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Trends in Nanotechnology & Material ScienceReview ArticleDispersability of carbon nanotubes in biopolymer-based fluids and their potential biotechnological applications.

Gianfranco Risuleo,1 and Camillo La Mesa,2,*

Keywords: Single-walled carbon nanotubes, biopolymers, phase separation, liquid crystals, gels, dispersions, repulsive/attractive interactions, biomedical implications.

Corresponding author:

1 Dept. of Biology and Biotechnologies "Charles Darwin", La Sapienza University2 Dept. of Chemistry, Cannizzaro Building, La Sapienza University,P.le A. Moro 5, I-00185 Rome, Italy.

Copyright: © 2016 Gianfranco Risuleo, and Camillo La Mesa. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,and reproduction in any medium, provided the original author and source are credited.Citation: © Gianfranco Risuleo, and Camillo La Mesa (2016) Dispers-ability of carbon nanotubes in biopolymer-based fluids and their potential biotechnological applications. Insights in trends-in-nanotechnology ma-terial science. 1: 1-7Charles Darwin, Dept. of Chemistry, Cannizzaro Building, La Sapienza

University, P.le A. Moro 5, I-00185 Rome, Italy. Tel: +39 06 4991 3707 Fax :+39 06 4906 31, Email:camillo.lamesa@uniroma1.it. Received Date: July 14, 2016

Accepted Date: : July 19, 2016Published Date: July 29, 2016

AbstractThe dispersability of carbon nanotubes in aqueous solutions containing proteins or nucleic acids is discussed. In the mentioned systems different dispersion modes occur, depending on the type and concentration of biopolymer, and on the amount of dispersed nanotubes, as well. The phase behavior depends on how much biopolymers are adsorbing, and, naturally, on their molecular details. Modulation of the nanotube/biopolymer interactions helps switching between repulsive and attractive regimes between surface-functionalized particles. Accordingly, dispersion or phase separation take place, and the formation of nematic phases, or gels, may prevail with respect to dispersions. We report on systems containing DNA-stabilized and lysozyme-stabilized carbon nanotubes, since the two cases are representative of largely different adsorption modes. In the former case, ss-DNA uniformly rolls around CNTs and ensures significant surface coverage; conversely, lysozyme poorly, randomly and non-cooperatively adsorb onto nanotubes. A fine tuning of temperature, nano-tube content, added polymer, pH and/or ionic strength conditions induces the formation of different dispersion modes and different phases, as well.

IntroductionThe combination of their outstanding mechanical, optical, thermal and electrical conductive properties[1-3] makes nanoparticles, NPs, useful in the preparation of composites for advanced materials. This holds true, in particular, for carbon nanotubes, CNTs. Their peculiar features can be properly tuned to build devices, sensors,[4] actuators, drug delivery systems, [5] and scaffolds for tissue engineering.[6] Despite the huge number of possible applications suggested to date, the poor dispersability of CNTs in aqueous media drastically limits the possibility to get bio-compatible materials. This is because π-π orbitals lying on the outer CNT surface generate an extended aromatic character, and do not allow favourable interactions with water-based media. Therefore, stabilization methods are required; some of them imply chemical modification of nanotubes, via oxidation and subsequent covalent functionalization.[7] The mentioned procedure largely increases CNTs solubility, at the expense of significant modifications in their size; it also decreases some physically relevant performances. Functionalization favors the onset of specific groups on CNTs and offers the possibility to anchor thereon species that are potentially bio-active. It is expected, therefore, that the resulting composites can be relevant in bio-oriented applications.

If the original properties of CNTs must be retained, non-covalent func-tionalization procedures are preferred to others. These involve the adsorp-

tion of polymers,[8]surfactants,[9] and combinations thereof. Polymers must have the appropriate conformation for an effective stabilization; moreover, they must maintain, or improve, the peculiarities of pristine CNTs. To get effective bio-intended applications, the adsorbing polymers must be fully compatible with the hosting tissues; however, many of them do not fulfill these requirements. This is why attention from the scientific community oriented towards biopolymers. Accordingly, polypeptides,[10]proteins,[11,12]polysaccharides,[13,14] and nucleic acids,[15] were con-sidered as possible reactants for preparing hybrids with CNTs.

The philosophical foundations underlying the deep interest towards organic-inorganic hybrids, including the ones reported below, are many folds. An appealing aspect is the possibility to obtain nano-composites showing hybrid hard-soft character. The CNT-based core is hard and serves as anchoring site for biopolymers; the latter may form random or locally ordered domains onto particles. The wonderful structures observed in sea urchins and shellfishes, for instance, are made of systems bearing in the same time inorganic and biological moieties which self-help and self-complement.[16] A detailed knowledge of processes occurring at the interfaces between inorganic materials, such as CNTs, and biological macromolecules is, thus, of fundamental relevance. The molecular/functional details governing interactions between NPs and biopolymers allow to design materials for drug delivery, implants and devices. Surface functionalization of orthopedic and dental implants is an example.[17]

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Perhaps, the interactions effectively taking place at the biological/inorganic interfaces are not fully understood.

The effectiveness of CNT in adsorbing biopolymers cannot be envisaged a priori, since their surfaces are characterized by peculiar topographies at the nanometer scale.[18] In addition to chemical functionalization, thermal cycling, or milling, the presence of kinks, defects, disclinations, and heterogeneity at a local level plays a substantial role in polymer adsorption.[19-21] Also the acidity and hydrophobicity of CNTs are relevant in the interactions with biopolymers. The latter are endowed with complex and deformable architectures, with many functional groups available to binding. However, only certain conformations permit to biopolymers an extensive and homogeneous adsorption onto nano-particles. Sometimes, the biopolymers must be modified for interactions to be significant. For instance, DNA must transformed in its single-strand form, ss-DNA, to get significant interactions with CNTs.[22]As a rule, biopolymer location onto the given surfaces is the sum of several different contributions to the binding energy.

Some items related to the formation of stable CNT/biopolymer adducts are described below. We present and discuss previous results, relative to the adsorption of selected proteins and/or DNA onto CNTs.[23]The above data must be critically evaluated, in case of possible biomedical applications. Our efforts try to find links between the properties of biopolymer/CNT complexes and the fate they may encounter in biological matrices. Among biopolymers considered here, LYSO and ss-DNA, are representative of random (poor) and uniform (significant) CNT coverage, respectively. The stability of such complexes are substantially different and find application for selected purposes, depending on practical demand. Along this line, focus is on:

A) Physico-chemical properties of dispersed CNTs;

B) Dispersions of CNTs in Protein Solutions;

C) Dispersions of CNTs in DNA and RNA; and

D) Interaction with biological structures and biocompatibility assess-ment.

A critical analysis of experiments may give rise to new perspectives and allow to forecast which properties are to be expected in the resulting systems. We report on different aspects and discuss available information on CNT-biopolymer systems formerly dealt with. The final aim of this contribution is to shed more light on points under debate, to indicate which research lines are substantial and deserve more investigation. After discussing the physico-chemical properties of CNTs, in the following sections focus on the strategies required for an efficient surface coverage, the structure and supra-molecular organization modes met in the mentioned systems, the interaction with biological structures, and their biocompatibility. These are, in fact, the problems to face with when CNTs interact with biopolymers.

A) Physico-chemical properties of CNTs dispersions

Since Iijima discovered CNTs [24] efforts were devoted to optimize the procedures giving these materials. Focus is mostly on nano-tubes physical properties, such as their structural features, electronic conductivity, elas-ticity, thermal/mechanical stability, dispersability, etc.[25,26] Nowadays, it is possible to obtain CNTs in many forms and purity degree, depending on the synthetic conditions. We shall not come into detail on the ubiquitous presence of iron or metallic residues deriving from the catalysts used in the preparation of CNTs. Metal clusters are often embedded into nano-tubes,[27,28] operate as junctions between different CNT sub-units, and are, very presumably, the places where oxidation preferentially takes place first.

Carbon nanotubes are single-, SWCNTs, Figure 1, or multi-walled, MWCNTs; they are substantially different in terms of properties and reactivity; unless otherwise stated we refer to SWCNTs. The two categories have different reactivity, solubility, electrical/thermal conductivity, and elasticity. MWCNTs have relatively large diameters, D; the possibility

to distinguish them from carbon-based fibers is cumbersome when D is high. SWCNTs are thin and characterized by high aspect ratios, L/D, where L is their average length.

Figure 1. Schematic view of indefinitely long CNTs left, with indicated the related length, L, and diameter, D. In the right part of the figure is indicated what happens when different portions of CNTs are joined by metallic clusters, in red. For the sake of simplicity, units in the right hand side of the figure are supposed to have the same length.

Given the dispersity in both quantities, estimates of L/D ratios are sub-ject to large uncertainties, and must be considered with due caution. To estimate L/D ratios[29] DLS-based investigations require evaluating two different diffusive components. This is possible if the rotational dr and translational dt diffusion coefficients are measured independently, using polarized and non-polarized scattering. Altenatives to the above proce-dure imply determining dr and dt by independent methods, such as DLS and transmission electron microscopy, TEM.[30] Such evaluation allows to forecast quantities related to L/D. The following quantities can be in-ferred: a) surface area per unit volume; b) solubility in a given medium; c) possible bundling, d) elasticity, and e) phase behavior. Combination of CNTs and polymers gives the possibility to predict whether such mixtures form bundles or, when properly functionalized, they form dispersions, gels or nematic fluids, Figure 2.

Figure 2. Formation of strictly packed bundles (when CNTS are naked), A, bicontinuous gels (CNTs are partly surface covered), B, and nematic phases, C. In the latter picture is indicated the hexagonal structure, in red, the distance between rods, in blue, and the rod section, in yellow. In C rods are envaginated in continuous layers, not shown, allowing them to stay separated.

Bundles occur when CNTs are not adequately surface stabilized, dispersions when surface-stabilized entities are present in small amounts. Gels do form upon three-dimensional entanglement of functionalized CNTs; gelation depends on CNT volume fraction and on L/D ratios, as well. The formation of nematic phases also depends on the above quanties. In classical theories for the phase separation of an-isometric entities, the isotropic-nematic phase boundary is proportional to L/D. [31] It is possible, on this regard, to compare experiments and theory, knowing that the phase sequence for CNTs aqueous systems fulfills the scheme

Dispersions (Disp) → two-phase systems ( 2Ph) → nematic fluids (Nem)

Disp/2Ph 2Ph/Nem

( N.B. CNTs concentration increases according to the above phase

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sequence. The arrows indicate the location of the corresponding phase transitions.)

The mentioned phase boundaries are predicted by well-known relations,[32-34] which depend on the number of entities and by a an twisting parameter, related to the reciprocal orientation of CNTs. Usually, a shift to higher concentrations is found comparing values predicted from theory with experimental ones; this is because DNA/CNT complexes are not rigid and L/D ratios lower than expected, as also occurs in relatively rigid polymers[32].

Another relevant effect met in building the phase diagrams of polymer-NP complexes is depletion,[35,36] which is concomitant to the formation of bundles and macroscopic phase separation. Depletion occurs because the partition of stabilizers between bulk and nano-particles surface is controlled by their affinity for two such phases. Polymers preferentially located on the particles’ surfaces are also present in the bulk. The partition between such phases gives rise to an unbalanced osmotic effect when the volume fraction of the polymer in the solvent reaches a critical value. Both polymers and/or surfactants are responsible for the onset of unbalanced osmotic effects. To our knowledge, no detailed studies reported on depletion in CNT/polymer mixtures; however, evidence arising from surfactant/SWCNTs demonstrated that depletion is effective.[37] The critical depletion threshold decreases in inverse proportion to the volume fraction of free micelles, their size, and is sensitive to ionic strength. That is the reason why CNTs dispersability in surfactant systems is moderate and superiorly limited. Similar conclusions apply to polymer solutions; convincing evidence exists on the depletion of silica particles covered by PEO polymers in presence of the same polymer in the bulk.[38] In biopolymer-based systems depletion may depend on concentration, pH, and, eventually, ionic strength. Knowledge of the aforementioned effects is relevant when we want to know what may happen when functionalized CNTs are located in biological matrices. There CNTs find peculiar pH and ionic strength conditions, which, in addition to the presence of other biopolymers and particles, do not ensure a disperse state. Knowledge of the physical conditions that may occur in biological matrices is, therefore, precondition to avoid undesired effects, such as bundling and precipitation. The physics underlying depletion is based on fundamental studies by De Gennes.[36]

B) Dispersions of CNTs in Protein Solutions

B1) Generalities and dilute regime conditions

Proteins and/or synthetic polypeptides are suitable CNT dispersants.[39-41] The most used proteins are LYSO and BSA, which are globular in normal conditions. Their compact structure does not allow extensive interactions with CNTs.[42] The interactions essentially take place because of hydrophobic interactions. Protein conformation depends on their secondary structure and on that of particles on which adsorption takes place. Protein adsorption efficiency depends on whether SWCNTs or MWCNTs are used; this is because L/D ratios in the two cases are substantially different. In systems containing globular proteins adsorption onto surfaces reduces the amount of α-helix in favor of the β-sheet one; [43] the tertiary structure of proteins adsorbed onto CNTs is presumably lost. The adsorbent capacity of proteins depends on their hydrophobic character, and is quantified by an empirical α/β ratio. Available studies refer only to pH-driven lysozyme binding in dilute dispersions of pristine, or oxidized SWCNTs.[42] In the latter case, binding is essentially induced by electrostatic interactions of lysozyme with carboxylate functionalities present on nanotubes. It is cumbersome separating pH-induced conformational changes from contributions due the ionization of carboxylate groups lying on oxidized SWCNTs. The pH-dependent conformational state of adsorbed proteins is strictly related to its adsorbent quality (and to solvent properties, as well), as observed in biological studies on the transfection of BSA/CNT adducts in cell cultures.[44] Studies considered so far refer to dilute regimes; it is expected that CNTs properties may change when they are in presence of considerable amounts of proteins.

B2) Dispersions in Protein-based Gels

The combination of the dispersing and gelling ability of globular proteins with the mechanical properties inherent to CNTs are relevant for practical applications. Understanding the optimal working conditions is fundamental in designing gel-based composites with tunable properties. The possibility to disperse CNTs in protein-based gels is, therefore, of substantial interest.

Globular proteins form gels at low pH, in semi-dilute regime and mild temperature conditions. Gelation implies the following steps: a) partial exposure to water of hydrophobic protein residues; b) clustering in seeds; and c) coalescence. A three-dimensional network is achieved when the protein volume fraction is higher than a critical threshold, ΦC,P. Optimal working conditions 8 require operating at temperatures higher than the gelation one, Tgel.[45] The latter is controlled by protein volume fraction, ionic strength and pH. In LYSO-based systems, for instance, optimal gelling conditions are in a pH range 2.0-3.0 and concentrations in the mmol kg-1 range.

Protein-based gels embed CNTs; the more prominent properties of such composites depend on nanotube/protein weight ratios. Added CNTs modify the gelation pathway, its kinetics, and shift Tgel. This fact helps forming hydrogels with over 90 wt% water, and significantly improves the mechanical performances of the composites. The maximum concentration of SWCNT that is effectively dispersed is < 1.0 wt.%.[46] To shed light on these processes, Tgel and the kinetics of gelation were determined. When T>Tgel, the gelation kinetics scales with the amount of dispersed nanotubes, Figure 3. Tiny amounts of carbon nanotubes do not disturb gel properties, and Tgel does not change. When ΦV,CNT > 0.3 wt%, gels become continuous in both protein and nanotubes; percolating and interconnected networks do form, and Tgel changes. Thus, despite SWCNTs dispersability is moderate, it offers the opportunity to modulate gel elasticity and resiliency. It is expected that the above composites may allow to prepare scaffolds with tunable properties.

Figure 3. A. DLS determination of gelation temperature for a 6.50 wt% LYSO solution, at pH 2.50. B. The light scattering decay as a function of measuring time, in s, for a pure LYSO gel, blue symbols, and for the same with 0.20 wt% SWCNTs in red. The working temperature is 47.0°C, LYSO content and pH are as before.

C) Dispersions of CNTs in DNA and RNA

C1) Dilute regimes

ss-DNA filaments, and RNA, have polar/non-polar moieties, are flexible, deformable, and may form spires around CNTs because of hydrophobic interactions. These complexes are stabilized by PO4

- units facing towards bulk waterand acting as kinetic barriers to bundling. These complexes are thermodynamically stable, and it is practically impossible to retrieve the isolated components by dilution, since the number of links between CNTs and the a-polar residues of DNA/RNA can be in the range of thousand units. Therefore, ss-DNA/CNT complexes are endowed with significant stability; in dilute regimes, they form stable dispersions. Their solubility is orders of magnitude higher than pristine CNTs and can be some wt% high![47].

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Figure 4. Titration of ss-DNA/CNT complexes, reported in differential form as δζ/δξ, as a function of didodecyldimethylammonium bromide, DDAB, content, ξ, at 25.0°C. The amount of complex in the mixture is 0.01 wt%. Complexes are made of 1/1 ss-DNA and CNT, in mass ratios.

In water ss-DNA/CNTs interact with oppositely charged species, Figure 4,[48] which titrate the complexes and favor precipitation. Proper titrants are calcium, cationic surfactants and proteins. If the reactants are surfactants, the alkyl chains facing outward the complexes become nucleation sites for aggregation of micelles or vesicles, operating as surface-bound hydrophobic reservoirs. Presumably, ss-DNA/CNT and RNA/CNT complexes may find extended application in biomedicine, since they are potentially the locii where biochemical reactions onto such residues take place.

C2) Formation of Nematic Liquid Crystalline Phases

In relatively concentrated regimes, ds-DNA may form liquid crystalline phases; such relatively rigid rods align parallel in an hexagonally ordered assembly.[49] It is also possible to form nematic fluids by adsorbing single strand DNA onto CNTs. In these complex systems, the phase transitions leading to pure nematic fluids depend on ionic strength, addition of polymer and/or combination thereof. The nematic order is made easy by adding anionic polyelectrolytes, which induce a segregative phase separation.[50] This fact gives the opportunity to concentrate dispersions containing ss-DNA/CNT complexes and form a pure nematic phase. The phase behavior of ss-DNA/CNT complexes can be inferred by rheology, Figure 5.[51,52]

Figure 5. Dependence of ηrel, the zero shear viscosity, on the volume fraction of (1/1 ss-DNA/SWCNT), ϕ (times102) in water, at 25.0°C. Data are normalized with respect to the solvent. The dispersion is in yellow, the two-phase are in white, the nematic in green.

The onset of nematic order is concomitant with significant changes in shear viscosity. This is because ordered rods have less possibility to disturb the reciprocal motions than in disperse form.[47]

In addition, entanglement of the complexes is much less significant. Nematic phases are orientationally ordered, as inferred by polarizing microscopy and 2H NMR, Figure 6, and by SAXS. 2H-NMR spectra indicate the average orientational order (which is retained for long times), whereas SAXS indicates the reciprocal arrangement of the complexes in an hexagonal array and the average distance between the single ss-DNA/SWCNT units. Optical textures inferred by microscopy allow to detect the reciprocal orientation of anisotropic domains and modifications induced by shear; such deformations are retained for long times. Shear-induced ordering gives rise to large domains, and most ss-DNA/SWCNTs oriented along the same direction.

Figure 6. A. Optical microscopy for a nematic sample made of 1/1 ss-DNA/SWCNT mass ratio, at 20.0°C. The overall mass content is 3.76 wt%. B. Optical microscopy for the same sample soon after shearing. The latter effect orients the domains along a preferred direction. C. NMR deuterium spectral profile of D2O in the same mixture, at 300 K.

Nematic droplets made of the mentioned complexes can be dispersed in solutions containing cationic surfactants, or LYSO. As a result, a peel confines them, and order inside droplets is retained for indefinite long times.[53] The mentioned preparation procedures give the possibility to prepare ordered particles and/or films with controlled sizes, respectively. The possibility to get hybrid films with diverse morphologies and functionalities is a key point in advanced research, whereas clustering in droplets is potentially useful in the preparation of macroscopic materials with anisotropic character.

D) Interaction with biological structures and assessment of biocompatibility

Assessment of cellular viability represents an essential step to ascertain the nanocarriers’biocompatibility. In a recent work,[54] administration to cultured cells of two functionalized Pt(IV)@CNTs differing in diameter and length was evaluated. In these experiments, non-phagocytic cancer cells (human cervix) and murine macrophages were considered. These types of cells have different cytological features and embryonal origin, with the second cell population being fully competent phagocytic. Results showed, that there was no significant difference when comparing the behavior of CNTs over a broad concentration range. Therefore, one can infer that nanotube length is of moderate relevance as far as biocompatibility is concerning. However, the active form of the drug is released more slowly by shorter CNTs compared to longer ones. In any case, phagocytic cells showed a higher sensitivity. In fact, dose-dependent cytotoxicity was observed at lower concentrations, suggesting a higher sensitivity of this specific cell population. In conclusion, CNTs intrinsic cytotoxicity is always an important issue to be investigated. The effect on cell viability suggests a potential usage of these nanovectors for anticancer treatment. The diameter of CNTs plays also a determinant role in drug release rates. Acute toxicity induced by BSA-CNTs was also investigated.[55] With respect to this, it should be pointed put that apoptosis, cell viability or other cell functions (such as immune cell activation) represent important parameters to assess the cytotoxicity of different treatments to

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cultured cells.[56] In particular, the cell death inducing effect by apoptosis of CNTs was analyzed on two different cells lines, Figure 7.

The cell membrane is the first barrier that nanoparticles encounter prior to cell penetration. An evaluation of the putative membrane damage should be, therefore, taken into account. To this end we investigated the cell membrane structure/ function by electrorotation. This method was adopted in our and other laboratories, to investigate cell toxicity mechanisms in a number of biological model systems.[57]

Figure 7. Quantitative analysis of apoptosis by fluorescence microscopy of TUNEL assay on3T6 (A) or HEK 293 cells (B) exposed to 50 or 100 µg•ml-1 of BSA-CNTs for 48 h (Top panel). At the bottom the TUNEL-positive cells are shown as fluorescent signals (Re-drawn from 56).

Supramolecular aggregates, interact with cell membranes and are internalized by endocytosis and/or passive diffusion. It is legitimate to assume that nanotubes bound to the surface or penetrating the cell may perturb the lipid bilayer. This would, in turn, lead to changes in the dielectric properties of plasma membranes.[56,60] Electrorotation shows that the specific capacitance and specific conductance are not significanltly altered in cells exposed to CNTs. Exemplyfing data are reported in Table 1.

Table 1. Cell membrane specific parameters capacitance (C) and conductance (G) measured in control or in cells exposed to BSA-CNTs (100 µg x mL-1 for 24 hours. SD=Standard Deviation. Data refer only to 3T6 cells.

Since capacitance and conductance of plasma membranes are directly related to its structure and ion permeability one can conclude that the interaction between CNTs and cells has limited effect on the plasma membrane properties. This represents an important result since the impact of nanotubes on plasma membranes had not been studied to any relevant extent. However, apoptosis is not the sole mechanism of cell death.[60] Other routes leading to cellular death such as necrosis, necroptotsis and autophagy were also described. Nevertheless, we can confidently conclude that our results tend to rule out the possibility that CNTs-dependent toxicity derives from a severe membrane damage

Diverse nanocarrier tools, such as liposomes, micelles, dendrimers, polymeric nanoparticles and carbon nanotubes have been used for the delivery of anticancer drugs. In lour laboratory, for instance, the delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles was quantitatively evaluated. These results further support the idea that supramolecular nano-carriers may have a potential use in nanobiotechnology. [61]

Conclusions

In this review, we focused on systems obtained by mixing CNTs (mostly single-walled ones) and biopolymers. There are substantial possibilities

for the surface functionalization by proteins and/or nucleic acids. Non-covalent functionalization is reversible or not, depending on the substances involved; for LYSO in dilute regimes, for instance, functionalization is practically reversible upon dilution. The resulting mixtures give homogeneous dispersions, gels and/or liquid crystalline materials. The physical forces operating in such media compel the systems towards one possible state with respect to another. This is the result of a delicate balance between attractive/repulsive forces active among the composites formed by CNTs and biopolymers. Uniform CNT coverage ensures the onset of dispersions. Formation of gels occurs in stabilized CNT dispersions and in presence of proteins; their surface covering ability is moderate with respect to ss-DNA. Protein-based gels dissolve moderate amounts of CNTs.

A detailed explanation of the forces acting in such systems is beyond current knowledge in the field, but preliminary information is at hand. Binding efficiency is a prerequisite getting uniform coverage and formation of dispersions, gels and long-range order phases. This condition holds in ss-DNA/CNT complexes, which behave as long rigid rods and undergo isotropic-nematic phase transitions when their concentration is significant. Conversely, mild and heterogeneous coverage ensures dispersion. These preliminary results offer the opportunity for more substantial studies, and give the opportunity to experience the molecular/functional details responsible for one organization mode with respect to others.

It is worth noting that the fine details leading to the formation of bio-polymer/CNT complexes find their origin in the molecular structure of adsorbing entities. In words, binding is limited and not extended in case of globular LYSO, which presumably adsorbs in a sort of “dot-by dot” mechanism on small regions of CNTs. ss-DNA, conversely, efficiently binds onto nanotubes, thanks to the formation of an extended hydrophobic network, which rolls around CNTs. The effect of ss-DNA binding scales in proportion to the number of binding units, and is, presumably, dependent on the nature of base pairs. In other words, binding efficiency is controlled by the number of hydrophobic residues that effectively take place in the interactions. Supposedly, the mentioned biopolymers are the extreme cases of hydrophobic interaction modes with CNTs that is weak for LYSO and strong for ss-DNA, respectively.

Nanomaterials represent a stimulating tool for the delivery of therapeutic molecules with anti-tumor or anti-proliferative effects. Delivery of poorly water-soluble drugs may benefit of CNTs as cargo vehicles. This could, incidentally, reduce accumulation and toxicity in non-specific target tissues, improving both pharmacokinetics and bioavailability, as well as increase local drug concentrations. One of the most attractive properties of CNTS and functionalized ones resides in their ability to cross the cell membrane, with consequent accumulation in the cytoplasm. In addition, their high surface/volume ratio provides multiple binding sites for molecules useful in selective targeting. In fact, CNTs have been adopted in a variety of different fields, i.e. bio-sensing, regenerative medicine and vehicles of bioactive molecules. These therapeutic molecules can either interact with the outer surface of CNTs or being encapsulated within their inner lumen. This avoids premature inactivation of the drug and enables controlled drug release.

Acknowledgments

Thanks are due to La Sapienza for partly financing the present research line, through a University project.

Conflict of interest. The authors declare no conflict of interest.

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