This is the author’s version of a work that was submitted/accepted for pub-lication in the following source:

Zhao, Yufeng, Sugiyama, Sadahiro, Miller, Thomas, & Miao, Xigeng (2008)Nanoceramics for blood-borne virus removal. Expert Review of MedicalDevices, 5(3), pp. 395-405.

This file was downloaded from: http://eprints.qut.edu.au/13252/

c© Copyright 2008 Future Drugs

Notice: Changes introduced as a result of publishing processes such ascopy-editing and formatting may not be reflected in this document. For adefinitive version of this work, please refer to the published source:

http://dx.doi.org/10.1586/17434440.5.3.395

-1-

Yufeng Zhao, Sadahiro Sugiyama, Xigeng Miao, "Nanoceramics for Blood Borne Virus Removal", Expert Review of Medical Devices, accepted, 11 March 2008. Copyright 2008 Future Drugs 5 Nanoceramics for Blood Borne Virus Removal

Yufeng Zhao1, Sadahiro Sugiyama2, Xigeng Miao2*

10 1Centre for Material and Fibre Innovation, Deakin University, Geelong Campus, Victoria

3217, Australia

2Institute of Health and Biomedical Innovation & School of Engineering Systems, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia. 15

*Corresponding Author. Tel.: +61 7 3138 6237

E-mail address: x.miao@qut.edu.au

Abstract

20

The development of nanoscience and nanotechnology in the field of ceramics has brought new opportunities for the development of virus removal techniques. A number of nanoceramics, including nanostructured alumina, titania, zirconia etc. have been introduced for the applications in virus removal/separation. Filtration or adsorption of viruses and thus the removal of viruses through nanoceramics, such as nanoporous/mesoporous ceramic 25 membranes, ceramic nanofibres, and ceramic nanoparticles will make it possible to produce an efficient system for virus removal from blood and with excellent chemical/thermal stability. Currently nanoceramic membranes and filters based on sol-gel alumina membranes and NanoCeram® nanofibre filters have been commercialized and applied to remove viruses from the blood. Nevertheless, filtration using nanoprous filters is limited to the removal of 30 only free viruses in the bloodstream. Keywords: Nanoceramics, nanoporous, membranes, nanofibres, nanoparticles, viruses, blood 35

1. Introduction

The risks associated with blood and blood products contaminated with viruses, such as human immunodeficiency virus (HIV) and hepatitis, have been an ongoing concern in the 40 health care industry. As reported by the World Health Organization (WHO), HIV has infected about 39.5 million people in the world, and more than 350 million people are infected with hepatitis B virus (HBV), which kills more than one million each year due to acute and chronic hepatitis and hepatocellular carcinoma.

-2-

Numerous attempts have been made to prevent, or eliminate, viral contamination in solutions by inactivating the viruses. Viruses in many aqueous solutions can easily be inactivated by pasteurization or thermal inactivation [1], radiation [2] and/or chemical [3] sterilization. Hydrophobic interaction chromatography has also been investigated to remove viruses from a solution. However, all of these methods have a common disadvantage, i.e. the loss of the 5 protein functional activity after the treatment. Although antibody-based chromatography can avoid this, it is too specific and always leads to failure in binding the target viruses [4].

It should be noted that the problems encountered in viral inactivation in blood and protein solutions are distinct from the problems of viral inactivation in other aqueous solutions. In fact, either whole blood, or some separated blood fractions, or other protein solutions from a 10 blood are sensitive to the physical or chemical treatments. Thus, novel, safe, and effective methods for virus removal from blood are required.

Contaminated human blood, or blood from patients with HIV/ HBV, can be purified by filtration of the freely moving viruses. In this aspect, nanomaterials become increasingly important. The aim of this review is to summarise research related to the virus removal 15 methods developed to date. A particular focus is on the potential applications of nanoceramics (e.g. nanoparticles, nanofibres, nanoporous coatings or membranes) for the removal of viruses from blood.

2. Blood constituents and viruses in blood 20

2.1 Blood constituents

Blood is a living tissue composed of blood cells suspended in plasma and accounts for 7% of the human body weight, with an average density of approximately 1060 kg/m³; very close to pure water's density of 1000 kg/m3 [101]. The average adult has a blood volume of roughly 5 litres. By far the most abundant cells in blood are erythrocytes (red blood cells), which 25 constitute about 45% of the whole blood by volume. The iron-containing heme portion of hemoglobin facilitates hemoglobin-bound transportation of oxygen and carbon dioxide by selectively binding to these respiratory gasses, thereby increasing their solubility in blood. Leukocytes (white blood cells) help to resist infections and thrombocytes (platelets) are important in the clotting of blood. Plasma, which is 92% water, makes up the remaining 55% 30 of the whole blood by volume [102].

2.2 Size of Viruses and Viruses in Blood

The majority of viruses which have been studied have a capsid diameter between 10 and 300 nm (nanometres). Some filoviruses have a total length of up to 1400 nm, although their capsid diameters are only about 80 nm. A notable exception to the normal viral size range is 35 the recently discovered mimivirus, with a diameter of 750 nm, which is larger than a Mycoplasma bacterium [5,6]. The mimivirus also holds the record for the largest viral genome size, possessing about 1000 genes (some bacteria only possess 400) on a genome approximately 1.2 megabases in length [7]. The severe acute respiratory syndrome (SARS) corona virus is 80–200 nm in size while the diameter of avian flu virus is 80-120 nm [8]. 40

-3-

The blood-borne viruses are those that are transmitted through contact with infected blood, e.g. HIV and several hepatitis viruses. HIV is a retrovirus that can lead to acquired immunodeficiency syndrome (AIDS), a condition in humans where the immune system begins to fail, leading to life-threatening opportunistic infections. Hepatitis implies injury to liver characterised by presence of inflammatory cells in the liver tissue. There are various 5 types of hepatitis viruses; the blood-borne hepatitis viruses include Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), etc. Among these, Hepatitis B virus has caused current epidemics in parts of Asia and Africa [103]. The diameter of HIV virus is between 100-120 nm, and the size of HBV is approximately 40 nm, surrounded by a membrane envelope [104]. Parvovirus B19 (B19 virus) was the first (and until 2005 the only) 10 known human parvovirus. It was discovered by chance in 1975 by Australian virologist Yvonne Cossart [1]. It gained its name because it was discovered in well B19 of a large series of petri dishes apparently numbered in this way [2]. Figure 1 illustrates the size range of viruses in comparison with atoms and proteins.

15

Figure 1. Illustration of the size range of atoms, proteins, and viruses.

3. Current Methods for Virus Removal

3.1 Filtration of Viruses

Filtration of viruses from the blood, or other solutions, is believed to be an effective way to 20 remove the viruses. A porcelain filter to remove viruses from solutions was firstly developed by Charles Chamberland in the late 19th century. However it could only filter out bacteria rather than smaller viable viruses [9]. Wang et al. [201] invented an improved method and an adsorbent for implementing the method to remove the viruses. 25 Filters generally include membrane filters and depth filters [105]. Depth filters have a fibrous, granular or sintered matrix that produces a random porous structure. Particles to be removed will be trapped in the connected pore channels. The principal retention mechanisms are random adsorption and mechanical entrapment throughout the depth of the matrix. On the other hand, membrane filters are porous structures where the particles to be removed are 30 retained on the surface or within a given fraction of the membrane thickness. Most of the particles and micro-organisms larger than the pore size of the membrane filters will be retained and separated. In other words, complete removal of the virus through simple

1nm

Atoms Proteins Viruses

10nm

100n

m

1μm

HBV HIV Human

Parvovirus B19

-4-

filtration is hard to achieve, as there is always a distribution of pore sizes. For the safety reason, two orthogonal viral clearance steps are required by the regulatory authorities. Membrane filters, which are generally polymeric, screen particles using pores smaller than the particles to be removed [10]. Membrane filters are being increasingly used for the direct 5 treatment of drinking water. Over 300 large water treatment facilities located in the USA and Europe are currently based on membrane filters [11]. Depth filters, on the other hand, have a random array of irregularly shaped pores that are generally larger than the particles to be removed. Thin layers of such depth filters are generally ineffective in filtering out small particles. However, by thickening the depth filter, i.e. by using a subsequent layer to 10 counteract the larger pores of the previous layer, the average pore size will decrease as the depth filter is thickened. A depth filter is generally capable of removing approximately 85-95% of particles; however its use for complete sanitization of viruses has never been achieved due to the limitation of its porous structure. Membrane filters can be used for final filtration, whereas a depth filter can be used as a prefilter to prolong the life of a downstream 15 membrane. In other words, membrane filters and depth filters offer certain advantages and limitations and can complement each other when used together in a filtration system. The key parameters used to assess nanofiltration/ultrafiltration are permeate flux, virus LRV (log reduction value) and protein sieving coefficient. The latter two are defined as follows [12]: 20

LRV = ionconcentrat virusfeed

ionconcentrat viruspermeatelog10−

Sieving coefficient = ionconcentrat protein feed

ionconcentrat protein permeate

3.2 Filtration with organic filters 25 Due to the ease of manufacturing and the low costs, organic or plastic material-based membrane filters are commonly used nowadays for microfiltration (MF) and nanofiltration (NF). MF is the process of removing contaminants in the 0.025 to 10.0 µm range from fluids by passage through a microporous medium, such as a membrane filter. On the other hand, NF involves the filtration of minute particles using a membrane filter with extremely small 30 pores and is especially effective in the removal of non-enveloped viruses that are resistant to inactivation methods such as heat or solvent/detergent treatments [13-17]. The NF method uses a pressure driven separation process, with electrolyte selectivity dependent on the pore size provided by the membrane structure and the membrane pore potential characteristics. 35 Kang and Shah reported [18] microporous polypropylene membrane filters modified with a cationic surfactant for separating particles of about 60 nm. A class of organic polymer membranes has been commercialized owing to the low production costs [19]. Van Holten et al. [19] evaluated the ability of the ViresolveTM 180 (Millipore Corp, Bedford, MA) size-exclusion filter, to remove blood-borne pathogens over a wide range of sizes and 40 physiochemical characteristics from resuspended precipitates. It is reported that, with a nominal pore size of about 12–18 nm, this size-exclusion membrane could clear HCV (hepatitis C virus) RNA to undetectable levels in the permeate [20].

Aethlon Medical is developing a hollow-fiber hemodialysis device designed to remove viruses and toxins from blood, which include HIV and pox-viruses [21]. Another 45 commercial product for virus removal is known as Planova, developed by Asahi Kasei

-5-

Corporation. Planova is made of Bemberg Microporous Membrane (BMM) hollow fibre membranes, which are composed of naturally hydrophilic cuprammonium regenerated cellulose with a narrow pore distribution. Each membrane has a multilayered pore structure, approximately 150 layers thick, consisting of large, bulky void pores connected by fine capillary pores. Planova 35N and 15N have nominal mean pore sizes of 35 and 15 nm, 5 respectively. Planova 35N is suitable for removing viruses ranging from 35 to 100 nm, such as HIV, HCV, etc., and Planova 15N is effective for removing viruses of less than 35nm, such as parvoviruses [22].

Menon et al. [21] have developed a hollow-fiber affinity dialysis cartridge designed to efficiently remove HIV viral proteins from the blood of HIV-infected patients. More recently, 10 in order to separate the viruses from some proteins by nanofiltration, Yokoyama et al. [23] have reported some enhanced nanofiltration methods to remove the small non-lipid-enveloped viruses such as HAV or human parvovirus B19 (B19V) during the manufacture of plasma derivatives, by which the LRV after filtration of B19 in glycine reached 7·5 log.

15

4. Nanoceramics for Virus Removal Although NF and MF through organic polymer membranes have achieved great success in the viral removal, these membranes are susceptible to flux reduction because the high pressures associated with the separation processes result in a membrane compaction. Organic 20 membranes are also vulnerable to oxidizers, acids, or bases in a liquid medium. This sensitivity inhibits regeneration and/or disinfection of the membrane once fouling occurs from organic debris and inorganic scaling. More chemically resistant organic membranes have recently been introduced, but their lack of long term durability still poses problems in many scenarios [24]. 25 Inorganic membranes can offer high thermal stability, high chemical stability and excellent biocompatibility; however the high costs of manufacturing these membranes have prevented their widespread uses [25]. Fortunately, research and technological advance has alleviated the membrane manufacturing problems and reduced the manufacturing costs so that a few 30 inorganic NF membranes are now commercially available. Recently, an Australian research group led by Zhu et al. [106] has announced that HIV may one day be able to be filtered out from the human blood, thus saving the lives of millions of people infected with the virus.

Several types of nanoceramic materials with different porous structures and/or geometries will be introduced in the following sections. They have all been used, or have the potential to 35 be used, for virus removal from blood.

4.1 Nanoporous Ceramic Membranes

Ceramic membranes have attracted considerable attention in many separation processes owing to their chemical stability and thermal stability [26-28]. It is reported that these ceramic membranes are able to function unaffected within organic and biological systems, 40 are easily sterilized by using steam treatment and exhibit long operational lives [29,30]. Usually, the porous ceramic membranes have an unsymmetrical layered structure, consisting of a macroporous (pore size > 400 nm) support, a porous intermediate layer, and a nanoporous top layer [31]. Figure 2 schematically illustrates the structure of a ceramic

-6-

membrane. The selectivity of this type of membrane is controlled solely by the pore size of the top layer and the flux passing through the filter depends, predominantly, on the thickness of the top layer. Therefore, the top layer fulfils the actual separation function of the membrane [32,33]. Matsushita et al. [34] have studied the relationship between the pore size and the filtration efficiency using a bacteriophage Qβ (NBRC 20012) as the model virus, and 5 proved that the membranes with pore sizes 0.5 and 1.0 μm showed about 1 log less removal than the 0.1μm pore-size membrane.

Figure 2. A schematic illustration of a ceramic membrane.

Sol–gel technology can be used to prepare well-designed ceramic membrane separation 10 layers. Suspensions of metal oxides or so-called sols are typically used in the sol-gel technology [35]. The pore size of the membrane is controlled by the metal oxide primary particle size, the packing status of the particles, as well as the temperature used to sinter the particles. Ke et al. [8] have developed specially designed ceramic membranes for nanofiltration that have the potential to remove viruses from water, air and blood. 15

Ceramic filtration membranes are already commercially available for some time and are offered in different materials and configurations [36]. Schaep et al. [37] produced a nanoporous γ-Al2O3 membrane for nanofiltration by a sol gel dipping technique with a microporous α-Al2O3 as the support material. 20 Titania ceramic membranes have a unique no neurological toxicity, as well as higher stability in organic solvents and caustic media, compared to commercial Al2O3 membranes [38] and hence have numerous potential applications in sensitive fields, e.g. food, biotechnology and pharmaceutical industries. As early as the 1980s, Gieselmann et al. [39] produced supported titania membranes much more permeable to water than alumina 25 membranes. However, Gestel et al. [40] demonstrated the amphoteric (either acidic or basic) character of the TiO2 nanofiltration membranes, meaning the dependence of the permeability on the pH level. Titania ceramic membranes with high fluxes can be obtained using the asymmetric multiplayer configuration. 30 A wide variety of materials (alumina, titania, zirconia, etc.) can be employed to alter the chemical, electrostatic, and/or physical nature of the pore structure and the membrane surface [41]. Takagi et al. [42] developed a composite TiO2/Al2O3 membrane through a sol-gel method and studied the effect of Al2O3 support on the electrical properties of the membrane. Aust et al. [43] have used ceramic membranes for many years and have 35 synthesized TiO2/ZrO2 mixed-oxide membranes through a colloidal sol-gel process [44-46]. They reported that the mixed-oxide membranes showed a decrease of the average pore size and an increase of the specific surface area compared to the pure oxides. Meanwhile the thermal stability of the nanopores was improved, especially for Ti-rich membranes [44].

-7-

Shojai and Mäntylä [47] found an obvious weight loss of yttria-doped zirconia (3Y–ZrO2) microfiltration membranes in aqueous solutions of acid and base at room temperature (25oC) and 80oC. Chang et al. [48] made a comparative study on the thermal stability, hydrothermal stability, and chemical stability of alumina, titania and zirconia membranes, and found that the stability of the membranes varied with their compositions. Wang et al. [49], on the other 5 hand, indicated that titania membranes showed higher corrosion resistance than γ-alumina.

4.2 Nanofibre Filters

Conventional ceramic membrane filters made through sol-gel technology or by sintering of colloidal particles, encounter the problem of pinhole or crack formation, resulting in a 10 dramatic loss of filtration efficiency. The pinhole or crack problem can be absent in newly developed nanofibre filters, where the nanofibres are dispersed in a liquid medium, and allowed to form a green sheet through a filtration or sedimentation process, followed by sintering of the nanofibres. The nanofibre filters can be made to have pores smaller than ~ 5 nm, which would be effective for filtering viruses (70-1000 nm in diameter) and bacteria. 15 Various ceramic nanofibres have been recently synthesized such as alumina [50, 51], silica [52, 53], titania [54, 55], and rare-earth oxides [56], of which the dimensions can be tailored from several nanometers to hundreds of nanometers by simply adjusting the synthesis conditions. For example, the thickness of titanate fibres can be adjusted from 10 to 100 nm, and the length from 100 nm to 30 μm [54]. 20

A commercial product named NanoCeram® composed of alumina (AlOOH) fibres is electropositive and can attract and retain electronegative particles. Alumina nanofibres were developed in Argonide Corp. in Russia. Argonide immobilized the fibers into a filter and subsequently was awarded a contract with NASA to develop a filter for space cabins. The 25 filters were commercialized in January 2003 in the form of laboratory size filters for biotech applications. The filters are highly electropositive as a result of the high surface area, covered by hydroxyl groups and attract and retain pathogens and viruses that are principally electronegative. The nanoalumina fibers are approximately 2 nm in diameter and several hundred nm in length. The filters are rather resistant to clogging, particularly for small (e.g. 30 virus size) particles, having a typical surface area of 350-500 m2 per gram (see Figure 3).

35

100 nm

Formatted: Normal, Left,Line spacing: single

Formatted: Line spacing: single

-8-

Figure 3. Microimage of NanoCeram® alumina fibres [57]. NanoCeram® fibers can be combined with submicron glass fibers to form composites that will filter sub-micron and nanosize electronegative particles, such as viruses and colloids. NanoCeram® fibers have been coated onto carbon and other substrates and may provide a 5 breathable biological barrier for collective protection. Now, through collaborative research a novel fibrous depth filter, comprising alumina nanofibres and microglass has been developed that is capable of achieving >6 log retention for viruses [107]. Electroadhesively grafting of nano-alumina fibers to a microglass fiber has been studied by 10 Kaledin et al. [108]. The resulting filters are capable of at least 7 logs of virus clearance in a near 1 mm thick layer. The highly porous structure of NanoCeram® could effectively filter viruses at flow rates at least two orders of magnitude greater than membranes that have pores small enough to exclude viruses. Meanwhile, the nanofibre filters are also far more resistant to clogging by sub-micron and nanosize particles than equivalent membranes. 15 Most nanofibres of ceramics can be produced by a sol-gel process variation, with subsequent heat treatment to a cut-off temperature. Mention should be made here of the electrospinning process, which is one of the most simple and effective ways of producing one-dimensional nanostructures, such as nanofibres/nanowires, nanotubes, and nanorods. Electrospinning has 20 been commonly used for producing polymeric nanofibres. However, using the electrospining technique, Kim et al. [58] were able to produce ceramic nanofibres of hydroxyapatite and fluorhydroxyapatite. Xia et al. [59] also produced nonofibers of bioactive glasses. While these bioactive nanofibres are useful for bone tissue scaffolds, they lack chemical stability and thus may not be suitable for use as long-life nanofibre filters. Nevertheless, the 25 electrospinning has been used to produce bioinert ceramic nanofibres such as alumina, titania, etc [60] [61]. 4.3 Nanofibre/porous substrate hierarchical filters 30 To achieve ceramic membranes with high filtration efficiency, hierarchically structured nanoceramic filters consisting of both nanofibres and a porous substrate have been developed. The use of ceramic nanofibres instead of particulates with irregular shapes to fabricate ceramic membranes is a new direction in developing high-performance ceramic membranes. Ke et al. [8] constructed a hierarchically structured separation layer of randomly 35 oriented fibers (LROF) on a porous ceramic substrate as the separation layer. The hierarchically structured layer contained titanate and boehmite (AlOOH) nanofibres to improve the filtration efficiency of the ceramic membranes; on top of the titanate fiber layer, a layer of γ-alumina fibers was formed using boehmite nanofibres. The randomly oriented titanate nanofibres could completely cover the rough surface of the porous substrate of 40 micrometer α-alumina particles, leaving no pinholes or cracks (Figure 4). The resulting membranes can effectively filter out species larger than 60 nm at flow rates orders of magnitude greater than with conventional membranes, and they do not have the structural deficiencies of conventional ceramic membranes. The resulting membranes with such a structure should possess the merits of ceramic membranes, being able to withstand steam 45 cleaning and regeneration at high temperatures. These properties are crucial for the applications of the ceramic membranes [35]. It is necessary to mention that, this review tends to include the latest development in this field, this novel porous filter reported by Ke et al. has the potential to filter the virus beyond 60 nm from blood, the economics factor and the practical application is not considered yet. 50

Formatted: Line spacing: single

-9-

Figure 4. Schematic profile of the ceramic membrane with randomly orientated titanate and 5 alumina nanofibres [8]. 4.4 Nanoporous alumina from anodization of aluminium films 10 Anodized aluminium oxide (AAO) membranes with a uniform pore size have been studied for the separation of viruses [62]. Porous alumina films are formed by anodic oxidation of aluminum in an acidic solution [109]. The porous alumina contains cylindrical pores of a uniform diameter oriented perpendicular to the surface and approximately arranged in a hexagonal array (Figure 5). Porous alumina with a pore density of 1012 cm-2 and pore 15 diameters ranging from about 5 nm up to 300 nm can be produced. One advantage of the nanoporous alumina is that a high temperature process can be employed for sterilization.

Figure 5. Highly ordered nanoporous anodic alumina [109]. 20

4.5 Mesoporous ceramics Ordered mesoporous materials (pore sizes between 2 nm and 50 nm [63]) have gone through a stage of very rapid development, after the first successful synthesis of meso-structured materials by Mobil Oil R&D Corporation through supramolecular templating in 1992 [64]. 25 To date, these materials have been well accepted in electronic, optical, environmental, and biomedical applications, due to their regularly arranged 3-dimensional pores. Periodic mesoporous silicas were purposely modified to be used as absorbents for pollutants in wastewaters. Thiol-functionalized large pores in MCM-41 (an ordered mesoporous silica) can remove the metal ions such as those in actinides and methylmercury for the remarkable 30 adsorption capacity of 600 Hg/g [65]. More recently, thiolated SBA-15 (an ordered

Macroporous alumina membrane

Alumina Nanofibre

Titania Nanofibre

Formatted: Line spacing: single

Formatted: Line spacing: single

-10-

mesoporous silica) was found to exhibit high complexation affinity to mercury cations, while aminated SBA-15 showed a high binding ability to copper, zinc, chromium, and nickel cations [66]. The demand for bioapplications, such as encapsulation and separation of proteins, drove the development of ordered mesoporous silicas towards very large pore sizes of near 30 nm [67-69]. Stucky et al. [69, 70] have synthesized a series of mesoporous silicas, 5 termed SBA-n (n = 1–3, 8, 11, 12, 14–16) structures. Figure 6 shows the TEM (transmission electron microscopy) images of mesoporous SBA-15 silicas with different pore sizes.

Figure 6. TEM images of calcined hexagonal SBA-15 mesoporous silicas with different average pore sizes: (A) 60Å, (B) 89Å, and (C) 200 Å [67]. 10 The uniquely ordered porous structure and the extremely high surface area of the mesoporous ceramics provide them with highly selective and excellent absorbent abilities, which enable them to act as both specific virus filters and adsorbents potential virus removal application. In recent years, Zhao et al. [71] and Xia et al. [72] have reported hexagonally 15 ordered biocompatible mesoporous materials, i.e. SiO2-CaO-P2O5 based mesoporous bioactive glasses (MBGs), which have been investigated for drug delivery.

4.6 Ceramic Nanoparticles

Ceramic nanoparticles, as a special type of nanoceramics, provide another efficient means 20 for the virus removal from human blood. One viable method to attack HIV is to target and drug the virus using nanoparticles to assist in the removal or destruction of the virus from the human body. With a size ranging from 5 to 20nm, inorganic metal oxide nanoparticles are so “unnaturally” small that they simply cross biological barriers and diffuse into living systems [73]. In 2007, Link et al. [74] reported that the flame-spray synthesized ceramic 25 nanoparticles, such as aluminum, cerium and zirconium oxides are ready to bind nucleic acids at up to 4 plasmids per nanoparticle, without any special treatment during or after the production process. Thus, nanoparticles can carry therapeutic genes and can enter cells infected with viruses to counter-act the viruses. On the other hand, it was also found that, inorganic metal oxide nanoparticles were able to efficiently bind and sediment a variety of 30 viruses including the human-pathogenic adenovirus and HIV type 1. Another “super star” nanoceramic material is amorphous Ca3(PO4)2. The use of amorphous Ca3(PO4)2 for virus removal would offer an ultra low cost method for disinfection while exclusively using harmless substances (calcium and phosphate) [74]. When combined with a filtration technology to remove infectious prokaryotes, the unique nucleic acid and virus-binding 35 capacity of the ceramic nanoparticles may represent an affordable and straightforward way to remove key pathogenic compounds from contaminated water and ready for medical applications [74].

Formatted: Line spacing: single

-11-

5. Conclusions Nanotechnology is paving the way for us one day to physically remove viruses in the human 5 blood by using ceramic nanomembrane/ nanofibre filters or ceramic nanoparticles. These nanoceramic filters/ nanoparticles can filter out/ absorb the unwanted viruses and thus purify the blood, leading to a new and very novel way of tackling the ever increasing spread of viruses in blood. Meanwhile, the rapid development of nanotechnology allows the fine design and control of pore size/ particle size of the nanoceramic materials, which will in turn 10 create some more accurate means for the virus removal. Targeted delivery of drugs with the above mentioned nanoceramics, or sometimes coupled to biosensors, has also been recently applied to kill the viruses in a very specific way. Most of the above virus removal techniques have been used for water treatment, which could provide valuable experience for the virus removal from human blood. However, while filtration technology should be effective in 15 removing free viruses in “blood products”, whole blood cells infected with viruses are another matter. For example, HIV predominately does not just float around the bloodstream. In reality, HIV invades macrophages and T cells where it either replicates or goes latent by incorporating its sequences into the genome of theses cells. Without the destruction of macrophages and T cells there is simply no way to purge HIV from blood. In other words, 20 while one can physically remove the extracellular viruses from blood and thus mitigate the viral contamination of blood, those intracellular viruses which replicate within blood cells cannot be attached by the filtration technology. 25 6. Expert commentary & five-year view Some viruses in blood are freely moving in the plasma and other viruses are engulfed by the blood cells. Viruses can also infect other cells and the tissues in the human body. The 30 removal of free viruses in the blood is relatively easy to achieve by filtration. Due to the small sizes (in nanometers) of the viruses such as HIV and hepatitis B, the filters for the filtration must have pore sizes smaller that the sizes of the viruses. Thus nanoporous membranes and filters have been developed over the last years. While sol-gel technology has been used to produce nanoporous membranes, there is a risk of pinhole or crack formation, 35 which results in the failure of viral removal by filtration. On the other hand, nanofibres have been used to produce nanoporous membranes through nanofibre assembly and partial sintering of the packed nanofibre sheets. Nanofibre membranes have solved the problem of pinhole or crack formation, but it is difficult to prepare the nanofibres with suitable diameters and aspect ratios. Virus removal through nanoporous membranes and filters is at 40 present delivered by the manufacturer as sterile devices for single use, which demands to be tested for integrity before and after use. Failure of the test before the use of the filter is easy to deal with; failure after use forces a rework of the batch, if a validated method is available for this purpose. Besides, there is another challenge i.e. their tendency to clog because of the presence of protein aggregates in solution. Proteins are subjected to shear forces during 45 filtration, which may have some deleterious effects on protein integrity, efforts need to be made to overcome these challenges in the future study. Free viruses can also be captured by nanoparticles with a binding/ targetting capability. Nanoparticles can also be used to deliver genes or therapeutic proteins to the cells infected by the viruses. However, it is still challenging to target the infected cells and find effective therapeutic proteins. 50

Deleted: Currently, m

Deleted: While

Deleted:

Deleted: but

Deleted:

Deleted: removed

-12-

In the coming years, new and modified nanofibres will be manufactured. Based on toxicity evaluation of nanofibres, more environmentally friendly and biologically compatible nanofibres will be produced. Titania and fluorohydroxyapatite nanofibres may prove to be favourable. Some new methods may be introduced; at the moment, the nanofibres cannot be 5 aligned under control and thus nanotools may be used to do the nanofibre handling. There will be more research on the functionalization of nanoparticles for targeted delivery of drugs or genes to inactivate and damage viruses and restore the functions of virus-infected cells. Nanoparticles and nanofibre membranes will be used jointly to construct medical devices for the capture and removal of viruses from blood. Blood purification will become common 10 practice like water purification does and will form a profitable biomedical industry. Eventually, the problem of infection by HIV and hepatitis B viruses will be alleviated. The development of nanoparticles and nanofibre filters will go hand in hand with the advance of nanoscience and nanotechnology, which holds a great potential for the 21th century. 15 7. Key issues

• Human immunodeficiency virus (HIV) has infected about 39.5 million people in the 20 world, and more than 350 million people are infected with hepatitis B virus (HBV).

• Through the application of nanomaterials, contaminated human blood or blood from patients with HIV/ HBV can be purified by filtration of the freely moving viruses or by genetic modification of the living cells infected with the viruses.

• Organic membranes are susceptible to flux reduction and sensitive to chemical attack, 25 which inhibits regeneration and/or disinfection of the membranes once fouling occurs from organic debris and inorganic scaling.

• Ceramic membranes can offer high thermal stability, high chemical stability and excellent biocompatibility, which will facilitate the sterilization of the medical devices, and bring new opportunities for virus removal. 30

• A wide range of nanoceramics have been recently developed such as alumina, titania, zirconia, etc. and various forms of the nanoceramics have been applied for virus removal including nanoporous/mesoporous membranes, nanofibres, ceramic nanoparticles, etc.

• Nanoceramic composites have been introduced to improve the comprehensive 35 properties and the study of nanocomposite filters or particles will remain to be one of the research focuses in the future.

• Nanoparticles, porous membranes and nanofibre membranes will be used jointly to construct medical devices for the capture and removal of viruses from blood.

• With the development of nanoscience and nanotechnology, the size and the structure 40 of the nanoparticles, the nanofibers, and the nanoporous membranes will be better controlled for improved properties.

8. Acknowledgements 45 The authors wish to express their gratitude to Prof. Peter Damian Hodgson from the Deakin University and Prof. Huai Yong Zhu from the Queensland University of Technology for their kind support and assistance during the preparation of this review paper. 50

-13-

9. References [1] Bridonneau P, Marcilly H, Vernois M et al. Vox Sang. 1996, 70 (4), 203-209 [2] Smith RA, Ingels J, Lochemes JJ, Dutkowsky JP, Pifer LL. Gamma irradiation of HIV-1. J. Orthop. Res. 5 19 (5), 815-819 (2001). [3] Gallagher MJ, Gutsol A., Fridman A., Friedman G, Dolgopolsky A. Non-thermal plasma Applications in air sterilization. Presented at: The 31st IEEE International Conference on Plasma Science. Baltimore, MD, United States, 28 June -1 July, 2004). [4] Konz JO, Lee AL, Lewis JA, Sagar SL. Development of a purification process for adenovirus: Controlling 10 virus aggregation to improve the clearance of host cell DNA. Biotech. Prog. 21 (2), 466-472 (2005). [5] Robertson J, Gomersall M, Gill P. Mycoplasma hominis: growth, reproduction, and isolation of small viable cells. J. Bacteriol. 124 (2), 1007-1018 (1975). [6] Claverie J, Ogata H, Audic S, Abergel C, Suhre K, Fournier P. Mimivirus and the emerging concept of "giant" virus. Virus. Res. 117 (1), 133-144 (2006). 15 [7] Raoult D, Audic S, Robert C, et al. The 1,2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350 (2004). **[8] Ke XB, Zhu HY, Gao XP, Liu JW, Zheng ZF. High-Performance Ceramic membranes with a separation layer of metal oxide nanofibres. Advan. Mat. 19 (6), 785-790 (2007). [9] Horzinek MC. The birth of virology. Anton. van Leeuwen. 71(1-2), 15-20 (1997). 20 [10] Tepper F, Rivkin T, Lukasic G, Novel nanofibre filter medium attracts waterborne pathogens. Filtr. Separ. 39 (6), 16-19 (2002). [11] Gitis V, Haught RC, Clark RM, Gun J, Ley O. Nanoscale probes for the evaluation of the integrity of ultrafiltration membranes. J. Membr. Sci. 276 (1-2), 199-207 (2006). [12] Bellara SR, Cui ZF, MacDonald SL, Pepper DS, Virus removal from bioproducts using ultrafiltration 25 membranes modified with latex particle pretreatment. Biosepar, 7, 79-88 (1998). [13] Prowse C, Ludlam CA, Yap PL. Human parvovirus B19 and blood products. Vox Sang. 72 (1), 1-10 (1997). [14] Ng PK, Dobkin MB. Pasteurization of antihemophilic factor and model virus inactivation studies. Thromb. Res. 39 (4), 439-447 (1985). 30 [15] Santagostino E, Mannucci PM, Gringeri A, et al. Transmission of parvovirus B19 by coagulation factor concentrates exposed 100 degrees heat after lyophilization. Transfus. 37 (5), 517-522 (1997). [16] Horowitz B. Investigations into the application of tri(n-butyl) phosphate/detergent mixtures to blood derivatives. Curr. Stud. Hematol. Transfus. 56, 83-96 (1989). [17] Luban NL. Human parvoviruses: implications for transfusion medicine. Transfus. 34 (9), 821-827 (1994). 35 [18] Kang PK, Shah DO. Filtration of nanoparticles with dimethyldioctadecyl-ammonium bromide treated microporous polypropylene filters. Langmuir. 13 (6), 1820-1826 (1997). [19] Skluzacek JM, Tejedor MI, Anderson MA. An iron-modified silica nanofiltration membrane: Effect of solution composition on salt rejection. Microp. Mesop. Mater. 94 (1-3), 288-294 (2006). [20] Van Holten RW, Ciavarella D, Oulundsen G, Harmon F, Riester S. Incorporation of an additional viral-40 clearance step into a human immunoglobulin manufacturing process. Vox Sang. 83 (3), 227-233 (2002). [21] Menon JG, Duffin RP, Tullis RH, Jacobitz FG. Hollow-fiber cartridges: Model systems for virus removal from blood. Presented at American Society of Mechanical Engineers, New Yoprk, United States, Irvine, CA, United States, 2006. [22] Naito Y, Fukutomi I, Masada Y et al. Virus removal from hemoglobin solution using planova membrane. 45 J. Artif. Organ. 5 (2), 141-145 (2002). *[23] Yokoyama T, Murai K, Murozuka T et al. Removal of small non-enveloped viruses by nanofiltration. Vox Sang. 86 (4), 225-229 (2004). [24] Balandin AA, Bergou JA. Phonon engineering in nano-devices and virus-based nano-templates. Proceedings of SPIE, 5846, 82-91 (2005). 50 [25] Gieselmann MJ, Anderson MA, Moosemiller MD, Hill CG. Physico-chemical properties of supported and unsupported γ-Al2-O3 and TiO2 ceramic membranes. Separ. Sci. Tech. 23 (12-13) 1695-1714 (1988). [26] De Vos RM, Verweij H. High-selectivity, high-flux silica membranes for gas separation. Science 279 (5357), 1710-1711 (1998). [27] Verweij H. Ceramic membranes: morphology and transport. J. Mater. Sci. 38 (23), 4677-4695 (2003). 55 [28] Cot L, Ayral A, Durand J, Guizard C, Hovnanian N, Julbe A. Inorganic membranes and solid state sciences. Solid State Sci. 2 (3), 313-334 (2000). [29] Nunes SP, Sforça ML, Peinemann K. Dense hydrophilic composite membranes for ultrafiltration. J. Membr. Sci. 106, 49-56 (1995). [30] Baker RW. Membrane Technology and Applications, 2nd ed., Wiley, Chichester, UK, 2004. 60

-14-

*[31] Benfer S, Arki P, Tomandl G. Ceramic membranes for filtration applications-preparation and characterization. Adv. Eng. Mater. 6 (7), 495-500 (2004). [32] Harper T, Vas Roman C, Holister P. Fueling the chemical industry's future. Chem. Eng. Prog. 99(11), 34S-8S (2003). [33] Sun DH, Chang C, Li S, Lin L. Near-field electrospining, nano Lett. 6(4), 839-842 (2006). 5 **[34] Matsushita T, Matsui Y, Shirasaki N, Kato Y. Effect of membrane pore size, coagulation time, and coagulant dose on virus removal by a coagulation-ceramic microfiltration hybrid system. Desalination 178, 21-26 (2005). [35] Larbot A, Fabre JP, Guizard C, Cot L. Inorganic membranes obtained by sol–gel techniques. J. Membr. Sci. 39 (8), 203-212 (1988). 10 [36] Bhave RR, Inorganic Membranes: Synthesis, Characteristics and Applications, Van Nostrand-Rheinhold, New York, 1991. *[37] Schaep J, Vandecasteele C, Peeters B, Luyten J, Dotremont C, Roels D. Characteristics and retention properties of a mesoporous γ-Al2O3 membrane for nanofiltration, J. Membr. Sci. 163 (2) 229-237 (1999). [38] Lescoche P, Bergel JY. Filtamitm: The 100% Titania dioxide ceramic membrane, in: Akin FT, Lin YS 15 (Ed.), Proceedings of the 8th International Conferences on Inorganic Embranes (ICIM 8th-2004), Cincinnati, Ohio, USA, July, 2004. [39] Gieselmann MJ, Hill CG, Anderson MA, Moosemiller MD. Physico-chemical properties of supported and unsupported γ-Al2O3 and TiO2 ceramic membranes, Sep. Sci. Technol. 23 (12-13), 1695-1714 (1988). [40] Van Gestel T, Vandecasteele C, Buekenhoudt A. et al. Salt retention in nanofiltration with multiplayer 20 ceramic TiO2 membranes. J. Membr. Sci. 209(2), 379-389 (2002). [41] Xu Q, Anderson MA. Sol-gel route to synthesis of microporous ceramic membranes: thermal stability of TiO2-ZrO2 mixed oxides. J. Amer. Ceram. Soc. 77 (7), 1939-1945 (1994). *[42] Takagi R, Larbot A, Cotb L, Nakagaki M, Effect of Al2O3 support on electrical properties of TiO2/Al2O3 membrane formed by sol-gel method. J. Membr. Sci. 177 (1-2) 33-40 (2000). 25 *[43] Aust U, Benfer S, Dietze M, Rost A, Tomandl G. Development of microporous ceramic membranes in the system TiO2/ZrO2. J. Membr. Sci. 281 (1-2), 463-471 (2006). [44] Benfer S, Popp U, Richter H, Siewert C, Tomandl G. Development and characterization of ceramic nanofiltration membranes. Sep. Purif. Tech. 22/23 (1-3), 231-237 (2001). [45] Richter H, Piorra A, Tomandl G. Developing of ceramic membranes for nanofiltration. Key Eng. Mater. 30 132-136, 1715-1718 (1997). [46] Benfer S, Arki P, Tomandl G. Ceramic Membranes for Filtration Applications-Preparation and Characterization. Adv. Eng. Mater. 6 (7), 495-500 (2004). [47] Shojai F, Mäntylä TA. Structural stability of yttria-doped zirconia membranes in acid and basic aqueous solutions. J. Eur. Ceram. Soc. 21 (1), 37-44 (2001). 35 *[48] Chang CH, Gopalan R, Lin YS. A comparative study on thermal and hydrothermal stability of alumina, titania and zirconia membranes. J. Membr. Sci. 91(1-2), 27-45 (1994). [49] Wang YH, Tian TF, Liu XQ, Meng GY. Titania membrane preparation with chemical stability for very hash environments applications. J. Membr. Sci. 280 (1-2), 261-269 (2006). [50] Zhu HY, Riches JD, Barry C. γ-Alumina nanofibres prepared from aluminum hydrate with poly (ethylene 40 oxide) surfactant. Chem. Mater. 14 (5), 2086-2093 (2002). *[51] Zhu HY, Gao XP, Song DY et al. Growth of boehmite nanofibres by assembling nanoparticles with surfactant micelles. J. Phys. Chem. B 108 (14), 4245-4247 (2004). [52] Wang J, Zhang J, Asoo BY, Stucky GD, Structure-selective synthesis of mesostructured/ mesoporous silica nanofibres. J. Am. Chem. Soc. 125 (46), 13966-13967 (2003). 45 [53] Wang B, Chi C, Shan W et al. Chiral Mesostructured Silica Nanofibres of MCM-41. Angew. Chem. Int. Ed. 45 (13), 2088-2090 (2006). **[54] Zhu HY, Gao XP, Lan Y, Song DY, Xi YX, Zhao JC. Hydrogen titanate nanofibres covered with anatase nanocrystals: A delicate structure achieved by the wet chemistry reaction of the titanate nanofibres. J. Am. Chem. Soc. 126 (27), 8380-8381 (2004). 50 **[55] Zhu HY, Lan Y, Gao XP, Ringer SP et al. Phase transition between nanostructures of titanate and titanium dioxide via simple wet-chemical reactions. J. Am. Chem. Soc. 127 (18), 6730-6736 (2005). [56] Huang PX, Wu F, Zhu BL et al. CeO2 nanorods and gold nanocrystals supported on CeO2 nanorods as catalyst. J. Phys. Chem. B. 109(41), 19169-19174 (2005). [57] Tepper F, Lerner M, Ginley D. Nanosized Alumina Fibers, Amer. Ceram. Soc. Bull., 80 (6) 57-60 (2001).. 55 *[58] Kim HW, Kim HE, Nanofibre generation of hydroxyapatite and fluor-hydroxyapatite bioceramics, J. Biomed. Mater. Res. Part B Appl. Biomater. 77(B), 323-328 (2006). *[59] Xia W, Zhang D, Chang J. Fabrication and in vitro biomineralization of bioactive glass (BG) nanofibres, Nanotechnology 18 135601 (7pp) (2007). [60] Li D., Xia Y. Fabrication of titania nanofibers by electrospinning, Nano Letters, 3, 555-560 (2003) 60

-15-

[61] Li D., McCann J.T., Xia Y. Electrospinning: A simple and versatile technique for producing ceramic nanofibers and nanotubes, J. Am. Ceram. Soc., 89 (6) 1861-1869 (2006) [62] Urase T, Yamamoto K, Ohgaki S. Effect of pore structure of membranes and module configuration on virus retention, J. Membr. Sci. 115 (1), 21-29 (1996). [63] IUPAC Manual of Symbols and Terminology, Appendix 2, Part I, Colloid and Surface Chemistry. Pure 5 Appl. Chem. 31 578-563 (1972). [64] Selvam P, Bhatia SK, Sonwane CG. Recent advances in processing and characterization of periodic mesoporous MCM-41 silicate molecular sieves. Ind. Eng. Chem. Res. 40 (15), 3237-3261 (2001). [65] Feng X, Fryxell GE, Wang LQ, Kim AY, Liu J, Kemner KM, Functionalized monolayers on ordered mesoporous supports. Science 276 (5314), 923-926 (1997). 10 [66] Liu AM, Hidajat K, Kawi S, Zhao DY. A new class of hybrid mesoporous materials with functionalized organic monolayers for selective adsorption of heavy metal ions. Chem. Commun. 13, 1145-1146 (2000). [67] Zhao DY, Feng JL, Huo QS et al. Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 Angstrom pores. Science 279 (5350), 548-552 (1998). [68] Zhao DY, Huo QS, Feng JL, Chmelka BF, Stucky GD. Nonionic triblock and star diblock copolymer and 15 oligomeric surfactant syntheses of highly ordered, hydrothermally stable, mesoporous silica structures. J. Am. Chem. Soc. 120 (24), 6024-6036 (1998). [69] Fan J, Yu C, Lei J, Zhang Q et al. Low-temperature strategy to synthesize highly ordered mesoporous silicas with very large pores. J. Am. Chem. Soc. 127(31), 10794-10795 (2005). [70] Yasuhiro S, Isabel D, Osamu T et al. Three-dimensional cubic mesoporous structures of SBA-12 and 20 related materials by electron crystallography, J. Phys. Chem. B 106 (12), 3118-3123 (2002). *[71] Zhao YF, Loo SCJ, Chen YZ., Boey FYC, Ma J. In situ SAXRD study of sol-gel induced well-ordered mesoporous bioglasses for drug delivery. J. Biomed. Mater. Res. A , Published Online: 15 Oct 2007, DOI: 10.1002/jbm.a.31545. [72] Xia W., Chang J. Well ordered mesoporous bioactive glasses (MBG)：a promising bioactive drug 25 delivery system, J. Control. Rel. 110 522-530 (2006) [73] Limbach LK, Li Y, Grass RN et al. Oxide nanoparticle uptake in human lung fibroblasts: Effects of particle size, agglomeration, and diffusion at low concentrations. Environ. Sci. Technol. 39 (23), 9370-9376 (2005). **[74] Link N, Brunner TJ, Dreesen IAJ, Stark WJ, Fussenegger M. Inorganic nanoparticles for transfection of 30 mammalian cells and removal of viruses from aqueous solutions. Biotech. Bioeng. 98 (5), 1083-1093 (2007). Websites [101] Elert G. Density of Blood. The Physics Factbook, 2004 <http://hypertextbook.com/facts/2004/MichaelShmukler.shtml> 35 [102] Wikipedia, the free encyclopedia, <http://en.wikipedia.org/wiki/Blood> [103] Clostridium, Wikipedia, the free encyclopedia,< http://en.wikipedia.org/wiki/Clostridium> [104] Virus, Wikipedia, the free encyclopedia, <http://en.wikipedia.org/w/index.php?title=Virus&oldid=158038916> [105] Tepper F, Kaledin L, Nano Fiber Biological Filter, Argonide Corp., 40 http://www.ssc.army.mil/soldier/jocotas/ColPro_Papers/Tepper.pdf [106] Foresight Nanotech Institute Weekly News Digest: 2007, <http://www.foresight.org/publications/weekly0088.html#challenge3c> [107] Tepper F, Kaledin L. Virus and protein separation using nano alumina fiber media. Argonide.com. <www.argonide.com/Paper%20PREP%2007-final.pdf> 45 [108] Fabrication of highly ordered nanoporous anodic alumina and template synthesis of nanomaterials. http://www.pcpm.ucl.ac.be/themes/template.php [109] http://www.pcpm.ucl.ac.be/themes/template.php Patents 50 [201] Henry Y, Wang IFT. Cellular adsorbents for removal of viral contaminants from blood and complex protein solutions. US4869826 (1989).

Formatted: Line spacing: single