significantly the water


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dispersibility of the modified CNTs. Such treatment provides the dispersibility in aqueous media required to use CNTs as gene delivery vectors.

3.3. Study of the cell viability and transfection efficiency of CNT 3.3.1. Formation of the gene vector As a model system, to demonstrate the ability of these materials to act as gene vectors, the ppAA coated CNTs were complexed with a plasmid reporter of green fluorescence protein (GFP) as described elsewhere.26 The complexation takes place through ionic interactions between the negatively charged phosphate groups on the plasmid DNA backbone, and the positively charged amines on the surface of the modified CNTs at pH 5. Gel retardation assay was used to prove the CNTs complexation ratio, when the same amount of plasmid GFP (pGFP) was exposed to increasing amounts of CNTs. Complete complexation was observed at a CNT-toDNA ratio of 150:1 for rCNT-ppAA, which was reduced to 25:1 when s-rCNT-ppAA were used (Figure S4). One of the advantages of using CNTs for delivery applications is their high surface area, allowing higher loading capacities than other nanovectors.20,

40

Noteworthy, this can be

seen in our results, where a lower quantity of CNTs was required to load the same amount of pGFP when the shortened version of rigid CNTs was employed. Because s-rCNTs are shorter than rCNTs, they offer a higher specific area per unit mass, allowing a greater loading efficiency. fCNT-ppAA exhibited the same complexation efficiency as s-rCNT (25:1). The difference in thickness between the fCNT and rCNT, is likely to be the cause of the higher loading efficiency observed for the thinner fCNT-ppAA. The lower diameter and length of the fCNTs enabled a higher pGFP loading capacity, which is increased again with the length reduction of the s-fCNTs (10:1 ratio).


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3.3.2 In vitro cytotoxicity and cell transfection assays In vitro testing of the CNT-mediated gene delivery systems was undertaken to evaluate their cytotoxicity and GFP transfection efficiency. The final complexes were diluted in DMEM and were incubated with 3T3 cells at different final concentrations (4, 10 and 30 µg/mL). The quantity of CNTs was kept constant across all samples, which due to different loading efficiencies led to a range of plasmid loadings of 4 to 450 µg/well (Table S2). The complexes exhibited a decrease in cell viability with the increase of CNT concentration as measured by MTT

(3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyletrazoliumbromide)

cell

viability

assay

(Figure 3). In general, toxicity of the four types of CNTs tends to increase as a function of CNT concentration. This may be caused by (i) the aggregation tendency of the CNTs at higher concentrations, and (ii) the fCNT tendency to tangle and form large aggregates. Optical microscopy images (Figure S5) of the 3T3 cells after incubation with the CNTs shows the presence of very large CNT agglomerates in the fCNT experiments, and smaller aggregates in the s-fCNTs, which are not present in both rCNTs and s-rCNTs experiments. We have demonstrated that the strong aggregation tendency of CNTs is specifically observed in the presence of serum proteins, as shown in Figure S5, which was not seen in the acetate buffer solutions (Figure 2). It can be seen visually (Figure S5B) and from the DLS size measurements (Figure S5C) that the CNTs flocculate in the presence of serum proteins, which is likely due to the formation of a protein corona. This effect is consistent to what has been observed in our previous work.41


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Figure 3. Cell viability of the pGFP complexes varying the CNT concentration. Data obtained after 48 h incubation in 3T3 cells. The aggregation caused by the protein corona formation, especially for flexible CNTs, may complicate their internalization into the cell with dramatic consequences at high concentrations.42 This is why cells incubated with low concentration (4 µg/mL) of rCNTs and s-rCNTs were the only ones to show GFP transfection (Figure 4). The observed green fluorescence was attributed to the production of GFP as CNTs are known to have minimal autofluorescence background.43 By using rigid CNTs at low concentration, 3T3 cells were successfully transfected due to their better dispersibility behavior in biological media compared to the fCNTs. This confirms that aggregation avoids CNT internalization and subsequent GFP transfection, decreasing also the cell viability. In order to confirm that the transfection ability is limited by the amount of plasmid, a dose matched positive control (50 ng of pGFP per well) was performed. This result shows that the transfection efficiency is consistent to the amount of plasmid loaded in the CNTs. These results are sustained when other fibroblast-like cells were transfected (COS-7), showing that pGFP transfection by rigid CNTs is not dependent on the cell type employed.44 32


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Figure 4. Fluorescence microscopy images of 3T3 and cos-7 cells incubated with 4 µg/mL of rCNTs, s-rCNTs, and with Gene Juice (GJ) as a positive control (50 ng/well of pGFP). Scale bar:100 µm.

3.4. In vivo biodistribution of the CNTs in mice In vitro studies are useful to reduce complexity when studying nanomaterial-cell interactions. However, the overall effect and behavior of these nanoparticles may change when introduced in living animals.45 For example, the formation of a protein corona around nanoparticles is difficult to avoid and can dramatically change the surface chemistry and properties of the nanomaterial. Recent reports suggest that nanoparticles with controlled geometrical features and appropriate surface modification can present preferential tissue and organ targeting when administered systemically.46-48 Such features are highly desirable since it implies that rational design of nanoparticles may address basic strategies for maximizing target accumulation while minimizing non-specific uptake.49


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Therefore, elucidating the in vivo biodistribution profile of the CNTs studied in this work is of vital importance in understanding how these systems behave under physiological conditions, and to begin understanding both their clearance and toxicological behavior. In general, nanoparticles are known to be cleared rapidly from the circulation by the mononuclear phagocyte system (MPS).50-51 However, tailoring the size, shape and surface characteristics of nanocarriers may influence the blood circulation time, clearance and organ biodistribution of the resulting delivery system, known as geometrical targeting.52-60 This in vivo study would enable the selection of the most promising candidate to maximize the CNT uptake when targeting particular organs for therapeutic delivery. 3.4.1. Labelling CNTs with a fluorescent dye Modern fluorescent imaging techniques have significantly enhanced the ability to study nanoparticle biodistribution in vivo in a facile and more cost-effective way. In order to utilize this approach, nanoparticles must be intrinsically fluorescent or labelled using an exogenous agent. Amino groups of ppAA provide a convenient chemical handle for functionalisation with amine reactive fluorophores, such as IRdye-NHS (IRDye® Cy5.5 Dye NHS Ester). The NHS groups react to form a covalent linkage to the CNTs, providing a fluorescently labelled nanomaterial. After several washing steps, the fluorescently labelled CNTs were analysed by UV-Vis (Figure S6). The dye concentration on the fluorescently labelled CNT varied across the samples ranging from 51 to 94 nmol/mg (Table S3). Once the fluorescent properties of the four types of CNTs were confirmed, the nanomaterials could be fluorescently tracked when administered systemically. 3.4.2. In vivo and ex vivo fluorescence imaging study of fluorescently labelled CNT


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In vivo and ex vivo fluorescence imaging can provide a semi-quantitative measurement of the different behavior of CNTs upon intravenous administration and allows monitoring of the same subject at multiple time points. The effect of the CNT length and diameter was therefore examined after well-dispersed fluorescently labeled CNTs (1.5 µg/g mouse) were administered intravenously to each mouse (n = 3). One mouse for each CNT group was imaged after 24 hours post injection (Figure 5). After the final time point the mice were culled and the major organs (liver, gut, lungs, kidneys, spleen and heart) were immediately harvested and imaged. No side effects were apparent in the mice injected with fluorescently labeled CNTs during the 24-hour time period of the study. In vivo imaging (Figure 5) shows some signs of accumulation of all CNTs (rCNTs, s-rCNTs, fCNTs and s-fCNTs) in the major abdominal organs. Others have also reported this behavior, confirming that the CNTs are able to circulate throughout the mouse enabling the subsequent accumulation within various tissues.54, 60

Figure 5. Comparison of in vivo biodistribution of the fluorescently-labeled (A) rigid CNTs and (B) flexible CNTs injected in mice. Fluorescence images acquired 24 h after the injection of (A1) rCNTs, (A2) s-rCNTs, (B1) fCNTs and (B2) s-fCNTs. The calibration bar on the left correspond


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to the A images and for mice B the one in the right applies. Prior to injection, ppAA-modified CNTs were fluorescently labeled with a IR dye through the amine group on the CNT surface. Images are normalized for quantity of injected fluorophore. In this study, it should be noted that the fluorescence images were significantly affected by the high levels of melanin present in the C57/bl 6 black mice, which greatly reduced the amount of detectable signal. To overcome this issue, we utilized ex vivo fluorescence imaging to determine the relative accumulation of CNTs in each organ (Figure 6).

Figure 6. Ex vivo organ biodistribution of the fluorescently labeled (A) rigid CNTs and (B) flexible CNTs after 24 h of injection. X-ray images overlaid with fluorescent images corresponding to the accumulation of (A1) rCNTs, (A2) s-rCNTs, (B1) fCNTs and (B2) s-fCNTs in the gut, liver, kidneys, lungs, spleen and heart. Images are normalized for quantity of injected fluorophore.


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Ex vivo fluorescence imaging is able to provide a reliable semi-quantitative assessment of the amount of fluorophore present in each organ.61 Results (Figure 7 and Figure S7A) show that lung accumulation of s-fCNTs was statistically higher than the observed in almost all the other organs (except for the gut). fCNT show similar behavior with statistically higher lung signal than the other organs (except no statistical difference compared to liver and gut). Others have reported similar results, specifically when SWNT or thin MWNT (10-30 nm) are used.62-64 Lung signal was reported to represent large and possibly aggregated particles being caught in the fine capillary beds.63, 65 This is consistent with our dispersibility studies described previously, where it was demonstrated that flexible CNTs tend to tangle and form larger aggregates (Figure 2B and Figure S5). In the fluorescence images for both types of flexible CNTs, similar accumulation was observed in the gut, liver and kidneys. The first two (gut and liver), indicate that flexible CNTs are likely being excreted through the biliary pathway. These results match with what was also observed by others, where CNTs were excreted by the gastrointestinal tract after being accumulated in the liver.59, 66-67 Both lung and liver accumulation are slightly higher for fCNT than for s-fCNT. This may be caused by the higher aggregation tendency of the fCNT compared to s-fCNT as discussed before. Kidney signal was also high, which can be explained by less dispersed CNTs that are not able to translocate through the renal filtration barrier and accumulate in the glomerular capillaries.55, 64 The lower accumulation of s-fCNT in the liver is also likely due to the slightly better dispersibility observed in biological media (Figure S5). When examining the ex vivo fluorescence images for rigid type nanoparticles, the level of accumulation in most of the organs is statistically similar for both rCNTs and the s-rCNTs (Figure 7 and Figure S7B). The highest signal is observed in the gut and liver, which suggests that rigid CNTs are also excreted through the biliary pathway. There is also a relatively strong


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signal detected in the kidneys, again suggesting that shorter fragments of rigid CNTs also accumulate in the kidney due to their larger diameter preventing translocation across the glomerular filtration system.55

Figure 7. Ex vivo semi-quantification of CNT distribution. Accumulation of fluorescently labelled rCNTs, s-rCNTs, fCNTs and s-fCNTs in different organs of mice measured 24 h postinjection. Data is presented as the mean ± SEM (n=3). Total normalised fluorescence is the raw integrated density of pixel gray values, divided by the ROI area for each individual organ and normalised for the quantity of injected Cy5.5 fluorophore. Asterisks on the top of each bar highlight the significance levels from the two-way ANOVA, followed by Tukey’s multiple comparisons, of the fluorescence signal of each class of CNTs in individual organs ***, P < 0.001; ****, P < 0.0001). 3.4.3. Comparison of the biodistribution between rigid and flexible CNTs When comparing ex vivo fluorescence images, flexible CNTs have a statistically larger accumulation in the lungs than rigid CNTs (Figure 7). The differences in dispersibility, as


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discussed previously in the in vitro experiments, are thought to be responsible for the remarkable difference in lung accumulation.63, 65 We have demonstrated that flexible CNTs tend to tangle and form larger aggregates in water (Figure 2B), which is exacerbated in the presence of serum proteins (Figure S5). The rigidity of the thicker CNTs (rigid CNTs) allows the adequate individualization of the CNTs prior to injection and prevents agglomeration in vivo, even after the formation of a protein corona. Thinner CNTs, on the contrary, form large aggregates after in vivo administration, which could result in the higher accumulation in the lungs. The same phenomenon is probably causing the higher signal observed in liver and kidneys for flexible CNTs. Lacerda and co-workers proved that individualized, well-dispersed MWNTs between 2030 nm are able to cross the endothelial fenestrations. However, the in vivo aggregation of the flexible CNT used in this study (15 nm diameter) would avoid translocation across the glomerular filter, instead most likely accumulating in the lumen of glomerular capillary.55 This study confirms that, rather than the CNT length, the diameter and dispersibility of the CNTs in biological environments is the key parameter that will directly affect biodistribution and cytotoxicity. Importantly, the various physicochemical properties of the CNTs, including surface chemistry, diameter and the rigidity will all influence the degree of dispersibility, and in term, the biodistribution. Diameter and rigidity are closely related traits - the thicker the CNT, the more rigid it will be. Therefore, both parameters must be taken into account when selecting a CNT-based delivery system. Overall, this work shows that using rigid CNTs will prevent undesired accumulation in organs such as the lungs.

4. Conclusions


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We have demonstrated that rigid (thick) and flexible (thin) CNTs can be shortened through mild oxidation treatments, without affecting their morphology, and leading to more hydrophilic surfaces. These differences in length, rigidity, and surface hydrophobicity, have a direct impact on the dispersibility, toxicity and interaction with DNA material. In general terms, rigid nanotubes studied in this work, (rCNTs and s-rCNTs) show a suitable degree of dispersion and higher cell viability. On the other hand, fCNTs are more prone to tangle in solution, which causes the formation of larger aggregates. Therefore, their stability decreases regardless of their surface functionalization, and increases their cytotoxicity. Although the dispersibility issues are reduced when fCNTs are shortened, their tangled behavior still induces aggregation and the formation of large supramolecular complexes. The different behavior between flexible and rigid studied nanotubes in the presence of serum also affects their ability to be used as gene vectors. Despite the ppAA-coated flexible nanotubes have been proven to bind higher amounts of pGFP than rigid tubes, their tangled behavior complicates the internalization of the complex into the cell and the successful release of the loaded DNA. In addition, this work shows semi-quantitative information about the distinct in vivo biodistribution profile of the different types of CNTs assessed. We have demonstrated that the physicochemical properties of the CNTs characterized, also influence their behavior inside the body. More specifically, we demonstrated that CNT rigidity (rigid vs flexible CNT) leads to differences in tissue accumulation. Further investigations should allow the total quantification of CNT accumulation in the organ tissues by in vivo radiotracing of radiolabeled systems. Furthermore, deeper research into the clearance half-life and blood circulation should be considered in order to identify the pharmacological profile of the in vivo administrated CNTs.


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These studies will provide a better understanding of the uptake mechanism and organ biodistribution of these systems, which is essential for targeted gene delivery. Overall, we have demonstrated that by choosing rigid CNTs over flexible ones, aggregation can be avoided, which is vital for the successful use of CNTs in drug and gene delivery.

ASSOCIATED CONTENT The following files are available free of charge. Supporting Information. Additional characterization of the shortened and/or functionalized CNTs and subsequent dispersibility data in vitro and quantification of organ-specific accumulation ex vivo (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Present Addresses †Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia ‡School of Chemistry, Physics and Mechanical Engineering, Faculty of Science and Engineering, Queensland University of Technology, Brisbane 4001, Australia


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Author Contributions All authors have given approval to the final version of the manuscript. Funding Sources Funding support from Generalitat de Catalunya (SGR 2009) granted to Grup d’Enginyeria de Materials (GEMAT) is kindly acknowledged. ACR was supported by a PhD fellowship from the IQS School of Engineering. The authors would also like to acknowledge the Australian National Health and Medical Research Council (GNT1112432 ACR). Notes The authors declare no competing financial interest. REFERENCES 1.

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In Vivo Fate of Carbon Nanotubes with Different Physicochemical

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