PEG-Modified Carbon Nanotubes in Biomedicine: Current Status and

Review pubs.acs.org/Biomac

PEG-Modified Carbon Nanotubes in Biomedicine: Current Status and Challenges Ahead Massimo Bottini,*,†,‡ Nicola Rosato,‡,§ and Nunzio Bottini⊥ †

Sanford Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy § IRCCS-Neuromed Institute, Via Atinense 18, 86077 Pozzilli, Isernia, Italy ⊥ La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, California 92037, United States ‡

ABSTRACT: Since their discovery at the end of the previous millennium, carbon nanotubes (CNTs) have been the object of thousands of papers describing their applications in fields ranging from physics to electronics, photonics, chemistry, biology, and medicine. The development of chemical approaches to modify their graphitic sidewalls enabled the generation of poly(ethylene glycol) (PEG)-modified CNTs and their exploration in multiple biomedical applications. Studies at the cellular and organism level revealed that PEGmodified CNTs have favorable pharmacokinetic and toxicology profiles. Recently, PEG-modified CNTs have been successfully tested in preclinical studies in the fields of oncology, neurology, vaccination, and imaging, suggesting that they are well suited for the generation of novel multifunctional nanodrugs. Here we will review published data about the application of PEG-modified CNTs as in vitro and in vivo therapeutic and imaging tools and describe what is known about the interaction between PEG-modified CNTs and biological systems. Although several pieces of the puzzle are still missing, we will also attempt to formulate a preliminary structure−function model for PEG-modified CNT cellular trafficking, disposition, and side effects.

structure characterized by a high aspect ratio (ratio between length and diameter). CNTs can be produced as single-walled (SWCNTs) or multiwalled (MWCNTs). The former consist of a single graphene sheet, whereas the latter consist of several concentric and nested graphene sheets. Both SWCNTs and MWCNTs are characterized by nanometric dimensions. In this regard, SWCNTs have diameters from 0.3 to 2 nm and lengths up to 1 μm, whereas MWCNTs have diameters from a few to tens of nanometers and lengths up to several micrometers. Approximately two-thirds of as-produced SWCNTs are semiconducting, and their particular band gap structure gives rise to intrinsic fluorescence in the near-infrared (NIR) range which is stable (no blinking) and far from the characteristic background of biological and environmental specimens.5−7 SWCNTs are also characterized by distinctive resonance-enhanced Raman signatures.8 These properties make SWCNTs suitable for imaging applications in a wide variety of biomedical fields. The main obstacle to utilization of as-produced unfunctionalized (pristine) CNTs (both SWCNTs and MWCNTs) is their scarce solubility in any aqueous environment due to the graphitic nature of their sidewalls. To overcome this problem, CNT surfaces have been modified by noncovalent coating with

1. INTRODUCTION Nanomedicine is a young discipline that recently emerged from the marriage of nanotechnology and medicine.1 By enabling controlled spatial and temporal delivery of traditional drugs, novel nanomedicine-based drugs (nanodrugs) hold the promise to dramatically improve disease therapy and human quality of life. Nevertheless, the development of a nanodrug is a complex process that involves the identification of a specific cell and receptor target related to a clinical condition and the assembly of the appropriate combination of components in order to minimize cost and complexity of the final adduct (Figure 1). Among nanotechnology-derived nanoparticles, carbon nanotubes (CNTs) have stimulated a great interest for biomedical applications because of their unique properties. 2−4 CNTs are characterized by unique spectroscopic, thermal, and electric properties that facilitate their visualization and tracking in biological environments and have a high surface area that enables loading of multiple moieties at high density. Moreover, CNTs are spontaneously internalized by a wide variety of cell types. The combination of these properties confers to CNTs a high potential as scaffold for the fabrication of nanodrugs. Indeed, an increasing number of publications describe the use of CNTs to deliver diagnostic and therapeutic relevant molecules in specific tissues/cells both in vitro and in vivo. A CNT can be thought as a graphene sheet rolled up and capped at the ends with hemifullerenes to form a cylindrical © 2011 American Chemical Society

Received: July 22, 2011 Revised: September 7, 2011 Published: September 14, 2011 3381

dx.doi.org/10.1021/bm201020h | Biomacromolecules 2011, 12, 3381−3393

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Figure 1. The nanotechnology approach. As a result of a bottom-up process, novel multifunctional nanoparticles (nanodrugs) are composed by a nanotechnology-derived carrier (nanocarrier) loaded with payloads (cargos) and agents (enhancers). The arsenal of nanocarriers is mostly composed by particles synthesized by condensation of polymers (biodegradable and block copolymers), amphiphile-based particles (liposomes and micelles), organic (dendrimers, peptide- and/or protein-conjugates, carbon nanotubes, carbon dots, etc.), and inorganic (metallic nanoparticles and quantum dots) particles. Small molecules, peptides, proteins, and nucleic acids have been encapsulated into or loaded onto nanocarriers as therapeutic agents. Enhancers are added to help the nanodrug to reach the site of interest (for instance the nucleus of a cell subpopulation) while avoiding the biological and biophysical barriers encountered following the administration.

the particles are recognized and ingested by the RES macrophages. Following phagocytosis, the particles are trafficked into specific vesicles (lysosomes) and attacked by digestive enzymes (over 60 lysosomal enzymes are known; these include esterases, lipases, glycoside hydrolases, proteases, nucleases, and sulfatases) and/or oxidative-reactive chemical species (such as nitric oxide, hydrogen peroxide, superoxides, and oxyhalide molecules) for degradation. However, several of the scaffolds used for nanodrugs are nonbiodegradable and, once internalized by macrophages, are transported and stored mainly into the spleen, liver, lymph nodes, and bone marrow. Lungs may also represent the final (unintentional) target for systemically distributed nanodrugs due to the fact that the respiratory system receives the whole cardiac output. As such, there is potential that several organs may be unintentionally exposed and accumulate nanodrugs, thereby leading to decreased blood circulation half-life of the administered nanoparticles and to potential undesired effects, such as oxidative stress and inflammation.10 Anaphylatoxins produced by activation of the complement system may trigger the release of pro-inflammatory mediators and induce anaphylaxis.

oligonucleotides and amphiphilic molecules or by covalent functionalization approaches.9 By the addition of appropriate molecules or chemical groups, modified CNTs are more hydrophilic, less aggregated, and amenable to further derivatization. Every nanodrug following administration must elude multiple biological and biophysical barriers in order to achieve the final therapeutic and/or diagnostic goal (Figure 1). Among those barriers, removal by the mononuclear phagocytic systemalso known as the reticuloendothelial system (RES)can dramatically hamper the efficiency of a nanodrug. RES macrophages can remove foreign particles from the bloodstream within seconds by recognizing blood serum components (opsonins) bound to the surface of the particles. Common opsonins are components of the complement system, immunoglobulins, fibronectin, type I collagen, and C-reactive proteins. Opsonization is a two-step process. First, opsonins come into contact with foreign particles by random Brownian motions, and then they bind the surface of the particles driven by specific attractive forces (such as electrostatic, hydrophobic, van der Waals, and others). After opsonization has occurred, 3382


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Figure 2. PEG-modified CNTs used in biomedicine. PEG-coated SWCNTs (cSWCNT1−7) are obtained by adsorption of PEG-modified amphiphilic polymers onto SWCNT sidewalls (the lipophilic parts of the polymers are drawn in red), whereas PEG-functionalized SWCNTs and MWCNTs are made of pristine materials that have undergone either strong acid treatment to introduce carboxylic acid groups (fSWCNT1s and fMWCNT1s) or 1,3 dipolar cycloaddition reactions (fSWCNT2s and fMWCNT2s). CNTs have been modified with both linear and branched PEG chains (n is the degree of polymerization of the chain) carrying different reactive functional groups (R) at the distal end of the projected PEG chains.

ethylene glycol units −(CH2−CH2−O)n− where the integer n is the degree of polymerization. A theory has been formulated to explain how the hydrophilic and flexible nature of PEG chains imparts resistance against opsonization to the surface of particles.12 When opsonins are attracted to the surface of PEGmodified particles, PEG chains are compressed and forced to shift to a higher energy conformation. This creates an opposing repulsive force thus balancing the attractive force between the opsonin and the surface. It has been reported that PEG chains of a 2 kDa molecular weight (MW) are required to achieve sufficient protection against opsonization.13,14 In light of the unique properties of CNTs and the ability of PEG chains to shield against opzonization, scientists have been testing PEG-modified CNTs in several biomedical applications, including imaging, vaccines, and tumor treatment. In this review, we will examine the state-of-the-art in the use of PEGmodified CNTs for imaging and therapeutics. The challenges that must be confronted in order to translate these particles into efficient and safe nanodrugs will also be discussed. After reviewing the published data about the use of PEG-modified CNTs as in vitro and in vivo delivery systems, we will describe what is known about the interaction of PEG-modified CNTs with the living matter, at the cellular and organism level. Finally, we will try to formulate a preliminary general structure−

Furthermore, it is of note that RES macrophages usually do not recognize nonopsonized particles which will then persist in the bloodstream and/or will be cleared through the urinary route. 11 Removal by the renal system represents an additional hurdle to nanodrug blood circulation half-life and usually occurs only for molecules with molecular weights smaller than 5 kDa but can be as high as hundreds of kDa for nanoparticles made of dense polymers (for instance, dendrimers). The disposition of a nanodrug is governed by several physical and chemical factors (such as dimension, chemical composition, shape, size, surface charge, etc.) and still under investigation for several nanoparticles that have been testing as scaffolds for nanodrugs. Since opsonization of particles is critical for triggering recognition by the RES macrophages, which results in decreased blood circulation half-life and potential side effects, several strategies have been developed for camouflaging particle surfaces. Opsonins in the blood interact with foreign particles mostly through hydrophobic and electrostatic interactions; thus, a widely used approach to shield particles against the attack of opsonins is to modify their surface with neutrally charged chemical moieties, such as hydrophilic polymers and nonionic surfactants. Among them, poly(ethylene glycol) (PEG) is one of most effective because it is highly hydrophilic and flexible. PEG is a compound composed of repeating 3383


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function model for cellular trafficking and disposition of PEGmodified CNTs.

peptides) were covalently linked to the distal ends of the PEG chains. cSWCNT1s and fSWCNT1s showed similar degrees of doxorubicin loading/binding. The loading/binding of doxorubicin was pH dependent; the CNT−RGD−doxorubicin nanodrugs were stable under physiological conditions and were able to release the cargo once the nanodrug had reached the lysosomes of integrin α Vβ 3-positive U87MG cells. The authors showed that other aromatic molecules (daunorubicin and fluoresceinamine) can be loaded onto pre-PEGylated CNTs using the “conjugation partitioning” approach. The in vitro intracellular delivery of anticancer and antifungal drugs by means of fSWCNT2s and fMWCNT2s has been investigated by Prato and collaborators.23−25 In a recent report they provided proof of concept that fSWCNT2s and fMWCNT2s can be used to transport amphotericin B (AMB) into a collection of reference and clinical fungal strains.25 AMB is an antifungal drug used in free form or is often encapsulated into liposomes to overcome its poor water solubility and nephrotoxicity. fSWCNT2−AMB and fMWCNT2−AMB complexes showed higher antifungal activity than AMB in both free and encapsulated forms. 2.2. PEG-Modified CNTs for in Vitro Delivery of Biomacromolecules. A problem commonly faced in biological studies and human therapy is to enable intracellular transport of biomacromolecules that do not spontaneously pass the plasma membrane such as peptides, proteins, and oligonucleotides. For the delivery of genetic material into cells, two approaches are widely used, namely viral vectors 26,27 and nonviral delivery reagents.28−30 Despite their excellent transfection efficiency, problems associated with viral vectors include immunogenicity, cytotoxicity, oncogenicity, limited size of genetic cargo, difficulty to functionalize with targeting ligands, and high cost. Among the existing arsenal of nonviral systems, those based on synthetic cationic reagents (polymers, peptides, and lipids) are widely used. However, those systems cannot match the efficiency of viral vectors plus they show relatively low stability in the bloodstream. PEG-modified CNTs are theoretically a promising type of nonviral delivery systems due to their favorable pharmacokinetic and toxic profiles. Few studies have been published so far limited to nonspecific transfection of plasmid DNA in cultured cells. Pantarotto et al. used fSWCNT2s and fMWCNT2s to electrostatically condense β-galactosidase plasmid DNA and transfect CHO cells.31 They observed a 10 times higher gene expression when compared to cells treated with naked plasmids. cSWCNT1s have been also used as nonviral vectors for small interfering RNA (siRNA)32,33 and antisense oligomers (ASOs).34 The use of ASOs is a highly appealing strategy to selectively suppress the expression of disease-related proteins.35,36 However, the use of ASOs in vivo has been severely hampered so far by their inefficient penetration of the cell membrane. T cells are among the most refractory cell types to siRNA and ASO internalization by nonviral agents.37 It has been described that PEG-modified CNTs are spontaneously internalized by T cells through nonspecific endocytosis triggered by interactions between hydrophobic domains on the plasma membrane and exposed portions of CNT sidewall.33 In light of this finding, our research group has investigated the use of cSWCNT1s as carriers of ASOs into T cells to silence a type 1 diabetes predisposing gene encoding the protein tyrosine phosphatase N22 (PTPN22).34,38−40 ASOs were conjugated to cSWCNT1s through a cleavable disulfide bond and once delivered into T cells led to a 1 order of magnitude higher knockdown of

2. PEG-MODIFIED CNTS AS THERAPEUTIC AND IMAGING SYSTEMS Figure 2 illustrates the PEG-modified CNTs that have been used in biomedical studies. PEG-coated SWCNTs are obtained by adsorption of amphiphilic polymers functionalized with activated PEG chains onto pristine SWCNTs using a noncovalent procedure based on ultrasonication, followed by ultracentrifugation and ultrafiltration (cSWCNT1−7).15 Polymers bind to SWCNTs through hydrophobic interactions between the lipophilic moieties and the graphitic SWCNT sidewalls, leaving the PEG chains and other hydrophilic groups projecting from the sidewall. PEG-coated SWCNTs are mostly composed by individual/small bundles of SWCNTs and form a stable suspension in high saline solutions and in serum. PEGfunctionalized SWCNTs and MWCNTs are generally fabricated by amidation of the carboxylic groups of oxidized CNTs (fSWCNT1s and fMWCNT1s).16,17 Oxidation is carried out by refluxing CNTs in oxiding agents such as piranha solution (mixture of sulfuric acid and hydrogen peroxide) or nitric acid. As well as PEG-coated SWCNTs, fSWCNT1s and fMWCNT1s are mostly composed by individual/small bundles of CNTs and form stable suspensions in high saline solutions and in serum. PEG-functionalized SWCNTs and MWCNTs are also made of pristine materials that have undergone 1,3 dipolar cycloaddition of azomethine ylides, generated by thermal condensation of α-amino acids and aldehydes (fSWCNT2s and fMWCNT2s).18 This reaction adds a large number of pyrrolidine rings to the CNT sidewalls. This imparts high solubility and the ability to stay in most biological environments as individual/bundles with nanometric diameters of SWCNTs and individual MWCNTs. PEG-modified CNTs have been used as delivery systems for a variety of drugs including small molecules (typically anticancer19−23,101 and antifungal24,25 drugs) and biomacromolecules (peptides,52,53,55 proteins,56 DNA,31,32 siRNA,32,33,51 and antisense oligomers34). Targeting ligands (vitamins,20,58 peptides,22,41 and antibodies54,62) have been also added to the CNT-based nanodrugs to enable delivery into a specific tissue or cell subpopulation. Imaging of PEG-modified CNTs was obtained by either adding probes (radionuclides41,48−50 and fluorochromes54,57,58,62,63) or exploiting the properties of CNTs arising from their quasi-1-D nature (NIR photoluminescence,42 Raman scattering,43,44 and photoacoustic signal45). 2.1. PEG-Modified CNTs for in Vitro Delivery of Small Molecules. SWCNTs coated with PEG-modified phospholipids (cSWCNT1s) have been pioneered by the Dai group and validated as targeted delivery systems of anticancer molecules (platinum(IV) complexes and paclitaxel).19−21 The cargo molecules were tethered to the PEG chains through bonds (such as peptidic19,20 and ester21) which were cleaved by the digestive enzymes once the nanodrugs were translocated into the lysosomal environment. Besides covalent conjugation, the same group investigated the functionalization of cSWCNT1s and fSWCNT1s through “conjugation partitioning”, that is, the use of diverse chemistries to load chemical species with different functionalities onto the same CNT. 22 Aromatic anticancer molecules (doxorubicin) were noncovalently loaded via π-stacking onto graphitic portions of the CNT sidewall not covered by the PEG chains, whereas targeting ligands (RGD 3384


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PTPN22 expression compared to ASOs internalized by electroporation. 2.3. PEG-Modified CNTs in Preclinical in Vivo Studies. The use of PEG-modified CNTs for in vivo studies is still in an exploratory stage. However interesting applications have been published focused on oncology (imaging and therapy), neurology, vaccination, and targeted delivery. The Dai group explored the use of cSWCNT1s in oncology. In an imaging study, the distal end of the projected PEG chains were functionalized with targeting ligands (RGD sequence) and radiolabels (64Cu-DOTA), systemically administered into tumor (U87MG human glioblastoma or HT-29 human colorectal cancer)-bearing mice, and imaged by microPET. 41 The accumulation of cSWCNT1-based nanodrugs in tumors has been also imaged by NIR photoluminescence,42 Raman scattering,43,44 or photoacoustic technology.45 In a therapeutic study, the anticancer drug Paclitaxel has been conjugated to branched PEG chains through a cleavable ester bond and led to reduced tumor volume in 4T1 breast cancer-bearing mice following systemic administration.20 cSWCNT1-based nanodrugs showed improved efficacy over Taxol (a formulation based on Paclitaxel dissolved in Cremophor EL and ethanol). However, the authors did not investigate the efficacy of CNTs as nanocarriers compared with approved therapeutic formulations (such as the albumin-based Abraxane and the liposomebased Myocet). These comparisons would be extremely important to establish whether new delivery systems offer advantages over approved ones. Other two types of PEG-coated CNTs have been used as therapeutic tools in oncology. SWCNTs coated with PEGgrafted poly(maleic anhydride-alt-1-octadecene) (cSWCNT2s) have been optimized by Liu et al. to display balanced tumor-tonormal organ uptake ratio and tested in cancer photothermal therapy applications following systemic administration.46 The strong absorption of cSWCNT2s in the NIR range was exploited by the authors to ablate a 4T1 tumor in the shoulder of female Balb/c mice by a low-power 808 nm NIR laser irradiation. Unfortunately, this approach can be used only for superficial tumors due to the minimal tissue penetration (maximum ∼2 cm) of NIR light. This limitation have been avoided by Gannon et al. by using a 13.56 MHz radiofrequency irradiation to heat cSWCNT3s (SWCNTs coated with Kentera, a PEG-modified polymer based on polyphenylene ethynylene) intratumorally injected into rabbits bearing hepatic VX2 tumors.47 Preclinical applications of PEG-modified CNTs have been reported also in the field of neurology. SWCNTs have physical dimensions on a scale similar to those of neuronal processes and electric conductivity that can be easily manipulated by changing the disposition of carbon atoms in the graphitic lattice. These properties suggested that these nanoparticles can be used to fabricate substrates, mimicking the topography of the native central nervous system tissue.48,49 In particular, fSWCNT1s have been recently reported to stimulate neurite outgrowth in vitro because of their inhibitory action on regulated (but not constitutive) endocytosis, as opposed to exocytosis.50,51 Furthermore, PEG has been widely used as a therapeutic agent per se to repair damaged nerve membranes in animal models of spinal cord injury (SCI).52−54 In light of these finding, Floyd et al. investigated the capacity of fSWCNT1s to promote tissue repair and functional recovery in a SCI animal model.55 The authors reported that delayed post-SCI administration of fSWCNT1s induced axonal regeneration

into the lesion cavity and recovery of locomotor function, without inducing reactive astrogliosis and hyperalgesia. Although the observed functional recovery was modest and the authors did not compare fSWCNT1s to free PEG, this study strongly suggests that PEG-modified CNTs are effective in promoting recovery and regeneration after SCI. Additional studies are warranted to optimize fSWCNT1s and investigate the neurological effects of other kinds of PEG-modified CNTs. Preclinical applications of fSWCNT2s and fMWCNT2s have been published in both the oncology (including imaging using the radionuclides 111In and 86Y56−58 and therapy using RNA interference59) and vaccination fields.60,61 Studies by Pantarotto et al. demonstrated a high production of antibodies with virusneutralizing capacity in response to immunization with fSWCNT2s functionalized with a peptide derived from the foot-and-mouth disease virus. Interestingly, fSWCNT2s devoid of the peptide moiety were not immunogenic, as antibodies against these particles were not detected in this study. 61 Targeted delivery of nanoparticles into a specific cell subpopulation in vivo is another area of interest, which would open great opportunities for the development of new nanodrugs. Our research group investigated if cSWCNT1 could specifically target T cells in vivo.62 We functionalized cSWCNT1s with monoclonal antibodies specific for receptors expressed on the T cell plasma membrane (CD3 and CD28) and a fusogenic polymer to enable release from the endolysosomal compartments. After injection in mice, the particles were internalized by T cells through receptor-induced endocytosis with high specificity (more than 60% of T cells were CNT-positive) and showed a largely cytoplasmic localization. On the other hand, nanoparticles devoid of the fusogenic polymer mainly showed perinuclear localization in T cells. Although sequestration in the lysosomes was escaped with the aid of specific enhancers, our study demonstrated that cSWCNT1s could efficiently deliver cargoes into the cytoplasm of specific cell populations in vivo.

3. CELLULAR INTERNALIZATION AND TRAFFICKING The interest in biological applications of surface-modified CNTs was spurred by two independent reports by Pantarotto et al.63 and Kam et al.64 showing that CNTs modified with macromolecules (proteins and peptides) can spontaneously penetrate cells. Understanding the mechanisms of cell internalization, trafficking, and excretion of surface-modified CNTs is of pivotal importance to develop them as safe nanodrugs. Our research group and others have used cSWCNT1s as delivery systems in T cells. cSWCNT1s conjugated with DyLight488 to the distal end of the projected 2 kDa MW PEG chains accumulated in the perinuclear vesicles of treated Jurkat leukemia T cells.62,65 Release from the vesicles was obtained by conjugating cSWCNT1s with a fusogenic polymer.62 The Dai group reported that coating cSWCNT1s with longer PEG chains (5 kDa vs 2 kDa MW) reduced the uptake by primary blood mononuclear cells.33 The internalization was probed by Raman spectroscopy. Cherukuri et al. used the intrinsic NIR fluorescence to find that J774.1A mouse peritoneal macrophage-like cells actively ingested cSWCNT4s (SWCNTs coated with a poloxamer, Pluronic F108).7 Once internalized, the CNTs were transported into small phagosomes where the PEG coating was displaced by proteins in the vesicles as suggested by the significant broadening and red-shifting of the NIR emission peaks. The nonreceptor-mediated uptake of cSWCNT1s and cSWCNT4s by immune cells (T cells and macrophages) likely 3385


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Figure 3. Features influencing the biological performances of PEG-modified CNTs. Numerous publications describe the relative and synergistic influence of structural and chemical features (such as homogeneity, density, length, nature, etc.) of PEG on the degree of steric stabilization, the behavior after administration, and, ultimately, the biological performances of PEG-modified CNTs. As well as other kinds of modified CNTs (for instance, DNA-coated CNTs), the biological performances of PEG-modified CNTs may also be influenced by such features of CNTs as length, diameter, chirality, and electronic type. However, these relationships have not been extensively investigated yet.

depends on nonspecific endocytosis triggered by the interaction of hydrophobic domains on cell membranes to exposed parts of the CNT sidewalls. Two studies also reported the receptormediated endocytosis of folate-conjugated cSWCNT1s by folate receptor-positive cancer cells (HeLa15 and SKOV-366). cSWCNT1s were found in the cytoplasm and did not translocate to the nucleus of these cells. However, in both studies the exact cytoplasmic location of cSWCNT1s was not investigated, although the punctuate nature of the CNT fluorescence suggested that the tubes were mostly entrapped in the endolysosomal vesicles. Cheng et al. reported that fSWCNT1s functionalized with 1.5 kDa MW PEG chains reversibly accumulated in the nucleus and more weakly in the cytoplasm of different mammalian epithelial cell lines (HeLa, U2OS, HT1080, MEF, C33A, and HEK293) through an energy-dependent mechanism.67 In another study, the authors found that only individually distributed or small bundles of short fMWCNT1s were able to translocate across the nuclear membrane of multidrug resistant cells (HepG2-DR), whereas cytoplasmic fMWCNT1s were organized in large aggregates.68 Pantarotto et al. reported that fMWCNT2s were able to pass the plasma membrane through energy-independent passive diffusion which was followed by translocation to the nucleus of human 3T6 and murine 3T3 fibroblasts.63 Kostarelos et al. recently observed the same (energy-independent) mechanism of internalization of both fSWCNT2s and fMWCNT2s for several mammalian cells (A549, HeLa, MOD-K, and Jurkat) as well as prokaryotic cells.69 However, the authors did not exclude that energy-dependent internalization might take place in concomitance with direct passive penetration. It is of note that Mu et al. formulated a mechanistic structure−function model for the interaction of plasma proteincoated non-PEGylated CNTs with HEK293 cells based on TEM analyses.70 They identified two mechanisms for cell uptake. Individual CNTs directly penetrated the plasma membrane, whereas aggregated ones followed an energydependent pathway and were transported into endosomal

vesicles. Once the aggregated CNTs were transported in the acidic environment of the lysosomes, individual CNTs could detach from the bundles, pass through the vesicle membrane, and translocate to the cytoplasm. Only short and individual CNTs were able to translocate to the nucleus. On the basis of the model generated for non-PEGylated CNTs, we can generate a model that fits the known data about internalization and trafficking of PEG-modified CNTs and highlights the deep relationship between the type of chemical modification and the biological performance of CNTs. As reported by Dumortier et al., only functionalization through 1,3 dipolar cycloaddition results in highly soluble CNTs, whereas CNTs fabricated through oxidation/amidation mainly form a stable suspension.71 This can be explained by considering that the cycloaddition reaction uniformly attacks the whole surface of the CNTs, whereas oxidation introduces carboxylic groups (used to link the PEG chains through amidation) mainly at the ends of the CNTs and at the site of defects of the sidewalls. 16 The extent of defects on the sidewalls of pristine CNTs is usually limited and can be slightly increased if certain oxidizing agents are used.72 Thus, a possible model is that fSWCNT2s and fMWCNT2s are homogeneously and densely functionalized, which keeps them solubilized as individual/small bundles of SWCNTs and individual MWCNTs, respectively, and facilitates their passive diffusion through membranes, whereas fSWCNT1s and fMWCNT1s are characterized by lower PEG coverage than fSWCNT2s and fMWCNT2s. Furthermore, also the process of coating CNTs with amphiphilic polymers [such as phospholipids (cSWCNT1s) or poloxamers (cSWCNT4s)] would lead to inhomogeneous coverage of CNT sidewalls. Cross sections of AFM images of cSWCNT1s coated with 2 kDa MW PEG chains showed exposed portions of the graphitic CNT sidewall extending for several tens of nanometers.65 Exposed portions in cSWCNT1s, cSWCNT4s, fSWCNT1s, and fMWCNT1s would be responsible for triggering nonspecific endocytosis by interaction with hydrophobic domains on the plasma membrane. Longer and branched PEG chains 3386


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may extend over the gaps in the PEG layer, thus decreasing cellular uptake by endocytosis. Interestingly, cSWCNT1s, fSWCNT1s, and fMWCNT1s were internalized by T cells through an energy-dependent mechanism but showed a different intracellular fate. cSWCNT1s were sequestered in the lysosomal vesicles of T cells, whereas fSWCNT1s and fMWCNT1s were found in the nucleus of several mammalian epithelial cell lines. Lysosomal trapping of cSWCNT1s may be explained by considering that individual CNTs cannot detach from aggregated cSWCNT1s because physically entangled in the wrapping alkyl chains of the phospholipids. Aggregated fSWCNT1s and fMWCNT1s are kept together only by hydrophobic and van der Waals forces; thus, peripheral PEG-modified CNTs may detach from the bundle and escape through the lysosomal membrane. The mechanisms of cell internalization, trafficking, excretion and, ultimately, toxicity of engineered nanoparticles depend on their physical and chemical properties (such as dimension, shape, size, surface charge, chemical functionalities, etc.). There are numerous publications describing the relative and synergistic influence of physicochemical properties of nanoparticles on their interaction with the living matter, at the organism and cellular level.73,74 However, that information is often contradictory or incomplete for some nanoparticles. Particularly in the case of CNTs, which are currently synthesized (and used) in a wide distribution of diameters, lengths, chiralities, and electronic types, making the exact description of the role of the structural and chemical features of CNTs on their interactions with the living matter a still unresolved challenge. However, recent success in separating CNTs into fractions with well-defined length, chiralities, and electronic type has made it possible to explore the role of those features on the cellular performances of CNTs.75−77 For instance, Becker et al. have reported a length-dependent uptake for DNA-modified SWCNTs, with SWCNTs shorter than ∼200 nm consumed more rapidly than longer tubes by several cell types. 78 Furthermore, Strano et al. developed a deterministic kinetic model for cellular uptake and validated it for DNA-modified SWCNTs.79 Although the lengthdependent efficiency of cellular uptake may be general for CNTs (as well as other kinds of nanoparticles), the exact threshold and efficiency of uptake would be dependent on other features of the nanotubes, especially the chemical makeup. Similar exhaustive investigations into the dependence of cellular uptake and trafficking of PEG-modified CNTs on CNT features have not been carried out yet (Figure 3). These works would complement the ones described in section 3 about the relationship between the type of chemical modification and the biological performance of CNTs and would also be of pivotal importance to shed light on the true potentiality of different PEG-modified CNTs as efficient and safe delivery systems.

administration of PEG-modified CNTs, by means of a variety of approaches. The Dai group reported that cSWCNT1s modified with 5 kDa MW linear PEG chains displayed higher blood circulation times (approximately 2 h vs 0.5 h) and lower accumulation into RES organs, such as liver and spleen, compared to those coated with 2 kDa MW PEG chains.41 On the contrary, fSWCNT2s and fMWCNT2s have been independently reported by two groups to be rapidly cleared (within 3 h) through the glomerular filtration systems with no uptake by the RES organs.56,58 Radiolabels were used in these in vivo investigations to trace PEG-modified CNTs in living animals and extracted organs. However, this approach may be biased by the metabolism of the labeled CNTs and detachment of the radioisotopes. For example, defunctionalization at the amide linkages between the PEG chains and the tubes of fSWCNT1s accumulated in the liver of injected mice has been reported. 80 The Dai group investigated the disposition of CNTs coated with different PEG-modified amphiphilic polymers using the intrinsic strong Raman scattering of the CNTs.81,82 In one study they found longer blood circulation half-life for cSWCNT1s coated with 7 kDa MW branched PEG chains compared to tubes coated with linear PEG chains of the same molecular weight (approximately 7 h vs 2 h).81 The clearance of cSWCNT1s was very slow (within months) and mainly occurred through the biliary-intestinal route, whereas only a very small portion, hypothesized to be composed by short CNTs, was excreted through the urinary route. In another report they investigated the disposition of SWCNTs coated with 5 kDa MW linear PEG-modified polymers based upon poly(maleic anhydride-alt-1-octadecene) (cSWCNT2) and poly(γ-glutamic acid) (cSWCNT5 and cSWCNT6). 82 cSWCNT2s and cSWCNT5s exhibited very long blood circulation half-lives (approximately 19 and 22 h, respectively), which were far longer than the values recorded for cSWCNT1s. On the contrary, cSWCNT6s showed a blood circulation halflife of ∼2 h, which was very similar to the value showed by cSWCNT1s coated with equal MW (5 kDa) linear PEG chains. The authors also described that reduced blood circulation times were observed for CNTs coated with polymers carrying shorter PEG chains (0.75 kDa vs 5 kDa) or with lower PEG density (PEG/backbone ratio). A more detailed investigation about the role of PEG length and density on the disposition of cSWCNT2s has been recently reported by Liu et al.46 Blood circulation and biodistribution measurements were carried out using the Raman scattering of the CNTs injected into Balb/c mice bearing 4T1 murine breast tumor. cSWCNT2s with increasing PEG length (from 2 to 5 kDa) and density (from 10% to 100%) showed longer blood circulation times, lower uptake in the RES organs (liver and spleen), and higher uptake in the tumor and skin. Although a long blood circulation might be advantageous for biomedical applications, high skin accumulation of certain cSWCNT2 formulations could be a problem. Long blood circulating nanocarriers (such as PEGmodified liposomes) showed skin accumulation and toxicity.83 Furthermore, high skin uptake of nanoparticles may impede the use of phototherapy in oncology, in which light must pass through the skin to reach the tumor, thereby leading to skin damage and reduced treatment efficacy. In light of these considerations, the authors tuned the PEG length and density to obtain cSWCNT2s with balanced blood circulation, RES organ uptake, and skin accumulation and successfully performed in vivo tumor photothermal ablation. Similar

4. DISPOSITION OF PEG-MODIFIED CNTS In the contest of the development of a new drug, the analysis of its disposition in preclinical studies is important in order to evaluate the actual therapeutic potential of the drug and solve potential problems of low biocompatibility. Information on the biodistribution profile of a drug following systemic administration is also necessary to elucidate the structure−function relationship and the likelihood of toxic effects. Recently, studies have been conducted to elucidate the blood circulation half-life, tissue accumulation, and excretion routes following intravenous 3387


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disposition characteristics (long blood circulation, uptake in the RES organs, and the skin) have been reported by Yang et al. for fSWCNT1s functionalized with 1.5 kDa MW PEG chains.84 CNTs were 13C-enriched, and their content in blood and extracted organs was determined by isotope ratio mass spectrometry. The authors described a blood circulation halflife of ∼15 h, using a first-order exponential decay model, and half-life values equal to 20 min and 22 h by fitting the experimental data to a two-compartment model. The longer blood circulation times of cSWCNT2s and cSWCNT5s compared to cSWCNT1s have been explained as due to their higher surface PEG density resulting from CNTcoating with polymers with multiple anchoring points respect to conventional phospholipids, which have single anchoring points. Furthermore, in agreement with this hypothesis, the long blood circulation time of fSWCNT1s may be explained by a denser surface PEG layer resulting per se from the oxidation/ amidation approach, compared to the noncovalent approach based on adsorption of phospholipids. Although the study of Liu et al. support a correlation between PEG density and blood circulation time of PEG-modified CNTs,46 two observations suggest that, as a general rule, circulation time is not only dependent on the degree of surface PEG density. A report by Prencipe et al. showed that cSWCNT6s (SWCNTs coated using a polymer with multiple anchoring points) had the same blood circulation times of cSWCNT1s coated with equal MW (5 kDa) linear PEG chains.82 Moreover, cSWCNT1s and fSWCNT1s have similar degrees of doxorubicin loading through π-stacking, thereby suggesting similar degrees of surface PEGylation.22 Along with surface PEG density, exposed surface charges might also explain differences in blood circulation. It has been reported that negatively charged liposomes incorporating PEG-modified phospholipids are activators of the complement cascade (through the classical and alternative pathways, see below) and that methylation of the phosphate−oxygen moiety prevented complement activation.99 Therefore, although the effects of PEG-modified CNTs on the complement system in vivo are still unknown, the presence of the negative charges on cSWCNT1s and cSWCNT6s, due to the phosphodiester moiety of the phospholipids, may trigger opsonization and uptake by the RES organs and be responsible for their inferior pharmacokinetic profiles respect to the CNTs lacking of such moiety (cSWCNT2s, cSWCNT5s, and fSWCNT1s). Future experiments with cSWCNT1s and cSWCNT6s fabricated using phospholipids with methylated phosphate−oxygen moiety could help validating this hypothesis. Two additional CNTs coated with amphiphilic polymers have been intravenously administered into animals and their disposition investigated. cSWCNT4s (SWCNTs coated with a poloxamer, Pluronic F108) displayed ∼1 h blood circulation half-life and accumulated mainly in the liver of treated rabbits. 85 Red shifts in the NIR emission peaks suggested that blood proteins displaced the poloxamer coating within a period of seconds following administration. In another report, in order to investigate the effects of purified and pristine CNTs in vivo, Yang et al. used Tween 80 as coating agent for CNTs (cSWCNT7s).86 The authors found accumulation mainly in the liver, spleen, and lung of treated mice. Both Pluronic F108 and Tween 80 are amphiphilic polymers with a single alkyl chain, which provides just a weak anchor to the CNT sidewalls. Although the displacement of those polymers by the blood proteins from CNT sidewalls may not lead to CNT

aggregation, as revealed by the retention of cSWCNT4 NIR fluorescence in vivo, the instability of the surface PEG coating may pose an obstacle to the use of cSWCNT4s and cSWCNT7s for biomedical applications. Although with differences in the blood circulation time and the accumulation in the major organs, cSWCNT1−7s and fSWCNT1s were mainly uptaken by the RES organs and just minimally excreted through the renal route, following their intravenous administration. On the contrary, TEM analyses of liver and kidney slices and urine samples from mice treated with fSWCNT2s and fMWCNT2s showed that these highly soluble CNTs can avoid uptake by the RES organs and be rapidly cleared through the renal route.56,87 Classically, it has been believed that low uptake of PEGylated nanoparticles by the RES organs is due to the ability of PEG to reduce opsonization; these results would suggest that 1,3 dipolar cycloaddition leads to CNTs with homogeneously PEG-covered sidewalls and to good protection against opsonization. Incidentally, the fSWCNT2s used by Lacerda et al.56 and the fSWCNT2s and fMWCNT2s used by Singh et al.58 were functionalized with very short PEG chains. The fact that fSWCNT2s and fMWCNT2s were not opsonized is somehow nonconcordant with previous models that a minimum surface PEG molecular weight of 2 kDa is required to achieve sufficient shielding against plasma protein binding.5,6 However, it has also been proposed that a homogeneous PEG layer with a minimum thickness of ∼5% of the particle’s diameter is effective against opsonization.88 Since CNTs have nanometric diameter, a subnanometric PEG layer could ensure sufficient repulsion of opsonins from CNT sidewalls. Moreover, in recent years there has been a growing body of evidence that PEGylation does not completely preclude opsonization and that reduced accumulation of PEGylated nanoparticles in the RES organs is also related to other PEG-mediated effects. A proposed alternative mechanism by which PEGylation could improve nanoparticle blood circulation time is the steric stabilization and minimization of interactions that lead to sequestration in the RES organs. For instance, this could include the interactions with Fc receptors and/or the prevention of nanoparticle aggregation and consequent phagocyotosis due to the large size of aggregates. Bally et al. reported that liposomes functionalized with 550 and 2 kDa MW PEG chains were similarly opsonized while exhibiting equally increased circulation time with respect to non-PEGylated nanoparticles. The authors concluded that the primary effect of PEGylation to prolong circulation longevity was due to steric stabilization that prevented liposome aggregation while not blocking plasma protein binding.89 Although the question of whether plasma proteins adsorb onto fSWCNT2s and fMWCNT2s remains unsolved, we can hypothesize that CNTs functionalized through 1,3 dipolar cycloaddition, by virtue of sterical stabilization due to homogeneously PEG-covered sidewalls, are able to avoid interactions (for instance CNT aggregation) that lead to sequestration in the RES organs and hamper the ability to cross the glomerular filter.

5. BIOCOMPATIBILITY AND TOXICITY PROFILE OF PEG-MODIFIED CNTS PEG is commonly perceived to be immunologically safe,90 and it is widely used as a therapeutic agent per se52−54 or to passivate the surface of drug delivery formulations to limit the uptake by RES organs. However, PEG can be immunogenic and has revealed its “Janus-faced” nature in several occasions. 3388


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Figure 4. The complement system. The complement system is composed by more than 30 proteins and is part of the innate immune system. Its activation leads to the recruitment of inflammatory and immune cells to the tissue, opsonization (and subsequent phagocytosis) of foreign material, and formation of pores on the membrane of pathogens. Complement activation can be triggered by the classical, lectin, or alternative pathway and proceeds through the effector pathway that is irrespective of the initial way of activation. Complement activation produces several anaphylatoxins and chemoattractants (C3a, C4a, and C5a) that have a potent activity on blood vessels, increasing vascular permeabilization and upregulating celladhesion molecules, and may induce anaphylaxis in sensitive individuals.

FDA-approved PEG-modified formulations (liposomes and medicines containing high PEG content) have triggered anaphylaxis in veterinary cases (pigs, cattle, sheep, etc.) and in human patients.91−93 The likely mechanism of these reaction was complement activation and production of anaphylatoxins (C3a and C5a) and pro-inflammatory mediators (Figure 4).94 It is noteworthy the case of Doxil, an approved chemotherapy drug composed by doxorubicin encapsulated into PEGmodified liposomes and used to treat AIDS-related Kaposi’s sarcoma, breast cancer, refractory ovarian carcinoma, and other solid tumors. Intravenous administration of Doxil in pigs induced dose-dependent cardiopulmonary and electrocardiogram anomalies caused by increased plasma level of the anaphylatoxin C5a.93 Doxil has also been reported to activate complement in human serum95 and elicit acute allergic reaction in cancer patients.96 Hamad et al. reported that the mechanism of complement activation by linear PEG is dependent on its concentration and length.97 Complement activation by short PEG chains (molecular weight less than 4 kDa) occurred exclusively through the lectin pathway (initiated by the ficolins/ MASP-2 complex), whereas longer PEG also triggered the alternative pathway turnover. However, the molecular mechanism of the interaction between PEG and ficolins remains unclear, since ficolins have specificity for sugars with Nacetylated groups, which are not present on PEG.98 One

hypothesis brought forward by the same authors is that ficolins may interact with hydroxylated compounds as efficiently as with acetylated ones. Beside PEG length and concentration, triggering of the complement cascade by PEG-modified formulations also depends on their structure and chemical makeup. A remarkable example is given by PEG-modified phospholipids (PEG-PLs). In micellar form, PEG-PLs were ineffective in triggering complement.99 This suggests that monomers of PEG-PLs do not affect complement as well because monomers and micelles are present at the same concentration in a solution of PEG-PLs at the critical micellar concentration. However, PEG-PLs incorporated in liposomes activated complement in human serum through the classical and the alternative pathways.95 Complement activation through the classical pathway has been explained with the presence of naturally occurring anti-PEG antibodies. These antibodies directed against 4−5 PEG units have IgG2 and IgM isotypes and are present in ∼25% of the general populations.100,101 It is noteworthy that also cleavage of C4 may be triggered by linear PEG through the classical pathway (instead of the lectin pathway) in sera with high titer of anti-PEG antibodies. These observations suggest that naturally occurring anti-PEG antibodies may play a role in the side effects triggered by PEGmodified formulations and mediated by complement activation. 3389


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As previously described, PEG-PLs have also been used to modify the surface of CNTs. cSWCNT1s coated with 5 kDa MW linear PEG chains triggered human complement in vitro through the lectin pathway initiated by both the MBL/MASPs and the ficolins/MASP-2 complexes. 102 Precisely how cSWCNT1s trigger the lectin pathway remains unclear because these nanoparticles lack the structures (in particular sugars with N-acetylated groups) necessary for MBL and ficolin binding.98,103,104 A couple of hypotheses have been formulated. Complement activation may be triggered by either direct or indirect (mediated by plasma proteins such as apo-lipoproteins) adsorption of MBL and/or ficolins on exposed portions of the graphitic CNT sidewall.65 Alternatively, activation may be triggered through binding of MBL and/or ficolins to the projecting PEG chains. Complement activation by cSWCNT1s is consistent with their accumulation in RES organs following intravenous administration.41,81 However, in vivo investigations on complement activation by cSWCNT1s coated with 5 kDa MW linear PEG were inconclusive. Moghimi et al. reported that also fSWCNT1s were able to activate the complement cascade in vitro.105 However, this was mentioned as an unpublished observation with no further detail provided. Moreover, in vivo investigations on complement activation by fSWCNT1s have not been reported yet as well as in vitro and in vivo studies on cSWCNT2−7s, fMWCNT1s, fSWCNT2s, and fMWCNT2s. However, the lack of immunogenicity61 and uptake by RES organs56,58 suggests that fSWCNT2s and fMWCNT2s do not activate complement and that naturally occurring anti-PEG antibodies are not able to bind the short PEG decorating fSWCNT2s and fMWCNT2s. More studies on the effects of PEG-modified CNTs on complement activation in vivo are warranted. Although certain PEG-modified CNTs were able to activate the complement system in vitro, no serious acute or chronic toxicity of these materials has been observed as a consequence of complement activation or other mechanisms. At the cellular level, exposition of several cell types to PEG-modified CNTs did not lead to any detectable apoptosis or necrosis.24,33,62,71,106 However, PEG-modified CNTs can affect the functionality of cells of the immune systems (T and B cells, macrophages, etc.) in function of their chemical makeup. Dumortier et al. reported that fSWCNT1s and fSWCNT2s had a different impact on primary immune cells.71 fSWCNT2s did not affect the functionality of B and T lymphocytes and macrophages, whereas fSWCNT1s elicited secretion of pro-inflammatory cytokines [tumor necrosis factor α (TNFα) and IL-6] by macrophages. However, the molecular basis of these observations has not been elucidated yet. No serious immune and/or inflammatory responses have been observed on mice administered with PEG-modified CNTs. A recent study of Mutlu et al. pointed out that individualization has a pivotal importance on the lung toxicity of intratracheally administered CNTs, more than their aspect ratio.107 The authors described that cSWCNT4s did not elicit any lung inflammation or fibrosis, whereas pristine nonPEGylated CNTs elicited areas of chronic inflammation in the bronchi and fibrosis. Furthermore, cSWCNT4s did not induce frustration of alveolar macrophages and were cleared from lungs over time although the clearance mechanism has not been described. The authors also did not investigate the metabolism of the material in the alveolar macrophages. As we already described, poloxamers can be displaced by proteins from CNT sidewalls once in the bloodstream or uptaken by

macrophages and the displacement does not lead to CNT aggregation.7,85 Individual protein-coated CNTs are able to cross biological membranes and can be excreted after intracellular trafficking.70 In light of these findings, cSWCNT4s might be able to escape from alveolar macrophages following poloxamer displacement, thus avoiding long-term lung accumulation and potential side effects. The role of CNT individualization on CNT toxicity is confirmed by the findings of Yang et al.86 In their study they investigated the effects of long-term accumulation in liver, spleen, and lungs of intravenously administered cSWCNT7s (SWCNTs coated with Tween 80) and found that, although not eliciting any apoptosis or fibrosis, there was hepatic stress and inflammation in areas surrounding CNTs trapped in the lung. The proposed main toxicological pathway was oxidative stress. Unlike other amphiphilic polymers, Tween 80 has a single unsaturated alkyl chain which provides a very weak anchor to the CNT sidewalls. TEM images showed that cSWCNT7s were mainly organized in bundles of long nanotubes, thereby supporting a model where CNTs are accumulated in RES organs and lungs and can elicit side effects proportionally to their degree of aggregation. Schipper et al. reported the long-term accumulation of CNTs into Kupffer cells through Raman spectroscopy following a single intravenous administration of high doses of cSWCNT1s coated with 5 kDa MW linear PEG and of fSWCNT1s functionalized with 10 kDa MW linear PEG. 108 The accumulation was not associated with any evident pathology. As previously mentioned, fSWCNT1s accumulated in the liver of treated mice can be partially PEG-defunctionalized.74 Thus, the study suggest that accumulated CNTs do not elicit significant liver damage even after partially loosing the protective PEG layer. These results are encouraging; however, the study was performed using small mice numbers. More extensive studies are needed using CNTs modified with PEG chains of different length and nature and generated using different protocols before a general structure−function model for biocompatibility and toxicity of PEG-modified CNTs can be formulated.

6. CONCLUSIONS AND FUTURE PERSPECTIVES In conclusion, multiple studies have shown the critical role of the structural and chemical features of PEG chains (density, length, homogeneity, etc.) on the performances of PEGmodified CNT at the cellular and organism level. Exhaustive studies about the role of CNT features (length, diameter, chirality, etc.) on the biological performances of PEG-modified CNT have not been carried out yet (Figure 3). However, although several pieces of the puzzle are still missing, we attempt to formulate a preliminary structure−function model for PEG-modified CNT cellular trafficking, disposition and side effects. A homogeneous surface PEG modification, as the one obtained through 1,3 dipolar cycloaddition, is necessary and sufficient to obtain sterically stabilized and highly soluble PEGmodified CNTs. These CNTs have the ability to stay as individual/small bundles with nanometric diameters of SWCNTs and individual MWCNTs in most biological environments. They have good resistance to opsonization and uptake from the RES organs. They are biocompatible and do not affect the functionality of immune cells. They are excreted mainly through the urinary system and able to pass through the plasma membrane and escape from lysosomes. If they are short 3390


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Table 1. Main Properties of the Interaction of PEG-Modified CNTs with the Living Matter at the Cellular and Organism Level cSWCNT1 modification approach composition solubility in PBS and serum blood circulation half-life accumulation in RES organs fate in RES organs excretion complement activation in vitro in vivo effects on T cells B cells macrophages cellular uptake intracellular fate

adsorption of phospholipids individual and small bundles of SWCNTs stable suspension

cSWCNT2

fSWCNT1

adsorption of poly(maleic anhydride-alt-1octadecene)-based polymer individual and small bundles of SWCNTs

fSWCNT2/fMWCNT2

oxidation/amidation

1,3 dipolar cycloaddition

stable suspension

individual and small bundles of SWCNTs stable suspension

small bundles of SWCNTs, individual MWCNTs stable solution

∼0.5−7 ha,b

∼2.5−21 ha,b

15.3 ha (22.5 hc)

3h

yes

yes

yes

no

− slow, mainly feces

− −

PEG-defunctionalization slow, mainly urines

− fast, mainly urines

yes (lectin pathway) −

− −

yesd −

− −

− − − endocytosis perinuclear vesicles

− − − − −

none none secretion of TNFα and IL-6 endocytosis cytoplasm and nucleus

nonee nonee nonee passive diffusion cytoplasm and nucleus

a

Blood circulation half-life values were calculated by using a first-order exponential decay model. bHigher blood circulation half-life values were obtained increasing the PEG surface density, length, and branched nature. cBlood circulation half-life value was calculated by using a twocompartmental model. dThe pathway for complement activation has not been described. eExperiments were carried out for fSWCNT2s.

desired application of the nanodrug. For example, we mentioned that gaps in the protective PEG layer may reduce the pharmacokinetic profile. However, it has been shown that these gaps enable adsorption of polyaromatic cargoes onto the CNT sidewalls, thereby increasing the loading capacity of PEGmodified CNTs. Functionalization approaches have advantages over coating techniques to decorate CNT sidewalls because they stably attach PEG chains and avoid undesired detachment of molecules or exchange with serum proteins following administration. However, the currently used functionalization approaches also dramatically change the ordered π-electron structure of CNTs, leading to reduced intrinsic NIR fluorescence and Raman scattering. Thus, coating approaches might be preferred because they are gentler and preserve the intactness of CNT sidewalls. To make things more complicated, even coating approaches may produce CNTs with suboptimal optical properties. Indeed some phospholipids might be able to “scrape” the CNT sidewalls during prolonged ultrasonication, thus damaging the ordered tubular structure and leading to CNTs with low NIR emission intensity.42 An important open question is whether the ability of fSWCNT2s and fMWCNT2s to avoid uptake from the RES organs and to pass through membranes is exclusively due to their physical properties (high solubility and sterically stabilization) without any contribution from the short length of the PEG chains used. Experiments on fSWCNT2s and fMWCNT2s with long (linear and branched) PEG chains could shed light on this aspect. Another open question is whether the translocation into the nucleus observed for epithelial cells and fibroblasts is applicable to other cell types, especially immune cells. Finally, it is important to clarify whether and how PEGmodified CNTs trigger the activation of the complement in vivo. Analyzing the effects of CNTs on complement activation and more exhaustively studying the toxicity of PEG-modified CNTs will help shed light on the true potential of these particles as future nanodrugs.

enough, fSWCNT2s and fMWCNT2s are able to translocate to the nucleus of certain cell types. On the other hand, a nonhomogeneous surface PEG layer, as the one obtained through absorption of amphiphilic polymers and oxidation/amidation, leads to individual/small bundles of CNTs mostly forming a stable suspension in high saline solutions. These CNTs most likely aggregate once administered due to nonperfect steric stabilization, which leads to lower blood circulation time, sequestration in the RES organs, and inability to cross the glomerular filter. Improved steric stabilization can be obtained by increasing the surface PEG density, using longer and more branched PEG chains or avoiding certain modification approaches (for example, the ones that leave exposed negative charges). The presence of gaps in the protective PEG layer enables the interaction of these CNTs with biomacromolecules in the bloodstream and on the plasma membrane. Interactions with blood serum components trigger opsonization and complement activation. Interactions between gaps in the protective PEG layer and lipids/proteins on the plasma membrane trigger endocytosis and transport of the CNTs into the endolysosomal vesicles. The ability to escape from vesicle sequestration (as well as to cross the glomerular filter) is a function of the degree of individualization of CNTs and of the tendency of CNTs to stay in small bundles. Data on the intracellular localization of PEG-modified CNTs suggest that small bundles of oxidized CNTs have a higher tendency to release individual CNTs than bundles of CNTs kept together by the alkyl chains of amphiphilic polymers. In Table 1 we reported the main properties for the PEGmodified CNTs, whose interactions with the living matter have been more accurately investigated. These data suggest that fSWCNT2s and fMWCNT2s rank as best followed by fSWCNT1s and cSWCNT2s, and by cSWCNT1s. However, it should be kept in mind that the fabrication of a nanodrug is a complex process and the choice of a specific modification approach and/or of the PEG chemistry is also dictated by the 3391


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AUTHOR INFORMATION

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ACKNOWLEDGMENTS

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REFERENCES

Review

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Corresponding Author *E-mail: [email protected]. Ph: (858) 646-3100 ext 3063. Fax: (858) 795-9225.

This work was supported by the Juvenile Diabetes Research Foundation and the Arthritis National Research Foundation.

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