Bowen Tian

Antitumor activity and prolonged survival by carbon-nanotube-mediated therapeutic siRNA silencing in a human lung xenograft model

Carbon nanotubes are novel nanomaterials that are thought to offer potential benefits to a variety of biomedical and clinical applications. In this study, the treatment of a human lung carcinoma model in vivo using siRNA sequences leading to cytotoxicity and cell death is carried out using either cationic liposomes (DOTAP:cholesterol) or amino-functionalized multi-walled carbon nanotubes (MWNT - NH(+)(3)). Validation for the most cytotoxic siRNA sequence using a panel of human carcinoma and murine cells reveals that the proprietary siTOX sequence is human specific and can lead to significant cytotoxic activities delivered both by liposome or MWNT - NH(+)(3) in vitro. A comparative study using both types of vector indicates that only MWNT - NH(+)(3):siRNA complexes administered intratumorally can elicit delayed tumor growth and increased survival of xenograft-bearing animals. siTOX delivery via the cationic MWNT - NH(+)(3) is biologically active in vivo by triggering an apoptotic cascade, leading to extensive necrosis of the human tumor mass. This suggests that carbon-nanotube-mediated delivery of siRNA by intratumoral administration leads to successful and statistically significant suppression of tumor volume, followed by a concomitant prolongation of survival of human lung tumor-bearing animals. The direct comparison between carbon nanotubes and liposomes demonstrates the potential advantages offered by carbon nanotubes for the intracellular delivery of therapeutic agents in vivo. The present work may act as the impetus for further studies to explore the therapeutic capacity of chemically functionalized carbon nanotubes to deliver siRNA directly into the cytoplasm of target cells and achieve effective therapeutic silencing in various disease indications where local delivery is feasible or desirable.

Asbestos-like Pathogenicity of Long Carbon Nanotubes Alleviated by Chemical Functionalization

Carbon nanotubes (CNTs) are considered one of the most popular types of nanomaterials and in the last few years have gained tremendous interest in a wide range of applications due to their unique physical, chemical, and electronic properties. Multi-walled carbon nanotubes (MWNTs) consist of sheets of carbon atoms rolled up into multiple concentric hollow tubular structures. The lack of dispersibility of pristineMWNTs in most solvents is owing to strong inter-tube van der Waals forces and this has been an obstacle for their effective use in biological applications and material sciences (i.e. composites). This may be largely overcome by surface modification of the nanotube backbone, allowing application of CNTs in biomedical applications. Some types of chemically functionalized CNTs have shown great advantages for use as delivery systems because of their capacity to pierce cellular membranes and translocate directly into the cytoplasm, providing a method for effective drug and macromolecule intracellular transport. Moreover, chemical surface-functionalization strategies can improve the colloidal properties of the CNT dispersions and result in populations of individualized MWNTs in physiological environments that have the capacity for glomerular translocation, leading to rapid urinary excretion. Such biokinetic processes are also extremely important to determine the biopersistence and ultimately the potential risk from medical use of carbon nanotubes. The use of CNTs—particularly in mass-scale, industrial applications—is currently considered with apprehension owing to their yet undefined safety profile and their potential environmental and health risks, especially given their structural resemblance to asbestos fibers. Several research groups have attempted to determine the carcinogenic risks that may be associated with intended or unintentional exposure to CNTs using various in vivo models. The first study that highlighted the importance of carbon nanotube length characteristics was carried out by Poland et al. using pristine (non-functionalized), long CNTs in a structure– toxicity study, which was originally validated with asbestos fibers. According to this method, which relates length and biopersistence of asbestos fibers to the development of mesothelioma (cancer of the pleural membrane), non-functionalized MWNTs longer than 20 mm were found to trigger an inflammatory response and result in granuloma formation seven days after intra-peritoneal exposure, similar to long asbestos fibers (LFA, long fiber amosite). This was thought to be due to induction of a process termed “frustrated phagocytosis” as resident and recruited macrophages attempt unsuccessfully to remove the long fibers from the mesothelium. Similar conclusions regarding the risk of unwanted

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.

Enhanced cellular internalization and gene silencing with a series of cationic dendron-multiwalled carbon nanotube:siRNA complexes

One of the major obstacles to the clinical development of gene silencing by small interfering RNA (siRNA) is its effective cytoplasmic delivery. Carbon nano‐tubes have been proposed as novel nanomaterials that can offer significant advantages for the intracellular delivery of nucleic acids, such as siRNA. We recently demonstrated in a proof‐of‐principle study that amino‐functionalized multiwalled carbon nanotubes (f‐MWNT) can effectively deliver in vivo an siRNA sequence, triggering cell apoptosis that results in human lung xenograft eradication and prolonged survival. In the present study, we demonstrate how a newly synthesized series of polycationic dendron‐MWNT constructs with a precisely tailored number of amino functions (dendron generations) can complex and effectively deliver double‐stranded siRNA to achieve gene silencing in vitro. A systematic comparison between the f‐MWNT series in terms of cellular uptake, cytotoxicity, and siRNA complexation is offered. Significant improvement in siRNA delivery with the dendron‐MWNT conjugates is shown, and gene silencing was obtained in 2 human cell lines using 2 different siRNA sequences. The study reveals that through f‐MWNT structure‐biological function analysis novel nanotube‐based siRNA transfer vectors can be designed with minimal cytotoxicity and effective delivery and gene‐silencing capabilities.—Al‐Jamal, K. T., Toma, F. M., Yilmazer, A., Ali‐Boucetta, H., Nunes, A., Herrero, M.‐A., Tian, B., Eddaoudi, A., Al‐Jamal, W. T., Bianco, A., Prato, M., Kostarelos, K. Enhanced cellular internalization and gene silencing with a series of cationic dendron‐multiwalled carbon nanotube:siRNA complexes. FASEB J. 24, 4354–4365 (2010). www.fasebj.org

Artificial envelopment of nonenveloped viruses: enhancing adenovirus tumor targeting in vivo

Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad by systemic administration is limited due to rapid clearance from blood circulation, transfection of nontarget tissues, toxicity, and immunogenicity. To address these limitations, we developed an artificially enveloped Ad vector prepared by self‐assembly of lipid bilayers around the Ad capsid. The physicochemical and structural features of the enveloped Ad vector can be altered according to the type of lipid used without the need for genetic modification or conjugation chemistry. Here we engineered 4 different types of artificially enveloped Ad using cationic, neutral, fusogenic, and PEGylated lipids to form the envelopes and obtained extended blood circulation times following i.v. administration and reduced vector immunogenicity. Moreover, the PEGylated lipid‐enveloped Ad was capable of specifically delivering genes via the systemic circulation to solid tumors subcutaneously implanted in the absence of high levels of gene transfer to the liver and spleen. This provides the basis for the development of a novel vector platform for systemic delivery of Ad to disseminated targets. Furthermore, the artificial envelopment of Ad presented herein is an illustration of a general strategy to modulate the biological function of nonenveloped viruses and may have implications broader than gene therapy.—Singh, R., Tian, B., Kostarelos, K. Artificial envelopment of nonenveloped viruses: enhancing adenovirus tumor targeting in vivo. FASEB J. 22, 3389–3402 (2008)

Intracellular trafficking and gene expression of pH-sensitive, artificially enveloped adenoviruses in vitro and in vivo

Recombinant adenovirus (Ad) has shown great promise in gene therapy. Artificial envelopment of adenovirus within lipid bilayers has previously been shown to decrease the immunogenicity and hepatic affinity of naked Ad in vivo. Unfortunately, this also resulted in a significant reduction of gene expression, which we attributed to poor endosomal release of the Ad from its artificial lipid envelope. In this work, we explored the artificial envelopment of Ad within pH-sensitive DOPE:CHEMS bilayers and characterized this vector by TEM, AFM, dot blot, dynamic light scattering and zeta potential measurements. The artificially enveloped viral vectors exhibited good stability at physiological pH but immediately collapsed and released naked Ad virions at pH 5.5. Intracellular trafficking using confocal laser scanning microscopy (CLSM) revealed that Cy3-labelled Ad enveloped in DOPE:CHEMS bilayers exhibited the characteristic Ad distribution within the cytoplasm that led to virion accumulation around the nuclear membrane, indicating endosomal release of Ad. We obtained equivalent levels of gene expression as those of naked Ad in a series of CAR-positive (CAR+) and CAR-negative (CAR-) cell lines. This suggested that the mechanism of infection for the artificially enveloped Ad remained dependent on the presence of CAR receptors. Finally, the pH-sensitive enveloped Ad were injected intratumorally in human cervical carcinoma xenograft-bearing nude mice, also illustrating their capacity for efficient in vivo marker gene expression. This study is a step forward toward the engineering of functional, artificially enveloped adenovirus vectors for gene transfer applications.

Efficient receptor-independent intracellular translocation of aptamers mediated by conjugation to carbon nanotubes

We have covalently grafted aptamers onto carboxylated carbon nanotubes to design a novel vector system that can easily translocate into the cytosol of different cell types independent of receptor-mediated uptake. We propose the use of carbon nanotubes for the efficient intracellular delivery of biologically active aptamers for potential therapeutic applications.

Design and Engineering of Multifunctional Quantum Dot-Based Nanoparticles for Simultaneous Therapeutic-Diagnostic Applications

Quantum dots (QD) are semiconducting nanocrystals that have recently received a lot of interest because of their efficacy as fluorescent probes. They offer a significant increase in photostability and fluorescence lifetime compared to more traditional organic dye-based probes; however, some concern from their potential in vivo use due to their chemical composition and their nanoscale dimensions exists. Within this chapter, we discuss current knowledge about the fluorescence properties and pharmacological profile of quantum dots, showing how these characteristics can be altered after incorporation within previously established drug delivery systems for the formation of novel hybrid systems. The incorporation of therapeutic agents into such hybrid systems can further result in the construction of theranostic devices, namely, QD-based theranostics. Alternatively, conjugation of quantum dots to therapeutic moieties can be used as a scaffold for theranostic device design. This chapter will discuss the current progress in QD-based theranostics.

The engineering of doxorubicin-loaded liposome-quantum dot hybrids for cancer theranostics

Many studies have recently attempted to develop multifunctional nanoconstructs by integrating the superior fluorescence properties of quantum dots (QD) with therapeutic capabilities into a single vesicle for cancer theranostics. Liposome-quantum dot (L-QD) hybrid vesicles have shown promising potential for the construction of multifunctional nanoconstructs for cancer imaging and therapy. To fulfil such a potential, we report here the further functionalization of L-QD hybrid vesicles with therapeutic capabilities by loading anticancer drug doxorubicin (Dox) into their aqueous core. L-QD hybrid vesicles are first engineered by the incorporation of TOPO-capped, CdSe/ZnS QD into the lipid bilayers of DSPC:Chol:DSPE-PEG2000, followed by Dox loading using the pH-gradient technique. The loading efficiency of Dox into L-QD hybrid vesicles is achieved up to 97%, comparable to liposome control. All these evidences prove that the incorporation of QD into the lipid bilayer does not affect Dox loading through the lipid membrane of liposomes using the pH-gradient technique. Moreover, the release study shows that Dox release profile can be modulated simply by changing lipid composition. In conclusion, the Dox-loaded L-QD hybrid vesicles presented here constitute a promising multifunctional nanoconstruct capable of transporting combinations of therapeutic and diagnostic modalities.

Development of dual-activity vectors by co-envelopment of adenovirus and SiRNA in artificial lipid bilayers.

Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.