Chapter 4
Cellulose Nanocrystals for Drug Delivery Downloaded by DUKE UNIV on June 26, 2012 | http://pubs.acs.org Publication Date (Web): December 20, 2009 | doi: 10.1021/bk-2009-1017.ch004
Maren Roman1, Shuping Dong1, Anjali Hirani2, and Yong Woo Lee2 1
Macromolecules and Interfaces Institute and Department of Wood Science and Forest Products, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 2 Department of Biomedical Sciences and Pathobiology, School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Since the first investigation of liposomes as drug carrier systems in chemotherapy, nanoscale carrier systems have attracted increasing attention in therapeutic and diagnostic medicine. Cellulose nanocrystals have attractive properties as nanoscale carriers for bioactive molecules in biomedical applications. To test whether cellulose nanocrystals could be used as carriers in the targeted delivery of therapeutics, their toxicity to human brain microvascular endothelial cells was measured. Cellulose nanocrystals were found to be non-toxic to the cells. For cellular uptake studies, cellulose nanocrystals were labeled with fluorescein-5'-isothiocyanate. The uptake studies showed minimal uptake of untargeted cellulose nanocrystals. The lack of toxicity and untargeted uptake support the potential of cellulose nanocrystals as carriers in targeted drug delivery applications.
© 2009 American Chemical Society In Polysaccharide Materials: Performance by Design; Edgar, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.
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Introduction Since the first investigation of liposomes as drug carrier systems in chemotherapy in 1974 (1), nanoscale carrier systems have attracted increasing attention in therapeutic and diagnostic medicine. In search of the “magic bullet”, an expression coined by Nobel laureate Paul Ehrlich in the early 1900s, many different types of nanoscale systems are under evaluation, including metal, inorganic, and polymer nanoparticles, quantum dots, carbon nanotubes, polymer micelles, dendrimers, and liposomes (2). To achieve the desired benefits, nanoscale carrier systems have to be non-toxic, biodegradable, able to overcome the physiological barriers in the body, and able to withstand the immune system long enough to carry out their mission. Neither of the currently studied systems is optimal. Frequently encountered problems include toxicity, toxic degradation products, low stability in the bloodstream, and accumulation over time in certain organs such as the kidneys, liver, or spleen. According to Couvreur and Vauthier, “we are still far from the magic bullet”(2). This study investigates a novel nanoscale carrier system that is based on an abundant, benign biopolymer, namely cellulose, and that has great potential to be the “magic bullet” for nanobiomedicine. Cellulose nanocrystals are ideally suited as nanoscale carriers for bioactive molecules in biomedical applications due to the following attributes: (i) Cellulose is biocompatible and does not trigger an immune response when embedded in bodily tissue (3). (ii) Cellulose nanocrystals are rodlike and have a size range between 50 and 200 nm with the majority of the particles between 100 and 150 nm long. Thus, they are too large for removal from the bloodstream by the renal system (i.e. the kidneys) but still small enough that the rate of clearance from the bloodstream by the mononuclear phagocytic system is sufficiently delayed (4). (iii) Being entirely composed of polysaccharide molecules, cellulose nanocrystals are highly hydrophilic in nature. A hydrophilic surface has been shown to impede adsorption of opsonin proteins, a critical step before phagocytosis during removal of nanoparticles from the bloodstream (5). Thus, cellulose nanocrystals are expected to have an inherently prolonged blood circulation half-life as compared to hydrophobic nanoparticles. (iv) The surface chemistry of cellulose nanocrystals is governed by hydroxyl groups, which can be easily converted into other functional groups for covalent and non-covalent binding of bioactive molecules at high densities to the surface of the nanoparticles. Also noteworthy in this context is the fact that cellulose can be broken down by certain fungal and bacterial enzymes to glucose (6, 7), a readily metabolized biochemical. Thus, though not an endogenous compound per se, it might be possible to achieve endogenous removal of cellulose from the body through e.g. systemic administration of cellulolytic enzymes and in situ degradation to glucose. Many human diseases, including Alzheimer’s disease, hypertension, and type 2 diabetes, are associated with an inflammation of the blood vessels (8, 9). Vascular inflammation in the brain plays a role in the pathogenesis of multiple sclerosis and traumatic brain injury, to name a few (10, 11). Inflammation of the
In Polysaccharide Materials: Performance by Design; Edgar, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.
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blood vessels is characterized by a dysfunction of vascular endothelial cells, the cells that line the inside of the blood vessels. The presented work is part of a larger effort to develop a drug delivery system that selectively targets inflammation-activated human brain microvascular endothelial cells (HBMECs) in the therapy of cerebrovascular inflammatory diseases. Here we report the results of our preliminary studies on the in vitro effect of cellulose nanocrystals on resting, non-activated HBMECs. To test whether cellulose nanocrystals exhibit any toxicity towards HBMECs, the cytotoxicity of cellulose nanocrystals was measured by MTT assay. Cellular uptake studies, using nanocrystals labeled with fluorescein-5'-isothiocyanate (FITC), were carried out to assess the uptake of untargeted cellulose nanocrystals.
Materials and Methods Preparation of Cellulose Nanocrystals Cellulose nanocrystals were prepared by sulfuric acid hydrolysis of dissolving-grade softwood sulfite pulp. Lapsheets of the pulp (Temalfa 93A-A), kindly provided by Tembec, Inc., were cut into small pieces of approximately 1 cm by 1 cm and milled in a Wiley mill (Thomas Wiley Mini-Mill) to pass a 60 mesh screen. The milled pulp was hydrolyzed under stirring with 64 wt % sulfuric acid (10 mL/g cellulose) at 45 °C for 60 min. The hydrolysis was stopped by diluting the reaction mixture 10-fold with cold (~4 °C) deionized water (Millipore Direct-Q 5, 18.2 MΩ·cm). The nanocrystals were collected and washed once with deionized water by centrifugation for 10 min at 4 °C and 4,550 × g (Thermo IEC Centra-GP8R) and then dialyzed (Spectra/Por 4 dialysis tubing) against deionized water until the pH of fresh dialysis medium stayed constant over time. The nanocrystal suspension was sonicated (Sonics & Materials Model VC-505) for 10 min at 200 W under ice-bath cooling and filtered through a 0.45 μm polyvinylidene fluoride (PVDF) syringe filter (Whatman) to remove any aggregates present. Immediately prior to use, the cellulose nanocrystal suspension was sterilized by filtration through a 0.2 μm PVDF syringe filter (Whatman). Characterization of Cellulose Nanocrystals by Atomic Force Microscopy Atomic force microscopy (AFM) was performed with an Asylum Research MFP-3D mounted onto an Olympus IX 71 inverted fluorescence microscope. One drop of a 0.001 wt % suspension of cellulose nanocrystals in water was deposited onto a microscopy slide and allowed to dry in air under ambient conditions. Samples were scanned in intermittent contact mode in air with Olympus OMCL-AC160TS tips (nominal tip radius:
