Lara Lacerda

Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type

The development of nanomaterials for biomedical and biotechnological applications is an area of research that holds great promise and intense interest1, and carbon-based nanostructures in particular, such as carbon nanotubes (CNTs), are attracting an increasing level of attention2,3. One of the key advantages that CNTs offer is the possibility of effectively crossing biological barriers, which would allow their use in the delivery of therapeutically active molecules. Our laboratories have been investigating the use of CNTs in biomedical applications, and in particular as nanovectors for therapeutic agent delivery4,5,6,7,8. The interaction between cells and CNTs is a critical issue that will determine any future biological application of such structures. Here we show that various types of functionalized carbon nanotubes (f-CNTs) exhibit a capacity to be taken up by a wide range of cells and can intracellularly traffic through different cellular barriers.

Cell-penetrating CNTs for delivery of therapeutics

The use of carbon-based nanostructures, such as carbon nanotubes, in biomedicine is increasingly attracting attention. One key advantage of carbon nanotubes is their ability to translocate through plasma membranes, allowing their use for the delivery of therapeutically active molecules in a manner that resembles cell-penetrating peptides. Moreover, exploitation of their unique electrical, optical, thermal, and spectroscopic properties in a biological context is hoped to yield great advances in the detection, monitoring, and therapy of disease. Here we offer a speculative overview of the general principles behind the mechanism of carbon nanotube penetration of the plasma membrane and a snapshot of the different therapeutic modalities based on these fascinating nanostructures that are currently being investigated.

Translocation mechanisms of chemically functionalised carbon nanotubes across plasma membranes

Understanding the mechanisms responsible for carbon nanotube (CNT) internalisation into live cells is considered critical both from a fundamental point of view and for further engineering of CNT-based delivery systems to intracellular targets. While several studies are focused on the development of such CNT-based delivery systems, attempts to systematically elucidate the cellular uptake mechanisms of CNTs are still rather limited. The aim of the present study is to evaluate the cellular internalisation of chemically functionalised multi-walled carbon nanotubes (f-MWCNTs) in the presence of different well-known cellular uptake inhibitors. Our data reveal how f-MWCNTs are able to translocate across cell membranes of both phagocytic and non-phagocytic cell lines. We have evidenced that at least 30-50% of f-MWCNTs are taken up by cells through an energy-independent mechanism. This characteristic makes nanotubes loaded with therapeutic or diagnostic cargos extremely interesting as the release of active molecules directly into the cytoplasm increase their biological activity and therapeutic efficacy.

Carbon-nanotube shape and individualization critical for renal excretion

In the past few years there has been an accumulating amount of evidence to suggest that the structural features of nanoparticles are responsible for dramatically different pharmacokinetic and body-excretion profiles. For example, a recent study convincingly illustrated that the mean hydrodynamic diameter of quantum dots (spherically shaped nanocrystals) is a determinant factor in achieving effective urinary excretion through the renal filtration barrier. In that study, however, the effect of nanoparticle shape on renal filtration was not examined at all. Our group has investigated the pharmacokinetic and excretion profiles of nonspherical, fibrilous carbon nanotubes (CNTs) and, in the present study, we have attempted to elucidate the mechanism by which such cylindrical nanoparticles can be excreted through the renal route. We have previously reported that surface-functionalized, water-dispersible, single-walled carbon nanotubes (SWNTs; average diameter 1 nm; average length 300–1000 nm) were capable of rapid and effective renal clearance and urinary excretion with a blood-circulation half-life of a few hours. These observations were in agreement with other studies

Tissue histology and physiology following intravenous administration of different types of functionalized multiwalled carbon nanotubes.

BACKGROUND Carbon nanotubes (CNTs) constitute one of the most important types of nanomaterials, increasingly gaining interest as tools for nanomedicine applications, such as sensors, implants or delivery systems. Our groups have reported previously that chemical functionalization of CNTs can lead to their almost complete elimination from the body of animals through the urinary excretion route. The administration of CNTs may, however, impact the physiological function of organs through which CNTs traverse or accumulate. AIM The present study addresses the short-term impact (first 24 h) of intravenous administration of various types of multiwalled nanotubes (MWNTs) on the physiology of healthy mice. MATERIALS & METHODS Nonfunctionalized, purified MWNTs (pMWNTs) and different types of water-dispersible, functionalized MWNTs (f-MWNTs) were tail-vein injected. Histological examination of tissues (kidney, liver, spleen and lung) harvested 24 h post-administration indicated that organ accumulation depended on the degree of ammonium (NH(3)(+)) functionalization at the f-MWNT surface. RESULTS The higher the degree of functionalization of MWNT-NH(3)(+), the less their accumulation in tissues. pMWNTs coated with autologous serum proteins prior to injection accumulated almost entirely in the lung and liver in large dark clusters. Moreover, various indicators of serum and urine analyses also confirmed that MWNT-NH(3)(+) injections did not induce any physiological abnormality in all major organs within the first 24 h post-injection. Interestingly, no abnormalities were observed either for f-MWNTs highly functionalized with carboxylate groups (diethylentriaminepentaacetic-functionalized MWNTs) or by upscaling to the highest doses ever injected so far in vivo (20 mg/kg). CONCLUSION The high degree of f-MWNT functionalization responsible for adequate individualization of nanotubes and not the nature of the functional groups was the critical factor leading to less tissue accumulation and normal tissue physiology at least within the first 24 h post-administration, even at the highest carbon nanotube doses ever administered in any study today.

Lipid−Quantum Dot Bilayer Vesicles Enhance Tumor Cell Uptake and Retention in Vitro and in Vivo

We report the construction of lipid-quantum dot (L-QD) bilayer vesicles by incorporation of the smallest (2 nm core size) commercially available CdSe/ZnS QD within zwitterionic dioleoylphosphatidylcholine and cationic 1,2-dioleoyl-3-trimethylammonium-propane lipid bilayers, self-assembling into small unilamellar vesicles. The incorporation of QD in the acyl environment of the lipid bilayer led to significant enhancement of their optical stability during storage and exposure to UV irradiation compared to that of QD alone in toluene. Moreover, structural characterization of L-QD hybrid bilayer vesicles using cryogenic electron microscopy revealed that the incorporation of QD takes place by hydrophobic self-association within the biomembranes. The L-QD vesicles bound and internalized in human epithelial lung cells (A549), and confocal laser scanning microscopy studies indicated that the L-QD were able to intracellularly traffick inside the cells. Moreover, cationic L-QD vesicles were injected in vivo intratumorally, leading to enhanced retention within human cervical carcinoma (C33a) xenografts. The hybrid L-QD bilayer vesicles presented here are thought to constitute a novel delivery system that offers the potential for transport of combinatory therapeutic and diagnostic modalities to cancer cells in vitro and in vivo.

The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-epsilon-caprolactone-based nanoparticles

Strangles is a bacterial infection of the Equidae family that affects the nasopharynx and draining lymph nodes, caused by Streptococcus equi subspecies equi. This agent is responsible for 30% of all worldwide equine infections and is quite sensitive to penicillin and other antibiotics. However, prevention is still the best option because the current antibiotic therapy and vaccination is often ineffective. As S. equi induces very strong systemic and mucosal responses in convalescent horses, an effective and economic strangles vaccine is still a priority. In this study the humoral, cellular and mucosal immune responses to S. equi antigens encapsulated or adsorbed onto poly-epsilon-caprolactone nanospheres were evaluated in mice. Particles were produced by a double (w/o/w) emulsion solvent evaporation technique and contained mucoadhesive polymers (alginate or chitosan) and absorption enhancers (spermine, oleic acid). Their intranasal administration, particularly those constituted by the mucoadhesive polymers, increased the immunogenicity and mucosal immune responses (SIgA) to the antigen. The inclusion of cholera toxin B subunit in the formulations successfully further activated the paths leading to Th1 and Th2 cells. Therefore, those PCL nanospheres are potential carriers for the delivery of S.equi antigens to protect animals against strangles.

Carbon nanotube cell translocation and delivery of nucleic acids in vitro and in vivo

In the last few years, the carbon nanotube (CNT) field has seen a new direction of investigation growing rapidly, along with the interest of more researchers from diverse fields of expertise interested in this new material in an attempt to exploit their properties in biomedical applications. Here we describe the most recently reported work on the application of CNT for gene encoding nucleic acid (DNA and RNA) delivery purposes by using in vitro and in vivo models. Several groups have now successfully observed the cellular internalisation of nucleic acids with the aid of CNT following very different protocols. The main processes for the internalisation pathways and intracellular release of the nucleic acids are here reviewed. Furthermore, we have just started to see some initial studies of in vivo work using siRNA-CNT conjugates to achieve silencing in tumour tissue. Admittedly, it is still very early days for the technology, but future studies are necessary, and will surely appear, in order to determine the feasibility of bringing the CNT closer to the clinic.

Carbon nanotube-mediated delivery of peptides and genes to cells: translating nanobiotechnology to therapeutics

During the last few years, there has been a tremendous amount of optimism and expectation about nanotechnology and its impact on various fields including medicine and pharmaceutical development. One of the most promising materials being developed during the nanotechnological renaissance we are currently experiencing is the carbon nanotube. Before any biology-related application can even be envisaged, the aqueous solubility of carbon nanotubes has to be resolved. Recently, a variety of methodologies have been proposed which lead to biologically compatible carbon nanotubes. Covalent functionalization of their surface is one methodology, allowing the first attempts towards applications in the field of nanomedicine. The possibility of incorporating functionalized carbon nanotubes into cells and the biological milieu offers numerous advantages for potential applications in biology and pharmacology. One of the most promising is their utilization as a new carrier system for the delivery of therapeutic molecules. In the present article, the first attempts to transform carbon nanotubes from biologically incompatible nanomaterials to biologically relevant components of advanced therapeutics and the ensuing novel structures obtained in our laboratories are presented.

Nanoengineering artificial lipid envelopes around adenovirus by self-assembly

We have developed a novel, reproducible, and facile methodology for the construction of artificial lipid envelopes for adenoviruses (Ad) by self-assembly of lipid molecules around the viral capsid. No alteration of the viral genome or conjugation surface chemistry at the virus capsid was necessary, therefore difficulties in production and purification associated with generating most surface-modified viruses can be eliminated. Different lipid bilayer compositions produced artificially enveloped Ad with physicochemical and biological characteristics determined by the type of lipid used. Physicochemical characteristics such as vector size, degree of aggregation, stability, and surface charge of the artificially enveloped Ad were correlated to their biological (gene transfer) function. In monolayer cell cultures, binding to the coxsackie and adenovirus receptor (CAR) was blocked using a zwitterionic envelope, whereas enhanced binding to the cell membrane was achieved using a cationic envelope. Envelopment of Ad by both zwitterionic and cationic lipid bilayers led to almost complete ablation of gene expression in cell monolayers, due to blockage of virion endosomal escape. Alternatively, artificial Ad envelopes built from lipid bilayers at the fluid phase in physiological conditions led to enhanced penetration of the vectors inside a three-dimensional tumor spheroid cell culture model and delayed gene expression in the tumor spheroid compared to nonenveloped adenovirus. These results indicate that construction of artificial envelopes for nonenveloped viruses by lipid bilayer wrapping of the viral capsids constitutes a general strategy to rationally engineer viruses at the nanoscale with control over their biological properties.